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WO2025224308A1 - Qpctl inhibitors - Google Patents

Qpctl inhibitors

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Publication number
WO2025224308A1
WO2025224308A1 PCT/EP2025/061346 EP2025061346W WO2025224308A1 WO 2025224308 A1 WO2025224308 A1 WO 2025224308A1 EP 2025061346 W EP2025061346 W EP 2025061346W WO 2025224308 A1 WO2025224308 A1 WO 2025224308A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
mixture
μmol
residue
give
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/061346
Other languages
French (fr)
Inventor
Bastiaan EVERS
Marcel SCHEEPSTRA
Gavin Alistair Whitlock
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scenic Immunology BV
Original Assignee
Scenic Immunology BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scenic Immunology BV filed Critical Scenic Immunology BV
Publication of WO2025224308A1 publication Critical patent/WO2025224308A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • QPCTL INHIBITORS FIELD OF THE INVENTION The current invention relates to QPCTL inhibitors represented by formula (I), and corresponding compositions and uses.
  • the inhibitors and compositions are for use in the treatment of cancer, neurodegenerative diseases such as Alzheimer's disease, synucleinopathies, Huntington’s disease, bacterial infections such as periodontitis and related disorders, and inflammatory diseases.
  • QPCTL glycosyl-peptide cyclotransferase-like protein
  • enzymes belong to the family of aminoacyltransferases.
  • QPCTL enzymes catalyze the cyclization of N-terminal glutamine and glutamic acid residues in peptides into an N-terminal pyroglutamate residues. These proteins have been identified as clinically relevant, for example for their role in treating tumors (Yu, L., Zhao, P., Sun, Y. et al. Development of a potent benzonitrile-based inhibitor of glutaminyl-peptide cyclotransferase-like protein (QPCTL) with antitumor efficacy. Sig Transduct Target Ther 8, 454 (2023)). Nonetheless, a few potent and/or selective QPCTL inhibitors have been identified to the best of our knowledge.
  • One aspect of the invention pertains to certain N or C-substituted-3,4-(fused ring)- heteroaryl or heterocyclic compounds (also referred to herein as" QPCTL inhibitors"), as described herein.
  • Another aspect of the invention pertains to a composition (e.g., a pharmaceutical composition) comprising a QPCTL inhibitor, as described herein, and a pharmaceutically acceptable carrier or diluent.
  • Another aspect of the present invention pertains to use in a treatment of a disorder of the human or animal body associated with abnormal QPCTL enzyme activity, comprising administering to a subject in need of treatment a therapeutically effective amount of compound of formula I or a hydrate, solvate, or pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention pertains to use in the inhibition of QPCTL activity in a subject, comprising administering to said subject an effective amount of compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention pertains to use in the inhibition of QPCTL protein/activity comprising contacting the QPCTL protein/enzyme, in vitro or in vivo, with an effective amount of a compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention pertains to use in the inhibition of QPCTL protein/activity in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention pertains to use in a method of treatment of the human or animal body by therapy.
  • Another aspect of the present invention pertains to its use in a method of treating diseases or disorders associated with abnormal QPCTL activity or QPCTL protein.
  • Another aspect of the present invention pertains for use in the preparation of a medicament for treating conditions associated with abnormal QPCTL activity.
  • Another aspect of the present invention pertains for use in the manufacture of a medicament for the treatment of a disorder or disease of the human or animal body that is associated by the abnormal QPCTL activity.
  • Another aspect of the present invention pertains to a kit comprising (a) a compound of formula I, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound.
  • Another aspect of the present invention pertains to a compound of formula I obtainable by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein. Another aspect of the present invention pertains to a compound of formula I obtained by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein. Another aspect of the present invention pertains to novel intermediates, as described herein, which are suitable for use in the methods of synthesis described herein. Another aspect of the present invention pertains to the use of such novel intermediates, as described herein, in the methods of synthesis described herein. As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspects of the invention.
  • the invention provides a QPCTL inhibitor represented by compound of formula I : or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein: R1 is independently selected from a group consisting of hydrogen, methyl, carboxylic acid, -C(O)OCH3, difluoropyrrolidinyl, phenyl, dimethylamino, methylamino, -NH2, cyano, tetrazolyl, 5-oxo-1,2,4- oxadiazolinyl, trifluoromethyl, and oxygen; R 1b represents Ring ‘A’ is selected from a group consisting of 6-membered aryl and heteroaryl ring; Ring ‘B’ is selected from a group consisting of 6-membered aryl, heteroaryl ring and 5- membered heteroaryl ring; Ring ‘C’ represents a saturated or unsaturated ring system; X is independently selected from a group consisting of
  • the Ring A is selected from a group consisting of pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and phenyl; and the Ring B is selected from a group consisting of imidazolyl, pyridyl, isoxazolyl, triazolyl, thiazolyl, triazolyl, and pyrazolyl.
  • the invention provides compound of formula Ia : or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein R1 is independently selected from a group consisting of hydrogen, methyl, carboxylic acid, -C(O)OCH3, difluoropyrrolidinyl, phenyl, dimethylamino, methylamino, -NH 2 , cyano, tetrazolyl, 5-oxo-1,2,4- oxadiazolinyl, trifluoromethyl, oxygen; R 1b represents or Ring ‘A’ is selected from a group consisting of pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and phenyl; Ring ‘B’ is selected from a group consisting of imidazolyl, pyridyl, isoxazolyl, triazolyl, thiazolyl, triazolyl, and pyrazolyl; Ring ‘C’ represents a
  • R 1 is independently either Carboxylic acid or -C(O)OCH 3 ; Ring ‘A
  • stereoisomers are disclosed and encompassed, both individually (e.g., as isolated from the other stereoisomer(s)) and as mixtures (e.g., as equimolar or non-equimolar mixtures of two or more stereoisomers).
  • each of the (R) and (S) enantiomers are disclosed and encompassed, both individually (e.g., as isolated from the other enantiomer) and as a mixture (e.g., as equimolar or non-equimolar mixtures of the two enantiomers).
  • Ring A, Ring B, Ring C, and any substituents thereon Ring A, Ring B, and Ring C may be susceptible to tautomerism.
  • both tautomers are disclosed and encompassed, both individually (e.g., as isolated from the other tautomer) and as mixtures (e.g., as equimolar or non-equimolar mixtures of two tautomer).
  • the compound is of formula II:
  • compound of one of the following formulae 1-119, or a pharmaceutically acceptable salt, hydrate, or solvate thereof is of one of the following formulae 1-45, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
  • compositions in a further aspect, provides a composition comprising an inhibitor according to the invention and a pharmaceutically acceptable excipient, preferably for use as a medicament.
  • a related aspect pertains to a method of preparing such a composition. Such compositions are referred to in the current application as compositions according to or of the invention. All specific embodiments disclosed above for an inhibitor according to the invention may be applied accordingly for an inhibitor according to the invention comprised in a composition according to the invention.
  • the invention provides a pharmaceutical composition comprising a compound of formula I or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula Ia or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient.
  • Formula Ia wherein R, R 1 , R 1a , R 1b , q, X, Q, n, m, p, R 2 , R 3 , R 5 , R 6 , W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ia in above;
  • the invention provides a pharmaceutical composition comprising a compound of formula Ib or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula Ic or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula Id or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient.
  • a composition according to the invention may be presented or formulated as capsules, tablets, powders, granules, solutions, suspensions in aqueous or non-aqueous liquids, edible, oil-in-water liquid emulsions, water- in-oil liquid emulsions, solution, syrups and elixirs, in microencapsulated form, liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles, transdermal patches, ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, drops, sprays, aerosols, oils, lozenges, pastilles, mouth washes, suppositories, enemas, aqueous and non-aqueous sterile injection solutions, and so on.
  • liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multi
  • compositions may include other agents conventional in the art having regard to the type of formulation.
  • a pharmaceutically acceptable carrier comprised in a composition are saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Supplementary active compounds, besides an inhibitor according to the invention, can also be incorporated into the compositions.
  • a composition according to the invention formulated as solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists.
  • prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • a composition according to the invention formulated as oral compositions generally include an inert diluent or an edible carrier.
  • the inhibitor according to the invention can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a composition according to the invention may be formulated for administration by inhalation, the inhibitor according to the invention can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a composition according to the invention may be formulated for transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • transdermal administration may be performed by iontophoresis.
  • a composition according to the invention may comprise a carrier system such as a colloidal system.
  • the colloidal system can be a liposome, a phospholipid bilayer vehicle.
  • the inhibitor according to the invention is encapsulated in a liposome.
  • An inhibitor according to the invention can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes.
  • Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.
  • the present invention pertains to QPCTL inhibitors, as described herein, in substantially purified form and/or in a form substantially free from contaminants.
  • the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.
  • the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form.
  • the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds.
  • the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to a equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.
  • the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight.
  • the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.
  • the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure.
  • 60% optically pure i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer
  • at least 70% optically pure e.g., at least 80% optically pure, e.g., at least 90% optically pure, e
  • Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereoisomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso- forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and b-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as“isomers” (or“isomeric forms”).
  • Keto-Enol Tautomerism Note that specifically included in the term“isomer” are compounds with one or more isotopic substitutions.
  • H may be in any isotopic form, including 1 H, 2 H (D), and 3 H (T);
  • C may be in any isotopic form, including 12 C, 13 C, and 14 C;
  • O may be in any isotopic form, including 16 O and 18 O; and the like.
  • a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof.
  • Salts It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977,“Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol.66, pp.1 -19.
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ as well as the ammonium ion (i.e., NH 4 + ).
  • Suitable organic cations include, but are not limited to substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ), for example, where each R is independently linear or branched saturatedC1-18 alkyl, C3-8cycloalkyl, C3-8cycloalkyl-C1-6alkyl, and phenyl-C1- 6alkyl, wherein the phenyl group is optionally substituted.
  • substituted ammonium ions e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 +
  • each R is independently linear or branched saturatedC1-18 alkyl, C3-8cycloalkyl, C3-8cycloalkyl-C1-6alkyl, and phenyl-C1- 6alkyl, wherein the phenyl group is optionally substituted.
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH3)4 + .
  • a salt may be formed with a suitable anion.
  • a parent structure contains a cationic group (e.g., -NMe2 + ), or has a functional group, which upon protonation may become cationic (e.g., -NH 2 may become -NH 3 + )
  • a salt may be formed with a suitable anion.
  • a counter-anion is generally always present in order to balance the positive charge.
  • an inner salt also referred to as a zwitterion
  • a zwitterion an inner salt
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic, acetic, trifluoroacetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, 1,2-ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and va
  • Suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Unless otherwise specified, a reference to a particular compound also includes salt forms thereof. Solvates and Hydrates It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compound.
  • a QPCTL inhibitor according to the invention may be present as a pharmaceutically acceptable solvate or hydrate.
  • solvate is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent.
  • the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
  • a reference to a particular compound also includes solvate and hydrate forms thereof.
  • Chemically Protected Forms It may be convenient or desirable to prepare, purify, and/or handle the compound in a chemically protected form.
  • the term “chemically protected form” is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, reactive chemical reagents, and the like).
  • one or more reactive functional groups are in the form of a protected or protecting group (alternatively as a masked or masking group or a blocked or blocking group).
  • a protected or protecting group By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed or the masking group transformed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts; 4th Edition; John Wiley and Sons, 2006).
  • a wide variety of such “protecting,” “blocking,” or “masking” methods are widely used and well known in organic synthesis.
  • a compound which has two nonequivalent reactive functional groups both of which would be reactive under specified conditions, may be derivatized to render one of the functional groups “protected,” and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group.
  • the protected group may be “deprotected” to return it to its original functionality.
  • an amine group may be protected, for example, as an amide (-NRCO-R), for example: as an acetamide (-NHCO-CH 3 ); or as a carbamate (-NRCO-OR), for example: as a benzyloxy carbamate (-NHCO- OCH 2 C 6 H 5 , -NH-Cbz), as a t-butoxy carbamate (-NHCO-OC(CH 3 ) 3 , -NH-Boc); as a 2-biphenyl-2-propoxy carbamate (-NHCO-OC(CH3)2C6H4C6H5, -NH-Bpoc), as a 9-fluorenylmethoxy carbamate (-NH-Fmoc), as a 6- nitroveratryloxy carbamate (-NH-Nvoc), as a 2-trimethylsilylethyloxy carbamate (-NH-Teoc), a 2,2,2- trichloroethyloxy carbamate (-
  • Prodrugs It may be convenient or desirable to prepare, purify, and/or handle the compound in the form of a prodrug.
  • the term “prodrug,” as used herein, pertains to a compound, which yields the desired active compound in vivo. Typically, the prodrug is inactive, or less active than the desired active compound, but may provide advantageous handling, administration, or metabolic properties.
  • some prodrugs are activated enzymatically to yield the active compound, or a compound, which, upon further chemical reaction, yields the active compound (for example, as in antibody directed enzyme prodrug therapy (ADEPT), gene directed enzyme prodrug therapy (GDEPT), lipid directed enzyme prodrug therapy (LIDEPT), etc.).
  • the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
  • the invention provides the use of a QPCTL inhibitor according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
  • the binding of QPCTL results in in the inhibition of QPCTL.
  • the inhibition of QPCTL is described in more detail above. All specific embodiments disclosed above for an inhibitor according to the invention and a composition according to the invention may be applied accordingly for the uses and therapeutic uses described below.
  • the invention provides the use of a compound of formula I according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
  • R, R 1 , R 1a , R 1b , q, X, Q, n, m, p, R 2 , R 3 , R 5 , R 6 , W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ia in above;
  • the invention provides the use of a compound of formula Ib according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
  • the invention provides the use of a compound of formula Ic according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
  • the invention provides the use of a compound of formula Id according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
  • a QPCTL inhibitor according to the invention or a composition according to the invention is for specifically binding and/or specifically inhibiting QPCTL and isoQPCTL.
  • the binding or inhibition stems from the amino acid sequence dependent molecular interaction between the inhibitor, or the inhibitor comprised in the composition, and QPCTL or isoQPCTL.
  • the inhibitor or composition is not able to significantly bind or inhibit other enzymes that may share a similar function but a different amino acid structure.
  • the use of a QPCTL inhibitor according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL comprises a QPCTL inhibitor which specifically binds or inhibits QPCTL or isoQPCTL.
  • the inhibition of QPCTL can be expressed as the half maximal inhibitory concentration (IC50), as known to the skilled person. Lower IC 50 values correspond with higher potencies to inhibit QPCTL.
  • IC 50 can be determined using Fluorescent Enzymatic assay protocol of Example 2. Claimable summary of IC 50 are provided in Example 2.
  • a QPCTL inhibitor according to the invention or a composition according to the invention for (specifically) inhibiting isoQPCTL comprises a QPCTL inhibitor having an IC50 for QPCTL equal to or higher than 10 ⁇ m, 9.63 ⁇ m, 8.3 ⁇ m, 6.8 ⁇ m, 3.95 ⁇ m, 3.16 ⁇ m, 2.88 ⁇ m, 1.83 ⁇ m, 1.06 ⁇ m, 1.05 ⁇ m, 0.721 ⁇ m, 0.694 ⁇ m, 5.3 ⁇ m, 0.502 ⁇ m, 442 nm, 440 nm, 434 nm, 417 nm, 394 nm, 365 nm, 362 nm, 342 nm, 324 nm, 294 nm, 249 nm, 240 nm, 231 nm, 220 nm, 215 nm, 154 nm, 153 nm, 151 nm, 135
  • the use of a QPCTL inhibitor according to the invention or a composition according to the invention for (specifically) inhibiting QPCTL comprises a QPCTL inhibitor having an IC 50 for QPCTL equal to or higher than >10 ⁇ m, 9.54 ⁇ m, 2.37 ⁇ m, 0.653 ⁇ m, 1.49 ⁇ m, 0.623 ⁇ m, 451 nm, 142 nm, 103 nm, 70 nm, 66 nm, 65 nm, 61 nm, 57 nm, 52 nm, 49 nm, 48 nm, 42 nm, 38 nm, 34 nm, 31 nm, 30 nm, 25 nm, 23 nm, 22 nm, 18 nm, 17 nm, 16 nm, 14 nm, 10 nm or 1 nm.
  • kits comprising (a) a compound of formula I, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound.
  • a composition e.g., a pharmaceutical composition
  • instructions for use for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound.
  • a disorder e.g., a disease
  • the invention pertains to a kit comprising (a) a compound of formula Ia, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound.
  • a disorder e.g., a disease
  • the invention pertains to a kit comprising (a) a compound of formula Ib, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound.
  • a disorder e.g., a disease
  • the invention pertains to a kit comprising (a) a compound of formula Ic, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound.
  • a disorder e.g., a disease
  • the invention pertains to a kit comprising (a) a compound of formula Id, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound.
  • a disorder e.g., a disease
  • the invention provides a QPCTL inhibitor according to the invention or a composition according to the invention for use as a medicament.
  • a QPCTL inhibitor according to the invention or a composition according to the invention for use as a medicament.
  • all specific embodiments for an inhibitor according to the invention, a composition according to the invention, and the uses presented above may be applied accordingly.
  • an inhibitor or a composition for use as a medicament is disclosed, a corresponding method for the manufacture or the production of a medicament comprising such an inhibitor or such a composition, a corresponding method of treatment comprising the administration of the inhibitor or composition to a subject in need thereof, and a corresponding use of such an inhibitor or such a composition as a medicament are also disclosed.
  • the inhibitor according to the invention and the composition according to the invention may be referred to as a medicament according to the invention.
  • a medicament according to the invention may be administered orally, nasally, buccally, sublingually, vaginally, parenterally, topically, systemically, intravenously, subcutaneously, intraperitoneally, intramuscularly, intrathecally, by inhalation or epidurally.
  • a medicament according to the invention may be administered separately, sequentially or simultaneously in combination with another medicaments.
  • the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
  • the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
  • the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients.
  • treating refers to therapeutic treatment, wherein the object is to prevent, reduce, alleviate or slow down (lessen), respectively and as applicable, the targeted pathologic disorder or disease and/or its progression in a subject.
  • said terms relate to a treatment which has the object of improving one or more symptoms and/or physiological parameters that are caused by, associated with and/or characteristic of the disease or disorder that is to be treated, and/or the object to preventing that such symptom(s) to arise and/or that such symptom(s) or physiological parameter(s) further deteriorate.
  • prevention or "preventing” of a disorder or disease refers to a compound that, in a statistical sample, reduces the occurrence of symptoms of a disorder or disease in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • a medicament according to the invention is administered to a subject in need thereof in an effective amount (i.e., amount that have desired therapeutic effect).
  • an effective amount refers to an amount of an inhibitor according to the invention comprised in said medicament.
  • the dose and dosage regimen will depend upon the degree of the infection in the subject, the characteristics of the particular inhibitor according to the invention, e.g., its therapeutic index, the subject, and the subject's history. Certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of the medicaments according to the invention can include a single treatment or a series of treatments.
  • the compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in a treatment of a disorder of the human or animal body associated with abnormal QPCTL enzyme activity comprising administering to a subject in need of treatment a therapeutically -effective amount of the said compound.
  • the compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in the inhibition of QPCTL activity in a subject comprising administering to said subject an effective amount of said compound.
  • the compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in the inhibition of QPCTL protein/activity comprising contacting the QPCTL protein/enzyme, in vitro or in vivo, with an effective amount of said compound.
  • the invention provides a QPCTL inhibitor according to the invention or a composition according to the invention, for use as a medicament.
  • the QPCTL inhibitor or the composition is for use in the treatment of a disease characterized by lysosomal dysregulation. Below, preferred diseases characterized by lysosomal dysregulation, and other preferred features of the treatment are disclosed.
  • Said diseases include cancer, neurodegenerative diseases such as Alzheimer's disease, synucleinopathies, Huntington’s disease, bacterial infections such as periodontitis and related disorders, and inflammatory diseases.
  • Treatment options Wherever an inhibitor or a composition for use as a medicament is disclosed, a corresponding method for the manufacture or the production of a medicament comprising such an inhibitor or such a composition, a corresponding method of treatment comprising the administration of the inhibitor or composition to a subject in need thereof, and a corresponding use of such an inhibitor or such a composition as a medicament are also disclosed.
  • the inhibitor according to the invention and the composition according to the invention may be referred to as a medicament according to the invention.
  • a medicament according to the invention may be administered orally, nasally, buccally, sublingually, vaginally, parenterally, topically, systemically, intravenously, subcutaneously, intraperitoneally, intramuscularly, intrathecally, by inhalation or epidurally.
  • a medicament according to the invention may be administered separately, sequentially or simultaneously in combination with another medicaments.
  • the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
  • the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
  • sequential therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
  • treating or “treatment” refer to therapeutic treatment, wherein the object is to prevent, reduce, alleviate or slow down (lessen), respectively and as applicable, the targeted pathologic disorder or disease and/or its progression in a subject.
  • said terms relate to a treatment which has the object of improving one or more symptoms and/or physiological parameters that are caused by, associated with and/or characteristic of the disease or disorder that is to be treated, and/or the object to preventing that such symptom(s) to arise and/or that such symptom(s) or physiological parameter(s) further deteriorate.
  • the skilled person and in particular, the treating physician
  • prevention or “preventing” of a disorder or disease refers to a compound that, in a statistical sample, reduces the occurrence of symptoms of a disorder or disease in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • a medicament according to the invention is administered to a subject in need thereof in an effective amount (i.e., amount that have desired therapeutic effect).
  • an effective amount refers to an amount of an inhibitor according to the invention comprised in said medicament.
  • the dose and dosage regimen will depend upon the degree of the infection in the subject, the characteristics of the particular inhibitor according to the invention, e.g., its therapeutic index, the subject, and the subject's history. Certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of the medicaments according to the invention can include a single treatment or a series of treatments. The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.
  • An effective amount of a peptide useful in the methods may be administered to a subject in need thereof by any of a number of well-known methods for administering pharmaceutical compounds.
  • Dosage, toxicity and therapeutic efficacy of a medicament according to the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Medicaments that exhibit high therapeutic indices are preferred.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • compositions in a further aspect, provides a composition comprising an inhibitor according to the invention and a pharmaceutically acceptable excipient, preferably for use as a medicament, more preferably for use in the treatment of a disease characterized by lysosomal dysregulation.
  • a related aspect pertains to a method of preparing such a composition. Such compositions are referred to in the current application as compositions according to or of the invention.
  • a composition according to the invention may be presented or formulated as capsules, tablets, powders, granules, solutions, suspensions in aqueous or non-aqueous liquids, edible, oil-in-water liquid emulsions, water-in-oil liquid emulsions, solution, syrups and elixirs, in microencapsulated form, liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles, transdermal patches, ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, drops, sprays, aerosols, oils, lozenges, pastilles, mouth washes, suppositories, enemas, aqueous and non-aqueous sterile injection solutions, and so on.
  • compositions may include other agents conventional in the art having regard to the type of formulation.
  • a pharmaceutically acceptable carrier comprised in a composition are saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Supplementary active compounds, besides an inhibitor according to the invention, can also be incorporated into the compositions.
  • a composition according to the invention formulated as solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists.
  • prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • a composition according to the invention formulated as oral compositions generally include an inert diluent or an edible carrier.
  • the inhibitor according to the invention can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a composition according to the invention may be formulated for administration by inhalation, the inhibitor according to the invention can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a composition according to the invention may be formulated for transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • transdermal administration may be performed by iontophoresis.
  • a composition according to the invention may comprise a carrier system such as a colloidal system.
  • the colloidal system can be a liposome, a phospholipid bilayer vehicle.
  • the inhibitor according to the invention is encapsulated in a liposome.
  • An inhibitor according to the invention can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes.
  • Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.
  • the invention provides the use of a QPCTL inhibitor according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
  • the binding of QPCTL results in in the inhibition of QPCTL.
  • the inhibition of QPCTL is described in more detail above.
  • a QPCTL inhibitor according to the invention or a composition according to the invention is for specifically binding and/or specifically inhibiting QPCTL.
  • the binding or inhibition stems from the amino acid sequence dependent molecular interaction between the inhibitor, or the inhibitor comprised in the composition, and QPCTL.
  • the inhibitor or composition is not able to significantly bind or inhibit other enzymes that may share a similar function but a different amino acid structure.
  • the use of a QPCTL inhibitor according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL comprises a QPCTL inhibitor which specifically bind and/or specifically inhibit QPCTL.
  • the inhibition of QPCTL can be expressed as the half maximal inhibitory concentration (IC 50 ), as known to the skilled person. Lower IC 50 values correspond with higher potencies to inhibit QPCTL. IC 50 can be determined using the protocol of Example 2.
  • a QPCTL inhibitor according to the invention or a composition according to the invention for (specifically) inhibiting isoQPCTL comprises a QPCTL inhibitor having an IC 50 for QPCTL equal to or higher than 10 ⁇ m, 9.63 ⁇ m, 8.3 ⁇ m, 6.8 ⁇ m, 3.95 ⁇ m, 3.16 ⁇ m, 2.88 ⁇ m, 1.83 ⁇ m, 1.06 ⁇ m, 1.05 ⁇ m, 0.721 ⁇ m, 0.694 ⁇ m, 5.3 ⁇ m, 0.502 ⁇ m, 442 nm, 440 nm, 434 nm, 417 nm, 394 nm, 365 nm, 362 nm, 342 nm, 324 nm, 294 nm, 249 nm, 240 nm, 231 nm, 220 nm, 215 nm, 154 nm, 153 nm, 151 nm, 135
  • the use of a QPCTL inhibitor according to the invention or a composition according to the invention for (specifically) inhibiting QPCTL comprises a QPCTL inhibitor having an IC50 for QPCTL equal to or higher than >10 ⁇ m, 9.54 ⁇ m, 2.37 ⁇ m, 0.653 ⁇ m, 1.49 ⁇ m, 0.623 ⁇ m, 451 nm, 142 nm, 103 nm, 70 nm, 66 nm, 65 nm, 61 nm, 57 nm, 52 nm, 49 nm, 48 nm, 42 nm, 38 nm, 34 nm, 31 nm, 30 nm, 25 nm, 23 nm, 22 nm, 18 nm, 17 nm, 16 nm, 14 nm, 10 nm or 1 nm, preferably as measured by the Fluorescent Enzymatic assay as described herein.
  • the uses above are for use in vitro. In embodiments, the uses above are for use in vivo.
  • the effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.
  • An effective amount of a peptide useful in the methods may be administered to a subject in need thereof by any of a number of well-known methods for administering pharmaceutical compounds.
  • Dosage, toxicity and therapeutic efficacy of a medicament according to the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Medicaments that exhibit high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays. Definitions All documents cited in the present specification are hereby incorporated by reference in their entirety.
  • physiological conditions mean a temperature from 25°C up to 45°C, more preferably from 30°C up to 40°C.
  • a concentration is preferably a molar concentration, preferably a molar concentration per weight or per volume, most preferably measured under physiological conditions.
  • a subject is defined in the context of this application as a (living) organism, unless explicitly stated otherwise.
  • a subject may be any organism, including invertebrates and vertebrates.
  • a subject is a vertebrate. More preferably, a vertebrate is a starfish or a mammal. Even more preferably, a mammal is a rat, a mouse, a rabbit or a human. Most preferably, a mammal is a human.
  • a subject is a non-human animal, more preferably a non-human vertebrate, most preferably a non-human mammal.
  • a pseudohalogen is -CN, -CP, -NC, -OH, -SH, -SeH,-TeH, -OCN, -SCN, -NCS, -SeCN, -TeCN, -N3, -NO, or -NO2.
  • a pseudohalogen is -CN, -NC, -OH, -SH, -OCN, -SCN, -NCS, -N3, -NO, or -NO2.
  • An organelle is preferably a lysosome or an endosome, more preferably a lysosome.
  • An endosome is preferably a late endosome.
  • An increase of a parameter by a factor equal to or lower than X is defined in the context of this application as a change of said parameter from its initial value A to a value equal to or lower than A*X.
  • a decrease of a parameter by a factor equal to or lower than X is defined in the context of this application as a change of said parameter from its initial value A to a value equal to or lower than A*X.
  • a decrease of a parameter by a factor equal to or higher than X is defined in the context of this application as a change of said parameter from its initial value A to a value equal to or higher than A*X.
  • a parameter that is essentially the same as in a corresponding composition, organelle, cell fraction, cell, membrane, tissue or organ derived from a healthy subject or as in a corresponding healthy subject preferably means that the value of said parameter cannot be distinguished by a skilled person from the value of a corresponding parameter in a corresponding composition, organelle, cell fraction, cell, membrane, tissue or organ derived from a healthy subject or in a corresponding healthy subject, and/or that the value of said parameter would be interpreted by a skilled person as measured in a corresponding composition, organelle, cell fraction, cell, membrane, tissue or organ derived from a healthy subject or in a corresponding healthy subject.
  • An alteration of a parameter which is significantly smaller after introduction of an inhibitor in a composition, organelle, cell fraction, cell, membrane, tissue, organ or subject preferably means that the absolute difference between the value of said parameter and the value of a corresponding parameter in a corresponding composition, organelle, cell fraction, cell, membrane, tissue or organ derived from a healthy subject or in a corresponding healthy subject is decreased by a factor equal to or lower than 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 after said introduction.
  • any parameter referred to herein is preferably determined using the specific method, assay or methodology described herein. Where the present specification does not mention or describe a specific method, assay or methodology for determining said parameter, said parameter can be measured in a manner suitable per se, as will be clear to the skilled person based upon reading the present disclosure.
  • Each amino acid sequence described herein by virtue of its identity or similarity percentage (at least 60%) with a given amino acid sequence respectively has in a further specific aspect an identity or a similarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity or similarity with the given amino acid sequence respectively.
  • sequence identity or similarity is determined by comparing the whole length of the sequences as identified herein. Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO means identity or similarity based on the full length of said sequence (i.e. over its whole length or as a whole). Sequence identity is defined in the context of this application as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The identity between two amino acid sequences is preferably defined by assessing their identity within a whole SEQ ID NO as identified herein or part thereof.
  • sequence identity also means the degree of sequence relatedness between amino acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • Sequence similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Sequence identity and similarity can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • Preferred computer program methods to determine sequence identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, FASTA, BLASTN, and BLASTP (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990)), EMBOSS Needle (Madeira, F., et al., Nucleic Acids Research 47(W1): W636-W641 (2019)).
  • the BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol.215:403-410 (1990)).
  • the EMOSS program is publicly available from EMBL-EBI. The well-known Smith Waterman algorithm may also be used to determine identity.
  • the EMBOSS Needle program is the preferred program used. Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48 (3):443-453 (1970); Comparison matrix: BLOSUM62 from Henikoff and Henikoff, Proc. Natl. Acad.
  • a program useful with these parameters is publicly available as the EMBOSS Needle program from EMBL -EBI.
  • the aforementioned parameters are the default parameters for a Global Pairwise Sequence alignment o f proteins (along with no penalty for end gaps).
  • Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: DNAfull; Gap Open Penalty: 10; Gap Extend Penalty: 0.5.
  • a program useful with these parameters is publicly available as the EMBOSS Needle program from EMBL-EBI.
  • the aforementioned parameters are the default parameters for a Global Pairwise Sequence alignment of nucleotide sequences (along with no penalty for end gaps).
  • the skilled person may also take into account so-called “conservative” amino acid substitutions, as will be clear to the skilled person.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having acidic side chains is aspartate and glutamate; and a group of amino acids having sulphur - containing side chains is cysteine and methionine.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys or Gln; Asn to Asp, His or Ser; Asp to Glu or Asn; Gln to Glu, Lys or Arg; Glu to Lys, Asp, Gln; His to Tyr or Asn; Ile to Leu, Val, or Met; Leu to Ile, Met or Val; Lys to Arg, Gln or Glu; Met to Val, Leu or Ile; Phe to Trp or Tyr; Ser to Thr, Ala or Asn; Thr to Ser; Trp to Tyr or Phe; Tyr to His, Trp or Phe; and Val to Ile, Leu or Met.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative. Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.
  • the embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating one or more of the above-described embodiments.
  • Example 1 Synthetic Scheme Compound 1: 6- -5- 3- To a mixture of methyl 6-chloro-5-nitro-pyridine-3-carboxylate (1 g, 4.62 mmol, 1 eq) and pentan-3-amine (402.46 mg, 4.62 mmol, 538.04 uL, 1 eq) in MeCN (10 mL) was added K2CO3 (1.28 g, 9.23 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue.
  • DIAD 118.59 mg, 586.46 ⁇ mol, 114.02 uL, 1.5 eq
  • the mixture was stirred at 65 °C for 16 h.
  • the mixture was concentrated in vacuum to give a residue.
  • the residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50mm*3 um;mobile phase: [water(0.225%FA)-ACN];B%: 22%-42%,10min) followed by lyophilization to give a residue.
  • Synthetic Scheme Compound 8 Synthesis of methyl 6-(1-ethylpropylamino)-5-nitro-pyridine-3-carboxylate (2) To a mixture of methyl 6-chloro-5-nitro-pyridine-3-carboxylate (3.86 g, 17.82 mmol, 1 eq) and pentan-3-amine (1.55 g, 17.82 mmol, 2.08 mL, 1 eq) in CH3CN (15 mL) was added K2CO3 (4.92 g, 35.63 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The mixture was filtered and the filtrate was concentrated in vacuum to give a residue.
  • the mixture was stirred at 60 °C for 16 h.
  • the mixture was concentrated at reduced pressure to give a residue.
  • the reaction mixture was purified by prep- HPLC(column: Phenomenex luna C18150*40mm* 15um;mobile phase: [water(0.225%FA)-ACN];B%: 7%-37%,10min) to give methyl 3-(1-ethylpropyl)-1-(1H-imidazol-2-ylmethyl)-2-oxo-imidazo [4,5-b]pyridine-6-carboxylate (120 mg, 349.47 ⁇ mol, 18.40% yield) as yellow oil.
  • Synthetic Scheme Compound 11 Synthesis of compound 2 A solution of compound 1 (2 g, 13.93 mmol, 1 eq) and CDI (2.71 g, 16.72 mmol, 1.2 eq) in CH 3 CN (20 mL) was stirred at 80 °C for 16 h. The mixture was filtered and the filter cake was washed with water (10 mL) and MeCN (10 mL). Then the filter cake was dried at reduced pressure to give a residue to give compound 2 (2.3 g, 13.56 mmol, 97.37% yield) as brown solid.
  • the residue was diluted with water (10 mL) and the pH was adjusted to 5 with 1M HCl, then the mixture was concentrated at reduced pressure to give a residue.
  • the residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um;mobile phase: [water (FA) -ACN];B%: 1%-30%, 10 min) to give Compound 11 (9.26 mg, 25.91 ⁇ mol, 32.08% yield) as yellow gum.
  • the residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 8%- 38%, 15 min) followed by lyophilization to give a residue.
  • the residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 8%- 38%, 15 min) followed by lyophilization to give a residue.
  • Synthetic Scheme Compound 14 Synthesis of compound 3 To a mixture of compound 1 (100 mg, 379.81 ⁇ mol, 1 eq), compound 2 (42.59 mg, 379.81 ⁇ mol, 1 eq) and PPh 3 (149.43 mg, 569.71 ⁇ mol, 1.5 eq) in THF (1 mL) was added DIAD (115.20 mg, 569.71 ⁇ mol, 110.77 ⁇ L, 1.5 eq) dropwise at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue.
  • the residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%- 30%, 15 min) followed by lyophilization to give Compound 14 (27.68 mg, 79.00 ⁇ mol, 21.72% yield, 98% purity) as white solid.
  • the residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 8%- 38%, 10 min) followed by lyophilization to give a residue.
  • the residue was triturated with MeCN at 60 °C for 16 h. Then the mixture was filtered and the filter cake was collected and followed by lyophilization to give Compound 18 (14.1 mg, 37.79 ⁇ mol, 12.07% yield, 99% purity) as white solid.
  • Synthetic Scheme Compound 24 Synthesis of compound 2 To a solution of compound 1 (500 mg, 1.86 mmol, 1 eq) and 1, 4-dibromobutane (482.99 mg, 2.24 mmol, 269.83 ⁇ L, 1.2 eq) in DMSO (6 mL) was added K2CO3 (515.27 mg, 3.73 mmol, 2 eq), the mixture was stirred at 30 °C for 16 h. The reaction mixture was diluted with ethyl acetate (100 mL). The organic layer was washed with brine (30 mL*3), dried over Na 2 SO 4 , filtered and the filtrate was concentrated under vacuum to give a residue.
  • Synthetic Scheme Compound 25 Synthesis of compound 2 A mixture of compound 1 (150 mg, 529.81 ⁇ mol, 1 eq), 5-(chloromethyl) -1-ethyl-imidazole (91.93 mg, 635.77 ⁇ mol, 1.2 eq) and Cs 2 CO 3 (517.87 mg, 1.59 mmol, 3 eq) in DMF (2 mL) was stirred at 40 °C for 16 h. The reaction mixture was diluted with ethyl acetate (50 mL). The organic layer was washed with brine (30 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue.
  • Synthetic Scheme Compound 27 Experimental Procedure for Compound 27: Synthesis of compound 2 To a mixture of compound 1 (200 mg, 1.49 mmol, 1 eq, HCl) and IMIDAZOLE (303.55 mg, 4.46 mmol, 3 eq) in DMF (5 mL) was added TBSCl (268.82 mg, 1.78 mmol, 218.55 ⁇ L, 1.2 eq) in portions at 0 °C under N 2 . The mixture was stirred at 25 °C for 16 h.
  • reaction mixture was diluted with ethyl acetate (50 mL) and washed with brine (20 mL*3), dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuum to give a residue.
  • the residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 10%-40%, 15 min) followed by lyophilization to give compound 4A (30 mg, 197.12 ⁇ mol, 19.93% yield) as light yellow gum and compound 4A_isomer (30 mg, 197.12 ⁇ mol, 19.93% yield) as white gum.
  • the residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%-35%, 10 min) followed by lyophilization to give a residue.
  • Synthetic Scheme Compound 28 Synthesis of compound 3 and compound 3A To a mixture of compound 1 (1 g, 7.93 mmol, 1 eq) and compound 2 (2.04 g, 23.79 mmol, 3 eq) in DCE (20 mL) was added Na2CO3 (2.52 g, 23.79 mmol, 3 eq), 2 - (2 -pyridyl) pyridine (1.24 g, 7.93 mmol, 1 eq) and Cu(OAc)2 (1.44 g, 7.93 mmol, 1 eq). The mixture was stirred at 70 °C for 3 h. The reaction mixture was concentrated in vacuum to give a residue.
  • the residue was diluted with water (10 mL) and the pH was adjusted to 5 with 1M HCl, then the mixture was concentrated at reduced pressure to give a residue.
  • the residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um;mobile phase: [water (FA) -ACN];B%: 1%-25%, 10 min) to give Compound 32 (9.16 mg, 22.65 ⁇ mol, 28.04% yield, FA) as yellow solid.
  • Synthetic Scheme Compound 34 Synthesis of compound 1 To a mixture of compound 1 (2 g, 8.40 mmol, 1 eq) and TEA (1.28 g, 12.61 mmol, 1.75 mL, 1.5 eq) in DMF (20 mL) was added compound 2 (769.13 mg, 8.82 mmol, 1.03 mL, 1.05 eq) at 0 °C under N 2 . The mixture was stirred at 25 °C for 16 h. The mixture was diluted with ethyl acetate (100 mL) and the resulting mixture was washed with water (30 mL*3), brine (20 mL*2), dried over Na2SO4, filtered and concentrated at reduced pressure to give a residue.
  • the reaction mixture was concentrated in vacuum to remove THF, then the mixture was adjusted to pH about 3-4 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue.
  • the residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%-35%, 10 min) followed by lyophilization to give Compound 34 (13.35 mg, 35.66 ⁇ mol, 17.31% yield, 100% purity) as white solid.
  • the colorless gum was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (ammonia hydroxide v/v) -ACN]; B%: 40%-70%, 9 min) to give compound 10 (55 mg, 134.94 ⁇ mol, 6.99% yield) as colorless gum.
  • reaction mixture was added into the water (10 mL) and the resulting mixture was extracted with ethyl acetate (10 mL*3), the combined organic phase was washed with saturated NaHCO3 aqueous solution (10 mL*3), dried with anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuum to give residue.
  • the residue was diluted with water (2 mL) and the pH was adjusted to 5 with 1M HCl, then the mixture was concentrated at reduced pressure to give a residue.
  • the residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um;mobile phase: [water (FA) -ACN];B%: 1%-20%, 7 min) to give Compound 37 (7.54 mg, 19.06 ⁇ mol, 11.32% yield, 98.2% purity, FA) as yellow solid.
  • Synthetic Scheme Compound 39 Experimental Procedure for Compound 39: Synthesis of compound 2 A mixture of compound 1 (9.5 g, 49.23 mmol, 1 eq), PMBNH 2 (12.15 g, 88.61 mmol, 11.47 mL, 1.8 eq) and DIPEA (11.45 g, 88.61 mmol, 15.43 mL, 1.8 eq) in THF (100 mL) was stirred at 0 °C for 0.5 h. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (100 mL* 3). The combined organic phase were collected, dried over Na 2 SO 4 , filtered and the filtrate was concentrated under reduced pressure to give a residue.
  • Synthetic Scheme Compound 40 Experimental To a mixture of compound 8 (50 mg, 134.61 ⁇ mol, 1 eq) and NH 2 OH•HCl (37.42 mg, 538.46 ⁇ mol, 4 eq) in MeOH (1 mL) was added DIEA (86.99 mg, 673.07 ⁇ mol, 117.24 ⁇ L, 5 eq). The mixture was stirred at 70 °C for 16 h.
  • reaction mixture was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-24%, 7 min) followed by lyophilization to give Compound 40 (4.74 mg, 12.47 ⁇ mol, 9.27% yield, 98% purity) as off-white solid.
  • the residue was purified by prep-HPLC (column: Unisil 3-100 C18 ⁇ Ltra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-26%, 7 min) to give a residue. Then the residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (NH4HCO3) -ACN]; B%: 5%-35%, 9 min) to give Compound 43 (4.09 mg, 9.41 ⁇ mol, 11.21% yield, 100% purity) as off-white solid.
  • Synthetic Scheme Compound 44 Synthesis of compound 2 To a solution of compound 1 (200 mg, 494.68 ⁇ mol, 1 eq) in DMA (1 mL) was added CuCN (53.17 mg, 593.62 ⁇ mol, 129.67 ⁇ L, 1.2 eq) and Pd(PPh 3 ) 4 (57.16 mg, 49.47 ⁇ mol, 0.1 eq) under N 2 . The mixture was stirred at 130 °C for 16 h under N2. The reaction mixture was quenched with NH3•H2O (1 mL) and diluted with Ethyl acetate (50 mL).
  • Example 2 Activity Studies and Data Assay 1 -isoQC Potency -Fluorescent Enzymatic Assay
  • the Golgi luminal, enzymatically active region of human isoQC (S53-L382; see, e.g., Huang et al., 2011) was obtained by expression in E. coli of an N-terminally GST Enterokinase and C-terminally 6xHis tagged construct in the pET41(+) vector and subsequent purification using Ni-NTA IMAC. Enterokinase digestion ensued, followed by another Ni-NTA IMAC purification, Superdex75 based size exclusion and finally a spin concentration using a 10 kDa molecular weight cutoff.
  • the final protein is formulated in 50 mM Tris-HCI, 150 mM NaCl, 50% Glycerol, pH 7.8. Pyroglutamylation was measured by conversion of glutamine-4-amino-7-methylcoumarine (H-Gln-AMC, Bachem) to pyroglutamyl-AMC, which is subsequently a specific substrate for pyroglutamylaminopeptidase (pGAPase, Qiagen), releasing free AMC which can be fluorescently detected.
  • pGAPase pyroglutamylaminopeptidase
  • Qiagen pyroglutamylaminopeptidase
  • pGAPase enzyme diluted in 50 mM Tris-HCI pH 8.0 containing 10 mM dithiothreitol (DTT) solution was prepared. 25 ⁇ L of the pyroglutamylation reaction mixture was transferred into an empty plate and to that, 25 ⁇ L of the pGAPase enzyme solution was added. Plates were then incubated for 25 minutes at room temperature before fluorescence readings were taken on an Envision2 plate reader with excitation set at 380 nm and emission at 450 nm.
  • DTT dithiothreitol
  • protein was purified by Ni-NTA IMAC, dialysed in the presence of Factor Xa, further purified by Ni-NTA reverse IMAC and finally filtrated for size on a Superdex 20016/60 column. Final protein was concentrated using a spin concentrator at 10 kDa molecular weight cutoff and formulated in 150 mM NaCl, 50 mM Tris-HCI, 50% Glycerol, pH 8.0.
  • Pyroglutamylation was measured by conversion of glutamine-4-amino-7-methylcoumarine (H-Gln-AMC, Bachem) to pyroglutamyl-AMC, which is subsequently a specific substrate for pyroglutamylaminopeptidase (pGAPase, Qiagen), releasing free AMC which can be fluorescently detected.
  • 7.5 ⁇ L aliquots of threefold dilutions of test compound in 50 mM Tris pH8 buffer were plated in 384-well plates.15 ⁇ L 20 ⁇ M h-Gln-AMC (diluted from a 40 mM stock dissolved in 25 mM HEPES using 50 mM Tris-HCI pH 8.0) was added to each well. Finally, 7.5 ⁇ L, 3 ng/ ⁇ L recombinant QC enzyme was added and the reaction mixture was incubated for 1 hour at 37°C. A 5 minute incubation at 98°C was then performed to denature the QC enzyme and stop the reaction.
  • pGAPase enzyme diluted in 50 mM Tris-HCI pH 8.0 containing 10 mM dithiothreitol (DTT) solution was prepared. 25 ⁇ L of the pyroglutamylation reaction mixture was transferred into an empty plate and to that, 25 ⁇ L of the pGAPase enzyme solution was added. Plates were then incubated for 25 minutes at room temperature before fluorescence readings were taken on an Envision2 plate reader with excitation set at 380 nm and emission at 450 nm.
  • DTT dithiothreitol

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Abstract

The current invention relates to QPCTL inhibitors represented by formula (I), and corresponding compositions and uses. Preferably, the inhibitors and compositions are for use in the treatment of cancer, neurodegenerative diseases such as Alzheimer's disease, synucleinopathies, Huntington's disease, bacterial infections such as periodontitis and related disorders, and inflammatory diseases.

Description

QPCTL INHIBITORS FIELD OF THE INVENTION The current invention relates to QPCTL inhibitors represented by formula (I), and corresponding compositions and uses. Preferably, the inhibitors and compositions are for use in the treatment of cancer, neurodegenerative diseases such as Alzheimer's disease, synucleinopathies, Huntington’s disease, bacterial infections such as periodontitis and related disorders, and inflammatory diseases. BACKGROUND OF THE INVENTION QPCTL (glutaminyl-peptide cyclotransferase-like protein) enzymes belong to the family of aminoacyltransferases. QPCTL enzymes catalyze the cyclization of N-terminal glutamine and glutamic acid residues in peptides into an N-terminal pyroglutamate residues. These proteins have been identified as clinically relevant, for example for their role in treating tumors (Yu, L., Zhao, P., Sun, Y. et al. Development of a potent benzonitrile-based inhibitor of glutaminyl-peptide cyclotransferase-like protein (QPCTL) with antitumor efficacy. Sig Transduct Target Ther 8, 454 (2023)). Nonetheless, a few potent and/or selective QPCTL inhibitors have been identified to the best of our knowledge. Hence, there is a continuing need in the art for novel potent and/or selective QPCTL inhibitors. SUMMARY OF THE INVENTION One aspect of the invention pertains to certain N or C-substituted-3,4-(fused ring)- heteroaryl or heterocyclic compounds (also referred to herein as" QPCTL inhibitors"), as described herein. Another aspect of the invention pertains to a composition (e.g., a pharmaceutical composition) comprising a QPCTL inhibitor, as described herein, and a pharmaceutically acceptable carrier or diluent. Another aspect of the present invention pertains to use in a treatment of a disorder of the human or animal body associated with abnormal QPCTL enzyme activity, comprising administering to a subject in need of treatment a therapeutically effective amount of compound of formula I or a hydrate, solvate, or pharmaceutically acceptable salt thereof. Another aspect of the present invention pertains to use in the inhibition of QPCTL activity in a subject, comprising administering to said subject an effective amount of compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof. Another aspect of the present invention pertains to use in the inhibition of QPCTL protein/activity comprising contacting the QPCTL protein/enzyme, in vitro or in vivo, with an effective amount of a compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof. Another aspect of the present invention pertains to use in the inhibition of QPCTL protein/activity in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof. Another aspect of the present invention pertains to use in a method of treatment of the human or animal body by therapy. Another aspect of the present invention pertains to its use in a method of treating diseases or disorders associated with abnormal QPCTL activity or QPCTL protein. Another aspect of the present invention pertains for use in the preparation of a medicament for treating conditions associated with abnormal QPCTL activity. Another aspect of the present invention pertains for use in the manufacture of a medicament for the treatment of a disorder or disease of the human or animal body that is associated by the abnormal QPCTL activity. Another aspect of the present invention pertains to a kit comprising (a) a compound of formula I, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound. Another aspect of the present invention pertains to a compound of formula I obtainable by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein. Another aspect of the present invention pertains to a compound of formula I obtained by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein. Another aspect of the present invention pertains to novel intermediates, as described herein, which are suitable for use in the methods of synthesis described herein. Another aspect of the present invention pertains to the use of such novel intermediates, as described herein, in the methods of synthesis described herein. As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspects of the invention. DETAILED DESCRIPTION QPCTL inhibitors In a first aspect, the invention provides a QPCTL inhibitor represented by compound of formula I : or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein: R1 is independently selected from a group consisting of hydrogen, methyl, carboxylic acid, -C(O)OCH3, difluoropyrrolidinyl, phenyl, dimethylamino, methylamino, -NH2, cyano, tetrazolyl, 5-oxo-1,2,4- oxadiazolinyl, trifluoromethyl, and oxygen; R1b represents Ring ‘A’ is selected from a group consisting of 6-membered aryl and heteroaryl ring; Ring ‘B’ is selected from a group consisting of 6-membered aryl, heteroaryl ring and 5- membered heteroaryl ring; Ring ‘C’ represents a saturated or unsaturated ring system; X is independently selected from a group consisting of -CH2-, -O- and keto ( ); or X is a bond; Y represents -N- or keto ( ); W represents ‘C’ or - R represents -CH2- or - ; represents a single bond or double bond; n = 0 or 1 ; q = 0 or 1 ; m = 1, 2 or 3; p =0, 1 or 2; Z=C or N, wherein when Z is N, R4 is absent; Q=C or N, wherein when p=2, Q is not ‘N’; R2 is either absent or independently selected from a group consisting of hydrogen, methyl, trifluoromethyl, ethyl, -OC2H5, hexafluoropropanyl, perfluoroethyl/ pentafluoroethyl, trifluoroethyl, difluoroethyl and ; R3 or from a group consisting of hydrogen, methyl, ring or phenyl ring; a group isopentyl, methyl, propyl, tert-butyl, trifluoromethyl, and difluoro ethyl; or R3 and R4 are joined together to form a cyclopentyl ring or tetrahydropyranyl ring; R5 represents hydrogen or methyl; R6 is independently selected from a group consisting of hydrogen, methyl, ethyl, isopropyl, cyclopentyl, cyclopropyl, trifluoromethyl, difluoroethyl, perfluoroethyl/ pentafluoroethyl, In or heteroaryl ring is selected from a group consisting of pyridyl, pyrazinyl, pyridazinyl, and pyrimidinyl; the 6-membered aryl is phenyl; and the 5- membered heteroaryl ring is selected from a group consisting of imidazolyl, isoxazolyl, triazolyl, thiazolyl, triazolyl, and pyrazolyl. In embodiments, the Ring A is selected from a group consisting of pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and phenyl; and the Ring B is selected from a group consisting of imidazolyl, pyridyl, isoxazolyl, triazolyl, thiazolyl, triazolyl, and pyrazolyl. In a further aspect, the invention provides compound of formula Ia : or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein R1 is independently selected from a group consisting of hydrogen, methyl, carboxylic acid, -C(O)OCH3, difluoropyrrolidinyl, phenyl, dimethylamino, methylamino, -NH2, cyano, tetrazolyl, 5-oxo-1,2,4- oxadiazolinyl, trifluoromethyl, oxygen; R1b represents or Ring ‘A’ is selected from a group consisting of pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and phenyl; Ring ‘B’ is selected from a group consisting of imidazolyl, pyridyl, isoxazolyl, triazolyl, thiazolyl, triazolyl, and pyrazolyl; Ring ‘C’ represents a saturated or unsaturated ring system; ‘X’ is independently selected from a group consisting of -CH2-, -O- and keto ( ); Y represents -N- or keto ( ); W represents ‘C’ or - R represents -CH2- or - ; represents a single bond or double bond; n = 0 or 1 ; q = 0 or 1 ; m = 1, 2 or 3; p =0 or 1; Z=C or N, wherein when Z is N, R4 is absent; Q=C or N; R2 is either absent or independently selected from a group consisting of hydrogen, methyl, ethyl, trifluoromethyl, pentafluoroethyl and trifluoroethyl; and R3 is either absent or independently selected from a group consisting of hydrogen, methyl, ethyl, isopropyl, trifluoromethyl, or R6 is independently selected from a group consisting of hydrogen, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, trifluoroethyl, difluoroethyl, pentafluoroethyl In a further aspect, the invention provides compound of formula Ib : or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein R1 is independently either Carboxylic acid or -C(O)OCH3; Ring ‘A’ is pyridyl; R2 is either absen ; R3 is either absent or R3 and R2 are joined together to form a cyclohexyl ring, pyridyl ring, cyclopentyl ring or phenyl ring; Ring ‘B’ is selected from a group consisting of imidazolyl, isoxazolyl, triazolyl and thiazolyl; R5 represents hydrogen or methyl; R6 represents hydrogen, methyl, isopropyl or ethyl; Ring ‘C’ represents a saturated or unsaturated ring system; Y represents keto ( ); W represents -N-; R represents -CH2-; q = 1 ; m = 1; p =0, 1 or 2; Z= N; and Q=C or N; wherein when p=2, Q is not ‘N’. In a further aspect, the invention provides a compound of formula Ic: or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein R1 is independently either Carboxylic acid or -C(O)OCH3; Ring ‘A’ is pyridyl; Ring ‘B’ is either Imidazolyl or triazolyl ; R5 is hydrogen; R6 is selected from a group consisting of methyl, ethyl, cyclopentyl and isopropyl; Ring ‘C’ represents a saturated or unsaturated ring system; Y represents -N- or keto ( ); W represents -N-; q = 1 ; Z=C or N; and R4 is independently selected from a group consisting of ethoxyethyl, isopentyl, methyl, propyl, tert-butyl, trifluoromethyl, and difluoro ethyl. In a further aspect, the invention provides a compound of formula Id: or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein: R1 is independently selected from a group consisting of carboxylic acid, tetrazolyl, trifluoromethyl, Ring ‘B’ is imidazolyl; R5 represents hydrogen; R6 is independently selected from a group consisting of ethyl, trifluoromethyl, difluoroethyl, and pentafluoroethyl, trifluoroethyl; q = 1 ; R3 is independently selected from a group consisting of hydrogen, methyl and phenyl; R4 is independently selected from a group consisting of hydrogen, flouro, and methyl; and alternatively, R3 and R4 are joined together to form a cyclopentyl ring or tetrahydropyranyl ring. Unless otherwise indicated, where a compound is shown or described which has one or more chiral centres, and two or more stereoisomers are possible, all such stereoisomers are disclosed and encompassed, both individually (e.g., as isolated from the other stereoisomer(s)) and as mixtures (e.g., as equimolar or non-equimolar mixtures of two or more stereoisomers). For example, unless otherwise indicated, where a compound has one chiral centre, each of the (R) and (S) enantiomers are disclosed and encompassed, both individually (e.g., as isolated from the other enantiomer) and as a mixture (e.g., as equimolar or non-equimolar mixtures of the two enantiomers). Note that, depending upon the identity of Ring A, Ring B, Ring C, and any substituents thereon, Ring A, Ring B, and Ring C may be susceptible to tautomerism. Unless otherwise indicated, where a compound is shown or described which is susceptible to tautomerism, and two tautomers are possible, both tautomers are disclosed and encompassed, both individually (e.g., as isolated from the other tautomer) and as mixtures (e.g., as equimolar or non-equimolar mixtures of two tautomer). In embodiments, the compound is of formula II:
Formula II. In embodiments, compound of one of the following formulae 1-119, or a pharmaceutically acceptable salt, hydrate, or solvate thereof. Preferably, the compound is of one of the following formulae 1-45, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
ı7
IJ3
Compoun 9 These compounds maybe also be denoted using the following “III-number” notation: III-1 Compound 43 III-16 Compound 11 III-31 Compound 35 III-2 Compound 14 III-17 Compound 25 III-32 Compound 37 III-3 Compound 26 III-18 Compound 40 III-33 Compound 30 III-4 Compound 12 III-19 Compound 34 III-34 Compound 31 III-5 Compound 27 III-20 Compound 23 III-35 Compound 7 III-6 Compound 1 III-21 Compound 38 III-36 Compound 4 III-7 Compound 10 III-22 Compound 32 III-37 Compound 5 III-8 Compound 15 III-23 Compound 22 III-38 Compound 6 III-9 Compound 21 III-24 Compound 13 III-39 Compound 8 III-10 Compound 33 III-25 Compound 16 III-40 Compound 9 III-11 Compound 44 III-26 Compound 18 III-41 Compound 19 III-12 Compound 17 III-27 Compound 28 III-42 Compound 36 III-13 Compound 20 III-28 Compound 45 III-43 Compound 39 III-14 Compound 24 III-29 Compound 3 III-44 Compound 41 III-15 Compound 29 III-30 Compound 2 III-45 Compound 42 In embodiments, the compound is of formula III-1 to III-45, or III-1 to III-44, or III-1 to III-43, or III-1 to III-42, or III-1 to III-41, or III-1 to III-40, or III-1 to III-39, or III-1 to III-38, or III-1 to III-37, or III- 1 to III-36, or III-1 to III-35, or III-1 to III-34, or III-1 to III-33, or III-1 to III-32, or III-1 to III-31, or III-1 to III-30, or III-1 to III-29, or III-1 to III-28, or III-1 to III-27, or III-1 to III-26, or III-1 to III-25, or III-1 to III-24, or III-1 to III-23, or III-1 to III-22, or III-1 to III-21, or III-1 to III-20, or III-1 to III- 19, or III-1 to III-18, or III-1 to III-17, or III-1 to III-16, or III-1 to III-15, or III-1 to III-14, or III-1 to III-13, or III-1 to III-12, or III-1 to III-11, or III-1 to III-10, or III-1 to III-9, or III-1 to III-8, or III-1 to III-7, or III-1 to III-6, or III-1 to III-5, or III-1 to III-4, or III-1 to III-3, or III-1 to III-2. Compositions In a further aspect, the invention provides a composition comprising an inhibitor according to the invention and a pharmaceutically acceptable excipient, preferably for use as a medicament. A related aspect pertains to a method of preparing such a composition. Such compositions are referred to in the current application as compositions according to or of the invention. All specific embodiments disclosed above for an inhibitor according to the invention may be applied accordingly for an inhibitor according to the invention comprised in a composition according to the invention. In a further aspect, the invention provides a pharmaceutical composition comprising a compound of formula I or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient. wherein R, R1, R1a, R1b, q, X, Q, n, m, p, R2, R3, R4, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula I in above; In a further aspect, the invention provides a pharmaceutical composition comprising a compound of formula Ia or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient. Formula Ia wherein R, R1, R1a, R1b, q, X, Q, n, m, p, R2, R3, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ia in above; In a further aspect, the invention provides a pharmaceutical composition comprising a compound of formula Ib or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient. wherein R, R1, q, Q, m, p, R2, R3, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ib in above; In a further aspect, the invention provides a pharmaceutical composition comprising a compound of formula Ic or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient. wherein R1, q, R4, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ic in above; In a further aspect, the invention provides a pharmaceutical composition comprising a compound of formula Id or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined above, and a pharmaceutically acceptable excipient. wherein R1, q, R3, R4, according to Formula Id in above; A composition according to the invention may be presented or formulated as capsules, tablets, powders, granules, solutions, suspensions in aqueous or non-aqueous liquids, edible, oil-in-water liquid emulsions, water- in-oil liquid emulsions, solution, syrups and elixirs, in microencapsulated form, liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles, transdermal patches, ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, drops, sprays, aerosols, oils, lozenges, pastilles, mouth washes, suppositories, enemas, aqueous and non-aqueous sterile injection solutions, and so on. It will be appreciated that the compositions may include other agents conventional in the art having regard to the type of formulation. Non-limiting examples of a pharmaceutically acceptable carrier comprised in a composition are saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds, besides an inhibitor according to the invention, can also be incorporated into the compositions. A composition according to the invention formulated as solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. A composition according to the invention formulated as compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. In a composition according to the invention prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. A composition according to the invention formulated as oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the inhibitor according to the invention can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. A composition according to the invention may be formulated for administration by inhalation, the inhibitor according to the invention can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. A composition according to the invention may be formulated for transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one aspect, transdermal administration may be performed by iontophoresis. A composition according to the invention may comprise a carrier system such as a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one aspect, the inhibitor according to the invention is encapsulated in a liposome. An inhibitor according to the invention can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems. Combinations It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the chemical groups represented by the variables (e.g. -R, -R1, -R1a, -R1b, -q, -X, -Q, -n, -m, -p, -R2, -R3, -R4, -R5, -R6, -W, -Y, - Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ etc.) are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace compounds that are stable compounds (i.e., compounds that can be isolated, characterised, and tested for biological activity). In addition, all sub-combinations of the chemical groups listed in the embodiments describing such variables are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination of chemical groups was individually and explicitly disclosed herein. Substantially Purified Forms In a further aspect, the present invention pertains to QPCTL inhibitors, as described herein, in substantially purified form and/or in a form substantially free from contaminants. In one embodiment, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight. Unless otherwise specified, the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to a equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer. In one embodiment, the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight. Unless specified, the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer. In one embodiment, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure. Isomers Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereoisomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso- forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and b-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as“isomers” (or“isomeric forms”). A reference herein to one tautomer is intended to encompass both tautomers. Keto-Enol Tautomerism Note that specifically included in the term“isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like. Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner. Salts It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977,“Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol.66, pp.1 -19. For example, if the compound is anionic, or has a functional group, which may be anionic (e.g., -COOH may be -COO-), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca2+ and Mg2+, and other cations such as Al3+ as well as the ammonium ion (i.e., NH4 + ). Examples of suitable organic cations include, but are not limited to substituted ammonium ions (e.g., NH3R+, NH2R2 +, NHR3 +, NR4 +), for example, where each R is independently linear or branched saturatedC1-18 alkyl, C3-8cycloalkyl, C3-8cycloalkyl-C1-6alkyl, and phenyl-C1- 6alkyl, wherein the phenyl group is optionally substituted. Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+. If the compound is cationic, or has a functional group, which upon protonation may become cationic (e.g., -NH2 may become -NH3 +), then a salt may be formed with a suitable anion. For example, if a parent structure contains a cationic group (e.g., -NMe2+ ), or has a functional group, which upon protonation may become cationic (e.g., -NH2 may become -NH3 +), then a salt may be formed with a suitable anion. In the case of a quaternary ammonium compound a counter-anion is generally always present in order to balance the positive charge. If, in addition to a cationic group (e.g., -NMe2 +, -NH3 +), the compound also contains a group capable of forming an anion (e.g., -COOH), then an inner salt (also referred to as a zwitterion) may be formed. For example, in the QPCTL inhibitors described herein and a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic, acetic, trifluoroacetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, 1,2-ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Unless otherwise specified, a reference to a particular compound also includes salt forms thereof. Solvates and Hydrates It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compound. A QPCTL inhibitor according to the invention may be present as a pharmaceutically acceptable solvate or hydrate. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc. Unless otherwise specified, a reference to a particular compound also includes solvate and hydrate forms thereof. Chemically Protected Forms It may be convenient or desirable to prepare, purify, and/or handle the compound in a chemically protected form. The term “chemically protected form” is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, reactive chemical reagents, and the like). In practice, well-known chemical methods are employed to reversibly render unreactive a functional group, which otherwise would be reactive, under specified conditions. In a chemically protected form, one or more reactive functional groups are in the form of a protected or protecting group (alternatively as a masked or masking group or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed or the masking group transformed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts; 4th Edition; John Wiley and Sons, 2006). A wide variety of such “protecting,” “blocking,” or “masking” methods are widely used and well known in organic synthesis. For example, a compound which has two nonequivalent reactive functional groups, both of which would be reactive under specified conditions, may be derivatized to render one of the functional groups “protected,” and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group. After the desired reaction (involving the other functional group) is complete, the protected group may be “deprotected” to return it to its original functionality. For example, a hydroxy group may be protected as an ether (-OR) or an ester (-OC(=O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t - butyldimethylsilyl ether; or an acetyl ester (-OC(=O)CH3, -OAc). For example, an amine group may be protected, for example, as an amide (-NRCO-R), for example: as an acetamide (-NHCO-CH3); or as a carbamate (-NRCO-OR), for example: as a benzyloxy carbamate (-NHCO- OCH2C6H5, -NH-Cbz), as a t-butoxy carbamate (-NHCO-OC(CH3)3, -NH-Boc); as a 2-biphenyl-2-propoxy carbamate (-NHCO-OC(CH3)2C6H4C6H5, -NH-Bpoc), as a 9-fluorenylmethoxy carbamate (-NH-Fmoc), as a 6- nitroveratryloxy carbamate (-NH-Nvoc), as a 2-trimethylsilylethyloxy carbamate (-NH-Teoc), a 2,2,2- trichloroethyloxy carbamate (-NH-Troc), as an allyloxy amide (-NH-Alloc), or as a 2(-phenylsulfonyl)ethyloxy carbamate (-NH-Psec); or, in suitable cases (e.g., cyclic amines), as a nitroxide radical (>N-O●); or, in suitable cases (e.g., heterocyclic nitrogens), as a 2-trimethylsilylethoxymethyl (N-SEM). Prodrugs It may be convenient or desirable to prepare, purify, and/or handle the compound in the form of a prodrug. The term “prodrug,” as used herein, pertains to a compound, which yields the desired active compound in vivo. Typically, the prodrug is inactive, or less active than the desired active compound, but may provide advantageous handling, administration, or metabolic properties. For example, some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). During metabolism, the ester group (-C(=O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(=O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Also, some prodrugs are activated enzymatically to yield the active compound, or a compound, which, upon further chemical reaction, yields the active compound (for example, as in antibody directed enzyme prodrug therapy (ADEPT), gene directed enzyme prodrug therapy (GDEPT), lipid directed enzyme prodrug therapy (LIDEPT), etc.). For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative. General Chemical Synthesis Several methods for the chemical synthesis of the FRPPO compounds are described herein. These and/or other well-known methods may be modified and/or adapted in known ways in order to facilitate the synthesis of additional compounds described herein. Uses and therapeutic uses In a further aspect, the invention provides the use of a QPCTL inhibitor according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL. Preferably, the binding of QPCTL results in in the inhibition of QPCTL. The inhibition of QPCTL is described in more detail above. All specific embodiments disclosed above for an inhibitor according to the invention and a composition according to the invention may be applied accordingly for the uses and therapeutic uses described below. In a further aspect, the invention provides the use of a compound of formula I according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
Formu a I wherein R, R1, R1a, R1b, q, X, Q, n, m, p, R2, R3, R4, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula I in above; In a further aspect, the invention provides the use of a compound of formula Ia according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL. wherein R, R1, R1a, R1b, q, X, Q, n, m, p, R2, R3, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ia in above; In a further aspect, the invention provides the use of a compound of formula Ib according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
Formu a Ib wherein R, R1, q, Q, m, p, R2, R3, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ib in above; In a further aspect, the invention provides the use of a compound of formula Ic according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL. wherein R1, q, R4, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ic in above; In a further aspect, the invention provides the use of a compound of formula Id according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL.
wherein R1, q, R3, R4, R5, 6, ng , an ng are as e ne according to Formula Id in above; In embodiments, the use of a QPCTL inhibitor according to the invention or a composition according to the invention is for specifically binding and/or specifically inhibiting QPCTL and isoQPCTL. Specifically means that the binding or inhibition stems from the amino acid sequence dependent molecular interaction between the inhibitor, or the inhibitor comprised in the composition, and QPCTL or isoQPCTL. As such, the inhibitor or composition is not able to significantly bind or inhibit other enzymes that may share a similar function but a different amino acid structure. In embodiments, the use of a QPCTL inhibitor according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL comprises a QPCTL inhibitor which specifically binds or inhibits QPCTL or isoQPCTL. The inhibition of QPCTL can be expressed as the half maximal inhibitory concentration (IC50), as known to the skilled person. Lower IC50 values correspond with higher potencies to inhibit QPCTL. IC50 can be determined using Fluorescent Enzymatic assay protocol of Example 2. Claimable summary of IC50 are provided in Example 2. In embodiments, the use of a QPCTL inhibitor according to the invention or a composition according to the invention for (specifically) inhibiting isoQPCTL comprises a QPCTL inhibitor having an IC50 for QPCTL equal to or higher than 10 µm, 9.63 µm, 8.3 µm, 6.8 µm, 3.95 µm, 3.16 µm, 2.88 µm, 1.83 µm, 1.06 µm, 1.05 µm, 0.721 µm, 0.694 µm, 5.3 µm, 0.502 µm, 442 nm, 440 nm, 434 nm, 417 nm, 394 nm, 365 nm, 362 nm, 342 nm, 324 nm, 294 nm, 249 nm, 240 nm, 231 nm, 220 nm, 215 nm, 154 nm, 153 nm, 151 nm, 135 nm, or 52 nm. In embodiments, the use of a QPCTL inhibitor according to the invention or a composition according to the invention for (specifically) inhibiting QPCTL comprises a QPCTL inhibitor having an IC50 for QPCTL equal to or higher than >10 µm, 9.54 µm, 2.37 µm, 0.653 µm, 1.49 µm, 0.623 µm, 451 nm, 142 nm, 103 nm, 70 nm, 66 nm, 65 nm, 61 nm, 57 nm, 52 nm, 49 nm, 48 nm, 42 nm, 38 nm, 34 nm, 31 nm, 30 nm, 25 nm, 23 nm, 22 nm, 18 nm, 17 nm, 16 nm, 14 nm, 10 nm or 1 nm. In embodiments, the uses above are for use in vitro. In embodiments, the uses above are for use in vivo. Kits: In a further aspect, the invention pertains to a kit comprising (a) a compound of formula I, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound. wherein R, R1, R1a, R1b, q, X, Q, n, m, p, R2, R3, R4, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula I in above; In a further aspect, the invention pertains to a kit comprising (a) a compound of formula Ia, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound.
Formula Ia wherein R, R1, R1a, R1b, q, X, Q, n, m, p, R2, R3, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ia in above; In a further aspect, the invention pertains to a kit comprising (a) a compound of formula Ib, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound. wherein R, R1, q, Q, m, p, R2, R3, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ib in above; In a further aspect, the invention pertains to a kit comprising (a) a compound of formula Ic, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound. wherein R1, q, R4, R5, R6, W, Y, Z, Ring ‘A’, Ring ‘B’ and Ring ‘C’ are as defined according to Formula Ic in above; In a further aspect, the invention pertains to a kit comprising (a) a compound of formula Id, as described herein, preferably provided as a composition (e.g., a pharmaceutical composition) and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder (e.g., a disease) as described herein, for example, written instructions on how to administer the compound. wherein R1, q, R3, R4, R5, R6, Ring ‘A’, and Ring ‘B’ are as defined according to Formula Id in above; In a further aspect, the invention provides a QPCTL inhibitor according to the invention or a composition according to the invention for use as a medicament. Likewise, all specific embodiments for an inhibitor according to the invention, a composition according to the invention, and the uses presented above may be applied accordingly. Wherever an inhibitor or a composition for use as a medicament is disclosed, a corresponding method for the manufacture or the production of a medicament comprising such an inhibitor or such a composition, a corresponding method of treatment comprising the administration of the inhibitor or composition to a subject in need thereof, and a corresponding use of such an inhibitor or such a composition as a medicament are also disclosed. In all these contexts, the inhibitor according to the invention and the composition according to the invention may be referred to as a medicament according to the invention. A medicament according to the invention may be administered orally, nasally, buccally, sublingually, vaginally, parenterally, topically, systemically, intravenously, subcutaneously, intraperitoneally, intramuscularly, intrathecally, by inhalation or epidurally. A medicament according to the invention, may be administered separately, sequentially or simultaneously in combination with another medicaments. As used herein, the term "simultaneous" therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time. The term "separate" therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes. The term "sequential" therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case. In the context of this application, the terms treating" or "treatment" refer to therapeutic treatment, wherein the object is to prevent, reduce, alleviate or slow down (lessen), respectively and as applicable, the targeted pathologic disorder or disease and/or its progression in a subject. In particular, said terms relate to a treatment which has the object of improving one or more symptoms and/or physiological parameters that are caused by, associated with and/or characteristic of the disease or disorder that is to be treated, and/or the object to preventing that such symptom(s) to arise and/or that such symptom(s) or physiological parameter(s) further deteriorate. Based on his general knowledge and the further disclosure herein, the skilled person (and in particular, the treating physician) will be able to suitably determine and measure said symptom(s) or physiological parameter(s), depending on the specific disease involved. In the context of this application, the terms "prevention" or "preventing" of a disorder or disease refers to a compound that, in a statistical sample, reduces the occurrence of symptoms of a disorder or disease in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. A medicament according to the invention is administered to a subject in need thereof in an effective amount (i.e., amount that have desired therapeutic effect). Preferably, an effective amount refers to an amount of an inhibitor according to the invention comprised in said medicament. The dose and dosage regimen will depend upon the degree of the infection in the subject, the characteristics of the particular inhibitor according to the invention, e.g., its therapeutic index, the subject, and the subject's history. Certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the medicaments according to the invention can include a single treatment or a series of treatments. The compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in a treatment of a disorder of the human or animal body associated with abnormal QPCTL enzyme activity , comprising administering to a subject in need of treatment a therapeutically -effective amount of the said compound. The compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in the inhibition of QPCTL activity in a subject, comprising administering to said subject an effective amount of said compound. The compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in the inhibition of QPCTL protein/activity comprising contacting the QPCTL protein/enzyme, in vitro or in vivo, with an effective amount of said compound. The compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in the inhibition of QPCTL protein/activity in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of said compound. The compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in a method of treatment of the human or animal body by therapy. The compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for use in a method of treating diseases or disorders associated with abnormal QPCTL activity or QPCTL protein. The compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I for the preparation of a medicament for treating conditions associated with abnormal QPCTL activity. Use of the compound of formula I as defined above and the pharmaceutical composition comprising the compound of formula I in the manufacture of a medicament for the treatment of a disorder or disease of the human or animal body that is associated by the abnormal QPCTL activity. Therapy Diseases In an aspect, the invention provides a QPCTL inhibitor according to the invention or a composition according to the invention, for use as a medicament. In embodiments, the QPCTL inhibitor or the composition is for use in the treatment of a disease characterized by lysosomal dysregulation. Below, preferred diseases characterized by lysosomal dysregulation, and other preferred features of the treatment are disclosed. Said diseases include cancer, neurodegenerative diseases such as Alzheimer's disease, synucleinopathies, Huntington’s disease, bacterial infections such as periodontitis and related disorders, and inflammatory diseases. Treatment options Wherever an inhibitor or a composition for use as a medicament is disclosed, a corresponding method for the manufacture or the production of a medicament comprising such an inhibitor or such a composition, a corresponding method of treatment comprising the administration of the inhibitor or composition to a subject in need thereof, and a corresponding use of such an inhibitor or such a composition as a medicament are also disclosed. In all these contexts, the inhibitor according to the invention and the composition according to the invention may be referred to as a medicament according to the invention. A medicament according to the invention may be administered orally, nasally, buccally, sublingually, vaginally, parenterally, topically, systemically, intravenously, subcutaneously, intraperitoneally, intramuscularly, intrathecally, by inhalation or epidurally. A medicament according to the invention, may be administered separately, sequentially or simultaneously in combination with another medicaments. As used herein, the term "simultaneous" therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time. The term "separate" therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes. The term "sequential" therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case. In the context of this application, the terms treating" or "treatment" refer to therapeutic treatment, wherein the object is to prevent, reduce, alleviate or slow down (lessen), respectively and as applicable, the targeted pathologic disorder or disease and/or its progression in a subject. In particular, said terms relate to a treatment which has the object of improving one or more symptoms and/or physiological parameters that are caused by, associated with and/or characteristic of the disease or disorder that is to be treated, and/or the object to preventing that such symptom(s) to arise and/or that such symptom(s) or physiological parameter(s) further deteriorate. Based on his general knowledge and the further disclosure herein, the skilled person (and in particular, the treating physician) will be able to suitably determine and measure said symptom(s) or physiological parameter(s), depending on the specific disease involved. In the context of this application, the terms "prevention" or "preventing" of a disorder or disease refers to a compound that, in a statistical sample, reduces the occurrence of symptoms of a disorder or disease in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. A medicament according to the invention is administered to a subject in need thereof in an effective amount (i.e., amount that have desired therapeutic effect). Preferably, an effective amount refers to an amount of an inhibitor according to the invention comprised in said medicament. The dose and dosage regimen will depend upon the degree of the infection in the subject, the characteristics of the particular inhibitor according to the invention, e.g., its therapeutic index, the subject, and the subject's history. Certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the medicaments according to the invention can include a single treatment or a series of treatments. The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of a peptide useful in the methods may be administered to a subject in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. Dosage, toxicity and therapeutic efficacy of a medicament according to the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Medicaments that exhibit high therapeutic indices are preferred. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any medicament according to the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Compositions In a further aspect, the invention provides a composition comprising an inhibitor according to the invention and a pharmaceutically acceptable excipient, preferably for use as a medicament, more preferably for use in the treatment of a disease characterized by lysosomal dysregulation. A related aspect pertains to a method of preparing such a composition. Such compositions are referred to in the current application as compositions according to or of the invention. All specific embodiments disclosed above for an inhibitor according to the invention may be applied accordingly for an inhibitor according to the invention comprised in a composition according to the invention. A composition according to the invention may be presented or formulated as capsules, tablets, powders, granules, solutions, suspensions in aqueous or non-aqueous liquids, edible, oil-in-water liquid emulsions, water-in-oil liquid emulsions, solution, syrups and elixirs, in microencapsulated form, liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles, transdermal patches, ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, drops, sprays, aerosols, oils, lozenges, pastilles, mouth washes, suppositories, enemas, aqueous and non-aqueous sterile injection solutions, and so on. It will be appreciated that the compositions may include other agents conventional in the art having regard to the type of formulation. Non-limiting examples of a pharmaceutically acceptable carrier comprised in a composition are saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds, besides an inhibitor according to the invention, can also be incorporated into the compositions. A composition according to the invention formulated as solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. A composition according to the invention formulated as compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. In a composition according to the invention prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. A composition according to the invention formulated as oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the inhibitor according to the invention can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. A composition according to the invention may be formulated for administration by inhalation, the inhibitor according to the invention can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. A composition according to the invention may be formulated for transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one aspect, transdermal administration may be performed by iontophoresis. A composition according to the invention may comprise a carrier system such as a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one aspect, the inhibitor according to the invention is encapsulated in a liposome. An inhibitor according to the invention can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems. Uses In a further aspect, the invention provides the use of a QPCTL inhibitor according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL. Preferably, the binding of QPCTL results in in the inhibition of QPCTL. The inhibition of QPCTL is described in more detail above. All specific embodiments disclosed above for an inhibitor according to the invention and a composition according to the invention may be applied accordingly for the uses and therapeutic uses described below. In embodiments, the use of a QPCTL inhibitor according to the invention or a composition according to the invention is for specifically binding and/or specifically inhibiting QPCTL. Specifically means that the binding or inhibition stems from the amino acid sequence dependent molecular interaction between the inhibitor, or the inhibitor comprised in the composition, and QPCTL. As such, the inhibitor or composition is not able to significantly bind or inhibit other enzymes that may share a similar function but a different amino acid structure. In embodiments, the use of a QPCTL inhibitor according to the invention or a composition according to the invention for binding and/or inhibiting QPCTL comprises a QPCTL inhibitor which specifically bind and/or specifically inhibit QPCTL. The inhibition of QPCTL can be expressed as the half maximal inhibitory concentration (IC50), as known to the skilled person. Lower IC50 values correspond with higher potencies to inhibit QPCTL. IC50 can be determined using the protocol of Example 2. In embodiments, the use of a QPCTL inhibitor according to the invention or a composition according to the invention for (specifically) inhibiting isoQPCTL comprises a QPCTL inhibitor having an IC 50 for QPCTL equal to or higher than 10 µm, 9.63 µm, 8.3 µm, 6.8 µm, 3.95 µm, 3.16 µm, 2.88 µm, 1.83 µm, 1.06 µm, 1.05 µm, 0.721 µm, 0.694 µm, 5.3 µm, 0.502 µm, 442 nm, 440 nm, 434 nm, 417 nm, 394 nm, 365 nm, 362 nm, 342 nm, 324 nm, 294 nm, 249 nm, 240 nm, 231 nm, 220 nm, 215 nm, 154 nm, 153 nm, 151 nm, 135 nm, or 52 nm, preferably as measured by the Fluorescent Enzymatic assay as described herein. In embodiments, the use of a QPCTL inhibitor according to the invention or a composition according to the invention for (specifically) inhibiting QPCTL comprises a QPCTL inhibitor having an IC50 for QPCTL equal to or higher than >10 µm, 9.54 µm, 2.37 µm, 0.653 µm, 1.49 µm, 0.623 µm, 451 nm, 142 nm, 103 nm, 70 nm, 66 nm, 65 nm, 61 nm, 57 nm, 52 nm, 49 nm, 48 nm, 42 nm, 38 nm, 34 nm, 31 nm, 30 nm, 25 nm, 23 nm, 22 nm, 18 nm, 17 nm, 16 nm, 14 nm, 10 nm or 1 nm, preferably as measured by the Fluorescent Enzymatic assay as described herein. In embodiments, the uses above are for use in vitro. In embodiments, the uses above are for use in vivo. The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of a peptide useful in the methods may be administered to a subject in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. Dosage, toxicity and therapeutic efficacy of a medicament according to the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Medicaments that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any medicament according to the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Definitions All documents cited in the present specification are hereby incorporated by reference in their entirety. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is made to the standard handbooks, to the general background art referred to above and to the further references cited therein. As used herein, the singular forms 'a', 'an', and 'the' include both singular and plural referents unless the context clearly dictates otherwise. The terms 'comprising', 'comprises' and 'comprised of' as used herein are synonymous with 'including', 'includes' or 'containing', 'contains', and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. Physiological conditions are defined in the context of this application as typical environmental conditions in a vertebrate, mammalian or human cell or tissue that is in homeostasis and is not subject to extraordinary external stress. Preferably, physiological conditions mean a temperature from 25°C up to 45°C, more preferably from 30°C up to 40°C. A concentration is preferably a molar concentration, preferably a molar concentration per weight or per volume, most preferably measured under physiological conditions. A subject is defined in the context of this application as a (living) organism, unless explicitly stated otherwise. A subject may be any organism, including invertebrates and vertebrates. Preferably, a subject is a vertebrate. More preferably, a vertebrate is a starfish or a mammal. Even more preferably, a mammal is a rat, a mouse, a rabbit or a human. Most preferably, a mammal is a human. In an alternative specific aspect, a subject is a non-human animal, more preferably a non-human vertebrate, most preferably a non-human mammal. A pseudohalogen is -CN, -CP, -NC, -OH, -SH, -SeH,-TeH, -OCN, -SCN, -NCS, -SeCN, -TeCN, -N3, -NO, or -NO2. Preferably, a pseudohalogen is -CN, -NC, -OH, -SH, -OCN, -SCN, -NCS, -N3, -NO, or -NO2. An acyl derivative bound to a parent molecule is a group comprising a C(=O)-XCO moiety, wherein XCO is a heteroatom, wherein the group is bound to the parent molecule via a bond to the explicit C in C(=O)-XCO. An organelle is preferably a lysosome or an endosome, more preferably a lysosome. An endosome is preferably a late endosome. An increase of a parameter by a factor equal to or higher than X is defined in the context of this application as a change of said parameter from its initial value A to a value equal to or higher than A*X. An increase of a parameter by a factor equal to or lower than X is defined in the context of this application as a change of said parameter from its initial value A to a value equal to or lower than A*X. A decrease of a parameter by a factor equal to or lower than X is defined in the context of this application as a change of said parameter from its initial value A to a value equal to or lower than A*X. A decrease of a parameter by a factor equal to or higher than X is defined in the context of this application as a change of said parameter from its initial value A to a value equal to or higher than A*X. A parameter that is essentially the same as in a corresponding composition, organelle, cell fraction, cell, membrane, tissue or organ derived from a healthy subject or as in a corresponding healthy subject, preferably means that the value of said parameter cannot be distinguished by a skilled person from the value of a corresponding parameter in a corresponding composition, organelle, cell fraction, cell, membrane, tissue or organ derived from a healthy subject or in a corresponding healthy subject, and/or that the value of said parameter would be interpreted by a skilled person as measured in a corresponding composition, organelle, cell fraction, cell, membrane, tissue or organ derived from a healthy subject or in a corresponding healthy subject. An alteration of a parameter which is significantly smaller after introduction of an inhibitor in a composition, organelle, cell fraction, cell, membrane, tissue, organ or subject preferably means that the absolute difference between the value of said parameter and the value of a corresponding parameter in a corresponding composition, organelle, cell fraction, cell, membrane, tissue or organ derived from a healthy subject or in a corresponding healthy subject is decreased by a factor equal to or lower than 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 after said introduction. Any parameter referred to herein is preferably determined using the specific method, assay or methodology described herein. Where the present specification does not mention or describe a specific method, assay or methodology for determining said parameter, said parameter can be measured in a manner suitable per se, as will be clear to the skilled person based upon reading the present disclosure. Each amino acid sequence described herein by virtue of its identity or similarity percentage (at least 60%) with a given amino acid sequence respectively has in a further specific aspect an identity or a similarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity or similarity with the given amino acid sequence respectively. In a specific aspect, sequence identity or similarity is determined by comparing the whole length of the sequences as identified herein. Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO means identity or similarity based on the full length of said sequence (i.e. over its whole length or as a whole). Sequence identity is defined in the context of this application as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The identity between two amino acid sequences is preferably defined by assessing their identity within a whole SEQ ID NO as identified herein or part thereof. Part thereof may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%. In the art, sequence identity also means the degree of sequence relatedness between amino acid sequences, as the case may be, as determined by the match between strings of such sequences. Sequence similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Sequence identity and similarity can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determine sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine sequence identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, FASTA, BLASTN, and BLASTP (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990)), EMBOSS Needle (Madeira, F., et al., Nucleic Acids Research 47(W1): W636-W641 (2019)). The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol.215:403-410 (1990)). The EMOSS program is publicly available from EMBL-EBI. The well-known Smith Waterman algorithm may also be used to determine identity. The EMBOSS Needle program is the preferred program used. Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48 (3):443-453 (1970); Comparison matrix: BLOSUM62 from Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Open Penalty: 10; and Gap Extend Penalty: 0.5. A program useful with these parameters is publicly available as the EMBOSS Needle program from EMBL -EBI. The aforementioned parameters are the default parameters for a Global Pairwise Sequence alignment o f proteins (along with no penalty for end gaps). Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: DNAfull; Gap Open Penalty: 10; Gap Extend Penalty: 0.5. A program useful with these parameters is publicly available as the EMBOSS Needle program from EMBL-EBI. The aforementioned parameters are the default parameters for a Global Pairwise Sequence alignment of nucleotide sequences (along with no penalty for end gaps). Optionally, in determining the degree of amino acid (sequence) similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having acidic side chains is aspartate and glutamate; and a group of amino acids having sulphur - containing side chains is cysteine and methionine. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys or Gln; Asn to Asp, His or Ser; Asp to Glu or Asn; Gln to Glu, Lys or Arg; Glu to Lys, Asp, Gln; His to Tyr or Asn; Ile to Leu, Val, or Met; Leu to Ile, Met or Val; Lys to Arg, Gln or Glu; Met to Val, Leu or Ile; Phe to Trp or Tyr; Ser to Thr, Ala or Asn; Thr to Ser; Trp to Tyr or Phe; Tyr to His, Trp or Phe; and Val to Ile, Leu or Met. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating one or more of the above-described embodiments. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein. Examples Several methods for the chemical synthesis of the QPCTL inhibitors are described herein. Examples of such methods are illustrated in the following schemes. Example 1: Synthetic Scheme Compound 1: 6- -5- 3- To a mixture of methyl 6-chloro-5-nitro-pyridine-3-carboxylate (1 g, 4.62 mmol, 1 eq) and pentan-3-amine (402.46 mg, 4.62 mmol, 538.04 uL, 1 eq) in MeCN (10 mL) was added K2CO3 (1.28 g, 9.23 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 10/1,TLC(Petroleum ether : Ethyl acetate=5:1,Rf=0.58)) to give methyl 6-(1-ethylpropylamino)-5-nitro-pyridine-3-carboxylate (1.18 g, 4.41 mmol, 95.62% yield) as yellow oil. Synthesis of methyl 5-amino-6-(1-ethylpropylamino)pyridine-3-carboxylate (3) To a solution of methyl 6-(1-ethylpropylamino)-5-nitro-pyridine-3-carboxylate (1.18 g, 4.41 mmol, 1 eq) in EtOAc (20 mL) was added Pd/C (120 mg, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give methyl 5-amino-6-(1-ethylpropylamino)pyridine-3-carboxylate (1 g, 4.21 mmol, 95.45% yield) as light yellow oil. Synthesis of methyl 3-(1-ethylpropyl)-2-oxo-1H-imidazo[4,5-b]pyridine-6-carboxylate (4) To a mixture of methyl 5-amino-6-(1-ethylpropylamino)pyridine-3-carboxylate (1 g, 4.21 mmol, 1 eq) in MeCN (30 mL) was added CDI (683.32 mg, 4.21 mmol, 1 eq). The mixture was stirred at 80 °C for 16 h. LCMS showed 56% of methyl 5-amino-6-(1-ethylpropylamino) pyridine-3-carboxylate remained and 35% of peak with desired mass. Then CDI (1.37 g, 8.43 mmol, 2 eq) was added to the reaction mixture. The mixture was stirred at 80 °C for 5 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 3/1, TLC (Petroleum ether : Ethyl acetate=3:1,Rf=0.23)) to give methyl 3-(1- ethylpropyl)-2-oxo-1H -imidazo[4,5-b]pyridine-6-carboxylate (1.1 g, 4.18 mmol, 99.14% yield) as white solid. Synthesis of [(3R)-1-[(2-fluoro-5-phenyl-phenyl)methyl]pyrrolidin-3-yl]-(3,4,6,7-tetra hydroimidazo[4,5-c]pyridin- 5-yl)methanone (5) To a mixture of methyl 3-(1-ethylpropyl)-2-oxo-1H-imidazo[4,5-b]pyridine-6-carboxylate (300 mg, 1.14 mmol, 1 eq), (3-ethylimidazol-4-yl)methanol (143.74 mg, 1.14 mmol, 1 eq) and PPh3 (448.28 mg, 1.71 mmol, 1.5 eq) in THF (4 mL) was added DIAD (345.60 mg, 1.71 mmol, 332.31 uL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 65 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed- phase HPLC (0.1% FA condition) followed by concentration give methyl 1-[(3- ethylimidazol-4-yl)methyl]-3-(1- ethylpropyl)-2-oxo-imidazo[4,5-b]pyridine-6-carboxylate (450 mg, crude) as yellow oil. Synthesis of 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo[4,5-b] pyridine-6-carboxylic acid (Compound 1) To a mixture of methyl 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo [4,5-b]pyridine-6- carboxylate (150 mg, 403.84 µmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH•H2O (33.89 mg, 807.68 µmol, 2 eq). The mixture was stirred at 25 °C for 2 h.. The reaction mixture was concentrated in vacuum to remove THF, the mixture was adjusted to pH about 3 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC(column: Unisil 3-100 C18 Ultra 150*50mm*3 um;mobile phase: [water(0.225%FA)-ACN];B%: 8%-38%,10min) followed by lyophilization to give 1-[(3-ethylimidazol-4-yl)methyl]-3- (1-ethylpropyl)-2-oxo-imidazo[4,5-b]pyridine-6-carboxylic acid (19.36 mg, 53.08 µmol, 13.14% yield, 98% purity) as white solid; m/z = (M + H)+ = 358.2; 1H NMR (400 MHz, DMSO-d6) δ = 13.23 - 12.97 (m, 1H), 8.57 (d, J = 1.9 Hz, 1H), 7.90 (d, J = 1.8 Hz, 1H), 7.67 (s, 1H), 6.96 (s, 1H), 5.20 (s, 2H), 4.27 (tt, J = 5.0, 10.1 Hz, 1H), 3.98 (q, J = 7.3 Hz, 2H), 2.22 - 2.09 (m, 2H), 1.89 - 1.76 (m, 2H), 1.16 (t, J = 7.2 Hz, 3H), 0.74 (t, J = 7.4 Hz, 6H) Synthetic Scheme Compound 2: Synthesis of methyl 6-anilino-5-nitro-pyridine-3-carboxylate (2) To a mixture of methyl 6-chloro-5-nitro-pyridine-3-carboxylate (1 g, 4.62mmol, 1 eq) and aniline (429.99 mg, 4.62 mmol, 421.56 uL, 1 eq) in MeCN (10 mL) was added K2CO3 (1.28 g, 9.23 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The mixture was filtered and the filtrate was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=200/1, 20/1, (TLC: Petroleum ether/Ethyl acetate=5/1)) to give methyl 6-anilino-5-nitro-pyridine-3-carboxylate (1 g, 3.66 mmol, 79.26% yield) as yellow solid. Synthesis of methyl 5-amino-6-anilino-pyridine-3-carboxylate (3) To a solution of methyl 6-anilino-5-nitro-pyridine-3-carboxylate (1 g, 3.66 mmol, 1 eq) in EtOAc (10 mL) was added Pd/C (100 mg, 365.97 µmol, 10% purity, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 25 °C for 16 h. The mixture was filtered and the filtrate was concentrated in vacuum to give methyl 5-amino-6-anilino-pyridine-3-carboxylate (900 mg, crude) as yellow oil. Synthesis of methyl 2-oxo-3-phenyl-1H-imidazo[4,5-b]pyridine-6-carboxylate (4) To a mixture of methyl 5-amino-6-anilino-pyridine-3-carboxylate (800 mg, 3.29 mmol, 1 eq) in MeCN (25 mL) was added CDI (533.25 mg, 3.29 mmol, 1 eq). The mixture was stirred at 60 °C for 2 h. LCMS showed 57% of methyl 5- amino-6-anilino-pyridine-3-carboxylate remained and 47% of peak with desired mass. Then CDI (266.63 mg, 1.64 mmol, 0.5 eq) was added to the reaction mixture. The mixture was stirred at 60 °C for 16 h. LCMS showed 38% of methyl 5- amino-6-anilino-pyridine-3-carboxylate remained and 60% of peak with desired mass. Then CDI (266.63 mg, 1.64 mmol, 0.5 eq) was added to the reaction mixture. The mixture was stirred at 60 °C for 5 h. LCMS showed 16% of methyl 5- amino-6-anilino-pyridine-3-carboxylate remained and 83% of peak with desired mass. Then CDI (266.63 mg, 1.64 mmol, 0.5 eq) was added to the reaction mixture. The mixture was stirred at 60 °C for 16 h. The reaction mixture was filtered and the filter cake was collected, dried under reduced pressure to give methyl 2-oxo-3-phenyl-1H-imidazo[4,5-b]pyridine- 6-carboxylate (600 mg, 2.23 mmol, 67.76% yield) as light purple solid. Synthesis of methyl 1-[(3-ethylimidazol-4-yl)methyl]-2-oxo-3-phenyl-imidazo[4,5-b] pyridine-6-carboxylate (5) To a mixture of methyl 2-oxo-3-phenyl-1H-imidazo[4,5-b]pyridine-6-carboxylate (100 mg, 371.40 µmol, 1 eq), (3- ethylimidazol-4-yl)methanol (46.85 mg, 371.40 µmol, 1 eq) and PPh3 (146.12 mg, 557.09 µmol, 1.5 eq) in THF (1 mL) was added DIAD (112.65 mg, 557.09 µmol, 108.32 uL, 1.5 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=0/1 to Ethyl acetate : Methanol=3/1) to give methyl 1-[(3-ethylimidazol-4-yl)methyl]-2-oxo-3-phenyl-imidazo[4,5-b]pyridine-6-carboxylate (40 mg, 105.99 µmol, 28.54% yield) as yellow solid. Synthesis of 1-[(3-ethylimidazol-4-yl)methyl]-2-oxo-3-phenyl-imidazo[4,5-b]pyridine-6- carboxylic acid (Compound 2) To a mixture of methyl 1-[(3-ethylimidazol-4-yl)methyl]-2-oxo-3-phenyl-imidazo[4,5-b] pyridine-6-carboxylate (40 mg, 105.99 µmol, 1 eq) in THF (1 mL) and H2O (0.3 mL) was added LiOH•H2O (8.90 mg, 211.98 µmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF. Then the mixture was acidified by 1N HCl and lots of solid formed. The resulting mixture was filtered and purified by prep-HPLC. The filter cake was purified by prep-HPLC(column: Unisil 3-100 C18 Ultra 150*50mm*3 um;mobile phase: [water(0.225%FA)-ACN];B%: 13%-33%,10min) and the filtrate was purified by prep-HPLC(column: Unisil 3-100 C18 Ultra 150*50mm*3 um;mobile phase: [water(0.225%FA)-ACN];B%: 10%-30%,10min),the two part were combined and followed by lyophilization to give 1-[(3-ethylimidazol-4-yl)methyl]-2-oxo-3-phenyl-imidazo[4,5-b]pyridine-6- carboxylic acid (22.16 mg, 58.55 µmol, 55.24% yield, 96% purity) as white solid; m/z = (M + H)+ = 364.1; 1H NMR (400 MHz, DMSO-d6) δ = 8.56 (d, J = 1.6 Hz, 1H), 8.02 (d, J = 1.8 Hz, 1H), 7.69 (s, 1H), 7.67 - 7.63 (m, 2H), 7.60 - 7.54 (m, 2H), 7.49 - 7.44 (m, 1H), 7.10 (s, 1H), 5.26 (s, 2H), 4.05 (q, J = 7.3 Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H) Synthetic Scheme Compound 3: 6- -5- 3- To a mixture of methyl 6-chloro-5-nitro-pyridine-3-carboxylate (500 mg, 2.31 mmol, 1 eq) and phenylmethanamine (247.38 mg, 2.31 mmol, 251.65 uL, 1 eq) in MeCN (10 mL) was added K2CO3 (638.13 mg, 4.62 mmol, 2 eq). The mixture was stirred at 80 °C for 16 h. The mixture was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 20/1,TLC(Petroleum ether : Ethyl acetate=10:1)) to give methyl 6-(benzylamino)-5-nitro-pyridine-3- carboxylate (630 mg, 2.19 mmol, 94.99% yield) as yellow oil. Synthesis of methyl 5-amino-6-(benzylamino)pyridine-3-carboxylate (3) To a solution of methyl 6-(benzylamino)-5-nitro-pyridine-3-carboxylate (630 mg, 2.19 mmol, 1 eq) in EtOAc (6 mL) was added Pd/C (63 mg, 219.31 µmol, 10% purity, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 2 h. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give methyl 5-amino-6-(benzylamino)pyridine-3-carboxylate (530 mg, 2.06 mmol, 93.93% yield) as white solid. Synthesis of methyl 3-benzyl-2-oxo-1H-imidazo[4,5-b]pyridine-6-carboxylate (4) To a mixture of methyl 5-amino-6-(benzylamino)pyridine-3-carboxylate (530 mg, 2.06 mmol, 1 eq) in MeCN (20 mL) was added CDI (334.02 mg, 2.06 mmol, 1 eq). The mixture was stirred at 60 °C for 2 h. LCMS showed 80% of methyl 5-amino-6-(benzylamino)pyridine-3-carboxylate remained and 19% of peak with desired mass. Then CDI (334.02 mg, 2.06 mmol, 1 eq) was added to the reaction mixture, the mixture was stirred at 60 °C for 16 h. The mixture was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 2/1) to give methyl 3-benzyl-2-oxo-1H- imidazo[4,5-b]pyridine-6-carboxylate (700 mg, crude) as white solid. Synthesis of methyl 3-benzyl-1-[(3-ethylimidazol-4-yl)methyl]-2-oxo-imidazo[4,5-b] pyridine-6-carboxylate (5) To a mixture of methyl 3-benzyl-2-oxo-1H-imidazo[4,5-b]pyridine-6-carboxylate (700 mg, 2.47 mmol, 1 eq), (3- ethylimidazol-4-yl)methanol (311.74 mg, 2.47 mmol, 1 eq) and PPh3 (972.18 mg, 3.71 mmol, 1.5 eq) in THF (10 mL) was added DIAD (749.50 mg, 3.71 mmol, 720.67 uL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 60 °C for 16 h. The mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA condition) followed by concentration to give methyl 3-benzyl-1-[(3-ethylimidazol-4-yl) methyl]-2-oxo- imidazo[4,5-b]pyridine-6-carboxylate (90 mg, 229.93 µmol, 9.31% yield) as yellow solid. Synthesis of 3-benzyl-1-[(3-ethylimidazol-4-yl)methyl]-2-oxo-imidazo[4,5-b]pyridine-6- carboxylic acid (Compound 3) To a mixture of methyl 3-benzyl-1-[(3-ethylimidazol-4-yl)methyl]-2-oxo-imidazo [4,5-b]pyridine-6-carboxylate (90 mg, 229.93 µmol, 1 eq) in THF (3 mL) was added LiOH•H2O (9.65 mg, 229.93 µmol, 1 eq) in H2O (1 mL). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated in vacuum to remove THF, the resulting mixture was adjusted pH to about 3 with 1M HCl and lots of solid formed. The mixture was filtered and the filter cake was collected, followed by lyophilization to give 3-benzyl-1-[(3-ethylimidazol-4-yl)methyl]-2- oxo-imidazo[4,5-b]pyridine-6-carboxylic acid (27.01 mg, 71.57 µmol, 31.13% yield, 100% purity) as white solid; m/z = (M + H)+ = 378.2; 1H NMR (400 MHz, DMSO- d6) δ = 8.60 (d, J = 1.5 Hz, 1H), 7.93 (d, J = 1.5 Hz, 1H), 7.67 (br s, 1H), 7.38 - 7.22 (m, 5H), 7.02 (br s, 1H), 5.22 (s, 2H), 5.12 (s, 2H), 3.98 (q, J = 7.2 Hz, 2H), 1.11 (t, J = 7.2 Hz, 3H) Synthetic Scheme Compound 4: 5- N- -3- 2- To a mixture of 5-bromo-2-fluoro-3-nitro-pyridine (1 g, 4.53 mmol, 1 eq) and pentan-3-amine (394.43 mg, 4.53 mmol, 527.31 uL, 1 eq) in MeCN (10 mL) was added K2CO3 (1.25 g, 9.05 mmol, 2 eq). The mixture was stirred at 80 °C for 16 h. The mixture was filtered and the filtrate was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 20/1) followed by concentration to give 5-bromo-N-(1-ethylpropyl)- 3-nitro-pyridin-2-amine (1.25 g, 4.34 mmol, 95.87% yield) as yellow oil. Synthesis of N-(1-ethylpropyl)-3-nitro-5-phenyl-pyridin-2-amine (3) To a mixture of 5-bromo-N-(1-ethylpropyl)-3-nitro-pyridin-2-amine (1.25 g, 4.34 mmol, 1 eq) and phenylboronic acid (634.74 mg, 5.21 mmol, 1.2 eq) in dioxane (9 mL) and H2O (3 mL) was added Pd(dppf)Cl2 (317.43 mg, 433.82 µmol, 0.1 eq) and Na2CO3 (919.59 mg, 8.68 mmol, 2 eq) under N2. The mixture was stirred at 80 °C for 16 h under N2. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 20/1,TLC(Petroleum ether : Ethyl acetate=10:1)) to give N-(1-ethylpropyl)-3-nitro-5- phenyl-pyridin-2-amine (1.2 g, 4.21 mmol, 96.94% yield) as yellow oil. Synthesis of N2-(1-ethylpropyl)-5-phenyl-pyridine-2,3-diamine (4) To a solution of N-(1-ethylpropyl)-3-nitro-5-phenyl-pyridin-2-amine (1.2 g, 4.21 mmol, 1 eq) in EtOAc (12 mL) was added Pd/C (120 mg, 420.55 µmol, 10% purity, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give N2-(1-ethylpropyl)-5-phenyl-pyridine-2,3-diamine (1 g, 3.92 mmol, 93.12% yield) as yellow oil. Synthesis of 3-(1-ethylpropyl)-6-phenyl-1H-imidazo[4,5-b]pyridin-2-one (5) To a mixture of N2-(1-ethylpropyl)-5-phenyl-pyridine-2,3-diamine (200 mg, 783.22 µmol, 1 eq) in MeCN (10 mL) was added CDI (127.00 mg, 783.22 µmol, 1 eq). The mixture was stirred at 60 °C for 2 h. LCMS showed 30% of N2-(1- ethylpropyl)-5-phenyl-pyridine-2,3-diamine remained and 67% of peak with desired mass. Then CDI (127.00 mg, 783.22 µmol, 1 eq) was added to the reaction mixture, the mixture was stirred at 60 °C for 16 h. The mixture was filtered and the filter cake was collected, dried under reduce pressure to give 3-(1-ethylpropyl)-6-phenyl-1H- imidazo[4,5-b]pyridin-2- one (110 mg, 390.97 µmol, 49.92% yield) as white solid. Synthesis of 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-6-phenyl-imidazo[4,5-b] pyridin-2-one (Compound 4) To a mixture of 3-(1-ethylpropyl)-6-phenyl-1H-imidazo[4,5-b]pyridin-2-one (110 mg, 390.97 µmol, 1 eq) and (3- ethylimidazol-4-yl)methanol (49.32 mg, 390.97 µmol, 1 eq) in THF (1 mL) was added PPh3 (153.82 mg, 586.46 µmol, 1.5 eq). Then DIAD (118.59 mg, 586.46 µmol, 114.02 uL, 1.5 eq) was added to the reaction mixture in portions at 0 °C. The mixture was stirred at 65 °C for 16 h. The mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50mm*3 um;mobile phase: [water(0.225%FA)-ACN];B%: 22%-42%,10min) followed by lyophilization to give a residue. The residue was purified by prep-HPLC(column: Phenomenex Gemini-NX C1875*30mm*3um;mobile phase: [water (0.05% ammonia hydroxide v/v)-ACN];B%: 37%- 67%,11.5min) followed by lyophilization to give 1-[(3-ethylimidazol-4-yl)methyl]-3-(1- ethylpropyl)-6-phenyl- imidazo[4,5-b]pyridin-2-one (12.46 mg, 31.99 µmol, 8.18% yield, 100% purity) as white solid; m/z = (M + H)+ = 390.2; 1H NMR (400 MHz, DMSO-d6) δ = 8.27 (d, J = 1.9 Hz, 1H), 7.78 (d, J = 2.0 Hz, 1H), 7.68 - 7.60 (m, 3H), 7.48 (t, J = 7.6 Hz, 2H), 7.42 - 7.35 (m, 1H), 7.08 (s, 1H), 5.18 (s, 2H), 4.24 (td, J = 5.0, 10.1 Hz, 1H), 4.02 (q, J = 7.3 Hz, 2H), 2.24 - 2.11 (m, 2H), 1.89 - 1.76 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H), 0.75 (t, J = 7.3 Hz, 6H) Synthetic Scheme Compound 5, Compound 6 and Compound 7: 6- -5- 3- To a mixture of methyl 6-chloro-5-nitro-pyridine-3-carboxylate (1 g, 4.62 mmol, 1 eq) and pentan-3-amine (402.46 mg, 4.62 mmol, 538.04 uL, 1 eq) in MeCN (10 mL) was added K2CO3 (1.28 g, 9.23 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The mixture was filtered and the filtrate was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 5/1,TLC(Petroleum ether : Ethyl acetate=5:1)) to give methyl 6-(1-ethylpropylamino)-5-nitro- pyridine-3-carboxylate (1.2 g, 4.49 mmol, 97.24% yield) as yellow oil. Synthesis of methyl 5-amino-6-(1-ethylpropylamino)pyridine-3-carboxylate (3) To a solution of methyl 6-(1-ethylpropylamino)-5-nitro-pyridine-3-carboxylate (1.2 g, 4.49 mmol, 1 eq) in EtOAc (12 mL) was added Pd/C (120 mg, 448.97 µmol, 10% purity, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 16 h. The mixture was filtered and the filtrate was concentrated in vacuum to give methyl 5-amino-6-(1-ethylpropylamino)pyridine-3-carboxy late (570 mg, 2.40 mmol, 53.50% yield) as yellow oil. Synthesis of methyl 3-(1-ethylpropyl)-2-oxo-1H-imidazo[4,5-b]pyridine-6-carboxylate (4) To a mixture of methyl 5-amino-6-(1-ethylpropylamino)pyridine-3-carboxylate (570 mg, 2.40 mmol, 1 eq) in MeCN (18 mL) was added CDI (389.49 mg, 2.40 mmol, 1 eq). The mixture was stirred at 80 °C for 2 h. LCMS showed 44% of methyl 5-amino-6-(1-ethylpropylamino) pyridine-3-carboxylate remained and 51% of peak with desired mass. Then CDI (389.49 mg, 2.40 mmol, 1 eq) was added to the reaction mixture. The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 3/1) to give methyl 3-(1- ethylpropyl)-2-oxo-1H-imidazo [4,5-b]pyridine-6-carboxylate (600 mg, 2.28 mmol, 94.87% yield) as white solid. Synthesis of methyl 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo [4,5-b]pyridine-6- carboxylate (5) To a mixture of methyl 3-(1-ethylpropyl)-2-oxo-1H-imidazo[4,5-b]pyridine-6-carboxylate (500 mg, 1.90 mmol, 1 eq), (3-ethylimidazol-4-yl)methanol (239.57 mg, 1.90 mmol, 1 eq) and PPh3 (747.14 mg, 2.85 mmol, 1.5 eq) in THF (1 mL) was added DIAD (576.00 mg, 2.85 mmol, 553.85 uL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 65 °C for 5 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed- phase HPLC (0.1% FA condition) followed by lyophilization to give methyl 1-[(3- ethylimidazol-4-yl)methyl]-3-(1- ethylpropyl)-2-oxo-imidazo[4,5-b]pyridine-6-carboxylate (400 mg, 1.08 mmol, 56.71% yield) as yellow oil. Synthesis of 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo[4,5-b] pyridine-6-carboxamide (Compound 7) To a mixture of methyl 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo [4,5-b]pyridine-6- carboxylate (100 mg, 269.23 µmol, 1 eq) in THF (1 mL) was added NH3•H2O (2.76 g, 25.97 mmol, 3.03 mL, 33% purity, 96.45 eq). The mixture was stirred at 100 °C for 16 h in a sealed tube. The mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC(column: Unisil 3-100 C18 Ultra 150*50mm*3 um;mobile phase: [water(0.225%FA)-ACN];B%: 13%-33%,10min) followed by lyophilization to give 1-[(3-ethylimidazol-4-yl)methyl]-3- (1-ethylpropyl)-2-oxo-imidazo[4,5-b]pyridine-6-carboxamide (10.97 mg, 27.70 µmol, 10.29% yield, 90% purity) as white solid; m/z = (M + H)+ = 357.2; 1H NMR (400 MHz, CD3OD) δ = 8.58 (s, 1H), 8.34 - 8.18 (m, 1H), 7.90 (br s, 2H), 7.23 (br s, 1H), 5.25 (br s, 2H), 4.43 - 4.30 (m, 1H), 4.16 (br d, J = 6.7 Hz, 2H), 2.37 - 2.21 (m, 2H), 1.94 - 1.84 (m, 2H), 1.32 (br t, J = 7.1 Hz, 3H), 0.82 (br t, J = 6.7 Hz, 6H). Synthesis of 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo[4,5-b] pyridine-6-carboxylic acid (6) To a mixture of methyl 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo [4,5-b]pyridine-6- carboxylate (300 mg, 807.68 µmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH•H2O (67.79 mg, 1.62 mmol, 2 eq). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC(0.1% FA condition) followed by concentration to give 1-[(3-ethylimidazol-4- yl)methyl]-3- (1-ethylpropyl)-2-oxo-imidazo[4,5-b]pyridine-6-carboxylic acid (200 mg, 559.59 µmol, 69.28% yield) as yellow oil. Synthesis of 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-N,N-dimethyl-2-oxo- imidazo[4,5-b]pyridine-6- carboxamide (Compound 5) To a mixture of 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo[4,5-b] pyridine-6-carboxylic acid (100 mg, 279.79 µmol, 1 eq) and N-methylmethanamine (27.38 mg, 335.75 µmol, 30.76 uL, 1.2 eq, HCl) in DMF (1 mL) was added HATU (159.58 mg, 419.69 µmol, 1.5 eq) and DIEA (108.48 mg, 839.38 µmol, 146.21 uL, 3 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was purified by prep-HPLC(column: Phenomenex Gemini- NX C1875*30mm*3um;mobile phase: [water (0.05% ammonia hydroxide v/v)-ACN];B%: 13%-43%,7min) followed by lyophilization to give 1-[(3-ethylimidazol-4-yl)methyl]-3- (1-ethylpropyl)-N,N-dimethyl-2-oxo-imidazo[4,5-b]pyridine- 6-carboxamide (18.07 mg, 47.00 µmol, 16.80% yield, 100% purity) as yellow solid; m/z = (M + H)+ = 385.2; 1H NMR (400 MHz, DMSO-d6) δ = 8.05 (d, J = 1.7 Hz, 1H), 7.66 (d, J = 0.9 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.02 (s, 1H), 5.14 (s, 2H), 4.23 (td, J = 5.1, 10.1 Hz, 1H), 4.00 (q, J = 7.2 Hz, 2H), 2.96 (br s, 6H), 2.21 - 2.09 (m, 2H), 1.82 (ddd, J = 5.1, 7.4, 13.7 Hz, 2H), 1.17 (t, J = 7.3 Hz, 3H), 0.74 (t, J = 7.3 Hz, 6H) Synthesis of 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-N-methyl-2-oxo-imidazo [4,5-b]pyridine-6- carboxamide (Compound 6) To a mixture of 1-[(3-ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-2-oxo-imidazo[4,5-b] pyridine-6-carboxylic acid (100 mg, 279.79 µmol, 1 eq) and MeNH2 (2 M, 209.85 uL, 1.5 eq) in DMF (1 mL) was added HATU (127.66 mg, 335.75 µmol, 1.2 eq) and DIEA (72.32 mg, 559.59 µmol, 97.47 uL, 2 eq). The mixture was stirred at 25 °C for 2 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30mm*3um;mobile phase: [water (0.05% ammonia hydroxide v/v)-ACN];B%: 10%-40%,7min) followed by lyophilization to give 1-[(3- ethylimidazol-4-yl)methyl]-3-(1-ethylpropyl)-N-methyl-2-oxo-imidazo[4,5-b]pyridine-6-carboxamide (18.37 mg, 49.59 µmol, 17.72% yield, 100% purity) as yellow oil; m/z = (M + H)+ = 371.2; 1H NMR (400 MHz, DMSO-d6) δ = 8.53 - 8.41 (m, 2H), 7.85 (d, J = 1.8 Hz, 1H), 7.66 (d, J = 0.9 Hz, 1H), 7.00 (s, 1H), 5.15 (s, 2H), 4.25 (tt, J = 4.9, 10.1 Hz, 1H), 4.01 (q, J = 7.2 Hz, 2H), 2.79 (d, J = 4.4 Hz, 3H), 2.22 - 2.06 (m, 2H), 1.82 (ddd, J = 5.1, 7.4, 13.8 Hz, 2H), 1.17 (t, J = 7.2 Hz, 3H), 0.72 (t, J = 7.3 Hz, 6H)
Synthetic Scheme Compound 8: Synthesis of methyl 6-(1-ethylpropylamino)-5-nitro-pyridine-3-carboxylate (2) To a mixture of methyl 6-chloro-5-nitro-pyridine-3-carboxylate (3.86 g, 17.82 mmol, 1 eq) and pentan-3-amine (1.55 g, 17.82 mmol, 2.08 mL, 1 eq) in CH3CN (15 mL) was added K2CO3 (4.92 g, 35.63 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The mixture was filtered and the filtrate was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate=20/1, 10/1, TLC (Petroleum ether : Ethyl acetate=5:1; Rf=0.7)) to give methyl 6-(1-ethylpropylamino)- 5-nitro-pyridine-3-carboxylate (3.3 g, 12.35 mmol, 69.30% yield) as yellow oil. Synthesis of methyl 5-amino-6-(1-ethylpropylamino)pyridine-3-carboxylate (3) To a solution of methyl 6-(1-ethylpropylamino)-5-nitro-pyridine-3-carboxylate (3.3 g, 12.35 mmol, 1 eq) in EtOAc (20 mL) was added (wet) Pd/C (300 mg, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 16 h. The mixture was filtered and the filtrate was dried under reduced pressure to give methyl 5-amino-6-(1-ethylpropylamino)pyridine-3-carboxylate (3.4 g, crude) as yellow oil. Synthesis of methyl 3-(1-ethylpropyl)-2-oxo-1H-imidazo [4,5-b]pyridine-6-carboxylate (4) To a mixture of methyl 5-amino-6-(1-ethylpropylamino) pyridine-3-carboxylate (3.4 g, 14.33 mmol, 1 eq) in MeCN (50 mL) was added CDI (4.65 g, 28.66 mmol, 2 eq). The mixture was stirred at 80 °C for 2 h. The mixture was diluted with EtOAc (40 mL) and the resulting mixture was washed with water (30 mL*2) and brine (30 mL), dried over Na2SO4, filtered and concentrated at reduced pressure to give methyl 3-(1-ethylpropyl)-2-oxo-1H-imidazo [4,5-b]pyridine-6- carboxylate (3.4 g, 12.91 mmol, 90.13% yield) as yellow solid. Synthesis of methyl 3-(1-ethylpropyl)-1-(1H-imidazol-5-ylmethyl)-2-oxo-imidazo [4,5-b] pyridine-6-carboxylate (5) To a solution of methyl 3-(1-ethylpropyl)-2-oxo-1H-imidazo[4,5-b]pyridine-6-carboxylate (200 mg, 759.61 µmol, 1 eq) and 1H-imidazol-5-ylmethanol (81.97 mg, 835.57 µmol, 1.1 eq) in THF (3 mL) was added PPh3 (298.85 mg, 1.14 mmol, 1.5 eq) and DIAD (230.40 mg, 1.14 mmol, 221.54 uL, 1.5 eq) under N2. The mixture was stirred at 60 °C for 16 h. The mixture was concentrated at reduced pressure to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA condition) followed by lyophilization to give methyl 3-(1-ethylpropyl)-1-(1H-imidazol-5-ylmethyl)-2-oxo- imidazo [4,5-b]pyridine-6-carboxylate (300 mg, crude) as white solid. Synthesis of 3-(1-ethylpropyl)-1-(1H-imidazol-5-ylmethyl)-2-oxo-imidazo [4,5-b] pyridine-6-carboxylic acid (Compound 8) To a solution of methyl 3-(1-ethylpropyl)-1-(1H-imidazol-5-ylmethyl)-2-oxo-imidazo[4,5-b] pyridine-6-carboxylate (100 mg, 291.22 µmol, 1 eq) in THF (2 mL) and H2O (1 mL) was added LiOH (13.95 mg, 582.45 µmol, 2 eq). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated at reduced pressure to give a residue. The residue was diluted with water (20 mL) and the pH was adjusted to 5 with 1M HCl. Then the resulting mixture was extracted with EtOAc (10 mL*3). The combined organic phase was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated at reduced pressure to give a residue. The reaction mixture was purified by prep-HPLC (column: Shim-pack C18 150*25*10um; mobile phase: [water (0.225%FA)-ACN];B%: 11%-31%,10min) to give 3-(1-ethylpropyl)-1-(1H- imidazol- 5-ylmethyl)- 2-oxo-imidazo [4,5-b]pyridine-6-carboxylic acid (5.82 mg, 17.67 µmol, 6.07% yield, 100% purity) as white solid; m/z = (M + H)+ = 330.4; 1H NMR (400 MHz, CD3OD) δ = 8.66 (s, 1H), 7.90 (s, 1H), 7.67 (s, 1H), 7.11 (s, 1H), 5.11 (s, 2H), 4.36 (dd, J = 5.2, 9.7 Hz, 1H), 2.34 - 2.19 (m, 2H), 1.96 - 1.83 (m, 2H), 0.82 (t, J = 7.5 Hz, 6H). Synthetic Scheme Compound 9: Synthesis of methyl 3-(1-ethylpropyl)-1-(1H-imidazol-2-ylmethyl)-2-oxo-imidazo [4,5-b]pyridine-6-carboxylate (2) To a solution of methyl 3-(1-ethylpropyl)-2-oxo-1H-imidazo[4,5-b]pyridine-6-carboxylate (500 mg, 1.90 mmol, 1 eq) and 1H-imidazol-2-ylmethanol (204.93 mg, 2.09 mmol, 1.1 eq) in THF (10 mL) was added DIAD (576.01 mg, 2.85 mmol, 553.85 uL, 1.5 eq) and PPh3 (747.15 mg, 2.85 mmol, 1.5 eq) under N2. The mixture was stirred at 60 °C for 16 h. The mixture was concentrated at reduced pressure to give a residue. The reaction mixture was purified by prep- HPLC(column: Phenomenex luna C18150*40mm* 15um;mobile phase: [water(0.225%FA)-ACN];B%: 7%-37%,10min) to give methyl 3-(1-ethylpropyl)-1-(1H-imidazol-2-ylmethyl)-2-oxo-imidazo [4,5-b]pyridine-6-carboxylate (120 mg, 349.47 µmol, 18.40% yield) as yellow oil. Synthesis of 3-(1-ethylpropyl)-1-(1H-imidazol-2-ylmethyl)-2-oxo-imidazo[4,5-b]pyridine -6-carboxylic acid (Compound 9) To a solution of methyl 3-(1-ethylpropyl)-1-(1H-imidazol-2-ylmethyl)-2-oxo-imidazo [4,5-b]pyridine-6-carboxylate (100 mg, 291.22 µmol, 1 eq) in THF (2 mL) and H2O (1 mL) was added LiOH (13.95 mg, 582.45 µmol, 2 eq). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated at reduced pressure to give a residue. The residue was diluted with water (2 mL) and the pH was adjusted to 5 with 1M HCl, the mixture was concentrated at reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Shim-pack C18 150*25*10um;mobile phase: [water(0.225%FA)-ACN];B%: 10%-30%,10min) to give 3-(1-ethylpropyl)-1-(1H-imidazol-2-ylmethyl)-2-oxo- imidazo[4,5-b]pyridine-6-carboxylic acid (37.55 mg, 114.01 µmol, 39.15% yield, 100% purity) as white solid; m/z = (M + H)+ = 330.2; 1H NMR (400 MHz, CD3OD) δ = 8.67 (d, J = 1.6 Hz, 1H), 7.83 (d, J = 1.6 Hz, 1H), 7.06 (s, 2H), 5.22 (s, 2H), 4.36 (tt, J = 5.2, 10.1 Hz, 1H), 2.34 - 2.18 (m, 2H), 1.96 - 1.83 (m, 2H), 0.84 (t, J = 7.5 Hz, 6H). Synthetic Scheme Compound 10: 3 To a mixture of compound 1 (0.5 g, 2.51 mmol, 1 eq) and compound 2 (218.85 mg, 2.51 mmol, 292.58 μL, 1 eq) in MeCN (10 mL) was added K2CO3 (694.03 mg, 5.02 mmol, 2 eq). The mixture was stirred at 60 °C for 5 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 9/1) to give compound 3 (650 mg, 2.44 mmol, 97.22% yield) as yellow oil. LCMS: RT = 0.943 min, m/z = 267.1 (M+H)+. Synthesis of compound 4 To a solution of compound 3 (650 mg, 2.44 mmol, 1 eq) in EtOAc (7 mL) was added Pd/C (65 mg, 10%) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 25 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give compound 4 (570 mg, 2.41 mmol, 98.82% yield) as red oil. LCMS: RT = 0.743 min, m/z = 237.2 (M+H)+. Synthesis of compound 5 To a mixture of compound 4 (570 mg, 2.41 mmol, 1 eq) in MeCN (6 mL) was added CDI (391.12 mg, 2.41 mmol, 1 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was dissolved in ethyl acetate (10 mL) and washed with H2O (3 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 5 (600 mg, 2.29 mmol, 94.83% yield) as red solid. LCMS: RT = 0.771 min, m/z = 263.1 (M+H)+. Synthesis of compound 7 To a mixture of compound 5 (100 mg, 381.24 μmol, 1 eq), compound 6 (48.10 mg, 381.24 μmol, 1 eq) and PPh3 (149.99 mg, 571.86 μmol, 1.5 eq) in THF (2 mL) was added DIAD (115.63 mg, 571.86 μmol, 111.19 μL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA) followed by concentration to give compound 7 (130 mg, 350.93 μmol, 92.05% yield) as yellow oil. LCMS: RT = 0.705 min, m/z = 371.2 (M+H)+. Synthesis of Compound 10 To a mixture of compound 7 (130 mg, 350.93 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (16.81 mg, 701.86 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF, the mixture was acidified to pH =6 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC(column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%- 35%, 10 min) followed by lyophilization to give Compound 10 (18.45 mg, 50.73 μmol, 14.46% yield, 98% purity) as yellow solid.1H NMR: (400 MHz, CD3OD) δ = 7.91 (br s, 1H), 7.87 - 7.81 (m, 2H), 7.35 (d, J = 8.3 Hz, 1H), 7.20 (br s, 1H), 5.26 (s, 2H), 4.26 (tt, J = 5.0, 10.2 Hz, 1H), 4.16 (q, J = 7.3 Hz, 2H), 2.12 (qdd, J = 7.2, 10.3, 14.3 Hz, 2H), 1.97 - 1.83 (m, 2H), 1.31 (t, J = 7.3 Hz, 3H), 0.83 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.685 min, m/z = 357.2 (M+H)+. Synthetic Scheme Compound 11: Synthesis of compound 2 A solution of compound 1 (2 g, 13.93 mmol, 1 eq) and CDI (2.71 g, 16.72 mmol, 1.2 eq) in CH3CN (20 mL) was stirred at 80 °C for 16 h. The mixture was filtered and the filter cake was washed with water (10 mL) and MeCN (10 mL). Then the filter cake was dried at reduced pressure to give a residue to give compound 2 (2.3 g, 13.56 mmol, 97.37% yield) as brown solid. Synthesis of compound 3 To a solution of compound 2A (2.05 g, 13.56 mmol, 1.68 mL, 1 eq) in DMF (20 mL) was added Cs2CO3 (13.26 g, 40.69 mmol, 3 eq) and compound 2 (2.3 g, 13.56 mmol, 1 eq). The mixture was stirred at 100 °C for 16 hr. The reaction mixture was diluted with H2O (50 mL) and was extracted with ethyl acetate (50 mL*3), the combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 5/1, 3/1. TLC (Petroleum ether : Ethyl acetate = 1:1; Rf = 0.49) ) to give yellow solid. The solid was purified by reversed-phase HPLC (0.1% FA condition)) to give compound 3 (300 mg, 1.25 mmol, 9.23% yield) as yellow solid.1H NMR: (400 MHz, DMSO-d6,) δ = 7.60 (d, J = 8.2 Hz, 1H), 7.04 (d, J = 8.1 Hz, 1H), 4.07 (tt, J = 5.1, 10.2 Hz, 1H), 1.97 - 1.83 (m, 2H), 1.82 - 1.68 (m, 2H), 0.72 (t, J = 7.4 Hz, 6H). Synthesis of compound 4 To a solution of compound 3A (168.91 mg, 1.17 mmol, 1 eq) in DMF (5 mL) was added Cs2CO3 (1.14 g, 3.50 mmol, 3 eq) and compound 3 (280 mg, 1.17 mmol, 1 eq). The mixture was stirred at 40 °C for 4 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (10 mL*3) and the combined organic phase was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated at reduced pressure to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 1/1, 0/1. TLC (Petroleum ether : Ethyl acetate = 1:1; Rf = 0.21) ) to give compound 4 (250 mg, crude) as colorless oil. Synthesis of compound 5 To a solution of compound 4 (150 mg, 431.23 μmol, 1 eq), TEA (130.91 mg, 1.29 mmol, 180.07 μL, 3 eq) in MeOH (10 mL) was added Pd(dppf)Cl2 (31.55 mg, 43.12 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (50 psi) at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 1/1, 0/1. TLC (Dichloromethane : Methanol = 10:1; Rf = 0.38)) to give a brown oil. The oil was purified by prep-TLC to give compound 5 (30 mg, 80.77 μmol, 18.73% yield) as brown oil. Synthesis of Compound 11 To a mixture of compound 5 (30 mg, 80.77 μmol, 1 eq) in THF (2 mL) and H2O (1 mL) was added LiOH (3.87 mg, 161.54 μmol, 2 eq). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated at reduced pressure to give a residue. The residue was diluted with water (10 mL) and the pH was adjusted to 5 with 1M HCl, then the mixture was concentrated at reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um;mobile phase: [water (FA) -ACN];B%: 1%-30%, 10 min) to give Compound 11 (9.26 mg, 25.91 μmol, 32.08% yield) as yellow gum. LCMS: RT = 0.636 min, m/z = 358.2 (M+H)+; 1H NMR: (400 MHz, CD3OD) δ = 7.97 - 7.86 (m, 2H), 7.64 (d, J = 8.1 Hz, 1H), 7.17 (br s, 1H), 5.40 - 5.26 (m, 2H), 4.38 (q, J = 7.2 Hz, 2H), 4.30 (s, 1H), 2.13 - 1.97 (m, 2H), 1.95 - 1.82 (m, 2H), 1.37 (t, J = 7.3 Hz, 3H), 0.81 (t, J = 7.4 Hz, 6H). Synthetic Scheme Compound 12:
Experimental Procedure Compound 12: Synthesis of compound 2 A mixture of compound 1 (670 mg, 4.67 mmol, 1 eq) and CDI (908.02 mg, 5.60 mmol, 1.2 eq) in ACN (8 mL) was stirred at 80 °C for 16 h. The mixture was filtered and the filter cake was washed with water (10 mL) and MeCN (10 mL). Then the filter cake was dried at reduced pressure to give compound 2 (770 mg, 4.54 mmol, 97.31% yield) as brown solid.1H NMR: (400 MHz, DMSO-d6) δ = 11.33 (br s, 1H), 11.07 (br s, 1H), 7.90 (s, 1H), 7.00 (s, 1H). Synthesis of compound 3 To a solution of compound 2 (1.1 g, 6.49 mmol, 1 eq) in DMF (2 mL) was added NaH (570.81 mg, 14.27 mmol, 60% purity, 2.2 eq) at 0 °C, then 3-bromopentane (979.83 mg, 6.49 mmol, 803.14 μL, 1 eq) was added. The mixture was stirred at 80 °C for 16 h. The reaction mixture was diluted with Ethyl acetate (100 mL). The organic Layer was washed with brine (50 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18150*25 mm* 10um; mobile phase: [water (FA) - ACN]; B%: 30%-60%, 10 min) to give compound 3 (130 mg, 542.34 μmol, 8.36% yield) as brown solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.23 (s, 1H), 7.97 (s, 1H), 7.41 (s, 1H), 7.07 (s, 1H), 4.07 (tt, J = 5.1, 10.2 Hz, 1H), 1.93 (qdd, J = 7.3, 10.2, 14.2 Hz, 2H), 1.83 - 1.69 (m, 2H), 0.72 (t, J = 7.4 Hz, 6H); Synthesis of compound 4 To a solution of compound 3 (130 mg, 542.34 μmol, 1 eq) and Cs2CO3 (530.12 mg, 1.63 mmol, 3 eq) in DMF (3 mL) was added 5-(chloromethyl) -1-ethyl-imidazole (117.64 mg, 813.51 μmol, 1.5 eq). The mixture was stirred at 40 °C for 16 h. The reaction mixture was diluted with water (30 mL). The organic layer was extracted with ethyl acetate (50 mL) and washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by prep-TLC (ethyl acetate: ethyl alcohol = 30: 1 to give compound 4 (90 mg, 258.74 μmol, 47.71% yield) as yellow gum. Synthesis of compound 5 To a mixture of compound 4 (80 mg, 229.99 μmol, 1 eq), Et3N (69.82 mg, 689.97 μmol, 96.03 μL, 3 eq) in MeOH (5 mL) was added Pd(dppf)Cl2 (16.83 mg, 23.00 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (15 psi) at 80 °C for 16 hours. The reaction mixture was concentrated under vacuum to give a residue. The residue was diluted with ethyl acetate (50 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give compound 5 (100 mg, crude) as yellow gum. Synthesis of Compound 12 To a solution of compound 5 (100 mg, 269.23 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (19.34 mg, 807.68 μmol, 3 eq). The mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was adjusted pH = 5 with 1 M HCl. Then the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-15%, 10 min) to give compound 12 (16.04 mg, 44.88 μmol, 16.67% yield, 100% purity) as yellow solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.71 - 8.55 (m, 1H), 7.94 - 7.84 (m, 1H), 7.71 - 7.62 (m, 1H), 6.93 - 6.85 (m, 1H), 5.21 (s, 2H), 4.27 - 4.14 (m, 1H), 4.03 - 3.94 (m, 2H), 2.04 - 1.65 (m, 4H), 1.23 - 1.10 (m, 3H), 0.81 - 0.57 (m, 6H); LCMS: RT = 0.259 min, m/z = 358.2 (M+H) +. Synthetic Scheme Compound 13: Synthesis of compound 3 To a mixture of compound 1 (0.5 g, 1.99 mmol, 1 eq) and compound 2 (173.31 mg, 1.99 mmol, 231.70 μL, 1 eq) in MeCN (10 mL) was added K2CO3 (549.60 mg, 3.98 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 0/1 to 10/1) to give compound 3 (480 mg, 1.59 mmol, 79.89% yield) as yellow oil. LCMS: RT = 1.033 min, m/z = 302.0/304.0 (M+H)+. Synthesis of compound 4 To a solution of compound 3 (200 mg, 661.89 μmol, 1 eq) and TEA (200.93 mg, 1.99 mmol, 276.38 μL, 3 eq) in MeOH (5 mL) was added Pd(dppf) Cl2 (48.43 mg, 66.19 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO several times. The mixture was stirred under CO (15 psi) at 70 °C for 16 h. LCMS showed 25% of compound 3 remained and 37% of peak with desired mass. Then the mixture was stirred at 80 °C for 5 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 10/1) to give compound 4 (130 mg, 462.13 μmol, 69.82% yield) as yellow oil. LCMS: RT = 0.972 min, m/z = 282.2 (M+H)+. Synthesis of compound 5 To a solution of compound 4 (130 mg, 462.13 μmol, 1 eq) in EtOAc (3 mL) was added Pd/C (14 mg, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 25 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give compound 5 (120 mg, crude) as orange oil. LCMS: RT = 0.583 min, m/z = 252.5 (M+H)+. Synthesis of compound 6 To a mixture of compound 5 (120 mg, 477.47 μmol, 1 eq) in MeCN (3 mL) was added CDI (85.16 mg, 525.22 μmol, 1.1 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was dissolved in ethyl acetate (8 mL) and washed with H2O (2 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 6 (120 mg, 432.72 μmol, 90.63% yield) as red gum. LCMS: RT = 0.819 min, m/z = 278.1 (M+H)+. Synthesis of compound 8 To a mixture of compound 6 (120 mg, 432.72 μmol, 1 eq), compound 7 (54.59 mg, 432.72 μmol, 1 eq) and PPh3 (170.24 mg, 649.07 μmol, 1.5 eq) in THF (3 mL) was added DIAD (131.25 mg, 649.07 μmol, 126.20 μL, 1.5 eq) dropwise at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA) followed by concentration to give compound 8 (60 mg, 155.66 μmol, 35.97% yield) as yellow gum. LCMS: RT = 0.724 min, m/z = 386.2 (M+H)+. Synthesis of Compound 13 To a mixture of compound 8 (60 mg, 155.66 μmol, 1 eq) in THF (2 mL), MeOH (2 mL) and H2O (1 mL) was added LiOH (7.46 mg, 311.32 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF, the mixture was acidified to pH = 5 - 6 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 8%- 38%, 15 min) followed by lyophilization to give a residue. The residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 8%- 38%, 15 min) followed by lyophilization to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (NH4HCO3) -ACN]; B%: 80%- 38%, min) followed by lyophilization to give Compound 13 (7.76 mg, 20.89 μmol, 13.42% yield, 100% purity) as white solid. 1H NMR: (400 MHz, CD3OD) δ = 7.87 - 7.79 (m, 2H), 7.17 (s, 1H), 5.21 (s, 2H), 4.34 (td, J = 5.1, 10.3 Hz, 1H), 4.11 J = 7.3 Hz, 2H), 2.74 (s, 3H), 2.39 - 2.21 (m, 2H), 1.88 (ddd, J = 5.1, 7.4, 13.9 Hz, 2H), 1.29 (t, J = 7.3 Hz, 3H), 0.82 J = 7.4 Hz, 6H); LCMS: RT = 0.699 min, m/z = 372.1 (M+H)+. Synthetic Scheme Compound 14: Synthesis of compound 3 To a mixture of compound 1 (100 mg, 379.81 μmol, 1 eq), compound 2 (42.59 mg, 379.81 μmol, 1 eq) and PPh3 (149.43 mg, 569.71 μmol, 1.5 eq) in THF (1 mL) was added DIAD (115.20 mg, 569.71 μmol, 110.77 μL, 1.5 eq) dropwise at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA) followed by lyophilization to give compound 3 (130 mg, 363.73 μmol, 95.77% yield) as colorless gum. LCMS: RT = 0.694 min, m/z = 358.2 (M+H)+. Synthesis of Compound 14 To a mixture of compound 3 (130 mg, 363.73 μmol, 1 eq) in THF (2 mL), MeOH (2 mL) and H2O (1 mL) was added LiOH (17.42 mg, 727.46 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF, the mixture was acidified to pH = 5 - 6 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%- 30%, 15 min) followed by lyophilization to give Compound 14 (27.68 mg, 79.00 μmol, 21.72% yield, 98% purity) as white solid. 1H NMR: (400 MHz, CD3OD) δ = 8.68 (s, 1H), 7.95 (s, 1H), 7.80 (br s, 1H), 7.22 (br s, 1H), 5.26 (s, 2H), 4.42 - 4.30 (m, 1H), 3.72 (s, 3H), 2.28 (ddd, J = 7.3, 10.1, 14.1 Hz, 2H), 1.98 - 1.80 (m, 2H), 0.81 (t, J = 7.3 Hz, 6H); LCMS: RT = 0.632 min, m/z = 344.2 (M+H)+. Synthetic Scheme Compound 15: 3 To a mixture of compound 1 (0.5 g, 2.31 mmol, 1 eq) and compound 2 (168.85 mg, 2.31 mmol, 233.21 μL, 1 eq) in MeCN (10 mL) was added K2CO3 (638.13 mg, 4.62 mmol, 2 eq). The mixture was stirred at 60 °C for 5 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 9/1) to give compound 3 (580 mg, 2.29 mmol, 99.20% yield) as yellow oil. LCMS: RT = 0.900 min, m/z = 254.1 (M+H)+. Synthesis of compound 4 To a solution of compound 3 (580 mg, 2.29 mmol, 1 eq) in EtOAc (6 mL) was added Pd/C (58 mg, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 25 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give compound 4 (500 mg, 2.24 mmol, 97.78% yield) as colorless oil. LCMS: RT = 0.293 min, m/z = 224.2 (M+H)+. Synthesis of compound 5 To a mixture of compound 4 (500 mg, 2.24 mmol, 1 eq) in MeCN (6 mL) was added CDI (363.12 mg, 2.24 mmol, 1 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was dissolved in ethyl acetate (10 mL) and washed with H2O (3 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 5 (500 mg, 2.01 mmol, 89.57% yield) as white solid. LCMS: RT = 0.719 min, m/z = 250.1 (M+H)+. Synthesis of compound 7 To a mixture of compound 5 (100 mg, 401.18 μmol, 1 eq), compound 6 (50.61 mg, 401.18 μmol, 1 eq) and PPh3 (157.84 mg, 601.77 μmol, 1.5 eq) in THF (2 mL) was added DIAD (121.68 mg, 601.77 μmol, 117.00 μL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA) followed by concentration to give compound 7 (100 mg, 279.79 μmol, 69.74% yield) as yellow oil. LCMS: RT = 0.668 min, m/z = 358.2 (M+H)+. Synthesis of Compound 15 To a mixture of compound 7 (100 mg, 279.79 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (6.70 mg, 279.79 μmol, 1 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF, the mixture was acidified to pH = 6 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%- 30%, 10 min) followed by lyophilization to give Compound 15 (28 mg, 81.54 μmol, 29.14% yield, 100% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.58 (d, J = 1.7 Hz, 1H), 7.90 (d, J = 1.7 Hz, 1H), 7.73 (br s, 1H), 7.03 (br s, 1H), 5.19 (s, 2H), 4.49 (td, J = 6.4, 9.4 Hz, 1H), 4.00 (q, J = 7.1 Hz, 2H), 2.15 (ddd, J = 7.2, 9.5, 14.0 Hz, 1H), 1.90 - 1.78 (m, 1H), 1.50 (d, J = 6.8 Hz, 3H), 1.15 (t, J = 7.2 Hz, 3H), 0.76 (t, J = 7.3 Hz, 3H); LCMS: RT = 0.623 min, m/z = 344.1 (M+H)+. Synthetic Scheme Compound 16: 16: Synthesis of compound 1 A mixture of compound 1 (500 mg, 2.31 mmol, 1 eq), propan-1-amine (136.54 mg, 2.31 mmol, 189.91 μL, 1 eq) and K2CO3 (638.13 mg, 4.62 mmol, 2 eq) in MeCN (5 mL) was stirred at 60 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give compound 1 (540 mg, 2.26 mmol, 97.72% yield) as yellow solid. LCMS: RT =0.807 min, m/z =240.1 (M+H)+ . Synthesis of compound 3 A mixture of compound 2 (540 mg, 2.26 mmol, 1 eq), Pd/C (50 mg, 10% purity) in EtOAc (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 20 °C for 16 h under H2 (15 Psi) atmosphere. The mixture was filtered and the filtrate was concentrated at reduced pressure to give compound 3 (500 mg, crude) as colorless gum. LCMS: RT =0.241 min, m/z =210.1 (M+H)+ . Synthesis of compound 4 To a mixture of compound 3 (500 mg, 2.39 mmol, 1 eq) in ACN (10 mL) was added CDI (406.84 mg, 2.51 mmol, 1.05 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The reaction mixture was diluted with ethyl acetate (30 mL). The organic layer was washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give compound 4 (640 mg, crude) as grey solid. LCMS: RT =0.700 min, m/z =236.2 (M+H)+ . Synthesis of compound 5 To a mixture of compound 4 (100 mg, 425.10 μmol, 1 eq), (3-ethylimidazol-4-yl) methanol (53.63 mg, 425.10 μmol, 1 eq) and PPh3 (167.25 mg, 637.65 μmol, 1.5 eq) in THF (2 mL) was added DIAD (128.94 mg, 637.65 μmol, 123.98 μL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by reversed phase flash (0.1% FA) to give compound 5 (130 mg, 378.59 μmol, 89.06% yield) as yellow solid. LCMS: RT =0.643 min, m/z =344.2 (M+H)+ . Synthesis of Compound 16 To a solution of compound 5 (130 mg, 378.59 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (18.13 mg, 757.18 μmol, 2 eq), the mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-25%, 10 min) to give Compound 16 (22.84 mg, 69.35 μmol, 18.32% yield, 100% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.58 (d, J = 1.6 Hz, 1H), 7.89 (d, J = 1.6 Hz, 1H), 7.65 (s, 1H), 6.99 (s, 1H), 5.18 (s, 2H), 4.00 (q, J = 7.3 Hz, 2H), 3.88 (t, J = 7.1 Hz, 2H), 1.80 - 1.68 (m, 2H), 1.16 (t, J = 7.2 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H); LCMS: RT =0.561 min, m/z =330.1 (M+H)+ . Synthetic Scheme Compound 17:
E Synthesis of compound 2 A mixture of compound 2 (500 mg, 2.31 mmol, 1 eq), cyclopentanamine (196.69 mg, 2.31 mmol, 227.91 μL, 1 eq) and K2CO3 (638.13 mg, 4.62 mmol, 2 eq) in MeCN (5 mL) was stirred at 60 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give compound 2 (600 mg, 2.26 mmol, 97.92% yield) as yellow solid. LCMS: RT =0.874 min, m/z =266.1 (M+H)+ . Synthesis of compound 3 A mixture of methyl compound 2 (600 mg, 2.26 mmol, 1 eq), Pd/C (60 mg, 10% purity) in EtOAc (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 20 °C for 16 h under H2 (15 Psi) atmosphere. The mixture was filtered and the filtrate was concentrated at reduced pressure to give compound 3 (570 mg, crude) as colorless gum. LCMS: RT =0.503 min, m/z =236.1 (M+H)+ . Synthesis of compound 4 To a mixture of compound 3 (570 mg, 2.42 mmol, 1 eq) in ACN (10 mL) was added CDI (412.47 mg, 2.54 mmol, 1.05 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The reaction mixture was diluted with ethyl acetate (30 mL). The organic layer was washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give compound 4 (630 mg, 2.41 mmol, 99.53% yield) as grey solid. LCMS: RT =0.792 min, m/z =262.1 (M+H)+ . Synthesis of compound 5 To a mixture of compound 4 (100 mg, 382.74 μmol, 1 eq), (3-ethylimidazol-4-yl) methanol (48.28 mg, 382.74 μmol, 1 eq) and PPh3 (150.58 mg, 574.11 μmol, 1.5 eq) in THF (2 mL) was added DIAD (116.09 mg, 574.11 μmol, 111.62 μL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by reversed phase flash (0.1% FA) to give compound 5 (100 mg, 270.70 μmol, 70.73% yield) as yellow solid. LCMS: RT =0.703 min, m/z =370.1 (M+H)+ . Synthesis of Compound 17 To a solution of compound 5 (100 mg, 270.70 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (12.97 mg, 541.39 μmol, 2 eq), the mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%-35%, 10 min) to give Compound 17 (24.88 mg, 70.01 μmol, 25.86% yield, 100% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.58 (d, J = 1.8 Hz, 1H), 7.89 (d, J = 1.8 Hz, 1H), 7.65 (d, J = 0.7 Hz, 1H), 7.01 (s, 1H), 5.17 (s, 2H), 4.94 - 4.82 (m, 1H), 4.04 - 3.95 (m, 2H), 2.26 - 2.11 (m, 2H), 1.92 (br d, J = 2.7 Hz, 4H), 1.72 - 1.58 (m, 2H), 1.12 (t, J = 7.2 Hz, 3H); LCMS: RT =0.661 min, m/z =356.1 (M+H)+ . Synthetic Scheme Compound 18: 3 To a mixture of compound 1 (0.5 g, 2.31 mmol, 1 eq) and compound 2 (229.09 mg, 2.31 mmol, 264.39 μL, 1 eq) in MeCN (10 mL) was added K2CO3 (638.53 mg, 4.62 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 9/1) to give compound 3 (580 mg, 2.08 mmol, 89.90% yield) as yellow solid. LCMS: RT = 0.947 min, m/z = 280.1 (M+H)+. Synthesis of compound 4 To a solution of compound 3 (580 mg, 2.08 mmol, 1 eq) in EtOAc (6 mL) was added Pd/C (58 mg, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 25 °C for 5 hours. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give compound 4 (500 mg, 2.01 mmol, 96.57% yield) as colorless oil. LCMS: RT = 0.608 min, m/z = 250.2 (M+H)+. Synthesis of compound 5 To a mixture of compound 4 (500 mg, 2.01 mmol, 1 eq) in MeCN (10 mL) was added CDI (325.20 mg, 2.01 mmol, 1 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was filtered and the filter cake was collected and concentrated in vacuum to give compound 5 (500 mg, 1.82 mmol, 90.56% yield) as gray solid. LCMS: RT = 0.818 min, m/z = 276.1 (M+H)+. Synthesis of compound 7 To a mixture of compound 5 (100 mg, 363.24 μmol, 1 eq), compound 6 (45.82 mg, 363.24 μmol, 1 eq) and PPh3 (142.91 mg, 544.86 μmol, 1.5 eq) in THF (2 mL) was added DIAD (110.17 mg, 544.86 μmol, 105.94 μL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated in vacuum. The residue was purified by reversed-phase HPLC (0.1% FA) followed by concentration to give compound 7 (120 mg, 312.95 μmol, 86.16% yield) as white solid. LCMS: RT = 0.731 min, m/z = 384.1 (M+H)+. Synthesis of Compound 18 To a mixture of compound 7 (120 mg, 312.95 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (14.99 mg, 625.91 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF, the mixture was acidified to pH=6 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 8%- 38%, 10 min) followed by lyophilization to give a residue. The residue was triturated with MeCN at 60 °C for 16 h. Then the mixture was filtered and the filter cake was collected and followed by lyophilization to give Compound 18 (14.1 mg, 37.79 μmol, 12.07% yield, 99% purity) as white solid. 1H NMR: (400 MHz, DMSO-d6) δ = 8.56 (d, J = 1.1 Hz, 1H), 7.88 (s, 1H), 7.65 (br s, 1H), 7.01 (br s, 1H), 5.16 (s, 2H), 4.33 (ddd, J = 3.9, 8.4, 12.0 Hz, 1H), 3.99 (q, J = 7.0 Hz, 2H), 2.35 - 2.27 (m, 2H), 1.84 (br d, J = 11.8 Hz, 2H), 1.78 - 1.64 (m, 3H), 1.48 - 1.32 (m, 2H), 1.29 - 1.18 (m, 1H), 1.11 (t, J = 7.2 Hz, 3H); LCMS: RT = 0.683 min, m/z = 370.1 (M+H)+. Synthetic Scheme Compound 19, Compound 21 and Compound 22:
Experimental Procedure Compound 19, Compound 21 and Compound 22: Synthesis of compound 2 A mixture of compound 1 (500 mg, 2.72 mmol, 1 eq), pentan-3-amine (249.31 mg, 2.86 mmol, 333.30 μL, 1.05 eq) and K2CO3 (752.98 mg, 5.45 mmol, 2 eq) in MeCN (5 mL) was stirred at 60 °C for 6 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give compound 2 (630 mg, 2.69 mmol, 98.73% yield) as yellow gum. LCMS: RT =0.955 min, m/z =235.2 (M+H)+ . Synthesis of compound 3 A mixture of compound 2 (630 mg, 2.69 mmol, 1 eq), Pd/C (60 mg, 10% purity) in EtOAc (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 20 °C for 16 h under H2 (15 Psi) atmosphere. The mixture was filtered and the filtrate was concentrated at reduced pressure to give compound 3 (540 mg, 2.64 mmol, 98.30% yield) as yellow solid.1H NMR: (400 MHz, DMSO-d6) δ = 7.74 (d, J = 2.0 Hz, 1H), 6.76 (d, J = 2.1 Hz, 1H), 6.12 (br d, J = 7.9 Hz, 1H), 5.23 (s, 2H), 4.07 - 3.97 (m, 1H), 1.61 - 1.52 (m, 2H), 1.51 - 1.42 (m, 2H), 0.84 (t, J = 7.4 Hz, 6H) Synthesis of compound 4 To a mixture of compound 3 (540 mg, 2.64 mmol, 1 eq) in ACN (10 mL) was added CDI (471.52 mg, 2.91 mmol, 1.1 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The reaction mixture was diluted with ethyl acetate (30 mL). The organic layer was washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give compound 4 (650 mg, crude) as yellow solid. LCMS: RT =0.770 min, m/z =231.2 (M+H)+ . Synthesis of Compound 19 To a mixture of compound 4 (650 mg, 2.82 mmol, 1 eq), (3-ethylimidazol-4-yl) methanol (356.12 mg, 2.82 mmol, 1 eq) and PPh3 (1.11 g, 4.23 mmol, 1.5 eq) in THF (10 mL) was added DIAD (856.21 mg, 4.23 mmol, 823.28 μL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by reversed phase flash (0.1% FA) to give 980 mg yellow solid.100 mg yellow solid was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (ammonia hydroxide v/v) -ACN]; B%: 28%-58%, 9 min) to Compound 19 (17.11 mg, 49.04 μmol, 1.74% yield, 97% purity) as yellow solid.1H NMR: (400 MHz, CD3OD) δ = 8.38 (d, J = 1.7 Hz, 1H), 7.80 - 7.69 (m, 2H), 7.21 - 7.13 (m, 1H), 5.22 (s, 2H), 4.36 (tt, J = 5.2, 10.3 Hz, 1H), 4.15 - 4.05 (m, 2H), 2.32 - 2.16 (m, 2H), 1.95 - 1.81 (m, 2H), 1.34 - 1.25 (m, 3H), 0.81 (t, J = 7.4 Hz, 6H); LCMS: RT =0.802 min, m/z =339.1 (M+H)+ . Synthesis of Compound 21 To a solution of Compound 19 (100 mg, 295.50 μmol, 1 eq) in 78-83-1 (5 mL) was added ZnBr2 (66.55 mg, 295.50 μmol, 14.79 μL, 1 eq) and NaN3 (19.21 mg, 295.50 μmol, 1 eq), the mixture was stirred at 100 °C for 16 h. HCl (1 N, 1 mL) and ethyl acetate (30 mL) were added, the organic layer was isolated and the aqueous layer extracted with ethyl acetate (20 mL*3). The organic layer was washed with brine (10 mL) and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%-35%, 10 min) to give Compound 21 (14.64 mg, 38.38 μmol, 12.99% yield, 100% purity) as off- white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.63 (d, J = 1.7 Hz, 1H), 8.05 (d, J = 1.6 Hz, 1H), 7.85 (s, 1H), 7.13 (s, 1H), 5.23 (s, 2H), 4.27 (td, J = 5.2, 10.2 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.25 - 2.10 (m, 2H), 1.92 - 1.77 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 0.76 (t, J = 7.3 Hz, 6H); LCMS: RT =0.726 min, m/z =382.1 (M+H)+ . Synthesis of compound 5 To a solution of NH2OH. HCl (92.41 mg, 1.33 mmol, 1.5 eq) in i-PrOH (5 mL) was added NaHCO3 (163.84 mg, 1.95 mmol, 75.85 μL, 2.2 eq), the mixture was stirred at 20 °C for 0.25 h. Then Compound 19 (300 mg, 886.51 μmol, 1 eq) was added and the mixture was stirred at 80 °C for 4 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was purified by column chromatography (100 mesh silica gel, ethyl acetate: methanol = 1: 0 to 10: 1) to give compound 5 (100 mg, 269.23 μmol, 30.37% yield) as yellow gum. LCMS: RT =0.718 min, m/z =357.2 (M+H)+ . Synthesis of compound 6 To a solution of compound 5 (100 mg, 269.23 μmol, 1 eq) and Py (25.56 mg, 323.08 μmol, 26.08 μL, 1.2 eq) in DMAC (2 mL) was added methyl carbonochloridate (300 mg, 3.17 mmol, 245.90 μL, 11.79 eq) at 0 °C under N2, the mixture was stirred at 20 °C for 3 h. The reaction mixture was quenched with NH4Cl aqueous solution (5 mL) and diluted with ethyl acetate (30 mL). The organic layer was washed with brine (10 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to compound 6 (70 mg, 162.99 μmol, 60.54% yield) as yellow gum. Synthesis of Compound 22 A mixture of compound 6 (65 mg, 151.35 μmol, 1 eq) in toluene (1 mL) was stirred at 110 °C for 16 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 3%-33%, 10 min) to give Compound 22 (22.62 mg, 55.78 μmol, 36.85% yield, 98% purity) as white solid. 1H NMR: (400 MHz, CD3OD) δ = 8.43 (d, J = 1.8 Hz, 1H), 8.07 (br s, 1H), 7.83 (d, J = 1.8 Hz, 1H), 7.34 - 7.30 (m, 1H), 5.28 (s, 2H), 4.43 - 4.31 (m, 1H), 4.19 (q, J = 7.3 Hz, 2H), 2.35 - 2.19 (m, 2H), 1.97 - 1.82 (m, 2H), 1.35 (t, J = 7.3 Hz, 3H), 0.83 (t, J = 7.4 Hz, 6H); LCMS: RT =0.748 min, m/z =398.1 (M+H)+ . Synthetic Scheme Compound 20:
Experimental Procedure Compound 20: Synthesis of compound 3 To a mixture of compound 1 (0.5 g, 2.31 mmol, 1 eq) and compound 2 (136.46 mg, 2.31 mmol, 198.35 μL, 1 eq) in MeCN (10 mL) was added K2CO3 (638.13 mg, 4.62 mmol, 2 eq). The mixture was stirred at 60 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 9/1) to give compound 3 (460 mg, 1.92 mmol, 83.29% yield) as yellow oil. LCMS: RT = 0.851 min, m/z = 240.2 (M+H)+. Synthesis of compound 4 To a solution of compound 3 (460 mg, 1.92 mmol, 1 eq) in EtOAc (5 mL) was added Pd/C (46 mg, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 25 °C for 5 hours. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give compound 4 (400 mg, 1.91 mmol, 99.42% yield) as colorless oil. Synthesis of compound 5 To a mixture of compound 4 (400 mg, 1.91 mmol, 1 eq) in MeCN (6 mL) was added CDI (309.97 mg, 1.91 mmol, 1 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was dissolved in ethyl acetate (10 mL) and washed with H2O (3 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 5 (440 mg, 1.87 mmol, 97.84% yield) as white solid. LCMS: RT = 0.720 min, m/z = 236.2 (M+H)+. Synthesis of compound 7 To a mixture of compound 5 (100 mg, 425.10 μmol, 1 eq), compound 6 (53.63 mg, 425.10 μmol, 1 eq) and PPh3 (167.25 mg, 637.65 μmol, 1.5 eq) in THF (2 mL) was added DIAD (128.94 mg, 637.65 μmol, 123.98 μL, 1.5 eq) in portions at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA) followed by concentration to give compound 7 (100 mg, 291.22 μmol, 68.51% yield) as yellow gum. LCMS: RT = 0.658 min, m/z = 344.1 (M+H)+. Synthesis of Compound 20 To a mixture of compound 7 (100 mg, 291.22 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (13.95 mg, 582.45 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF, the mixture was acidified to pH = 6 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3 - 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%- 30%, 10 min) followed by lyophilization to give Compound 20 (33.22 mg, 98.85 μmol, 33.94% yield, 98% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.58 (d, J = 1.7 Hz, 1H), 7.88 (d, J = 1.7 Hz, 1H), 7.65 (s, 1H), 7.01 (s, 1H), 5.16 (s, 2H), 4.75 (quin, J = 6.9 Hz, 1H), 4.00 (q, J = 7.2 Hz, 2H), 1.52 (d, J = 7.0 Hz, 6H), 1.12 (t, J = 7.2 Hz, 3H); LCMS: RT = 0.606 min, m/z = 330.2 (M+H)+. Synthetic Scheme Compound 23: 2 To a solution of diethyl propanedioate (4.07 g, 25.39 mmol, 3.84 mL, 1.1 eq) in DMF (50 mL) was added NaH (1.11 g, 27.70 mmol, 60% purity, 1.2 eq) at 0 °C, the mixture was stirred at 0 °C for 0.5 h. Then compound 2 (5 g, 23.09 mmol, 1 eq) which was dissolved in DMF (5 mL) was added. The mixture was stirred at 80 °C for 16 h. The reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (100 mL*3). The combined organic phase was washed with brine (100 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1) to compound 2 (6.8 g, crude) as yellow solid. Synthesis of compound 3 To a solution of compound 2 (7.2 g, 21.16 mmol, 1 eq) in DMSO (50 mL) and H2O (2 mL) was added LiCl (1.35 g, 31.74 mmol, 650.00 μL, 1.5 eq), the mixture was stirred at 100 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by reversed phase flash (0.1% FA) to give compound 3 (1.8 g, 6.71 mmol, 31.72% yield) as red gum.1H NMR: (400 MHz, CDCl3) δ = 9.34 (d, J = 1.9 Hz, 1H), 8.97 (d, J = 1.9 Hz, 1H), 4.40 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 4.03 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H). Synthesis of compound 4 To a solution of compound 3 (200 mg, 745.65 μmol, 1 eq) in DMF (5 mL) was added NaH (65.61 mg, 1.64 mmol, 60% purity, 2.2 eq) at 0 °C, the mixture was stirred at 0 °C, for 15 min, then MEI (232.84 mg, 1.64 mmol, 102.12 μL, 2.2 eq) was added, the mixture was stirred at 20 °C for 2 h. The reaction mixture was diluted with ethyl acetate (100 mL). The organic layer was washed with brine (30 mL*3), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give compound 4 (170 mg, 573.79 μmol, 76.95% yield) as colorless oil. Synthesis of compound 5 To a mixture of compound 4 (170 mg, 573.79 μmol, 1 eq) in AcOH (3 mL) was added Fe (160.23 mg, 2.87 mmol, 5 eq). The mixture was stirred at 100 °C for 2 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was diluted with MeOH (20 mL), filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 1: 1) to give compound 5 (120 mg, 544.90 μmol, 94.96% yield) as brown solid. Synthesis of compound 6 A mixture of compound 5 (70 mg, 317.86 μmol, 1 eq), compound 5A (55.16 mg, 381.43 μmol, 1.2 eq) and Cs2CO3 (310.69 mg, 953.57 μmol, 3 eq) in DMF (2 mL) was stirred at 40 °C for 16 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give compound 6 (120 mg, crude) as yellow oil. Synthesis of Compound 23 To a solution of compound 6 (120 mg, 365.45 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (17.50 mg, 730.89 μmol, 2 eq). The mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was adjusted pH = 5 with 1 M HCl. Then the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-15%, 15 min) to give compound 23 (69.03 mg, 219.60 μmol, 60.09% yield, 100% purity) as yellow solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.71 (d, J = 1.7 Hz, 1H), 7.81 (d, J = 1.7 Hz, 1H), 7.67 (s, 1H), 6.92 (s, 1H), 5.04 (s, 2H), 3.97 (q, J = 7.3 Hz, 2H), 1.34 (s, 6H), 1.25 (t, J = 7.3 Hz, 3H); LCMS: RT = 0.267 min, m/z = 315.2 (M+H) +. Synthetic Scheme Compound 24: Synthesis of compound 2 To a solution of compound 1 (500 mg, 1.86 mmol, 1 eq) and 1, 4-dibromobutane (482.99 mg, 2.24 mmol, 269.83 μL, 1.2 eq) in DMSO (6 mL) was added K2CO3 (515.27 mg, 3.73 mmol, 2 eq), the mixture was stirred at 30 °C for 16 h. The reaction mixture was diluted with ethyl acetate (100 mL). The organic layer was washed with brine (30 mL*3), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give compound 2 (250 mg, 775.64 μmol, 41.61% yield) as colorless oil. Synthesis of compound 3 To a mixture of compound 2 (250 mg, 775.64 μmol, 1 eq) in AcOH (3 mL) was added Fe (216.60 mg, 3.88 mmol, 5 eq). The mixture was stirred at 100 °C for 2 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was diluted with MeOH (20 mL), filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 1: 1) to give compound 3 (140 mg, 568.50 μmol, 73.29% yield) as white solid. Synthesis of compound 4 A solution of compound 3 (70 mg, 284.25 μmol, 1 eq), compound 3A (49.32 mg, 341.10 μmol, 1.2 eq) and Cs2CO3 (277.84 mg, 852.75 μmol, 3 eq) in DMF (2 mL) was stirred at 40 °C for 3 h. The reaction mixture was diluted with ethyl acetate (50 mL). The organic layer was washed with brine (30 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 1 to ethyl acetate: methanol = 5: 1) to give compound 4 (55 mg, crude) as colorless gum. Synthesis of Compound 24 To a solution of compound 4 (55 mg, 155.19 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (11.15 mg, 465.57 μmol, 3 eq). The mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was adjusted pH = 5 with 1 M HCl. Then the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-23%, 10 min) to give Compound 24 (31.11 mg, 91.40 μmol, 58.89% yield, 100% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.69 (d, J = 1.6 Hz, 1H), 7.78 (d, J = 1.6 Hz, 1H), 7.68 (s, 1H), 6.95 (s, 1H), 5.09 - 4.99 (m, 2H), 3.96 (q, J = 7.2 Hz, 2H), 2.04 - 1.91 (m, 8H), 1.21 (t, J = 7.2 Hz, 3H); LCMS: RT = 0.640 min, m/z = 341.0 (M+H) +. Synthetic Scheme Compound 25: Synthesis of compound 2 A mixture of compound 1 (150 mg, 529.81 μmol, 1 eq), 5-(chloromethyl) -1-ethyl-imidazole (91.93 mg, 635.77 μmol, 1.2 eq) and Cs2CO3 (517.87 mg, 1.59 mmol, 3 eq) in DMF (2 mL) was stirred at 40 °C for 16 h. The reaction mixture was diluted with ethyl acetate (50 mL). The organic layer was washed with brine (30 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by reversed phase flash (0.1% FA) to give compound 2 (220 mg, crude) as yellow gum. Synthesis of compound 3 To a mixture of compound 2 (200 mg, 511.17 μmol, 1 eq), Et3N (155.17 mg, 1.53 mmol, 213.45 μL, 3 eq) in MeOH (10 mL) was added Pd(dppf)Cl2 (37.40 mg, 51.12 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (15 psi) at 80 °C for 16 hours. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to ethyl acetate: methanol = 10: 1) to give compound 3 (100 mg, crude) as yellow gum. Synthesis of Compound 25 To a solution of methyl compound 3 (100 mg, 269.98 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (19.40 mg, 809.93 μmol, 3 eq), the mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was adjusted pH = 5 with 1 M HCl. Then the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-15%, 10 min) to give Compound 25 (24.66 mg, 67.81 μmol, 25.12% yield, 98% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.72 (d, J = 1.5 Hz, 1H), 7.82 (d, J = 1.5 Hz, 1H), 7.67 (s, 1H), 6.91 (s, 1H), 5.03 (s, 2H), 4.08 (ddd, J = 3.4, 8.0, 11.3 Hz, 2H), 4.00 - 3.92 (m, 4H), 1.89 - 1.81 (m, 2H), 1.79 - 1.71 (m, 2H), 1.25 (t, J = 7.3 Hz, 3H); LCMS: RT = 0.476 min, m/z = 357.2 (M+H) +. Synthetic Scheme Compound 26:
Ex Synthesis of compound 2 To a solution of compound 1 (4.5 g, 21.80 mmol, 1 eq), TEA (6.62 g, 65.39 mmol, 9.10 mL, 3 eq) in MeOH (10 mL) was added Pd(dppf)Cl2 (1.59 g, 2.18 mmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (15 psi) at 50 °C for 16 hours. The reaction mixture was concentrated under vacuum to give a residue. The mixture was diluted with ethyl acetate (100 mL), the organic layer was filtered and the filtrate was washed with brine (50 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1) to give compound 2 (4.2 g, crude) as white solid. 1H NMR: (400 MHz, METHANOL-d4) δ = 7.95 (s, 2H), 3.96 (s, 3H), 2.66 (s, 3H) Synthesis of compound 3 A solution of compound 2 (1 g, 5.39 mmol, 1 eq), pentan-3-amine (563.53 mg, 6.47 mmol, 753.38 μL, 1.2 eq), XPhos (513.68 mg, 1.08 mmol, 0.2 eq) and Cs2CO3 (3.51 g, 10.78 mmol, 2 eq) in toluene (10 mL) was added Pd2 (dba)3 (493.36 mg, 538.77 μmol, 0.1 eq) under N2. The mixture was stirred at 100 °C for 16 h. A solution of compound 2 (3.2 g, 17.24 mmol, 1 eq), pentan-3-amine (2.25 g, 25.86 mmol, 3.01 mL, 1.5 eq), XPhos (1.64 g, 3.45 mmol, 0.2 eq) and Cs2CO3 (11.23 g, 34.48 mmol, 2 eq) in toluene (10 mL) was added Pd2 (dba)3 (1.58 g, 1.72 mmol, 0.1 eq) under N2. The mixture was stirred at 100 °C for 16 h. The combined reaction mixtures were diluted with ethyl acetate (100 mL), the organic Layer was filtered and the filtrate was washed with brine (50 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 3: 1) to give compound 3 (2.4 g, crude) as yellow gum. 1H NMR: (400 MHz, METHANOL-d4) δ = 7.84 (d, J = 8.6 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 3.88 (s, 3H), 3.38 6.3 Hz, 1H), 2.41 (s, 3H), 1.71 - 1.53 (m, 4H), 0.95 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.645 min, m/z = 237.2 +. Synthesis of compound 4 A solution of compound 3 (2.4 g, 10.16 mmol, 1 eq) and NBS (1.63 g, 9.14 mmol, 0.9 eq) in ACN (40 mL) was stirred at 0 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was diluted with ethyl acetate (100 mL). The organic layer was washed with saturated NaHCO3 aqueous solution (50 mL) and brine (50 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 1: 1) to give compound 4 (1.1 g, 3.49 mmol, 34.36% yield) as yellow gum. LCMS: RT = 0.959 min, m/z = 315.1 (M+H) +. Synthesis of compound 5 A solution of compound 4 (400 mg, 1.27 mmol, 1 eq), tert-butyl carbamate (222.99 mg, 1.90 mmol, 1.5 eq), XPhos (120.99 mg, 253.80 μmol, 0.2 eq) and Cs2CO3 (1.24 g, 3.81 mmol, 3 eq) in dioxane (5 mL) was added Pd2(dba)3 (116.21 mg, 126.90 μmol, 0.1 eq) under N2. The mixture was stirred at 100 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 3: 1) to give compound 5 (70 mg, 199.18 μmol, 15.70% yield) as yellow gum. LCMS: RT = 0.779 min, m/z = 352.2 (M+H) +. Synthesis of compound 6 A solution of compound 5 (70 mg, 199.18 μmol, 1 eq) and HCl/dioxane (4 M, 2 mL) was stirred at 20 °C for 2 h. The reaction mixture was concentrated under vacuum to give compound 6 (55 mg, 191.11 μmol, 95.95% yield, HCl) as yellow solid. Synthesis of compound 7 A solution of compound 6 (50 mg, 173.74 μmol, 1 eq, HCl), TEA (52.74 mg, 521.22 μmol, 72.55 μL, 3 eq) and CDI (42.26 mg, 260.61 μmol, 1.5 eq) in ACN (1 mL) was stirred at 80 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 1: 1) to give compound 7 (35 mg, 126.21 μmol, 72.64% yield) as colorless gum. LCMS: RT = 0.613 min, m/z = 278.2 (M+H) +. Synthesis of compound 8 To a solution of compound 7 (35 mg, 126.21 μmol, 1 eq) and CS2CO3 (123.36 mg, 378.63 μmol, 3 eq) in DMF (2 mL) was added 5-(chloromethyl) -1-ethyl-imidazole (27.38 mg, 189.31 μmol, 1.5 eq). The mixture was stirred at 40 °C for 2 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give compound 8 (45 mg, 116.74 μmol, 92.50% yield) as yellow gum. LCMS: RT = 0.627 min, m/z = 386.2 (M+H) +. Synthesis of Compound 26 To a solution of compound 8 (45 mg, 116.74 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (5.59 mg, 233.49 μmol, 2 eq). The mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. Then the residue was adjusted pH = 5 with 1 M HCl and the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-20%, 10 min) to give Compound 26 (31.6 mg, 84.23 μmol, 72.15% yield, 99% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 7.81 (s, 1H), 7.66 (s, 1H), 6.87 (s, 1H), 5.17 (s, 2H), 4.43 - 4.32 (m, 1H), 4.00 (q, J = 7.1 Hz, 2H), 2.74 (s, 3H), 2.31 - 2.15 (m, 2H), 1.84 - 1.71 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H), 0.79 (t, J = 7.3 Hz, 6H); LCMS: RT = 0.371 min, m/z = 372.2 (M+H) +. Synthetic Scheme Compound 27: Experimental Procedure for Compound 27: Synthesis of compound 2 To a mixture of compound 1 (200 mg, 1.49 mmol, 1 eq, HCl) and IMIDAZOLE (303.55 mg, 4.46 mmol, 3 eq) in DMF (5 mL) was added TBSCl (268.82 mg, 1.78 mmol, 218.55 μL, 1.2 eq) in portions at 0 °C under N2. The mixture was stirred at 25 °C for 16 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with brine (20 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 2 (210 mg, 988.87 μmol, 66.53% yield) as colorless gum. Synthesis of compound 4A To a mixture of compound 2 (210 mg, 988.87 μmol, 1 eq) and compound 3 (133.50 mg, 988.87 μmol, 94.68 μL, 1 eq) in DMF (5 mL) was added K2CO3 (410.00 mg, 2.97 mmol, 3 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with brine (20 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 10%-40%, 15 min) followed by lyophilization to give compound 4A (30 mg, 197.12 μmol, 19.93% yield) as light yellow gum and compound 4A_isomer (30 mg, 197.12 μmol, 19.93% yield) as white gum. Compound 4A: 1H NMR: (400 MHz, CD3OD) δ = 7.93 (br s, 1H), 7.01 (br s, 1H), 4.63 (s, 2H), 3.95 (d, J = 7.1 Hz, 2H), 1.39 - 1.25 (m, 1H), 0.69 - 0.62 (m, 2H), 0.46 - 0.39 (m, 2H) Compound 4A_isomer: 1H NMR: (400 MHz, CD3OD) δ = 7.69 (s, 1H), 7.15 (s, 1H), 4.52 (s, 2H), 3.85 (d, J = 7.2 Hz, 2H), 1.29 - 1.17 (m, 1H), 0.67 - 0.60 (m, 2H), 0.43 - 0.36 (m, 2H) Synthesis of compound 6 To a mixture of compound 5 (51.90 mg, 197.12 μmol, 1 eq), compound 4A (30 mg, 197.12 μmol, 1 eq) and PPh3 (77.55 mg, 295.68 μmol, 1.5 eq) in THF (2 mL) was added DIAD (59.79 mg, 295.68 μmol, 57.49 μL, 1.5 eq) dropwise at 0 °C under N2. The mixture was stirred at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA) followed by concentration to give compound 6 (50 mg, 125.80 μmol, 63.82% yield) as yellow gum. Synthesis of Compound 27 To a mixture of compound 6 (50 mg, 125.80 μmol, 1 eq) in THF (2 mL), MeOH (2 mL) and H2O (1 mL) was added LiOH (6.03 mg, 251.59 μmol, 2 eq). The reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF and MeOH, the mixture was acidified to pH = 5-6 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%-35%, 10 min) followed by lyophilization to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (NH4HCO3) -ACN]; B%: 10%-40%, 8 min) followed by lyophilization to give Compound 27 (4.91 mg, 12.81 μmol, 10.18% yield, 100% purity) as white solid.1H NMR: (400 MHz, CD3OD) δ = 8.69 (d, J = 1.5 Hz, 1H), 8.00 (s, 1H), 7.95 (d, J = 1.5 Hz, 1H), 7.25 (s, 1H), 5.28 (s, 2H), 4.37 (td, J = 5.1, 10.1 Hz, 1H), 3.95 (d, J = 7.1 Hz, 2H), 2.28 (ddd, J = 7.3, 10.2, 14.1 Hz, 2H), 1.96 - 1.83 (m, 2H), 1.19 - 1.06 (m, 1H), 0.81 (t, J = 7.4 Hz, 6H), 0.66 - 0.56 (m, 2H), 0.38 (q, J = 5.0 Hz, 2H); LCMS: RT = 0.691 min, m/z = 384.2 (M+H)+. Synthetic Scheme Compound 28: Synthesis of compound 3 and compound 3A To a mixture of compound 1 (1 g, 7.93 mmol, 1 eq) and compound 2 (2.04 g, 23.79 mmol, 3 eq) in DCE (20 mL) was added Na2CO3 (2.52 g, 23.79 mmol, 3 eq), 2 - (2 -pyridyl) pyridine (1.24 g, 7.93 mmol, 1 eq) and Cu(OAc)2 (1.44 g, 7.93 mmol, 1 eq). The mixture was stirred at 70 °C for 3 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 0/1) to give compound 3 (180 mg, 1.08 mmol, 13.66% yield) as yellow oil and compound 3A (290 mg, 1.75 mmol, 22.01% yield) as yellow oil. Synthesis of compound 4 To a mixture of compound 3 (180 mg, 1.08 mmol, 1 eq) in THF (2 mL) was added LiAlH4 (49.33 mg, 1.30 mmol, 1.2 eq) in portions at 0 °C under N2. The mixture was stirred at 25 °C for 2 h. Na2SO4•10H2O was added to the reaction mixture in portions. Then the mixture was dried with Na2SO4, filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was purified by reversed-phase HPLC (0.1% NH3•H2O) followed by concentration to give compound 4 (90 mg, 651.39 μmol, 60.14% yield) as colorless gum.1H NMR: (400 MHz, CD3OD) δ = 7.61 (s, 1H), 6.89 (s, 1H), 4.68 (s, 2H), 3.41 - 3.34 (m, 1H), 1.08 - 1.00 (m, 4H) Synthesis of compound 6 To a mixture of compound 5 (171.50 mg, 651.39 μmol, 1 eq), compound 4 (90 mg, 651.39 μmol, 1 eq) and PPh3 (256.27 mg, 977.08 μmol, 1.5 eq) in THF (3 mL) was added DIAD (197.57 mg, 977.08 μmol, 189.97 μL, 1.5 eq) dropwise at 0 °C under N2. The mixture was stirred at 60 °C for 16 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% FA) followed by concentration to give compound 6 (150 mg, 391.19 μmol, 60.06% yield) as yellow oil. Synthesis of Compound 28 To a mixture of compound 6 (150 mg, 391.19 μmol, 1 eq) in THF (1 mL), MeOH (1 mL) and H2O (0.5 mL) was added LiOH (18.74 mg, 782.38 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF and MeOH and the mixture was acidified to pH = 5-6 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Synergi C18150*25 mm* 10um; mobile phase: [water (FA) -ACN]; B%: 3%- 33%, 10 min) followed by lyophilization to give Compound 28 (30.73 mg, 81.52 μmol, 20.84% yield, 98% purity) as white solid. 1H NMR: (400 MHz, CD3OD) δ = 8.69 (br s, 1H), 7.91 (br s, 1H), 7.80 (br s, 1H), 7.09 (br s, 1H), 5.35 (br s, 2H), 4.36 (br dd, J = 5.3, 8.5 Hz, 1H), 3.30 - 3.25 (m, 1H), 2.37 - 2.17 (m, 2H), 2.00 - 1.79 (m, 2H), 1.13 - 0.94 (m, 4H), 0.82 (br t, J = 7.0 Hz, 6H) 1H NMR: (400 MHz, DMSO-d6) δ = 8.56 (d, J = 1.8 Hz, 1H), 7.84 (d, J = 1.8 Hz, 1H), 7.60 (s, 1H), 6.85 (s, 1H), 5.27 (s, 2H), 4.27 (td, J = 5.0, 10.0 Hz, 1H), 3.22 (br d, J = 2.9 Hz, 1H), 2.21 - 2.11 (m, 2H), 1.88 - 1.77 (m, 2H), 0.94 - 0.84 (m, 4H), 0.74 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.660 min, m/z = 370.2 (M+H)+. Synthetic Scheme Compound 29:
Exper Synthesis of compound 2 To a solution of compound 1 (200 mg, 1.19 mmol, 1 eq) in DMF (5 mL) was added NaH (56.95 mg, 1.42 mmol, 60% purity, 1.2 eq) at 0 °C, the reaction mixture was stirred at 0°C for 15 min, then SEM-Cl (237.35 mg, 1.42 mmol, 251.97 μL, 1.2 eq) was added dropwise at 0 °C, the mixture was stirred at 20 °C for 3 h. The mixture was quenched with H2O (20 mL), and extracted with ethyl acetate (50 mL). The combined organic layer was washed with brine (50 mL*2), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 3: 1) to give compound 2 (100 mg, 334.63 μmol, 28.21% yield) as yellow gum. LCMS: RT = 0.868 min, m/z = 299.1 (M+H) +. Synthesis of compound 3 A solution of 1, 4-dibromobutane (156.06 mg, 722.80 μmol, 87.18 μL, 1.2 eq), compound 2 (180 mg, 602.33 μmol, 1 eq) and K2CO3 (166.50 mg, 1.20 mmol, 2 eq) in DMSO (3 mL) was stirred at 30 °C for 2 h. The mixture was diluted with ethyl acetate (50 mL). The organic layer was washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1) to give compound 3 (130 mg, 368.34 μmol, 61.15% yield) as yellow gum. LCMS: RT = 0.972 min, m/z = 353.1 (M+H) +. Synthesis of compound 4 To a solution of compound 3 (120 mg, 340.01 μmol, 1 eq), TEA (68.81 mg, 680.02 μmol, 94.65 μL, 2 eq) in MeOH (4 mL) was added Pd(dppf)Cl2 (24.88 mg, 34.00 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (15 psi) at 80 °C for 16 hours. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 3: 1) to give compound 4 (100 mg, 265.59 μmol, 78.11% yield) as yellow gum. 1H NMR: (400 MHz, METHANOL-d4) δ = 8.46 (s, 1H), 7.85 (s, 1H), 5.23 (s, 2H), 3.99 (s, 3H), 3.62 - 3.56 (m, 2H), 2.18 - 2.07 (m, 6H), 2.01 - 1.94 (m, 2H), 0.94 - 0.89 (m, 2H), -0.05 (s, 9H); LCMS: RT = 0.947 min, m/z = 376.9 (M+1) +. Synthesis of compound 5 To a solution of compound 4 (100 mg, 265.59 μmol, 1 eq) in DCM (2 mL) was added TFA (1 mL). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under vacuum to give compound 5 (120 mg, crude, TFA) as brown gum. LCMS: RT = 0.727 min, m/z = 247.1 (M+H) +. Synthesis of compound 6 To a solution of compound 5 (60 mg, 243.64 μmol, 1 eq) and Cs2CO3 (238.15 mg, 730.93 μmol, 3 eq) in DMF (1 mL) was added 5-(chloromethyl) -1-ethyl-imidazole (52.85 mg, 365.47 μmol, 1.5 eq). The mixture was stirred at 40 °C for 16 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give compound 6 (80 mg, crude) as yellow gum. LCMS: RT = 0.559 min, m/z = 355.2 (M+H) +. Synthesis of Compound 29 To a solution of compound 6 (80 mg, 225.73 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (10.81 mg, 451.46 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was adjusted pH = 5 with 1 M HCl. Then the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (NH4HCO3) -ACN]; B%: 7%-37%, 9 min) to give Compound 29 (10.43 mg, 30.34 μmol, 13.44% yield, 99% purity) as white solid.1H NMR: (400 MHz, METHANOL-d4) δ = 8.62 - 8.23 (m, 1H), 7.93 - 7.51 (m, 2H), 7.16 (br s, 1H), 5.04 (br s, 2H), 4.11 (q, J = 7.2 Hz, 2H), 2.28 - 1.70 (m, 8H), 1.34 (br t, J = 7.3 Hz, 3H); LCMS: RT = 1.083 min, m/z = 339.2 (M+H) +. Synthetic Scheme Compound 30: 2 To a solution of compound 1 (1 g, 5.98 mmol, 1 eq) in ACN (10 mL) was added CDI (1.45 g, 8.97 mmol, 1.5 eq). The mixture was stirred at 80 °C for 16 h. The mixture was diluted with water (20 mL) and the resulting mixture was filtered. The filter cake was collected and dried at reduced pressure to give compound 2 (1.1 g, 5.69 mmol, 95.20% yield) as white solid. Synthesis of compound 3 To a solution of compound 2 (1.05 g, 5.44 mmol, 1 eq) in DMF (15 mL) was added NaH (456.57 mg, 11.42 mmol, 60% purity, 2.1 eq) at 0 °C. The mixture was stirred at 20 °C for 0.25 h. Then 3-bromopentane (821.07 mg, 5.44 mmol, 673.01 μL, 1 eq) was added. The mixture was stirred at 80 °C for 16 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was diluted with water (50 mL). Then extracted with ethyl acetate (50 mL), the organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 10%-30%, 10 min) to give compound 3 (140 mg, crude) as white solid. Synthesis of compound 4 To a mixture of compound 3A (22.49 mg, 170.91 μmol, 1.5 eq) and compound 3 (30 mg, 113.94 μmol, 1 eq) in DMF (1 mL) was added Cs2CO3 (111.37 mg, 341.83 μmol, 3 eq). The mixture was stirred at 40 °C for 2 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give a compound 4 (45 mg, crude) as colorless gum. Synthesis of compound 30 To a solution of compound 4 (45 mg, 125.56 μmol, 1 eq) in THF (1.5 mL) and H2O (0.5 mL) was added LiOH (3.01 mg, 125.56 μmol, 1 eq). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was adjusted pH = 5 with 1 M HCl. Then the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-28%, 10 min) to give Compound 30 (17.67 mg, 51.31 μmol, 40.87% yield, 100% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.63 (br s, 1H), 8.44 (s, 1H), 7.97 (s, 1H), 5.37 (s, 2H), 4.25 - 4.17 (m, 1H), 3.68 (s, 3H), 2.07 - 1.95 (m, 2H), 1.90 - 1.80 (m, 2H), 0.76 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.211 min, m/z = 345.2 (M+H) +. Synthetic Scheme Compound 31: Synthesis of compound 2 To a mixture of compound 1 (1 g, 7.09 mmol, 1 eq) in MeCN (10 mL) was added MeI (4.02 g, 28.34 mmol, 1.76 mL, 4 eq) and K2CO3 (1.96 g, 14.17 mmol, 2 eq). The mixture was stirred at 50 °C for 16 h. The reaction mixture was quenched with H2O (20 mL) and extracted with ethyl acetate (20 mL*3), the combined organic phase was washed with H2O (20 mL), brine (20 mL) and dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 5/1) to give compound 2 (210 mg, 1.35 mmol, 19.10% yield) as light yellow oil. 1H NMR: (400 MHz, CDCl3) δ = 8.13 (s, 1H), 4.40 (q, J = 7.1 Hz, 2H), 4.34 (s, 3H), 1.41 (t, J = 7.2 Hz, 3H) Synthesis of compound 3 To a mixture of compound 2 (290 mg, 1.87 mmol, 1 eq) in THF (3 mL) was added LAH (78.03 mg, 2.06 mmol, 1.1 eq) in portions at 0 °C under N2. The mixture was stirred at 25 °C for 2 h. Na2SO4•10H2O was added to the reaction mixture in portions. Then the mixture was dried with Na2SO4, filtered and the filtrate was concentrated at reduced pressure to give compound 3 (170 mg, 1.50 mmol, 80.41% yield) as colorless oil.1H NMR: (400 MHz, CDCl3) δ = 7.56 (s, 1H), 4.78 (s, 2H), 4.11 (s, 3H) Synthesis of compound 4 To a solution of compound 3 (50 mg, 442.02 μmol, 1 eq) in DCM (1.5 mL) and DMF (0.05 mL) was added SOCl2 (78.88 mg, 663.03 μmol, 48.10 μL, 1.5 eq). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated in vacuum to give compound 4 (50 mg, crude) as brown oil. 1H NMR: (400 MHz, DMSO-d6) δ = 7.80 (s, 1H), 5.02 (s, 2H), 4.05 (s, 3H) Synthesis of compound 5 To a solution of compound 4A (30 mg, 113.94 μmol, 1 eq) and compound 4 (22.49 mg, 170.91 μmol, 1.5 eq) in DMF (2 mL) was added Cs2CO3 (111.37 mg, 341.83 μmol, 3 eq). The mixture was stirred at 40 °C for 16 h. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with brine (8 mL*3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue 1. The aqueous phase was collected and followed by lyophilization to give a residue 2. The residue 2 was triturated with ethyl acetate (30 mL) at 25 °C for 15 min. The resulting mixture was filtered and the filtrate was combined with residue 1 and concentrated in vacuum to give compound 5 (30 mg, 83.71 μmol, 73.46% yield) as light yellow gum. Synthesis of Compound 31 To a mixture of compound 5 (30 mg, 83.71 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (4.01 mg, 167.41 μmol, 2 eq). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated in vacuum to remove THF, then the mixture was adjusted to pH about 3-4 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%-35%, 10 min) followed by lyophilization to give Compound 31 (12.21 mg, 35.46 μmol, 42.36% yield, 100% purity) as yellow solid.1H NMR: (400 MHz, CD3OD) δ = 8.54 (br s, 1H), 8.07 (br s, 1H), 7.74 (s, 1H), 5.42 (s, 2H), 4.36 - 4.23 (m, 1H), 4.15 (s, 3H), 2.15 - 1.85 (m, 4H), 0.91 - 0.74 (m, 6H); LCMS: RT = 0.692 min, m/z = 345.2 (M+H)+. Synthetic Scheme Compound 32: Synthesis of compound 2 To a mixture of compound 1 (2 g, 10.36 mmol, 1 eq) and compound 1A (1.08 g, 12.44 mmol, 1.45 mL, 1.2 eq) in dioxane (10 mL) was added DIEA (2.68 g, 20.73 mmol, 3.61 mL, 2 eq). The mixture was stirred at 40 °C for 16 h. The reaction mixture was purified by reversed-phase HPLC (0.1% FA condition) to give compound 2 (0.9 g, 3.69 mmol, 35.64% yield) as yellow solid. LCMS: RT = 0.562 min, m/z = 244.4 (M+H)+. Synthesis of compound 3 To a solution of compound 2 (200 mg, 820.72 μmol, 1 eq) in dioxane (2 mL) was added TEA (249.14 mg, 2.46 mmol, 342.70 μL, 3 eq) and DPPA (271.03 mg, 984.86 μmol, 213.41 μL, 1.2 eq). The mixture was stirred at 20 °C for 1 h. Then the mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 3/1, 1/1. TLC (Petroleum ether : Ethyl acetate = 1:1; Rf = 0.43) ) to give compound 3 (100 mg, 415.47 μmol, 50.62% yield) as yellow solid. LCMS: RT = 0.678 min, m/z = 241.1 (M+H)+. Synthesis of compound 4 To a solution of compound 3A (60.08 mg, 415.47 μmol, 1 eq) in DMF (2 mL) was added Cs2CO3 (406.11 mg, 1.25 mmol, 3 eq) and compound 3 (100 mg, 415.47 μmol, 1 eq). The mixture was stirred at 40 °C for 16 h. The reaction mixture was diluted with water (10 mL) and the resulting mixture was extracted with EtOAc (10 mL*3) and the combined organic phase was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated at reduced pressure to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 1/1, 0/1. TLC (Petroleum ether : Ethyl acetate = 1:1; Rf = 0.1)) to give compound 4 (120 mg, 344.01 μmol, 82.80% yield) as yellow oil. LCMS: RT = 0.630 min, m/z = 349.2 (M+H)+. Synthesis of compound 5 To a solution of compound 4 (100 mg, 286.67 μmol, 1 eq), TEA (87.03 mg, 860.02 μmol, 119.70 μL, 3 eq) in MeOH (0.5 mL) was added Pd(dppf)Cl2 (20.98 mg, 28.67 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (50 psi) at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 1/1, 0/1. TLC (Dichloromethane : Methanol = 10:1; Rf = 0.38)) to give compound 5 (50 mg, 134.26 μmol, 46.83% yield) as brown oil. Synthesis of Compound 32 To a mixture of compound 5 (30.08 mg, 80.77 μmol, 1 eq) in THF (2 mL) and H2O (1 mL) was added LiOH (3.87 mg, 161.54 μmol, 2 eq). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated at reduced pressure to give a residue. The residue was diluted with water (10 mL) and the pH was adjusted to 5 with 1M HCl, then the mixture was concentrated at reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um;mobile phase: [water (FA) -ACN];B%: 1%-25%, 10 min) to give Compound 32 (9.16 mg, 22.65 μmol, 28.04% yield, FA) as yellow solid. 1H NMR: (400 MHz, CD3OD) δ= 8.31 (s, 1H), 7.91 (s, 1H), 7.83 (br s, 1H), 7.26 (br s, 1H), 5.26 (s, 2H), 4.26 (br s, 1H), 4.13 (q, J = 7.3 Hz, 2H), 2.15 - 1.93 (m, 2H), 1.71 (br s, 2H), 1.32 (t, J = 7.3 Hz, 3H), 0.67 (br s, 6H); LCMS: RT = 0.249 min, m/z = 359.2 (M+H)+. Synthetic Scheme Compound 33: 2 To a mixture of compound 1 (0.5 g, 2.30 mmol, 1 eq) and TEA (349.53 mg, 3.45 mmol, 480.78 uL, 1.5 eq) in DCM (5 mL) was added pentan-3-amine (210.76 mg, 2.42 mmol, 281.76 uL, 1.05 eq) at 0 °C under N2. The mixture was stirred at 0 °C for 1 h. Then the mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate=80/1, 10/1) to give compound 2 (430 mg, 1.51 mmol, 65.68% yield) as yellow solid. Synthesis of compound 3 To a mixture of compound 2 (180 mg, 633.17 µmol, 1 eq) in H2O (2 mL) and EtOH (2 mL) was added Fe (176.81 mg, 3.17 mmol, 5 eq) and NH4Cl (203.21 mg, 3.80 mmol, 6 eq). The mixture was stirred at 80 °C for 2 h. The reaction mixture was diluted with H2O (20 mL) and the resulting mixture was extracted with ethyl acetate (20 mL*3).Then combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 3 (160 mg, crude) as brown oil. Synthesis of compound 4 To a solution of compound 3 (160 mg, 629.18 µmol, 1 eq) in MeCN (3 mL) was added CDI (153.03 mg, 943.77 µmol, 1.5 eq). The mixture was stirred at 80 °C for 16 h. The mixture was concentrated at reduced pressure to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate=10/1, 3/1; TLC (Petroleum ether : Ethyl acetate=3:1; Rf=0.12)) to give compound 4 (0.2 g, crude) as pale solid. Synthesis of compound 5 To a solution of compound 1A (103.18 mg, 713.54 μmol, 1 eq) in DMF (3 mL) was added Cs2CO3 (697.45 mg, 2.14 mmol, 3 eq) and compound 4 (200 mg, 713.54 μmol, 1 eq). The mixture was stirred at 40 °C for 4 h. The reaction mixture was diluted with water (10 mL) and the resulting was extracted with EtOAc (10 mL*3), the combined organic phase was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated at reduced pressure to give compound 5 (200 mg, 514.89 μmol, 72.16% yield) as brown oil. Synthesis of Compound 33 To a mixture of compound 5 (200 mg, 514.89 μmol, 1 eq) in THF (2 mL) and H2O (1 mL) was added LiOH (24.66 mg, 1.03 mmol, 2 eq). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated at reduced pressure to give a residue. The residue was diluted with water (10 mL) and the pH was adjusted to 5 with 1M HCl, then the mixture was concentrated at reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um;mobile phase: [water (FA) -ACN];B%: 3%-33%, 10 min) to give Compound 33 (94.52 mg, 252.45 μmol, 49.03% yield) as white solid.1H NMR: (400 MHz, CD3OD) δ = 7.89 (br s, 1H), 7.65 (d, J = 6.1 Hz, 1H), 7.17 (d, J = 11.1 Hz, 2H), 5.22 (s, 2H), 4.26 - 4.18 (m, 1H), 4.14 (q, J = 7.3 Hz, 2H), 2.16 - 2.02 (m, 2H), 1.94 - 1.81 (m, 2H), 1.31 (t, J = 7.3 Hz, 3H), 0.83 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.668 min, m/z = 375.2 (M+H)+. Synthetic Scheme Compound 34: Synthesis of compound 1 To a mixture of compound 1 (2 g, 8.40 mmol, 1 eq) and TEA (1.28 g, 12.61 mmol, 1.75 mL, 1.5 eq) in DMF (20 mL) was added compound 2 (769.13 mg, 8.82 mmol, 1.03 mL, 1.05 eq) at 0 °C under N2. The mixture was stirred at 25 °C for 16 h. The mixture was diluted with ethyl acetate (100 mL) and the resulting mixture was washed with water (30 mL*3), brine (20 mL*2), dried over Na2SO4, filtered and concentrated at reduced pressure to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 1/0, 200/1) to give compound 3 (550 mg, 1.80 mmol, 21.45% yield) as yellow oil. 1H NMR: (400 MHz, CDCl3) δ = 7.43 (dd, J = 7.0, 9.5 Hz, 1H), 6.54 (dd, J = 1.7, 9.5 Hz, 1H), 3.43 - 3.34 (m, 1H), (s, 3H), 1.57 - 1.53 (m, 1H), 0.95 (t, J = 7.5 Hz, 6H). Synthesis of compound 4 To a solution of compound 3 (550 mg, 1.80 mmol, 1 eq) in H2O (2 mL) and EtOH (2 mL) was added Fe (503.28 mg, 9.01 mmol, 5 eq) and NH4Cl (578.49 mg, 10.81 mmol, 6 eq). The mixture was stirred at 80 °C for 2 h. The reaction mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (8 mL*3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 4 (520 mg, crude) as yellow oil. Synthesis of compound To a solution of compound 4 (520 mg, 1.89 mmol, 1 eq) in MeCN (6 mL) was added CDI (459.65 mg, 2.83 mmol, 1.5 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with H2O (8 mL*3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 7/3, TLC (Petroleum ether: Ethyl acetate = 3: 1, Rf = 0.32)) to give compound 5 (270 mg, 896.55 μmol, 47.44% yield) as yellow solid. Synthesis of compound 7 To a solution of compound 5 (100 mg, 332.06 μmol, 1 eq) and compound 6 (72.02 mg, 498.08 μmol, 1.5 eq) in DMF (2 mL) was added Cs2CO3 (324.57 mg, 996.17 μmol, 3 eq). The mixture was stirred at 40 °C for 2 h. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with brine (8 mL*3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 0/1, TLC (Dichloromethane: Methanol = 10: 1, Rf = 0.37)) to give compound 7 (90 mg, 219.89 μmol, 66.22% yield) as colorless gum. Synthesis of compound 8 To a solution of compound 7 (90 mg, 219.89 μmol, 1 eq) in MeOH (3 mL) was added Pd(dppf)Cl2 (16.09 mg, 21.99 μmol, 0.1 eq) and TEA (44.50 mg, 439.78 μmol, 61.21 μL, 2 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture was stirred under CO (15 psi) at 80 °C for 48 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 0/1, TLC (Dichloromethane: Methanol = 10: 1, Rf = 0.38)) to give compound 8 (80 mg, 205.95 μmol, 93.66% yield) as brown gum. Synthesis of Compound 34 To a mixture of compound 8 (80 mg, 205.95 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (9.87 mg, 411.91 μmol, 2 eq). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated in vacuum to remove THF, then the mixture was adjusted to pH about 3-4 with 1N HCl. The resulting mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%-35%, 10 min) followed by lyophilization to give Compound 34 (13.35 mg, 35.66 μmol, 17.31% yield, 100% purity) as white solid.1H NMR: (400 MHz, CD3OD) δ = 7.91 (s, 1H), 7.67 (dd, J = 7.0, 8.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.06 - 6.94 (m, 1H), 5.31 (s, 2H), 4.34 - 4.16 (m, 3H), 2.18 - 2.01 (m, 2H), 1.95 - 1.79 (m, 2H), 1.39 (t, J = 7.3 Hz, 3H), 0.82 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.790 min, m/z = 375.2 (M+H)+. Synthetic Scheme Compound 35: 35: Synthesis of compound 3 To a mixture of compound 1 (1 g, 3.91 mmol, 1 eq) and DIEA (757.35 mg, 5.86 mmol, 1.02 mL, 1.5 eq) in DMF (10 mL) was added compound 2 (340.51 mg, 3.91 mmol, 455.23 μL, 1 eq) slowly at 0 °C under N2. The mixture as stirred at 25 °C for 16 h. The mixture was diluted with ethyl acetate (100 mL) and the resulting mixture was washed with water (20 mL*3), brine (20 mL*2), dried over Na2SO4, filtered and concentrated at reduced pressure to give a residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 1/0, 80/1) to give compound 3 (0.35 g, 1.08 mmol, 27.73% yield) as yellow oil. 1H NMR: (400 MHz, CDCl3) δ = 6.44 (dd, J = 2.0, 11.5 Hz, 1H), 3.38 - 3.28 (m, 1H), 1.79 - 1.59 (m, 4H), 0.98 (t, J = 7.4 Hz, 6H). Synthesis of compound 4 To a solution of compound 3 (0.35 g, 1.08 mmol, 1 eq) in H2O (2 mL) and EtOH (2 mL) was added Fe (302.47 mg, 5.42 mmol, 5 eq) and NH4Cl (347.62 mg, 6.50 mmol, 6 eq). The mixture was stirred at 80 °C for 2 h. The reaction mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (8 mL*3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 4 (390 mg, crude) as yellow oil. Synthesis of compound 5 To a mixture of compound 4 (390 mg, 1.33 mmol, 1 eq) in MeCN (4 mL) was added CDI (323.58 mg, 2.00 mmol, 1.5 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with H2O (8 mL*3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 7/3, TLC (Petroleum ether: Ethyl acetate = 3: 1, Rf = 0.34)) to give compound 5 (210 mg, 658.01 μmol, 49.46% yield) as white solid. Synthesis of compound 7 To a solution of compound 5 (100 mg, 313.34 μmol, 1 eq) and 5-(chloromethyl) -1-ethyl-imidazole (67.96 mg, 470.01 μmol, 1.5 eq) in DMF (2 mL) was added Cs2CO3 (306.27 mg, 940.01 μmol, 3 eq). The mixture was stirred at 40 °C for 2 h. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with brine (8 mL*3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 0/1, TLC (Dichloromethane: Methanol = 10: 1, Rf = 0.52)) to give compound 7 (86 mg, 201.27 μmol, 64.23% yield) as colorless gum. Synthesis of compound 8 To a solution of compound 7 (86 mg, 201.27 μmol, 1 eq) in MeOH (5 mL) was added TEA (40.73 mg, 402.54 μmol, 56.03 μL, 2 eq) and Pd(dppf)Cl2 (14.73 mg, 20.13 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (15 psi) at 80 °C for 48 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to 0/1, TLC (Dichloromethane: Methanol = 10: 1, Rf = 0.49)) to give compound 8 (75 mg, 184.54 μmol, 91.69% yield) as brown gum. Synthesis of Compound 35 To a solution of compound 8 (75 mg, 184.54 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (8.84 mg, 369.07 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF, then the mixture was adjusted to pH about 3-4 with 1N HCl. The resulting mixture was concentrated in vacuum. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 5%-35%, 10 min) followed by lyophilization to give Compound 35 (12.95 mg, 33.00 μmol, 17.88% yield, 100% purity) as off-white solid.1H NMR: (400 MHz, CD3OD) δ = 8.45 (br s, 1H), 7.29 (br s, 1H), 7.02 (d, J = 9.0 Hz, 1H), 5.31 (s, 2H), 4.32 (q, J = 7.3 Hz, 2H), 4.22 - 4.11 (m, 1H), 2.15 - 2.01 (m, 2H), 1.86 (ddd, J = 5.3, 7.2, 14.2 Hz, 2H), 1.43 (t, J = 7.3 Hz, 3H), 0.82 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.826 min, m/z = 393.2 (M+H)+. Synthetic Scheme Compound 36:
Synthesis of compound 2 To a mixture of compound 1 (5.9 g, 20.93 mmol, 1 eq) and DIEA (5.41 g, 41.86 mmol, 7.29 mL, 2 eq) in THF (80 mL) was added PMBNH2 (3.16 g, 23.02 mmol, 2.98 mL, 1.1 eq) at 0 °C. The mixture was stirred at 20 °C for 3 h. The reaction mixture was diluted with ethyl acetate (150 mL). The organic layer was washed with brine (50 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1) to give compound 2 (7 g, 20.70 mmol, 98.90% yield) as yellow solid.1H NMR (400 MHz, DMSO-d6) δ = 8.98 (br t, J = 6.0 Hz, 1H), 8.82 (s, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.10 (s, 1H), 6.92 (d, J = 8.6 Hz, 2H), 4.58 (d, J = 6.1 Hz, 2H), 3.73 (s, 3H). Synthesis of compound To a mixture of compound 2 (7.59 g, 22.45 mmol, 1 eq) and NH4Cl (1.44 g, 26.93 mmol, 1.2 eq) in EtOH (90 mL) and H2O (30 mL) was added Fe (6.27 g, 112.23 mmol, 5 eq), the mixture was stirred at 80 °C for 2 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was diluted with Ethyl acetate (200 mL). The organic layer was washed with brine (80 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 1: 1) to give compound 3 (6.8 g, 22.07 mmol, 98.31% yield) as yellow gum. Synthesis of compound 4 To a solution of compound 3 (6.8 g, 22.07 mmol, 1 eq) in MeCN (80 mL) was added CDI (5.37 g, 33.10 mmol, 1.5 eq). The mixture was stirred at 80 °C for 16 h. The mixture was filtered and the filter cake was washed with water (20 mL) and MeCN (50 mL). Then the filter cake was dried at reduced pressure to give compound 4 (6 g, 17.96 mmol, 81.37% yield) as white solid.1H NMR (400 MHz, DMSO-d6) δ = 11.43 (br s, 1H), 7.97 (s, 1H), 7.44 (s, 1H), 7.32 - 7.26 (m, 2H), 6.93 - 6.87 (m, 2H), 4.94 (s, 2H), 3.71 (s, 3H). Synthesis of compound 6 To a solution of compound 4 (6 g, 17.96 mmol, 1 eq) in DMF (80 mL) was added NaH (861.84 mg, 21.55 mmol, 60% purity, 1.2 eq) at 0 °C, then compound 5 (4.07 g, 26.93 mmol, 3.33 mL, 1.5 eq) was added. The mixture was stirred at 60 °C for 16 h. The reaction mixture was poured into water (500 mL) and extracted with ethyl acetate (100 mL*3). The combined organic layers were washed with brine (100 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1) to give compound 6 (7.4 g, crude) as yellow gum.1H NMR (400 MHz, DMSO-d6) δ = 8.31 (s, 1H), 7.51 (s, 1H), 7.25 (d, J = 8.6 Hz, 2H), 6.93 - 6.87 (m, 2H), 5.01 (s, 2H), 4.15 (tt, J = 5.1, 10.2 Hz, 1H), 3.71 (s, 3H), 1.98 - 1.90 (m, 2H), 1.85 - 1.74 (m, 2H), 0.72 (t, J = 7.3 Hz, 6H) Synthesis of compound 7 To a solution of compound 6 (5.5 g, 13.60 mmol, 1 eq) in MeCN (80 mL) and H2O (80 mL) was added CAN (22.37 g, 40.81 mmol, 20.34 mL, 3 eq) at 0 °C. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was diluted with Ethyl acetate (200 mL). The organic Layer was washed with brine (100 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 3: 1) to give compound 7 (3.2 g, 11.26 mmol, 82.78% yield) as yellow solid. 1H NMR (400 MHz, DMSO-d6) δ = 11.57 (s, 1H), 8.24 (s, 1H), 7.18 (s, 1H), 4.13 - 4.01 (m, 1H), 2.01 - 1.85 (m, 2H), 1.84 - 1.70 (m, 2H), 0.72 (t, J = 7.4 Hz, 6H). Synthesis of compound 8 To a solution of compound 7 (3.2 g, 11.26 mmol, 1 eq) in DMF (50 mL) was added NaH (540.50 mg, 13.51 mmol, 60% purity, 1.2 eq) at 0 °C, then SEM-Cl (2.25 g, 13.51 mmol, 2.39 mL, 1.2 eq) was added. The mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was diluted with ethyl acetate (200 mL). The organic layer was washed with brine (100 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1) to give compound 8 (4.1 g, 9.89 mmol, 87.85% yield) as yellow solid. Synthesis of compound 8 To a mixture of compound 8 (800 mg, 1.93 mmol, 1 eq) and compound 9 (153.02 mg, 2.12 mmol, 1.1 eq) in THF (15 mL) was added n-BuLi (2.5 M, 926.62 μL, 1.2 eq) (in THF) slowly at -70 °C under N2, The resulting mixture was stirred at -70 °C for 1 h. The mixture was quenched with saturated NH4Cl (2 mL) and the resulting mixture was concentrated under vacuum to give a residue. The residue was diluted with Ethyl acetate (100 mL). The organic layer was washed with brine (50 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 3: 1) to give 310 mg colorless gum. The colorless gum was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (ammonia hydroxide v/v) -ACN]; B%: 40%-70%, 9 min) to give compound 10 (55 mg, 134.94 μmol, 6.99% yield) as colorless gum.1H NMR (400 MHz, CD3OD) δ = 8.49 (s, 1H), 7.62 (s, 1H), 5.39 (s, 2H), 5.04 (d, J = 6.5 Hz, 2H), 4.83 (s, 2H), 4.26 (tt, J = 5.1, 10.4 Hz, 1H), 3.61 (t, J = 8.0 Hz, 2H), 2.17 - 2.02 (m, 2H), 1.99 - 1.84 (m, 2H), 0.93 - 0.87 (m, 2H), 0.84 (t, J = 7.3 Hz, 6H), -0.07 (s, 8H). Synthesis of compound 11 To a solution of compound 10 (50 mg, 122.68 μmol, 1 eq) in THF (1 mL) was added TBAF (1 M, 1.23 mL, 10 eq). The mixture was stirred at 60 °C for 36 h. The reaction mixture was diluted with Ethyl acetate (50 mL). The organic layer was washed with brine (30 mL*3), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 0: 1, TLC (dichloromethane: methanol = 10: 1) to give compound 11 (35 mg, crude) as white solid. 1H NMR (400 MHz, CD3OD) δ = 8.43 (s, 1H), 7.44 (s, 1H), 5.03 (d, J = 6.6 Hz, 2H), 4.83 (d, J = 6.6 Hz, 2H), 4.22 (tt, J = 5.1, 10.4 Hz, 1H), 2.14 - 2.03 (m, 2H), 1.96 - 1.85 (m, 2H), 0.84 (t, J = 7.4 Hz, 6H) Synthesis of Compound 36 To a solution of compound 11 (30 mg, 108.18 μmol, 1 eq) in DMF (1 mL) was added Cs2CO3 (105.74 mg, 324.54 μmol, 3 eq) and 5-(chloromethyl) -1-ethyl-imidazole (23.46 mg, 162.27 μmol, 1.5 eq). The mixture was stirred at 40 °C for 16 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was purified prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%- 21%, 7 min) to give Compound 36 (13.07 mg, 33.57 μmol, 31.03% yield, 99% purity) as off-white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.56 (s, 1H), 8.21 - 8.10 (m, 1H), 7.65 (s, 1H), 7.45 (s, 1H), 6.90 (s, 1H), 6.63 - 6.44 (m, 1H), 5.13 (s, 2H), 4.90 (d, J = 6.0 Hz, 2H), 4.62 (d, J = 6.0 Hz, 2H), 4.24 - 4.13 (m, 1H), 4.06 - 3.97 (m, 2H), 2.09 - 1.93 (m, 2H), 1.89 - 1.75 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H), 0.74 (t, J = 7.3 Hz, 6H); LCMS: RT = 0.316 min, m/z = 386.3 (M+H)+. Synthetic Scheme Compound 37: 2 To a solution of compound 1 (300 mg, 1.78 mmol, 1 eq), sodium;triacetoxyboranuide (3.77 g, 17.80 mmol, 10 eq) and CH3COOH (21.37 mg, 355.91 μmol, 20.35 μL, 0.2 eq) in DCE (5 mL) was added acetaldehyde (391.94 mg, 8.90 mmol, 499.29 μL, 5 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to give residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 5/1, 3/1) to give compound 2 (170 mg, 756.60 μmol, 42.52% yield) as yellow solid. LCMS: RT = 0.741 min, m/z = 225.1 (M+H)+. Synthesis of compound 3 To a solution of compound 2 (170 mg, 756.60 μmol, 1 eq) in DMF (2 mL) was added CS2CO3 (739.55 mg, 2.27 mmol, 3 eq) and compound 2A (109.41 mg, 756.60 μmol, 1 eq). The mixture was stirred at 40 °C for 16 h. The reaction mixture was added into the water (10 mL) and the resulting mixture was extracted with ethyl acetate (10 mL*3), the combined organic phase was washed with saturated NaHCO3 aqueous solution (10 mL*3), dried with anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuum to give residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 1/1, 0/1) to give compound 3 (100 mg, 300.45 μmol, 39.71% yield) as yellow oil.1H NMR: (400 MHz, DMSO-d6) δ = 8.76 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.62 (s, 1H), 5.40 (s, 2H), 4.06-4.11 (m, 2H), 3.51 (q, J = 6.9 Hz, 4H), 1.11-1.20 (m, 9H). Synthesis of compound 4 To a solution of compound 3 (100 mg, 300.45 μmol, 1 eq), TEA (91.21 mg, 901.36 μmol, 125.46 μL, 3 eq) in MeOH (0.5 mL) was added Pd(dppf)Cl2 (21.98 mg, 30.05 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (50 psi) at 70 °C for 16 h. The reaction mixture was concentrated in vacuum to give residue. The residue was purified by silica gel chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 1/1, 0/1. TLC (Dichloromethane : Methanol = 10:1; Rf = 0.38)) to give compound 4 (60 mg, 168.34 μmol, 56.03% yield) as brown solid. Synthesis of Compound 37 To a mixture of compound 4 (60 mg, 168.34 μmol, 1 eq) in THF (2 mL) and H2O (1 mL) was added LiOH (8.06 mg, 336.68 μmol, 2 eq). The mixture was stirred at 25 °C for 1 h. The mixture was concentrated at reduced pressure to give a residue. The residue was diluted with water (2 mL) and the pH was adjusted to 5 with 1M HCl, then the mixture was concentrated at reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um;mobile phase: [water (FA) -ACN];B%: 1%-20%, 7 min) to give Compound 37 (7.54 mg, 19.06 μmol, 11.32% yield, 98.2% purity, FA) as yellow solid. 1H NMR: (400 MHz, CD3OD) δ = 8.92 (brs, 1H), 8.40 (s, 1H), 8.16 (brs, 1H), 7.74 (s, 1H), 7.12 (s, 1H), 5.57 (brs, 2H), 4.22-4.18 (m, 2H), 3.68-3.51 (m, 4H), 1.28-1.32 (m, 3H), 1.32-1.03 (m, 6H); LCMS: RT = 0.644 min, m/z = 343.4 (M+H)+. Synthetic Scheme Compound 38: Experimental Procedure Compound 38: Synthesis of compound 3 To a mixture of compound 1 (200 mg, 1.15 mmol, 1 eq) and Cs2CO3 (1.13 g, 3.46 mmol, 3 eq) in DMF (4 mL) was added compound 2 (250.38 mg, 1.38 mmol, 1.2 eq, HCl). The mixture was stirred at 40 °C for 2 h. The mixture was diluted with ethyl acetate (50 mL) and the resulting mixture was washed with brine (20 mL*2), dried over Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 0: 1) to give compound 3 (170 mg, 603.48 μmol, 52.37% yield) as yellow gum. LCMS: RT = 0.802 min, m/z = 281.9 (M + H)+. Synthesis of compound 4 To a mixture of compound 3 (170 mg, 603.48 μmol, 1 eq) and NH4Cl (38.74 mg, 724.18 μmol, 1.2 eq) in EtOH (3 mL) and H2O (1 mL) was added Fe (168.51 mg, 3.02 mmol, 5 eq), the mixture was stirred at 80 °C for 2 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was purified by column chromatography (100 mesh silica gel, dichloromethane: methanol = 1: 0 to 5: 1 to give compound 4 (110 mg, 437.00 μmol, 72.41% yield) as yellow solid. Synthesis of compound 5 A mixture of compound 4 (100 mg, 397.28 μmol, 1 eq) and CDI (96.63 mg, 595.91 μmol, 1.5 eq) in ACN (2 mL) was stirred at 80 °C for 16 h. The mixture was filtered and the filter cake was washed with water (5 mL) and ACN (5 mL). Then the filter cake was dried at reduced pressure to give compound 5 (50 mg, 180.04 μmol, 45.32% yield) as white solid. 1H NMR: (400 MHz, DMSO-d6) δ = 11.49 (s, 1H), 7.99 (s, 1H), 7.68 (s, 1H), 7.30 (s, 1H), 7.06 (s, 1H), 5.05 (s, 2H), 4.01 (q, J = 7.2 Hz, 2H), 1.18 (t, J = 7.3 Hz, 3H). Synthesis of compound 7 To a mixture of compound 5 (30 mg, 108.03 μmol, 1 eq) and Cs2CO3 (105.59 mg, 324.08 μmol, 3 eq) in DMF (1 mL) was added compound 6 (30.09 mg, 129.63 μmol, 1.2 eq). The mixture was stirred at 40 °C for 2 h. The reaction mixture was diluted with Ethyl acetate (50 mL). The resulting mixture was washed with brine (30 mL*2), dried over Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 0: 1; TLC (petroleum ether: ethyl acetate = 0: 1) to give compound 7 (35 mg, 97.29 μmol, 90.06% yield) as white solid. LCMS: RT = 0.744 min, m/z = 360.1 (M + H)+. Synthesis of compound 8 To a mixture of compound 7 (30 mg, 83.40 μmol, 1 eq), TEA (25.32 mg, 250.19 μmol, 34.82 μL, 3 eq) in MeOH (3 mL) was added Pd(dppf) Cl2 (6.10 mg, 8.34 μmol, 0.1 eq) under N2. The suspension was degassed under vacuum and purged with CO for 3 times. The reaction mixture stirred under CO (15 Psi) at 80 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 0: 1 to ethyl acetate: methanol = 10: 1) to give compound 8 (30 mg, crude) as yellow solid. LCMS: RT = 0.700 min, m/z = 384.1 (M + H)+. Synthesis of Compound 38 To a solution of compound 8 (30 mg, 78.26 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (5.62 mg, 234.79 μmol, 3 eq). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under vacuum to give a residue. Then the residue was adjusted pH = 5 with 1 M HCl and the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (The residue was purified by prep-HPLC (column: Unisil 3- 100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-20%, 5 min) to give Compound 38 (16.4 mg, 40.41 μmol, 51.64% yield, 91% purity) as white solid.1H NMR: (400 MHz, DMSO-d6) δ = 8.58 (s, 1H), 7.94 (s, 1H), 7.66 (s, 1H), 6.98 (s, 1H), 5.23 (s, 2H), 4.95 (q, J = 9.2 Hz, 2H), 3.99 (q, J = 7.2 Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H); LCMS: RT = 0.170 min, m/z = 368.1 (M - H)-. Synthetic Scheme Compound 39: Experimental Procedure for Compound 39: Synthesis of compound 2 A mixture of compound 1 (9.5 g, 49.23 mmol, 1 eq), PMBNH2 (12.15 g, 88.61 mmol, 11.47 mL, 1.8 eq) and DIPEA (11.45 g, 88.61 mmol, 15.43 mL, 1.8 eq) in THF (100 mL) was stirred at 0 °C for 0.5 h. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (100 mL* 3). The combined organic phase were collected, dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1/0 to 4/1, TLC (Petroleum ether: Ethyl acetate = 5: 1; Rf = 0.27)) to give compound 2 (12 g, 40.86 mmol, 83.00% yield) as yellow solid.1H NMR: (400 MHz, CDCl3) δ = 9.06 (s, 1H), 8.43 (br s, 1H), 7.28 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.7 Hz, 2H), 6.80 (s, 1H), 4.48 (d, J = 5.3 Hz, 2H), 3.85 (s, 3H). Synthesis of compound 3 A mixture of compound 2 (12 g, 40.86 mmol, 1 eq), Fe (11.41 g, 204.29 mmol, 5 eq) and NH4Cl (13.11 g, 245.14 mmol, 6 eq) in EtOH (70 mL) and H2O (70 mL) was stirred at 80 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated to remove EtOH. The resulting mixture was diluted with water (200 mL) and extracted with EtOAc (150 mL x 3), the combined organic phase were collected, dried over Na2SO4, filtered and the filterate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1/0 to 4/1, TLC (Petroleum ether: Ethyl acetate = 1: 1; Rf = 0.20)) to give compound 3 (9 g, 34.13 mmol, 83.53% yield) as purple solid.1H NMR: (400 MHz, CD3OD) δ = 7.47 (s, 1H), 7.28 (d, J = 8.6 Hz, 2H), 6.92 - 6.88 (m, 2H), 6.36 (s, 1H), 4.34 (s, 2H), 3.78 (s, 3H). Synthesis of compound 4 A mixture of compound 3 (9.62 g, 36.48 mmol, 1 eq) and CDI (8.87 g, 54.72 mmol, 1.5 eq) in ACN (100 mL) was stirred at 80 °C for 16 h. The reaction mixture was triturated with water (100 mL) at 25 °C for 10 min. The resulting mixture was filtered and the filtered cake was collected, concentrated in vacuum to give compound 4 (9 g, 31.06 mmol, 85.16% yield) as purple solid.1H NMR: (400 MHz, DMSO-d6) δ = 11.43 (br s, 1H), 7.96 (s, 1H), 7.49 - 7.24 (m, 3H), 6.89 (br d, J = 8.3 Hz, 2H), 4.94 (br s, 2H), 3.71 (s, 3H). Synthesis of compound 5 To a mixture of compound 4 (7 g, 24.16 mmol, 1 eq) in DMF (100 mL) was added NaH (1.16 g, 28.99 mmol, 60% purity, 1.2 eq) in portions at 0 °C under N2. Then then 3-bromopentane (5.47 g, 36.24 mmol, 4.48 mL, 1.5 eq) was added to the reaction mixture. The mixture was stirred at 60 °C for 16 h. LCMS showed 20% of compound 4 remained and 75% of peak with desired mass was detected. NaH (483.20 mg, 12.08 mmol, 60% purity, 0.5 eq) was added at 0 °C, then 3- bromopentane (1.82 g, 12.08 mmol, 1.50 mL, 0.5 eq) was added. The mixture was stirred at 60 °C for 16 h. The reaction mixture was poured into water (500 mL) slowly and extracted with EtOAc (100 mL*3). The combined organic phase was washed with brine (100 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1; TLC (petroleum ether: ethyl acetate = 3: 1, Rf = 0.30)) to give compound 5 (7.4 g, 20.56 mmol, 85.12% yield) as red oil.1H NMR: (400 MHz, DMSO-d6) δ = 8.31 (s, 1H), 7.40 (s, 1H), 7.26 (d, J = 8.8 Hz, 2H), 6.96 - 6.85 (m, 2H), 5.01 (s, 2H), 4.16 (td, J = 5.1, 10.3 Hz, 1H), 3.71 (s, 3H), 1.97 (ddd, J = 7.4, 10.2, 14.1 Hz, 2H), 1.80 (ddd, J = 5.3, 7.3, 14.1 Hz, 2H), 0.72 (t, J = 7.3 Hz, 6H) Synthesis of compound 6 A mixture of compound 5 (6.2 g, 17.23 mmol, 1 eq), Pd(dppf) Cl2 (2.52 g, 3.45 mmol, 0.2 eq) and TEA (5.23 g, 51.69 mmol, 7.19 mL, 3 eq) in MeOH (60 mL) was stirred under CO (277.89 μmol) (50 psi) at 80 °C for 72 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1; TLC (Petroleum ether: Ethyl acetate = 3: 1; Rf = 0.13)) to give compound 6 (6 g, 15.65 mmol, 90.82% yield) as black oil. 1H NMR: (400 MHz, CDCl3) δ = 8.43 (s, 1H), 7.76 (s, 1H), 7.25 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 5.05 (s, 2H), 4.29 (tt, J = 5.2, 10.1 Hz, 1H), 3.98 (s, 3H), 3.77 (s, 3H), 2.04 - 1.86 (m, 4H), 0.86 (t, J = 7.4 Hz, 6H). Synthesis of compound 7 To a mixture of compound 6 (6 g, 15.65 mmol, 1 eq) in MeCN (50 mL) and H2O (50 mL) was added CAN (25.74 g, 46.94 mmol, 23.40 mL, 3 eq) in portions at 0 °C. The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated in vacuum to remove MeCN, then the aqueous phase was extracted with ethyl acetate (30 mL*3), the combined organic phase was collected, dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 1/0 to DCM: MeOH = 10 /1) to give compound 7 (3.3 g, 12.53 mmol, 80.10% yield) as yellow solid.1H NMR: (400 MHz, DMSO-d6) δ = 11.66 (s, 1H), 8.57 (s, 1H), 7.66 (s, 1H), 4.22 - 4.08 (m, 1H), 3.85 (s, 3H), 1.98 (s, 2H), 1.88 - 1.75 (m, 2H), 0.74 (t, J = 7.4 Hz, 6H). Synthesis of compound 8 To a mixture of compound 7 (1 g, 3.80 mmol, 1 eq) and 5-(chloromethyl) -1-ethyl-imidazole (823.81 mg, 5.70 mmol, 1.5 eq) in DMF (10 mL) was added Cs2CO3 (3.71 g, 11.39 mmol, 3 eq). The mixture was stirred at 40 °C for 2 h. LCMS showed one main peak of compound 7 remained and desired mass detected. Then the mixture was stirred at 40 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give a residue. The residue was purified by reversed-phase HPLC (0.1% NH3•H2O) followed by lyophilization to give compound 8 (590 mg, 1.59 mmol, 41.86% yield) as brown gum. Synthesis of Compound 39 To a mixture of compound 8 (100 mg, 269.23 μmol, 1 eq) in THF (1 mL) was added MeMgBr (3 M, 206.41 μL, 2.3 eq) at -30 °C under N2. The mixture was stirred at 25 °C for 2 h. LCMS showed compound 8 remained and desired mass detected. Then MeMgBr (3 M, 224.36 μL, 2.5 eq) was added to the mixture at -30 °C under N2. The mixture was stirred at 25 °C for 16 h. The reaction mixture was quenched with H2O (0.5 mL) and concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Synergi C18150*25 mm* 10um; mobile phase: [water (FA) -ACN]; B%: 1%-31%, 10 min) followed by lyophilization to give Compound 39 (2.35 mg, 6.26 μmol, 2.33% yield, 99% purity) as off-white solid.1H NMR: (400 MHz, CD3OD) δ = 8.34 (s, 1H), 7.78 (s, 1H), 7.54 (s, 1H), 7.13 (s, 1H), 5.23 (s, 2H), 4.25 (td, J = 5.1, 10.2 Hz, 1H), 4.14 (br d, J = 7.3 Hz, 2H), 2.13 - 2.04 (m, 2H), 1.96 - 1.84 (m, 2H), 1.53 (s, 6H), 1.31 (t, J = 7.3 Hz, 3H), 0.84 (t, J = 7.3 Hz, 6H); LCMS: RT = 0.724 min, m/z = 372.2 (M+H)+. Synthetic Scheme Compound 40: Experimental To a mixture of compound 8 (50 mg, 134.61 μmol, 1 eq) and NH2OH•HCl (37.42 mg, 538.46 μmol, 4 eq) in MeOH (1 mL) was added DIEA (86.99 mg, 673.07 μmol, 117.24 μL, 5 eq). The mixture was stirred at 70 °C for 16 h. The reaction mixture was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-24%, 7 min) followed by lyophilization to give Compound 40 (4.74 mg, 12.47 μmol, 9.27% yield, 98% purity) as off-white solid.1H NMR: (400 MHz, CD3OD) δ = 8.48 (s, 1H), 7.91 (s, 1H), 7.84 (br d, J = 4.3 Hz, 1H), 7.19 (s, 1H), 5.28 (s, 2H), 4.37 - 4.24 (m, 1H), 4.16 (d, J = 7.3 Hz, 2H), 2.09 (ddd, J = 7.3, 10.4, 14.2 Hz, 2H), 1.93 (ddd, J = 5.3, 7.4, 14.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H), 0.84 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.565 min, m/z = 373.2 (M + H)+. Synthetic Scheme Compound 41:
Experimental Procedure for Compound 41: Synthesis of compound 9 To a mixture of compound 8 (100 mg, 269.23 μmol, 1 eq) in MeOH (2 mL) was added NaBH4 (95 mg, 2.51 mmol, 9.33 eq) in portions. The reaction mixture was stirred at 25 °C for 2 h. LCMS showed one main peak of compound 8 remained and desired mass detected. Then NaBH4 (95 mg, 2.51 mmol, 9.33 eq) was added to the reaction mixture in portions. The mixture was stirred at 40 °C for 16 h. LCMS showed compound 8 remained and desired mass detected. The mixture was stirred at 60 °C for 2 h. The reaction mixture was quenched with NH4Cl (10 mL) and extracted with ethyl acetate (5 mL*3), the combined organic phase were collected, dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give compound 9 (90 mg, crude) as yellow gum. Synthesis of compound 10 To a mixture of compound 9 (90 mg, 262.07 μmol, 1 eq) in THF (1 mL) was added MnO2 (68.35 mg, 786.20 μmol, 3 eq). The mixture was stirred at 60 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuum. The residue was purified by reversed-phase HPLC (0.1% NH3•H2O) followed by concentration to give compound 10 (20 mg, 58.58 μmol, 22.35% yield) as light yellow gum. Synthesis of Compound 41 To a mixture of compound 10 (15 mg, 43.94 μmol, 1 eq) and CsF (13.35 mg, 87.87 μmol, 3.24 μL, 2 eq) in THF (1 mL) was added TMSCF3 (9.37 mg, 65.90 μmol, 1.5 eq). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 0%-30%, 7 min) followed by lyophilization to give Compound 41 (4.72 mg, 11.36 μmol, 25.85% yield, 99% purity) as off-white solid. 1H NMR: (400 MHz, CD3OD) δ = 8.43 (s, 1H), 7.90 (s, 1H), 7.52 (s, 1H), 7.19 (s, 1H), 5.27 (s, 2H), 5.07 (q, J = 6.8 Hz, 1H), 4.27 (td, J = 5.1, 10.3 Hz, 1H), 4.15 (q, J = 7.2 Hz, 2H), 2.14 - 2.02 (m, 2H), 1.92 (td, J = 6.6, 13.1 Hz, 2H), 1.32 (t, J = 7.3 Hz, 3H), 0.89 - 0.78 (m, 6H); LCMS: RT = 0.617 min, m/z = 412.2 (M+H)+. Synthetic Scheme Compound 42:
Synthesis of compound 2 To a mixture of compound 1 (100 mg, 247.34 μmol, 1 eq), CuI (2.36 mg, 12.37 μmol, 0.05 eq) and NaI (74.15 mg, 494.68 μmol, 2 eq) in dioxane (1 mL) was added N, N'-dimethylethane-1, 2-diamine (2.18 mg, 24.73 μmol, 2.66 μL, 0.1 eq). The mixture was stirred at 110 °C for 16 h under N2. The reaction mixture was diluted with ethyl acetate (50 mL). The resulting mixture was washed with brine (20 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1) to give compound 2 (100 mg, 221.58 μmol, 89.59% yield) as colorless gum. Synthesis of compound 3 To a solution of compound 2 (80 mg, 177.27 μmol, 1 eq), CuI (50.64 mg, 265.90 μmol, 1.5 eq) in DMF (2 mL) was added DIEA (183.28 mg, 1.42 mmol, 247.01 μL, 8 eq) and methyl 2, 2-difluoro-2-fluorosulfonyl-acetate (272.44 mg, 1.42 mmol, 180.42 μL, 8 eq). The mixture was stirred at 80 °C for 16 h under N2. The reaction mixture was diluted with ethyl acetate (50 mL). The resulting mixture was washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1) to give compound 3 (75 mg, crude) as yellow gum. Synthesis of compound 4 To a solution of compound 3 (75 mg, 190.64 μmol, 1 eq) in ACN (1 mL) and H2O (1 mL) was added CAN (313.55 mg, 571.93 μmol, 285.04 μL, 3 eq) at 0 °C. The mixture was stirred at 20 °C for 2 h. The reaction mixture was diluted with ethyl acetate (100 mL). The organic layer was washed with brine (30 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 5: 1 to give compound 4 (35 mg, 128.09 μmol, 67.19% yield) as white solid. Synthesis of Compound 42 To a solution of compound 4 (35 mg, 128.09 μmol, 1 eq) in DMF (1 mL) was added Cs2CO3 (125.20 mg, 384.26 μmol, 3 eq) and 5-(chloromethyl) -1-ethyl-imidazole (27.78 mg, 192.13 μmol, 1.5 eq). The mixture was stirred at 40 °C for 2 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 6%- 36%, 7 min) to give Compound 42 (36.6 mg, 95.96 μmol, 74.92% yield, 100% purity) as yellow gum. 1H NMR: (400 MHz, METHANOL-d4) δ = 8.57 (s, 1H), 7.82 (s, 1H), 7.67 (s, 1H), 7.16 (s, 1H), 5.29 (s, 2H), 4.31 (tt, J = 5.0, 10.1 Hz, 1H), 4.13 (q, J = 7.2 Hz, 2H), 2.10 (qdd, J = 7.2, 10.2, 14.4 Hz, 2H), 2.00 - 1.87 (m, 2H), 1.31 (t, J = 7.3 Hz, 3H), 0.85 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.477 min, m/z = 382.1 (M+H)+. Synthetic Scheme Compound 43: 2 To a solution of methyl compound 8 (50 mg, 134.61 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (6.45 mg, 269.23 μmol, 2 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated in vacuum to remove THF, then the mixture was adjusted pH to about 3-4 with 1N HCl. The resulting mixture was purified by reversed-phase HPLC (0.1% FA) followed by lyophilization to give compound 2 (30 mg, 83.94 μmol, 62.35% yield) as colorless gum. Synthesis of Compound 43 To a solution of compound 1 (30 mg, 83.94 μmol, 1 eq) in DMF (1 mL) was added CDI (17.69 mg, 109.12 μmol, 1.3 eq) and the resulting mixture was stirred at 25 °C for 1 h. Then methanesulfonamide (10.70 mg, 109.12 μmol, 1.3 eq) and DBU (16.61 mg, 109.12 μmol, 16.45 μL, 1.3 eq) was added and the mixture was stirred at 25 °C for 4 h. The mixture reaction was concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 μLtra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-26%, 7 min) to give a residue. Then the residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (NH4HCO3) -ACN]; B%: 5%-35%, 9 min) to give Compound 43 (4.09 mg, 9.41 μmol, 11.21% yield, 100% purity) as off-white solid.1H NMR: (400 MHz, CD3OD) δ = 8.57 (s, 1H), 8.03 (s, 1H), 7.87 (brs, 1H), 7.20 (s, 1H), 5.30 (s, 2H), 4.31 (td, J = 5.0, 10.0 Hz, 1H), 4.15 (q, J = 7.3 Hz, 2H), 3.33 (s, 3H), 2.18 - 2.03 (m, 2H), 2.01 - 1.88 (m, 2H), 1.32 (t, J = 7.3 Hz, 3H), 0.85 (t, J = 7.4 Hz, 6H); LCMS: RT = 0.589 min, m/z = 435.2 (M+H)+. Synthetic Scheme Compound 44: Synthesis of compound 2 To a solution of compound 1 (200 mg, 494.68 μmol, 1 eq) in DMA (1 mL) was added CuCN (53.17 mg, 593.62 μmol, 129.67 μL, 1.2 eq) and Pd(PPh3)4 (57.16 mg, 49.47 μmol, 0.1 eq) under N2. The mixture was stirred at 130 °C for 16 h under N2. The reaction mixture was quenched with NH3•H2O (1 mL) and diluted with Ethyl acetate (50 mL). The organic layer was washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 10: 1) to give compound 2 (100 mg, 285.38 μmol, 57.69% yield) as colorless gum. Synthesis of compound 3 To a solution of compound 2 (100 mg, 285.38 μmol, 1 eq) in MeCN (1 mL) and H2O (1 mL) was added CAN (469.35 mg, 856.13 μmol, 426.68 μL, 3 eq). The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was diluted with Ethyl acetate (50 mL). The organic layer was washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by column chromatography (100 mesh silica gel, petroleum ether: ethyl acetate = 1: 0 to 3: 1) to give compound 3 (70 mg, crude) as white solid. Synthesis of compound 4 To a solution of compound 3 (70 mg, 304.00 μmol, 1 eq) and Cs2CO3 (297.15 mg, 911.99 μmol, 3 eq) in DMF (2 mL) was added 5-(chloromethyl) -1-ethyl-imidazole (65.94 mg, 456.00 μmol, 1.5 eq). The mixture was stirred at 40 °C for 2 h. The reaction mixture was diluted with ethyl acetate (30 mL). The resulting mixture was washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give compound 4 (50 mg, 147.75 μmol, 48.60% yield) as yellow gum. Synthesis of Compound 44 To a solution of compound 4 (50 mg, 147.75 μmol, 1 eq) in 2-methylpropan-1-ol (2 mL) was added NaN3 (9.61 mg, 147.75 μmol, 1 eq) and ZnBr2 (33.27 mg, 147.75 μmol, 7.39 μL, 1 eq), the mixture was stirred at 100 °C for 16 h. HCl (1 N, 1 mL) and ethyl acetate (30 mL) were added, the resulting mixture was washed with brine (10 mL) and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 1%-26%, 7 min) to give Compound 44 (3.46 mg, 8.98 μmol, 6.08% yield, 99% purity) as yellow solid.1H NMR: (400 MHz, CD3OD) δ = 8.71 - 8.44 (m, 1H), 8.15 - 8.09 (m, 1H), 8.08 - 7.90 (m, 1H), 7.43 (s, 1H), 5.33 (s, 2H), 4.34 - 4.19 (m, 3H), 2.17 - 2.03 (m, 2H), 2.00 - 1.87 (m, 2H), 1.37 (t, J = 7.3 Hz, 3H), 0.86 (br t, J = 7.3 Hz, 6H); LCMS: RT = 0.670 min, m/z = 382.0 (M+H)+. Synthetic Scheme Compound 45:
Synthesis of compound 2 To a solution of compound 1 (1 g, 7.43 mmol, 1 eq) in DMF (15 mL) was added imidazole (1.52 g, 22.29 mmol, 3 eq) and TBSCl (1.34 g, 8.92 mmol, 1.09 mL, 1.2 eq) at 0 °C under N2, the mixture was stirred at 20 °C for 16 h. The reaction mixture was diluted with Ethyl acetate (100 mL). The resulting mixture was washed with brine (50 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give compound 2 (1.5 g, 7.06 mmol, 95.05% yield) as yellow gum. Synthesis of compound 3 To a solution of compound 2 (500 mg, 2.35 mmol, 1 eq) in DMF (8 mL) was added NaH (113.00 mg, 2.83 mmol, 60% purity, 1.2 eq) at 0 °C, the mixture was stirred at 0 °C for 0.25 h. Then 2, 2, 2-trifluoroethyl trifluoromethanesulfonate (655.77 mg, 2.83 mmol, 1.2 eq) was added, the mixture was stirred at 20 °C for 16 h. The reaction mixture was added into water (20 mL) and extracted with ethyl acetate (50 mL*2). The combined organic phase was washed with brine (20 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by reversed phase flash (0.1% NH3•H2O). The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm* 5um; mobile phase: [water (ammonia hydroxide v/v) -ACN]; B%: 45%-75%, 9 min) to give compound 3 (150 mg, 509.53 μmol, 21.64% yield) as colorless gum.1H NMR: (400 MHz, DMSO-d6) δ = 7.64 (s, 1H), 7.08 (s, 1H), 5.00 (q, J = 9.4 Hz, 2H), 4.54 (s, 2H), 0.86 (s, 9H), 0.04 (s, 6H) Synthesis of compound 4 To a solution of compound 3 (150 mg, 509.53 μmol, 1 eq) in DCM (1 mL) was added TFA (1.54 g, 13.51 mmol, 1 mL, 26.51 eq), the mixture was stirred at 40 °C for 16 h. The reaction mixture was concentrated under vacuum to give compound 4 (90 mg, 499.65 μmol, 98.06% yield) as yellow gum. Synthesis of compound 5 To a solution of compound 4 (90 mg, 499.65 μmol, 1 eq) in DCM (3 mL) and DMF (0.1 mL) was added SOCl2 (89.16 mg, 749.47 μmol, 54.37 μL, 1.5 eq) at 0 °C, the mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under vacuum to give compound 5 (100 mg, crude) as yellow solid.1H NMR: (400 MHz, DMSO-d6) δ = 9.30 (s, 1H), 7.90 (s, 1H), 5.42 (q, J = 8.8 Hz, 2H), 5.03 (s, 2H). Synthesis of compound 7 To a mixture of compound 6 (50 mg, 189.90 μmol, 1 eq) and Cs2CO3 (185.62 mg, 569.71 μmol, 3 eq) in DMF (1 mL) was added compound 5 (56.56 mg, 284.86 μmol, 1.5 eq). The mixture was stirred at 40 °C for 2 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to give compound 7 (60 mg, 141.04 μmol, 74.27% yield) as yellow gum. Synthesis of Compound 45 To a solution of compound 7 (60 mg, 141.04 μmol, 1 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (6.76 mg, 282.08 μmol, 2 eq), the mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under vacuum to give a residue. The residue was adjusted pH = 5 with 1 M HCl. Then the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (FA) -ACN]; B%: 0%-30%, 7 min) to give Compound 45 (20.56 mg, 49.48 μmol, 35.08% yield, 99% purity) as yellow solid.1H NMR: (400 MHz, METHANOL-d4) δ = 8.52 (s, 1H), 8.10 (s, 1H), 7.83 (s, 1H), 7.29 (s, 1H), 5.33 (s, 2H), 5.10 (q, J = 8.9 Hz, 2H), 4.36 - 4.23 (m, 1H), 2.16 - 2.01 (m, 2H), 2.00 - 1.86 (m, 2H), 0.85 (br t, J = 7.0 Hz, 6H); LCMS: RT = 0.575 min, m/z = 412.2 (M+H)+. Example 2: Activity Studies and Data Assay 1 -isoQC Potency -Fluorescent Enzymatic Assay The Golgi luminal, enzymatically active region of human isoQC (S53-L382; see, e.g., Huang et al., 2011) was obtained by expression in E. coli of an N-terminally GST Enterokinase and C-terminally 6xHis tagged construct in the pET41(+) vector and subsequent purification using Ni-NTA IMAC. Enterokinase digestion ensued, followed by another Ni-NTA IMAC purification, Superdex75 based size exclusion and finally a spin concentration using a 10 kDa molecular weight cutoff. The final protein is formulated in 50 mM Tris-HCI, 150 mM NaCl, 50% Glycerol, pH 7.8. Pyroglutamylation was measured by conversion of glutamine-4-amino-7-methylcoumarine (H-Gln-AMC, Bachem) to pyroglutamyl-AMC, which is subsequently a specific substrate for pyroglutamylaminopeptidase (pGAPase, Qiagen), releasing free AMC which can be fluorescently detected. For a typical inhibition assay, 7.5 µL aliquots of threefold dilutions of test compound in 50mM Tris pH8 buffer were plated in 384-well plates. 15 µL 20 µM hGln-AMC (diluted from a 40 mM stock dissolved in 25 mM HEPES using 50 mM Tris-HCI 25 pH 8.0) was added to each well. Finally, 7.5 µL, 3 ng/µL recombinant isoQC enzyme was added and the reaction mixture was incubated for 1 hour at 37°C. A 5 minute incubation at 98°C was then performed to denature the isoQC enzyme and stop the reaction. To detect the amount of pyroglutamyl-AMC formed, a 0.125 U/mL pGAPase enzyme diluted in 50 mM Tris-HCI pH 8.0 containing 10 mM dithiothreitol (DTT) solution was prepared. 25 µL of the pyroglutamylation reaction mixture was transferred into an empty plate and to that, 25 µL of the pGAPase enzyme solution was added. Plates were then incubated for 25 minutes at room temperature before fluorescence readings were taken on an Envision2 plate reader with excitation set at 380 nm and emission at 450 nm. DMSO concentrations during the assay never exceeded 1% and activities were compared to controls containing only the same amount of DMSO and no test compound. Controls were taken along containing pyroglutamylated-AMC (Bachem, dissolved at 10 mM in DMSO) instead of H-Gln-AMC to control for pGAPase inhibition. IC50 values are calculated using non-linear regression analysis with GraphPad Prism software Assay 2 -QC Potency -Fluorescent Enzymatic Assay Recombinant QC enzyme was produced by bacterial expression of residues 33-361 of uniprot ID Q16769, fused to a His tag, codon optimized and cloned into the pET32a plasmid backbone. After scale-up, expression and lysis, protein was purified by Ni-NTA IMAC, dialysed in the presence of Factor Xa, further purified by Ni-NTA reverse IMAC and finally filtrated for size on a Superdex 20016/60 column. Final protein was concentrated using a spin concentrator at 10 kDa molecular weight cutoff and formulated in 150 mM NaCl, 50 mM Tris-HCI, 50% Glycerol, pH 8.0. Pyroglutamylation was measured by conversion of glutamine-4-amino-7-methylcoumarine (H-Gln-AMC, Bachem) to pyroglutamyl-AMC, which is subsequently a specific substrate for pyroglutamylaminopeptidase (pGAPase, Qiagen), releasing free AMC which can be fluorescently detected. For a typical inhibition assay, 7.5 µL aliquots of threefold dilutions of test compound in 50 mM Tris pH8 buffer were plated in 384-well plates.15 µL 20 µM h-Gln-AMC (diluted from a 40 mM stock dissolved in 25 mM HEPES using 50 mM Tris-HCI pH 8.0) was added to each well. Finally, 7.5 µL, 3 ng/µL recombinant QC enzyme was added and the reaction mixture was incubated for 1 hour at 37°C. A 5 minute incubation at 98°C was then performed to denature the QC enzyme and stop the reaction. To detect the amount of pyroglutamyl-AMC formed, a 0.125 U/mL pGAPase enzyme diluted in 50 mM Tris-HCI pH 8.0 containing 10 mM dithiothreitol (DTT) solution was prepared. 25 µL of the pyroglutamylation reaction mixture was transferred into an empty plate and to that, 25 µL of the pGAPase enzyme solution was added. Plates were then incubated for 25 minutes at room temperature before fluorescence readings were taken on an Envision2 plate reader with excitation set at 380 nm and emission at 450 nm. DMSO concentrations during the assay never exceeded 1% and activities were compared to controls containing only the same amount of DMSO and no test compound. Controls were taken along containing pyroglutamylated-AMC (Bachem, dissolved at 10 mM in DMSO) instead of H-Gln-AMC to control for pGAPase inhibition. IC50 values are calculated using non-linear regression analysis with GraphPad Prism software. Table 1: IC50 values obtained from activity studies of compounds of formula I. Molecule isoQC IC50 (nM) QC IC50 (nM) Compound 1 190 49 Compound 2 3160 Compound 3 2880 Compound 4 >10000 Compound 5 >10000 Compound 6 >10000 Compound 7 9630 Compound 8 >10000 Compound 9 >10000 Compound 10 215 22 Compound 11 365 16 Compound 12 153 23 Compound 13 694 52 Compound 14 135 16 Compound 15 220 38 Compound 16 721 66 Compound 17 294 25 Compound 18 1050 103 Compound 19 >10000 9540 Compound 20 324 42 Compound 21 231 10 Compound 22 551 18 Compound 23 440 57 Compound 24 342 48 Compound 25 394 34 Compound 26 151 1 Compound 27 154 1490 Compound 28 1060 142 Compound 29 362 61 Compound 30 6800 653 Compound 31 8300 2370 Compound 32 502 51 Compound 33 240 30 Compound 34 434 65 Compound 35 3950 229 Compound 36 >10000 >10000 Compound 37 5300 451 Compound 38 442 31 Compound 39 >10000 >10000 Compound 40 417 70 Compound 41 >10000 >10000 Compound 42 >10000 >10000 Compound 43 52 17 Compound 44 249 14 Compound 45 1830 623 The foregoing has described the principles, preferred embodiments, and modes of operation of the present invention. However, the invention should not be construed as limited to the particular embodiments discussed. Instead, the above-described embodiments should be regarded as illustrative rather than restrictive. It should be appreciated that variations may be made in those embodiments by workers skilled in the art without departing from the scope of the present invention.

Claims

CLAIMS 1. A compound of formula I: or a hydrate, solvate, or wherein: R1 is independently selected from a group consisting of hydrogen, a C1-3 alkyl, a halogen, a pseudohalogen, an acyl derivative and a 4-, 5- or 6-membered ring, wherein each C1-3 alkyl may be substituted with one or more halogens or pseudohalogens, wherein each pseudohalogen may be optionally substituted with one or more methyls; preferably R1 is independently selected from a group consisting of hydrogen, methyl, carboxylic acid, - C(O)OCH3, difluoropyrrolidinyl, phenyl, dimethylamino, methylamino, -NH2, cyano, tetrazolyl, 5-oxo- 1,2,4-oxadiazolinyl, trifluoromethyl,
R1a is independently selected from a group consisting of hydrogen, flouro, methyl and oxygen (=O); R1b represents hydrogen or flouro; Ring ‘A’ is selected from a group consisting of 6-membered aryl and heteroaryl ring; Ring ‘B’ is selected from a group consisting of 6-membered aryl, heteroaryl ring and 5- membered heteroaryl ring; Ring ‘C’ represents a saturated or unsaturated ring system; X is independently selected from a group consisting of -CH2-, -O- and keto ( ); or X is a bond; Y represents -N- or keto ( ); W represents ‘C’ or - R represents -CH2- or - ; represents a single bond or double bond; n = 0 or 1 ; q = 0 or 1 ; m = 1, 2 or 3; p =0, 1 or 2; Z=C or N, wherein when Z is N, R4 is absent; Q=C or N, wherein when p=2, Q is not ‘N’; R2 is either absent or independently selected from a group consisting of hydrogen, a C1-3 alkyl, a O-C1-3 alkyl and a cyclic moiety, wherein each C1-3 alkyl may be substituted with one or more halogens or pseudohalogens, preferably a group consisting of hydrogen, methyl, trifluoromethyl, ethyl, -OC2H5, hexafluoropropanyl, perfluoroethyl/ pentafluoroethyl, trifluoroethyl, ; R3 is either absent or independently selected from a group consisting of hydrogen, a C1-3 alkyl, a O-C1-3 alkyl and a cyclic moiety, wherein each C1-3 alkyl may be substituted with one or more halogens or pseudohalogens, preferably a group consisting of hydrogen, methyl, ethyl, phenyl or R2 and R3 ar ntyl ring or phenyl ring; R4 is independently selected from a group consisting of hydrogen, a C1-4 alkyl, a halogen, a pseudohalogen, ethoxyethyl and a cyclic moiety, wherein each C1-3 alkyl may be substituted with one or more halogens or pseudohalogens, preferably a group consisting of hydrogen, flouro, ethoxyethyl, isopentyl, methyl, propyl, tert-butyl, trifluoromethyl, and difluoro ethyl; or R3 and R4 are joined together to form a cyclopentyl ring or tetrahydropyranyl ring; R5 represents hydrogen or methyl; R6 is independently selected from a group consisting of a C1-3 alkyl, a halogen, a pseudohalogen and a cyclic moiety, wherein each C1-3 alkyl may be substituted with one or more halogens or pseudohalogens, preferably a group consisting of hydrogen, methyl, ethyl, isopropyl, cyclopentyl, cyclopropyl, trifluoromethyl, difluoroethyl, perfluoroethyl/ pentafluoroethyl, trifluoroethyl, , wherein each -CN, -CP, -NC, -OH, -SH, -SeH,-TeH, -OCN, -SCN, - NCS, -SeCN, -TeCN, -N3, -NO, or -NO2. 2. The compound of claim 1, wherein the 6-membered aryl or heteroaryl ring is selected from a group consisting of pyridyl, pyrazinyl, pyridazinyl, and pyrimidinyl; the 6-membered aryl is phenyl; and the 5- membered heteroaryl ring is selected from a group consisting of imidazolyl, isoxazolyl, triazolyl, thiazolyl, triazolyl, and pyrazolyl. 3. The compound of claim 1 or 2, wherein the Ring A is selected from a group consisting of pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and phenyl; and the Ring B is selected from a group consisting of imidazolyl, pyridyl, isoxazolyl, triazolyl, thiazolyl, triazolyl, and pyrazolyl. 4. The compound according to any of claim 1-3, of formula Ia :
or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein: R1 is independently selected from a group consisting of hydrogen, methyl, carboxylic acid, -C(O)OCH3, difluoropyrrolidinyl, phenyl, dimethylamino, methylamino, -NH2, cyano, tetrazolyl, 5-oxo-1,2,4- oxadiazolinyl, trifluoromethyl, R1b represents hydrogen or flouro; Ring ‘A’ is selected from a group consisting of pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and phenyl; Ring ‘B’ is selected from a group consisting of imidazolyl, pyridyl, isoxazolyl, triazolyl, thiazolyl, triazolyl, and pyrazolyl; Ring ‘C’ represents a saturated or unsaturated ring system; ‘X’ is independently selected from a group consisting of -CH2-, -O- and keto ( ); Y represents -N- or keto ( ); W represents ‘C’ or - R represents -CH2- or - ; represents a single bond or double bond; n = 0 or 1 ; q = 0 or 1 ; m = 1, 2 or 3; p =0 or 1; Z=C or N, wherein when Z is N, R4 is absent; Q=C or N; R2 is either absent or independently selected from a group consisting of hydrogen, methyl, ethyl, trifluoromethyl, pentafluoroethyl and trifluoroethyl; and R3 is either absent or independently selected from a group consisting of hydrogen, methyl, ethyl, isopropyl, trifluoromethyl, or R6 is independently selected from a group consisting of hydrogen, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, trifluoroethyl, difluoroethyl, pentafluoroethyl 5. The compound according to any of claims 1-4, A compound of formula Ib:
or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein R1 is independently either Carboxylic acid or -C(O)OCH3; Ring ‘A’ is pyridyl; R2 is either ; R3 is either or R3 R2 are to a cyclohexyl ring, pyridyl ring, cyclopentyl ring or phenyl ring; Ring ‘B’ is selected from a group consisting of imidazolyl, isoxazolyl, triazolyl and thiazolyl; R5 represents hydrogen or methyl; R6 represents hydrogen, methyl, isopropyl or ethyl; Ring ‘C’ represents a saturated or unsaturated ring system Y represents keto ( ); W represents - R represents - ; q = 1 ; m = 1; p =0, 1 or 2; Z= N; and Q=C or N; wherein when p=2, Q is not ‘N’. 6. The compound according to any of claims 1-5, of formula Ic: Ic or a hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein R1 is independently either Carboxylic acid or -C(O)OCH3; Ring ‘A’ is pyridyl; Ring ‘B’ is either Imidazolyl or triazolyl ; R5 is hydrogen; R6 is selected from a group consisting of methyl, ethyl, cyclopentyl and isopropyl; Ring ‘C’ represents a saturated or unsaturated ring system; Y represents -N- or keto ( ); W represents -N-; q = 1 ; Z=C or N; and R4 is independently selected from a group consisting of ethoxyethyl, isopentyl, methyl, propyl, tert-butyl, trifluoromethyl, and difluoro ethyl. 7. The compound according to any one of claims 1-6, of formula Id: or a hydrate, solvate, or phar wherein: R1 is independently selected from a group consisting of carboxylic acid, tetrazolyl, trifluoromethyl, Ring ‘B’ is imidazolyl; R5 represents hydrogen; R6 is independently selected from a group consisting of ethyl, trifluoromethyl, difluoroethyl, pentafluoroethyl, and trifluoroethyl; q = 1 ; R3 is independently selected from a group consisting of hydrogen, methyl and phenyl; R4 is independently selected from a group consisting of hydrogen, flouro, and methyl; and alternatively, R3 and R4 are joined together to form a cyclopentyl ring or tetrahydropyranyl ring. 8. The compound according to any one of claim 1 to 7, of formula II:
Formula II. 9. The compound of claim 1, which is compound 1-119, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, preferably compound 1-45, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
ı36
Compoun 9 10. A pharmaceutical composition comprising a compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1-9, and a pharmaceutically acceptable excipient. 11. The compound according to claims 1-9 or the pharmaceutical composition according to claim 10 for use in a treatment of a disorder of the human or animal body associated with abnormal QPCTL enzyme activity , comprising administering to a subject in need of treatment a therapeutically-effective amount of compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1-9 or the composition as defined in claim 10. 12. The compound according to claims 1-9 or the pharmaceutical composition according to claim 10 for use in the inhibition of QPCTL activity in a subject, comprising administering to said subject an effective amount of compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1 -9 or the composition as defined in claim 10. 13. The compound according to claims 1-9 or the pharmaceutical composition according to claim 10 for use in the inhibition of QPCTL protein/activity comprising contacting the protein/enzyme, in vitro or in vivo, with an effective amount of a compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1 to 9 or the composition as defined in claim 10. 14. The compound according to claims 1-9 or the pharmaceutical composition according to claim 10 for use in the inhibition of QPCTL protein/activity in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1-9 or the composition as defined in claim 10. 15. A compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1 -9 or the composition as defined in claim 10, for use in a method of treatment of the human or animal body by therapy.
16. A compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1 to 9 or the composition as defined in claim 10, for use in a method of treating diseases or disorders associated with abnormal QPCTL activity or QPCTL protein. 17. A kit comprising (a) a compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1 to 9 or the composition as defined in claim 10, preferably provided as a pharmaceutical composition and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, in a method of treatment of a disorder of the human or animal body. 18. A compound or a hydrate, solvate, or pharmaceutically acceptable salt thereof as defined in claims 1 to 9 or the composition as defined in claim 10, for use in a method of treatment of a disorder selected from a group consisting of cancer, neurodegenerative diseases such as Alzheimer's disease, synucleinopathies, Huntington’s disease, bacterial infections such as periodontitis and related disorders, and inflammatory diseases.
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