EP4698224A1 - Dérivés de saponine en tant qu'adjuvants et sondes de photoaffinité - Google Patents

Dérivés de saponine en tant qu'adjuvants et sondes de photoaffinité

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Publication number
EP4698224A1
EP4698224A1 EP24720517.2A EP24720517A EP4698224A1 EP 4698224 A1 EP4698224 A1 EP 4698224A1 EP 24720517 A EP24720517 A EP 24720517A EP 4698224 A1 EP4698224 A1 EP 4698224A1
Authority
EP
European Patent Office
Prior art keywords
compound
pharmaceutically acceptable
carbamate
certain embodiments
acceptable salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24720517.2A
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German (de)
English (en)
Inventor
Alberto FERNÁNDEZ TEJADA
Abhijit Saha
Priscila TONON
Juan Anguita
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centro de Investigacion Cooperativa en Biociencias
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Centro de Investigacion Cooperativa en Biociencias
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Application filed by Centro de Investigacion Cooperativa en Biociencias filed Critical Centro de Investigacion Cooperativa en Biociencias
Publication of EP4698224A1 publication Critical patent/EP4698224A1/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response

Definitions

  • the present application is encompassed within the field of chemical immunology. More specifically, it relates to synthetic adjuvants and vaccines based on a triterpene glycoside saponin scaffold and pharmaceutical compositions thereof as well as the use of said compounds and compositions in the treatment of and immunization for diseases such as neurodegenerative and infectious diseases and cancers.
  • the compounds are also useful as chemical probes in photoaffinity labeling (PAL) experiments.
  • Vaccines have been approved and have improved health care over the last several decades. Attenuated or inactivated pathogens and their toxins have been historically used as vaccines. Modern subunit vaccines based on homogeneous antigens offer more precise targeting and improved safety compared with traditional whole-pathogen vaccines. However, they are also less immunogenic and require an adjuvant to increase the immunogenicity of the antigen and potentiate the immune response (Pifferi, C. et al. Nat. Rev. Chem. 2021 , 25 (4), 3-7). Adjuvants enhance antigen-specific immune responses by modulating and enhancing the innate and adaptive (acquired) immunity when delivered together with an antigen (Bergmann-Leitner, E. et al.
  • Vaccines 2014, 2 (2), 252-296 allow the dose of expensive antigens to be decreased, reduce booster immunizations, generate more rapid and durable immune responses, and increase the effectiveness of vaccines in poor responders (Reed, S. G. et al. Nat. Med. 2013, 19 (12), 1597-1608).
  • Aluminum-containing adjuvants were the first human vaccine adjuvants approved in clinical use.
  • Aluminum salts, either alone (alum) or in proprietary mixtures (AS04), and oil-in- water emulsions containing squalene (MF59, AS03) have been used as adjuvants in a number of vaccines, but have relatively low potency and significant side effects, respectively.
  • QS-21 is one of the most promising adjuvants currently under investigation. Isolated from Quillaja saponaria tree bark, it is composed of four structural domains: a branched trisaccharide, a quillaic acid triterpene, a bridging linear tetrasaccharide, and a pseudodimeric acyl chain.
  • QS-21 is not a single molecule but a ⁇ 2:1 mixture of two isomers that differ at the terminal sugar of the linear tetrasaccharide domain, the first isomer having a terminal apiose (QS-21-Api) and the second one having a xylose (QS-21-Xyl).
  • Vaccines that contain QS-21 have been investigated or are under development for several cancers, and for infectious and neurodegenerative diseases (malaria, acquired immunodeficiency syndrome, hepatitis, tuberculosis and Alzheimer’s disease).
  • infectious and neurodegenerative diseases malaria, acquired immunodeficiency syndrome, hepatitis, tuberculosis and Alzheimer’s disease.
  • QS-21 suffers from several liabilities, including limited access from its natural source, toxic side effects and chemical instability through spontaneous hydrolysis of the acyl chain.
  • the scarcity, heterogeneity and dose-limiting toxicity of QS-21 have hampered its further use in human vaccines.
  • Triterpene glycoside saponin-derived adjuvants are disclosed in W02009/126737, WO2015/184451 , WO2017/079582, WO2017/106836, WO2018/191598, WO2018/200645 WO2018/200656 and WO2019/079160.
  • PAL photoaffinity labeling
  • photoaffinity probes involves the incorporation of three important functionalities; an affinity/specificity unit, in other words, the small molecule of interest, a photoreactive moiety and an identification/reporter tag.
  • the specificity unit is responsible for reversible binding to target proteins, the photoreactive moiety allows photo-inducible permanent attachment to targets, and the identification component is vital for the detection and isolation of probe-protein adducts.
  • Three types of photoactivating probes are mainly used in photoaffinity labeling: azides, benzophenones and diazirines; whereas biotin is commonly used as identification tag.
  • the probe-protein complex can be isolated via HPLC and analyzed by MS (Yu W et al. Curr Opin Chem Biol. 2022;69:102173; Kozoriz K et al. Acc. Chem. Res. 2023;56(1):25-36).
  • the present invention solves one or more of the aforementioned needs by the provision of new triterpene glycoside saponin adjuvants.
  • the active compounds of the present invention have found to induce significantly higher anti-OVA antibody titers and/or reduced toxicity than the natural QS-21.
  • the saponin compounds provided may be used as chemical probe in photoaffinity labeling (PAL) experiments since they bear an acyl chain comprising a photoreactive moiety selected from diazirine, benzophenone and azido as well as a terminal biotin that can be used as identification/reporter tag.
  • PAL photoaffinity labeling
  • One aspect of the present invention relates to a compound of general formula (I) or a pharmaceutically acceptable salt thereof wherein G is hydrogen, a branched trisacchride of formula (VI) or a stereoisomer of formula (VI) wherein each occurrence of R p is independently hydrogen or OR q ; wherein each occurrence of R q is independently hydrogen or an optionally substituted group selected from 6-10-membered aryl, benzyl, Ci-e aliphatic, or Ci-e heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; or two R q are taken together to form a 5-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur;
  • — is a single or double bond
  • V is H or OR X ; wherein R x is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates;
  • PRG is a moiety comprising a photoreactive group selected from diazirine, benzophenone and azido or a combination thereof;
  • Linker is a chemical spacer;
  • Biotin is a biotin moiety.
  • Another aspect of the present invention relates to a method of synthesizing a compound of general formula (I) or a pharmaceutically acceptable salt thereof which comprises reacting: wherein G, — , II and V take the meanings as previously defined in formula (I); with wherein PRG, linker and biotin take the meanings as previously defined in formula (I).
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of general formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier and an antigen.
  • Another aspect of the present invention relates to a compound of general formula (I) or a pharmaceutically acceptable salt thereof for use in medicine.
  • Another aspect of the present invention relates to a compound of general formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof for use in the treatment and/or prevention of cancer, an infectious disease or a neurodegenerative disease.
  • Another aspect of the present invention relates to a compound of general formula (I) or a pharmaceutically acceptable salt thereof for use as chemical probe in photoaffinity labeling (PAL) experiments.
  • PAL photoaffinity labeling
  • Figure 1 shows the toxicity assessment in mice by median weight loss after injection of OVA or one of adjuvants tested: QS-21 , BTS-25-BP and BTS-31-DAz.
  • Figure 2 shows antibody responses in mice induced by adjuvant BTS-31-DAz upon coadministration with OVA antigen.
  • A-B ELISA standard curves showing total anti-OVA IgG levels (represented as optical density [OD] measurements) in mouse sera on (A) day 21 and (B) day 35.
  • C-D Anti-OVA total IgG titers on (C) day 21 and (D) day35. Data points correspond to individual mice (five animals per group) and horizontal bars indicate median titers.
  • Figure 3 shows IgG subtyping of anti-OVA antibodies at day 35 resulting from mouse vaccinations with adjuvant BTS-31-DAz coadministered with OVA antigen.
  • A-C ELISA standard curves showing anti-OVA IgG subtype levels (represented as optical density [OD] measurements) of (A) I gG 1 , (B) lgG2b, and (C) lgG2c (d) lgG3 antibodies on day 28 after first immunization.
  • Statistical significance across the different dose-response curves was assessed by comparing to the no-adjuvant (OVA alone) control group using two-way ANOVA Dunnett’s multiple comparisons test at the various dilutions.
  • Vaccines containing QS-21 either alone in purified form or as a major component of adjuvant mixtures (e.g., Quil A, ISCOMs, ISCOMATRIX, AS01 , AS02), have been investigated in clinical trials for cancers (melanoma, sarcoma, breast, prostate, ovarian, lung), infectious diseases (hepatitis, HIV, malaria, tuberculosis) and Alzheimer’s disease.
  • adjuvant mixtures e.g., Quil A, ISCOMs, ISCOMATRIX, AS01 , AS02
  • QS- 21 suffers from several limitations.
  • access to homogeneous QS-21 is limited due to an exceedingly low-yielding isolation and heterogeneity of crude extracts from Quillaja saponaria.
  • QS-21 is associated with clinical toxicity including swelling and erythema at the injection site, and systemic flu-like symptoms.
  • QS-21 undergoes spontaneous hydrolysis of the acyl chain domain ester linkages, producing adjuvant-inactive and hemolytic byproducts, complicating formulation and storage.
  • the mechanisms of action of QS-21 are poorly understood, hindering rational design of improved variants and optimal matching of adjuvants with vaccine antigens based on desired immunological end points.
  • the inherent liabilities of QS-21 highlight the need for improved analogues.
  • the compounds of the invention may be suitable for carrying out photoaffinity labeling (PAL) experiments, which is a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites.
  • PAL photoaffinity labeling
  • aliphatic or "aliphatic group” as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not cyclic (also referred to herein as "carbocycle,” “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms.
  • aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms.
  • cycloaliphatic refers to a monocyclic C3- Ce hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.
  • Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • lower alkyl refers to a C1.4 straight or branched alkyl group.
  • exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.
  • heteroatom means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2/7-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl)).
  • unsaturated means that a moiety has one or more units of unsaturation.
  • acyl used alone or a part of a larger moiety, refers to groups formed by removing a hydroxy group from a carboxylic acid.
  • halogen means F, Cl, Br, or I.
  • aralkyl and “arylalkyl” are used interchangeably and refer to alkyl groups (e.g. C1.6 alkyl) in which a hydrogen atom has been replaced with an aryl group (e.g. 6-10- membered aryl).
  • aryl group e.g. 6-10- membered aryl.
  • groups include, without limitation, benzyl, cinnamyl, and dihyrocinnamyl.
  • aryl used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic or bicyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members.
  • aryl may be used interchangeably with the term “aryl ring”.
  • aryl refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents.
  • aryl is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
  • heteroaryl and “heteroar-” used alone or as part of a larger moiety refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 TT electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms.
  • heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
  • heteroaryl and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
  • Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4/7-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1 ,4-oxazin- 3(4/-/)-one.
  • heteroaryl group may be mono- or bicyclic.
  • heteroaryl may be used interchangeably with the terms “heteroaryl ring", “heteroaryl group” or “heteroaromatic” any of which terms include rings that are optionally substituted.
  • heteroarylkyl and heteroarylalkyl refer to an alkyl group substituted by a heteroaryl moiety, wherein the alkyl and heteroaryl portions independently are optionally substituted.
  • heteroaliphatic as used herein, means aliphatic groups wherein one or two carbon atoms are independently replaced by one or more of oxygen, sulfur, nitrogen, or phosphorus. Heteroaliphatic groups may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and include “heterocycle”, “heterocyclyl”, “heterocycloaliphatic” or “heterocyclic” groups.
  • heterocycle As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical” and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
  • nitrogen includes a substituted nitrogen.
  • the nitrogen may be N (as in 3,4-dihydro-2/7-pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in N-substituted pyrrolidinyl).
  • a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
  • saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
  • heterocycle refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
  • the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond.
  • the term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation but is not intended to include aryl or heteroaryl moieties, as herein defined.
  • the present invention provides "pharmaceutically acceptable" compositions, which comprise a therapeutically effective amount of one or more of the compounds described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • the pharmaceutical compositions of the present invention may be specially formulated for administration by injection.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and
  • the term "pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
  • pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, tertiary, or quaternary amine.
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (Ci-4alkyl)4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • Organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra).
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each stereocenter, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • Provided compounds may comprise one or more saccharide moieties. Unless otherwise specified, both D- and L-configurations, and mixtures thereof, are within the scope of the invention. Unless otherwise specified, both a- and p-linked embodiments, and mixtures thereof, are contemplated by the present invention. If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, chiral chromatography, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • diastereomeric salts are formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
  • protecting group it is meant that a particular functional moiety, e.g., O, S, or N, is masked or blocked, permitting, if desired, a reaction to be carried out selectively at another reactive site in a multifunctional compound.
  • a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group is preferably selectively removable by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms a separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group will preferably have a minimum of additional functionality to avoid further sites of reaction.
  • oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.
  • Suitable carboxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.
  • hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p- AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy) methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiomethyl, methyl, methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxy
  • the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4- methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p- methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1 -methoxyethyl
  • Amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2 , 7-d i-f-b uty l-[9- ( 10, 10-dioxo-10, 10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1- adamantyl)-1 -methylethyl carbamate (Adpoc), 1 ,1-dimethyl-2-haloethyl
  • protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the method of the present invention. Additionally, a variety of protecting groups are described by Greene and Wuts (supra). As described herein, compounds of the invention may contain "optionally substituted” moieties. In general, the term “substituted” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an "optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen; -(CH2)o-4R°; -(CH2)o-40R°; -0(CH2)o-4R°, -O- (CH 2 )O-4C(0)OR°; -(CH 2 )O-4CH(OR°) 2 ; -(CH 2 ) o.
  • each R° may be substituted as defined below and is independently hydrogen, C1.6 aliphatic, -CH2Ph, -0(CH2)o-iPh, -CH2-(5-6-membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences
  • Suitable monovalent substituents on R° are independently halogen, -(CH 2 )o- 2 R A , -(haloR A ), -(CH 2 )O- 2 OH, -(CH 2 ) 0-2 OR A , -(CH 2 ) 0.2 CH(OR A ) 2 ; -O(haloR A ), -CN, -N 3 , -(CH 2 ) 0 .
  • each R A is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from Ci- 4 aliphatic, -CH 2 Ph, -0(CH 2 )o-iPh, or a 5- 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted” group include: -O(CR* 2 ) 2 .3O-, wherein each independent occurrence of R* is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of R* include halogen, -R A , -(haloR A ), -OH, -OR A , -O(haloR A ), -CN, -C(O)OH, -C(O)OR A , -NH 2 , -NHR A , -NR A 2 , or -NO 2 , wherein each R A is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci- 4 aliphatic, -CH 2 Ph, -0(CH 2 )o-i Ph, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -Rt, -NR ⁇ , -C(O)Rt, -C(O)ORt, -C ⁇ C ⁇ R -C ⁇ CH ⁇ O ⁇ , -S(O) 2 R t , -S(O) 2 NR t 2 , -C(S)NR t 2 , -C(NH)NR t 2 , or -N(R t )S(O) 2 R t ; wherein each R f is independently hydrogen, Ci- 6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R f , taken together with their intervening atom(s) form an unsubstitute
  • Suitable substituents on the aliphatic group of R f are independently halogen, -R A , -(haloR A ), -OH, -OR A , -O(haloR A ), -CN, -C(O)OH, -C(O)OR A , -NH 2 , -NHR A , -NR A 2 , or- NO 2 , wherein each R A is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C1.4 aliphatic, -CH 2 Ph, -0(CH 2 )o-i Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • Liposomes refer to closed bilayer membranes containing an entrapped aqueous volume. Liposomes may also be uni-lamellar vesicles possessing a single membrane bilayer or multi-lamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase.
  • Liposomes as they are ordinarily used, consist of smectic mesophases, and can consist of either phospholipid or nonphospholipid smectic mesophases. Smectic mesophase is most accurately described by Small, HANDBOOK OF LIPID RESEARCH, Vol. 4, Plenum, NY, 1986, pp. 49-50. According to Small, "[w]hen a given molecule is heated, instead of melting directly into an isotropic liquid, it may instead pass through intermediate states called mesophases or liquid crystals, characterized by residual order in some directions but by lack of order in others... In general, the molecules of liquid crystals are somewhat longer than they are wide and have a polar or aromatic part somewhere along the length of the molecule.
  • the molecular shape and the polarpolar, or aromatic, interaction permit the molecules to align in partially ordered arrays... These structures characteristically occur in molecules that possess a polar group at one end.
  • Liquid crystals with long-range order in the direction of the long axis of the molecule are called smectic, layered, or lamellar liquid crystals... In the smectic states the molecules may be in single or double layers, normal or tilted to the plane of the layer, and with frozen or melted aliphatic chains.”
  • enriched refers to a mixture having an increased proportion of one or more species.
  • the mixture is "enriched” following a process that increases the proportion of one or more desired species in the mixture.
  • the desired species comprise(s) greater than 10% of the mixture.
  • the desired species comprise(s) greater than 25% of the mixture.
  • the desired species comprise(s) greater than 40% of the mixture.
  • the desired species comprise(s) greater than 60% of the mixture.
  • the desired species comprise(s) greater than 75% of the mixture.
  • the desired species comprise(s) greater than 85% of the mixture.
  • the desired species comprise(s) greater than 90% of the mixture.
  • the desired species comprise(s) greater than 95% of the mixture.
  • Such proportions can be measured any number of ways, for example, as a molar ratio, volume to volume, or weight to weight.
  • a target compound refers to compounds that are substantially free of compounds of related non-target structure or chemical precursors (when chemically synthesized). This quality may be measured or expressed as "purity.”
  • a target compound has less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, and 0.1 % of non-target structures or chemical precursors.
  • the present application provides compounds of general formula (I) or a pharmaceutically acceptable salt therof which are useful as adjuvants and as photoaffinity probes.
  • the present application provides compounds of formula (I) or a pharmaceutically acceptable salt thereof wherein G is hydrogen or In certain embodiments, G is hydrogen. In certain embodiments, G is p-D-xylose
  • V is H or OH. In certain embodiments, V is H. In certain embodiments, V is OH.
  • — is a single or double bond
  • V is H or OR X ; and R x is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates.
  • — is a single or double bond
  • V is H or OH.
  • G is hydrogen or
  • — is a single or double bond
  • V is H or OH.
  • G is hydrogen or or
  • — is a single or double bond
  • V is OH.
  • G is
  • — is a single or double bond
  • V is OH.
  • G is
  • V is OH.
  • PRG represents a moiety comprising a photoreactive group selected from diazirine, benzophenone and azido or a combination thereof.
  • PRG is a moiety comprising a photoreactive group selected from an aryldiazirine, a benzophenone, an arylzide or a combination thereof.
  • PRG is a moiety comprising a photoreactive group selected from a phenyldiazirine, a benzophenone, a phenylazide or a combination thereof.
  • PRG is a moiety comprising a photoreactive group selected from diazirine including alkyl and aryl diazirines (e.g. phenyl diazirines) such as:
  • PRG is a moiety comprising a photoreactive group selected from a benzophenone such as:
  • PRG is a moiety comprising a photoreactive group selected from azido, preferably an aryl azide such as a phenyl azide.
  • phenyl azide is: wherein Y is independently selected from halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF 3 , CC , -CH2-CH3, -CH2-OH, -CH2NH2, CH 2 SH, CH2CI, CH 2 Br, CH 2 F, CHF 2 , CH 2 CN, CH2CF3, CH2CCI3, and CN. More particularly, Y is independently selected from F and CH3.
  • phenyl azides include, but are not limited to:
  • linker represents a chemical spacer, that is, a carbon chain, which may include other elements, that covalently attaches PRG and Biotin.
  • the linker is or comprises (CH2)I-2O or is a divalent PEG-based spacer, preferably a divalent PEG-based spacer comprising formula wherein n is a positive integer preferably selected from 1 to 20, more preferably 1 to 10 and even more preferably 1 to 5 such as 1 , 2, 3, 4 or 5.
  • n is 2, 3 or 4.
  • n is 2 or 4.
  • biotin represents a biotin moiety, preferably.
  • BTS-31-DAz and BTS-25- BP may be synthesized in total 30 and 27 steps starting from the semi-purified bark extract Quil A as shown in the experimental part.
  • Another aspect of the present invention relates to a method of synthesizing a compound of general formula (I) or a pharmaceutically acceptable salt thereof which comprises reacting: wherein G, — , II and V take the meanings as previously defined in formula (I); with wherein PRG, linker and biotin take the meanings as previously defined in formula (I).
  • Another aspect of the present invention relates to a method of synthesizing BTS-31-DAz or a pharmaceutically acceptable salt thereof which comprises at least one of the steps disclosed in the experimental part. In a particular embodiment, the method comprises reacting compounds 22 and 30.
  • Another aspect of the present invention relates to a method of synthesizing BTS-25- BP or a pharmaceutically acceptable salt thereof which comprises at least one of the steps disclosed in the experimental part.
  • the method comprises reacting compounds 22 and 24.
  • the pharmaceutical composition comprises a compound or a pharmaceutically acceptable salt thereof according to the present invention such as BTS-31-DAz or BTS-25-BP, a pharmaceutically acceptable carrier and optionally an antigen.
  • the pharmaceutical composition comprises one or both of BTS- 31-DAz and BTS-25-BP or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier and an antigen.
  • the term “antigen” refers to a substance that is able to generate a specific immune response and induce the formation of specific antibodies or specially sensitized T cells or both.
  • the antigen is therefore capable of activating lymphocytes, and as such, is a complete antigen, i.e., possesses antigenic properties de novo, being able to generate an immune response by themselves.
  • Said antigen is characterized by a molecular mass above 14 kDa, having a complex chemical composition and ideally contains aromatic radicals.
  • Said antigens belong to four main groups, proteins, polysaccharides, nucleic acids and lipids, preferably proteins.
  • an "immune response" to an antigen or immunogenic composition is the development in a subject of a humoral and/or a cell-mediated immune response to molecules present in the antigen or vaccine composition of interest.
  • a "humoral immune response” is an antibody-mediated immune response and involves the induction and generation of antibodies that recognize and bind with some affinity for the antigen in the immunogenic composition of the invention, while a "cell-mediated immune response” is one mediated by T-cells and/or other white blood cells.
  • a “cell-mediated immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
  • the ability of a particular antigen or composition to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, by assaying for T-lymphocytes specific for the antigen in a sensitized subject, or by measurement of cytokine production by T cells in response to re-stimulation with antigen.
  • assays are well known in the art. See, e.g., Erickson et al. (1993) J. Immunol. 151 :4189-4199; and Doe et al. (1994) Eur. J. Immunol. 24:2369-2376.
  • the antigen may comprise at least one B cell epitope and/or a T cell epitope.
  • B cell epitope refers to any antigen portion or region that is recognized by secreted antibodies or B-cell receptors and is able to trigger an immune response in a B cell. Methods and techniques to determine if a peptide/molecule is or contains a B cell epitope are well known to the skilled person in the art and described in the literature (Ahmad, T. A., et al.
  • the T cell epitope is selected from a helper T cell epitope or a CD8 epitope.
  • Helper T cell epitope refers to peptides derived from antigens and recognized by the Helper T-cell receptor (TCR) when bound to class II Major Histocompatibility Complex (MHC-II) molecules displayed on the cell surface of Antigen Presenting Cells (APCs), which lead to the activation of the Helper T cells.
  • Said peptides are the specific amino acid sequence of the antigen which is recognized by the TCR.
  • Said peptides can also be used as “helper epitopes”, epitopes which are known to be recognized by and activate Helper T cells, wherein said helper epitopes are fused to, or used in proximity of (such as co-expressed in the APC membrane), antigens of interest, more preferably B cell epitopes, being newly presented to Helper T cells in order to improve and enhance the immunological response of the Helper T cells.
  • helper epitopes epitopes which are known to be recognized by and activate Helper T cells, wherein said helper epitopes are fused to, or used in proximity of (such as co-expressed in the APC membrane), antigens of interest, more preferably B cell epitopes, being newly presented to Helper T cells in order to improve and enhance the immunological response of the Helper T cells.
  • CD8 cell epitope or “CD8 T cell epitope” as used herein refers to peptides derived from antigens recognized by CD8 T cell receptors when said antigens are bound to class I (MHC I) or class II (MHC II) Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC I class I
  • MHC II Major Histocompatibility Complex
  • APCs antigen presenting cells
  • CTLs cytotoxic T lymphocytes
  • the B cell epitope and/or the CD8 T cell epitope is selected from the group consisting of peptides, glycopeptides and carbohydrates capable of inducing an immune response against a neurodegenerative disease (e.g. the immunogenic region of a neurodegeneration-associated antigen), an infectious disease (e.g. the immunogenic region of a bacterial-, viral-, or protozoal-associated antigen) or a cancer cell (e.g. the immunogenic region of a cancer-associated antigen, also known as tumor-associated antigen or TAA).
  • a neurodegenerative disease e.g. the immunogenic region of a neurodegeneration-associated antigen
  • an infectious disease e.g. the immunogenic region of a bacterial-, viral-, or protozoal-associated antigen
  • a cancer cell e.g. the immunogenic region of a cancer-associated antigen, also known as tumor-associated antigen or TAA.
  • antigens useful in the present invention are, without limitation, tetanus toxoid, egg albumin, thyroglobulin, recombinant hemagglutinin B (rHagB) antigen, recombinant protein from H1 N1 influenza, protective BpOmpW from Burkholderia pseudomallei.
  • rHagB hemagglutinin B
  • the pharmaceutical composition includes a pharmaceutically acceptable amount of a compound of the present application.
  • the pharmaceutical composition includes an immunologically effective amount of an antigen.
  • the compounds of the application and an antigen form an active ingredient.
  • the compound of the present application alone forms an active ingredient.
  • the amount of active ingredient(s) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration.
  • the amount of active ingredient(s) that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • this amount will range from about 1 % to about 99 % of active ingredient, preferably from about 5 % to about 70 %, most preferably from about 10 % to about 30 %, or from about 1 % to 99 %, preferably from 10 % to 90 %, 20 % to 80 %, 30 % to 70 %, 40 % to 60 %, 45 % to 55 %, or about 50 %.
  • formulations of the present application include injectable formulations.
  • the present application provides formulations comprising a liposome formulation of MPL and a compound of the present invention.
  • the present application provides formuiations comprising MPL, a compound of the present invention and a squaiene emulsion.
  • the present application provides formulations comprising MPL, a compound of the present invention, and CpG 7909 or CpG 1018.
  • MPL is a heterogeneous mixture of molecules from a biological source including both agonists and antagonists for TLR4.
  • CpG 7909 is an immunomodulating synthetic oligonucleotide designed to specifically agonise the Toll-like receptor 9 (TLR9).
  • the present application provides formulations comprising immune stimulating complexes (ISCOM) or ISCOM matrices of a compound of the present invention.
  • ISCOM matrices of a compound of the present invention and an antigen.
  • ISCOMs are open cage-like nanoparticulate structures comprising a saponin (here, a compound of the present invention), cholesterol, phospholipid and an antigen.
  • ISCOM particules are typically spherical of approximately 40 nm diameter.
  • ISCOMs deliver antigen to the cytosol, and have been demonstrated to promote antibody response and induction of T helper cell as well as cytotoxic T lymphocyte responses in variety of experimental animal models.
  • ISCOM matrices formulations e.g. ISCOMATRIX and Matrix-M
  • ISCOMATRIX and Matrix-M contain the same components and have the same structure as the ISCOM but without the incorporated antigen.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Non-limiting examples of pharmaceutically-acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluen
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Non-limiting examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present application include water, alcohols (including but not limited to methanol, ethanol, butanol, etc.), polyols (including but not limited to glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain additives such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • additives such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chiorobufanoi, phenol sorbic acid, and the like, it may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, in addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the absorption of the drug in order to prolong the effect of a formulation, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form.
  • the compounds of the present application which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present application, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present application may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present application employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the present application employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and then gradually increasing the dosage until the desired effect is achieved.
  • a compound or pharmaceutical composition of the present application is provided to a subject chronically.
  • Chronic treatments include any form of repeated administration for an extended period of time, such as repeated administrations for one or more months, between a month and a year, one or more years, or longer.
  • a chronic treatment involves administering a compound or pharmaceutical composition of the present application repeatedly over the life of the subject.
  • Preferred chronic treatments involve regular administrations, for example one or more times a day, one or more times a week, or one or more times a month, in general, a suitable dose, such as a daily dose of a compound of the present application, will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • doses of the compounds of the present application for a patient when used for the indicated effects, will range from about 0.0001 to about 100 mg per kg of body weight per day.
  • the daily dosage will range from 0.001 to 50 mg of compound per kg of body weight, and even more preferably from 0.01 to 10 mg of compound per kg of body weight.
  • the dose administered to a subject may be modified as the physiology of the subject changes due to age, disease progression, weight, or other factors.
  • provided adjuvant compounds of the present application are administered as pharmaceutical compositions or vaccines.
  • the amount of adjuvant compound administered will be 1-2000 pg.
  • the amount of adjuvant compound administered will be 1-1000 pg.
  • the amount of adjuvant compound administered will be 1-500 pg.
  • the amount of adjuvant compound administered will be 1-250 pg.
  • the amount of adjuvant compound administered will be 100-1000 pg.
  • the amount of adjuvant compound administered will be 100-500 pg.
  • the amount of adjuvant compound administered will be 100-200 pg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 250-500 pg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 10-1000 pg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 500-1000 pg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 50-250 pg. in certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 50-500 pg. In some embodiments, provided adjuvant compounds of the present application are administered as pharmaceutical compositions or vaccines.
  • the amount of adjuvant compound administered will be 1-2000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-1000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-500 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-250 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-1000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-500 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-200 mg.
  • the amount of adjuvant compound administered will be 250-500 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 10-1000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 500-1000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 50-250 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 50-500 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 0.01-215.4 mg.
  • the amount of adjuvant administered will be 1000-5000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1000-4000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1000-3000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1000-2000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 2000-5000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 2000-4000 pg/kg.
  • the amount of adjuvant administered will be 2000-3000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 3000-5000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 3000-4000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 4000-5000 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1-500 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 500-1000 pg/kg.
  • the amount of adjuvant administered will be 1000-1500 pg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 2 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 3 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 4 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 5 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 0.0029-5 mg/kg.
  • the amount of adjuvant administered in females is less than the amount of adjuvant administered in males. In certain embodiments, the amount of adjuvant administered to infants is less than the amount of adjuvant administered to adults. In certain embodiments, the amount of adjuvant administered to pediatric recipients is less than the amount of adjuvant administered to adults. In certain embodiments, the amount of adjuvant administered to immunocompromised recipients is more than the amount of adjuvant administered to healthy recipients. In certain embodiments, the amount of adjuvant administered to elderly recipients is more than the amount of adjuvant administered to non-elderly recipients.
  • the effective dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present application to be administered alone, in certain embodiments the compound is administered as a pharmaceutical formulation or composition as described above.
  • the compounds according to the present application may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.
  • kits comprising pharmaceutical formulations or compositions of a compound of the present application.
  • such kits include the combination of a compound of formula (I) such as BTS-31-DAz and BTS-25-BP and an antigen.
  • the agents may be packaged separately or together.
  • the kit optionally includes instructions for prescribing the medication, in certain embodiments, the kit includes multiple doses of each agent.
  • the kit may include sufficient quantities of each component to treat one or more subject for a week, two weeks, three weeks, four weeks, or multiple months.
  • the kit may include a full cycle of immunotherapy.
  • the kit includes a vaccine comprising one or more bacterial-, viral-, protozoal-, neurodegenerative disease- or cancer-associated antigens, and one or more provided compounds.
  • Compounds of formula (I) such as BTS-31-DAz and BTS-25-BP or a pharmaceutically acceptable salt thereof may be used as adjuvant in vaccines to increase the immune response to an antigen or enhance certain activities of cells from the immune system.
  • Another aspect of the present application relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in medicine, and more particularly, for use in the treatment and/or prevention of cancer, an infectious disease or a neurodegenerative disease.
  • any reference to the medical use of a compound of formula (I) or a pharmaceutically acceptable salt thereof has to be understood as a reference to medical use of an immunogenic composition comprising a compound of formula (I) and an antigen.
  • any reference to a pharmaceutical composition of a compound of formula (I) or a pharmaceutically acceptable salt thereof has to be understood as a composition comprising an antigen.
  • Another aspect of the present application relates to a method for the treatment and/or prevention of a disorder in a subject said method comprising the administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical compositon thereof to the subject, wherein the disorder is cancer, an infectious disease or a neurodegenerative disease.
  • Another aspect of the present application relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical compositon thereof for use in the immunization of a subject.
  • Another aspect of the present application relates to a method for immunizing a subject, said method comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
  • a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.
  • Any animal that may experience the beneficial effects of the compositions of the present application is within the scope of subjects that may be treated, in some embodiments, the subjects are mammals. In some embodiments, the subjects are humans.
  • the vaccines of the present application may be used to confer resistance to infection by either passive or active immunization.
  • a vaccine of the present application is administered to an animal to elicit a protective immune response which either prevents or attenuates a proliferative or infectious disease.
  • the vaccines of the present application are used to confer resistance to infection through passive immunization, the vaccine is provided to a host animal (e.g., human, dog, or mouse), and the antisera elicited by this vaccine is recovered and directly provided to a recipient suspected of having an infection or disease or exposed to a causative organism.
  • the present application thus concerns and provides a means for preventing or attenuating a proliferative disease resulting from organisms or tumor cells which have antigens that are recognized and bound by antisera produced in response to the immunogenic antigens included in vaccines of the present application.
  • a vaccine is said to prevent or attenuate a disease if its administration to an animal results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or in the total or partial immunity of the animal to the disease.
  • the administration of the vaccine may be for either a "prophylactic" or "therapeutic" purpose.
  • the vaccine(s) are provided in advance of any symptoms of proliferative disease.
  • the prophylactic administration of the vaccine(s) serves to prevent or attenuate any subsequent presentation of the disease.
  • the vaccine(s) is provided upon or after the detection of symptoms which indicate that an animal may be infected with a pathogen or have a certain cancer.
  • the therapeutic administration of the vaccine(s) serves to attenuate any actual disease presentation.
  • the vaccines may be provided either prior to the onset of disease proliferation (so as to prevent or attenuate an anticipated infection or cancer) or after the initiation of an actual proliferation.
  • the present application provides vaccines comprising one or more antigens (e.g. one or more bacterial, viral, protozoal, neurodegenerative disease or tumor- related antigens) in combination with a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the vaccine comprises a single bacterial, viral, protozoal, neurodegenerative disease or tumor-related antigen in combination with a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the vaccine comprises two or more bacterial, viral, protozoal, neurodegenerative disease or tumor-related antigens in combination with a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • one or more antigens of provided vaccines are bacterial- associated antigens.
  • one or more antigens of provided vaccines are viral-associated antigens.
  • one or more antigens of provided vaccines are protozoal-associated antigens.
  • one or more antigens of provided vaccines are neurodegenerative disease-associated antigens.
  • one or more antigens of provided vaccines are cancer- or tumor-associated antigens.
  • vaccines may optionally include a pharmaceutically acceptable excipient or carrier.
  • provided vaccines may comprise one or more antigens that are optionally conjugated to a pharmaceutically acceptable excipient or carrier.
  • said one or more antigens are conjugated covalently to a pharmaceutically acceptable excipient.
  • said one or more antigens are non-covalentiy associated with a pharmaceutically acceptable excipient.
  • adjuvants may be used to increase the immune response to an antigen.
  • provided vaccines may be used to invoke an immune response when administered to a subject.
  • an immune response to an antigen may be potentiated by administering to a subject a provided vaccine in an effective amount to potentiate the immune response of said subject to said antigen.
  • the compounds of the present application may be used in cancer vaccines as adjuvants in combination with tumor-associated antigens such as those listed hereinbefore.
  • said vaccines may be used in the treatment or prevention of neoplasms.
  • the neoplasm is a benign neoplasm.
  • the neoplasm is a malignant neoplasm. Any cancer may be treated using compounds of the invention with an antigen.
  • the malignancy is a hematological malignancy.
  • cancers besides hematological malignancies may also be treated using a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the cancer is a solid tumor.
  • compounds and pharmaceutical compositions of the present application can be employed in combination therapies, that is, the compounds and pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
  • the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (e.g., an inventive compound may be administered concurrently with another antiproliferative agent), or they may achieve different effects (e.g., control of any adverse effects).
  • therapies or anticancer agents that may be used in combination with the inventive anticancer agents of the present application include surgery, radiotherapy (gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes, to name a few), endocrine therapy, biologic response modifiers (interferons, interleukins, and tumor necrosis factor (TNF) to name a few), hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g., antiemetics), and other approved chemotherapeutic drugs, including, but not limited to, alkylating drugs (mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites (Methotrexate), purine antagonists and pyrimidine antagonists (6-Mercaptopurine, 5- Fluorouracil, Cytarabile, Gemcitabine), spindle poisons (Vinblastine, Vincris
  • the present invention also encompasses the use of certain cytotoxic or anticancer agents currently in clinical trials and which may ultimately be approved by the FDA (including, but not limited to, epothilones and analogues thereof and geldanamycins and analogues thereof).
  • FDA cytotoxic or anticancer agents
  • epothilones and analogues thereof include, but not limited to, epothilones and analogues thereof and geldanamycins and analogues thereof.
  • the present application provides a method of treating infectious disease in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an antigen.
  • the infection is bacterial.
  • the infection is viral.
  • the infection is protozoal. Examples of infectiuous diseases include, but are not limited to, malaria, acquired immunodeficiency syndrome, hepatitis and tuberculosis.
  • the subject is human.
  • the present application provides a method of treating neurodegenerative diseases in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an antigen.
  • the present application can be used in the treatment of neurodegenerative diseases (e.g. Alzheimer’s disease).
  • the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use as chemical probe applying a chemical proteomics approach, which involves photoaffinity labeling (PAL) experiments for photocrosslinking followed by affinity purification and mass spectrometry (MS) analysis.
  • PAL photoaffinity labeling
  • MS mass spectrometry
  • PAL has become one of the most powerful strategies to study protein-protein interactions (PPIs) as well as to identify molecular targets of drugs or pharmaceutical agents for mechanistic elucidation.
  • the photoreactive group diazirine, benzophenone or azide
  • the biotin is used as a reporter group or tag for isolation/purification of the proteindrug covalent complex.
  • target identification the identity of the putative receptor is resolved using MS-based proteomic analysis.
  • TLC Thin-layer chromatography
  • F254 fluorescent indicator
  • TLC plates were visualized under UV light (254 nm) and by staining with cerium ammonium molybdenate (CAM), phosphomolybdic acid (PMA), or 5% sulfuric acid in ethanol solutions or ninhydrin solutions.
  • CAM cerium ammonium molybdenate
  • PMA phosphomolybdic acid
  • mice Groups of five mice (C57BL/6, female, 6-8 weeks old) were vaccinated subcutaneously three times every 10 days (days 0, 14, and 28) with endotoxin-free OVA (EndoFitTM Ovalbumin, Invitrogen) (10 pg/mouse) in phosphate-buffered saline (PBS, 100 pL) either alone (noadjuvant control group) or with the synthetic saponins (20 pg/mouse).
  • OVA EndoFitTM Ovalbumin, Invitrogen
  • mice were bled via the submandibular vein at the indicated pre- (day -1) and post-vaccination timepoint (day 21), and by cardiac puncture at the experimental endpoint (day 35). Blood was collected in BD Microtainer® tubes (Clot Activator/SSTTM Gel) and centrifuged at 7500g for 10 min, after which serum was harvested and stored at -20°C until further analysis.
  • ELISA enzyme-linked immunosorbent assay
  • KPL SureBlue reserveTM commercial solution 100 pl/well, SeraCare
  • TMB 3,3’,5,5’-tetramethylbenzidine
  • OD optical density
  • Antibody titer was defined as the highest serum dilution that showed an absorbance of 0.1 or greater over that of the pre-sera (1-3).
  • Vet-Sap/Quil A (1 .15 g) and potassium hydroxide (0.97 g, 17 mmol) were suspended in ethanol: water (1 :1) (50 mL), then the mixture is heated to 85 °C for 7 h. The reaction mixture was then cooled to 0 °C, neutralized with 1.0 N HCI, and concentrated to approximately one-half of the volume. The crude was frozen and lyophilized, and the resulting dry solid is purified by silica gel chromatography (CH2Cl2/MeOH/Water/AcOH, 15:9:2:1). The major spot observed by TLC in the above solvent system is isolated by concentrating the fractions.
  • the resulting solid is dried by azeotropic removal of solvents with toluene (2 x 20 mL) and lyophilized in MeCN/water (1 :1) (3 x 15 mL) to provide a mixture of prosapogenins (1 :2) as a light tan foam ( ⁇ 0.6 g, 50 % mass yield).
  • xylose and rhamnose-containing prosapogenins 1 and 2 correspond to the two most abundant trisaccharide-triterpene fragments found in QS saponins.
  • the solid mixture of prosapogenins 1 and 2 (-0.600 g) is azeotroped from pyridine (8 mL), kept in high vacuum for 1 h, then pyridine (8 mL) is added, followed by TESOTf (2.0 mL, 8.8 mmol) was added dropwise.
  • the reaction mixture is stirred for 3 days, TLC indicated incomplete conversion then TESOTf (1 mL, 4.4 mmol) is added.
  • the mixture is concentrated and passed through a short plug of silica gel eluted with hexanes/EtOAc (4:1 to 2:1). The eluate is concentrated, the resulting yellow oil is dissolved in MeOH/THF (1 :1) (20 mL), and the solution is stirred for another 3 days to remove the silyl esters by solvolysis.
  • the reaction mixture is concentrated and the resulting mixture of xylose- and rhamnose- containing (TES)g-protected prosapogenin diacids (1 and 2) is separated by silica gel chromatography (hexanes/EtOAc, 4:1 to 2:1) to afford purified xylose-containing protected prosapogenin 3 (-0.25 g, -21 % yield) as a white solid.
  • the prosapogenin diacid 3 (250 mg, 0.126 mmol, 1.0 equiv.) is dissolved in dry DCM (2 mL), and added pyridine (202 pL, 2.52 mmol, 20 equiv.), TBP (623 mg, 2.52 mmol, 20 equiv.), and benzyl chloroformate (80 pL, 0.630 mmol, 5 equiv.). The mixture is stirred at rt overnight. After 18 h, complete conversion of the starting material is indicated by TLC.
  • PhSeSePh (205 mg, 0.66 mmol, 10 equiv.) is taken and passed Ar/vacuum cycle x 3 and then dissolved in THF (5 mL) and H 3 PO 2 (50 % in water) (785 pL, 7.25 mmol, 110 equiv.).
  • the yellow solution is heated at 40 °C for 1-2 h until it turns transparent.
  • the reaction mixture is removed from the heat, diluted with toluene (5 mL) and distilled water (5 mL), and stirred vigorously for 5 min under Ar.
  • the organic layer was transferred via a syringe to another flask containing anhydrous MgSC under Ar atmosphere.
  • the prosapogenin-linear trisaccharide saponin amine 20 (100 mg, 0.03327 mmol, 1.0 equiv.) is azeotropically dried, and the activated acid is then cannula transferred to it at ice bath condition, stirred 0 °C - rt for 2 h.
  • the reaction is quenched by adding water, diluted with DCM and extracted with NaHCCh.
  • Organic layer was concentrated and purified by silica gel chromatography (toluene/EtOAc, 20:1 to 11 :1) to afford the aminoacyl, branched trisaccharide- containing saponin 21 (90 mg, 87 %) as a white glassy solid.
  • This lyophilized solid is dissolved in a solution of water/MeCN (4:1) and purified by RP-HPLC PREP column using a linear gradient of 20— >100% CH3CN in water (0.05 vol% TFA) in 30 min.
  • the purity of the product was checked by C18 analytical column using a linear gradient of 5 - ⁇ 100% CH3CN in water (0.05 vol% TFA) in 30 min the same gradient as shown below.
  • H 2 N-Glu(Biot)-OAII 27 (AS-lll-003): In a 10 mL round bottom flask, H2N-Glu(Biot)-OAII, 27 (95 mg, 0.154 mmol, 1 eqv.) was dissolved in 3.2 mL dry DCM. And then added DIPEA (35 pL, 0.201 mmol, 1.3 eqv.), Dazirine- COOH (55 mg, 0.238 mmol, 1.5 eqv.) and PyBOP (110 mg, 0.211 mmol, 1.37 eqv.) giving a clear solution. The mixture was then stirred at rt for 2-3 h giving a bright new spot in the TLC.
  • DIPEA 35 pL, 0.201 mmol, 1.3 eqv.
  • Dazirine- COOH 55 mg, 0.238 mmol, 1.5 eqv.
  • PyBOP 110 mg, 0.211 mmol, 1.37 eqv.
  • the mixture was concentrated by N2 flow and purified by silica gel chromatography using the solvent gradient DCM/MeOH 9.5:0.5 to 9:1 with 1 % TEA affording the desire product DAz- Glu(Biot)-OAII, 28 (120 mg, 94%) as glassy solid.
  • azeotropically dried DAz-Glu(Biot)-OAII, 28 (150 mg, 0.181 mmol, 1 eqv.) is dissolved in MeOH/DCM (5 mL, 4:1) and added 4-methylmorpholine (19 pL, 0.181 mmol, 1 eqv.).
  • the mixture is degassed by performing three freeze-thaw-pump cycles. After the degas, (triphenylphosphine)palladium (0) (41 mg, 0.036 mmol, 0.2 eqv.) was added and the mixture was degassed again by performing the same freeze-thaw cycle x 3. The mixture is stirred at rt overnight.
  • DAz-Glu(Biot)-OH, 29 13 mg, 0.0165 mmol, 1 eqv.
  • EDC 30 mg, 0.165 mmol, 10 eqv.
  • NHS 18.8 mg, 0.165 mmol, 10 eqv.
  • the mixture is stirred 0 °C - rt for 2h under argon atmosphere. After 2h, another 15 mg of EDC and 15 mg of NHS was added and stirred at rt for another 1 h.
  • Biotin-PEG5-NH2 (17 mg, 0.033 mmol, 1 eqv.) was dissolved in 1 mL DMF.
  • DIPEA 16 pL, 0.097 mmol, 2.5 eqv.
  • BP-NHS, 23 110 mg, 0.23 mmol, 7.18 eqv.
  • the reaction was quenched by adding 0.5 mL of acetonitrile with 0.05 % TFA.
  • aminoacyl Saponin 22 (3.38 mg, 2.24 pmol, 1 eqv.) was dissolved in 0.7 mL dry DMF and cooled to 0 °C. And then DIPEA (1 L, 5.6 pmol, 2.5 eqv.) was added to it.
  • the NHS activated ester 24 (2.29 mg, 2.7 pmol, 1.2 eqv.) was dissolved in 1 mL dry DMF and then transferred to the saponin mixture via a gas tight glass syringe and stirred at 0 °C - rt for 2-3 h.
  • the reaction was quenched by adding 1 mL of cold water in ice bath condition; purified by RP-HPLC PREP column using a linear gradient of 20 - ⁇ 70 % CH3CN in water (0.05 vol% TFA) in 30 min.
  • the purity of the product was checked by C18 analytical column using a linear gradient of 5 - ⁇ 100% CH3CN in water (0.05 vol % TFA) in 30 min.
  • OVA ovalbumin
  • BTS-31-DAz elicited significantly higher anti-OVA IgG levels compared to the OVA-alone (no adjuvant) negative control ( Figure 2a).
  • BTS-31-DAz showed slightly higher antibody response.
  • the above trend was maintained on day 35 as well (at the time of sacrifice) with higher IgG response elicited by BTS-31-DAz ( Figure 2b) over the QS-21 group.
  • IgG subtyping such as lgG1 , lgG2 and lgG3 subclasses of the anti- OVA antibodies (day 35) for the above saponins and compared to that of total IgG ( Figure 3).
  • the antibody response for the BTS-31-DAz over OVA alone was significantly higher.
  • the antibody response elicited by BTS-31-DAz over QS21 was higher.
  • BTS-31-DAz also showed significantly higher antibody levels for both lgG2b ( Figures 3b) and lgG2c ( Figures 3c) subclass than QS21.
  • lgG3 although the antibody response was lower, BTS-31-DAz showed higher antibody response than QS21.

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Abstract

La présente invention concerne des composés basés sur un échafaudage de saponine de glycoside triterpénique, qui portent une chaîne acyle comprenant une fraction photoréactive et une biotine terminale, de formule générale (I). La présente invention concerne également l'utilisation desdits composés et des compositions de ceux-ci dans le traitement et l'immunisation de maladies telles que des maladies neurodégénératives et infectieuses et des cancers ainsi que leur utilisation en tant que sonde chimique dans le marquage par photoaffinité (PAL) (pour la photoréticulation avec des partenaires d'interaction) suivi d'une purification par affinité du complexe covalent et de la protéomique à base de MS pour l'identification et l'élucidation cibles du récepteur de protéine.
EP24720517.2A 2023-04-20 2024-04-19 Dérivés de saponine en tant qu'adjuvants et sondes de photoaffinité Pending EP4698224A1 (fr)

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KR102256410B1 (ko) 2008-04-08 2021-05-26 슬로안-케테링인스티튜트퍼캔서리서치 트리테르펜 사포닌, 그의 합성 방법, 및 그의 용도
KR102424785B1 (ko) 2014-05-30 2022-07-25 메모리얼 슬로안 케터링 캔서 센터 최소 사포닌 유사체, 이의 합성법 및 용도
CN108778289B (zh) 2015-11-06 2020-11-17 佐剂技术公司 三萜皂苷类似物
ES2944947T3 (es) 2015-12-17 2023-06-27 Memorial Sloan Kettering Cancer Center Variantes de triterpeno-saponina, métodos de síntesis y uso de las mismas
WO2018191598A1 (fr) 2017-04-13 2018-10-18 Adjuvance Technologies, Inc. Synthèse de saponine triterpénique, intermédiaires et combinaisons d'adjuvants
CA3061205A1 (fr) 2017-04-25 2018-11-01 Memorial Sloan Kettering Cancer Center Analogues de saponine triterpenique
US20200164065A1 (en) 2017-04-25 2020-05-28 Adjuvance Technologies, Inc. Triterpene saponin analogues
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