WO2017070794A1 - Process for the preparation of 2-[2-(2-amino-2-carboxy-ethylamino)-2-carboxy-ethylamino]-succinic acid (am-a) and analogs and derivatives thereof - Google Patents

Process for the preparation of 2-[2-(2-amino-2-carboxy-ethylamino)-2-carboxy-ethylamino]-succinic acid (am-a) and analogs and derivatives thereof Download PDF

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WO2017070794A1
WO2017070794A1 PCT/CA2016/051259 CA2016051259W WO2017070794A1 WO 2017070794 A1 WO2017070794 A1 WO 2017070794A1 CA 2016051259 W CA2016051259 W CA 2016051259W WO 2017070794 A1 WO2017070794 A1 WO 2017070794A1
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compound
formula
nmr
mhz
amino
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PCT/CA2016/051259
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French (fr)
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Gerard Wright
Alfredo Capretta
Silvia ALBU
Kalinka Koteva
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Mcmaster University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
    • C07C227/20—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters by hydrolysis of N-acylated amino-acids or derivatives thereof, e.g. hydrolysis of carbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C303/00—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides
    • C07C303/36—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of amides of sulfonic acids
    • C07C303/40—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of amides of sulfonic acids by reactions not involving the formation of sulfonamide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C311/16—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom
    • C07C311/19—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups

Definitions

  • TITLE PROCESS FOR THE PREPARATION OF 2-[2-(2-AMINO-2-CARBOXY- ETHYLAMINO)-2-CARBOXY-ETHYLAMINO]-SUCCINIC ACID (AM-A) AND ANALOGS AND DERIVATIVES THEREOF
  • the present application relates to a process for the preparation of
  • AM-A Aspergillomarasmines A
  • present application describes a process for the preparation of AM-A and analogs and derivatives thereof.
  • the application also includes certain novel analogs of AM-A, compositions comprising these analogs and methods of use thereof.
  • AM-A and B are fungus-derived molecules that were discovered and reported in the early 1960s (1, 2) along with their wilting and necrotic activity in plant leaves. This molecule was refurbished in the 1980s as an inhibitor of angiotensin-converting enzyme (ACE) (3) and in the early 1990s as a pre-clinical candidate for the inhibition of activation of human endothelin (4, 5). The activation of human endothelin is initiated by the endothelin-converting enzyme, which, like angiotensin-converting enzyme, is a metalloproteinase.
  • ACE angiotensin-converting enzyme
  • Carbapenems, or "last resort” antibiotics have encountered bacterial resistance.
  • Two sources of resistance are the enzymes New Delhi metallo-P-lactamase 1 (NDM-1) and Verona integrin-encoded metallo-P-lactamase 2 (VIM-2).
  • AM-A was also reported as a rapid and potent inhibitor of both NDM and VIM-2 by removing the zinc from the metallo-P-lactamases (MBLs) without toxic side effects.
  • AM-A was used in combination with the "last resort” antibiotics to restore the activity of the bacterial resistant antibiotics in MBL-derived Gram-negative pathogens (7).
  • AM-A is harvested from its natural resource.
  • AM-A is purified from Aspergillus flavus oryzae, Aspergillus versicolor, ascomycete Pyrenophora teres, colletotrichum gloeosporioides and fusarium oxysporum (2, 8, 9).
  • this process can be tedious, time consuming, and expensive, as well being potentially unsustainable for the resource in larger quantities (10).
  • the present application relates to improved synthetic methods for the preparation of the aspergillomarasmine A (AM-A) compound and analogs and derivatives thereof.
  • the features of this process involves the use of a cyclic activated serine synthon in the construction of the poly-amino acid, AM-A.
  • the present application includes a process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof: C0 2 H C0 2 H
  • PG 1 and PG are suitable protecting groups.
  • the present application also includes a process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof, the process comprising: a) reacting the compound of Formula (II) with a compound of Formula (VII) under conditions to provide a compound of Formula (VIII):
  • PG and PG are suitable protecting groups.
  • the present application includes novel non-natural diastereomeric compounds of the application, along with pharmaceutical compositions comprising said compounds and a pharmaceutically acceptable carrier, and methods of use thereof .
  • the present applications also includes compounds of Formula A, and pharmaceutically acceptable salts and/or solvates thereof;
  • R 2 is selected from H, S0 2 Ar, CH 2 CH(C0 2 R 6 ) HR 7 , CH 2 C0 2 R 6 , CH 2 C(0) HR 7 and Ci. 6 alkyl;
  • R -R are independently selected from H and C 1-6 alkyl
  • R 6 and R 7 are independently selected from H, Ci ⁇ alkyl and S0 2 Ar; n is 1 or 2,
  • Ar is aryl that is unsubstituted or substituted with one or more of halo, N0 2 , C(0)C 1- 4 alkyl and C0 2 C 1-4 alkyl; and each alkyl group is optionally fluoro-substituted, provided that when n is 1, R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are not all H.
  • compositions comprising a compound of
  • Formula A and a pharmaceutically acceptable carrier.
  • the present application includes a method of treating a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more ⁇ -lactam antibiotics in combination with an effective amount of one or more compounds of Formula A as defined above, or pharmaceutically acceptable salts and/or solvates thereof.
  • the present application also includes a use of a ⁇ -lactam antibiotic in combination with one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for treating a bacterial infection in a subject; a use of a ⁇ -lactam antibiotic in combination with one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for treating a bacterial infection in a subject; and a ⁇ -lactam antibiotic and one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to treat a bacterial infection in a subject.
  • suitable means that the selection of the particular compound or conditions would depend on the specific synthetic manipulation to be performed, and the identity of the molecule(s) to be transformed, but the selection would be well within the skill of a person trained in the art. All process/method steps described herein are to be conducted under conditions sufficient to provide the product shown and for the reaction to proceed to a sufficient extent. A person skilled in the art would understand that all reaction conditions, including, for example, reaction solvent, reaction time, reaction temperature, reaction pressure, reactant ratio and whether or not the reaction should be performed under an anhydrous or inert atmosphere, can be varied to optimize the yield of the desired product and it is within their skill to do so.
  • the expression "proceed to a sufficient extent" as used herein with reference to the reactions or process steps disclosed herein means that the reactions or process steps proceed to an extent that conversion of the starting material or substrate to product is sufficient for the given reaction. Conversion may be sufficient when greater than about 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the starting material or substrate is converted to product.
  • the compounds described herein have at least one asymmetric centre. Where compounds possess more than one asymmetric centre, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (e.g. less than 50%, suitably less than 20%, suitably less than 10%), more suitably less than 5%) of compounds having alternate stereochemistry.
  • oxidant refers to any compound or combination of compounds that oxidizes a desired functional group.
  • An oxidizing agent results in the overall loss of electrons, or in the case of organic chemistry, loss of hydrogen atoms from the functional group.
  • reducing agent refers to any compound or combination of compounds that reduces a desired functional group.
  • a reducing agent results in the overall addition of electrons, or in the case of organic chemistry, hydrogen atoms to the functional group.
  • non-nucleophilic base refers to an organic base that is a poor nucleophile. Typically, non-nucleophilic bases are bulky which prevent the base from acting as a nucleophile.
  • inert solvent means a solvent that does not interfere with or otherwise inhibit a reaction. Accordingly, the identity of the inert solvent will vary depending on the reaction being performed. The selection of inert solvent is within the skill of a person in the art. Examples of inert solvents include, but are not limited to, benzene, toluene, tetrahydrofuran, acetone, dioxane, ethyl ether, dichloromethane, dichloroethane, ethyl acetate, dimethyl formamide (DMF), acetonitrile, C 1-6 alkylOH (e.g.
  • DMSO dimethylsulfoxide
  • aqueous solutions such as water and dilute acids and bases, and ionic liquids, provided that such solvents do not interfere with the reaction.
  • solvent includes both a single solvent and a mixture comprising two or more solvents.
  • alkyl as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups.
  • Ci. 6 alkyl means an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms.
  • t-Boc refers to the group t-butyloxycarbonyl.
  • Ac refers to the group acetyl.
  • Ts (tosyl) as used herein refers to the group ⁇ -toluenesulfonyl.
  • oNosyl as used herein refers to the group orthonitrobenzenesulfonyl.
  • Bn as used herein refers to the group benzyl.
  • Trityl refers to the group triphenylmethyl.
  • Fmoc refers to the group fluorenylmethoxycarbonyl.
  • Me as used herein refers to the group methyl.
  • Et as used herein refers to the group ethyl.
  • Ph as used herein refers to the group phenyl.
  • Ns as used herein refers to the group nitrobenzylsulfonyl.
  • TFA as used herein refers to the compound trifluoroacetic acid.
  • HC1 as used herein refers to the compound hydrochloric acid.
  • PhSH as used herein refers to the compound of thiophenol.
  • DIPEA as used herein refers to the compound of N,N diisopropylethylamine.
  • TMTOH as used herein refers to the compound of trimethyltin hydroxide.
  • CAN as used herein refers to the compound of cerium(IV) ammonium nitrate.
  • TcBoc refers to group trichloro-t-butyloxy-carbonyl.
  • Cbz as used herein refers to the group benzyl oxycarbonyl.
  • Pmb as used herein refers to the group para-methoxybenzyl.
  • Bn as used herein refers to the group benzyl.
  • protecting group or "PG” or the like as used herein refer to a chemical moiety which protects or masks a reactive portion of a molecule to prevent side reactions in those reactive portions of the molecule, while manipulating or reacting a different portion of the molecule. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portions of the molecule.
  • PG protecting group
  • the selection of a suitable protecting group can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in "Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W.
  • protecting groups include, but are not limited to t-Boc, Ac, Ts, methyl esters, t-butyl esters, silyl esters, o-Ns, p-Ns, Bn, Fmoc, benzoyl, dimethoxytrityl, p- methyoxybenzyl ether, trityl, carbooxybenzyl, benzoyl and the like.
  • suitable deprotecting agent as used herein is intended to include any of the standard deprotecting agents used in the deprotection procedures (i.e. removal of protecting groups) as outlined in the application.
  • Many conventional deprotecting agents are known in the art, for example as described in "Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and
  • deprotecting agents include, but are not limited to TFA, HC1, PhSH/DIPEA, H 2 /Pd/C, TMTOH, piperidine, H 3( aq), CAN and the like.
  • activating group means any substituent that increases the reactivity of its adjacent or nearby atoms, relative to the atom's reactivity in the presence of hydrogen.
  • natural aspergillomarasmine A “natural AM- A”, “naturally occurring aspergillomarasmine A” and “naturally occurring AM -A” all refer to AM-A as produced in nature, for example by the fungus, Aspergillus versicolour.
  • natural isomers of Formula I refers to compounds of Formula I having the same relative stereochemistry as found in naturally occurring AM-A (i.e. L,L,L).
  • non-natural isomers of Formula I refers to compounds of Formula I having a relative stereochemistry other than that found in naturally occurring AM-A (i.e. L,L,L).
  • amino acids contained in compounds of the formulae (II), (Ilia), (Illb) and (VII) can be of either the D- or L- configurations or can be mixtures of the D- and L- isomers, including racemic mixtures.
  • pharmaceutically acceptable salt means an acid addition salt or a basic addition salt suitable for, or compatible with, the treatment of subjects.
  • pharmaceutically acceptable salts embraces salts commonly used to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically acceptable acid addition salts are prepared from an inorganic acid or an organic acid. Examples of such inorganic acids include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.
  • organic acids include, for example, aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include, without limitation, formic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, ⁇ -hydroxybutyric, malonic, galactic, and galacturonic acid.
  • organic acids include, for example, aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include, without limitation, formic, ace
  • Suitable pharmaceutically-acceptable base addition salts include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from ⁇ , ⁇ '- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine, lysine and procaine.
  • a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
  • solvates refers to complexes formed between a compound and a solvent from which the compound is precipitated or in which the compound is made. Accordingly, the term “solvate” as used herein means a compound, or a salt of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice. Examples of suitable solvents include, but are not limited to ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate”. The formation of solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions to form a particular solvate can be made by a person skilled in the art.
  • pharmaceutically acceptable solvate means a solvate suitable for, or compatible with, the treatment of subjects.
  • a suitable solvent is physiologically tolerable at the dosage used or administered.
  • disease, disorder or condition arising from a bacterial infection refers to any disease, disorder or condition that is directly or indirectly caused by the presence of a bacterial infection in a subject.
  • subject includes all members of the animal kingdom including mammals, such as equines and humans. Other examples of subjects include, companion animals, such as felines and canines.
  • composition refers to a composition of matter for pharmaceutical use.
  • pharmaceutically acceptable means compatible with the treatment of subjects.
  • parenteral means taken into the body or administered in a manner other than through the gastrointestinal tract.
  • administered means administration of an effective amount of a compound, including for example, the antibiotic and compound of Formula A (such as a compound of Formula I), or a salt and/or solvate thereof, to a cell either in cell culture or in a subject.
  • a compound including for example, the antibiotic and compound of Formula A (such as a compound of Formula I), or a salt and/or solvate thereof, to a cell either in cell culture or in a subject.
  • an effective amount of the antibiotic and/or compound of Formula A (such as a compound of Formula I), or a salt and/or solvate thereof, is an amount that, for example, reduces the bacterial infection compared to the bacterial infection without administration of the antibiotic and the compound of Formula A (or compound of Formula I), or a salt and/or solvate thereof.
  • an effective amount of the compound of Formula A (or compound of Formula I), or a salt and/or solvate thereof is, for example, an amount that, for example, reduces the bacterial infection compared to the reduction of the bacterial infection with administration of the antibiotic alone.
  • reducing the infection it is meant, for example, reducing the amount of the infectious agent in the subject and/or reducing the symptoms of the infection. Effective amounts may vary according to factors such as the disease state, age, sex and/or weight of the subject.
  • the amount of a given compound or composition that will correspond to such an amount will vary depending upon various factors, such as the given compound or composition, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • the terms "to treat”, “treating” and “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, diminishment of extent of bacterial infection, stabilization (i.e.
  • "Palliating" an infection, disease, disorder and/or condition means that the extent and/or undesirable clinical manifestations of an infection, disease, disorder and/or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the infection, disease, disorder and/or condition.
  • prevention or “prophylaxis” and the like as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with a bacterial infection and/or a disease, disorder and/or condition arising from a bacterial infection or manifesting a symptom associated with a bacterial infection and/or a disease, disorder and/or condition arising from a bacterial infection.
  • a subject for example a subject "in need thereof is a subject who has been diagnosed with, is suspected of having, may come in to contact with, and/or was previously treated for a bacterial infection or a disease, disorder or condition arising from a bacterial infection.
  • the present application includes a process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof:
  • n 1 or 2;
  • R 1 is a direct bond or 0-S0 2 ;
  • PG 1 and PG are suitable protecting groups.
  • the compound of Formula (III) is a compound of
  • PG 1 is a suitable protecting group for carboxylic acids
  • PG is a suitable protecting and/or activating group for amines.
  • the compounds of Formula (Ilia) are available using methods known in the art.
  • compounds of Formula (Ilia) are prepared by combining the commercially available D-/L-serine with methanesulfonyl chloride in the presence of an organic non-nucleophilic base in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • trityl protected L-serine is used.
  • non-limiting reaction temperatures are about 30°C to about 80°C, about 40°C to about 75°C, or about 50°C to about 70°C.
  • Examples of non-limiting reaction times are about 30 hours to about 80 hours, about 40 hours to about 70 hours, or about 50 hours to about 60 hours.
  • suitable bases include, but are not limited to, organic amines, such as triethylamine.
  • PG 1 is a suitable protecting group for carboxylic acids.
  • PG 1 is selected from methyl, ethyl, t-butyl, benzyl, trialkylsilyl and triarylsilyl. In a further embodiment, PG 1 is selected from methyl, ethyl, t-butyl, benzyl and trimethylsilyl. In yet a further embodiment, PG 1 is selected from methyl, t- butyl and benzyl.
  • PG is a suitable protecting and/or activating group for amines.
  • PG is selected from t-Boc, TcBoc, Fmoc, Bn, benzoyl, Cbz, 4-nitrobenzyloxy carbonyl, Pmb, o-nosyl, p-nosyl and trityl.
  • PG is selected from t-Boc, Fmoc, Bn, benzoyl, Cbz, Pmb, o-nosyl and trityl.
  • PG is an activating group on the compound of
  • Formula (Ilia) which activates the carbon adjacent to the nitrogen for nucleophilic attack by the compound of Formula (II).
  • PG is an electron- withdrawing group.
  • PG is o-nosyl.
  • the protecting PG 1 group is a functional group that is both an electron- withdrawing group as well as a protecting group which could be removed under conditions suitable to not remove PG 1 .
  • the o-nosyl moiety is selected for its versatility as both an activating group and a protecting group.
  • the compounds of Formula (Ilia) wherein PG is o- nosyl are prepared by treating a compound of Formula (Ilia) wherein PG is a bulky, acid labile protecting group, such as trityl, in the presence of an acid in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • a compound of Formula (Ilia) wherein PG is a bulky, acid labile protecting group, such as trityl is a bulky, acid labile protecting group, such as trityl
  • Examples of non-limiting reaction temperatures are about -10°C to about 15°C, about - 5°C to about 10°C, or about 0°C to about 5°C.
  • Examples of non-limiting reaction times are about 10 minutes to about 1 hour or about 30 minutes to about 45 minutes.
  • suitable acids include, but are not limited to, organofluorine compounds, such as trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • o-nitrobenzylsulfonyl chloride is added in the same pot in an inert solvent at room temperature and for a time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction times are about 10 hours to about 30 hours or about 15 hours to about 24 hours.
  • the compound of Formula (II) is L-aspartic acid-di- t-butyl ester or L-aspartic acid di-methyl ester.
  • the compounds of Formula (II) are prepared using methods known in the art.
  • the conditions to provide the compound of Formula (IV) comprise combining the compound of Formula (II) with the compounds of Formula (Ilia) or (Illb) in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • non-limiting reaction temperatures are about 10°C to about 80°C, about 15°C to about 70°C, or about 20°C to about 60°C.
  • non-limiting reaction times are about 1 hour to about 35 hours, about 5 hours to about 30 hours, or about 10 hours to about 24 hours.
  • the conditions to provide the compound of Formula (V) comprise combining the compound of Formula (IV) with any
  • the conditions to provide the compound of Formula (V) comprise treating the compound of Formula (IV) with a suitable deprotecting agent in the presence of a non- nucleophilic base in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • suitable deprotecting agent in the presence of a non- nucleophilic base in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • non-limiting reaction temperatures are about 20°C to about 40°C or about 23°C to about 30°C.
  • Examples of non-limiting reaction times are about 15 minutes to about 2 hours or about 30 minutes to about 1 hour.
  • Suitable deprotecting agents include, but are not limited to, trifluoroacetic acid (TFA), hydrochloric acid (HC1), thiophenol (PhSH), hydrogenolysis (H 2 /Pd/C) and piperidine.
  • suitable bases include, but are not limited to, organic amines, such as diisopropylethylamine.
  • the conditions to provide the compound of Formula (VI) comprise combining the compound of Formula (V) with the compounds of Formula (Ilia) or (Illb) in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • non-limiting reaction temperatures are about 10°C to about 80°C, about 15°C to about 70°C, or about 20°C to about 60°C.
  • non-limiting reaction times are about 1 hour to about 35 hours, about 5 hours to about 30 hours, or about 10 hours to about 24 hours.
  • the conditions to provide the compound of Formula (IV) and the compound of Formula (VI) comprise basic conditions.
  • the basic conditions comprise an organic non- nucleophilic base.
  • non-nucleophilic bases include, but are not limited to, triethylamine, ⁇ , ⁇ -diisopropylethylamine and l,8-diazabicycloundec-7-ene (DBU).
  • the conditions to provide the compound of Formula (IV) and the compound of Formula (VI) further comprise combining compounds of Formula (II) and (V) with the compound of Formula (Illb) in the presence of a soluble salt in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • a soluble salt in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • non-limiting reaction temperatures are about 20°C to about 80°C, about 30°C to about 70°C, or about 40°C to about 60°C.
  • Examples of non -limiting reaction times are about 1 hour to about 30 hours, about 5 hours to about 20 hours, or about 10 hours to about 15 hours.
  • a soluble salt include, but are not limited to, monopotassium phosphate and/or sodium phosphate.
  • Formula (V) and the compound of Formula (VI) to the compound of Formula (I) is carried out using any deprotecting agent suitable for removing PG 1 and PG 2 protecting groups.
  • Any deprotecting agent suitable for removing PG 1 and PG 2 protecting groups are known in the art, for example as described in "Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).
  • Suitable deprotecting agents include, but are not limited to trifluoroacetic acid (TFA); hydrochloric acid (HC1); a mixture of thiophenol and N,N-diisopropylethylamine (PhSH/DIPEA); hydrogenolysis (H 2 , Pd/C); trimethyltin hydroxide (TMTOH); piperidine; 3 ⁇ 4(aq); and the like.
  • TFA trifluoroacetic acid
  • HC1 hydrochloric acid
  • PhSH/DIPEA a mixture of thiophenol and N,N-diisopropylethylamine
  • H 2 , Pd/C hydrogenolysis
  • TMTOH trimethyltin hydroxide
  • piperidine 3 ⁇ 4(aq); and the like.
  • the compound of Formula (I) or esters, amides, salts and/or solvates thereof are prepared on a solid support, such as a solid support resin or bead.
  • the compound of Formula (I) or esters, amides, salts and/or solvates thereof is prepared on a Wang resin.
  • the compound of Formula (II) attached to the Wang resin is commercially available in either the D-/L- configuration wherein the carboxylic acids and amine are protected with suitable protecting groups.
  • the compound of Formula (II) is Fmoc protected L- aspartic acid-di-t-butyl ester.
  • the support-bound compounds of Formula (II) are prepared using methods known in the art.
  • the amine protecting group of the support-bound compound of Formula (II) is removed with a suitable deprotecting agent for a time sufficient for the conversion to proceed to a sufficient extent.
  • suitable deprotecting agents include, but are not limited to, piperidine.
  • the support is subsequently washed with in inert solvent.
  • the conditions to provide the support-bound compound of Formula (IV) comprise combining the support-bound compound of Formula (II) with the compound of Formula (Ilia) in an inert solvent at room temperature and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction times are about 1 hour to about 35 hours, about 5 hours to about 30 hours, or about 10 hours to about 24 hours. In an embodiment, the resin is drained and washed with an inert solvent. [0097] In an embodiment of the application, the conditions to provide the support-bound compound of Formula (V) comprise combining the support-bound compound of Formula (IV) with any deprotecting agent suitable to selectively remove
  • the conditions to provide the support-bound compound of Formula (V) comprise treating the support-bound compound of Formula (IV) with a suitable deprotecting agent in the presence of a non-nucleophilic base in an inert solvent at room temperature and time sufficient for the conversion to proceed to a sufficient extent.
  • a suitable deprotecting agent include, but are not limited to, thiophenol (PhSH).
  • suitable bases include, but are not limited to, organic amines, such as diisopropylethylamine. The resin was drained and washed with an inert solvent.
  • the conditions to provide the support-bound compound of Formula (VI) comprise combining the support-bound compound of Formula (V) with the compound of Formula (Ilia) of (Illb) in an inert solvent at room temperature and time sufficient for the conversion to proceed to a sufficient extent.
  • Examples of non -limiting reaction times are about 1 hour to about 35 hours, about 5 hours to about 30 hours, or about 10 hours to about 24 hours.
  • the resin was drained and washed with an inert solvent.
  • the conditions to provide the support-bound compound of Formula (I) comprise combining the support-bound compound of Formula (VI) with any deprotecting agent or combindation of deprotecting
  • the conditions to provide the compound of Formula (I) comprise treating the support-bound compound of Formula
  • a suitable deprotecting agent to remove PG and not PG in the presence of a non-nucleophilic base in an inert solvent at room temperature and time sufficient for the conversion to proceed to a sufficient extent.
  • suitable deprotecting agents include, but are not limited to thiophenol (PhSH).
  • suitable bases include, but are not limited to, organic amines, such as diisopropylethylamine.
  • the support is drained and washed with an inert solvent and dried under vacuum for time period of about 3 hours to about 5 hours.
  • the compound of Formula (I) solid support is then treated to remove PG simultaneously with the liberation of the for example, using a mixture of suitable acids, a trialkylsilyl agent and water at room temperature and time sufficient for the conversion to proceed to a sufficient extent.
  • suitable acids include, but are not limited to, organofluorine compounds, such as trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • trialkyl silyl agents include, but are not limited to, triisopropylsilane.
  • deprotection/liberationthe conditions comprises a ratio of 95:2.5:2.5 of a suitable acid:trialkyl silyl agent: water.
  • the compound of Formula (I) has the following relative stereochemistry:
  • the present application encompasses both natural and non-natural isomers of Formula (I). Accordingly, in some embodiments the compound of Formula (I) has the same stereochemistry as the corresponding natural producted isolated from nature, eg. from the fungus, Aspergillus versicolour.
  • the present application also includes a process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof, the process comprising: a) reacting the compound of Formula (II) with a compound of Formula (VII) nder conditions to provide a compound of Formula (VIII):
  • n 1 or 2;
  • PG and PG are suitable protecting groups.
  • PG 1 is a suitable protecting group for carboxylic acids.
  • PG 1 is selected from methyl, ethyl, t-butyl, benzyl, trialkylsilyl and triarylsilyl. In a further embodiment, PG 1 is selected from methyl, ethyl, t-butyl, benzyl and trimethylsilyl. In yet a further embodiment, PG 1 is selected from methyl, t- butyl and benzyl. In yet a further embodiment, PG 1 is methyl.
  • PG is a suitable protecting group for amines.
  • PG is selected from t-Boc, TcBoc, Fmoc, Bn, benzoyl, Cbz, 4- nitrobenzyloxy carbonyl, Pmb, o-nosyl, p-nosyl and trityl.
  • PG is selected from t-Boc, Fmoc, Bn, benzoyl, Cbz, Pmb, o-nosyl and trityl.
  • PG is t-Boc.
  • the compound of Formula (II) is L-aspartic acid-di- t-butyl ester or L-aspartic acid di -methyl ester.
  • the compounds of Formula (II) are prepared using methods known in the art.
  • the compound of Formula (VII) is commercially available.
  • the compound of Formula (VII) is (R)-ter t-butyl 4-formyl-2,2- dimethyloxazolidine-3-carboxylate (R-Garner' s aldehyde).
  • the compound of Formula (VII) is prepared using methods known in the art.
  • the conditions to provide the compound of Formula (VIII) comprise reductive amination.
  • the conditions to provide the compound of Formula (VIII) comprise combining the compound of Formula (II) with the compound of Formula (VII) in the presence of a reducing agent and in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • a reducing agent include, but are not limited to, reducing metal salts.
  • the reducing metal salts include, but are not limited to, sodium borohydride, sodium cyanoborohydride and sodium triacetoxyborohydride.
  • the conditions to provide the compound of Formula (IX) comprise combining the compound of Formula (VIII) with any deprotecting agent suitable to selectively remove PG 2 over PG 1.
  • the conditions to provide the compound of Formula (IX) comprise treating the compound of Formula (VIII) with a suitable deprotecting agent in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 20°C to about 40°C or about 23°C to about 30°C. Examples of non-limiting reaction times are about 1 hour to about 30 hours, about 5 hours to about 20 hours or about 10 hours to about 16 hours. Examples of suitable deprotecting agents include, but are not limited to, trifluoroacetic acid (TFA).
  • the conditions to provide the compound of Formula (X) comprise reductive amination.
  • the conditions to provide the compound of Formula (X) comprise combining the compound of Formula (IX) with the compound of Formula (VII) in the presence of a reducing agent in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • a reducing agent examples include, but are not limited to, reducing metal salts.
  • the reducing metal salts include, but are not limited to, sodium borohydride, sodium cyanoborohydride and sodium tri acetoxyb orohy dri de .
  • Formula (X) is further protected with protecting groups suitable for amines.
  • protecting groups suitable for amines can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in "Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).
  • suitable protecting groups include, but are not limited to t-Boc, Ac, Ts, o-Ns, p-Ns, Bn, Fmoc, benzoyl, dimethoxytrityl, p- methyoxybenzyl ether, trityl, carbooxybenzyl, benzoyl and the like.
  • Formula (XI) is suitably carried out in the presence of an oxidant in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • an oxidant in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent.
  • non-limiting reaction temperatures are about 15°C to about 40°C, about 20°C to about 35°C, or about 23°C to about 30°C.
  • non-limiting reaction times are about 10 minutes to about 5 hours, about 30 minutes to about 4 hours, or about 1 hour to about 3 hours.
  • suitable oxidants include, but are not limited to, chromium trioxide, potassium permanganate, pyridinium dichromate or ruthenium tetroxide.
  • Formula (IX) and the compound of Formula (XI) to the compounds of Formula (I) is carried out using any deprotecting agent suitable for removing both PG 1 protecting groups.
  • Any deprotecting agent suitable for removing both PG 1 protecting groups are known in the art, for example as described in "Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).
  • Suitable deprotecting agents include, but are not limited to trifluoroacetic acid (TFA); hydrochloric acid (HC1); a mixture of thiophenol and N,N-diisopropylethylamine (PhSH/DIPEA); hydrogenolysis (H 2 , Pd/C); trimethyltin hydroxide (TMTOH); piperidine; H 3 ( a q); and the like.
  • the compound of Formula (I) has the following relative stereochemistry:
  • the preparation of a compound of Formula (I), or esters, amides and/or solvates thereof using a compound of Formula (II) and compound of Formula (VIII) is also carried out using a solid support, such as support bound compound of Formula (II).
  • esters or amides of the compound of Formula (I) include, but are not limited to, esters, amides and salts thereof.
  • the method of preparing esters or amides of the compound of Formula (I) comprises combining the compound of Formula (I) prepared using any of the above methods with an esterification agent or amidiation agent respectively.
  • the present application includes novel non-natural diastereomeric compounds of the application. Accordingly, the application includes compounds of Formula (I) having the relative stereochemistry:
  • the present applications also includes compounds of Formula A, and pharmaceutically acceptable salts and/or solvates thereof;
  • R 2 is selected from H, S0 2 Ar, CH 2 CH(C0 2 R 6 )NHR 7 , CH 2 C0 2 R 6 , CH 2 C(0)NHR 7 and Ci. 6 alkyl;
  • R -R are independently selected from H and C 1-6 alkyl
  • R 6 and R 7 are independently selected from H, Ci ⁇ alkyl and S0 2 Ar; n is 1 or 2,
  • Ar is aryl that is unsubstituted or substituted with one or more of halo, N0 2 , C(0)C . 4 alkyl and C0 2 C 1-4 alkyl; and each alkyl group is optionally fluoro-substituted, provided that when n is 1, R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are not all H.
  • R is selected from CH 2 CH(C0 2 R 6 )NHR 7 , CH 2 C0 2 R 6 and CH 2 C(0)NHR 7 , in which R 6 and R 7 are independently selected from H, C 1-4 alkyl and S0 2 Ar, and aryl that is unsubstituted or substituted with N0 2 , suitable 2-nitro. 3 5
  • R -R are independently selected from H and C 1-4 alkyl.
  • n is 2.
  • the compounds of Formula A contain several enantiomeric atoms.
  • the application includes all stereoisomers of the compounds of Formula A, including mixtures thereof.
  • the stereochemistry of the compounds of Formula A is the same as the stereochemistry found in naturally occurring AM-A or in toxin A, for example AM-A isolated, for example by extraction, from a fungus, such as Aspergillus versicolor.
  • the compounds of Formula A are selected from:
  • the present application also includes all uses of the one or more of the above non-natural compounds of Formula (I) and Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, including their use as a medicament.
  • the one or more non-natural compounds of Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof are suitably formulated into pharmaceutical compositions for administration into a subject thereof.
  • the present application further includes a pharmaceutical composition comprising one or more non-natural compounds of Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, and a pharmaceutically acceptable carrier and/or diluent.
  • Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof are further modified to increase cell permeability and target delivery. Modifications include, but are not limited to, derivatization of the one or more non- natural compounds of Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, with one or more of fatty acids, vitamins (e.g. folates), a single- chain antigen binding molecule, cell -penetrating peptides (CPPs), nanoparticles,
  • further modifications include, encapsulation of the one or more non-natural compounds of Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, within drug delivery vehicles.
  • drug delivery vehicles include, but are not limited to, liposome- or
  • the present application includes a method of treating a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more ⁇ -lactam antibiotics in combination with an effective amount of one or more compounds of Formula A, or pharmaceutically acceptable salts and/or solvates thereof:
  • R 2 is selected from H, S0 2 Ar, CH 2 CH(C0 2 R 6 ) HR 7 , CH 2 C0 2 R 6 , CH 2 C(0) HR 7 and Ci 6 alkyl;
  • R 3 -R 5 are independently selected from H and C 1-6 alkyl
  • R 6 and R 7 are independently selected from H, C 1-6 alkyl and S0 2 Ar; n is 1 or 2, Ar is aryl that is unsubstituted or substituted with one or more of halo, N0 2 , C(0)C 1- 4 alkyl and C0 2 C 1-4 alkyl; and each alkyl group is optionally fluoro-substituted, provided that when n is 1, R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are not all H.
  • the present application also includes a use of a ⁇ -lactam antibiotic in combination with one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for treating a bacterial infection in a subject; a use of a ⁇ -lactam antibiotic in combination with one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for treating a bacterial infection in a subject; and a ⁇ -lactam antibiotic and one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to treat a bacterial infection in a subject.
  • the present application includes a method of treating or preventing a disease, disorder or condition arising from a bacterial infection in a subject comprising administering, to the subject, an effective amount of one or more ⁇ - lactam antibiotics in combination with an effective amount of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof.
  • the present application also includes a use of a ⁇ -lactam antibiotic in combination with of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for treating a disease, disorder or condition arising from a bacterial infection in a subject; a use of a ⁇ -lactam antibiotic in combination with of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for treating disease, disorder or condition arising from a bacterial infection in a subject; and a ⁇ -lactam antibiotic and of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to treat a disease, disorder or condition arising from a bacterial infection in a subject.
  • the present application includes a method of improving the efficacy of a B-lactam antibiotic for treating a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof in combination with the antibiotic.
  • the present application also includes a use of one or more compounds of Formula II as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for improving the efficacy of a B-lactam antibiotic for treating a bacterial infection in a subject; a use of of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for improving the efficacy of a B-lactam antibiotic for treating a bacterial infection in a subject; and of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to improve the efficacy of a B-lactam antibiotic to treat a bacterial infection in a subject.
  • the present application also includes a method of improving the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof.
  • the present application also includes a use of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for improving the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection in a subject; a use of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for improving the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection in a subject; and one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to improve the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection in a subject.
  • the therapeutic methods and uses described above are also applicable to the combination of an effective amount of one or more non-natural compounds of Formula I and an effective amount
  • the bacterial infection is an infection of at least one metallo-B-lactamase (MBL)-expressing bacterium
  • the disease, disorder or condition arising from a bacterial infection is a disease, disorder or condition arising from at least one MBL-expressing bacterial infection.
  • the MBL is an IMP -type, a Verona integron-encoded metallo-P-lactamase (VEVI) or a New Delhi metallo- ⁇ - lactamase (NDM).
  • VEVI Verona integron-encoded metallo-P-lactamase
  • NDM New Delhi metallo- ⁇ - lactamase
  • the MBL is VEVI or NDM.
  • the bacterial infection is an infection of at least one carbapenem-resistant Gram-negative bacteria.
  • the bacterial infection is an infection of at least one bacterium belonging to the family Enter obacteriaceae, Acinetobacter, Pseudomonas.
  • the Enterobacteriaceae bacterium is a Klebsiella species, such as Klebsiella pneumonia or Escherichia coli.
  • the Pseudomonas bacterium is Pseudomonas aeruginosa.
  • the bacterium causing infection is selected from
  • Staphylococcus aureus Staphylococcus epidermidis and other coagulase-negative staphylococci
  • Streptococcus pyogenes Streptococcus pneumoniae
  • Streptococcus agalactiae Enterococcus species
  • Coryne bacterium diphtheriae Listeria monocytogenes
  • Bacillus anthracis Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Vibrio cholerae, and Campylobacter jejuni.
  • the bacterium causing infection is selected from selected from Enterobacteriaceae (includes: Escherichia, Salmonella, Klebsiella, Enterobacter), Pseudomonas aeruginosa, Acinetobacter species, Haemophilus influenzae, Clostridium tetani, Clostridium botulinum, Bacteroides species, Prevotella species, Porphyromonas species, Fusobacterium species, Mycobacterium tuberculosis, and Mycobacterium leprae.
  • the bacterium causing infection is from the
  • the bacterium causing infection is Klebsiella pneumoniae.
  • the diseases, disorders or conditions arising from a bacterial infection include all such pathogeneses that are common to infections of MBL-expression bacteria. These are well known to those skilled in the art. Some of the more common examples are listed below for the better known MBL-expressing bacteria, however, a person skilled in the art would appreciate that these lists are non-exhaustive and many of the diseases, disorders and conditions listed for one MBL-expression bacterium will be common to other MBL- expressing bacteria.
  • the disease, disorder or condition arising from a bacterial infection is for example, but not limited to, pneumonia (for example bronchopneumonia or bronchitis), thrombophlebitis, urinary tract infection (UTI), cholecystitus, diarrhea, upper respiratory tract infection, lower biliary tract infection, wound infection, surgical wound infection, osteomyelitis, meningitis, bacteremia, septicemia, sepsis, septic shock, rhinoscleroma, ozena, ankylosing spondylitis, destructive changes to human lungs via inflammation and hemorrhage with cell death (necrosis), lung abscesses, cavitations, empyemas, or ural adhesions, or a combination thereof.
  • pneumonia for example bronchopneumonia or bronchitis
  • UTI urinary tract infection
  • cholecystitus cholecystitus
  • diarrhea upper respiratory tract infection
  • lower biliary tract infection wound infection
  • the disease, disorder or condition arising from bacterial infection is for example, but not limited to, cystic fibrosis, pneumonia, bacteremia, endocarditis, meningitis, brain abscesses, septic shock, UTI, gastrointestinal infection (e.g. diarrhea, enteritis, or enterocolitis), skin infections (e.g.
  • ecthyma gangrenosum soft tissue infections, infections of burn injuries, infections of the outer ear, bacterial keratitis, endophthalmitis, infections due to the presence of a medical device, infections due to hospitalization, infections caused by low water quality, postoperative infections, or osteomyelitis, or a combination thereof.
  • the disease, disorder or condition arising from bacterial infection is for example, but not limited to, enteric infections (e.g. diarrhea), intra-abdominal infections, cholecy stilus, bacteremia, cholangitis, UTI, meningitis, pneumonia, septic arthritis, endophthalmitis, suppurative thyroiditis, osteomyelitis, endocarditis, skin infections or soft tissue infections, or a combination thereof.
  • enteric infections e.g. diarrhea
  • intra-abdominal infections e.g. diarrhea
  • cholecy stilus bacteremia
  • cholangitis cholangitis
  • UTI meningitis
  • endophthalmitis suppurative thyroiditis
  • osteomyelitis suppurative thyroiditis
  • endocarditis skin infections or soft tissue infections, or a combination thereof.
  • the subject is a human. In a further embodiment, the subject is an animal, such as a companion animal or livestock.
  • the companion animal or livestock is cat, dog, horse, pig, bird, cow or chicken.
  • the one or more antibiotics are selected from any antibiotic which treats metallo-B-lactamase-expressing bacterial infections.
  • one or more antibiotics are B-lactam antibiotics.
  • the B-lactam antibiotic is selected from penicillin derivatives (penems), cephalosporins (cephems), monobactams and carbapenems.
  • the B-lactam antibiotic is selected from imipenem, ertapenem, meropenem, doripenem, biapenem, panipenem, ticarcillin, ampicillin, amoxicillin, carbenicillin, piperacillin, azlocillin, mezlocillin, ticarcillin, cefoperazone, cefotaxime, ceftriaxone and ceftazidime.
  • the one or more antibiotics are carbapenem antibiotics.
  • the carbapenem antibiotic is selected from meropenem, biapenm, doripenem, ertapenem, panipenem and imipenem.
  • the one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, and the one or more ⁇ -lactam antibiotics in the compositions and kits of the present application are formulated as separate pharmaceutical compositions, for separate administration to, or use in, subjects.
  • the separate pharmaceutical compositions are formulated independently of each other and in accordance with the desired mode of administration for each active.
  • the one or more ⁇ -lactam antibiotics are formulated for administration, or use, by oral delivery or for delivery by injection.
  • the one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof are formulated for administration, or use, by oral delivery or for delivery by injection.
  • the one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, and the one or more ⁇ -lactam antibiotics in the compositions and kits of the present application are formulated as a single pharmaceutical composition, for combined, simultaneous administration to, or use in, subjects.
  • the single pharmaceutical composition is formulated for administration, or use, by oral delivery or by injection.
  • the methods and uses as described in this section for the one or more non- natural compounds of Formula (I) or Formula A, or a pharmaceutically acceptable salts and/or solvates thereof further comprise the modification of one or more non-natural compounds of Formula (I) or Formula A as defined above, or a pharmaceutically acceptable salts and/or solvates thereof, through derivatization or encapsulation in drug delivery vehicles as described above, to increase cell permeability and target delivery. Selection for the type of modification used is dependent, for example, upon the disease, disorder or condition being treated and the administrative route, and is well within the knowledge of a person skilled in the art.
  • Trifluoroacetic acid (TFA) (11 ml) was added dropwise over 30 min. to a solution of 2 (2.0 g, 5.8 mmol) in DCM (11ml) and methanol (11 ml) at 0°C.
  • the reaction mixture was stirred for 30min at 0°C at which time the solvents were removed under reduced pressure.
  • the residue was partitioned between diethyl ether (100 ml) and water (100 ml).
  • the ether layer was further extracted with water (3 x 20ml).
  • the combined aqueous layer was treated with NaHCC>3 (6 g, 69 mmol) at 0°C until pH 8 was achieved.
  • N-trityl-Z>-serine methyl ester (9).
  • 13 C NMR (176 MHz, CDC1 3 ) ⁇ 167.19, 135.03, 132.71, 131.87, 131.83, 124.87, 53.21, 37.77, 34.42.
  • the water layer was separeated and extracted with ethylacetate (2 x 10 ml) and dichlorom ethane (2 x 10 ml).
  • the combined organic layers were evaporated to dryness and used directly in the next step without further purification.
  • LL-25h was obtained following the general procedure for sulfamidates opening by reacting sulfamidate L-24i (0.25 mmol, 84 mg) with L-aspartic acid dimethyl ester in 71% yield (89 mg).
  • Procedure A Compound 25 (0.25 mmol) was stirred in a mixture of trifluoroacetic acid and DCM (2 mL, 6: 1 ratio) at room temperature for 16 h. The solvents were evaporated, then co-evaporated with MeOH (3 x 10 mL) and dried under high vacuum. The resulting salt was dissolved in saturated potassium carbonate solution and extracted with chloroform (5 x 10 mL). The combined organic layers were dried (MgS0 4 ) and concentrated in vacuo to give the free amino ester. The crude product was purified by flash column chromatography (using a gradient of 0-40% CH 2 C1 2 : MeOH). For compound LD-25a during flash chromatography demethylation of one ester group occurred and compound LD-26c was obtained in 62% yield (38mg). Compound LL-26b was obtained in 78% yield (65 mg).
  • Procedure B Compound 25 (0.5 mmol) was stirred in 4M HC1 in dioxane (2 ml) at room temperature for 0.5 h. The solvents were evaporated and then co- evaporated with MeOH (3 x 10 mL) and dried under high vacuum. The resulting salt was dissolved in saturated potassium carbonate solution, and extracted with chloroform (5 ⁇ 10 mL). The combined organic layers were dried (MgS0 4 ) and concentrated in vacuo to give the free amino ester. The crude product was used in the next step without further purification. Compound LD-26a was obtained in 64% yield (83 mg). Compound LL- 26b was obtained in 77% yield (130 mg).
  • LLL-27d was prepared starting from L-24e according to the synthetic scheme.
  • LLL-27e was obtained by reacting sulfamidate L-24g (0.25 mmol, 90 mg) with LL-26d (prepared above) in 43% yield (56 mg).
  • DLL-27f was obtained as described for LLL-27e. Yield 40%.
  • the oNs group was further deprotected using thiophenol (5eq), DIPEA (4 eq) in acetonitrile for 2 h at room temperature. After draining and resin wash as already described, small part was taken for cleavage to confirm the product formation. Next ring opening reaction was carried out with another oNs-Azi-OMe to yield the protected AM-A attached to a solid support. After the nosyl group deprotection using the conditions described above, the resin was washed with DCM, methanol , dried under vacuum for 4 h, and the cleaved with a mixture of TFA/TIS/H 2 0 (95:2.5:2.5) for 2 hours at room temperature. The resulting solution was concentrated under reduced pressure and purified using reverse phase chromatography. 20
  • AM-A, derivatives and analogues synthesized according to Examples 1 to 13 were explored for their biological activity through dose-response assays using purified recombinant NDM-1 as well as cell-based assays to assess in vivo synergy with meroprenem in an NDM-1 expressing strain of E.coli.
  • NDM-1 (final concentration: 4 nM) was incubated with AM-A compounds in serial 1/2 dilutions from a maximum concentration of 256 ⁇ for 10 minutes at 37°C.
  • the buffer used was 50 mM HEPES pH 7.5 supplemented with 50 ng/ ⁇ bovine serum albumin and 10 ⁇ ZnS0 4 for enzyme stability. Tween20 was also added to a final concentration of 0.01% to prevent spurious inhibition due to compound insolubility. Addition of substrate (nitrocefin, final concentration 30 ⁇ ) initiated reaction.
  • Progress curves of hydrolysis at 37°C were monitored at 490 nm in 96-well microplate format using a Spectramax reader (Molecular Devices).
  • IC 50 curves were generated using GraFit version 4.0.10 and a 4-parameter fit. The results are shown in Table 4, column 2.
  • RC Rescue concentration
  • AM-A can be depicted as follows with the three a-carbons labeled as positions 3, 6, and 9, and the three amino acid units as Asp, APA1 and APA2:
  • the N-terminal amine is therefore dispensable.
  • protection with the bulky nosyl group weakens enzyme inhibition activity and in-cell activity.
  • Alternate spacing of the C- terminal carboxyl (C2 of AM-B vs. C3 AM-A) is accommodated with retention of activity, nevertheless, a free carboxyl rather than an amide is optimal for activity in the cell, perhaps reflecting access and transport to the periplasm.
  • APA2 toxin A, ZX-26e
  • toxin A showed unusual behavior in enzyme inhibition studies. Rather than precipitous dose dependence from full activity to inactivation, toxin A IC 50 curves showed a shallow inhibition gradient and did not result in complete loss of enzyme action. Furthermore, toxin A had no ability to rescue meropenem activity in cell potentiation assays.
  • the APA2 unit is therefore optimal for AM-A activity, but is tolerant of modification. Protection of the carboxylates of toxin A (compounds ZX-30a and LL-26d) or replacement of N-terminal Asp with Glu abolishes activity.
  • Compounds with good enzyme inhibition activity but little cell-based activity can still represent viable drug candidates.
  • Structural modifications and/or formulation modifications to improve cell permeation are known in the art and can be readily applied here.
  • the compounds can be modified by conjugation with fatty acids, vitamins (e.g. folates), single chain antigen, binding molecules, cell penetrating peptides, nanoparticles, antibodies and/or protein.
  • the compounds can be encapsulated within drug delivery vehicles, such as liposomes, micelles, dendrimers or polyethylene glycol carriers.

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Abstract

The present application relates to processes for the preparation of the aspergillomarasmine A (AM-A) compound and analogs and derivatives thereof. The features of this process involves the use of a cyclic activated serine synthon in the construction of the poly-amino acid backbone. Also included are certain novel analogs of AM-A along with compositions comprising these compounds and uses thereof.

Description

TITLE: PROCESS FOR THE PREPARATION OF 2-[2-(2-AMINO-2-CARBOXY- ETHYLAMINO)-2-CARBOXY-ETHYLAMINO]-SUCCINIC ACID (AM-A) AND ANALOGS AND DERIVATIVES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority from co-pending
U.S. provisional patent application No. 62/248,648, filed on October 30, 2015, copending U.S. provisional patent application No. 62/266,258, filed on December 11, 2015 and co-pending U.S. provisional patent application No. 62/374,970, filed on August 15, 2016, the contents of each of which are incorporated herein by reference in their entirety.
FIELD
[0002] The present application relates to a process for the preparation of
Aspergillomarasmines A (AM-A). In particular, the present application describes a process for the preparation of AM-A and analogs and derivatives thereof. The application also includes certain novel analogs of AM-A, compositions comprising these analogs and methods of use thereof.
BACKGROUND
[0003] Aspergillomarasmines A (AM-A) and B (AM-B) are fungus-derived molecules that were discovered and reported in the early 1960s (1, 2) along with their wilting and necrotic activity in plant leaves. This molecule was refurbished in the 1980s as an inhibitor of angiotensin-converting enzyme (ACE) (3) and in the early 1990s as a pre-clinical candidate for the inhibition of activation of human endothelin (4, 5). The activation of human endothelin is initiated by the endothelin-converting enzyme, which, like angiotensin-converting enzyme, is a metalloproteinase. This previous work demonstrated that AM-A was well-tolerated and had low toxicity in mice (LD50 159.8 mg/kg, i.v. compared to EDTA at 28.5 mg/kg) and had no effect on mean atrial blood pressure (6).
[0004] Carbapenems, or "last resort" antibiotics have encountered bacterial resistance. Two sources of resistance are the enzymes New Delhi metallo-P-lactamase 1 (NDM-1) and Verona integrin-encoded metallo-P-lactamase 2 (VIM-2). AM-A was also reported as a rapid and potent inhibitor of both NDM and VIM-2 by removing the zinc from the metallo-P-lactamases (MBLs) without toxic side effects. Specifically, AM-A was used in combination with the "last resort" antibiotics to restore the activity of the bacterial resistant antibiotics in MBL-derived Gram-negative pathogens (7).
[0005] With studies establishing the importance of AM-A as a potent inhibitor for several clinically relevant protein and bacterial targets, there is a need for larger quantities of AM-A to be produced and consequently a need for efficient avenues in which to produce them. Like most natural products, AM-A is harvested from its natural resource. Routinely, AM-A is purified from Aspergillus flavus oryzae, Aspergillus versicolor, ascomycete Pyrenophora teres, colletotrichum gloeosporioides and fusarium oxysporum (2, 8, 9). However, this process can be tedious, time consuming, and expensive, as well being potentially unsustainable for the resource in larger quantities (10). Furthermore, the number of structural analogs that can be obtained from harvesting is limited. Most often, one turns to total synthesis as the only viable means to produce a given natural product as a drug without a huge economical expense, although, total synthesis has the added imposition that an industrially and economically viable total synthesis ideally comprises no more than 20 steps with acceptable overall yield. There are several total syntheses of natural products which have been implemented on an economically viable large scale synthesis, including the synthesis of the carbapenem-type antibiotic, thienamycin (11).
[0006] Ohfune et al. used the C3 amino acid equivalents, 2-amino-3-butenol derivatives, as a masked serine moiety for the synthesis of the AM-A poly amino acid (12). The authors, however, were unsuccessful in their last synthetic step to produce the AM-A poly amino acid with the appropriate stereochemistry.
SUMMARY
[0007] The present application relates to improved synthetic methods for the preparation of the aspergillomarasmine A (AM-A) compound and analogs and derivatives thereof. The features of this process involves the use of a cyclic activated serine synthon in the construction of the poly-amino acid, AM-A.
[0008] Accordingly, the present application includes a process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof: C02H C02H
(I)
the process comprising:
a) reacting a compound of Formula (II) with a compound of Formula (III) under conditions to provide a compound of Formula (IV):
1
Figure imgf000004_0001
(II) (HI) (IV) b) treating the compound of Formula (IV) with a suitable deprotecting agent under conditions to provide a compound of Formula (V):
Figure imgf000004_0002
(V) c) reacting the compound of Formula (V) with a compound of Formula (III) under conditions to provide a compound of Formula (VI):
Figure imgf000004_0003
(V) (III) VI)
d) deprotecting the compound of Formula (VI) under conditions to provide the compound of Formula (I),
wherein
n is 1 or 2; R1 is a direct bond or 0-S02; and
1 2
PG1 and PG are suitable protecting groups.
[0009] The present application also includes a process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof, the process comprising: a) reacting the compound of Formula (II) with a compound of Formula (VII) under conditions to provide a compound of Formula (VIII):
Figure imgf000005_0001
(Π) (VII) (VIII) b) treating the compound of Formula (VIII) with a suitable deprotecting agent under conditions to provide a compound of Formula (IX):
Figure imgf000005_0002
c) reacting the compound of Formula (IX) with the compound of Formula (VII) under conditions to provide a compound of Formula (X):
Figure imgf000005_0003
d) oxidizing the compound of Formula (X) under conditions to provide a compound of Formula (XI):
Figure imgf000006_0001
(XI) and e) deprotecting the compound of Formula (XI) under conditions to provide the compound of Formula (I), wherein n is 1 or 2;
1 2
PG and PG are suitable protecting groups.
[0010] The present application includes novel non-natural diastereomeric compounds of the application, along with pharmaceutical compositions comprising said compounds and a pharmaceutically acceptable carrier, and methods of use thereof .
[0011] The present applications also includes compounds of Formula A, and pharmaceutically acceptable salts and/or solvates thereof;
Figure imgf000006_0002
A
wherein:
R2 is selected from H, S02Ar, CH2CH(C02R6) HR7, CH2C02R6, CH2C(0) HR7 and Ci. 6alkyl;
3 5
R -R are independently selected from H and C1-6alkyl;
R6 and R7 are independently selected from H, Ci^alkyl and S02Ar; n is 1 or 2,
Ar is aryl that is unsubstituted or substituted with one or more of halo, N02, C(0)C1- 4alkyl and C02C1-4alkyl; and each alkyl group is optionally fluoro-substituted, provided that when n is 1, R2, R3, R4, R5, R6 and R7 are not all H.
[0012] Also included are pharmaceutical compositions comprising a compound of
Formula A and a pharmaceutically acceptable carrier.
[0013] The present application includes a method of treating a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more β-lactam antibiotics in combination with an effective amount of one or more compounds of Formula A as defined above, or pharmaceutically acceptable salts and/or solvates thereof.
[0014] The present application also includes a use of a β-lactam antibiotic in combination with one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for treating a bacterial infection in a subject; a use of a β-lactam antibiotic in combination with one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for treating a bacterial infection in a subject; and a β-lactam antibiotic and one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to treat a bacterial infection in a subject.
[0015] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.
DETAILED DESCRIPTION
I. Definitions
[0016] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.
[0017] In understanding the scope of the present disclosure, the term
"comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.
[0018] As used in this application and claim(s), the word "consisting" and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
[0019] The term "consisting essentially of, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and/or steps.
[0020] The terms "about", "substantially" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.
[0021] The present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.
[0022] As used in this application, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise. For example, an embodiment including "a compound" should be understood to present certain aspects with one compound or two or more additional compounds. [0023] In embodiments comprising an "additional" or "second" component, such as an additional or second compound, the second component as used herein is chemically different from the other components or first component. A "third" component is different from the other, first, and second components, and further enumerated or "additional" components are similarly different.
[0024] The term "and/or" as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that "at least one of or "one or more" of the listed items is used or present.
[0025] The term "suitable" as used herein means that the selection of the particular compound or conditions would depend on the specific synthetic manipulation to be performed, and the identity of the molecule(s) to be transformed, but the selection would be well within the skill of a person trained in the art. All process/method steps described herein are to be conducted under conditions sufficient to provide the product shown and for the reaction to proceed to a sufficient extent. A person skilled in the art would understand that all reaction conditions, including, for example, reaction solvent, reaction time, reaction temperature, reaction pressure, reactant ratio and whether or not the reaction should be performed under an anhydrous or inert atmosphere, can be varied to optimize the yield of the desired product and it is within their skill to do so.
[0026] The expression "proceed to a sufficient extent" as used herein with reference to the reactions or process steps disclosed herein means that the reactions or process steps proceed to an extent that conversion of the starting material or substrate to product is sufficient for the given reaction. Conversion may be sufficient when greater than about 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the starting material or substrate is converted to product.
[0027] In embodiments of the application, the compounds described herein have at least one asymmetric centre. Where compounds possess more than one asymmetric centre, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (e.g. less than 50%, suitably less than 20%, suitably less than 10%), more suitably less than 5%) of compounds having alternate stereochemistry.
[0028] The term "oxidant" as used herein refers to any compound or combination of compounds that oxidizes a desired functional group. An oxidizing agent results in the overall loss of electrons, or in the case of organic chemistry, loss of hydrogen atoms from the functional group.
[0029] The term "reducing agent" as used herein refers to any compound or combination of compounds that reduces a desired functional group. A reducing agent results in the overall addition of electrons, or in the case of organic chemistry, hydrogen atoms to the functional group.
[0030] The term "non-nucleophilic base" as used herein refers to an organic base that is a poor nucleophile. Typically, non-nucleophilic bases are bulky which prevent the base from acting as a nucleophile.
[0031] The term "inert solvent" as used herein means a solvent that does not interfere with or otherwise inhibit a reaction. Accordingly, the identity of the inert solvent will vary depending on the reaction being performed. The selection of inert solvent is within the skill of a person in the art. Examples of inert solvents include, but are not limited to, benzene, toluene, tetrahydrofuran, acetone, dioxane, ethyl ether, dichloromethane, dichloroethane, ethyl acetate, dimethyl formamide (DMF), acetonitrile, C1-6alkylOH (e.g. methanol, ethanol, n-propanol, 2-propanol, n-butanol, butan-2-ol and 2- methyl- 1-propanol), diethyl carbonate, hexane and dimethylsulfoxide (DMSO). Further examples, can include aqueous solutions, such as water and dilute acids and bases, and ionic liquids, provided that such solvents do not interfere with the reaction.
[0032] The term "solvent" includes both a single solvent and a mixture comprising two or more solvents.
[0033] The term "alkyl" as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups. The term Ci. 6alkyl means an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms.
[0034] t-Boc as used herein refers to the group t-butyloxycarbonyl. [0035] Ac as used herein refers to the group acetyl.
[0036] Ts (tosyl) as used herein refers to the group ^-toluenesulfonyl.
[0037] oNosyl as used herein refers to the group orthonitrobenzenesulfonyl.
[0038] Bn as used herein refers to the group benzyl.
[0039] Trityl (or "Trt") as used herein refers to the group triphenylmethyl.
[0040] Fmoc as used herein refers to the group fluorenylmethoxycarbonyl.
[0041] Me as used herein refers to the group methyl.
[0042] Et as used herein refers to the group ethyl.
[0043] Ph as used herein refers to the group phenyl.
[0044] Ns as used herein refers to the group nitrobenzylsulfonyl.
[0045] TFA as used herein refers to the compound trifluoroacetic acid.
[0046] HC1 as used herein refers to the compound hydrochloric acid.
[0047] PhSH as used herein refers to the compound of thiophenol.
[0048] DIPEA as used herein refers to the compound of N,N diisopropylethylamine.
[0049] TMTOH as used herein refers to the compound of trimethyltin hydroxide.
[0050] CAN as used herein refers to the compound of cerium(IV) ammonium nitrate.
[0051] TcBoc as used herein refers to group trichloro-t-butyloxy-carbonyl.
[0052] Cbz as used herein refers to the group benzyl oxycarbonyl.
[0053] Pmb as used herein refers to the group para-methoxybenzyl.
[0054] Bn as used herein refers to the group benzyl.
[0055] The terms "protecting group" or "PG" or the like as used herein refer to a chemical moiety which protects or masks a reactive portion of a molecule to prevent side reactions in those reactive portions of the molecule, while manipulating or reacting a different portion of the molecule. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portions of the molecule. The selection of a suitable protecting group can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in "Protective Groups in Organic Chemistry" McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas). Examples of suitable protecting groups include, but are not limited to t-Boc, Ac, Ts, methyl esters, t-butyl esters, silyl esters, o-Ns, p-Ns, Bn, Fmoc, benzoyl, dimethoxytrityl, p- methyoxybenzyl ether, trityl, carbooxybenzyl, benzoyl and the like.
[0056] The term "suitable deprotecting agent" as used herein is intended to include any of the standard deprotecting agents used in the deprotection procedures (i.e. removal of protecting groups) as outlined in the application. Many conventional deprotecting agents are known in the art, for example as described in "Protective Groups in Organic Chemistry" McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and
rd
Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas). Examples of suitable deprotecting agents include, but are not limited to TFA, HC1, PhSH/DIPEA, H2/Pd/C, TMTOH, piperidine, H3(aq), CAN and the like.
[0057] The term "activating group" as used herein means any substituent that increases the reactivity of its adjacent or nearby atoms, relative to the atom's reactivity in the presence of hydrogen.
[0058] The term "aspergillomarasmine A" or "AM- A" as used herein refers to the naturally occurring compound having the following relative stereochemistry:
Figure imgf000012_0001
[0059] The terms "natural aspergillomarasmine A", "natural AM- A", "naturally occurring aspergillomarasmine A" and "naturally occurring AM -A" all refer to AM-A as produced in nature, for example by the fungus, Aspergillus versicolour.
[0060] The term "natural isomers of Formula I" as used herein refers to compounds of Formula I having the same relative stereochemistry as found in naturally occurring AM-A (i.e. L,L,L).
[0061] The term "non-natural isomers of Formula I" as used herein refers to compounds of Formula I having a relative stereochemistry other than that found in naturally occurring AM-A (i.e. L,L,L).
[0062] The amino acids contained in compounds of the formulae (II), (Ilia), (Illb) and (VII) can be of either the D- or L- configurations or can be mixtures of the D- and L- isomers, including racemic mixtures.
[0063] The term "pharmaceutically acceptable salt" means an acid addition salt or a basic addition salt suitable for, or compatible with, the treatment of subjects. The term "pharmaceutically acceptable salts" embraces salts commonly used to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically acceptable acid addition salts are prepared from an inorganic acid or an organic acid. Examples of such inorganic acids include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Examples of appropriate organic acids include, for example, aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include, without limitation, formic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic, galactic, and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from Ν,Ν'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine, lysine and procaine.
[0064] The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
[0065] The term "solvates" as used herein refers to complexes formed between a compound and a solvent from which the compound is precipitated or in which the compound is made. Accordingly, the term "solvate" as used herein means a compound, or a salt of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice. Examples of suitable solvents include, but are not limited to ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate". The formation of solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions to form a particular solvate can be made by a person skilled in the art.
[0066] The term "pharmaceutically acceptable solvate" means a solvate suitable for, or compatible with, the treatment of subjects. For pharmaceutically acceptable solvates, a suitable solvent is physiologically tolerable at the dosage used or administered.
[0067] The expression "disease, disorder or condition arising from a bacterial infection" as used herein refers to any disease, disorder or condition that is directly or indirectly caused by the presence of a bacterial infection in a subject.
[0068] The term "subject" as used herein includes all members of the animal kingdom including mammals, such as equines and humans. Other examples of subjects include, companion animals, such as felines and canines.
[0069] The term "pharmaceutical composition" as used herein refers to a composition of matter for pharmaceutical use. [0070] The term "pharmaceutically acceptable" means compatible with the treatment of subjects.
[0071] The term "parenteral" as used herein means taken into the body or administered in a manner other than through the gastrointestinal tract.
[0072] The term "administered" as used herein means administration of an effective amount of a compound, including for example, the antibiotic and compound of Formula A (such as a compound of Formula I), or a salt and/or solvate thereof, to a cell either in cell culture or in a subject.
[0073] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve a desired result. For example, in the context of treating a bacterial infection, or a disease, disorder or condition arising from a bacterial infection, an effective amount of the antibiotic and/or compound of Formula A (such as a compound of Formula I), or a salt and/or solvate thereof, is an amount that, for example, reduces the bacterial infection compared to the bacterial infection without administration of the antibiotic and the compound of Formula A (or compound of Formula I), or a salt and/or solvate thereof. Further, in the context of improving the efficacy of an antibiotic for the treatment of a bacterial infection or a disease, disorder or condition arising from a bacterial infection an effective amount of the compound of Formula A (or compound of Formula I), or a salt and/or solvate thereof, is, for example, an amount that, for example, reduces the bacterial infection compared to the reduction of the bacterial infection with administration of the antibiotic alone. By "reducing the infection", it is meant, for example, reducing the amount of the infectious agent in the subject and/or reducing the symptoms of the infection. Effective amounts may vary according to factors such as the disease state, age, sex and/or weight of the subject. The amount of a given compound or composition that will correspond to such an amount will vary depending upon various factors, such as the given compound or composition, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. [0074] The terms "to treat", "treating" and "treatment" as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, diminishment of extent of bacterial infection, stabilization (i.e. not worsening) of the state of the bacterial infection, preventing spread of the bacterial infection, delay or slowing of infection progression, amelioration or pallitating of the bacterial infectious state, diminishment of the reoccurrence of bacterial infection, diminishment, stabilization, alleviation or amelioration of one or more diseases, disorders or conditions arising from the bacterial infection, diminishment of the reoccurrence of one or more diseases, disorders or conditions arising from the bacterial infection, and remission of the bacterial infection and/or one or more symptoms or conditions arising from the bacterial infection, whether partial or total, whether detectable or undetectable. "To treat", "treating" and "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "To treat", "treating" and "treatment" as used herein also include prophylactic treatment. For example, a subject with an early bacterial infection is treated to prevent progression, or alternatively a subject in remission is treated to prevent recurrence.
[0075] "Palliating" an infection, disease, disorder and/or condition means that the extent and/or undesirable clinical manifestations of an infection, disease, disorder and/or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the infection, disease, disorder and/or condition.
[0076] The term "prevention" or "prophylaxis" and the like as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with a bacterial infection and/or a disease, disorder and/or condition arising from a bacterial infection or manifesting a symptom associated with a bacterial infection and/or a disease, disorder and/or condition arising from a bacterial infection.
[0077] When used, for example, with respect to the methods of treatment, uses, compositions and kits of the application, a subject, for example a subject "in need thereof is a subject who has been diagnosed with, is suspected of having, may come in to contact with, and/or was previously treated for a bacterial infection or a disease, disorder or condition arising from a bacterial infection.
II. Methods of the Application
[0078] The present application includes a process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof:
Figure imgf000017_0001
(I) the process comprising:
a) reacting a compound of Formula (II) with a compound of Formula (III) under
Figure imgf000017_0002
(Π) (III) (IV)
conditions to provide a compound of Formula (IV): b) treating the compound of Formula (IV) with a suitable deprotecting agent under conditions to provide a compound of Formula (V):
Figure imgf000017_0003
(V) c) reacting the compound of Formula (V) with a compound of Formula (III) under conditions to provide a compound of Formula (VI):
Figure imgf000018_0001
(V) (III) (VI)
and
d) deprotecting the compound of Formula (VI) under conditions to provide the compound of Formula (I),
wherein
n is 1 or 2;
R1 is a direct bond or 0-S02; and
1 2
PG1 and PG are suitable protecting groups.
[0079] In an embodiment, the compound of Formula (III) is a compound of
Formula (Ilia) or a compound of Formula (Illb):
Figure imgf000018_0002
wherein
PG1 is a suitable protecting group for carboxylic acids; and
PG is a suitable protecting and/or activating group for amines.
[0080] In an embodiment of the application, the compounds of Formula (Ilia) are available using methods known in the art. For example, compounds of Formula (Ilia) are prepared by combining the commercially available D-/L-serine with methanesulfonyl chloride in the presence of an organic non-nucleophilic base in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. In an embodiment, trityl protected L-serine is used. Examples of non-limiting reaction temperatures are about 30°C to about 80°C, about 40°C to about 75°C, or about 50°C to about 70°C. Examples of non-limiting reaction times are about 30 hours to about 80 hours, about 40 hours to about 70 hours, or about 50 hours to about 60 hours. Examples of suitable bases include, but are not limited to, organic amines, such as triethylamine.
[0081] In an embodiment, PG1 is a suitable protecting group for carboxylic acids.
In another embodiment, PG1 is selected from methyl, ethyl, t-butyl, benzyl, trialkylsilyl and triarylsilyl. In a further embodiment, PG1 is selected from methyl, ethyl, t-butyl, benzyl and trimethylsilyl. In yet a further embodiment, PG1 is selected from methyl, t- butyl and benzyl.
[0082] In an embodiment, PG is a suitable protecting and/or activating group for amines. In another embodiment, PG is selected from t-Boc, TcBoc, Fmoc, Bn, benzoyl, Cbz, 4-nitrobenzyloxy carbonyl, Pmb, o-nosyl, p-nosyl and trityl. In a further embodiment, PG is selected from t-Boc, Fmoc, Bn, benzoyl, Cbz, Pmb, o-nosyl and trityl.
[0083] In an embodiment, PG is an activating group on the compound of
Formula (Ilia) which activates the carbon adjacent to the nitrogen for nucleophilic attack by the compound of Formula (II). In another embodiment, PG is an electron- withdrawing group. In a further embodiment, PG is o-nosyl.
[0084] In some embodiments, to facilitate efficient ring opening of the compound of Formula (Ilia), the protecting PG1 group is a functional group that is both an electron- withdrawing group as well as a protecting group which could be removed under conditions suitable to not remove PG1. In some embodiments, the o-nosyl moiety is selected for its versatility as both an activating group and a protecting group.
2
[0085] In an embodiment, the compounds of Formula (Ilia) wherein PG is o- nosyl are prepared by treating a compound of Formula (Ilia) wherein PG is a bulky, acid labile protecting group, such as trityl, in the presence of an acid in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about -10°C to about 15°C, about - 5°C to about 10°C, or about 0°C to about 5°C. Examples of non-limiting reaction times are about 10 minutes to about 1 hour or about 30 minutes to about 45 minutes. Examples of suitable acids include, but are not limited to, organofluorine compounds, such as trifluoroacetic acid (TFA). Following the removal of the bulky, acid labile protecting group, and without isolation of the intermediate, unstable aziridine, o-nitrobenzylsulfonyl chloride, for example, is added in the same pot in an inert solvent at room temperature and for a time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction times are about 10 hours to about 30 hours or about 15 hours to about 24 hours.
[0086] Compounds of Formula (Illb) are either commercially available or are prepared using methods known in the art.
[0087] The compounds of Formula (II) are commercially available in either the
D-, L- or D/L-configuration wherein the carboxylic acids are protected with suitable protecting groups. In an embodiment, the compound of Formula (II) is L-aspartic acid-di- t-butyl ester or L-aspartic acid di-methyl ester. Alternatively, the compounds of Formula (II) are prepared using methods known in the art.
[0088] In an embodiment of the application, the conditions to provide the compound of Formula (IV) comprise combining the compound of Formula (II) with the compounds of Formula (Ilia) or (Illb) in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 10°C to about 80°C, about 15°C to about 70°C, or about 20°C to about 60°C. Examples of non-limiting reaction times are about 1 hour to about 35 hours, about 5 hours to about 30 hours, or about 10 hours to about 24 hours.
[0089] In an embodiment of the application, the conditions to provide the compound of Formula (V) comprise combining the compound of Formula (IV) with any
2 1
deprotecting agent suitable to selectively remove PG over PG . In an embodiment of the application, the conditions to provide the compound of Formula (V) comprise treating the compound of Formula (IV) with a suitable deprotecting agent in the presence of a non- nucleophilic base in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 20°C to about 40°C or about 23°C to about 30°C. Examples of non-limiting reaction times are about 15 minutes to about 2 hours or about 30 minutes to about 1 hour. Examples of suitable deprotecting agents include, but are not limited to, trifluoroacetic acid (TFA), hydrochloric acid (HC1), thiophenol (PhSH), hydrogenolysis (H2/Pd/C) and piperidine. Examples of suitable bases include, but are not limited to, organic amines, such as diisopropylethylamine.
[0090] In an embodiment of the application, the conditions to provide the compound of Formula (VI) comprise combining the compound of Formula (V) with the compounds of Formula (Ilia) or (Illb) in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 10°C to about 80°C, about 15°C to about 70°C, or about 20°C to about 60°C. Examples of non-limiting reaction times are about 1 hour to about 35 hours, about 5 hours to about 30 hours, or about 10 hours to about 24 hours.
[0091] In an embodiment of the application, the conditions to provide the compound of Formula (IV) and the compound of Formula (VI) comprise basic conditions. In another embodiment, the basic conditions comprise an organic non- nucleophilic base. Examples of non-nucleophilic bases include, but are not limited to, triethylamine, Ν,Ν-diisopropylethylamine and l,8-diazabicycloundec-7-ene (DBU).
[0092] In another embodiment of the application, the conditions to provide the compound of Formula (IV) and the compound of Formula (VI) further comprise combining compounds of Formula (II) and (V) with the compound of Formula (Illb) in the presence of a soluble salt in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 20°C to about 80°C, about 30°C to about 70°C, or about 40°C to about 60°C. Examples of non -limiting reaction times are about 1 hour to about 30 hours, about 5 hours to about 20 hours, or about 10 hours to about 15 hours. Examples of a soluble salt include, but are not limited to, monopotassium phosphate and/or sodium phosphate.
[0093] The deprotection of the compound of Formula (IV) to the compound of
Formula (V) and the compound of Formula (VI) to the compound of Formula (I) is carried out using any deprotecting agent suitable for removing PG 1 and PG 2 protecting groups. Many conventional deprotecting agents are known in the art, for example as described in "Protective Groups in Organic Chemistry" McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas). Examples of suitable deprotecting agents include, but are not limited to trifluoroacetic acid (TFA); hydrochloric acid (HC1); a mixture of thiophenol and N,N-diisopropylethylamine (PhSH/DIPEA); hydrogenolysis (H2, Pd/C); trimethyltin hydroxide (TMTOH); piperidine; ¾(aq); and the like.
[0094] In an embodiment of the application, the compound of Formula (I) or esters, amides, salts and/or solvates thereof are prepared on a solid support, such as a solid support resin or bead. In another embodiment, the compound of Formula (I) or esters, amides, salts and/or solvates thereof is prepared on a Wang resin. The compound of Formula (II) attached to the Wang resin is commercially available in either the D-/L- configuration wherein the carboxylic acids and amine are protected with suitable protecting groups. In an embodiment, the compound of Formula (II) is Fmoc protected L- aspartic acid-di-t-butyl ester. Alternatively, the support-bound compounds of Formula (II) are prepared using methods known in the art.
[0095] In an embodiment of the application, the amine protecting group of the support-bound compound of Formula (II) is removed with a suitable deprotecting agent for a time sufficient for the conversion to proceed to a sufficient extent. Examples of non- limiting reactions times are about 5 minutes to about 30 minutes or about 10 minutes to about 20 minutes. Examples of suitable deprotecting agents include, but are not limited to, piperidine. In an embodiment, the support is subsequently washed with in inert solvent.
[0096] In an embodiment of the application, the conditions to provide the support-bound compound of Formula (IV) comprise combining the support-bound compound of Formula (II) with the compound of Formula (Ilia) in an inert solvent at room temperature and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction times are about 1 hour to about 35 hours, about 5 hours to about 30 hours, or about 10 hours to about 24 hours. In an embodiment, the resin is drained and washed with an inert solvent. [0097] In an embodiment of the application, the conditions to provide the support-bound compound of Formula (V) comprise combining the support-bound compound of Formula (IV) with any deprotecting agent suitable to selectively remove
2 1
PG over PG . In an embodiment of the application, the conditions to provide the support-bound compound of Formula (V) comprise treating the support-bound compound of Formula (IV) with a suitable deprotecting agent in the presence of a non-nucleophilic base in an inert solvent at room temperature and time sufficient for the conversion to proceed to a sufficient extent. Examples of non -limiting reaction times are about 30 minutes to about 3 hours or about 1 hour to about 2 hours. Examples of suitable deprotecting agents include, but are not limited to, thiophenol (PhSH). Examples of suitable bases include, but are not limited to, organic amines, such as diisopropylethylamine. The resin was drained and washed with an inert solvent.
[0098] In an embodiment of the application, the conditions to provide the support-bound compound of Formula (VI) comprise combining the support-bound compound of Formula (V) with the compound of Formula (Ilia) of (Illb) in an inert solvent at room temperature and time sufficient for the conversion to proceed to a sufficient extent. Examples of non -limiting reaction times are about 1 hour to about 35 hours, about 5 hours to about 30 hours, or about 10 hours to about 24 hours. The resin was drained and washed with an inert solvent.
[0099] In an embodiment of the application, the conditions to provide the support-bound compound of Formula (I) comprise combining the support-bound compound of Formula (VI) with any deprotecting agent or combindation of deprotecting
2 1
agents suitable to remove PG and PG and optionally to liberate the compound of Formula (I) from the support.
[00100] In an embodiment of the application, the conditions to provide the compound of Formula (I) comprise treating the support-bound compound of Formula
2 1
(VI) with a suitable deprotecting agent to remove PG and not PG in the presence of a non-nucleophilic base in an inert solvent at room temperature and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction times are about 30 minutes to about 3 hours or about 1 hour to about 2 hours. Examples of suitable deprotecting agents include, but are not limited to thiophenol (PhSH). Examples of suitable bases include, but are not limited to, organic amines, such as diisopropylethylamine. In an embodiment, the support is drained and washed with an inert solvent and dried under vacuum for time period of about 3 hours to about 5 hours.
[00101] embodiment of the application, the compound of Formula (I) solid support is then treated to remove PG simultaneously with the liberation of the for example, using a mixture of suitable acids, a trialkylsilyl agent and water at room temperature and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction times are about 30 minutes to about 3 hours or about 1 hour to about 2 hours. Examples of suitable acids include, but are not limited to, organofluorine compounds, such as trifluoroacetic acid (TFA). Examples of trialkyl silyl agents include, but are not limited to, triisopropylsilane. In some embodiments deprotection/liberationthe conditions comprises a ratio of 95:2.5:2.5 of a suitable acid:trialkyl silyl agent: water.
[00102] Other options for providing compounds of Formula I, for example by
1 2
removing PG and PG and liberation, individually or simultaneously are possible and could be selected by a person skilled in the art.
[00103] In an embodiment, with reference to the formula of AM- A, the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000024_0001
(I) (I)
[00104] In another embodiment, again with reference to the formula of AM- A, the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000024_0002
CO- [00105] In another embodiment, again with reference to the formula of AM-A, the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000025_0001
[00106] The present application encompasses both natural and non-natural isomers of Formula (I). Accordingly, in some embodiments the compound of Formula (I) has the same stereochemistry as the corresponding natural producted isolated from nature, eg. from the fungus, Aspergillus versicolour.
[00107] The present application also includes a process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof, the process comprising: a) reacting the compound of Formula (II) with a compound of Formula (VII) nder conditions to provide a compound of Formula (VIII):
Figure imgf000025_0002
b) treating the compound of Formula (VIII) with a suitable deprotecting agent under conditions to provide a compound of Formula (IX):
Figure imgf000026_0001
(IX) c) reacting the compound of Formula (IX) with the compound of Formula (VII) under conditions to provide a compound of Formula X):
Figure imgf000026_0002
(IX) (VII) (X)
d) oxidizing the compound of Formula (X) under conditions to provide a compound of Formula (XI):
Figure imgf000026_0003
(XI)
and
e) deprotecting the compound of Formula (XI) under conditions to provide the compound of Formula (I),
wherein
n is 1 or 2;
1 2
PG and PG are suitable protecting groups.
[00108] In an embodiment, PG1 is a suitable protecting group for carboxylic acids.
In another embodiment, PG1 is selected from methyl, ethyl, t-butyl, benzyl, trialkylsilyl and triarylsilyl. In a further embodiment, PG1 is selected from methyl, ethyl, t-butyl, benzyl and trimethylsilyl. In yet a further embodiment, PG1 is selected from methyl, t- butyl and benzyl. In yet a further embodiment, PG1 is methyl.
[00109] In an embodiment, PG is a suitable protecting group for amines. In another embodiment, PG is selected from t-Boc, TcBoc, Fmoc, Bn, benzoyl, Cbz, 4- nitrobenzyloxy carbonyl, Pmb, o-nosyl, p-nosyl and trityl. In a further embodiment, PG is selected from t-Boc, Fmoc, Bn, benzoyl, Cbz, Pmb, o-nosyl and trityl. In yet a further embodiment, PG is t-Boc.
[001 10] The compound of Formula (II) is commercially available in either the D-,
L- or D/L-configuration wherein the carboxylic acids are protected with suitable protecting groups. In an embodiment, the compound of Formula (II) is L-aspartic acid-di- t-butyl ester or L-aspartic acid di -methyl ester. Alternatively, the compounds of Formula (II) are prepared using methods known in the art.
[001 1 1] The compound of Formula (VII) is commercially available. In an embodiment, the compound of Formula (VII) is (R)-ter t-butyl 4-formyl-2,2- dimethyloxazolidine-3-carboxylate (R-Garner' s aldehyde). Alternatively, the compound of Formula (VII) is prepared using methods known in the art.
[001 12] In an embodiment of the application, the conditions to provide the compound of Formula (VIII) comprise reductive amination. In an embodiment, the conditions to provide the compound of Formula (VIII) comprise combining the compound of Formula (II) with the compound of Formula (VII) in the presence of a reducing agent and in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 10°C to about 50°C, about 15°C to about 40°C, or about 23°C to about 35°C. Examples of non-limiting reaction times are about 1 hour to about 30 hours, about 5 hours to about 25 hours, or about 10 hours to about 20 hours. Examples of a reducing agent include, but are not limited to, reducing metal salts. In an embodiment, the reducing metal salts include, but are not limited to, sodium borohydride, sodium cyanoborohydride and sodium triacetoxyborohydride.
[001 13] In an embodiment of the application, the conditions to provide the compound of Formula (IX) comprise combining the compound of Formula (VIII) with any deprotecting agent suitable to selectively remove PG 2 over PG 1. In an embodiment of the application, the conditions to provide the compound of Formula (IX) comprise treating the compound of Formula (VIII) with a suitable deprotecting agent in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 20°C to about 40°C or about 23°C to about 30°C. Examples of non-limiting reaction times are about 1 hour to about 30 hours, about 5 hours to about 20 hours or about 10 hours to about 16 hours. Examples of suitable deprotecting agents include, but are not limited to, trifluoroacetic acid (TFA).
[00114] In an embodiment of the application, the conditions to provide the compound of Formula (X) comprise reductive amination. In an embodiment, the conditions to provide the compound of Formula (X) comprise combining the compound of Formula (IX) with the compound of Formula (VII) in the presence of a reducing agent in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 10°C to about 50°C, about 15°C to about 40°C, or about 23°C to about 35°C. Examples of non-limiting reaction times are about 1 hour to about 30 hours, about 5 hours to about 25 hours, or about 10 hours to about 20 hours. Examples of a reducing agent include, but are not limited to, reducing metal salts. In an embodiment, the reducing metal salts include, but are not limited to, sodium borohydride, sodium cyanoborohydride and sodium tri acetoxyb orohy dri de .
[00115] In some embodiments, at least one of the H moieties in the compound of
Formula (X) is further protected with protecting groups suitable for amines. The selection of a suitable protecting group can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in "Protective Groups in Organic Chemistry" McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas). Examples of suitable protecting groups include, but are not limited to t-Boc, Ac, Ts, o-Ns, p-Ns, Bn, Fmoc, benzoyl, dimethoxytrityl, p- methyoxybenzyl ether, trityl, carbooxybenzyl, benzoyl and the like.
[00116] The conversion of the compound of Formula (X) to the compound of
Formula (XI) is suitably carried out in the presence of an oxidant in an inert solvent at temperatures and time sufficient for the conversion to proceed to a sufficient extent. Examples of non-limiting reaction temperatures are about 15°C to about 40°C, about 20°C to about 35°C, or about 23°C to about 30°C. Examples of non-limiting reaction times are about 10 minutes to about 5 hours, about 30 minutes to about 4 hours, or about 1 hour to about 3 hours. Examples of suitable oxidants, include, but are not limited to, chromium trioxide, potassium permanganate, pyridinium dichromate or ruthenium tetroxide.
[00117] The deprotection of the compound of Formula (VIII) to compound of
Formula (IX) and the compound of Formula (XI) to the compounds of Formula (I) is carried out using any deprotecting agent suitable for removing both PG1 protecting groups. Many conventional deprotecting agents are known in the art, for example as described in "Protective Groups in Organic Chemistry" McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas). Examples of suitable deprotecting agents include, but are not limited to trifluoroacetic acid (TFA); hydrochloric acid (HC1); a mixture of thiophenol and N,N-diisopropylethylamine (PhSH/DIPEA); hydrogenolysis (H2, Pd/C); trimethyltin hydroxide (TMTOH); piperidine; H3(aq); and the like.
[00118] In an embodiment, with reference to the formula of AM- A, the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000029_0001
(I)· [00119] In another embodiment, again with reference to the formula of AM-A, the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000030_0001
[00120] In an embodiment, the preparation of a compound of Formula (I), or esters, amides and/or solvates thereof using a compound of Formula (II) and compound of Formula (VIII) is also carried out using a solid support, such as support bound compound of Formula (II).
[00121] In an embodiment of the application, analogs of compound of Forumula
(I) include, but are not limited to, esters, amides and salts thereof. In an embodiment, the method of preparing esters or amides of the compound of Formula (I) comprises combining the compound of Formula (I) prepared using any of the above methods with an esterification agent or amidiation agent respectively.
III. Compounds of the Application
[00122] The present application includes novel non-natural diastereomeric compounds of the application. Accordingly, the application includes compounds of Formula (I) having the relative stereochemistry:
Figure imgf000030_0002
Figure imgf000031_0001
[00123] The present applications also includes compounds of Formula A, and pharmaceutically acceptable salts and/or solvates thereof;
Figure imgf000031_0002
A
wherein:
R2 is selected from H, S02Ar, CH2CH(C02R6)NHR7, CH2C02R6, CH2C(0)NHR7 and Ci. 6alkyl;
3 5
R -R are independently selected from H and C1-6alkyl;
R6 and R7 are independently selected from H, Ci^alkyl and S02Ar; n is 1 or 2,
Ar is aryl that is unsubstituted or substituted with one or more of halo, N02, C(0)C . 4alkyl and C02C1-4alkyl; and each alkyl group is optionally fluoro-substituted, provided that when n is 1, R2, R3, R4, R5, R6 and R7 are not all H.
[00124] In some embodiments of the compounds of Formula A, R is selected from CH2CH(C02R6)NHR7, CH2C02R6 and CH2C(0)NHR7, in which R6 and R7 are independently selected from H, C1-4alkyl and S02Ar, and aryl that is unsubstituted or substituted with N02, suitable 2-nitro. 3 5
[00125] In some embodiments of the compounds of Formula A, R -R are independently selected from H and C1-4alkyl.
[00126] In some embodiments of the compounds of Formula A, n is 2.
[00127] The compounds of Formula A contain several enantiomeric atoms. The application includes all stereoisomers of the compounds of Formula A, including mixtures thereof. In some embodiments, the stereochemistry of the compounds of Formula A is the same as the stereochemistry found in naturally occurring AM-A or in toxin A, for example AM-A isolated, for example by extraction, from a fungus, such as Aspergillus versicolor.
[00128] In some embodiments, the compounds of Formula A are selected from:
Figure imgf000032_0001
H
H02C 9<¾H NH2
H
C02H C02H
Figure imgf000032_0002
C02H C02H
Figure imgf000033_0001
C02H
Figure imgf000033_0002
C02H
,ΝΗ 2
N H
C02tBu C°2H ; and
Figure imgf000033_0003
[00129] The present application also includes all uses of the one or more of the above non-natural compounds of Formula (I) and Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, including their use as a medicament.
[00130] The one or more non-natural compounds of Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, are suitably formulated into pharmaceutical compositions for administration into a subject thereof. Accordingly, the present application further includes a pharmaceutical composition comprising one or more non-natural compounds of Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, and a pharmaceutically acceptable carrier and/or diluent.
[00131] In some embodiments, the one or more non -natural compounds of
Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, are further modified to increase cell permeability and target delivery. Modifications include, but are not limited to, derivatization of the one or more non- natural compounds of Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, with one or more of fatty acids, vitamins (e.g. folates), a single- chain antigen binding molecule, cell -penetrating peptides (CPPs), nanoparticles,
25
antibodies and proteins. In some embodiments, further modifications include, encapsulation of the one or more non-natural compounds of Formula (I) or Formula (A), and pharmaceutically acceptable salts and/or solvates thereof, within drug delivery vehicles. Examples of drug delivery vehicles include, but are not limited to, liposome- or
25
micelle-based carriers, dendrimers and polyethylene glycol carriers. Selection for the type of modification used is dependent, for example, upon the disease, disorder or condition being treated and the administrative route, however would be within the knowledge of a person skilled in the art.
IV. Methods of the Treatment
[00132] The present application includes a method of treating a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more β-lactam antibiotics in combination with an effective amount of one or more compounds of Formula A, or pharmaceutically acceptable salts and/or solvates thereof:
Figure imgf000034_0001
wherein:
R2 is selected from H, S02Ar, CH2CH(C02R6) HR7, CH2C02R6, CH2C(0) HR7 and Ci 6alkyl;
R 3 -R 5 are independently selected from H and C1-6alkyl;
R6 and R7 are independently selected from H, C1-6alkyl and S02Ar; n is 1 or 2, Ar is aryl that is unsubstituted or substituted with one or more of halo, N02, C(0)C1- 4alkyl and C02C1-4alkyl; and each alkyl group is optionally fluoro-substituted, provided that when n is 1, R2, R3, R4, R5, R6 and R7 are not all H.
[00133] The present application also includes a use of a β-lactam antibiotic in combination with one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for treating a bacterial infection in a subject; a use of a β-lactam antibiotic in combination with one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for treating a bacterial infection in a subject; and a β-lactam antibiotic and one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to treat a bacterial infection in a subject.
[00134] In another embodiment, the present application includes a method of treating or preventing a disease, disorder or condition arising from a bacterial infection in a subject comprising administering, to the subject, an effective amount of one or more β- lactam antibiotics in combination with an effective amount of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof.
[00135] The present application also includes a use of a β-lactam antibiotic in combination with of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for treating a disease, disorder or condition arising from a bacterial infection in a subject; a use of a β-lactam antibiotic in combination with of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for treating disease, disorder or condition arising from a bacterial infection in a subject; and a β-lactam antibiotic and of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to treat a disease, disorder or condition arising from a bacterial infection in a subject. [00136] In another embodiment, the present application includes a method of improving the efficacy of a B-lactam antibiotic for treating a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof in combination with the antibiotic.
[00137] The present application also includes a use of one or more compounds of Formula II as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for improving the efficacy of a B-lactam antibiotic for treating a bacterial infection in a subject; a use of of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for improving the efficacy of a B-lactam antibiotic for treating a bacterial infection in a subject; and of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to improve the efficacy of a B-lactam antibiotic to treat a bacterial infection in a subject.
[00138] The present application also includes a method of improving the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof.
[00139] The present application also includes a use of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for improving the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection in a subject; a use of one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for preparation of a medicament for improving the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection in a subject; and one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, for use to improve the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection in a subject. [00140] In an embodiment, the therapeutic methods and uses described above are also applicable to the combination of an effective amount of one or more non-natural compounds of Formula I and an effective amount of a B-lactam antibiotic.
[00141] In an embodiment, the bacterial infection is an infection of at least one metallo-B-lactamase (MBL)-expressing bacterium, and the disease, disorder or condition arising from a bacterial infection is a disease, disorder or condition arising from at least one MBL-expressing bacterial infection. In an embodiment, the MBL is an IMP -type, a Verona integron-encoded metallo-P-lactamase (VEVI) or a New Delhi metallo-β- lactamase (NDM). In a further embodiment, the MBL is VEVI or NDM.
[00142] In an embodiment, the bacterial infection is an infection of at least one carbapenem-resistant Gram-negative bacteria.
[00143] In an embodiment, the bacterial infection is an infection of at least one bacterium belonging to the family Enter obacteriaceae, Acinetobacter, Pseudomonas.
[00144] In an embodiment the Enterobacteriaceae bacterium is a Klebsiella species, such as Klebsiella pneumonia or Escherichia coli. In another embodiment, the Pseudomonas bacterium is Pseudomonas aeruginosa.
[00145] In an embodiment, the bacterium causing infection is selected from
Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus species, Coryne bacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Vibrio cholerae, and Campylobacter jejuni.
[00146] In an embodiment, the bacterium causing infection is selected from selected from Enterobacteriaceae (includes: Escherichia, Salmonella, Klebsiella, Enterobacter), Pseudomonas aeruginosa, Acinetobacter species, Haemophilus influenzae, Clostridium tetani, Clostridium botulinum, Bacteroides species, Prevotella species, Porphyromonas species, Fusobacterium species, Mycobacterium tuberculosis, and Mycobacterium leprae. [00147] In an embodiment, the bacterium causing infection is from the
Enterobacteriaceae family.
[00148] In an embodiment, the bacterium causing infection is Klebsiella pneumoniae.
[00149] The diseases, disorders or conditions arising from a bacterial infection include all such pathogeneses that are common to infections of MBL-expression bacteria. These are well known to those skilled in the art. Some of the more common examples are listed below for the better known MBL-expressing bacteria, however, a person skilled in the art would appreciate that these lists are non-exhaustive and many of the diseases, disorders and conditions listed for one MBL-expression bacterium will be common to other MBL- expressing bacteria.
[00150] In an embodiment, the disease, disorder or condition arising from a bacterial infection, such as an infection of K. pneumoniae, is for example, but not limited to, pneumonia (for example bronchopneumonia or bronchitis), thrombophlebitis, urinary tract infection (UTI), cholecystitus, diarrhea, upper respiratory tract infection, lower biliary tract infection, wound infection, surgical wound infection, osteomyelitis, meningitis, bacteremia, septicemia, sepsis, septic shock, rhinoscleroma, ozena, ankylosing spondylitis, destructive changes to human lungs via inflammation and hemorrhage with cell death (necrosis), lung abscesses, cavitations, empyemas, or ural adhesions, or a combination thereof.
[00151] In an embodiment, the disease, disorder or condition arising from bacterial infection, such as an infection of Pseudomonas aeruginosa, is for example, but not limited to, cystic fibrosis, pneumonia, bacteremia, endocarditis, meningitis, brain abscesses, septic shock, UTI, gastrointestinal infection (e.g. diarrhea, enteritis, or enterocolitis), skin infections (e.g. ecthyma gangrenosum) , soft tissue infections, infections of burn injuries, infections of the outer ear, bacterial keratitis, endophthalmitis, infections due to the presence of a medical device, infections due to hospitalization, infections caused by low water quality, postoperative infections, or osteomyelitis, or a combination thereof.
[00152] In an embodiment, the disease, disorder or condition arising from bacterial infection, such as an infection of Escherichia coli, is for example, but not limited to, enteric infections (e.g. diarrhea), intra-abdominal infections, cholecy stilus, bacteremia, cholangitis, UTI, meningitis, pneumonia, septic arthritis, endophthalmitis, suppurative thyroiditis, osteomyelitis, endocarditis, skin infections or soft tissue infections, or a combination thereof.
[00153] In an embodiment, the subject is a human. In a further embodiment, the subject is an animal, such as a companion animal or livestock.
[00154] In an embodiment, the companion animal or livestock is cat, dog, horse, pig, bird, cow or chicken.
[00155] The one or more antibiotics are selected from any antibiotic which treats metallo-B-lactamase-expressing bacterial infections. In an embodiment, one or more antibiotics are B-lactam antibiotics. In an embodiment, the B-lactam antibiotic is selected from penicillin derivatives (penems), cephalosporins (cephems), monobactams and carbapenems. In an embodiment, the B-lactam antibiotic is selected from imipenem, ertapenem, meropenem, doripenem, biapenem, panipenem, ticarcillin, ampicillin, amoxicillin, carbenicillin, piperacillin, azlocillin, mezlocillin, ticarcillin, cefoperazone, cefotaxime, ceftriaxone and ceftazidime.
[00156] In another embodiment, the one or more antibiotics are carbapenem antibiotics. In an embodiment, the carbapenem antibiotic is selected from meropenem, biapenm, doripenem, ertapenem, panipenem and imipenem.
[00157] In an embodiment, the one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, and the one or more β-lactam antibiotics in the compositions and kits of the present application are formulated as separate pharmaceutical compositions, for separate administration to, or use in, subjects. In this embodiment, the separate pharmaceutical compositions are formulated independently of each other and in accordance with the desired mode of administration for each active. In an embodiment, the one or more β-lactam antibiotics are formulated for administration, or use, by oral delivery or for delivery by injection. In another embodiment, the one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof are formulated for administration, or use, by oral delivery or for delivery by injection. [00158] In an embodiment, the one or more compounds of Formula A as defined above, or a pharmaceutically acceptable salt and/or solvate thereof, and the one or more β-lactam antibiotics in the compositions and kits of the present application are formulated as a single pharmaceutical composition, for combined, simultaneous administration to, or use in, subjects. In an embodiment, the single pharmaceutical composition is formulated for administration, or use, by oral delivery or by injection.
[00159] The methods and uses as described in this section for the one or more non- natural compounds of Formula (I) or Formula A, or a pharmaceutically acceptable salts and/or solvates thereof, further comprise the modification of one or more non-natural compounds of Formula (I) or Formula A as defined above, or a pharmaceutically acceptable salts and/or solvates thereof, through derivatization or encapsulation in drug delivery vehicles as described above, to increase cell permeability and target delivery. Selection for the type of modification used is dependent, for example, upon the disease, disorder or condition being treated and the administrative route, and is well within the knowledge of a person skilled in the art.
EXAMPLES
[00160] The following non -limiting examples are illustrative of the present application:
[00161] Materials and instrumentation:
[00162] All solvents were purchased from Sigma-Aldrich (anhydrous grade) and were used without further purification. N-(Triphenylmethyl)-L-Serine Methyl ester was purchased from TCI America. Trimethyltinhydroxide was purchased from VWR. L- Aspartic acid di t-butyl ester hydrochloride and all other reagents were purchased from Sigma-Aldrich. Trt-Z)-Serine methyl ester1 was synthesized following a method already described.
[00163] Flash chromatography was performed on Teledyne Combi Rf200 system.
[00164] The purity of the final product was assessed using LC-MS (QTRAP 2000,
Sciex and Agilent 1100 HPLC). The structure of the compounds was confirmed by 1 -D and 2-D NMR experiments on a Bruker AVIII 700 MHz instrument in an appropriate deuterated solvent. Chemical shifts are reported in parts per million (ppm) relative to tetramethyl silane using the residual solvent signal as an internal signal. High resolution mass spectra (HRMS) were obtained using a Therm oFisher-XL-Orbitrap Hybrid mass spectrometer equipped with electrospray interface operated in positive ion mode. Optical rotation studies were carried out on a Rudolph Research Analytical Autopal III Automatic Polarimeter in 0.1N H3 in water.
Example 1: Total Synthesis of (L, L, L)-AM-A
Figure imgf000041_0001
L-aspartic
Figure imgf000041_0002
H C02H C02H
L H
NH,
.Bu02C^N^N^NH2 H02C N^N AMA
C02tBu H C02H C02H C02H
(a) MsCl/TEA, 60h at 65°C, 90%; (b) TFA/DCM/MeOH, 30min at 0°C then oNsCl,16h at RT, 71%; (c) L-Aspartic acid-di-t-butyl ester/THF, 16h at RT, 80%; (d) PhSH/DiPEA/AcNl, lh at RT, 80%; (e) oNs-L-Azi-OMe/THF, 30%; (f) TMTOH (6eq)/DCE, 3h at 80°C, 82%; (g) PhSH/DiPEA/AcNl, 5h, RT, 50%; (h) TFA/DCM (1 : 1), 24 h at 4°C, 23%.
(S)-methyl l-tritylaziridine-2-carboxylate (2)14
Figure imgf000041_0003
[00165] N-Trityl-J-serine methyl ester (1, 9.38 g, 25.9 mmol) was dissolved in anhydrous tetrahydrofuran (THF) (70 ml) at room temperature. Triethylamine (TEA) (8.3 mL, 57.4 mmol, 2.2 eq.) was added in nine portions, followed by gradual addition of methanesulfonyl chloride (MsCl) (2.9 g, 26.2 mmol, 1.01 eq.) over a period of 15 min. (ice-water bath was used to maintain the temperature of the reaction below 20°C, because of the heat generated upon addition of MsCl). The mixture was left to stir for 30 min. at room temperature. The temperature was then raised to 65°C and mixture was heated at reflux for 60h. The solvent was removed under reduced pressure. The residue was then dissolved in ethyl acetate (50 ml) and washed successively with 10% citric acid (3 x 10 ml) and saturated solution of sodium bicarbonate (3 x 10ml). The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The final product was purified by silica gel flash chromatography using 20% ethyl acetate in hexanes as the eluent. Yield of 2: 90%. C23H2iN02, Exact Mass: 343.15723, Found: [M+Na]+ 366.1467. 1H MR (700 MHz, CDC13) δ 7.48 (d, J= 7.7 Hz, 6H), 7.29 - 7.23 (m, 9H), 3.75 (d, J = 1.1 Hz, 3H), 2.24 (dd, J = 2.8, 1.4 Hz, 1H), 1.87 (dd, J = 6.2, 2.6 Hz, 1H), 1.40 (dd, J = 6.2, 1.5 Hz, 1H). 13C NMR (176 MHz, CDC13) δ 172.09, 143.75, 129.47, 127.80, 127.08, 52.26, 31.85, 28.84.
(S)-methyl l-((2-nitrophenyl)sulfonyl)aziridine-2-carboxylate (3)14.
Figure imgf000042_0001
[00166] Trifluoroacetic acid (TFA) (11 ml) was added dropwise over 30 min. to a solution of 2 (2.0 g, 5.8 mmol) in DCM (11ml) and methanol (11 ml) at 0°C. The reaction mixture was stirred for 30min at 0°C at which time the solvents were removed under reduced pressure. The residue was partitioned between diethyl ether (100 ml) and water (100 ml). The ether layer was further extracted with water (3 x 20ml). The combined aqueous layer was treated with NaHCC>3 (6 g, 69 mmol) at 0°C until pH 8 was achieved. Ethyl acetate (100 ml) was added to the aqueous solution followed by a solution of o-nitrobenzenesulfonyl chloride (1.2g, 5.8 mmol) in ethyl acetate (4 ml) at 0°C. The resulting mixture was stirred vigorously at room temperature for 24h. After completion of the reaction the two layers were separated and the aqueous layer was extracted with ethyl acetate (3 x 20 ml). The combined organic layers and extracts were washed with brine (3x 50 ml), dried over anhydrous magnesium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel using 40% ethyl acetate in hexanes as the eluent to yield 3 as clear oil. Yield 71%, CioHioN206S, Exact Mass: 286.02596, Found [M+H]+ 287.0331. 1H NMR (700 MHz, CDC13) δ 8.32 - 8.20 (m, 1H), 7.88 - 7.69 (m, 3H), 3.81 (s, 3H), 3.63 (dd, J = 7.1, 4.4 Hz, 1H), 3.09 (d, J= 7.1 Hz, 1H), 2.80 (d, J= 4.4 Hz, 1H). 13C NMR (176 MHz, CDCI3) δ 167.17, 135.00, 132.69, 131.81, 124.84, 53.19, 37.75, 34.40.
(S)-di-ieri-butyl-2-(((S)-3-methoxy-2-(2-nitrophenylsulfonamido)-3- oxopropyl)amino)succinate (4)15.
L-aspartic
Figure imgf000043_0001
[00167] To a solution of aziridine (3) (0.7 g, 2.45 mmol) in THF (10 ml) was added J-aspartic acid di-t-butyl ester (1.37g, 4.8 mmol, 2 eq.) and TEA (49.5 mg, 0.49 mmol, 0.2 eq.). The reaction was carried out for 20h at room temperature. After completion of the reaction, the solvent was removed under reduced pressure, and then the residue was redissolved in ethyl acetate (20 ml) and washed with brine (2 x 20 ml), dried over anhydrous magnesium sulfate (MgS04) and concentrated in vacuo. The crude product was purified using flash chromatography on silica gel using 20% ethyl acetate in hexanes as the eluent. Yield of 4 was 71%. C22H33N3O10S, Exact Mass: 531.18867, Found [M+H]+ 532.1943. 1H NMR (700 MHz, CDCI3) δ 8.16 - 8.03 (m, 1H), 7.88 (dt, J = 7.8, 3.9 Hz, 1H), 7.75 - 7.65 (m, 2H), 4.24 (t, J = 4.2 Hz, 1H), 3.51 (s, 3H), 3.39 (ddd, J = 16.8, 10.0, 4.5 Hz, 2H), 2.80 (dd, J = 12.8, 4.5 Hz, 1H), 2.59 (dd, J = 16.1, 5.1 Hz, 1H), 2.48 (dd, J = 16.2, 7.3 Hz, 1H), 1.45 (s, 10H), 1.44 (s, 9H). 13C NMR (176 MHz, CDCI3) δ 172.74, 170.64, 170.39, 147.84, 134.84, 133.48, 132.73, 130.66, 125.42, 81.23, 80.97, 59.07, 57.24, 52.60, 50.22, 39.51, 28.22, 28.18. (S)-di-ieri-butyl2-(((S)-2-amino-3-methoxy-3-oxopropyl)amino)succinate (5)
Figure imgf000044_0001
[00168] Compound 4 (0.27 mg, 0.51 mmol) was dissolved in dry acetonitrile (2 ml) and to this solution was added thiophenol (280.5 mg, 2.55 mmol, 5 eq.) and diisopropylethylamine (263.2 mg, 2.04 mmol, 4 eq.). The reaction was carried out for lh at room temperature at which time the solvent was evaporated in vacuo and the residue subjected to C18 flash chromatography (8% acetonitrile in water as the eluent) to afford final product. Yield 80%, C16H30N2O6, Exact Mass: 346.21039, Found: [M+H]+ 347.2170. 1H NMR (700 MHz, CDC13) δ 3.72 (s, 3H), 3.53 (dd, J = 7.0, 4.2 Hz, 1H), 3.47 (dd, J = 7.6, 5.5 Hz, 1H), 2.95 (dd, J = 12.0, 7.0 Hz, 1H), 2.79 (d, J = 4.3 Hz, 1H), 2.60 (dd, J= 15.7, 5.4 Hz, 1H), 2.50 - 2.43 (m, 1H), 1.46 (s, 9H), 1.45 (s, 9H). 13C NMR (176 MHz, CDC13) δ 175.32, 172.95, 170.30, 81.66, 81.12, 58.88, 54.87, 52.21, 51.29, 39.72, 28.23, 28.21.
(S)-di-teri-butyl2-(((S)-3-methoxy-2-(((S)-3-methoxy-2-(2
nitrophenylsulfonamido)-3-oxopropyl)amino)-3-oxopropyl)amino)succinate (6).
Figure imgf000044_0002
[00169] To a solution of 5 (100 mg, 0.290 mmol, 1.1 eq.) in THF (2 ml) was added
3 (75 mg, 0.26 mmol, 1 eq.) and TEA (5.25 mg, 0.05mmol, 0.2 eq.). The reaction solution was stirred for 20h at room temperature. Solvent was then removed in vacuo and the final product purified by flash chromatography on silica gel using 40% ethyl acetate in hexanes. Yield 41%, C26H4oN4012S, Exact Mass: 632.23635, [M+H]+ 633.2440. 1H NMR (700 MHz, Chloroform- ) δ 8.09 (dd, J = 6.0, 3.4 Hz, 1H), 7.94 - 7.85 (m, 1H), 7.76 - 7.66 (m, 3H), 4.24 (t, J = 4.5 Hz, 1H), 3.77 - 3.71 (m, 1H), 3.70 (d, J = 5.1 Hz, 3H), 3.56 (s, 3H), 3.46 (dd, J = 7.1, 5.9 Hz, 1H), 3.34 - 3.25 (m, 2H), 2.88 (dd, J = 12.0, 7.3 Hz, 1H), 2.82 (dd, J = 12.9, 4.5 Hz, 1H), 2.75 (dd, J= 12.0, 4.6 Hz, 1H), 2.60 (dd, J = 15.9, 6.0 Hz, 1H), 2.51 (dd, J = 15.9, 7.0 Hz, 1H), 1.46 (s, 9H), 1.44 (s, 9H).13C NMR (176 MHz, CDC13) δ 173.92, 172.70, 170.53, 170.21, 147.70, 134.70, 132.64, 130.47, 125.37, 81.51, 80.88, 61.85, 58.59, 57.13, 52.52, 52.02, 49.95, 49.70, 39.34, 28.05.
(S)-3-(((S)-l-carboxy-2-(((S)-l,4-di-teri-butoxy-l,4-dioxobutan-2-
18
yl)amino)ethyl)amino)-2-(2-nitrophenylsulfonamido)propanoic acid (7) .
Figure imgf000045_0001
[00170] To a solution of 6 (157 mg, 0.25 mmol) in anhydrous dichloroethane
(DCE) (2 ml) was added trimethyltin hydroxide (270 mg, 1.5 mmol, 6 eq). The reaction mixture was then heated at reflux for 2h. The solvent was removed in vacuo. Ethyl acetate (20ml) was added and washed with 5% hydrochloric acid solution in water (4 x 10ml), and then with brine. After drying over anhydrous MgS04 the solvent was removed in vacuo and the product subjected to CI 8 flash chromatography (50 % acetonitrile in water as the eluent) to afford final product 7. Yield 82%. C24H36N4012S, Exact Mass: 604.20505, Found [M+H]+ 605.2111. 1H NMR (700 MHz, Methanol-^) δ 8.20 (dt, J = 6.0, 3.5 Hz, 1H), 7.98 (dt, J = 5.2, 3.3 Hz, 1H), 7.85 (tq, J = 7.9, 4.5, 3.9 Hz, 3H), 3.92 (dd, J = 8.3, 5.8 Hz, 1H), 3.65 (dd, J = 7.1, 4.1 Hz, 1H), 3.55 (dd, J = 12.1, 5.9 Hz, 1H), 3.50 (t, J = 6.0 Hz, 1H), 3.22 (dd, J = 13.7, 7.2 Hz, 1H), 3.08 (dd, J = 13.7, 4.2 Hz, 1H), 2.70 (dt, J = 28.6, 6.0 Hz, 2H), 1.48 (d, J = 3.4 Hz, 12H), 1.44 (s, 13H).13C NMR (176 MHz, Methanol-^) δ 174.05, 172.82, 172.39, 171.94, 149.54, 135.38, 134.03, 133.59, 132.13, 126.83, 82.83, 82.31, 63.34, 58.66, 55.03, 50.50, 47.77, 39.67, 28.44, 28.35. (S)-2-amino-3-(((S)-l-carboxy-2-(((S)-l,4-di-teri-butoxy-l,4-dioxobutan-2- yl)amino)ethyl)amino)propanoic acid (8).
Figure imgf000046_0001
[00171] The nosyl group deprotection was carried out as described for 5 for 5h.
The final product was purified using reverse phase (CI 8) flash chromatography using 20% acetonitrile/water as the eluent. Yield 50%, C18H33N308, Exact Mass: 419.22677, Found [M+H]+ 420.2336. 1H NMR (700 MHz, Deuterium Oxide) δ 4.04 (td, J = 5.6, 2.0 Hz, 1H), 3.98 (t, J = 6.1 Hz, 1H), 3.66 (dd, J = 7.9, 5.3 Hz, 1H), 3.45 (ddd, J = 13.4, 5.8, 3.1 Hz, 1H), 3.34 - 3.28 (m, 1H), 3.28 - 3.19 (m, 2H), 3.05 - 2.99 (m, 1H), 2.99 - 2.93 (m, 1H), 1.51 (d, J = 2.9 Hz, 9H), 1.48 (d, J = 3.1 Hz, 9H). 13C NMR (176 MHz, Deuterium Oxide) δ 174.16, 171.94, 171.10, 169.91, 85.48, 84.12, 60.17, 56.82, 52.16, 46.97, 46.30, 35.94, 27.08.
(S)-2-(((S)-2-(((S)-2-amino-2-carboxyethyl)amino)-2-carboxyethyl)amino)succinic acid (LLL-AM-A).
Figure imgf000046_0002
8
[00172] Deprotection of di-t-butyl ester groups was carried out on a solution of compound 8 (29 mg, 0.07 mmol) in 4 ml of 50% TFA in DCM. After removal of the solvent in vacuo, the product was purified by ion-exchange chromatography on Dowex 1x8 and eluted with 2M acetic acid solution in water. Yield: 23%; Ο10Η17Ν3Ο8, Exact Mass: 307.10157, Found [M+H]+ 308.1090.1H NMR (700 MHz, 0.1M NH4OH in Deuterium Oxide ) δ 3.79 (dd, J = 6.5, 4.0 Hz, 1H), 3.75 (dd, J = 9.3, 3.9 Hz, 1H), 3.39 (dd, J= 9.7, 4.3 Hz, 1H), 3.19 (ddd, J= 12.7, 7.5, 5.4 Hz, 2H), 3.02 (dd, J= 12.8, 9.7 Hz, 1H), 2.90 (dd, J = 13.3, 4.0 Hz, 1H), 2.77 (dd, J = 17.0, 3.9 Hz, 1H), 2.62 (dd, J = 17.0, 9.3 Hz, 1H). C NMR (176 MHz, Deuterium Oxide ) δ 177.98, 177.91, 175.25, 175.22, 60.57, 59.75, 54.61, 48.30, 47.64, 37.13. [a]20 D -47 (c 1.00, 0.1M NH4OH in H20).
Example 2: Total Synthesis of (L, D, D)-AM-A
Trt-D-Ser(OMe)
HN..
9 "Trt Trt 10
L-aspart
Figure imgf000047_0001
15
H 9°- h L H 9<¾H Q
AMA
C02tBu H C02H C02H H C02H
16
(a) MsCl/TEA, 60h at 65°C, 90%; (b) TFA/DCM/MeOH, 30min at 0°C,oNsCl , 16h at RT, 71%; (c) L-Aspartic acid-di-t-butyl ester/THF, 16h at RT, 80%; (d) PhSH/DiPEA/AcNl, lh at RT, 80%; (e) oNs -L- Azi -OMe/THF, 30%; (f) TMTOH (6eq)/DCE, 3h at 80°C, 82%; (g) PhSH/DiPEA/AcNl, 5h, RT, 50%; (h) TFA/DCM (1 : 1), 24 h at 4°C, 23%.
(R)-methyl l-tritylaziridine-2-carboxylate (10).
Figure imgf000047_0002
[00173] The compound was synthesized using the procedure described for 2 using
N-trityl-Z>-serine methyl ester (9). The compound showed 1H NMR (700 MHz, CDC13) δ 7.52 (dt, J = 73, 2.4 Hz, 7H), 7.29 (m, 12H), 3.78 (d, J = 1.7 Hz, 3H), 2.27 (td, J = 2.8, 1.5 Hz, 1H), 1.91 (dt, J = 6.2, 3.1 Hz, 1H), 1.47 - 1.39 (m, 1H).13C NMR (176 MHz, CDC13) δ 167.19, 135.03, 132.71, 131.87, 131.83, 124.87, 53.21, 37.77, 34.42.
(R)-methyl l-((2-nitrophenyl)sulfonyl)aziridine-2-carboxylate (11).
Figure imgf000048_0001
[00174] The compound was synthesized using the procedure described for 3 using
10 as the starting material. The compound showed: 1H NMR (700 MHz, CDC13) 5 8.32 - 8.20 (m, 1H), 7.84 - 7.73 (m, 3H), 3.81 (d, J = 1.0 Hz, 3H), 3.69 - 3.58 (m, 1H), 3.09 (dd, J = 7.1, 0.9 Hz, 1H), 2.81 (dd, J = 4.5, 1.0 Hz, 1H).13C NMR (176 MHz, CDCI3) δ 167.20, 135.03, 132.72, 131.84, 124.87, 53.23, 37.78, 34.43.
(S)-di-ieri-butyl-2-(((R)-3-methoxy-2-(2-nitrophenylsulfonamido)-3- oxopropyl)amino)succinate (12).
L-
Figure imgf000048_0002
[00175] The compound was synthesized using the procedure described for 4 using
J-aspartic acid di-t-butyl ester and 11 as the starting materials. The compound showed: 1H NMR (700 MHz, CDCI3) δ 8.13 - 7.93 (m, 1H), 7.94 - 7.77 (m, 1H), 7.77 - 7.62 (m, 2H), 4.13 (td, J = 4.5, 1.5 Hz, 1H), 3.33 (d, J = 6.2 Hz, 1H), 3.25 - 3.08 (m, 1H), 3.03 - 2.89 (m, 1H), 2.51 (ddd, J= 16.0, 5.4, 1.6 Hz, 1H), 2.43 (ddd, J = 15.9, 7.0, 1.6 Hz, 1H), 1.43 (d, J = 1.8 Hz, 9H), 1.43 (d, J = 1.8 Hz, 9H).13C NMR (176 MHz, CDCI3) δ 172.23, 169.95, 147.69, 134.22, 133.42, 132.67, 130.55, 125.43, 81.23, 80.97, 58.59, 56.98, 52.54, 49.70, 39.08, 27.92. (S)-di-ieri-butyl-2-(((R)-2-amino-3-methoxy-3-oxopropyl)amino)succinate (13).
Figure imgf000049_0001
[00176] The compound was synthesized using the procedure described for 5 using
12 as the starting material. The compound showed: 1H MR (700 MHz, CDC13) δ 3.72 (s, 3H), 3.52 (dd, J = 6.8, 4.4 Hz, 1H), 3.45 (dd, J = 7.3, 5.5 Hz, 1H), 3.05 (dd, J = 12.0, 4.4 Hz, 1H), 2.68 (dd, J = 12.0, 6.8 Hz, 1H), 2.59 (dd, J = 15.7, 5.6 Hz, 1H), 2.47 (dd, J = 15.7, 7.3 Hz, 1H), 2.16 (s, 1H), 1.45 (s, PH), 1.44 (s, PH). 13C NMR (176 MHz, CDCI3) δ 175.00, 172.92, 170.28, 81.62, 81.06, 59.02, 55.01, 52.18, 51.64, 39.47, 28.21, 28.19.
(S)-di-teri-butyl-2-(((R)-3-methoxy-2-(((R)-3-methoxy-2-(2-nitrophenylsulfonamido)- 3-oxopropyl)amino)-3-oxopropyl)amino)succinate (14).
Figure imgf000049_0002
[00177] The compound was synthesized using the procedure described for 6 using
11 and 13 as the starting materials. The compound showed: 1H MR (700 MHz, CDC13) δ 8.20 - 8.02 (m, 1H), 7.93 - 7.79 (m, 1H), 7.79 - 7.67 (m, 2H), 3.70 (s, 3H), 3.68 (s, 3H), 3.45 (t, J = 5.8 Hz, 2H), 3.41 (dd, J = 12.5, 4.7 Hz, 1H), 3.31 (d, J = 5.4 Hz, 1H), 3.21 (dd, J = 12.5, 7.0 Hz, 1H), 2.97 (dd, J = 12.1, 4.6 Hz, 1H), 2.71 (dd, J = 12.1, 5.9 Hz, 1H), 2.59 (dd, J = 15.9, 5.6 Hz, 1H), 2.54 - 2.46 (m, 2H), 1.46 (s, 9H), 1.44 (s, 9H); 13C NMR (176 MHz, CDC13) δ 173.45, 172.74, 172.24, 170.37, 133.80, 133.64, 132.74, 131.17, 125.48, 60.22, 59.33, 58.81, 52.62, 52.30, 49.99, 45.51, 39.34, 28.23, 28.21. (R)-3-(((R)-l-carboxy-2-(((S)-l,4-di-teri-butoxy-l,4-dioxobutan-2- yl)amino)ethyl)amino)-2-(2-nitrophenylsulfonamido)propanoic acid (15).
Figure imgf000050_0001
[00178] The compound was synthesized using the procedure described for 7 using
14 as the starting material. 1H NMR (700 MHz, Methanol -<f4) δ 8.26 - 8.1 1 (m, 1H), 8.04 - 7.91 (m, 1H), 7.90 - 7.79 (m, 2H), 4.00 (d, J = 9.0 Hz, 1H), 3.68 - 3.58 (m, 1H), 3.60 - 3.52 (m, 1H), 3.49 (d, J = 4.8 Hz, OH), 3.40 (d, J = 12.8 Hz, 1H), 2.94 (d, J = 1 1.2 Hz, 1H), 2.85 - 2.68 (m, 2H), 1.52 - 1.47 (m, 9H), 1.47 - 1.42 (m, 8H). 13C NMR (176 MHz, Methanol-^) δ 173.87, 173.67, 172.71, 172.08, 149.51, 135.38, 134.02, 133.79, 132.08, 126.74, 83.09, 82.37, 63.47, 59.68, 55.18, 50.42, 48.40, 39.52, 28.40, 28.33. (R)-2-amino-3-(((R)-l-carboxy-2-(((S)-l,4-di-teri-butoxy-l,4-dioxobutan-2- yl)amino)ethyl)amino)propanoic acid (16).
tBuO
Figure imgf000050_0002
15 16
[00179] The compound was synthesized using the procedure described for 8 using
15 as the starting material. 1H NMR (700 MHz, Deuterium Oxide) δ 4.03 (t, J = 5.4 Hz, 1H), 3.97 (t, J = 6.0 Hz, 1H), 3.61 (t, J = 6.9 Hz, 1H), 3.44 (tt, J = 14.1, 6.9 Hz, 2H), 3.19 (ddd, J = 28.1, 14.6, 8.3 Hz, 2H), 3.05 (dd, J = 17.6, 5.1 Hz, 1H), 2.97 (dd, J = 17.6, 5.8 Hz, 1H), 1.51 (s, 9H), 1.49 (s, 9H).13C NMR (176 MHz, Deuterium Oxide) δ 171.73, 170.83, 169.10, 59.17, 57.35, 51.87, 47.15, 46.28, 42.52, 35.60, 27.18, 27.05.
(S)-2-(((R)-2-(((R)-2-amino-2-carboxyethyl)amino)-2-carboxyethyl)amino)succinic acid (LDD-AM-A).
[00180] The compound was synthesized using the procedure described for LLL-
AM-A using 16 as the starting material. The compound showed 1H NMR (700 MHz, 0.1 M NH4OH in Deuterium Oxide) δ 3.72 (dd, J = 6.8, 3.9 Hz, 1H), 3.67 (dd, J = 8.4, 4.0 Hz, 1H), 3.35 (dd, J = 9.4, 4.2 Hz, 1H), 3.23 (d, J= 12.4 Hz, 1H), 3.12 (dd, J = 13.3, 7.1 Hz, 1H), 2.93 - 2.87 (m, 2H), 2.78 - 2.71 (m, 1H), 2.60 (dd, J = 16.7, 8.7 Hz, 1H).13C NMR (176 MHz, 0.1M H4OH in Deuterium Oxide) δ 176.89, 176.11, 175.83, 174.87, 58.93, 58.02, 46.03, 37.67. [a]20 D -45 (c 1.00, 0.1M H4OH in H20). 2D NMR (HSQC, HMBC) confirms the structure of LDD-AM-A.
Example 3: Total Synthesis of AM-A (Starting with (S)-tert-butyl 4-f r my 1-2,2- dimethyloxazolidine-3-carboxylate)
O^OH
Me02l di
Figure imgf000051_0001
Boc C<¼H H C02H
Me02C — N X H02C L
C02Me ^00 NHBoc C02H NH;
AMA
(S)-dimethyl 2-((((R)-3-(teri-butoxycarbonyl)-2,2-dimethyloxazolidin-4 yl)methyl)amino)succinate (19).
Figure imgf000051_0002
[00181] To a stirred solution of J-aspartic acid dimethyl ester hydrochloride (18,
197.7 mg, 1 mmol) in dry MeOH (10 mL), at room temperature, was added (R)-tert-butyl 4-formyl-2,2-dimethyloxazolidine-3-carboxylate (R-Garner's aldehyde, 17, 230 mg, 1 mmol). The mixture was stirred at room temperature under an inert atmosphere for 15 min. The resulting imine was treated with Na(OAc)3BH (260 mg, 1.25 mmol) in two portions over 30 min. The reaction mixture was stirred at room temperature for 16 h, at which time the reaction was quenched with water (5 mL). The aqueous layer was extracted with ethyl acetate (3 x 15 mL) and dried over Na2S04. The solvent was evaporated under a reduced pressure and the crude product was purified by silica gel chromatography with a gradient of hexanes in ethyl acetate. Compound 19 was obtained as an oil (280 mg, 75 %). The NMR shows presence of rotamers due to the presence of tertiary amine therefore carried forward to the next step and characterized.
(S)-dimethyl 2-(((R)-2-amino-3-hydroxypropyl)amino)succinate (20).
Figure imgf000052_0001
[00182] Compound 19 (187 mg, 0.5 mmol) was stirred in a mixture of trifluoroacetic acid and DCM (2 mL, 6: 1 ratio) at room temperature for 16 h. The sample was evaporated to dryness, redissolved in methanol (10 ml) then evaporated. The evaporation process was repeated two additional times before the sample was dried under high vacuum to afford the desired product (1 14 mg) as a trifluoroacetate salt. 1H NMR (600 MHz, CD3OD): δ = 3.74 (s, 3H), 3.70 (s, 3H), 3.67 (dd, J = 8.5 Hz, J = 4.7 Hz, 1H), 3.60 (dd, J = 8.2 Hz, J = 4.8 Hz, 1H), 2.87 (dd, J = 13.5 Hz, J = 8.5 Hz, 1H), 2.83 (m, 1H), 2.80 (dd, J = 13.5 Hz, J = 4.7 Hz, 1H), 2.68 (dd, J = 16.7 Hz, J = 8.2 Hz, 1H); HRMS (ES) for C9Hi8N205 calculated 235.1288 [M + H] + found 235.1289 [M + H]+.
(R)-2-amino-3-(((S)-l,4-dimethoxy-l,4-dioxobutan-2-yl)amino)propanoic acid (20a).
Figure imgf000052_0002
[00183] To a stirred solution of compound 20 (1 17 mg, 0.5 mmol) in 2ml acetone was added drop wise Jones reagent (0.52 mL of a 2 M solution, 106 mmol), over 5 min. The reaction mixture was stirred at room temperature for a further 2.5 h. The excess Jones reagent was quenched with z'PrOH (2 mL) and the solvent was evaporated under a reduced pressure. The residue was suspended in water (5mL) and extracted with CH2C12 (2 x 10 mL) followed by ethyl acetate (2 x lOmL). The combined organic extracts were dried over Na2S04 and evaporated. Purification by silica gel chromatography (0-40% CH2C12: MeOH) yielded the desired product (51 mg, 41 %). 1H NMR (600 MHz, CD3OD): δ = 3.74 (s, 3H), 3.69 (s, 3H), 3.67 (dd, J= 7.9 Hz, J = 5.0 Hz, 1H), 3.63 (dd, J = 7.9 Hz, J= 4.1 Hz, 1H), 3.09 (dd, J= 13.5 Hz, J= 7.9 Hz, 1H), 3.02 (dd, J= 13.5 Hz, J = 4.1 Hz, 1H), 2.80 (dd, J = 16.7 Hz, J = 5.0 Hz, 1H), 2.68 (dd, J = 16.7 Hz, J = 7.9 Hz, 1H); 13C NMR (150 MHz, CD3OD): δ = 175.1, 173.6, 173.0, 157.4, 57.8, 55.6, 52.7, 38.1; HRMS (ES) for C9H17N206 calculated 249.1087 [M + H] + found 249.1089 [M +
(S)-dimethyl-2-(((R)-2-((((R)-3-(teri-butoxycarbonyl)-2,2-dimethyloxazolidin-4- yl)methyl)amino)-3-hydroxypropyl)amino)succinate (21).
Figure imgf000053_0001
re uct ve am nat on
[00184] To a stirred solution of compound 20, (117.0 mg, 1 mmol) in dry MeOH
(5 mL), at room temperature, was added (R)-tert-buty\ 4-formyl-2,2- dimethyloxazolidine-3-carboxylate (R-Garner's aldehyde, 17, 115 mg, 0.5mmol) and the mixture was stirred at room temperature under an inert atmosphere for 15 min. The resulting imine was treated with Na(OAc)3BH (130 mg, 0.62 mmol) in two portions over 30 min. The reaction mixture was stirred at room temperature for 16 h, at which time the reaction was quenched with water (2.5 mL). The aqueous layer was extracted with ethyl acetate (2 x 10 mL) followed by CH2C12 (2 x lOmL). The combined organic extracts were dried over Na2S04 and evaporated. Purification by silica gel chromatography (0-40% CH2C12: MeOH) yielded the desired product. Compound 21 was obtained as an oil (107 mg, 48 %) and characterized by HRMS. The NMR shows presence of rotamers due to the presence of tertiary amine. HRMS (ES) for C20H38N3O8 calculated 448.2655 [M + H] + found 448.2663 [M + H]+. (S)-dimethyl-2-((teri-butoxycarbonyl)((R)-2-((teri-butoxycarbonyl)(((R)-3-(teri- butoxycarbonyl)-2,2-dimethyloxazolidin-4-yl)methyl)amino)-3- hydroxypropyl)amino)succinate (22).
Figure imgf000054_0001
[00185] To a solution of 21 (112 mg, 0.25 mmol) and K2C03 (34.5 mg, 0.25 mmol) in water (0.15mL), a solution of Boc20 (131 mg, 0.6 mmol, 2.2 equiv) in dioxane (0.15 mL) was added. The resulting solution was stirred for 16 h at room temperature. The crude reaction mixture was evaporated under reduce pressure and the residue redissolved in 5ml of H20 washed with 10 mL of a 9: 1 Hexane:Et20 solution. The water layer was separeated and extracted with ethylacetate (2 x 10 ml) and dichlorom ethane (2 x 10 ml). The combined organic layers were evaporated to dryness and used directly in the next step without further purification.
(R)-3-((teri-butoxycarbonyl)((R)-2-((teri-butoxycarbonyl)((S)-l,4-dimethoxy-l,4- dioxobutan-2-yl)amino)-l-carboxyethyl)amino)-2-((terf- butoxycarbonyl)amino)propanoic acid (23).
Figure imgf000054_0002
[00186] To a stirred solution of compound 22 (65 mg, 0.1 mmol) in acetone (lmL) was added dropwise Jones reagent (0.26 mL of a 2 M solution, 53 mmol). The reaction mixture was stirred at room temperature for a further 2.5 h. The excess Jones reagent was quenched with z'PrOH (1 mL) and the solvent was evaporated under a reduced pressure. The residue was suspended in water (5mL) and extracted with CH2C12 (2 x 10 mL) followed by ethyl acetate (2 x lOmL). The combined organic extracts were dried over Na2S04 and evaporated. Compound 23 was analyzed by LC- MS. Runs were performed using HPLC grade water containing 0.01% HCOOH (solvent A) and acetonitrile containing 0.01 % HCOOH (solvent B) as eluents, at 1 mL/min flow rate on a Synergi™ 4 μτη Hydro-RP 80 Λ, LC Column (250 x 4.6 mm). The desired products were eluted using a 95 to 5% gradient (Solvent A: Solvent B) over 30 min. Retention time 21.5 min. HRMS (ES) for C^H^O^ calculated 634.2818 [M - H] + found 634.2816 [M - H]+.
Example 4: Total Synthesis of AM-A (Starting with (R)-3-l,2,3-oxathiazolidine-4- carboxylate 2,2-dioxide).
. NH,
R"02C
Figure imgf000055_0001
D-24a: R = Me; R' = Boc LD-25a: R = Me; R* = Boc
D-24b: R = Me; R' = Cbz LD-25b: R = Me; R'■- Cbz
D-24c: R = Me; R' = Pmb LD-25c: R = Me; R' = Pmb
D-24d: R = Me; R' = benzyl LD-25d: R = Me; R' : benzyl
L-24e: R = benzyl; R' = Boc LL-25e: R = benzyl; R' = Boc
D-24f: R = benzyl; R' = Fmoc LD-25f: R = benzyl; R' = Fmoc
L-24g: R = tBu; R' = Cbz LL-25g: R = tBu; R' = Cbz; R"=tBu
L-24h: R = tBu; R' = H LL-25h: R = tBu; R' = CH2COOtBu; R"
L-24i: R = tBu; R' = CH2COOtBu LL-25i: R = H; R' = CH2COOH; R" = H
DL-25j: R = tBu; R' = Cbz; R" = tBu
Figure imgf000055_0002
LD-26a: R = Me; R" = Me
LDD-27a: R=Me; R -Boc; R"=Me
LL-26b: R = Bn; R" = Me
LDD-27b: R=H; R'=Boc; R"=Me
LD-26c: R = H; R" = Me
LDD-27c: R=Bn; R'=Boc; R"=Me
LL-26d: R = tBu; R" = tBu
LLL-27d: R=Bn; R'=Boc; R"=Me
LL-26e: R = H; R" = H
LLL-27e: R=tBu; R'=Cbz; R"=tBu
DL-26f: R = tBu; R" = tBu
DLL-27f: R=tBu; R'=Cbz R"=tBu deprotection
Figure imgf000055_0003
AM-A
General Procedure for Sulfamidate Synthesis.
[00187] The procedure used was a modification of a previously described literature synthesis.21 To a solution of SOCl2 (3.75 mmol) in dry CH3CN (10 mL cooled to -40°C) was added, dropwise over 30 min. under N¾ the protected serine (2.5 mmol) in dry CH3CN (8 mL). The mixture was stirred at -40°C for 30 min at which time dry pyridine (12.5 mmol) in CH3CN (3 mL) was added dropwise over 10 min. The reaction was slowly brought to room temperature and then stirred for 18 h. The reaction mixture was then quenched with ice water and extracted with ethyl acetate (2 x 10 mL). The organic phase was removed in vacuo then coevaporated with CH3CN. The crude sulfamidite was used without further purification in the next step.
[00188] To a cooled (ice-bath) solution of the crude sulfamidite (ca. 2.5 mmol) in
CH3CN (8 mL) was added ruthenium (III) chloride (10 mg), sodium periodate (3.75 mmol) and water (8 mL). The mixture was stirred for 3 hours at 0°C then extracted with ethyl acetate. The organic layer was dried over MgS04, filtered and evaporated in vacuo. The sulfamidates were purified using a gradient hexane ethyl acetate 0 - 60%.
[00189] The physical properties of sulfamidates D-24a, D-24d and L-24e were identical to those reported previously.
Compound L-24g
[00190] Compound L-24g was obtained in 75% yield (669 mg) and showed 1H
NMR (600 MHz, CDCI3): δ 7.37 (m, 5H), 5.35 (d, J= 12.1 Hz, 1H), 4.76 (dd, J= 9.2, 6.6 Hz, 1H), 4.70 (apd, J = 6.6 Hz, 1H), 4.66 (dd, J = 9.2, 1.4 Hz, 1H), 1.42 (s, 9H); 13C NMR (150 MHz, CDC13): δ 165.7, 149.5, 134.4, 128.8, 128.3, 84.9, 69.8, 68.3, 58.4, 27.8; HRMS (ES) for Ci5Hi9N07S calculated 380.0774 [M+Na]+; found 380.0772 [M+Na]+.
Compound L-24h
[00191] A solution of compound L-24g (178.5mg, 0.5 mmol) in ethyl acetate /
DCM (1 : 1; 30mL total volume) was treated with 20% wt. Pd(OH)2 on carbon (80 mg). The reaction mixture was stirred at rt under an atmosphere of hydrogen for 18h then filtered through a Celite pad and rinsed with DCM (2 x 10 mL) and ethyl acetate (2 x 10 mL). The combined filtrate was concentrated in vacuo to provide L-24h in 80% yield (150 mg). The compound showed 1H NMR (600 MHz, CDC13): δ 5.13 (br s, 1H), 4.72 (dd, J = 8.8 Hz, J = 7.8 Hz 1H), 4.50 (dd, J = 8.8 Hz, J = 5.5 Hz, 1H), 4.35 (apt, J = 6.5 Ηζ,ΙΗ), 1.42 (s, 9H); 1JC NMR (150 MHz, CDC13): δ 167.2, 85.7, 69.9, 56.6, 28.1; HRMS (ES) for C7H13NO5S calculated 246.0407 [M+Na]+; found 246.0406 [M+Na]+.
(S)-tert-butyl 3-(2-(ieri-butoxy)-2-oxoethyl)-l,2,3-oxathiazolidine-4-carboxylate 2,2- dioxide (L-24i)
[00192] Sulfamidate L-24h (111 mg, 0.5 mmol) was dissolved in ether (2 ml) and treated with CSCO3 (325 mg, 1 mmol) followed by t-butyl bromoacetate (146 mg, 0.75mmol). The reaction mixture was stirred under nitrogen at 65°C for 18 h and then was concentrated in vacuo. The residue was purified by column chromatography (using a gradient of 0 - 40% EtOAc in hexanes) to afford the title compound in 24% yield (40 mg). 1H MR (600 MHz, CD3OD): δ 4.84 (dd, J = 8.8 Hz, J = 7.6 Hz 1H), 4.73 (dd, J = 8.8 Hz, J= 4.2 Hz, 1H), 4.68 (dd, J = 7.6 Hz, J = 4.2 Hz, 1H), 4.05 (d, J = 18.1 Hz, 1H), 4.01 (d, J = 18.1 Hz, 1H), 1.51 (s, 9H) 1.50 (s, 9H); 13C NMR (150 MHz, CD3OD): δ = 169.0, 168.9, 84.7, 83.8, 69.8, 61.3, 28.3, 28.2; HRMS (ES) for Ci3H23N07S calculated 360.1092 [M + Na]+, found 360.1087 [M + Na]+.
General procedure for sulfamidates opening (I)
Figure imgf000057_0001
24 25
[00193] The free amino ester 18 (1 mmol) was dissolved in AcCN (1 mL) and added to a solution of compound 24 (0.5 mmol) in AcCN (1 mL). The reaction mixture was stirred at 60°C for 12 h and treated with 1 M monopotassium phosphate (4 mL). The aqueous layer was extracted with ethyl acetate (2 x 10 mL), dried over (Mg2S04) and concentrated. The crude reaction mixture was purified by flash column chromatography (using a gradient of 0-60% EtOAc in hexanes).
(S)-dimethyl-2-(((R)-2-((ieri-butoxycarbonyl)amino)-3-methoxy-3- oxopropyl)amino)succinate (LD-25a).
[00194] Compound LD-25a was obtained in 62% (112 mg) yield. 1H NMR (600
MHz, CD3OD): δ = 4.21 (apt, J = 5.5 Hz, 1H), 3.72 (s, 6H), 3.67 (s, 3H), 3.62 (dd, J = 7.4 Hz, J= 5.8 Hz, 1H), 3.06 (dd, J= 12.6 Hz, J= 4.7 Hz, 1H), 2.84 (dd, J= 12.6 Hz, J = 6.4 Hz, 1H), 2.72 (dd, J = 16.0 Hz, J = 5.8 Hz, 1H), 2.62 (dd, J = 16.0 Hz, J = 7.4 Hz, 1H),1.45 (s, 9H); 13C NMR (150 MHz, CD3OD): δ = 175.1, 173.6, 173.0, 157.4, 80.7, 55.5, 52.7, 52.6, 52.4, 38.9, 28.7; HRMS (ES) for Ci5H27N208 calculated 363.1767 [M + H] + found 363.1763 [M + H]+.
(S)-dimethyl 2-(((R)-2-(benzylamino)-3-methoxy-3-oxopropyl)amino)succinate (LD- 25d)
[00195] Compound LD-25d was obtained by reacting sulfamidate D-24d (0.5 mmol, 136 mg) with L-aspartic acid dimethyl ester in 63% yield (111 mg). 1H MR (600 MHz, CD3OD): δ 7.33 (m, 5H), 3.81 (d, J = 12.8 Hz, 1H), 3.71 (s, 6H), 3.67 (d, J = 12.8 Hz, 1H), 3.64 (s, 3H), 3.60 (dd, J = 7.2 Hz, J = 5.7 Hz, 1H), 3.37 (dd, J = 6.8 Hz, J = 5.0 Hz, 1H), 3.02 (dd, J = 12.3 Hz, J = 5.0 Hz, 1H), 2.68 (dd, J = 12.3 Hz, J = 6.8 Hz, 1H), 2.70 (dd, J = 16.0 Hz, J = 5.7 Hz, 1H), 2.61 (dd, J = 16.0 Hz, J = 7.2 Hz, 1H); 13C NMR (150 MHz, CD30D): δ 175.2, 175.1, 173.0, 140.7, 129.6, 129.5, 128.3, 61.6, 58.9, 52.8, 52.6, 52.4, 52.3, 50.3, 38.4; HRMS (ES) for C17H25N206 calculated 353.1713 [M + H] + found 353.1721 [M + H] +.
(S)-dimethyl-2-(((R)-3-(benzyloxy)-2-((ieri-butoxycarbonyl)amino)-3- oxopropyl)amino)succinate (LL-25e)
[00196] Compound LL-25e was made using the L-sulfamidate L-24e. The product was obtained in 71% yield (155 mg). 1H NMR (600 MHz, CD3OD): δ = 7.34 (m, 5H), 5.18 (d, J= 12.4 Hz, 1H), 5.13 (d, J= 12.4 Hz, 1H), 4.24 (apt, J= 5.6 Hz, 1H), 3.69 (s, 3H), 3.65 (s, 3H), 3.63 (dd, J = 7.2 Hz, J= 5.9 Hz, 1H), 3.09 (dd, J = 12.5 Hz, J= 6.5 Hz, 1H), 2.85 (dd, J = 12.5 Hz, J = 4.8 Hz, 2H), 2.72 (dd, J = 16.1 Hz, J = 5.9 Hz, 1H), 2.60 (dd, J = 16.1 Hz, J = 7.2 Hz, 2H) 1.45 (s, 9H); 13C NMR (150 MHz, CD3OD): δ = 175.1, 173.1, 172.9, 157.9, 137.3, 129.6, 129.3, 129.2, 80.8, 58.8, 55.7, 52.7, 52.4, 38.3, 28.7; MS (ES) for C21H3iN208 calculated 439.2002 [M + H] + found 439.2 [M + H]+. (S)-di-terf-butyl 2-(((S)-2-(((benzyloxy)carbonyl)amino)-3-(ieri-butoxy)-3- oxopropyl)amino)succinate (LL-25g)
[00197] Compound LL-25g was obtained by reacting sulfamidate L-24g (0.5 mmol, 179 mg) with L-aspartic acid di-tert-butyl ester in 75% yield (195 mg). 1H NMR (600 MHz, CD3OD): δ 7.34 (m, 5H), 5.11 (s, 2H), 4.18 (dd, J = 6.4 Hz, J = 4.6 Hz, 1H), 3.49 (apt, J = 6.0 Hz, 1H), 3.09 (dd, J = 12.4 Hz, J = 6.4 Hz, 1H), 2.89 (dd, J = 12.4 Hz, J = 4.6 Hz, 1H), 2.62 (dd, J = 16.1 Hz, J = 5.7 Hz, 1H), 2.54 (dd, J = 16.1 Hz, J = 6.5 Hz, 1H), 1.48 (s, 9H), 1.46 (s, 9H), 1.44 (s, 9H); NMR (150 MHz, CD30D): δ 173.6, 171.9, 158.6, 138.2, 129.5, 129.0, 128.9, 83.2, 83.0, 82.4, 67.7, 59.4, 56.2, 49.6, 39.7, 28.4, 28.3, 28.3; HRMS (ES) for C27H42N208 calculated 523.3019 [M + H]+ found 523.3014 [M + H]+ .
(S)-di-terf-butyl 2-(((S)-3-(teri-butoxy)-2-((2-(teri-butoxy)-2-oxoethyl)amino)-3- oxopropyl)amino)succinate (LL-25h)
[00198] Compound LL-26d (194 mg, 0.5 mmol) was dissolved in dry EtOH /
DMF (1 : 1, 8 ml) and treated with TEA (101 mg, lmmol) followed by t-butyl bromoacetate (97 mg, 0.5 mmol). The reaction mixture was stirred under nitrogen at 65°C for 18 h at which time water was added and the solution extracted with ethyl acetate. The organic layer was evaporated in vacuo and the residue was purified by column chromatography (using a gradient of 0 - 40% EtOAc in hexanes) to afford the title compound as a white powder in 28% yield (70mg).
[00199] Alternatively, LL-25h was obtained following the general procedure for sulfamidates opening by reacting sulfamidate L-24i (0.25 mmol, 84 mg) with L-aspartic acid dimethyl ester in 71% yield (89 mg). Compound LL-25h showed 1H NMR (600 MHz, CD3OD): δ 3.45 (apt, 7 = 6.3 Hz, 1H), 3.36 (d, 7 = 17.0 Hz, 1H), 3.29 (dd, 7 = 7.2 Hz, 7 = 4.9 Hz 1H), 3.26 (d, 7 = 17.0 Hz, 1H), 2.83 (dd, 7 = 12.0 Hz, 7 = 7.2 Hz, 1H), 2.78 (dd, 7= 12.0 Hz, 7= 4.9 Hz, 1H), 2.61 (dd, 7= 16.0 Hz, 7= 6.3 Hz, 1H), 2.51 (dd, 7 = 16.0 Hz, 7 = 6.3 Hz, 1H), 1.48 (s, 18H), 1.46 (s, 9H), 1.45 (s, 9H); 13C NMR (150 MHz, CD3OD): δ 173.9, 173.6, 172.4, 171.9, 82.9, 82.7, 82.4, 82.2, 61.8, 59.2, 50.3, 39.9, 28.4. HRMS (ES) for C25H46N208 calculated 503.3332 [M + H]+, found 503.3321 [M + H]+. (R)-di-ferf-butyl 2-(((S)-2-(((benzyloxy)carbonyl)amino)-3-(ieri-butoxy)-3- oxopropyl)amino)succinate (DL-25j)
[00200] Compound DL-25j was obtained by reacting sulfamidate L-24g (0.4 mmol, 150 mg) with D-aspartic acid di-tert-butyl ester in 75% yield (165 mg). 1H NMR (700 MHz, Chloroform- ) δ 7.39 - 7.27 (m, 5H), 5.11 (s, 2H), 4.22 (s, 1H), 3.44 - 3.31 (m, 1H), 3.08 (dd, J = 12.4, 4.5 Hz, 1H), 2.94 (dd, J = 12.4, 4.3 Hz, 1H), 2.55 (dd, J = 15.9, 5.7 Hz, 1H), 2.48 (dd, J = 15.9, 6.9 Hz, 1H), 1.54 (s, 18H), 1.45 (s, 19H). 13C NMR (176 MHz, Chloroform- ) δ 170.22, 128.60, 128.18, 82.27, 81.73, 66.93, 58.68, 49.10, 39.34, 28.19. HRMS (ES) for C27H42N208 calculated 523.3019 [M + H]+ found 523.3043[M + H]+ .
General procedures for Boc deprotection
Figure imgf000060_0001
25 26
[00201] Procedure A: Compound 25 (0.25 mmol) was stirred in a mixture of trifluoroacetic acid and DCM (2 mL, 6: 1 ratio) at room temperature for 16 h. The solvents were evaporated, then co-evaporated with MeOH (3 x 10 mL) and dried under high vacuum. The resulting salt was dissolved in saturated potassium carbonate solution and extracted with chloroform (5 x 10 mL). The combined organic layers were dried (MgS04) and concentrated in vacuo to give the free amino ester. The crude product was purified by flash column chromatography (using a gradient of 0-40% CH2C12: MeOH). For compound LD-25a during flash chromatography demethylation of one ester group occurred and compound LD-26c was obtained in 62% yield (38mg). Compound LL-26b was obtained in 78% yield (65 mg).
[00202] Procedure B: Compound 25 (0.5 mmol) was stirred in 4M HC1 in dioxane (2 ml) at room temperature for 0.5 h. The solvents were evaporated and then co- evaporated with MeOH (3 x 10 mL) and dried under high vacuum. The resulting salt was dissolved in saturated potassium carbonate solution, and extracted with chloroform (5 χ 10 mL). The combined organic layers were dried (MgS04) and concentrated in vacuo to give the free amino ester. The crude product was used in the next step without further purification. Compound LD-26a was obtained in 64% yield (83 mg). Compound LL- 26b was obtained in 77% yield (130 mg).
(R)-2-amino-3-(((S)-l,4-dimethoxy-l,4-dioxobutan-2-yl)amino)propanoicacid (LD- 26c).
[00203] Compound LD-26c showed 1H NMR (600 MHz, CD3OD): δ = 4.09 (dd, J
= 4.5 Hz, J = 2.6 Hz 1H), 3.78 (s, 3H), 3.75 (dd, J = 8.1 Hz, J = 4.1 Hz, 1H), 3.68 (s, 3H), 3.39 (dd, J = 6.8 Hz, J = 2.6Hz, 1H), 3.21 (dd, J = 13.3 Hz, J = 4.5 Hz, 1H), 2.87 (dd, J = 16.6 Hz, J = 4.1 Hz, 1H), 2.64 (dd, J = 16.6 Hz, J = 8.1 Hz, 1H); 13C NMR (150 MHz, CD3OD): δ = 173.6, 173.1, 172.8, 56.6, 55.9, 53.1, 52.3, 45.1, 37.4; HRMS (ES) for C9Hi7N206 calculated 249.1087 [M + H] + found 249.1089 [M + H]+.
(S)-di-tert-butyl 2-(((S)-2-amino-3-(tert-butoxy)-3-oxopropyl)amino)succinate (LL- 26d)
[00204] A solution of compound LL-25g (261 mg, 0.5 mmol) in ethyl acetate /
DCM (1 : 1, 30 mL) was treated with 20% wt. Pd(OH)2 on carbon (80 mg). The reaction mixture was stirred at 60 °C under an atmosphere of hydrogen for 30 min and monitored via TLC. When the starting material had been consumed, the reaction mixture was filtered through a Celite pad and washed with DCM (2 x 10 mL) and ethyl acetate (2 x 10 mL). The combined filtrate was concentrated in vacuo. The crude product was used directly in the next step without further purification.
(S)-2-(((S)-2-amino-2-carboxyethyl)amino)succinic acid (LL-26e)
[00205] Compound LL-25g (42 mg, 0.08 mmol) was dissolved in DCM (0.8 mL), cooled to 0°C under a nitrogen atmosphere, treated with anisole (48 mg, 0.45 mmol) and trifluoromethanesulfonic acid (67 mg, 0.45 mmol) then stirred at 0°C for 30 min. and at room temperature for an additional lh. The reaction mixture was cooled down to 0°C treated with a solution of NaHC03 (10 eq.) in water (1.5 mL) and stirred for lh. The water layer was separated, washed with DCM (3 x 5 mL) and then freeze dried. The resulting solid was precipitated in a mixture of H20 : MeOH : AcOH (0.15 mL : 0.6 mL : 8.2 μί) to afford LL-26e in 82 % yield (14.4 mg). 1H NMR (700 MHz, D20): δ 3.74 (dd, J= 7.6 Hz, J = 5.6 Hz, 1H), 3.47 (dd, J= 10.5 Hz, J = 3.7 Hz, 1H), 2.99 (dd, J = 6.6 Hz, J = 2.7 Hz, 2H), 2.63 (dd, J = 15.7 Hz, J = 3.7 Hz, 1H) 2.33 (dd, J = 15.7 Hz, J = 10.5 Hz, 1H); 13C NMR (176 MHz, D20): δ 178.8, 177.4, 173.3, 60.3, 52.3, 46.8, 38.6; HRMS (ES) for C7Hi2N206 calculated 221.0773 [M + H]+, found 221.0765 [M + H]+; [a]D 20 -26 (c 0.21, 0.1 N H4OH).
(R)-di-ferf-butyl 2-(((S)-2-amino-3-(ieri-butoxy)-3-oxopropyl)amino)succinate (DL- 26f)
[00206] As described for LL-26d. Compound was used in the next step without further purification.
General procedure for sulfamidates opening (Π)
[00207] Compound 26 (0.10 mmol) was dissolved in AcCN (0.5 mL) and added to a solution of sulfamidate 24 (0.10 mmol) in AcCN (0.5 mL). The reaction mixture was stirred at 60°C for 12 h and treated with 1 M monopotassium phosphate (4 mL). The aqueous layer was extracted with ethyl acetate (2 x 10 mL), dried over (MgS04) and concentrated. The crude reaction mixture was purified by flash column chromatography (using a gradient of 0-60% EtOAc in hexanes). Compound LDD-27a lost 2 methyl groups during flash chromatography and, as a result, LDD-27b was obtained in 22% yield (9.5mg). Compound LDD-27c was obtained in 34 % yield (21 mg).
Compound (LDD-27b).
[00208] 1H NMR (600 MHz, CD3OD): δ = 4.25 (dd, J = 8.4 Hz, J = 4.5 Hz 1H),
4.18 (apt, J = 4.0 Hz, 1H), 3.78 (s, 3H), 3.69 (s, 3H), 3.48 (apt, J = 5.5 Hz, 1H), 3.38 (dd, J = 12.7 Hz, J = 4.5 Hz, 1H), 2.87 (m, 4H), 2.74 (dd, J = 15.6 Hz, J = 6.0 Hz, 1H), 1.45 (s, 9H); 13C NMR (150 MHz, CD3OD): δ = 173.4, 173.3, 172.3, 172.2 157.7, 80.1, 63.3, 55.3, 53.8, 53.2, 52.8, 52.5, 50.1, 49.4, 36.7, 28.7.
(6S,9S,12S)-6,9-dibenzyl-12-methyl-2,2-dimethyl-4,14-dioxo-3-oxa-5,8,ll- triazapentadecane-6,9,12-tricarboxylate (LLL-27d).
[00209] LLL-27d was prepared starting from L-24e according to the synthetic scheme. 1H NMR (600 MHz, CD3OD): δ = 7.35 (m, 10H), 5.18 (d, J = 5.7 Hz, 2H), 5.13 (d, J = 12.2 Hz, 2H), 4.27 (apt, J = 5.7 Hz, 1H), 3.69 (s, 3H), 3.64 (s, 3H), 3.62 (dd, J = 7.1 Hz, J = 5.9 Hz, 1H), 3.41 (dd, J= 7.3 Hz, J = 5.0 Hz, 1H), 3.03 (dd, J = 12.4 Hz, J = 6.7 Hz, 1H), 2.85 (dd, J= 12.6 Hz, J= 4.7 Hz, 1H), 2.82 (m, 1H), 2.77 (dd, J = 12.1 Hz, J= 5.0 Hz, 1H), 2.70 (dd, J = 16.2 Hz, J= 5.9 Hz, 1H), 2.59 (dd, J = 16.2 Hz, J= 7.1 Hz, 1H),1.45 (s, 9H); HRMS (ES) for C3iH41N3Oio calculated 616.2894 [M + H] + found 616.2870 [M + H]+.
(5S, SS, llS)-tetra-terf-butyl 3-oxo-l-phenyl-2-oxa-4,7,10-triazadodecane-5,8,ll,12- tetracarboxylate (LLL-27e)
[00210] Following the general procedure for sulfamidates opening, compound
LLL-27e was obtained by reacting sulfamidate L-24g (0.25 mmol, 90 mg) with LL-26d (prepared above) in 43% yield (56 mg). 1H NMR (600 MHz, CD3OD): δ 7.34 (m, 5H), 5.12 (d, J = 12.4 Hz, 1H), 5.09 (d, J = 12.4 Hz, 1H), 4.18 (dd, J = 6.8 Hz, J = 4.5 Hz, 1H), 3.42 (apt, J = 6.2 Hz, 1H), 3.27 (apt, J= 6.2 Hz, 1H), 3.00 (dd, J= 12.4 Hz, J= 6.8 Hz, 1H), 2.84 (dd, J= 12.4 Hz, J= 4.5 Hz, 1H), 2.75 (apd, J= 6.2 Hz, 1H), 2.59 (dd, J = 16.1 Hz, J = 6.2 Hz, 1H), 2.56 (dd, J = 16.1 Hz, J = 6.2 Hz, 1H), 1.48 (s, 9H), 1.47 (s, 9H), 1.43 (s, 9H); 13C NMR (150 MHz, CD3OD): δ = 174.1, 173.9, 172.0, 171.9, 158.7, 138.2, 129.5, 129.0, 83.1, 82.9, 82.8, 82.3, 67.7, 62.6, 59.2, 56.5, 50.6, 39.9, 28.4, 28.3, 28.2. HRMS (ES) for C34H55N3O10 calculated 666.3966 [M+H]+; found 666.3964 [M+H]+.
(5R, SS, llS)-tetra-terf-butyl 3-oxo-l-phenyl-2-oxa-4,7,10-triazadodecane-5,8,ll,12- tetracarboxylate (DLL-27f)
[00211] DLL-27f was obtained as described for LLL-27e. Yield 40%. 1H NMR (700 MHz, Chloroform- ) δ 7.41 - 7.29 (m, 5H), 5.11 (s, 2H), 4.28 (dd, J = 8.8, 4.7 Hz, 1H), 3.44 (t, J = 6.4 Hz, 1H), 3.19 - 3.10 (m, 2H), 2.95 - 2.84 (m, 1H), 2.80 - 2.70 (m, 1H), 2.60 (ddd, J = 31.3, 14.5, 6.5 Hz, 2H), 2.49 (d, J = 6.5 Hz, 1H), 1.51 - 1.39 (m, 36H). 13C NMR (176 MHz, Chloroform- ) δ 170.32, 156.45, 136.72, 128.57, 128.13, 82.21, 81.51, 80.97, 66.92, 62.39, 58.75, 55.00, 50.14, 49.50, 39.31, 28.25. HRMS (ES) for C34H55N3O10 calculated 666.3966 [M+H]+; found 666.3929[M+H]+. Example 5: Solid Phase Synthesis of AM-A
Reaction Scheme:
Fmoc-L-Asp(OtBu^ H-Asp(OtBu)-Q R=Me, tBu
Figure imgf000064_0001
COOR
N
Ns
Figure imgf000064_0002
AMA-A
Conditions: a) 20% Piped dine in DMF, 20 min, RT; b) oNos-Azi-OMe (2 eq ) in THF, o/n at room temperature; c) thiophenol (5eq), DiPEA (4 eq), acetonitrile, 2h at room temperature; d) TFA/TIS/H20 (95:2.5:2.5), 2h at room temperature.
[00212] The synthesis was carried out using Fmoc protected L-aspartic acid t-butyl ester attached to Wang resin. After Fmoc deprotection, the resin was drained, washed with DMF, iso-PrOH and THF. First ring opening reaction was carried out with oNs-Azi- OMe (2eq) in THF overnight at room temperature. The resin was drained and washed with THF, DMF, acetonitrile prior to oNs deprotection. Small part of the resin (22 mg wet weight) was taken for cleavage to confirm the product formation.
[00213] The oNs group was further deprotected using thiophenol (5eq), DIPEA (4 eq) in acetonitrile for 2 h at room temperature. After draining and resin wash as already described, small part was taken for cleavage to confirm the product formation. Next ring opening reaction was carried out with another oNs-Azi-OMe to yield the protected AM-A attached to a solid support. After the nosyl group deprotection using the conditions described above, the resin was washed with DCM, methanol , dried under vacuum for 4 h, and the cleaved with a mixture of TFA/TIS/H20 (95:2.5:2.5) for 2 hours at room temperature. The resulting solution was concentrated under reduced pressure and purified using reverse phase chromatography.20
Example 6: Total Synthesis of LLD and LDL AM-A
Synthetic scheme:
Figure imgf000065_0001
LLL-7 LLD-7 LLL-6 LLD-8
LDD-7 LDL-7 LDDS LDLS
Figure imgf000065_0002
(S)-3-(((S)-l-carboxy-2-(((D)-l,4-di-teri-butoxy-l,4-dioxobutan-2- yl)aniino)ethyl)aniii]o)-2-(2-niti ophenylsiilfonaniido)pi opanoic acid (LLD-7)6 .
[00214] The compound was synthesized using the procedure described for LLL-l using LLD-6 as the starting material. 1H MR (700 MHz, Methanol -d4) δ 8.14 (dd, J = 7.3, 2.0 Hz, 1H), 7.93 (dd, J = 7.4, 1.8 Hz, 1H), 7.87 - 7.80 (m, 3H), 4.41 (t, J = 6.2 Hz, 1H), 4.00 (t, J = 5.4 Hz, 1H), 3.93 (dd, J = 8.1 , 5.0 Hz, 1H), 3.53 - 3.45 (m, 1H), 3.40 (dd, J = 13.4, 4.9 Hz, 1H), 3. 19 (td, J = 12.9, 4.4 Hz, 1H), 3.07 (s, 1H), 2.94 - 2.88 (m, 2H), 1.51 (s, 8H), 1.48 (s, 7H). 13C NMR (176 MHz, MeOD) δ 175.05, 172.01, 171.53, 170.35, 149.31, 135.35, 134.73, 133.85, 131.77, 126.26, 85.07, 83.57, 59.26, 58.01, 56.47, 49.68, 47.48, 37.04, 35.69, 28.36, 28.18.
(S)-3-(((D)- l-carboxy-2-(((S)- 1 ,4-di-terf-butoxy- 1 ,4-dioxobutan-2- yl)amino)ethyl)amino)-2-(2-nitrophenylsulfonamido)propanoic acid (LDL-7)6 .
[00215] The compound was synthesized using the procedure described for LLL-l using LDL-6 as the starting material. 1H NMR (600 MHz, Methanol -<f4) δ 8.20 - 8.14 (m, 1H), 8.01 - 7.94 (m, 1H), 7.88 - 7.77 (m, 3H), 4.09 (s, 1H), 3.74 - 3.65 (m, 1H), 3.56 (t, J= 5.7 Hz, 1H), 3.51 (dd, J = 13.5, 4.5 Hz, 1H), 3.47 (dd, J = 12.7, 7.0 Hz, 1H), 3.41 - 3.33 (m, 2H), 3.04 (dt, J = 12.9, 8.2 Hz, 1H), 2.87 (dd, J = 7.4, 5.4 Hz, OH), 1.49 (s, 9H), 1.45 (s, 8H). 13C NMR (151 MHz, MeOD) δ 173.28, 172.57, 171.96, 149.44, 135.37, 134.02, 131.89, 126.67, 83.42, 82.59, 62.44, 59.35, 55.39, 50.18, 47.74, 39.09, 28.40. (S)-2-amino-3-(((S)-l-carboxy-2-(((D)-l,4-di-teri-butoxy-l,4-dioxobutan-2- yl)amino)ethyl)amino)propanoic acid (LLD-8)
[00216] The compound was synthesized using the procedure described for LLL-8 using LLD-l as the starting material. 1H NMR (700 MHz, Deuterium Oxide) δ 4.04 (t, J = 5.6 Hz, 1H), 3.90 (t, J = 6.2 Hz, 1H), 3.53 (dd, J = 8.0, 4.8 Hz, 1H), 3.35 (dd, J = 13.4,
5.5 Hz, 1H), 3.26 (dd, J = 13.0, 4.9 Hz, 1H), 3.21 - 3.13 (m, 2H), 3.07 - 2.91 (m, 2H), 1.55 - 1.45 (m, 17H). 13C NMR (176 MHz, D20) δ 172.49, 171.04, 85.54, 84.14, 56.87, 56.71, 53.00, 47.94, 46.87, 35.97, 27.20, 27.07.
(S)-2-amino-3-(((D)-l-carboxy-2-(((S)-l,4-di-teri-butoxy-l,4-dioxobutan-2- yl)amino)ethyl)amino)propanoic acid (LDL-8)
[00217] The compound was synthesized using the procedure described for LLL-8 using LDL-7 as the starting material. 1H NMR (600 MHz, Deuterium Oxide) δ 3.91 - 3.79 (m, 2H), 3.40 - 3.35 (m, 1H), 3.27 (dd, J = 13.5, 5.1 Hz, 1H), 3.24 - 3.15 (m, 2H),
3.06 (dd, J = 13.5, 7.2 Hz, 1H), 2.97 - 2.90 (m, 2H), 2.87 (dd, J = 17.2, 6.4 Hz, 1H), 1.49 (d, J = 14.4 Hz, 25H). 13C NMR (151 MHz, D20) δ 172.95, 171.48, 84.93, 83.86, 61.48, 57.44, 54.34, 53.56, 48.42, 47.25, 42.53, 27.1 1.
(S)-2-(((S)-2-(((D)-2-amino-2-carboxyethyl)amino)-2-carboxyethyl)amino) succinic acid (LLD-AM-A)
[00218] The compound was synthesized using the procedure described for LLL-
AM-A using LLD-8 as the starting material. The compound showed 1H NMR (600 MHz, 0.1 M H4OH in Deuterium Oxide) δ 3.75 (ddd, J = 10.7, 9.0, 4.1 Hz, 2H), 3.35 (dd, J = 9.6, 4.2 Hz, 1H), 3.29 (dd, J = 13.1, 4.0 Hz, 1H), 3.22 (dd, J = 12.6, 4.2 Hz, 1H), 3.01 (dd, J= 12.6, 9.7 Hz, 1H), 2.82 - 2.72 (m, 2H), 2.63 (dd, J= 17.0, 9.1 Hz, 1H). 13C NMR (151 MHz, D20) δ 176.19, 174.74, 61.36, 60.01, 55.00, 48.53, 48.39, 37.86.
(S)-2-(((D)-2-(((S)-2-amino-2-carboxyethyl)amino)-2-carboxyethyl)amino) succinic acid (LDL-AM-A)
[00219] The compound was synthesized using the procedure described for LLL-
AMA using LDL-S as the starting material. The compound showed 1H NMR (600 MHz 0.1 M H4OH in Deuterium Oxide) δ 3.73 - 3.63 (m, 2H), 3.34 - 3.23 (m, 3H), 2.88 (dd, J = 11.9, 9.2 Hz, 1H), 2.81 - 2.71 (m, 2H), 2.63 (ddd, J = 17.3, 8.6, 4.8 Hz, 1H). 13C NMR (151 MHz, 0.1 M NH4OH in Deuterium Oxide) δ 174.06, 171.02, 60.83, 59.73, 54.99, 54.12, 47.78, 39.83. Rt: 7.31 min (b)
Example 7: Total Synthesis of Lycomarasmine
Figure imgf000067_0001
R2=tBu
R2=tBu
Figure imgf000067_0002
LL-31a: x=CH2; R^tBu; R2=tBu LL-33a; R^Me; R2=tBu
LL-31b: x=CH2CH2; R1 = tBu; R2=tBu LL-33b: R^H, R2=tBu
LL-31c: x=CH2CH2; R^ Me; R2=tBu LL-33c (lycomarasmine): R-, =H; R2=H
H COOH
HOOC,
N ^ NH2
COOH
32 Conditions: (a) THF, 20h at RT; (b) PhSH (5eq), DiPEA (4eq), acetonitrile; (c) BrCH2CO H2 (2 eq), THF, 20h at RT; (d) Li OH (6.6 eq) , THF/H20 (1 :2), lh at 0°C; (e) CF3SO3H (5 eq), anisole (6 eq), DCM , lh at 0°C -> RT.
Tert-butyl (2S)-l-(2-nitrobenzene-l-sulfonyl)aziridine-2-carboxylate (L-28a)
[00220] (25)-l-(2-Nitrobenzene-l-sulfonyl) aziridine-2-carboxylic acid (0.7 g, 2.6 mmol), sulfuric acid (0.02 g, 0.02 mmol) and tert-butyl acetoacetate (2.6 g, 17 mmol) were charged into a glass pressure tube fitted with a threaded Teflon plug. The reaction was sealed and held at room temperature for 24h. After 24h, the reaction vessel was cooled to reduce any pressure that might have been generated during the reaction and the cap carefully removed. The mixture was transferred in a separately funnel and partitioned between ether, 1.25% NaOH and brine. The organic extract was then dried over anhydrous MgS04. The NaOH phase was acidified and extracted with ether to recover unreacted acid. Yield 50%; 1H NMR (700 MHz, Chloroform- ) δ 8.27 (dd, J = 7.3, 1.6 Hz, 1H), 7.78 (ddd, J = 15.8, 6.8, 2.4 Hz, 3H), 3.01 (d, J = 7.1 Hz, 1H), 2.73 (d, J = 4.5 Hz, 1H), 2.04 (s, 1H), 1.48 (s, 9H); 13C NMR (176 MHz, CDC13) δ 165.48, 134.69, 132.45, 131.55, 124.65, 77.19, 50.89, 38.69, 34.04, 27.89; HRMS: C13H16N206S calculated for [M+NH4]+ 346.1119, found [M+NH4]+ 346.1049.
(S)-di-terf-butyl 2-(((S)-3-ieri-butyl-2-(2-nitrophenylsulfonamido)-3-oxopropyl) amino)succinate (LL-30a)
[00221] The aziridine ring opening was carried out as described in Example 1 using compound L-28a and the di-t-butyl ester of aspartic acid. The final product LL- 30a was purified using normal phase chromatography in 20% ethyl acetate in hexanes. Rt: 7.8 min; Yield: 60 %; 1H NMR (700 MHz, Chloroform- ) δ 8.15 - 8.03 (m, 1H), 7.95 - 7.82 (m, 1H), 7.70 (q, J = 5.3, 4.1 Hz, 2H), 4.18 - 4.02 (m, 1H), 3.39 (t, J = 6.2 Hz, 1H), 3.29 (dd, J = 12.4, 4.2 Hz, 1H), 2.79 (dd, J = 12.6, 4.5 Hz, 1H), 2.59 (dd, J = 16.2, 5.5 Hz, 1H), 2.51 (dd, J = 16.1, 6.8 Hz, 1H), 1.45 (s, 18H), 1.27 (s, 9H); 13C NMR (176 MHz, Chloroform- ) δ 172.70, 170.26, 169.09, 135.01, 133.40, 132.73, 130.68, 125.49, 110.14, 82.70, 81.81, 81.34, 58.94, 57.76, 50.56, 50.55, 39.60, 28.24, 28.20, 27.89; HRMS: C25H39N3O10S calculated for [M+H]+ 574.2436, found [M+H]+ 574.2392. (2S)-2-({(2S)- 2-[2-tert-Butoxycarbonyl-2-(2-nitro-benzenesulfonylamino)- ethylamino]-pentanedioic acid di-tert-butyl ester (LL-30b)
[00222] Reaction was carried out as describedin Example 1 using L-glutamic acid- di-t-butyl ester instead of aspartic acid. The crude compound was purified using normal phase flash chromatography in 20% ethyl acetate in hexanes. Yield: 70%; Rt: 7.8 min; 1H MR (700 MHz, Chloroform- ) δ 8.11 - 8.03 (m, 1H), 7.95 - 7.85 (m, 1H), 7.78 - 7.62 (m, 2H), 4.14 (t, J = 4.4 Hz, 1H), 3.14 (dd, J = 12.2, 4.4 Hz, 1H), 3.05 (s, 1H), 2.77 - 2.62 (m, 1H), 2.35 (dddd, J = 47.4, 16.1, 9.2, 6.2 Hz, 2H), 1.97 - 1.80 (m, 1H), 1.76 - 1.62 (m, 1H), 1.46 (s, 18H), 1.29 (s, 9H); 13C NMR (176 MHz, Chloroform- ) δ 173.84, 172.62, 169.12, 134.99, 133.44, 132.80, 130.62, 125.61, 82.85, 81.68, 80.56, 61.60, 57.60, 50.06, 32.02, 28.77, 28.26, 28.24, 27.90; HRMS: C26H41N30ioS calculated for [M+H]+ 588.2593, found [M+H]+ 588.2565.
(S)- 2-(((S)- 2-(((S)- 2-[2-Methoxycarbonyl-2-(2-nitro-benzenesulfonylamino)- ethylamino]-pentanedioic acid di-tert-butyl ester (LL-30c)
[00223] The aziridine ring opening was carried out using the procedure describedin
Example 1. Compound LL-30c was obtained in pure form using normal phase flash chromatography in 20% ethyl acetate in hexanes. Yield: 85%; Rt: 7.17 min; 1H NMR (700 MHz, Chloroform- ) δ 8.11 - 8.03 (m, 1H), 7.97 - 7.86 (m, 1H), 7.77 - 7.67 (m, 2H), 4.25 (t, J = 4.4 Hz, 1H), 3.54 (s, 3H), 3.32 (dd, J= 8.2, 5.2 Hz, 1H), 3.28 - 3.19 (m, 1H), 3.03 (dd, J = 8.9, 5.1 Hz, 1H), 2.69 (dd, J = 12.5, 4.7 Hz, 1H), 2.44 - 2.24 (m, 4H), 1.98 (dtd, J = 15.6, 7.5, 5.2 Hz, 1H), 1.88 (dddd, J = 14.1, 9.0, 6.6, 5.1 Hz, 1H), 1.80 - 1.62 (m, 2H), 1.50 - 1.41 (m, 34H); 13C NMR (176 MHz, Chloroform- ) δ 175.00, 172.55, 170.47, 134.63, 133.40, 132.68, 130.44, 125.46, 81.64, 81.13, 80.45, 77.20, 61.64, 56.80, 54.37, 52.52, 49.54, 31.95, 30.13, 28.55, 28.09; HRMS: C^HssNsC oS calculated for [M+H]+ 546.2123, found [M+H]+ 546.2097.
(S)-di-terf-butyl 2-(((S)-2-amino-3-terf-butyl-3-oxopropyl)amino)succinate (ZX-31a)
[00224] Deprotection of the nosyl group was carried out using the procedure described in Example 1. Normal phase flash purification using methanol in chloroform (0 to 20%) afforded pure compound LL-31a. Yield: 80%; 1H NMR (700 MHz, Methanol- d4) δ 3.43 (t, J = 6.4 Hz, 1H), 3.38 (dd, J = 6.4, 4.6 Hz, 1H), 2.91 (dd, J = 12.2, 6.4 Hz, 1H), 2.74 (dd, J = 12.2, 4.6 Hz, 1H), 2.60 (dd, J = 15.9, 6.0 Hz, 1H), 2.51 (dd, J = 15.9, 6.7 Hz, 1H), 1.50 - 1.44 (m, 27H); 13C NMR (176 MHz, Methanol -d4) δ 173.20, 172.65, 170.49, 81.32, 81.16, 80.77, 58.24, 54.38, 50.60, 38.59, 26.90; HRMS: C19H36N206 calculated for [M+H]+ 389.2653, found [M+H]+ 389.2633.
(2S)-2-({(2S)- 2-(2-Amino-2-tert-butoxycarbonyl-ethylamino)}-pentanedioic acid di- tert-butyl ester (LL-31b)
[00225] Deprotection of the nosyl group was carried out using the procedure described in Example 1. Purification using reverse phase flash (CI 8) chromatography in 0-100% acetonitrile in water afforded pure compound LL-31b. Yield: 89%; Rt: 4.9 min; 1H NMR (700 MHz, Methanol-^) δ 3.37 (dd, J = 6.6, 4.6 Hz, 1H), 3.18 (dd, J = 8.0, 5.4 Hz, 1H), 3.15 - 3.09 (m, 1H), 2.82 (ddd, J = 12.0, 9.7, 5.5 Hz, 1H), 2.71 - 2.61 (m, 1H), 2.45 - 2.28 (m, 1H), 1.94 - 1.71 (m, 1H), 1.52 - 1.43 (m, 27H); 13C NMR (176 MHz, Methanol-^) δ 175.41, 175.16, 174.42, 174.20, 82.94, 82.67, 81.62, 63.76, 62.31, 61.43, 55.88, 52.02, 44.80, 32.97, 32.79, 29.78, 29.47, 28.37; HRMS: C20H38N2O6 calculated for [M+H]+403.2810, found [M+H]+403.2791.
(S)- 2-(((S)- 2-(((S)- 2-(2-Amino-2-methoxycarbonyl-ethylamino)-pentanedioic acid di-ferf-butyl ester (LLL-31c)
[00226] Deprotection of the nosyl group was carried out using the procedure described in Example 1. Yield: 86%; Rt: 4.5 min; 1H NMR (700 MHz, Chloroform-^ δ 3.71 (s, 3H), 3.49 (dd, J = 7.0, 4.4 Hz, 1H), 3.05 (dd, J = 8.7, 5.2 Hz, 1H), 2.85 (dd, J = 11.8, 6.9 Hz, 1H), 2.67 (dd, J = 11.8, 4.4 Hz, 1H), 2.39 - 2.24 (m, 2H), 1.92 - 1.83 (m, 1H), 1.43 (m, 18H); 13C NMR (176 MHz, Chloroform- ) δ 175.38, 174.18, 172.62, 81.39, 80.38, 61.63, 54.88, 52.17, 51.12, 32.21, 28.23, 28.21; HRMS: C17H32N206 calculated for [M+H]+ 361.2340, found [M+H]+ 361.2321.
(2S)-2-({(2S)- 2-(2-Amino-2-carboxy-ethylamino)}-pentanedioic acid (LL-32)
[00227] Deprotection of tert-butyl ester groups was carried out as previously
23
described. The pure final product was isolated by precipitation from water (1 ml), methanol (3 ml) and acetic acid (0.04 ml). Yield: 79%; 1H NMR (700 MHz, Deuterium Oxide) δ 3.71 (ddd, J = 7.8, 4.6, 2.0 Hz, 1H), 3.22 - 3.18 (m, 1H), 2.97 (dddd, J = 15.3, 13.2, 9.1, 2.9 Hz, 2H), 2.33 - 2.21 (m, 2H), 1.96 - 1.81 (m, 2H); 1JC NMR (176 MHz, Deuterium Oxide) δ 182.61, 180.44, 174.95, 63.32, 54.24, 48.14, 34.31, 29.07; HRMS: C8Hi4N206 calculated for [M+H]+ 235.0932, found [M+H]+ 235.0928.
(2S)-2-({(2S)- 2-[2-(Carbamoylmethyl-amino)-2-methoxycarbonyl-ethylamino]}- succinic acid di-terf-butyl ester (LL-33a)
[00228] Treatment of L-aspartic acid-di-t-butyl ester with (S)-methyl l-((2- nitrophenyl)sulfonyl)aziridine-2-carboxylatefollowed by the nosyl deprotection protocol as described in Example 1 allowed for the preparation of (^-di-tert-butyl 2-(((S)-2- amino-3-methoxy-3-oxopropyl)amino)succinate. The free amine (65 mg, 0.18 mmol) in THF (2ml) was added bromoacetamide (50 mg, 0.36 mmol) and the reaction was carried out overnight at room temperature. The final product LL-33a was then purified using normal phase chromatography in 20% methanol in chloroform. Yield: 75 %; Rt: 4.36 min; 1H NMR (700 MHz, Methanol-^) δ 3.82 - 3.76 (m, 1H), 3.73 (s, 3H), 3.48 (t, J = 6.3 Hz, 1H), 3.39 (s, 1H), 3.16 (d, J = 16.8 Hz, 1H), 2.86 (dd, J = 22.4, 6.2 Hz, 1H), 2.66 - 2.59 (m, 1H), 2.58 - 2.49 (m, 1H), 1.48 (s, 9H), 1.45 (s, 9H); 13C NMR (176 MHz, Methanol-^) δ 177.15, 174.96, 173.95, 171.98, 82.89, 82.30, 62.36, 59.41, 52.48, 28.38; HRMS: C18H33N307, calculated for [M+H]+ 404.2399, found [M+H]+ 404.2380 .
(2S)-2-({(2S) 2-[2-(Carbamoylmethyl-amino)-2-carboxy-ethylamino]}-succinic acid di-ferf-butyl ester (LL-33b)
[00229] To a solution of compound LL-33a (77 mg, 0.19 mmol) in THF (3.5 ml) was added a solution of lithium hydroxide (29 mg, 1.26 mmol) in water (6.3 ml) at 0°C. The reaction was carried out for lh at 0°C. Solvent was removed under reduced pressure and acidified to pH 3 with 5% hydrochloric acid and lyophilized. The product LL-33b was purified first using normal phase chromatography in 20% methanol in chloroform. Yield: 74%; Rt: 4.6 min; 1H NMR (700 MHz, Deuterium Oxide) δ 3.88 (d, J = 16.1 Hz, 1H), 3.80 - 3.70 (m, 1H), 3.70 - 3.63 (m, 2H), 3.11 (s, 2H), 2.85 - 2.70 (m, 2H), 1.50 (d, J = 1.9 Hz, 9H), 1.47 (d, J = 1.9 Hz, 9H); 13C NMR (176 MHz, Deuterium Oxide) δ 172.97, 172.09, 84.16, 83.52, 61.34, 57.33, 47.52, 46.89, 37.84, 27.23; HRMS: C17H3iN307, calculated for [M+H]+ 390.2242, found [M+H]+390.2230 . (2S)-2-({(2S)2-[2-(Carbamoylmethyl-amino)-2-carboxy-ethylamino]}-succinic acid (Lycomarasmine) (LL-33c)
[00230] Jert-Butyl ester deprotection of compound LL-33b was carried out as
23
previously described. The final product LL-33c was isolated by precipitation from a mixture of water (1 ml), methanol (3 ml) and acetic acid (0.04 ml). Yield: 10%; 1H NMR (700 MHz, Deuterium Oxide) 4.14 (dd, J = 8.4, 6.0 Hz, 1H), 3.95 - 3.85 (m, 2H), 3.60 - 3.53 (m, 2H), 3.53 - 3.44 (m, 1H), 2.90 (dd, J= 17.7, 3.6 Hz, 1H), 2.77 (ddd, J = 17.7, 11.0, 9.0 Hz, 1H); 13C NMR (176 MHz, D20) 176.80, 172.67, 170.91, 60.09, 59.81, 58.12, 49.78, 45.63, 35.39; HRMS: C9Hi5N307 calculated for [M+H]+ 278.0990, Found [M+H]+ 278.0982.
Example 8: Total Synthesis
Figure imgf000072_0001
Figure imgf000072_0002
(a) THF, 20h at RT; (b) PhSH (5eq), DiPEA (4eq), acetonitrile; (c) CF3S03H (5 eq), anisole (6 eq), DCM, lh at 0°C -> RT; (d) TMTOH (trimethyltin hydroxide) (6eq), DCE, 3h at 80°C.
(S)-di-ter^butyl-2-(((S)-3-ter^butyl-2-(((S)-3-methoxy-2-(2-nitrophenylsulfonamido)- 3-oxopropyl)amino)-3-oxopropyl)amino)succinate (LLL-34a)
[00231] LL-31a was reacted with (^-methyl l-((2-nitrophenyl)sulfonyl)aziridine-
2-carboxylate as described in Example 1 to afford compound LLL-34a. Purification using normal phase flash chromatography in 20% ethyl acetate in hexanes afforded pure compound LLL-34a. Rt: 6.14; Yield: 62%; 1H NMR (700 MHz, Chloroform 8.09 (dd, J = 5.9, 3.4 Hz, 1H), 7.89 (dd, J = 5.9, 3.3 Hz, 1H), 7.71 (dd, J = 6.0, 3.4 Hz, 2H),
4.24 (d, J = 4.3 Hz, 1H), 3.56 (s, 3H), 3.45 (t, J = 6.5 Hz, 1H), 3.29 (dd, J = 12.8, 4.4 Hz, 1H), 3.17 (dd, J = 7.4, 4.5 Hz, 1H), 2.79 (m, 2H), 2.76 - 2.67 (m, 1H), 2.56 (dd, J = 35.9, 6.5 Hz, 1H), 1.46 (s, 9H), 1.44 (d, J = 2.9 Hz, 18H).13C NMR (176 MHz, Chloroform- ) δ 172.88, 170.73, 170.37, 147.87, 133.44, 132.74, 130.62, 125.49, 81.81, 81.53, 80.95, 62.67, 58.74, 57.34, 52.63, 50.33, 49.87, 39.56, 28.24; HRMS: C29H46N4012S calculated for [M+H]+ 675.2913, found [M+H]+ 675.2883.
(S)- 2-(((S)- 2-(((S)- 2-{2-Methoxycarbonyl-2-[2-methoxycarbonyl-2-(2-nitro- benzenesulfonylamino)-ethylamino]-ethylamino}-pentanedioic acid di-terf-butyl ester (LLL-34b)
[00232] The aziridine ring opening was carried out using the procedure described in Example 1 Compound LLL-34b was obtained in pure form using normal phase flash chromatography in 20% ethyl acetate in hexanes. Yield: 68%; Rt: 5.9 min; 1H NMR (700 MHz, Chloroform- ) δ 8.12 - 8.04 (m, 1H), 7.92 - 7.86 (m, 1H), 7.76 - 7.67 (m, 2H),
4.25 (t, J = 4.5 Hz, 1H), 3.70 (s, 3H), 3.55 (s, 3H), 3.38 - 3.19 (m, 2H), 2.84 - 2.72 (m, 2H), 2.65 (dd, J = 1 1.7, 4.8 Hz, 1H), 2.39 - 2.23 (m, 2H), 1.89 (ddt, J = 14.6, 9.0, 6.0 Hz, 1H), 1.76 (dtd, J = 14.7, 8.8, 6.2 Hz, 1H), 1.49 - 1.39 (m, 18H); 13C NMR (176 MHz, Chloroform- ) δ 174.13, 172.66, 170.63, 147.85, 134.77, 133.51, 132.81, 130.61, 125.51, 81.48, 80.36, 52.66, 52.17, 49.98, 49.72, 32.21, 28.24; HRMS: C27H42N4Oi2S; calculated for [M+H]+ 647.2600; Found [M+H]+ 647.2570.
(S)- 2-(((S)- 2-(((S)- 2-[2-tert-Butoxycarbonyl-2-(2-nitro-benzenesulfonylamino)- ethylamino]-pentanedioic acid di-tert-butyl ester (LLL-35)
[00233] Compound LLL-34a was subjected sequentially to the methyl ester
23 hydrolysisas described in Example 1 followed by the t-butyl ester deprotection as previously described. Purification using reverse phase flash (C I 8) chromatography in 0- 100%) acetoniotrile in water afforded pure compound LLL-35. Yield: 75%>; Rt: 4.3 min; 1H NMR (700 MHz, Deuterium Oxide) δ 8.17 (dt, J = 8.2, 3.1 Hz, 1H), 8.12 - 8.01 (m, 1H), 7.91 (ddd, J = 9.1, 4.9, 1.9 Hz, 2H), 4.05 (dd, J = 8.1, 4.5 Hz, 1H), 3.84 (dd, J = 8.9, 3.8 Hz, 1H), 3.62 (d, J = 7.3 Hz, 1H), 3.31 (ddd, J = 44.6, 12.8, 6.9 Hz, 2H), 3.18 - 3.06 (m, 1H), 2.85 (dd, J = 17.5, 3.8 Hz, 1H), 2.73 (dd, J = 17.6, 8.8 Hz, 1H); 13C NMR (176 MHz, Deuterium Oxide) δ 179.33, 175.37, 149.01, 136.85, 135.72, 132.74, 127.79, 61.97, 60.88, 59.98, 51.27, 48.96, 37.77; HRMS: C16H2oN4012S calculated for [M+H]+ 493.0878, found [M+H]+493.0854 .
(S)-di-terf-butyl 2-(((S)-3-ieri-butyl-2-(((S)-3-methoxy-2-amino)-3-oxopropyl) amino)-3-oxopropyl)amino)succinate (LLL-36)
[00234] Deprotection of the nosyl group was carried out using the procedure described in Example 1. Purification using normal phase flash chromatography in 0-20% methanol in chloroform afforded pure compound LLL-36. Rt: 4.4 min; Yield: 62%; 1H MR (700 MHz, Methanol-^) δ 3.74 (s, 3H), 3.53 (dd, J = 6.5, 4.8 Hz, 1H), 3.44 (t, J = 6.2 Hz, 1H), 3.24 (dd, J = 7.1, 5.4 Hz, 1H), 2.92 (dd, J = 12.1, 6.5 Hz, 1H), 2.80 - 2.70 (m, 3H), 2.64 - 2.50 (m, 2H), 1.48 (d, J = 2.0 Hz, 18H), 1.46 (s, 9H); 13C NMR (176 MHz, Methanol-^) δ 175.87, 174.24, 173.87, 171.87, 82.75, 82.21, 63.16, 59.31, 55.52, 52.58, 51.89, 50.75, 39.91, 28.40; HRMS: C23H43N308 calculated for [M+H]+ 490.3130, found [M+H]+ 490.3108.
(S)- 2-(((S)- 2-(((S)- 2-{2-Carboxy-2-[2-carboxy-2-(2-nitro-benzenesulfonylamino) - ethylamino]-ethylamino}-pentanedioic acid di-terf-butyl ester (LLL-37)
[00235] Methyl ester hydrolysis was carried out using the procedure described in
Example 1. Compound LLL-37 was obtained in pure form using normal phase flash chromatography in 40% ethyl acetate in hexanes. Yield: 23%; Rt: 7.1min; 1H NMR (700 MHz, Methanol-^) δ 8.21 - 8.13 (m, 1H), 8.01 - 7.94 (m, 1H), 7.88 - 7.77 (m, 2H), 4.10 (t, J = 6.9 Hz, 1H), 3.74 - 3.65 (m, 1H), 3.46 (dd, J = 12.3, 6.6 Hz, 1H), 3.40 - 3.34 (m, 1H), 3.12 (dd, J = 13.5, 7.3 Hz, 1H), 3.01 (dd, J = 13.5, 4.4 Hz, 1H), 2.47 - 2.33 (m, 2H), 1.99 - 1.82 (m, 1H), 1.50 (s, 9H), 1.45 (s, 9H); 13C NMR (176 MHz, Methanol-^) δ 174.36, 172.83, 171.90, 149.47, 135.38, 133.99, 131.97, 126.65, 83.26, 81.85, 61.46, 50.16, 47.53, 32.79, 29.24, 28.36; HRMS: C25H38N4012S, calculated for [M+H]+ 619.2287 Found: [M+H]+619.2270 . (S)- 2-(((S)- 2-(((S)- 2-[2-(2-Amino-2-carboxy-ethylamino)-2-carboxy-ethylamino]- pentanedioic acid di-tert-butyl ester (LLL-38a)
[00236] Deprotection of the nosyl group was carried out using the procedure described in Example 1. Compound LLL-38a was obtained in pure form using normal phase flash chromatography in 0- 20% methanol in chloroform. Yield: 58 %; Rt: 4.4 min; 1H NMR (700 MHz, Deuterium Oxide) δ 3.93 (t, J = 5.5 Hz, 1H), 3.89 - 3.82 (m, 1H), 3.52 (d, J = 6.0 Hz, 1H), 3.38 (dd, J= 13.4, 5.5 Hz, 1H), 3.31 (d, J = 5.0 Hz, 1H), 3.18 - 3.03 (m, 2H), 2.53 (d, J = 30.3 Hz, 1H), 1.54 (d, J = 6.1 Hz, 9H), 1.48 (s, 9H); 13C NMR (176 MHz, Deuterium Oxide) δ 173.89, 172.53, 83.03, 59.68, 53.40, 47.53, 46.67, 31.07, 27.23; HRMS: C19H35N308 calculated for [M+H]+ 434.2504, found [M+H]+ 434.2483 .
(S)- 2-(((S)- 2-(((S)- 2-[2-(2-Amino-2-carboxy-ethylamino)-2-carboxy-ethylamino]- pentanedioic acid (LLL-38b)
[00237] Deprotection of tert-butyl ester groups was carried out as described in Example 1. The pure final product LLL-38b was isolated by precipitation from water (1 ml), methanol (3 ml) and acetic acid (0.04 ml). Yield: 69%; 1H NMR (700 MHz, Deuterium Oxide) δ 3.85 (q, J = 4.4, 3.7 Hz, 1H), 3.67 (t, J = 5.7 Hz, 1H), 3.39 (dd, J = 10.1, 3.8 Hz, 1H), 3.30 (dd, J= 12.9, 3.7 Hz, 1H), 3.24 (dd, J= 13.6, 6.3 Hz, 1H), 3.03 (t, J= 11.5 Hz, 1H), 2.94 - 2.87 (m, 1HDDL), 2.46 - 2.38 (m, 2H), 2.16 - 2.07 (m, 2H); 13C NMR (176 MHz, Deuterium Oxide) δ 181.53, 177.45, 173.82, 173.16, 62.68, 60.46, 54.56, 48.42, 47.08, 33.89, 25.95; HRMS: CiiHi9N308 calculated for [M+H]+ 322.1252, found [M+H]+ 322.1238.
Example 9: Natural-AM-A (NMR data)
[00238] 1H NMR (700 MHz, 0.1 M NH4OH in Deuterium Oxide ) 3.92 - 3.84 (m,
2H), 3.46 (dd, J = 9.7, 4.5 Hz, 1H), 3.32 (ddd, J = 16.9, 13.2, 5.1 Hz, 2H), 3.19 (dd, J = 12.9, 9.6 Hz, 2H), 2.92 (dd, J = 13.5, 4.0 Hz, 1H), 2.86 (dd, J = 17.6, 3.7 Hz, 1H), 2.73 (dd, J = 17.6, 9.3 Hz, 1H), 2.72 - 2.67 (m, 1H). 13C NMR (176 MHz, 0.1 M NH4OH in Deuterium Oxide) δ 178.62, 61.40, 60.27, 54.73, 48.69, 48.37, 38.59. [a]20 D -48 (c 1.00, 0.1M NH4OH in H20). 2D NMR data confirms the structure of natural AMA-A.1H NMR data for the L,L,L; L,D,D; L,D,L; and L,L,D stereoisomers are summarized in Table 1 and the corresponding C data is shown in Table 2. Optical rotation data for natural and synthetic AM-A molecules is shown in Table 3.
Example 10: Anhydro-(L, L, L)-AM-A (NMR data)
[00239] Following the procedure used for LL-26e, compound LLL-27e (56 mg,
0.11 mmol) was fully deprotected to produce LLL-AM-A in 79 % yield (27 mg). The compound showed identical physical and spectroscopic properties to those reported above. 1H NMR (700 MHz, Deuterium oxide) δ 4.24 - 4.19 (m, 1H), 3.87 - 3.81 (m, 1H), 3.67 (dd, J = 7.1, 3.8 Hz, 1H), 3.44 (dd, J = 13.8, 5.4 Hz, 1H), 3.18 (dd, J = 14.6, 4.7 Hz, 1H), 3.03 (dd, J = 13.8, 6.8 Hz, 1H), 2.79 (dd, J = 16.5, 7.1 Hz, 1H), 2.61 (dd, J = 16.5, 3.8 Hz, 1H). 13C NMR (176 MHz, Deuterium Oxide) δ 179.03, 176.61, 174.32, 62.62, 55.21, 53.63, 48.46, 43.60, 39.59.
Example 11: Anhydro-(L, D, D)-AM-A (NMR data)
[00240] 1H NMR (700 MHz, Deuterium Oxide) δ 4.14 (ddd, J = 13.9, 8.1, 2.4 Hz,
1H), 4.04 (dt, J = 4.6, 2.2 Hz, 1H), 3.88 (dt, J = 9.3, 3.2 Hz, 1H), 3.59 - 3.53 (m, 1H), 3.39 - 3.26 (m, 2H), 2.91 (ddd, J = 15.7, 4.3, 2.0 Hz, 1H), 2.83 (ddd, J = 13.8, 7.0, 2.0 Hz, 1H), 2.39 (ddd, J = 15.8, 9.5, 2.5 Hz, 1H). 13C NMR (176 MHz, Deuterium Oxide) δ 179.7, 179.1, 176.7, 172.4, 61.97, 56.39, 53.87, 51.51, 44.61, 40.03.
Example 12: (R)-2-(((S)-2-(((S)-2-amino-2-carboxyethyl)amino)-2- carboxyethyl)amino)succinic acid (DLL-AM-A)
[00241] Following the procedure used for LLL-AM-A, compound DLL-271 (20 mg, 0.11 mmol) was fully deprotected to produce DLL-AMA in 67 % yield (6 mg). 1H NMR (700 MHz, Deuterium Oxide) δ 3.69 (dd, 7 = 7.2, 4.1 Hz, 1H), 3.64 (dd, 7 = 8.7, 4.3 Hz, 1H), 3.34 (dd, 7 = 9.2, 4.3 Hz, 1H), 3.18 (dd, 7 = 12.5, 4.3 Hz, 1H), 3.08 (dd, 7 = 13.1, 7.3 Hz, 1H), 2.92 - 2.81 (m, 2H), 2.72 (dd, 7 = 16.6, 4.3 Hz, 1H), 2.57 (dd, 7 = 16.6, 8.7 Hz, 1H). 13C NMR (176 MHz, Deuterium Oxide) δ 178.57, 178.34, 61.48, 60.48, 54.85, 48.73, 48.65, 37.74. HRMS (ES) for Ο10Η18Ν3Ο8 calculated 308.1094 [M + H]+, found 308.1093 [M + H]+. Example 13: (S)-2-(((S)-2-carboxy-2-((carboxymethyl)amino)ethyl)amino) succinic acid (LL- 25i, LL-AM-B)
[00242] Following the procedure used for LL-26e, compound LL-25h (70 mg,
0.14 mmol) was fully deprotected to produce LL-25i or JJ-AM-B in 66 % yield (25mg). 1H NMR (700 MHz, D20): δ 3.72(m, 2H), 3.29 (dd, J = 9.8 Hz, J = 4.2 Hz, 1H), 3.16 (dd, J = 9.5 Hz, J = 4.0 Hz, 1H), 3.13 (dd, J = 13.7 Hz, J = 5.9 Hz, 1H), 3.00 (m, 1H), 2.76 (dd, J= 13.4 Hz, J= 3.9 Hz, 1H), 2.69 (dd, J= 17.5 Hz, J= 3.5 Hz, 1H), 2.56 (dd, J = 17.5 Hz, J = 9.4 Hz, 1H); 13C NMR (176 MHz, D20): δ 177.5, 177.4, 173.6, 173.3, 59.9, 59.4, 54.6, 48.0, 47.1, 36.0; HRMS (ES) for C9Hi4N208 calculated 279.0828 [M +H]+, found 279.0814 [M +H]+; [a]D 2° -28 (c 0.19, 0.1 N NH4OH).
Example 14: Biological Assays of AM-A and Derivatives
[00243] AM-A, derivatives and analogues synthesized according to Examples 1 to 13 were explored for their biological activity through dose-response assays using purified recombinant NDM-1 as well as cell-based assays to assess in vivo synergy with meroprenem in an NDM-1 expressing strain of E.coli.
Structure-Activity Relation (SAR) of AM-A and Derivatives in NDM-1 Inhibition Through Dose-Response Enzyme Assay
[00244] NDM-1 (final concentration: 4 nM) was incubated with AM-A compounds in serial 1/2 dilutions from a maximum concentration of 256 μΜ for 10 minutes at 37°C. The buffer used was 50 mM HEPES pH 7.5 supplemented with 50 ng/μΤ bovine serum albumin and 10 μΜ ZnS04 for enzyme stability. Tween20 was also added to a final concentration of 0.01% to prevent spurious inhibition due to compound insolubility. Addition of substrate (nitrocefin, final concentration 30 μΜ) initiated reaction. Progress curves of hydrolysis at 37°C were monitored at 490 nm in 96-well microplate format using a Spectramax reader (Molecular Devices). IC50 curves were generated using GraFit version 4.0.10 and a 4-parameter fit. The results are shown in Table 4, column 2. Cell-based Potentiation of Meropenem
24
[00245] Rescue concentration (RC) values were determined by standard methods setting up checkerboards with 8 concentrations of each meropenem and AM-A derivatives in serial ½ dilutions. RC is the concentration of compound needed to fully regain the activity of meropenem at the clinical breakpoint of 1 μg/mL (-2.5 μΜ). The results are shown in Table 4, column 3.
[00246] AM-A can be depicted as follows with the three a-carbons labeled as positions 3, 6, and 9, and the three amino acid units as Asp, APA1 and APA2:
Figure imgf000078_0001
I I I II I
Asp APA1 APA2
[00247] Altering the stereochemistry at positions 3, 6, and 9 had minimal effect on in vitro DM-1 inactivation and isomers retained synergy with meropenem though with some reduced efficiency. The stereochemistry of the a-carbon of Asp at position 3 is also flexible with no significant influence on activity. Therefore, there is tolerance in the stereochemistry at positions 3, 6, and 9, reflecting significant plasticity in biological activity.
[00248] NDM-1 IC50 curves for JJ-AM-B (LL-25i), where APA2 is replaced by Gly, and lycomarasmine (LL-33c), where APA2 is replaced by Gly-amide, exhibited complete enzyme inactivation, although the latter had only marginal activity in cells. The N-terminal amine is therefore dispensable. However protection with the bulky nosyl group weakens enzyme inhibition activity and in-cell activity. Alternate spacing of the C- terminal carboxyl (C2 of AM-B vs. C3 AM-A) is accommodated with retention of activity, nevertheless, a free carboxyl rather than an amide is optimal for activity in the cell, perhaps reflecting access and transport to the periplasm.
[00249] Complete removal of APA2 (toxin A, ZX-26e) has an impact on both in vitro enzyme activity and synergy with meropenem in cells. Unlike compounds with a Gly or APA equivalent unit in the APA2 position, toxin A showed unusual behavior in enzyme inhibition studies. Rather than precipitous dose dependence from full activity to inactivation, toxin A IC50 curves showed a shallow inhibition gradient and did not result in complete loss of enzyme action. Furthermore, toxin A had no ability to rescue meropenem activity in cell potentiation assays. The APA2 unit is therefore optimal for AM-A activity, but is tolerant of modification. Protection of the carboxylates of toxin A (compounds ZX-30a and LL-26d) or replacement of N-terminal Asp with Glu abolishes activity.
[00250] The role of the Asp position was explored by changing Asp to Glu (Compound ZX-38b). This analogue retained good in vitro enzyme inhibition activity, but lacked the ability to potentiate meropenem in cells.
[00251] Compounds with good enzyme inhibition activity but little cell-based activity can still represent viable drug candidates. Structural modifications and/or formulation modifications to improve cell permeation are known in the art and can be readily applied here. For example, the compounds can be modified by conjugation with fatty acids, vitamins (e.g. folates), single chain antigen, binding molecules, cell penetrating peptides, nanoparticles, antibodies and/or protein. Alternatively, or in addition, the compounds can be encapsulated within drug delivery vehicles, such as liposomes, micelles, dendrimers or polyethylene glycol carriers.
[00252] While the present application has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
[00253] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term. Table 1. 1H NMR data for Natural AM-A and the L,L,L; L,D,D; L,D,L; and L,L,D stereoisomers
Figure imgf000080_0001
10
Figure imgf000080_0002
Table 2. C- Carbon Data for Natural AM-A and the L,L,L; L,D,D; L,D,L; and L,L,D stereoisomers
Figure imgf000081_0001
Table 3. Optical Rotation data for Natural and Synthetic AM-A molecules
Figure imgf000082_0001
1) Rudolph Research Analytical Autopol III Automatic Polanmeter; 2)Perkin Elmer Polarimeter
Table 4. Concentration Response and Fractional Inhibitory Concentration Index Values for Sel
Figure imgf000083_0001
Figure imgf000084_0001
a IC50 measurements required < 20 % residual activity. b RC= Rescue Concentration is the concentration of compound needed to fully regain the activity of meropenem at the clinical breakpoint (2.5 μΜ). Not active (NA) indicates no inhibition or synergy observed at given concentrations. NT = not tested. Ns = 2- nitrobenzenesulfonyl.
REFERENCES
1. Robert, M., Barbier, M., Lederer, E., Roux, L., Biemann, K., Vetter, W. Two new natural phytotoxins: aspergillomarasmine A and B and their relation to lycomarasmine and its derivatives. Bulletin de la Societe Chimique de France 187-188 (1962).
2. Haenni, A.L. et al. Structure chimique des aspergillomarasmines A et B, Helv.
Chim. Acta 48, 729-750 (1965).
3. Mikami, Y., Suzuki, T. Novel microbial inhibitors of angiotensin-converting enzyme, aspergillomarasmine A and B. Agric. Biol. Chem. 47, 2693-2695 (1983).
4. Arai, K. et al. Aspergillomarasmine A and B, potent microbial inhibitors of
endothelin-converting enzyme. Biosci. Biotech. Biochem. 57, 1944 (1993).
5. Arai, Y., Nakakita, Y., Munakata, M., Ashizawa, N., Matsura, A.
Aspergillomarasmines as endothelin-converting enzyme inhibitors for treatment of hyperendothelinemia. JP 06192081, (1994).
6. Matsuura, A., et al. Pharmacological profiles of aspergillomarasmines as
endothelin-converting enzyme inhibitors. Jap. J. Pharmacol. 63, 187-193 (1993).
7. King, A.M., Reid-Yu, S.A., Wang, W., King, D.T., De Pascale, G., Strynadka, N.C., Walsh, T.R., Coombes, B.K., Wright, G.D. Aspergillomarasmine A overcomes metallo-P-lactamase antibiotic resistance. Nature. 510, 503-506, (2014).
8. Weiergang, I, Lyngs Jorgenson, H.J., Moller, I.M., Friis P., Smedegaard- Peterson, V. Optimization of in vitro growth conditions of Pyrenophora teres for production of the phytotoxin aspergillomarasmine A. Physiologicla and
Molecular Plant Pathology. 60, 131-130, (2002).
9. Wagman, G.H., Cooper, R. Natural products isolation: separation methods for antimicrobials, antivirals and enzyme inhibitors. Elsevier, p 499, (1988).
10. Lahlou, M. The success of natural product in drug discovery. Pharmacology and Pharmacy, 4, 17-31, (2013).
11. Hanessian, S. Reflections on the total synthesis of natural products: art, craft, logic, and the chiron approach. Pure & Appl. Chem. 65, 1189-1204, (1993). Ohfune, Y., Kurokawa, N. Synthesis of the serine equivalent, (2R) and (2S)- amino-3-butenol derivatives. Synthetic approaches to the metal chelating poly- amino acid, "Aspergillomarasmine A". Tet. Lett, 25, 1071-1074, (1984).
O'Brien, K. ό. P., K.; Kelleher, F., Studies on the Synthesis of Orthogonally Protected Azalanthionines and Routes towards β-Methyl Azalanthionines by ring opening of N-activated aziridine-2-carboxylates. Tetrahedron 2014, 70, 5082- 5092.
Zwanenburg, B. e. a., Synthesis and crystal structure of enantiopure N-trytyl-2-yl- methanols from L-serine and L-threonine. J. Chem. Soc, Perkin Trans.1 1997, 963-967.
Olsen, C. A. e. a., Aminolysis of Resin-Bound N-Nosylaziridine-2-carboxylic Acids. Organic Letters 2006, 8 (15), 3371-3374.
Turner, J. J. e. a., Synthesis of β-Lactams via Ring Opening of a Serine Derived Aziridine. Synlett 2001, J J, 1727-1730.
Konno, K. e. a., 2-Nitro- and 2,4-Dinitrobenzenesulfonamides as Protecting groups for Primary Amines. Synlett 2001, 7, 1 167-1 169.
Nicolaou, K. C; Estrada, A. A.; Zak, M.; Lee, S. H.; Safina, B. S., A mild and selective method for the hydrolysis of esters with trimethyltin hydroxide. Angew Chem Int Ed Engl 2005, 44 (9), 1378-82.
King, A. M.; Reid-Yu, S. A.; Wang, W.; King, D. T.; De Pascale, G.; Strynadka, N. C; Walsh, T. R.; Coombes, B. K.; Wright, G. D. Nature 2014, 510, 503.
Crestey F.; Witt, M.; Frydenvang, K.; Staerk, D.; Jaroszewski, J.W.; Franzyk, H. Microwave-Assisted Ring Opening of Activated Aziridines with Resin-Bound Amines, J. Org. Chem. 2008, 73, 3566-3569.
Rashid Baig, N. B.; Chandrakala, R. N.; Sudhir, V. S.; Chandrasekaran, S., Synthesis of Unnatural Selenocystines and β-Aminodiselenides via Regioselective Ring-Opening of Sulfamidates Using a Sequential, One-Pot, Multistep Strategy. J. Org. Chem. 2010, 75 (9), 2910-2921.
Koteva, K.; King, A. M.; Capretta, A.; Wright, G. D., Total Synthesis and Activity of the Metallo-P-lactamase Inhibitor Aspergillomarasmine A. Angew. Chem. Int. Ed. 2016, 55 (6), 2210-2212. Liao, D.; Yang, S.; Wang, J.; Zhang, J.; Hong, B.; Wu, F.; Lei, X., Total Synthesis and Structural Reassignment of Aspergillomarasmine A. Angew. Chem. Int. Ed. 2015, 54, 1-6.
Pillai, S. K.; Moellenng Jr, R. C; Eliopoulos, G. M., Antimicrobial combinations. In Antibiotics in Laboratory Medicine, Lorian, V., Ed. Williams & Wilkins: Philadelphia, PA, 2005; pp 365-440.
a) Scott, R.; Crabbe, D; et al. Expert Opin. Drug Deli, 2008, 5, 549. b) Saltzman, W.; Torchilin, V. Drug delivery systems, Access Science. McGraw-Hill Co., 2008.

Claims

1. A process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof:
Figure imgf000088_0001
(I)
the process comprising: a) reacting a compound of Formula (II) with a compound of Formula (III) under conditions to provide a compound of Formula (IV):
Figure imgf000088_0002
(Π) (III) (IV) b) treating the compound of Formula (IV) with a suitable deprotecting agent under conditions to provide a compound of Formula (V):
Figure imgf000088_0003
(V) c) reacting the compound of Formula (V) with a compound of Formula (III) under conditions to provide a compound of Formula (VI):
Figure imgf000088_0004
(V) (III) (VI) and d) deprotecting the compound of Formula (VI) under conditions to provide the compound of Formula (I), wherein n is 1 or 2;
R1 is a direct bond or 0-S02; and
1 2
PG1 and PG are suitable protecting groups.
2. The process of claim 1, wherein the compound of Formula (III) is a compound of Formula (Ilia) or a compound of Formula (Illb):
Figure imgf000089_0001
(Ilia) (Illb)
or wherein
PG1 is a suitable protecting group for carboxylic acids; and PG is a suitable protecting and/or activating group for amines.
3. The process of claim 1 or 2, wherein PG1 is selected from methyl, ethyl, t-butyl, benzyl, trialkylsilyl and triarylsilyl.
4. The process of any one of claims 1 to 3, wherein PG is selected from t-Boc, Fmoc, Bn, benzoyl, Cbz, Pmb, o-nosyl, p-nosyl and trityl.
5. The process of claim 1 or 2, wherein PG is an activating group which activates the carbon adjacent to the N for nucleophilic attack.
6. The process of any one of claims 1 to 5, wherein the conditions to provide the compound of Formula (IV) and the compound of Formula (VI) comprise basic conditions.
7. The process of claim 6, wherein the basic conditions are provided by an organic non-nucleophilic base.
8 The process of claim 7, wherein the organic non-nucleophilic base is a trialkylamine.
9. The process of claim 8, wherein the trialkylamine is triethylamine.
10. The process of any one of claims 1 to 9, wherein the conditions to provide the compound of Formula (IV) and the compound of Formula (VI) further comprise combining the compound of Formula (II) and the compound of Formula (V) with the compounds of Formula (Ilia) or (Illb) in the presence of a soluble salt.
11. The process of any one of claims 1 to 10, wherein the soluble salt is monopotassium phosphate or sodium phosphate.
12. The process of any one of claims 1 to 11, wherein the suitable deprotecting agent to provide the compound of Formula (V) selectively removes the protecting group PG over PG1.
13. The process of any one of claims 1 to 12, further comprising reacting the compound of Formula (I) with an esterification agent or amidation agent.
14. The process of claim 1, wherein the compound of Formula (I) or esters, amides, salts and/or solvates thereof is prepared on a solid support.
15. The process of any one of claims 1 to 14, wherein the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000090_0001
(I) (I)
16. The process of any one of claims 1 to 14, wherein the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000091_0001
(I)
17. A process for the preparation of a compound of Formula (I) or esters, amides, salts and/or solvates thereof, the process comprising: a) reacting the compound of Formula (II) with a compound of Formula (VII) under conditions to provide a compound of Formula VIII):
Figure imgf000091_0002
(II) (VII) (VIII)
b) treating the compound of Formula (VIII) with a suitable deprotecting agent under conditions to provide a compound of Formula (IX):
Figure imgf000091_0003
(ix) c) reacting the compound of Formula (IX) with the compound of Formula (VII) under conditions to provide a compound of Formula (X):
Figure imgf000091_0004
(ix) (vii) (x) d) oxidizing the compound of Formula (X) under conditions to provide a compound of Formula (XI):
Figure imgf000092_0001
(XI) and e) deprotecting the compound of Formula (XI) under conditions to provide the compound of Formula (I), wherein n is 1 or 2;
1 2
PG and PG are suitable protecting groups.
18. The process of claim 17, wherein the conditions to provide the compound of Formula (VIII) and the compound of Formula (X) comprise reductive amination.
19. The process of claim 17 or 18, wherein the oxidizing of the compound of Formula (X) is performed using an oxidant selected from chromium trioxide, potassium permanganate, pyridinium dichromate and ruthenium tetroxide.
20. The process of claim 19, wherein the oxidant is chromium trioxide.
21. The process of any one of claims 17 to 20, wherein PG1 is selected from methyl, ethyl, t-butyl, benzyl, trialkylsilyl and triarylsilyl.
22. The process of any one of claims 17 to 21, wherein PG is selected from t-Boc, Fmoc, Bn, benzoyl, Cbz, Pmb, o-nosyl, p-nosyl and trityl.
23. The process of any one of claims 17 to 22, wherein the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000092_0002
(I).
24. The process of any one of claims 17 to 22, wherein the compound of Formula (I) has the following relative stereochemistry:
Figure imgf000093_0001
(I)-
25. A compound of Formula A, or a pharmaceutically acceptable salt and/or solvate thereof;
Figure imgf000093_0002
A
wherein:
R2 is selected from H, S02Ar, CH2CH(C02R6)NHR7, CH2C02R6, CH2C(0)NHR7 and Ci. 6alkyl;
3 5
R -R are independently selected from H and C1-6alkyl;
R6 and R7 are independently selected from H, C1-6alkyl and S02Ar; n is 1 or 2,
Ar is aryl that is unsubstituted or substituted with one or more of halo, N02,
Figure imgf000093_0003
4alkyl and C02C1-4alkyl; and each alkyl group is optionally fluoro-substituted, provided that when n is 1, R2, R3, R4, R5, R6 and R7 are not all H.
26. The compound of claim 25, wherein R2 is selected from CH2CH(C02R6)NHR7, CH2C02R6 and CH2C(0)NHR7, in which R6 and R7 are independently selected from H, C1-4alkyl and S02Ar, and aryl that is unsubstituted or substituted with N02.
27. The compound of claim 25 or 26, wherein R 3 -R 5 are independently selected H and C1-4alkyl.
28. The compound of any one of claims 25 to 27, wherein n is 2.
29. The compound of claim 25 selected from:
Figure imgf000094_0001
C02H
Figure imgf000095_0001
30. A pharmaceutical composition comprising one of more compounds of any one of claims 25 to 29 and a pharmaceutically acceptable carrier.
31. A method of improving the efficacy of a B-lactam antibiotic for treating a disease, disorder or condition arising from a bacterial infection comprising administering, to a subject in need thereof, an effective amount of one or more compounds of Formula A of any one of claims 25 to 29, or a pharmaceutically acceptable salt and/or solvate thereof.
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