Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
  • C07D285/01—Five-membered rings
  • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
  • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
  • C07D285/08—1,2,4-Thiadiazoles; Hydrogenated 1,2,4-thiadiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/433—Thidiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• the present invention relates to novel compounds, and the use of such compounds in medicine.
• the present invention relates to compounds that are useful in treating a disorder or condition ameliorated by the activation of AMP-activated protein kinase.
• AMP-activated protein kinase is a protein kinase enzyme that consists of three protein sub-units and is activated by hormones, cytokines, exercise, and stresses that diminish cellular energy state (e.g. glucose deprivation).
• AMPK Activation of AMPK increases processes that generate adenosine 5'-triphosphate (ATP) (e.g., fatty-acid oxidation) and restrains others such as fatty acid-, glycerolipid- and protein-synthesis that consume ATP, but are not acutely necessary for survival. Conversely, when cells are presented with a sustained excess of glucose, AMPK activity diminishes and fatty acid-, glycerolipid- and protein-synthesis are enhanced. AMPK thus is a protein kinase enzyme that plays an important role in cellular energy homeostasis.
• ATP adenosine 5'-triphosphate
• AMPK is a preferred target for the treatment of the metabolic syndrome and especially type 2 diabetes.
• AMPK is also involved in a number of pathways that are important for many different diseases (e.g. AMPK is also involved in a number of pathways that are important in CNS disorders, fibrosis, osteoporosis, heart failure and sexual dysfunction).
• AMPK is also involved in a number of pathways that are important in cancer. Several tumour suppressors are part of the AMPK pathway.
• AMPK acts as a negative regulator of the mammalian TOR (mTOR) and EF2 pathway, which are key regulators of cell growth and proliferation.
• the deregulation may therefore be linked to diseases such as cancer (as well as diabetes).
• AMPK activators may therefore be of utility as anti- cancer drugs. It has been shown that AMPK activator drugs (e.g. metformin and 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide (i.e. the compound of formula II below)) are effective at treating pain.
• metformin and 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide i.e. the compound of formula II below
• hepatic steatosis may be regulated by AMPK (Zhao et al. J. Biol. Chem. 2020 295: 12279- 12289).
• AMPK Activation of AMPK inhibits de novo lipogenesis while promoting fatty acid oxidation ( ⁇ -oxidation) in the liver.
• AMPK activation also reduces free fatty acid release from adipose tissue and prevents hepatic steatosis.
• Pharmacological activation of AMPK in the liver was reported to promote beneficial effects on multiple aspects of non-alcoholic fatty liver disease (NAFLD).
• AMPK activators may be useful in the treatment of NAFLD and NASH.
• An example of an AMPK activator is 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazol-5-yl]benzamide (i.e. the compound of formula II), which was first disclosed in WO 2011/004162.
• an AMPK agonist i.e. an AMPK activator
• the compound of formula II is useful in the treatment of disorders or conditions which are ameliorated by the activation of AMPK.
• Such compounds may be useful in the treatment of cardiovascular disease (such as heart failure), diabetic kidney disease, type 2 diabetes, insulin resistance, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, pain, opioid addiction, obesity, cancer, inflammation (including chronic inflammatory diseases), autoimmune diseases, osteoporosis and intestinal diseases.
• cardiovascular disease such as heart failure
• diabetic kidney disease type 2 diabetes
• insulin resistance non-alcoholic fatty liver disease
• non-alcoholic steatohepatitis pain
• opioid addiction obesity
• cancer inflammation (including chronic inflammatory diseases)
• autoimmune diseases autoimmune diseases
• osteoporosis and intestinal diseases.
• R 1 and R 2 are independently selected from the group consisting of hydrogen, the sidechain of a proteinogenic amino acid, and C1-6 alkyl optionally substituted by one or more substituents selected from the group consisting of -OH, -NH 2 and -C(O)NH 2 ;
• R 3 and R 4 are independently selected from the group consisting of hydrogen, C 1-4 alkyl optionally substituted by a phenyl group and A 1 ; or R 1 (or R 2 ) and R 3 (or R 4 ) together with the carbon and nitrogen atoms to which they are bound form a 4- to 6-membered heterocycloalkyl group;
• a 2 represents hydrogen, C1-4 alkyl optionally substituted by a phenyl group, each A 3 independently represents hydrogen
• a 2 represents hydrogen
• each A 3 represents hydrogen
• the compounds of the invention have been found to metabolise in vivo to form 4- chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl] benzamide (referred to herein as a compound of formula II) which is known to be an AMPK activator.
• the compounds of the invention may be considered to be prodrugs of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5- yl]benzamide.
• Pharmaceutically acceptable salts of potential utility include those discussed in J. Pharmaceutical Sciences, 66: 1-19 (1977), by Berge et al.
• Pharmaceutically acceptable salts of the compound of formula I may be prepared in accordance with techniques that are well known to those skilled in the art.
• Examples of pharmaceutically acceptable addition salts include those derived from organic acids, such as citric, tartaric, acetic (including halogenated forms of acetic acid, such as trifluoroacetic acid), malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids; and from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids.
• the pharmaceutically acceptable salt is a citric acid salt or a trifluoroacetic acid salt of the compound of formula I.
• alkyl groups defined herein may be straight-chain or, when there is a sufficient number (i.e.
• cycloalkyl groups that may be mentioned include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• part cyclic alkyl groups (which may also be referred to as “part cycloalkyl” groups) that may be mentioned include cyclopropylmethyl.
• Heterocycloalkyl groups that may be mentioned include non-aromatic monocyclic heterocycloalkyl groups in which at least one (e.g.
• one to four) of the atoms in the ring system is other than carbon (i.e. a heteroatom, e.g. sulfur, oxygen or, particularly, nitrogen), and in which the total number of atoms in the ring system is from four to six.
• substituents for the avoidance of doubt, in cases in which the identity of two or more substituents in a compound of formula I may be the same, the actual identities of the respective substituents are not in any way interdependent. Where groups are referred to herein as being optionally substituted it is specifically contemplated that such optional substituents may be not present (i.e.
• Such solvents for the purpose of the invention may not interfere with the biological activity of the solute.
• suitable solvents include, but are not limited to, water, methanol, ethanol, and acetic acid.
• Compounds of formula I contain double bonds and may thus exist as E (ent ought) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
• Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.
• the present invention also embraces isotopically-labelled compounds of formula I which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (or the most abundant one found in nature). All isotopes of any particular atom or element as specified herein are contemplated within the scope of the invention.
• the compounds of formula I also include deuterated compounds, i.e. compounds of formula I in which one or more hydrogen atoms are replaced by the hydrogen isotope deuterium.
• deuterated compounds i.e. compounds of formula I in which one or more hydrogen atoms are replaced by the hydrogen isotope deuterium.
• compounds of the invention that are the subject of this invention include those that are stable.
• compounds of the invention include those that are sufficiently robust to survive isolation from, e.g., a reaction mixture to a useful degree of purity.
• structures may or may not be presented with chemical names. Where any question arises as to nomenclature, the structure prevails. Where it is possible for the compound to exist as a tautomer (e.g. in an alternative resonance form) the depicted structure represents one of the possible tautomeric forms, wherein the actual tautomeric form(s) observed may vary depending on environmental factors such as solvent, temperature or pH. All tautomeric (and resonance) forms and mixtures thereof are included within the scope of the invention.
• Amino acids are organic compounds that contain amino (–NH2) and carboxyl (–CO2H) functional groups, along with (in most cases) a sidechain.
• Alpha amino acids are those where the amino and carboxyl functional groups are bonded to the same carbon atom (i.e. the ⁇ -carbon atom).
• Particular alpha amino acids that might be mentioned are the proteinogenic amino acids.
• Proteinogenic amino acids are amino acids that are incorporated biosynthetically into proteins during translation.
• the proteinogenic amino acids are glycine (Gly), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), pyrrolysine (Pyl), selenocysteine (Sec), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
• sidechain of a proteinogenic amino acid refers to the group other than hydrogen that is bonded to the ⁇ -carbon atom of a proteinogenic amino acid.
• sidechains of one or more proteinogenic amino acids may be located at any of the R 1 , R 2 , R 5a or R 5b positions of the compound of formula I.
• the compounds of the invention may contain multiple different sidechains in these positions, i.e. the prodrug of compound II may contain a chain having two or three different amino acids linked to the thiadiazole ring.
• proteinogenic amino acid sidechains include methyl substituted with an imidazolyl or indolyl group (for histidine and tryptophan), propyl substituted with a guanidino group (for arginine) and butyl substituted with an amino group (for lysine).
• Particular compounds of the invention that may be mentioned are those where R 1 and R 2 are the same.
• R 1 and R 2 may both be hydrogen.
• Other compounds of the invention that may be mentioned are those where R 1 and R 2 are the different.
• R 1 may be the side chain of a proteinogenic amino acid and R 2 may be hydrogen.
• compounds of the first aspect of the invention may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism.
• compounds of formula I where R 1 and R 2 are different e.g. where R 1 is methyl and R 2 is hydrogen
• R 1 and R 2 may be such that the carbon substituted with R 1 and R 2 is in the L-configuration or the D-configuration, as understood by those skilled in the art.
• compounds contain an R 5a and/or an R 5b group the chiral centres to which those R 5a and R 5b groups are bound may also, independently, be in either the L-configuration or the D-configuration.
• when R 1 and R 2 are different the carbon substituted with R 1 and R 2 is in the L-configuration.
• the carbon substituted with R 1 and R 2 may be in the D-configuration.
• the compound of formula I may be a compound of formula I-A or a compound of formula I-B wherein R 1 , R 2 , R 3 and R 4 are as described herein (i.e. as described in the first aspect of the invention, including all embodiments and particular features, and combinations thereof).
• Particular examples of compounds of the invention are those in which R 1 and R 2 are both hydrogen or the carbon substituted with R 1 and R 2 is in the L-configuration, e.g. when one of R 1 and R 2 is hydrogen and the other is a sidechain of a proteinogenic amino acid.
• a 1 may represent A 2 may represent hydrogen, C1-4 alkyl optionally substituted by a phenyl group, wherein A 3 , R 5a and R 5b are as described herein (i.e. as described in the first aspect of the invention, including all embodiments and particular features, and combinations thereof).
• Particular examples of compounds of the invention are those in which A 1 represents a proteinogenic amino acid or two linked proteinogenic amino acids.
• Preferred examples of compounds of the invention that may be mentioned include those where: A 2 represents hydrogen, ; each A 3 represents hydrogen; and R 5b is as described herein.
• R 1 is selected from the group consisting of hydrogen, the sidechain of a proteinogenic amino acid, and C1-6 alkyl optionally substituted by one or more substituents selected from the group consisting of -OH, -NH2 and -C(O)NH2, or R 1 and R 3 are linked to form a pyrrolidine ring (as is found in proline); and R 2 is hydrogen.
• R 2 is hydrogen, and R 1 is hydrogen or the sidechain of a proteinogenic amino acid selected from the group consisting of Glu and, particularly, Arg, His, Lys, Ser, Thr, Asn, Gln, Cys, Sec, Ala, Ile, Leu, Met, Phe, Trp, Tyr, Asp and Val; or R 2 is hydrogen, and R 1 and R 3 together with the carbon and nitrogen atoms to which they are bound form a pyrrolidine ring.
• R 2 is hydrogen, and R 1 is hydrogen or the sidechain of an amino acid selected from the group consisting of Arg, His, Lys and Trp (e.g.
• R 2 is hydrogen, and R 1 and R 3 together with the carbon and nitrogen atoms to which they are bound form a pyrrolidine ring.
• R 2 is hydrogen, and R 1 is hydrogen or the sidechain of an amino acid selected from the group consisting of Lys, Ala, Ile, Phe, Leu, Ser and Asp; or R 2 is hydrogen, and R 1 and R 3 together with the carbon and nitrogen atoms to which Preferably, R 2 is hydrogen.
• Particular embodiments of the invention include compounds of formula I wherein: R 1 is hydrogen or the sidechain of an amino acid selected from the group consisting of Arg, His, Lys and Trp (e.g.
• R 3 and R 4 are independently selected from the group consisting of hydrogen, C 1-3 alkyl optionally substituted by one or more phenyl groups (e.g. a methyl or benzyl group), ; and R 5a is hydrogen or the sidechain of a proteinogenic amino acid.
• R 3 and R 4 are independently selected from the group consisting of hydrogen and C1-3 alkyl optionally substituted by one or more phenyl groups (e.g. R 3 and R 4 are independently hydrogen, methyl or benzyl).
• R 3 and R 4 are independently selected from the group consisting of hydrogen and methyl (e.g. R 3 and R 4 are both methyl).
• R 3 and R 4 are independently selected from the group consisting of hydrogen, C1-3 alkyl optionally substituted by one or more phenyl groups (e.g. a methyl or benzyl group)
• R 1 and R 3 together with the carbon and nitrogen atoms to which they are bound form a pyrrolidine ring
• R 4 is selected from the group consisting of hydrogen, C1-3 alkyl optionally substituted by one or more phenyl groups
• R 5a is the sidechain of a proteinogenic amino acid (e.g. Phe, Val, Lys, Asp or Arg).
• R 1 is hydrogen or the sidechain of a proteinogenic amino acid selected from the group consisting of Glu and, particularly, Arg, His, Lys, Ser, Thr, Asn, Gln, Cys, Sec, Ala, Ile, Leu, Met, Phe, Trp, Tyr, Asp and Val (e.g. Lys, Ala, Ile, Phe, Leu, Ser and Asp; or Arg, His, Lys and Trp);
• R 2 represents hydrogen;
• R 3 and R 4 are independently selected from the group consisting of hydrogen and C 1-3 alkyl optionally substituted by one or more phenyl groups (e.g.
• R 1 and R 3 together with the carbon and nitrogen atoms to which they are bound form a pyrrolidine ring.
• R 2 represents hydrogen
• R 1 is hydrogen or the sidechain of a proteinogenic amino acid selected from the group consisting of Glu, Arg, His, Lys, Ser, Thr, Asn, Gln, Cys, Sec, Ala, Ile, Leu, Met, Phe, Trp, Tyr, Asp and Val (e.g. Lys, Ala, Ile, Phe, Leu, Ser and Asp);
• R 3 and R 4 are independently selected from the group consisting of hydrogen, C1-3 alkyl optionally substituted by one or more phenyl groups (e.g.
• R 1 and R 3 together with the carbon and nitrogen atoms to which they are bound form a pyrrolidine ring
• R 4 is selected from the group consisting of hydrogen, C 1-3 alkyl optionally substituted by one or more phenyl groups (e.g. a methyl or benzyl group)
• R 5a is hydrogen or the sidechain of a proteinogenic amino acid selected from the group consisting of Glu, Arg, His, Lys, Ser, Thr, Asn, Gln, Cys, Sec, Ala, Ile, Leu, Met, Phe, Trp, Tyr, Asp and Val (e.g.
• a 2 represents R 5b is preferably hydrogen or the sidechain of a proteinogenic amino acid selected from the group consisting of Glu, Arg, His, Lys, Ser, Thr, Asn, Gln, Cys, Sec, Ala, Ile, Leu, Met, Phe, Trp, Tyr, Asp and Val (e.g. Phe, Val, Lys, Asp and Arg); and each A 3 is independently selected from the group consisting of hydrogen and C 1-3 alkyl (e.g. methyl).
• the compounds of the invention have been found to metabolise in vivo to form 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl] benzamide (referred to herein as a compound of formula II).
• the precise mechanisms by which the compounds of the invention are converted to the compound of formula II may nevertheless differ for different compounds of the invention.
• the compounds of the invention decompose into the compound of formula II in two steps. First the ester is hydrolysed (chemically or enzymically) to form the N-hydroxymethyl intermediate (referred to elsewhere herein as the compound of formula III), and then that intermediate is converted into the compound of formula II.
• the intermediate that is formed as a result of the hydrolysis i.e. Compound of formula III
• the intermediate that is formed as a result of the hydrolysis may be more readily taken up in the gut compared to Compound of formula II, thus the formation of Compound of formula III in the intestine may be advantageous for achieving optimal systemic exposure for the patient.
• compounds of the invention which contain a lipophilic (alkyl) group at R 1 /R 2 have a relatively high stability at pH 7.4. The stability is lower at this pH when R 1 /R 2 groups are H or a -NH2 containing amino acid side chain (e.g. glycine-based or lysine-based).
• R 1 /R 2 groups is an electron withdrawing amino acid side chain (e.g. aspartic acid-based, arginine-based or glutamic acid-based side chain).
• Particular compounds that may be mentioned in this respect are compounds of formula A: wherein: (i) R 1 represents hydrogen, R 2 represents the sidechain of aspartic acid, the sidechain of arginine or the sidechain of glutamic acid, R 3 is hydrogen or methyl, and R 4 hydrogen or methyl; or (ii) R 1 , R 2 and R 3 represent hydrogen, and R 4 represents A 1 , A 1 represents wherein A 2 is hydrogen or methyl, A 3 is hydrogen or methyl, and R 5a represents the sidechain of aspartic acid, the sidechain of arginine or the sidechain of glutamic acid; or a pharmaceutically acceptable salt or solvate thereof.
• R 1 represents hydrogen
• R 2 represents the sidechain of aspartic acid or the side chain of arginine
• R 3 is hydrogen or methyl
• R 4 hydrogen or methyl
• R 1 , R 2 and R 3 represent hydrogen
• R 4 represents A 1
• a 1 represents A 2 is hydrogen or methyl
• a 3 is hydrogen or methyl
• R 5a represents the sidechain of aspartic acid or the side chain of arginine; or a pharmaceutically acceptable salt or solvate thereof.
• Still further particular compounds that may be mentioned in this respect are compounds of formula C, wherein: (i) R 1 , R 3 and R 4 represent hydrogen, and R 2 represents the sidechain of aspartic acid, the sidechain of arginine or the sidechain of glutamic acid; or (ii) R 1 , R 2 and R 3 represent hydrogen, and R 4 represents A 1 , R 5a represents the sidechain of aspartic acid, the sidechain of arginine or the sidechain of glutamic acid; or a pharmaceutically acceptable salt or solvate thereof.
• the compound of the invention is a compound of formula I in which R 2 is hydrogen, and R 1 is the sidechain of aspartic acid, optionally wherein R 3 and R 4 are independently hydrogen or methyl.
• the invention also relates to compounds of formula I as defined herein (including compounds of formula I as defined according to any of the preferred embodiments set out herein) provided that: (i) the compound is not a compound of formula A; or (ii) the compound is not a compound of formula B; or (iii) the compound is not a compound of formula C; or (iv) the compound is not selected from the group consisting of:
• the compound is not a compound of formula I in which R 2 is hydrogen, and R 1 is the sidechain of aspartic acid (optionally wherein R 3 and R 4 are independently hydrogen or methyl); or (vi) the compound is not: .
• Particularly preferred compounds of the invention are:
• the compounds of the invention may be considered to be prodrugs of 4-chloro-N-[2- [(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide as they have been found to break down in vivo to form said compound.
• prodrug refers to a compound that, following oral or parenteral administration, is metabolised in vivo to yield a pharmalogically active compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).
• parenteral administration includes all forms of administration other than oral administration.
• General information on prodrugs may be found e.g. in Bundegaard, H.
• Sufficient bioavailability is important to achieve a therapeutically active concentration at the site of action.
• An improvement i.e. an increase
• bioavailability may be demonstrated by measuring the C max or the area under the curve (AUC) in the blood of a subject following administration of the compound (or pharmaceutical formulation thereof) to that subject.
• the compounds and formulations of the invention are useful in the therapies described herein in a subject in need of such therapy.
• Subjects that may be mentioned include animals, such as mammals. Particular mammals that might be mentioned include, for example, primates (e.g., humans, male or female), cows, horses, dogs and cats. Preferably the subject is a human.
• C max and “AUC” will be well understood by the person skilled in the art to refer, in the present context, to the peak plasma concentration of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide after administration (e.g. to a human subject) and the integral of the concentration/time curve for that substance following the administration of the compound of the invention (or formulation thereof), respectively.
• administration of the compounds of the invention is capable of increasing the bioavailability of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5- yl]benzamide compared to administration of 4-chloro-N-[2-[(4-chlorophenyl)methyl]- 3-oxo-1,2,4-thiadiazol-5-yl]benzamide.
• the phrase “increasing the bioavailability” means that administration of the compound of the invention results in a larger systemically available fraction of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3- oxo-1,2,4-thiadiazol-5-yl]benzamide in vivo compared to administration of 4-chloro- N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide.
• the increase oxo-1,2,4-thiadiazol-5-yl]benzamide may be at least about 10%, (at least) about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% (i.e. 2-fold), about 200% (i.e. 3-fold), about 300% (i.e. 4-fold), about 400% (i.e. 5-fold), about 500% (i.e. 6-fold), about 600% (i.e. 7-fold), about 700% (i.e. 8-fold), about 800% (i.e. 9-fold) or about 900% (i.e. 10-fold).
• the improvement of the bioavailability provided by the compounds of the invention may be demonstrated using suitable methods known in the art.
• the improvement in bioavailability may be demonstrated by comparing the pharmacokinetic data (e.g. AUC data) for a subject who has been administered a compound of the invention with the corresponding data for a subject who has been administered 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5- yl]benzamide.
• compounds of the invention have enhanced stability under acidic conditions (e.g. at pH 1.2).
• the compounds of the invention are suited to being prepared into pharmaceutical formulations for oral administration to patients where passage through the stomach is required.
• the compounds of the invention have also been found to be surprisingly effective at activating AMPK, as is evidenced by the data in the examples.
• the compounds of the invention may be useful in the treatment of disorders or conditions which are ameliorated by the activation of AMPK. Such compounds may therefore be useful in the treatment of the particular diseases described herein.
• the compounds of the invention may be prepared from 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide.
• 4-Chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide may, in turn, be prepared in accordance with techniques that are well known to those skilled in the art.
• 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5- yl]benzamide may be made in accordance with the techniques described in international patent application WO 2011/004162, and all of its content is hereby incorporated by reference.
• Compounds of the invention as described herein may be prepared in accordance with techniques that are well known to those skilled in the art, such as those described in Compounds of formula I may be obtained by analogy with the processes known in the literature, or by conventional synthetic procedures, in accordance with standard techniques, from available starting materials using appropriate reagents and reaction conditions. In this respect, the skilled person may refer to inter alia “Comprehensive Organic Synthesis” by B. M.
• a process for the preparation of a compound of the invention as hereinbefore defined comprises: (i) reaction of a compound of formula III, with a compound of formula IV, (ii) reaction of a compound of formula V, with a compound of formula IV, wherein R 1 and R 2 are as defined hereinabove and R 3 and R 4 are as defined hereinabove or independently represent a suitable protecting group (e.g. Boc), in the presence of a suitable base (e.g. 4-dimethylaminopyridine), a suitable amide coupling agent (e.g.
• N,N'-dicyclohexylcarbodiimide N,N'-dicyclohexylcarbodiimide
• a suitable solvent e.g. tetrahydrofuran
• the processes decribed herein for preparing compounds of the invention embrace those processes which comprise reaction of a compound of formula III with up to three (e.g. two) compounds of formula IV in a sequential manner.
• the compound of formula IV may be the same or different in each of steps (i), (iii) and (v).
• the functional groups of intermediate compounds may need to be protected by protecting groups.
• one of R 3 and R 4 may represent hydrogen and the other may represent a suitable protecting group (e.g. Boc).
• the protection and deprotection of functional groups may take place before or after the above-mentioned reactions.
• Protecting groups may be removed in accordance with techniques that are well known to those skilled in the art and as described hereinafter.
• protected compounds/intermediates described herein may be converted chemically to unprotected compounds using standard deprotection techniques. The use of protecting groups is fully described in “Protective Groups in Organic Synthesis”, 3rd edition, T.W.
• Specific transformation steps that may be employed in order to form compounds of formula I therefore include deprotection steps, such as deprotection of an N-Boc protecting group by reaction in the presence of an acid, or, a hydroxy group protected as a silyl ether (e.g. a tert-butyl-dimethylsilyl protecting group) may be deprotected by reaction with an acid or a source of fluoride ions, e.g. by employing the reagent tetrabutylammonium fluoride (TBAF).
• deprotection steps such as deprotection of an N-Boc protecting group by reaction in the presence of an acid, or, a hydroxy group protected as a silyl ether (e.g. a tert-butyl-dimethylsilyl protecting group) may be deprotected by reaction with an acid or a source of fluoride ions, e.g. by employing the reagent tetrabutylammonium flu
• the compound of formula III may be obtained by conventional synthetic procedures, in accordance with standard techniques, from available starting materials using appropriate reagents and reaction conditions.
• the compound of formula III (referred to in the examples as Compound 2) may be prepared by reaction of a compound of formula II with formaldehyde in the presence of a suitable base (e.g. triethylamine) and a suitable solvent (e.g. N,N-dimethylformamide) according to procedures know to the person skilled in the art.
• a suitable base e.g. triethylamine
• a suitable solvent e.g. N,N-dimethylformamide
• processes for preparation of compounds of the invention as described herein may include, as a final step, isolation and optionally purification of the compound of the invention.
• Pharmaceutical Formulations As indicated herein, the compounds of the invention are useful as therapeutic agents for treating a variety of medical disorders or conditions. Typically, compounds of the invention will be administered to a subject in need thereof in the form of a pharmaceutical formulation. According to a second aspect of the invention, there is provided a pharmaceutical formulation comprising the compound of formula I (or a pharmaceutically acceptable salt or solvate thereof). Such formulations are referred to herein as the formulations of the invention. All embodiments and particular features thereof described herein in respect of the first aspect of the invention are disclosed herein in respect of the second aspect of the invention.
• the pharmaceutical formulations of the second aspect of the invention may be prepared in accordance with standard and/or accepted pharmaceutical practice.
• the compound of the invention (or the pharmaceutically acceptable salt or solvate thereof) is the sole active pharmaceutical ingredient present in the formulation.
• the compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) is present in the formulation alongside one or more other active pharmaceutical ingredients, or may be administered as part of a combination therapy with one or more other active pharmaceutical ingredients.
• the formulations of the second aspect of the invention will generally be provided as a mixture comprising the compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) and one or more pharmaceutically acceptable excipients.
• the one or more pharmaceutically acceptable excipients may be selected with due regard to the intended route of administration in accordance with standard pharmaceutical practice.
• Such pharmaceutically acceptable excipients are preferably chemically inert to the active compound and preferably have no detrimental side effects or toxicity under the conditions of use.
• Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). A brief review of methods of drug delivery may also be found in e.g. Langer, Science 249, 1527 (1990).
• formulations of the invention may contain a lubricant, a binder, a filler, a surfactant, a diluent, an anti-adherent, a coating, a flavouring, a colourant, a glidant, a preservative, a sweetener, a disintegrant, an adsorbent, a buffering agent, an antioxidant, a chelating agent, a dissolution enhancer, a dissolution retardant and/or a wetting agent.
• Particular pharmaceutically acceptable excipients include colloidal silicon dioxide, anhydrous lactose, dicalcium phosphate, mannitol, pregelatinised starch, hydroxypropyl cellulose, povidone, low-substituted hydroxypropyl cellulose, croscarmellose sodium, sodium starch glycolate, sodium stearyl fumarate, talc, hydroxypropyl methylcellulose, polysorbate 80, sodium lauryl sulphate, poloxamer 188, poloxamer 407, propylene glycol, titanium dioxide, hypromellose phthalate, hypromellose acetate succinate, methacrylic acid, methyl methacrylate copolymer, Opadry® I, Opadry® II.
• the compounds of the invention may be mixed, either together or separately, with one or more of the pharmaceutical excipients listed above.
• Mixtures of a compound of the invention and one or more pharmaceutically acceptable excipients may be processed into pellets or granules, or compressed into tablets.
• pharmaceutical formulations of the invention include formulations that are provided in the form of a tablet, mini-tablets, blocks, pellets, particles, granules or a powder for oral administration.
• Mixtures of a compound of the invention and one or more pharmaceutically acceptable excipients may also be provided in a form that is suitable for subcutaneous or intramuscular delivery, such as an injectable solution or a freeze- dried powder suitable for reconstitution in a suitable fluid prior to administration.
• a suitable pharmaceutically acceptable dosage form such as an injectable solution or a freeze- dried powder suitable for reconstitution in a suitable fluid prior to administration.
• formulations described herein may act systemically, and may therefore be administered accordingly using suitable techniques known to those skilled in the art.
• Formulations as described herein will normally be administered orally, subcutaneously or intramuscularly in a suitable pharmaceutically acceptable dosage form.
• the pharmaceutical formulation of the second aspect of the invention is preferably an oral pharmaceutical formulation, particularly in view of the enhanced stability under acidic conditions that has been observed for compounds of the invention.
• Formulations of the invention may be prepared for oral administration in the form of a capsule.
• capsules such as soft gelatin capsules may be prepared containing the compound of the invention alone (or a pharmaceutically acceptable salt or solvate thereof), optionally together with a suitable vehicle, e.g. vegetable oil, fat etc.
• a suitable vehicle e.g. vegetable oil, fat etc.
• hard gelatin capsules may contain the compound of the invention alone (or a pharmaceutically acceptable salt or solvate thereof) alone, or in combination with solid powdered ingredients such as a disaccharide (e.g. lactose or saccharose), an starch), a polysaccharide (e.g. amylopectin or cellulose derivatives) or gelling agent (e.g. gelatin).
• Particular pharmaceutical formulations of the invention include formulations that may be mentioned are those provided in the form of a capsule or a tablet, e.g. for oral administration.
• Pharmaceutical formulations that may be mentioned include those in which the compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) is present in a total amount that is at least 1% (or at least 10%, at least 30% or at least 50%) by weight of the formulation and up to 99% by weight of the formulation.
• the weight ratio of the compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) to the totality of the components i.e. the compound of the invention and all pharmaceutical excipients, e.g.
• adjuvants, diluents and carriers of the pharmaceutical formulation is at least 1:99 (or at least 10:90, at least 30:70 or at least 50:50) and up to 99:1.
• a “therapeutically effective amount”, an “effective amount” and a “dosage” as used herein refer to an amount of a compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) that is sufficient to produce a desired clinical effect, which can be a therapeutic and/or beneficial effect.
• the effective amount or dosage will vary with the age or general condition of the subject (e.g.
• a “therapeutically effective amount”, “effective amount” or “dosage” in any individual case can be determined by one of skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
• compositions of the invention may be administered (for example, by way of one or more preparations as described herein) at varying doses, with suitable doses being readily determined by one of skill in the art.
• the total dosage of the compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) that is to be administered to a subject in need thereof may range from about 1 milligram per day (mg/day) to about 1000 mg/day (e.g. from about 10 mg/day to about 500 mg/day).
• Such dosages may be, for example, oral dosages of the formulations of the second aspect of the invention.
• treatment with such formulations may comprise administration of a unit dose formulation containing from about 1 mg to about 3000 mg of the compound of the invention, for example from about 2 mg to about 2000 mg, or from about 5 mg to about 1000 mg (e.g. from about 10 mg to about 500 mg), of the compound of the invention.
• treatment may comprise administration of the compound of the invention (e.g. in the form of one or more capsules containing said formulation) using a single daily dose.
• the total daily dosage of the compound of the invention may be administered in divided doses two, three or four times daily (e.g. twice daily with reference to the doses described herein, such as a dose of 100 mg, 250 mg, 500 mg or 1000 mg twice daily).
• the daily dose of the compound of the invention administered to a subject is in the range of from about 1 to about 3000 mg, preferably from about 1 to about 1000 mg.
• the term “about” as used herein when referring to a measurable value such as an amount of a compound, dose, time, temperature, and the like, refers to variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.
• the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage which will be most suitable for an individual subject.
• the above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
• Medical uses As indicated herein, the compounds of the invention are useful as pharmaceuticals.
• a compound of the invention as hereinbefore defined (i.e. a compound as defined in the first aspect of the invention), or a pharmaceutical formulation as defined in respect of the second aspect of the invention, for use in medicine.
• references to compounds as defined in the first aspect of the invention include references to compounds of formula I (including all embodiments thereof) and pharmaceutically acceptable salts and solvates thereof.
• a compound as defined in the first aspect of the invention may be particularly useful in treating a disorder or condition ameliorated by the activation of AMP-activated protein kinase (AMPK).
• AMPK AMP-activated protein kinase
• a compound of the invention, or a formulation comprising said compound for use in the treatment of a disorder or condition ameliorated by the activation of AMPK.
• a compound of the invention, or a formulation comprising said compound in the manufacture of a medicament for the treatment of a disorder or condition ameliorated by the activation of AMPK.
• a method of treating a disorder or condition ameliorated by the activation of AMPK comprising administering a compound of the invention (or a formulation comprising said compound) to a subject (e.g. a human) in need thereof.
• activate AMPK we mean that the steady state level of phosphorylation of the Thr- 172 moiety of the AMPK- ⁇ (AMPK-alpha) subunit is increased compared to the steady state level of phosphorylation in the absence of a compound of formula I or an active metabolite thereof (e.g.
• disorder or condition ameliorated by the activation of AMPK will be understood by those skilled in the art to include cardiovascular disease (such as heart failure), diabetic kidney disease, diabetes (such as type 2 diabetes), insulin resistance, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, pain, opioid addiction, obesity, cancer, inflammation (including chronic inflammatory diseases), autoimmune diseases, osteoporosis, intestinal diseases and hyperinsulinemia associated with obesity or cardiovascular disease.
• cardiovascular disease such as heart failure
• diabetes such as type 2 diabetes
• insulin resistance non-alcoholic fatty liver disease
• non-alcoholic steatohepatitis non-alcoholic steatohepatitis
• pain opioid addiction
• obesity cancer
• inflammation including chronic inflammatory diseases
• autoimmune diseases include osteoporosis, intestinal diseases and hyperinsulinemia associated with obesity or cardiovascular disease.
• Other diseases or conditions that may be ameliorated by the activation of AMPK include hyperinsulinemia and associated conditions, a condition/disorder where fibrosis plays a role, sexual dysfunction and neurodegenerative
• cancer will be understood by those skilled in the art to include one or more diseases in the class of disorders that is characterized by uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue through invasion, proliferation or by implantation into distant sites by metastasis.
• proliferation we include an increase in the number and/or size of cancer cells.
• metastasis we mean the movement or migration (e.g. invasiveness) of cancer cells from a primary tumor site in the body of a subject to one or more other areas within the subject's body (where the cells can then form secondary tumors).
• compounds of the invention may be suitable for use in the treatment of any cancer type, including all tumors (non-solid and, preferably, solid tumors, such as carcinoma, adenoma, adenocarcinoma, blood cancer, irrespective of the organ).
• the cancer cells may be selected from the group consisting of cancer cells of the breast, bile duct, brain, colon, stomach, reproductive organs, thyroid, hematopoietic system, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.
• the cancer is selected from the group consisting of colon cancer (including colorectal adenomas), breast cancer (e.g.
• the cancer is selected from the group consisting of colon, prostate and, particularly, breast cancer.
• the cancer is a non-solid tumor, it is preferably a hematopoietic tumor such as a leukemia (e.g. Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Acute Lymphocytic Leukemia (ALL), or Chronic Lymphocytic Leukemia (CLL).
• the cancer cells are breast cancer cells.
• diabetes i.e. diabetes mellitus
• type 1 diabetes and type 2 diabetes both of which involve the malfunction of glucose homeostasis.
• the compounds of the invention and formulations thereof may be particularly suitable for use in the treatment of type 1 diabetes and/or type 2 diabetes.
• the compounds of the invention are particularly suited for the treatment of type 2 diabetes.
• the compounds of the invention are also suitable for treating diabetic kidney disease (i.e. diabetic nephropathy).
• Diabetes kidney disease refers to kidney damage caused by diabetes and is a serious complication of type 1 diabetes and type 2 diabetes.
• Diabetic kidney disease affects the kidneys' ability to remove waste products from blood to be excreted as urine, and can lead to kidney failure.
• the compounds of the invention are also suitable for treating chronic kidney disease, including chronic kidney disease in the absence of type 2 diabetes.
• Chronic kidney disease is a condition characterised by a gradual loss of kidney function over time. Chronic kidney disease usually occurs as a result of one or more other diseases or conditions that affect the kidneys, such as high blood pressure, diabetes, high cholesterol, kidney infections, glomerulonephritis, polycystic kidney disease, obstruction of the urinary tract blockages in the flow of urine and long-term medication use.
• hyperinsulinemia or an associated condition will be understood by those skilled in the art to include hyperinsulinemia, type 2 diabetes, glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hyperinsulinism in childhood, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions such as stroke, systemic lupus polycystic ovary syndrome.
• Other disease states include progressive renal disease such as chronic renal failure.
• compounds of the invention and formulations thereof may be suitable for use in the treatment of obesity associated with hyperinsulinemia and/or cardiovascular disease associated with hyperinsulinemia.
• Compounds of the invention and formulations thereof may also be suitable for use in the treatment of cardiovascular disease, such as heart failure, wherein said cardiovascular disease is not associated with hyperinsulinemia.
• cardiovascular disease such as heart failure
• compounds of the invention and formulations thereof may also be suitable for use in the treatment of obesity which is not associated with hyperinsulinemia.
• the treatment of obesity and/or cardiovascular disease (such as heart failure) where AMPK activation may be beneficial is included within the scope of the invention.
• a condition/disorder where fibrosis plays a role includes (but is not limited to) scar healing, keloids, scleroderma, pulmonary fibrosis (including idiopathic pulmonary fibrosis), nephrogenic systemic fibrosis, and cardiovascular fibrosis (including endomyocardial fibrosis), systemic sclerosis, liver cirrhosis, eye macular degeneration, retinal and vitreal retinopathy, Crohn’s/inflammatory bowel disease, post-surgical scar tissue formation, radiation and chemotherapeutic-drug induced fibrosis, and cardiovascular fibrosis.
• the compounds of invention may also be useful in the treatment of sexual dysfunction (e.g. the treatment of erectile dysfunction).
• the compounds of invention may also be useful in the treatment of inflammation.
• Neurodegenerative diseases that may be mentioned include Alzheimer ⁇ s disease, Parkinson ⁇ s disease and Huntington ⁇ s disease, amyotrophic lateral sclerosis, polyglutamine disorders, such as spinal and bulbar muscular atrophy (SBMA), dentatorubral and pallidoluysian atrophy (DRPLA), and a number of spinocerebellar ataxias (SCA).
• SBA spinal and bulbar muscular atrophy
• DPLA dentatorubral and pallidoluysian atrophy
• SCA spinocerebellar ataxias
• Compounds of the invention may be useful in the treatment of a non-alcoholic fatty liver disease (NAFLD).
• NAFLD non-alcoholic fatty liver disease
• NAFLD is defined by excessive fat accumulation in greater than 5% of hepatocytes histologically).
• NAFLD is particularly prevalent in obese patients, with around 80% thought to have the disease. NAFLD may be diagnosed wherein alcohol consumption of the patient is not considered to be a main causative factor.
• a typical threshold for diagnosing a fatty liver disease as “not alcohol related” is a daily consumption of less than 20 g for female subjects and less than 30 g for male subjects.
• NAFLD Non-alcoholic steatohepatitis
• NASH non- alcoholic steatohepatitis
• AMPK activator compounds such as 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide (i.e. the compound of formula II) are capable of treating pain (Das V, et al.
• the compounds of the invention may be used to treat pain in place of an opioid-based therapy, as known by those skilled in the art. Accordingly, the compounds of the invention may be useful in treating opioid addiction.
• autoimmune diseases know to those skilled in the art include Crohn’s/inflammatory bowel disease, systemic lupus erythematosus and type 1 diabetes.
• Particular intestinal diseases that should be mentioned include Crohn’s/inflammatory bowel disease and cancer of gastrointestinal tract.
• references to the “treatment” of a particular condition will take their normal meanings in the field of medicine.
• the terms may refer to achieving a reduction in the severity and/or frequency of occurrence of one or more clinical symptom associated with the condition, as judged by a physician attending a subject having or being susceptible to such symptoms.
• Such treatment or prevention will be performed in a subject in need thereof.
• the need of a subject for such treatment or prevention may be assessed by those skilled the art using routine techniques.
• a “subject in need” of the compound of the invention includes a subject that is suffering a disorder or condition ameliorated by the activation of AMPK.
• the terms “disease” and “disorder” may be used interchangeably.
• the administration of a compound of the invention enhances the bioavailability of 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide in the systemic circulation.
• formulations comprising a compound of the invention has been shown to provide an approximately ten-fold increase in the plasma yl]benzamide under certain circumstances compared to administration of a formulation that comprises 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5- yl]benzamide.
• Compounds of the invention may have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g.
• Figure 3 shows comparative results of oral pharmacokinetic studies using Compounds 5, 6, 7, 9, 10, 13, 14, 16 and 17. Examples The present invention is explained in greater detail in the following non-limiting examples.
• the reaction schemes described below are intended to provide a general description of the methodology employed in the preparation of the compounds of the invention.
• the examples provided herein are offered to illustrate but not limit the compounds of the All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents and catalysts utilised to synthesise the compounds of the invention are either commercially available or can be routinely prepared by procedures described in the literature, for example, Houben-Weyl “Science of Synthesis” volumes 1-48, Georg Thieme Verlag, and subsequent versions thereof.
• a reaction may be carried out in the presence of a suitable solvent or diluent or of mixture thereof in a manner known to those skilled in the art of organic synthesis.
• a reaction may also be carried out, if needed, in the presence of an acid or a base, with cooling or heating, for example in a temperature range from about -30 °C to about 150 °C.
• a reaction is carried out in a temperature range from about 0 °C to about 100 °C, and more particularly, in a temperature range from room temperature to about 80 °C, in an open or closed reaction vessel and/or in the atmosphere of an inert gas, for example nitrogen.
• an inert gas for example nitrogen.
• AUC Area under the concentration-time curve aq: Aqueous b.w.: Body weight C max : Peak plasma concentration d: Doublet DCC: N,N'-dicyclohexylcarbodiimide DCM: Dichloromethane DMAP: 4-Dimethylaminopyridine DMSO: Dimethylsulfoxide eq.: equivalent ESI: Electrospray ionisation Et 3 N: Triethylamine EtOH: Ethanol g: Gram h: Hours HPLC: High performance liquid chromatography LC: Liquid chromatography LC-MS/MS: Liquid chromatography – (tandem) mass spectrometry LLOQ: Lower limit of quantification m: Multiplet MeOH: Methanol MeOD: Methanol-d 4 Min: Minutes mL: Millilitre MRT: Mean residence time ND: Not detected NMR: Nuclear magnetic resonance RT: Room temperature s: Singlet T
• Example 1 preparation of dimethylamino-acetic acid 5-[(Z)-4-chloro-benzoylimino]- 2-(4-chloro-benzyl)-3-oxo-[1,2,4]thiadiazolidin-4-ylmethyl ester (Compound 3)
• Compound 1 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5- yl]benzamide
• Compound 1 was prepared in accordance with the procedures described in WO 2011/004162.
• Example 2 preparation of citric acid salt of dimethylamino-acetic acid 5-[(Z)-4-chloro- benzoylimino]-2-(4-chloro-benzyl)-3-oxo-[1,2,4]thiadiazolidin-4-ylmethyl ester (Compound 4)
• To a stirring solution of dimethylamino-acetic acid 5-[(Z)-4-chloro-benzoylimino]-2- (4-chloro-benzyl)-3-oxo-[1,2,4]thiadiazolidin-4-ylmethyl ester (Compound 3; 550mg, 1.1 mmol) in acetone (30.0 v) was added citric acid (0.9 eq.).
• Step-2 1-tert-butyl 2-[5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl pyrrolidine-1,2-dicarboxylate (Compound A3).
• Step-3 2-( ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ carbonyl)pyrrolidin-1-ium trifluoroacetate (Compound 5): To a stirring solution of Compound A3 (0.5 g, 0.82 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL, 2.0 vol) at 0-5 °C. The reaction mixture was stirred for 16h at ambient temperature (25-30 °C). The reaction mixture was concentrated to remove the dichloromethane.
• Step 1 [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2,6-bis( ⁇ [(tert-butoxy)carbonyl]amino ⁇ )hexanoate (Compound B2)
• DMAP 0.074 g, 0.60 mmol
• DCC 0.50 g, 2.43 mmol
• Step-2 6- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ -6-oxohexane-1,5-bis(aminium) di-trifluoroacetate (6)
• dichloromethane 5 mL
• trifluoroacetic acid 1 mL, 2.0 vol
• the reaction mixture was stirred for 16h at ambient temperature (25-30 °C).
• the reaction mixture was concentrated to remove the dichloromethane.
• Example 5 Preparation of (6-azaniumyl-1- ⁇ [5-(4-chlorobenzamido)-2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -1-oxohexan-2- yl)dimethylazanium di-trifluoroacetate (Compound 7)
• Step-1 6- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -2-(dimethylamino)hexanoic acid
• Compound C2 To a solution of Boc-Lys-OH (C1) (2.0 g, 8.1 mmol) in methanol (20 mL), were added 37% formaldehyde (2.6 mL, 32.4 mmol) followed by Pd/C (0.2 g, 10% w/w) and the resulting mixture was hydrogenated in a parr apparatus (5 kg, H2 pressure) for 16h.
• Step-2 [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 6- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -2-(dimethyl amino)
• DMAP 0.35 g, 0.29 mmol
• DCC 0.50 g, 2.43 mmol
• Step-3 (6-azaniumyl-1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -1-oxohexan-2-yl)dimethylazanium di-trifluoroacetate (Compound 7): To a stirring solution of Compound C3 (0.18 g, 0.027 mmol) in dichloromethane (3.6 mL) was added trifluoroacetic acid (0.36 mL, 2.0 vol) at 0-5 °C. The reaction mixture was stirred for 3h at ambient temperature (25-30 °C).
• Step-1 [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2- ⁇ [(tert-butoxy)carbonyl](methyl)amino ⁇ acetate (D2): To a stirring solution of N-Boc sarcosine (Compound D1; 0.57 g, 3.04 mmol) in THF (12 mL, 20V) was added DMAP (0.044 g, 0.36 mmol) followed by DCC (0.62 g, 3.04 mmol) at 0-5 °C and stirred for 20 min.
• N-Boc sarcosine Compound D1; 0.57 g, 3.04 mmol
• DCC 0.62 g, 3.04 mmol
• Step-2 (2- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ -2-oxoethyl)(methyl)azanium trifluoroacetate (Compound 8): To a stirring solution of Compound D2 (0.2 g, 0.34 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (0.4 mL, 2.0 vol) at 0-5 °C. The reaction mixture was stirred for 16h at ambient temperature (25-30 °C). The reaction mixture was concentrated to remove the dichloromethane.
• Example 7 Preparation of 2- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3- oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -2-oxoethan-1- trifluoroacetate (Compound 9): Step-1: [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ acetate (Compound E2): To a stirring solution of N-Boc glycine (Compound E1; 0.53 g, 3.04 mmol) in THF (11 mL, 20V) was added DMAP (0.044 g, 0.36 mmol) followed by DCC (0.62 g, 3.04 mmol) at 0-5 °C and stirred for 20 min.
• Step-2 2- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ -2-oxoethan-1-aminium trifluoroacetate (Compound 9): To a stirring solution of Compound E2 (0.3 g, 0.52 mmol) in dichloromethane (3.0 mL) was added trifluoroacetic acid (0.6 mL, 2.0 vol) at 0-5 °C. The reaction mixture was stirred for 2h at ambient temperature (25-30 °C). The reaction mixture was concentrated to remove the dichloromethane.
• Example 8 Preparation of 2-( ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3- oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ carbonyl)-1-methylpyrrolidine (Compound 10): Step-1: 1-methylpyrrolidine-2-carboxylic acid (Compound F1): To a solution of L-Proline (Compound A1; 2.0 g, 17.37 mmol) in methanol (20 mL), were added 37% formaldehyde (1.54 mL, 19.1 mmol) followed by Pd/C (0.5 g, 25% w/w) and the resulting mixture was hydrogenated in a Parr apparatus (1 kg H2 pressure) for 16h After completion of the reaction the reaction mixture was filtered through Celite® and concentrated under reduced pressure.
• Compound F1 1-methylpyrrolidine-2-carboxylic acid
• Step-2 2-( ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ carbonyl)-1-methylpyrrolidin-1-ium trifluoroacetate (Compound 10): To a stirring solution of Compound F1 (0.39 g, 3.0 mmol) in THF (10 mL, 20V) was added DMAP (0.074 g, 0.60 mmol) followed by DCC (0.62 g, 3.04 mmol) at 0-5 °C and stirred for 20 min.
• DMAP 0.074 g, 0.60 mmol
• DCC (0.62 g, 3.04 mmol
• Step-1 [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ propanoate (Compound G2): To a stirring solution of N-Boc alanine (Compound G1) (0.57 g, 3.04 mmol) in THF (10 mL, 20V) was added DMAP (0.044 g, 0.36 mmol) followed by DCC (0.62 g, 3.04 mmol) at 0-5 °C and stirred for 20 min.
• Step-2 1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ -1-oxopropan-2-aminium trifluoroacetate (Compound 11): To a stirring solution of Compound G2 (0.3 g, 0.51 mmol) in dichloromethane (3.0 mL) was added trifluoroacetic acid (0.9 mL, 3.0 vol) at 0-5 °C. The reaction mixture was stirred for 2h at ambient temperature (25-30 °C). The reaction mixture was concentrated to remove the dichloromethane.
• Example 10 Preparation of 1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]- 3-oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -3-methyl-1-oxopentan-2-a trifluoroacetate
• Compound 12 Step-1: [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-methylpentanoate (Compound H2): To a stirring solution of N-Boc Isoleucine (Compound H1; 0.56 g, 3.04 mmol) in THF (10 mL, 20V) was added DMAP (0.044 g, 0.36 mmol) followed by DCC (0.62 g, 3.04 mmol) at 0-5 °C
• Step-2 1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl] -3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ -3-methyl-1-oxopentan-2-aminium trifluoroacetate
• dichloromethane 3.0 mL
• trifluoroacetic acid 1.2 mL, 3.0 vol
• the reaction mixture was stirred for 2h at ambient temperature (25-30 °C).
• the reaction mixture was concentrated to remove the dichloromethane.
• Example 11 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3- oxo-1,2,4-thiadiazolidin-4-yl]methyl 2-(dimethylamino)-3-phenylpropanoate (Compound 13): Step-1: 2-(dimethylamino)-3-phenylpropanoic acid (Compound I2): To a solution of Phe-Ala-OH (Compound I1; 1.0 g, 6.05 mmol) in methanol (20 mL), were added 37% formaldehyde (1.96 mL, 24.21 mmol) followed by Pd/C (0.1 g, 10% w/w) and the resulting mixture was hydrogenated in a parr apparatus (5 kg H2 pressure) for 16h.
• a parr apparatus 5 kg H2 pressure
• Step-2 [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2-(dimethylamino)-3-phenylpropanoate (Compound 13): To a stirring solution of Compound I2 (0.47 g, 2.43 mmol) in THF (10 mL, 20V) was added DMAP (0.044 g, 0.36 mmol) followed by DCC (0.62 g, 3.04 mmol) at 0-5 °C and stirred for 20 min.
• Step-1 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-phenylpropanoic acid (Compound J1): To a solution of Phe-Ala-OH (Compound I1; 1.0 g, 6.05 mmol) in DCM (10 mL), was added triethylamine (1.70 mL, 12.17 mmol) at 0 °C followed by di-tert-butyl pyrocarbonate (2.18 mL, 9.08 mmol) and the resulting reaction mixture was stirred for 16 h at room temperature. The reaction mixture was carefully acidified with 5% citric acid solution to pH 4.
• Step-2 [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-phenylpropanoate (Compound J2)
• DMAP 0.044 g, 0.36 mmol
• DCC 0.62 g, 3.04 mmol
• Step-3 1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ -1-oxo-3-phenylpropan-2-aminium trifluoroacetate (Compound 14)
• dichloromethane 3.0 mL
• trifluoroacetic acid 0.9 mL, 3.0 vol
• the reaction mixture was stirred for 2h at ambient temperature (25-30 °C).
• the reaction mixture was concentrated to remove the dichloromethane.
• Example 13 Preparation of 1- (4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]- 3-oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -4-methyl-1-oxopentan-2- trifluoroacetate
• Compound 15 Step-1: [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -4-methyl pentanoate (Compound K2): To a stirring solution of 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -4-methylpentanoic acid (Compound K1; 0.56 g, 2.43 mmol) in THF (10 mL, 20V) was added DMAP (0.044 g, 0.36 mmol) followed by DCC (0.62 g, 3.
• Step-2 1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ -4-methyl-1-oxopentan-2-aminium trifluoroacetate (Compound 15): To a stirring solution of Compound K2 (0.35 g, 0.56 mmol) in dichloromethane (3.0 mL) was added trifluoroacetic acid (1.05 mL, 3.0 vol) at 0-5 °C. The reaction mixture was stirred for 2h at ambient temperature (25-30 °C). The reaction mixture was concentrated to remove the dichloromethane.
• Step-1 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-[(tert-butyldimethylsilyl) oxy]propanoic acid (Compound L2)
• Boc-Ser-OH Compound L1; 1.0 g, 4.87 mmol
• DCM DCM
• Step-2 [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-[(tert-butyldimethyl silyl)oxy]propanoate (Compound L3)
• DMAP 0.044 g, 0.36 mmol
• DCC 0.62 g, 3.04 mmol
• Step-3 1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4- thiadiazolidin-4-yl]methoxy ⁇ -3-hydroxy-1-oxopropan-2-aminium trifluoroacetate (Compound 16): To a stirring solution of Compound L3 (0.4 g, 0.56 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (1.2 mL, 3.0 vol) at 0-5 °C. The reaction mixture was stirred for 2h at ambient temperature (25-30 °C).
• Example 15 Preparation of 3-carboxy-1- ⁇ [5-(4-chlorobenzamido)-2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -1-oxopropan-2- aminium trifluoroacetate (Compound 17): M 3 17 Step-1:4-(tert-butoxy)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -4-oxobutanoic acid (Compound M2): To a solution of L-Aspartic acid 4-tert-butyl ester (Compound M1; 2.0 g, 10.5 mmol) in dioxane:water (7:3, 20 mL), was added NaOH (0.84 g, 21.1 mmol) followed by di- was stirred for 16 h at room temperature.
• Step-2 tert-butyl 1-[5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ butanedioate (Compound M3): To a stirring solution of Compound M2 (0.88 g, 3.04 mmol) in THF (18 mL, 20V) was added DMAP (0.044 g, 0.36 mmol) followed by DCC (0.62 g, 3.04 mmol) at 0-5 °C and stirred for 20 min.
• Step-3 3-carboxy-1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -1-oxopropan-2-aminium trifluoroacetate (Compound 17): To a stirring solution of Compound M3 (0.45 g, 0.66 mmol) in dichloromethane (4.5 mL) was added trifluoroacetic acid (0.9 mL, 2.0 vol) at 0-5 °C. The reaction mixture was stirred for 6h at ambient temperature (25-30 °C). The reaction mixture was concentrated to remove the dichloromethane.
• Step-2 Preparation of methyl 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3- phenylpropanamido]acetate (N2): To a stirring solution of (2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-phenylpropanoic acid (J1, which was synthesized as described above; 2.6 g, 9.8 mmol) in DCM (52 mL, 20V) were added DCC (2.42 g, 11.76 mmol), DMAP (119 mg, 0.98 mmol) and triethyl amine (2.71 mL, 19.6 mmol) at 0-5 °C and stirred for 20 min.
• DCM 52 mL, 20V
• Step-3 Preparation of 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3- phenylpropanamido]acetic acid (Compound N3): To a stirring solution of Compound N2 (3.0 g, 8.91 mmol) in methanol (30 mL, 10V) was added 2M NaOH solution (12 mL, 4V) at 0-5 °C and the reaction mixture was stirred for 16 h at ambient temperature (25-30 °C). The reaction mass was t t d t l til dil t d ith t (50 L) d h d ith MTBE (100 mL).
• Step-4 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3- phenylpropanamido]acetate (Compound N4): To a stirring solution of compound N3 (1.6 g, 4.96 mmol) in THF (32 mL, 20V) was added DCC (1.53 g, 7.45 mmol) followed by DMAP (0.3 g, 2.48 mmol) at 0-5 °C and stirred for 20 min.
• Step-5 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl 2-[(2S)-2-azaniumyl-3-phenylpropanamido]acetate chloride (Compound 18): To a stirring solution of compound N4 (0.2 g, 0.279 mmol) in THF (1.0 mL, 5 vol), was added HCl in diethyl ether (4.0 mL, 20 vol) at 0-5 °C and the reaction mixture was stirred for 20 min at same temperature.
• Step-2 Preparation of (2S)-1- ⁇ [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]- 3-oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -3-methyl-1-oxobutan-2-aminium trifluoroacetate (Compound O3): To a stirring solution of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl (2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3- methylbutanoate (Compound O2; 6.0 g, 9.85 mmol) in dichloromethane (25 mL) was added trifluoroacetic acid (24 mL, 4.0 vol) at 0-5 °C and stirred for 4h at ambient temperature (25-30 °C).
• Step-3 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl (2S)-2-(2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3- methylbutanamido)-3-methylbutanoate (Compound O4): To a stirring solution of (2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-methylbutanoic acid (Compound O1; 1.30 g, 6.02 mmol) in THF (30 mL, 20V) was added DMAP (0.088 g, 0.72 mmol) followed by DCC (1.39 g, 6.75 mmol) at 0-5 °C and stirred
• Step-4 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl (2S)-2-[(2S)-2-azaniumyl-3-methylbutanamido]-3- methylbutanoate trifluoroacetate (Compound O5): To a stirring solution of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 12 4 thiadia olidin 4 l]meth l (2S) 2 (2 ⁇ [(tert b to )carbon l]amino ⁇ 3 methylbutanamido)-3-methylbutanoate (Compound O4; 0.66 g, 0.93 mmol) in dichloromethane (25 mL) was added trifluoroacetic acid (1.98 mL, 3.0 vol) at 0-5 °
• Step-5 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl (2S)-2-[(2S)-2-azaniumyl-3-methylbutanamido]-3- methylbutanoate chloride (Compound 19): To a stirring solution of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl (2S)-2-[(2S)-2-azaniumyl-3-methylbutanamido]-3- methylbutanoate trifluoroacetate (Compound O5; 0.45 g, 0.62
• Step-2 Preparation of 2-[(2S)-2,6-bis( ⁇ [(tert-butoxy)carbonyl]amino ⁇ ) hexanamido]acetic acid (Compound P3): To a stirring solution of methyl 2-[(2S)-2,6-bis( ⁇ [(tert- butoxy)carbonyl]amino ⁇ )hexanamido]acetate (Compound P2; 3.0 g, 7.19 mmol) in methanol (30 mL, 10V) was added 2M NaOH solution (12 mL, 4V) at 0-5 °C. The resulting reaction mixture was allowed to stir at ambient temperature (25-30 °C) for 16 h.
• Step-3 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl 2-[(2S)-2,6-bis( ⁇ [(tert-butoxy)carbonyl]amino ⁇ ) hexanamido] acetate (Compound P4): To a stirring solution of (2-[(2S)-2,6-bis( ⁇ [(tert- butoxy)carbonyl]amino ⁇ )hexanamido]acetic acid (Compound P3; 1.75 g, 4.34 mmol) in THF (35 mL, 20V) was added DMAP (0.26 g, 2.17 mmol) followed by EDC.HCl (1.24 g, 6.51 mmol) at 0-5 °C and stirred for 20 min.
• Step-4 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl 2-[(2S)-2,6-diazaniumylhexanamido]acetate di-trifluoroacetate (Compound P5): To a stirring solution of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl 2-[(2S)-2,6-bis( ⁇ [(tert-butoxy)carbonyl]amino ⁇ ) hexanamido] acetate (Compound P4; 0.3 g, 0.37 mmol) in dichloromethane (4.5 mL, 15V) was added trifluoroacetic acid (1.2 mL, 4 vol) at 0-5 °C and was stirred
• Step-1 Preparation of benzyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ acetate (Compound Q2): To a stirring solution of 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ acetic acid (Compound E1; 2.0 g, 11.42 mmol) in DCM (40 mL, 20V) was added DMAP (130 mg, 1.14 mmol) followed by EDC.HCl (2.65 g, 17.12 mmol) at 0-5 °C and the resulting mixture was stirred for 5 minutes.
• Step-2 Preparation of benzyl 2-aminoacetate (Compound Q3): To a stirring solution of benzyl 2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ acetate (Compound Q2; 2 g, 7.54 mmol) in dichloromethane (30 mL, 15V) was added trifluoroacetic acid (4 mL, 2 V) at 0-5 °C. The reaction mixture was allowed to stir for 3h at ambient temperature (25-30 °C). The reaction mass was concentrated to remove the volatiles and the residue obtained was triturated with diethyl ether to afford Compound Q3 as a white solid (1.8 g, 85% yield).
• Step-3 Preparation of tert-butyl (3S)-3- ⁇ [2-(benzyloxy)-2-oxoethyl]carbamoyl ⁇ -3- ⁇ [(tert-butoxy)carbonyl]amino ⁇ propanoate (Compound Q5): To a stirring solution of (2S)-4-(tert-butoxy)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -4- oxobutanoic acid (Compound Q4; 4.37 g, 15.1 mmol) in DCM (20 mL, 20V) was added DMAP (140 mg, 1.21 mmol) followed by EDC.HCl (3.23 g, 16.95 mmol) at 0-5 °C and stirred for 5 min.
• DCM 20 mL, 20V
• Step-4 Preparation of 2-[(2S)-4-(tert-butoxy)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -4- oxobutanamido] acetic acid (Compound Q6): To a solution of tert-butyl (3S)-3- ⁇ [2-(benzyloxy)-2-oxoethyl]carbamoyl ⁇ -3- ⁇ [(tert- butoxy)carbonyl] amino ⁇ propanoate (Compound Q5; 1.0 g, 2.29 mmol) in ethyl acetate (30 mL, 30V), was added Pd/C (0.1 g, 10% w/w) and the resulting mass was hydrogenated in a parr apparatus (5 kg H2 pressure) for 3h.
• a parr apparatus 5 kg H2 pressure
• Step-5 Preparation of tert-butyl (3S)-3- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-[(2- ⁇ [5- (4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4- yl]methoxy ⁇ -2-oxoethyl)carbamoyl]propanoate (Compound Q7): To a solution of 2-[(2S)-4-(tert-butoxy)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -4- oxobutanamido]acetic acid (Compound Q6; 0.64 g, 1.87 mmol) in THF (7 mL, 20V)
• Step-6 Preparation of (1S)-2-carboxy-1-[(2- ⁇ [5-(4-chlorobenzamido)-2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -2- oxoethyl)carbamoyl]ethan-1-aminium trifluoroacetate (Compound Q8): To a stirring solution of tert-butyl (3S)-3- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -3-[(2- ⁇ [5- yl]methoxy ⁇ -2-oxoethyl)carbamoyl]propanoate (Compound Q7; 0.5 g, 0.67 mmol) in dichloromethane (7.5 mL) was added trifluoroacetic acid (2 mL, 4.0 vol) at 0-5 °C and was stirred for 4h at ambient temperature (25-30 °C).
• Step-7 Preparation of (1S)-2-carboxy-1-[(2- ⁇ [5-(4-chlorobenzamido)-2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -2- oxoethyl)carbamoyl]ethan-1-aminium chloride (Compound 22): To a stirring solution (1S)-2-carboxy-1-[(2- ⁇ [5-(4-chlorobenzamido)-2-[(4- chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4-yl]methoxy ⁇ -2- oxoethyl)carbamoyl]ethan-1-aminium trifluoroacetate (Compound 22): To a stirring solution (1S)-2-carboxy-1-[(2- ⁇ [5-(4-chlorobenzamido)-2-[(4- chloroph
• Step-1 Preparation of (2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -5- ⁇ [(1E)- ⁇ [(tert- butoxy)carbonyl] amino ⁇ ( ⁇ [(tert-butoxy)carbonyl]imino ⁇ )methyl]amino ⁇ pentanoic acid (Compound R2): To a solution of (2S)-2-amino-5-carbamimidamidopentanoic acid (Compound R1; 8.8 g, 50.51 mmol) in t-Butanol (158 mL, 18V) and water (158 mL, 18V), were added NaOH (7.07 g, 176.80 mmol) and Boc2O (46.42 mL, 202.06 mmol) at 0 °C.
• reaction mass was allowed to stir for 48 h at ambient temperature (25-30 °C). Evaporated the volatiles from the reaction mass and acidified carefully with saturated citric acid solution to pH 3-4. The product was extracted with ethyl acetate, and the extract was dried over Na2SO4, filtered, evaporated in a vacuum and purified by reverse phase column chromatography to obtain Compound R2 as a white solid (3.7 g, 15% yield).
• Step-2 Preparation of benzyl 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -5- ⁇ [(1E)- ⁇ [(tert-butoxy) carbonyl]amino ⁇ ( ⁇ [(tert-butoxy)carbonyl]imino ⁇ )methyl]amino ⁇ pentanamido] acetate
• Compound R3 To a stirring solution of (2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -5- ⁇ [(1E)- ⁇ [(tert- butoxy)carbonyl] amino ⁇ ( ⁇ [(tert-butoxy)carbonyl]imino ⁇ )methyl]amino ⁇ pentanoic acid (Compound R2; 1.33 g, 2.15 mmol) in THF (8 mL, 20V) was added DMAP (0.052 g, 0.43 mmol) followed by EDC.HCl (0.54 g, 2.86 mmol) at 0-5
• Step-3 Preparation of 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -5- ⁇ [(1E)- ⁇ [(tert- butoxy)carbonyl] amino ⁇ ( ⁇ [(tert-butoxy)carbonyl]imino ⁇ )methyl]amino ⁇ pentanamido]acetic acid
• Compound R4 To a solution of benzyl 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -5- ⁇ [(1E)- ⁇ [(tert- butoxy)carbonyl]amino ⁇ ( ⁇ [(tert-butoxy)carbonyl]imino ⁇ )methyl]amino ⁇ pentanamido]acetate (Compound R3; 0.6 g, 0.96 mmol) in ethyl acetate (6 mL, 10V), was added Pd/C (0.06 g, 10% w/w) and hydrogenated in a parr apparatus (5 kg H
• Step-4 Preparation of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -5- ⁇ [(1E)- ⁇ [(tert-butoxy)carbonyl]amino ⁇ ( ⁇ [(tert-butoxy)carbonyl]imino ⁇ )methyl]amino ⁇ pentanamido] acetate (Compound R5): To a stirring solution of 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -5- ⁇ [(1E)- ⁇ [(tert- butoxy)carbonyl]amin
• Step-5 Preparation of [azaniumyl( ⁇ [(4S)-4-azaniumyl-4-[(2- ⁇ [5-(4- chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4- yl]methoxy ⁇ -2-oxoethyl)carbamoyl]butyl]amino ⁇ )methylidene]azanium trichloride (Compound 23): To a stirring solution of [5-(4-chlorobenzamido)-2-[(4-chlorophenyl)methyl]-3-oxo- 1,2,4-thiadiazolidin-4-yl]methyl 2-[(2S)-2- ⁇ [(tert-butoxy)carbonyl]amino ⁇ -5- ⁇ [(1E)- ⁇ [(tert-butoxy)carbonyl]amino ⁇ ( ⁇ [(tert- butoxy)carbonyl]imin
• the INS-1E cells were treated in serum free medium with increasing doses of Compound 3 for 4 hours in accordance with the methods described in Steneberg et al., JCI Insight. 2018;3(12):e99114. https://doi.org/10.1172/jci.insight.99114.
• Western Blot analysis Western blot analysis of the INS-1E cells was performed as described in Steneberg et. al., JCI Insight. 2018;3(12):e99114. https://doi.org/10.1172/jci.insight.99114.
• the cell lysates were passed through a 30-gauge needle, ⁇ 8-times and centrifuged for 10 min at +4 °C at 14000 rpm.
• the final concentration of Compound 1 in of the formulation was 10 mg/mL.
• the formulation was administered at 5 mL/kg body weight.
• Formulation of Compound 3 6 mL of 2% w/v methyl cellulose solution in phosphate buffer (pH 7.5) was added to a 25 mL of conical flask together with 1 g of 2 mm glass beads. The solution was vigorously stirred on a magnetic stirrer. 78 mg of Compound 3 was slowly added to the solution, and the solution was vigorously stirred for approximately 1h. The pH of the formulation was measured to be 7.41. Each formulation was freshly prepared prior to administration to the animals. The final concentration of Compound 3 in the formulation was 13 mg/mL, which is equivalent to 10 mg/mL of Compound 1.
• the formulation was administered at 5 mL/kg body weight. Dose selection and justification for selection The doses of 50 mg/kg and 65 mg/kg b.w. for Compound 1 and Compound 3, respectively, were selected for comparison of equimolar doses. Dose Administration Adult healthy male Sprague Dawley rats aged 8 to 10 weeks were used for experimentation after a minimum three days of acclimatisation. Fed state animals were administered the formulation of Compound 1 or Compound 3 orally via gavage at a dose of 50 mg/kg or 65 mg/kg body weight, respectively.
• Alkaline borate buffer - Boric acid (1.237 g) and potassium chloride (1.491 g) were dissolved in 100 mL of Ultrapure water type-1 to get 0.2 M solution. About 50 mL of 0.2 M solution was taken in beaker and the pH was adjusted to 9.20 using 0.2 M NaOH solution. The final volume was made up to 200 mL using Ultrapure water type-1 and stored at room temperature (22 ⁇ 3 °C). 5 NjM solutions of Compound 17 in each buffer were prepared with 5 mM DMSO stock solutions. The solutions were incubated at 22 ⁇ 3 °C for 120 minutes, with shaking at 400 rpm using a thermomixer.

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