DIMER OF BIGUANIDINES AND THEIR THERAPEUTIC USES
20230416196 · 2023-12-28
Inventors
- RAPHAËL RODRIGUEZ (IVRY-SUR-SEINE, FR)
- ANTOINE VERSINI (ETERVILLE, FR)
- TATIANA CAÑEQUE (PARIS, FR)
- SEBASTIAN MÜLLER (IVRY-SUR-SEINE, FR)
- STÉPHANIE SOLIER (PARIS, FR)
- LEEROY BARON (SAINT-MANDE, FR)
Cpc classification
International classification
Abstract
The present invention relates to compounds comprising two biguanidyl radicals that can be useful as anti-inflammatory agent, and also to new compounds comprising two biguanidyl radicals and their use as a drug, in particular for treating a cancer, a metabolic disease, a secondary mitochondrial disorder due to copper overload including Indian childhood cirrhosis, Wilson's disease and Idiopathic infantile copper toxicosis or due to iron overload, an infection by a virus such as a coronavirus or an influenza virus, a neurodegenerative disease or disorder and aging.
Claims
1-17. (canceled)
18. A method of treating an inflammatory disease or disorder or an autoimmune disease or disorder comprising administering a compound of formula (I) to a subject having said inflammatory or autoimmune disease or disorder, wherein the formula (I) is ##STR00080## with R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 being independently selected from the group consisting of H, a C.sub.1-C.sub.6alkyl, a C.sub.0-C.sub.6alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), a C.sub.3-C.sub.10cycloalkyl, a C.sub.3-C.sub.10cycloheteroalkyl, a C.sub.6-C.sub.12aryl, and a C.sub.5-C.sub.12heteroaryl, said alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl being optionally substituted by a R group or a R group; or R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 being independently selected from the group consisting of H, a C.sub.1-C.sub.6alkyl, a C.sub.0-C.sub.6alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), a C.sub.3-C.sub.10cycloalkyl, a C.sub.3-C.sub.10cycloheteroalkyl, a C.sub.6-C.sub.12aryl, and a C.sub.5-C.sub.12heteroaryl, said alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl being optionally substituted by a R group or a R group, and R.sub.1 and R.sub.8 forming together a linker -L-, -L- and -L-, if present, being independently a linear hydrocarbon chain of 8 to 16 carbons optionally interrupted by an oxygen atom; and/or a function selected from the group consisting of amide (C(O)NH or NHC(O)), carbonyl (C(O)), ester (C(O)O or OC(O)), sulfonyl (SO.sub.2), sulfinyl (S(O)), thiocarbonyl (C(S)), thioester (C(O)S or SC(O)), carbonyloxy (OC(O)O), S(O)NH, NHS(O), SO.sub.2NH, NHSO.sub.2, phosphate (OP(O)OHO) and phosphonate (P(O)OHO or OP(O)OH); and/or a 3-7-membered ring, optionally selected from the group consisting of a cycloalkyl, a heterocycloalkyl, an aryl, and a heteroaryl, said ring being optionally substituted by a group R or R; said linear hydrocarbon chain being optionally substituted by a group R or R; with R being selected from the group consisting of a C.sub.1-C.sub.6alkyl optionally substituted by at least one halogen, a C.sub.1-C.sub.6alkyloxy optionally substituted by at least one halogen, a C.sub.3-C.sub.10cycloalkyl, a C.sub.3-C.sub.10cycloheteroalkyl, a C.sub.6-C.sub.12aryl, and a C.sub.5-C.sub.12heteroaryl, the group being optionally substituted by a group R, and R being selected from the group consisting of a halogen, a hydroxyl, a thiol, a cyano, an ethynyl (CCH), a nitro, an amino (NH.sub.2), a phosphate (PO.sub.4.sup.3-), a C.sub.1-C.sub.6alkyl optionally substituted by at least one halogen, a C.sub.1-C.sub.6alkoxy optionally substituted by at least one halogen, a C.sub.1-C.sub.6thioalkyl optionally substituted by at least one halogen, C.sub.0-C.sub.3alkyl-NHC(O)R, C.sub.0-C.sub.3alkyl-C(O)NRR, C.sub.0-C.sub.3alkyl-NHC(O)OR, C.sub.0-C.sub.3alkyl-NHC(O)NRR, C.sub.0-C.sub.3alkyl-C(O)R, C.sub.0-C.sub.3alkyl-C(O)OR, C.sub.0-C.sub.3alkyl-NRR, C.sub.0-C.sub.3alkyl-SOR, C.sub.0-C.sub.3alkyl-SO.sub.2R, C.sub.0-C.sub.3alkyl-SONRR, C.sub.0-C.sub.3alkyl-SO.sub.2NRR, C.sub.0-C.sub.3alkyl-NHSO.sub.2R, -a C.sub.3-C.sub.10cycloalkyl, a C.sub.3-C.sub.10cycloheteroalkyl, a C.sub.6-C.sub.12aryl, and a C.sub.5-C.sub.12heteroaryl, with R being H or a C.sub.1-C.sub.3 or C.sub.1-C.sub.6alkyl optionally substituted by at least one halogen and said cycloalkyl, cycloheteroalkyl, aryl or heteroaryl being optionally substituted by a halogen, a hydroxyl, a cyano, a nitro, an amino, or a C.sub.1-C.sub.3alkoxy, or a pharmaceutically acceptable salt, stereoisomer, tautomer or solvate thereof.
19. The method according to claim 18, wherein said inflammatory disease or disorder or autoimmune disease or disorder is a systemic inflammatory response syndrome, a cytokine release syndrome (CRS), an Adult Respiratory Distress Syndrome (ARDS), a Macrophage Activation Syndrome (MAS), an Alveolar inflammatory response, a paediatric multisystem inflammatory syndrome, a Hemophagocytic lymphohistiocytosis (HLH), systemic lupus erythematosus, a sepsis, septic shock, Crohn's disease, ulcerative colitis, rheumatoid arthritis, or a hypercytokinemia.
20. The method according to claim 19, wherein the CRS is induced by a virus of Orthocoronavirinae subfamily.
21. The method according to claim 20, wherein said virus is Middle East respiratory syndrome-related coronavirus (MERS-CoV), -CoV, Severe acute respiratory syndrome coronavirus (SARS-CoV), -CoV or Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), -CoV.
22. The method according to claim 18, wherein said inflammatory disease or disorder or autoimmune disease or disorder is selected from the group consisting of a metabolic disease, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, insulin resistance, hyperglycemia, hyperinsulinemia, glucose intolerance, hypertension, NAFLD, NASH and obesity, polycystic ovary syndrome, metabolic syndrome, cardiovascular diseases, hypertension, atherosclerosis and arteriosclerosis, a mitochondrial dysfunction, a primary or a secondary mitochondrial dysfunction, a secondary mitochondrial disorder due to copper overload, Indian childhood cirrhosis, Wilson's disease, Idiopathic infantile copper toxicosis due to iron overload, Hereditary hemochromatosis, Juvenile Hemochromatosis, Neonatal iron storage disease, type I Tyrosinemia, Zellweger syndrome, mental disorders, schizophrenia, anxiety disorders, mild cognitive disorder, depressive disorder, bipolar disorder, autism spectrum disorder, Fragile X syndrome, an infection by a virus, a neurodegenerative disease or disorder and aging.
23. The method according to claim 22, wherein said infection by a virus is a infection by a coronavirus or an influenza virus.
24. The method according to claim 22, wherein said inflammatory disease or disorder or autoimmune disease or disorder is selected from the group consisting of diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, polycystic ovary syndrome, and glucose intolerance.
25. The method according to claim 18, wherein the compound is selected from the group consisting of ##STR00081## with n being an integer from 6 to 14; ##STR00082## with n being an integer from 8 to 16, and R being a C.sub.1-C.sub.6alkyl, or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-3CCH); ##STR00083## with n being an integer from 6 to 14; ##STR00084## with n being an integer from 6 to 14; ##STR00085## ##STR00086## with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 4 to 14, or from 4 to 12, or from 5 to 10, or from 6 to 10; and ##STR00087## with n being an integer selected from 8 to 16; wherein the dotted line being present or absent and being one or two atoms with covalent bonds, or a pharmaceutically acceptable salt, stereoisomer, tautomer or solvate thereof.
26. The method according to claim 18, wherein R.sub.1 and R.sub.8 are independently selected from the group consisting of H, a C.sub.1-C.sub.6alkyl and a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-3CCH), or they form together a linker -L-.
27. The method according to claim 18, wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are independently selected from the group consisting of H, a C.sub.1-C.sub.6alkyl and a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-3CCH).
28. The method according to claim 18, wherein R.sub.3 and R.sub.6 are H.
29. The method according to claim 18, wherein R.sub.2 and R.sub.7 are H.
30. The method according to claim 18, wherein L, and L if present, is independently (CH.sub.2).sub.fCR.sub.aCR.sub.eCR.sub.fCR.sub.b(CH.sub.2).sub.g, (CH.sub.2).sub.fCR.sub.aCHCHCR.sub.b(CH.sub.2).sub.g, with R.sub.a and R.sub.b being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), with R.sub.e and R.sub.f being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), f and g being an integer independently selected from 0 to 12 and the sum f and g being an integer selected from 0 to 12, or from 3 to 10, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.hCCCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 12 and the sum h and i being an integer selected from 0 to 12, or from 3 to 10, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.hCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 0 to 14, or from 3 to 12, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.n, with n being an integer selected from 4 to 16; or CR.sub.a(CH.sub.2).sub.nCR.sub.b, with R.sub.a and R.sub.b being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), and with n being an integer selected from 2 to 14; or (CH.sub.2).sub.pCHR.sub.cCR.sub.eCR.sub.fCHR.sub.d(CH.sub.2).sub.q, (CH.sub.2).sub.pCHR.sub.eCHCHCHR.sub.d(CH.sub.2).sub.q, with R.sub.c and R.sub.d being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), with R.sub.e and R.sub.f being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), p and q being an integer independently selected from 0 to 12 and the sum p and q being an integer selected from 0 to 12, or from 3 to 10, or from 4 to 10, or from 5 to 10, or from 6 to 10.
31. The method according to claim 18, wherein L, and L if present, is independently (CH.sub.2).sub.fCR.sub.aCR.sub.eCR.sub.fCR.sub.b(CH.sub.2).sub.g, (CH.sub.2).sub.fCR.sub.aCHCHCR.sub.b(CH.sub.2).sub.g, with R.sub.a and R.sub.b being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), with R.sub.e and R.sub.f being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), f and g being an integer independently selected from 0 to 12 and the sum f and g being an integer selected from 4 to 12, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.hCCCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 12 and the sum h and i being an integer selected from 4 to 12, or from 3 to 10, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.hCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 6 to 14, or from 6 to 10; or (CH.sub.2).sub.n, with n being an integer selected from 8 to 16; or CR.sub.a(CH.sub.2).sub.nCR.sub.b, with R.sub.a and R.sub.b being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), and with n being an integer selected from 6 to 14; or (CH.sub.2).sub.pCHR.sub.cCR.sub.eCR.sub.fCHR.sub.d(CH.sub.2).sub.q, or (CH.sub.2).sub.pCHR.sub.cCHCHCHR.sub.d(CH.sub.2).sub.q, with R.sub.c and R.sub.d being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), with R.sub.e and R.sub.f being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), p and q being an integer independently selected from 0 to 12 and the sum p and q being an integer selected from 4 to 12, or from 4 to 10, or from 5 to 10, or from 6 to 10.
32. The method according to claim 18, wherein L, and L if present, is independently (CH.sub.2).sub.fCR.sub.aCHCHCR.sub.b(CH.sub.2).sub.g, with R.sub.a and R.sub.b being H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), f and g being an integer independently selected from 0 to 12 and the sum f and g being an integer selected from 7 to 12, or from 7 to 11, or from 8 to 11, or from 9 to 10; or (CH.sub.2).sub.hCCCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 12 and the sum h and i being an integer selected from 7 to 12, or from 7 to 11, or from 8 to 11, or from 9 to 10; or (CH.sub.2).sub.hCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 9 to 14, or from 10 to 13, or from 10 to 12; or (CH.sub.2).sub.n, with n being an integer selected from 11 to 16; or CR.sub.a(CH.sub.2).sub.nCR.sub.b, with R.sub.a and R.sub.b being H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), and with n being an integer selected from 9 to 14; or (CH.sub.2).sub.pCHR.sub.cCHCHCHR.sub.a(CH.sub.2).sub.q, with R.sub.c and R.sub.d being H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), p and q being an integer independently selected from 0 to 12 and the sum p and q being an integer selected from 7 to 12, or from 7 to 11, or from 8 to 11, or from 9 to 10.
33. The method according to claim 18, wherein the compound is selected from the group consisting of ##STR00088## with n being an integer from 9 to 14; ##STR00089## with n being an integer from 11 to 16 and R being a C.sub.1-C.sub.6alkyl, or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-3CCH); ##STR00090## with n being an integer from 9 to 14; ##STR00091## with n being an integer from 9 to 14; ##STR00092## with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 4 to 14, or from 4 to 12, or from 5 to 10, or from 6 to 10; and ##STR00093## with n being an integer selected from 8 to 16; ##STR00094## with n being an integer from 2-14 or from 4-14 or from 8-14 or from 8-12 or from 8-10; and ##STR00095## with n being an integer from 6-16, from 8-16 or from 8-12 or from 8-10 and R being an ethyl, a propyl or CH2-CCH, or a pharmaceutically acceptable salt, stereoisomer, tautomer or solvate thereof.
34. A compound of formula (I) wherein the formula (I) is ##STR00096## wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 being independently selected from the group consisting of H, a C.sub.1-C.sub.6alkyl, a C.sub.0-C.sub.6alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), a C.sub.3-C.sub.10cycloalkyl, a C.sub.3-C.sub.10cycloheteroalkyl, a C.sub.6-C.sub.12aryl, and a C.sub.5-C.sub.12heteroaryl, said alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl being optionally substituted by a R group or a R group; and 1) R.sub.1 and R.sub.8 being independently selected from the group consisting of H, a C.sub.1-C.sub.6alkyl, a C.sub.0-C.sub.6alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), a C.sub.3-C.sub.10cycloalkyl, a C.sub.3-C.sub.10cycloheteroalkyl, a C.sub.6-C.sub.12aryl, and a C.sub.5-C.sub.12heteroaryl, said alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl being optionally substituted by a R group or a R group; and -L- being independently a linear hydrocarbon chain of 11 to 16 carbons optionally interrupted by an oxygen atom; and/or a function selected from the group consisting of amide (C(O)NH or NHC(O)), carbonyl (C(O)), ester (C(O)O or OC(O)), sulfonyl (SO.sub.2), sulfinyl (S(O)), thiocarbonyl (C(S)), thioester (C(O)S or SC(O)), carbonyloxy (OC(O)O), S(O)NH, NHS(O), SO.sub.2NH, NHSO.sub.2, phosphate (OP(O)OHO) and phosphonate (P(O)OHO or OP(O)OH); and/or a 3-7-membered ring, optionally selected from the group consisting of a cycloalkyl, a heterocycloalkyl, an aryl, and a heteroaryl, said ring being optionally substituted by a group R or R; said linear hydrocarbon chain being optionally substituted by a group R or R; or 2) R.sub.1 and R.sub.8 forming together a linker -L-; and -L- and -L- being independently a linear hydrocarbon chain of 4 to 16 carbons optionally interrupted by a heteroatom; and/or a function selected from the group consisting of amide (C(O)NH or NHC(O)), carbonyl (C(O)), ester (C(O)O or OC(O)), sulfonyl (SO.sub.2), sulfinyl (S(O)), thiocarbonyl (C(S)), thioester (C(O)S or SC(O)), carbonyloxy (OC(O)O), S(O)NH, NHS(O), SO.sub.2NH, NHSO.sub.2, phosphate (OP(O)OHO) and phosphonate (P(O)OHO or OP(O)OH); and/or a 3-7-membered ring, optionally selected from the group consisting of a cycloalkyl, a heterocycloalkyl, an aryl, and a heteroaryl, said ring being optionally substituted by a group R or R; said linear hydrocarbon chain being optionally substituted by a group R or R; or 3) the compound is selected from the group consisting of ##STR00097## with n being an integer from 2-14 or from 4-14 or from 8-14 or from 8-12 or from 8-10; and ##STR00098## with n being an integer from 6-16, from 8-16 or from 8-12 or from 8-10 and R being an ethyl, a propyl or CH.sub.2CCH, wherein in item 1) and 2) R being selected from the group consisting of a C.sub.1-C.sub.6alkyl optionally substituted by at least one halogen, a C.sub.1-C.sub.6alkyloxy optionally substituted by at least one halogen, a C.sub.3-C.sub.10cycloalkyl, a C.sub.3-C.sub.10cycloheteroalkyl, a C.sub.6-C.sub.12aryl, and a C.sub.5-C.sub.12heteroaryl, the group being optionally substituted by a group R, and R being selected from the group consisting of a halogen, a hydroxyl, a thiol, a cyano, an ethynyl (CCH), a nitro, an amino (NH.sub.2), a phosphate (PO.sub.4.sup.3-), a C.sub.1-C.sub.6alkyl optionally substituted by at least one halogen, a C.sub.1-C.sub.6alkoxy optionally substituted by at least one halogen, a C.sub.1-C.sub.6thioalkyl optionally substituted by at least one halogen, C.sub.0-C.sub.3alkyl-NHC(O)R, C.sub.0-C.sub.3alkyl-C(O)NRR, C.sub.0-C.sub.3alkyl-NHC(O)OR, C.sub.0-C.sub.3alkyl-NHC(O)NRR, C.sub.0-C.sub.3alkyl-C(O)R, C.sub.0-C.sub.3alkyl-C(O)OR, C.sub.0-C.sub.3alkyl-NRR, C.sub.0-C.sub.3alkyl-SOR, C.sub.0-C.sub.3alkyl-SO.sub.2R, C.sub.0-C.sub.3alkyl-SONRR, C.sub.0-C.sub.3alkyl-SO.sub.2NRR, C.sub.0-C.sub.3alkyl-NHSO.sub.2R, -a C.sub.3-C.sub.10cycloalkyl, a C.sub.3-C.sub.10cycloheteroalkyl, a C.sub.6-C.sub.12aryl, and a C.sub.5-C.sub.12heteroaryl, with R being H or a C.sub.1-C.sub.3 or C.sub.1-C.sub.6alkyl optionally substituted by at least one halogen and said cycloalkyl, cycloheteroalkyl, aryl or heteroaryl being optionally substituted by a halogen, a hydroxyl, a cyano, a nitro, an amino, or a C.sub.1-C.sub.3alkoxy, or a pharmaceutically acceptable salt, stereoisomer, tautomer or solvate of any of these compounds.
35. The compound according to claim 34, wherein R.sub.1 and R.sub.8 are independently selected from the group consisting of H, a C.sub.1-C.sub.6alkyl and a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-3CCH), or they form together a linker -L-.
36. The compound according to claim 34, wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are independently selected from the group consisting of H, a C.sub.1-C.sub.6alkyl and a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-3CCH).
37. The compound according to claim 34, wherein R.sub.3 and R.sub.7 are H.
38. The compound according to claim 34, wherein R.sub.2 and R.sub.7 are H.
39. The compound according to claim 34, wherein L, and L if present, is independently (CH.sub.2).sub.fCR.sub.aCR.sub.eCR.sub.fCR.sub.b(CH.sub.2).sub.g, (CH.sub.2).sub.fCR.sub.aCHCHCR.sub.b(CH.sub.2).sub.g, with R.sub.a and R.sub.b being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), with R.sub.e and R.sub.f being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), f and g being an integer independently selected from 0 to 12 and the sum f and g being an integer selected from 0 to 12, or from 3 to 10, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.hCCCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 12 and the sum h and i being an integer selected from 0 to 12, or from 3 to 10, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.hCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 0 to 14, or from 3 to 12, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.n, with n being an integer selected from 4 to 16; or CR.sub.a(CH.sub.2).sub.nCR.sub.b, with R.sub.a and R.sub.b being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), and with n being an integer selected from 2 to 14; or (CH.sub.2).sub.pCHR.sub.cCR.sub.eCR.sub.fCHR.sub.d(CH.sub.2).sub.q, (CH.sub.2).sub.pCHR.sub.eCHCHCHR.sub.d(CH.sub.2).sub.q, with R.sub.e and R.sub.d being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), with R.sub.e and R.sub.f being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), p and q being an integer independently selected from 0 to 12 and the sum p and q being an integer selected from 0 to 12, or from 3 to 10, or from 4 to 10, or from 5 to 10, or from 6 to 10.
40. The compound according to claim 34, wherein L, and L if present, is independently (CH.sub.2).sub.fCR.sub.aCR.sub.eCR.sub.fCR.sub.b(CH.sub.2).sub.g, (CH.sub.2).sub.fCR.sub.aCHCHCR.sub.b(CH.sub.2).sub.g, with R.sub.a and R.sub.b being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), with R.sub.e and R.sub.f being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), f and g being an integer independently selected from 0 to 12 and the sum f and g being an integer selected from 4 to 12, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.hCCCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 12 and the sum h and i being an integer selected from 4 to 12, or from 3 to 10, or from 4 to 10, or from 5 to 10, or from 6 to 10; or (CH.sub.2).sub.hCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 6 to 14, or from 6 to 10; or (CH.sub.2).sub.n, with n being an integer selected from 8 to 16; or CR.sub.a(CH.sub.2).sub.nCR.sub.b, with R.sub.a and R.sub.b being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), and with n being an integer selected from 6 to 14; or (CH.sub.2).sub.pCHR.sub.cCR.sub.eCR.sub.fCHR.sub.d(CH.sub.2).sub.q, or (CH.sub.2).sub.pCHR.sub.cCHCHCHR.sub.d(CH.sub.2).sub.q, with R.sub.c and R.sub.d being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), with R.sub.e and R.sub.f being independently H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), p and q being an integer independently selected from 0 to 12 and the sum p and q being an integer selected from 4 to 12, or from 4 to 10, or from 5 to 10, or from 6 to 10.
41. The compound according to claim 34, wherein L, and L if present, is independently (CH.sub.2).sub.fCR.sub.aCHCHCR.sub.b(CH.sub.2).sub.g, with R.sub.a and R.sub.b being H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), f and g being an integer independently selected from 0 to 12 and the sum f and g being an integer selected from 7 to 12, or from 7 to 11, or from 8 to 11, or from 9 to 10; or (CH.sub.2).sub.hCCCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 12 and the sum h and i being an integer selected from 7 to 12, or from 7 to 11, or from 8 to 11, or from 9 to 10; or (CH.sub.2).sub.hCC(CH.sub.2).sub.i, with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 9 to 14, or from 10 to 13, or from 10 to 12; or (CH.sub.2).sub.n, with n being an integer selected from 11 to 16; or CR.sub.a(CH.sub.2).sub.nCR.sub.b, with R.sub.a and R.sub.b being H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), and with n being an integer selected from 9 to 14; or (CH.sub.2).sub.pCHR.sub.cCHCHCHR.sub.a(CH.sub.2).sub.q, with R.sub.c and R.sub.d being H, a C.sub.1-C.sub.6alkyl or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-6CCH), p and q being an integer independently selected from 0 to 12 and the sum p and q being an integer selected from 7 to 12, or from 7 to 11, or from 8 to 11, or from 9 to 10.
42. The compound according to claim 34, wherein the compound is selected from the group consisting of ##STR00099## with n being an integer from 9 to 14; ##STR00100## with n being an integer from 11 to 16 and R being a C.sub.1-C.sub.6alkyl, or a C.sub.0-C.sub.3alkyl-ethynyl ((CH.sub.2).sub.0-3CCH); ##STR00101## with n being an integer from 9 to 14; ##STR00102## with n being an integer from 9 to 14; ##STR00103## with h and i being an integer independently selected from 0 to 14 and the sum h and i being an integer selected from 4 to 14, or from 4 to 12, or from 5 to 10, or from 6 to 10; and ##STR00104## with n being an integer selected from 8 to 16; ##STR00105## with n being an integer from 2-14 or from 4-14 or from 8-14 or from 8-12 or from 8-10; and ##STR00106## with n being an integer from 6-16, from 8-16 or from 8-12 or from 8-10 and R being an ethyl, a propyl or CH2-CCH, or a pharmaceutically acceptable salt, stereoisomer, tautomer or solvate thereof.
43. A method of treating a disease comprising administering a compound according to claim 34 to a subject in need of treatment, said disease being selected from the group consisting of a cancer, a metabolic disease, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, insulin resistance, hyperglycemia, hyperinsulinemia, glucose intolerance, hypertension, NAFLD, NASH and obesity, polycystic ovary syndrome, metabolic syndrome, cardiovascular diseases, hypertension, atherosclerosis and arteriosclerosis, a secondary mitochondrial disorder due to copper overload, Indian childhood cirrhosis, Wilson's disease, Idiopathic infantile copper toxicosis due to iron overload, Hereditary hemochromatosis, Juvenile Hemochromatosis, Neonatal iron storage disease, type I Tyrosinemia, Zellweger syndrome, mental disorders, schizophrenia, anxiety disorders, mild cognitive disorder, depressive disorder, bipolar disorder, autism spectrum disorder, Fragile X syndrome, an infection by a virus, a neurodegenerative disease or disorder and aging.
44. The method according to claim 43, wherein said infection by a virus is a infection by a coronavirus or an influenza virus.
45. The method according to claim 43, wherein said inflammatory disease or disorder or autoimmune disease or disorder is selected from the group consisting of diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, polycystic ovary syndrome, and glucose intolerance.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0521]
[0522]
[0523]
[0524]
[0525]
[0526] (A) Kaplan-Meier survival curves of mice challenged by cecal ligation and puncture (CLP) using a 21G needle and treated with LCC-12 (0.3 mg/kg, 4 h post-challenge, IP, n=10), dexamethasone (1.0 mg/kg at to) or saline solution (IP, n=10). (B) Kaplan-Meier survival curves of mice challenged by CLP using a 25G needle and treated with LCC-12 (0.3 mg/kg, 4 h post-challenge, IP, n=10) or saline solution (IP, n=10). Mantel-Cox Log-rank test for. Hazard ratio calculated using Mantel-Haenszel. n.d.=not determined. (C) Curves showing the average body weight of mice challenged by CLP using a 25G needle and treated with LCC-12 (0.3 mg/kg, 4 h post-challenge, IP, n=10) or saline solution (IP, n=10). (D) Curves showing the average gravity score of symptoms of mice challenged by CLP using a 25G needle and treated with LCC-12 (0.3 mg/kg, 4 h post-challenge, IP, n=10) or saline solution (IP, n=10). Clinical score are out of a total of 15, with 0 representing no symptoms and 15 representing maximal symptoms. Gravity score criteria are indicated. For (C) and (D) 2-way ANOVA. Mean valuesSEM.
[0527]
[0528] (A) CD4.sup.+ T cells, non-activated (naCD4) and activated (aCD4). Left: Flow cytometry of CD44 at the plasma membrane. Right: Flow cytometry of CD25 and CD69 cell surface markers of naCD4, aCD4 and aCD4 treated with LCC-12 (10 M). (B) CD8.sup.+ T cells, non-activated (naCD8) and activated (aCD8). Left: Flow cytometry of CD44 at the plasma membrane. Right: Flow cytometry of CD25 and CD69 cell surface markers of naCD8, aCD8 and aCD8 treated with LCC-12 (10 M). (C) Neutrophils, non-activated (naG) and activated (aG). Left: Flow cytometry of CD44 at the plasma membrane. Right: Flow cytometry of CD64 and CD66b cell surface markers of naG, aG and aG treated with LCC-12 (10 M). (D) Monocytes, non-activated (naMo) and activated (aMo). Left: Flow cytometry of CD44 at the plasma membrane. Right: Flow cytometry of CD25 and CD80 cell surface markers of naMo, aMo and aMo treated with LCC-12 (10 M). (E) Dendritic cells, non-activated (naDC) and activated (aDC). Left: Flow cytometry of CD44 at the plasma membrane. Right: Flow cytometry of CD40, CD83, CD80 and CD86 cell surface markers of naDC, aDC and aDC treated with LCC-12 (10 M).
[0529]
[0530] Cell viability curves of cells treated with 10 M LCC-12 on cell lines as indicated. Cells were co-treated with TGF- or OSM as indicated.
[0531]
[0532] (A) Flow cytometry of CD44 surface staining of cells treated with TGF- or OSM. (B) Box plots of ICP-MS of cells treated with TGF- or OSM, showing increase of copper in the mesenchymal state. Mann-Whitney test. (C) Western blot of cells treated with TGF- or OSM, showing increase of SOD2 in the mesenchymal state. (D) Western blot of mesenchymal and epithelial markers of cells treated with TGF- or OSM, showing that LCC-12 blocks EMT.
[0533]
[0534] Chemograms of biopsy-derived organoids of pancreatic ductal adenocarcinoma treated with LCC-12 with the IC.sub.50-values indicated.
[0535]
[0536] (A) Molecular structure of isotopologue .sup.15N-.sup.13C-LCC-12. (B) NanoSIMS images of .sup.15N and .sup.197Au in aMDM. Scale bar, 10 m. (C) Schematic illustration of click labeling of alkyne-containing LCC-12 in cells. (D) Fluorescence microscopy of labeled LCC-12 (0.1 M) in activated MDM (aMDM), showing localization in vicinity of cytochrome c (cyt c) in mitochondria. n=6 donors. 50 cells were quantified per donor. (E) Fluorescence microscopy of labeled LCC-12 (0.1 M) in aMDM co-treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Scale bar, 10 m. (F) Fluorescence microcopy images of labeled LCC-12 (0.1 M). Click labeling performed in the presence of ascorbate without added copper(II) in naMDM and aMDM. Scale bar, 10 m. (G) Fluorescence microcopy images of labeled LCC-12 (0.1 M). Click labeling performed with and without ascorbate (Asc) in absence of added copper(II) in aMDM. Scale bar, 10 m. (H) Schematic illustration of mitochondrial isolation and ICP-MS of mitochondrial copper in naMDM and aMDM. n=6 donors. (E)-(G) Student's T-test. Mean valuesSEM. Images of a representative donor are shown and at least 50 cells were quantified per condition. (H) Mann-Whitney test. Mean valuesSEM.
[0537]
[0538] (A) Reaction scheme of H.sub.2O.sub.2 biosynthesis from superoxide catalyzed by mitochondrial superoxide dismutase 2 (SOD2). (B) Fluorescence microcopy images of SOD2 in non-activated MDM (naMDM) and activated MDM (aMDM). Mitochondria were stained using an antibody against cyt c. Student's T-test. Mean valuesSEM. Images of a representative donor are shown and at least 50 cells were quantified per condition. (C) Flow cytometry analysis of mitochondrial H.sub.2O.sub.2 in naMDM and aMDM. n=6 donors. (D) and (E) Box plots of quantitative mass-spectrometry-based metabolomics of NAD.sup.+, NADH, KG and acetyl-CoA. (F) Heatmap of quantitative mass-spectrometry-based metabolomics of total cellular extracts of key metabolites whose biosynthesis depend on NAD(H). n=9 donors. MDM were activated with LPS and IFN (act. MDM), and co-treated with Met (10 mM) or LCC-12 (10 M) as indicated. Kruskal-Wallis test with Dunn's post-test. Mean valuesSEM.
EXAMPLES
Example 1: Synthesis of Compounds
[0539] Products were purified on a preparative HPLC Quaternary Gradient 2545 equipped with a Photodiode Array detector (Waters) fitted with a reverse phase column (XBridge Prep C18 5 m OBD 30150 mm). NMR spectroscopy was performed on Bruker spectrometers. Spectra were run in DMSO-d.sub.6 or Methanol-d.sub.6 at 298 K unless stated otherwise. .sup.1H-NMR spectra were recorded at 400 or 500 MHz, and chemical shifts are expressed in ppm using the residual non-deuterated solvent signal as internal standard. The following abbreviations are used: s, singlet; brs, broad signal; d, doublet; dd, doublet of doublet; t, triplet; m, multiplet; ex, exchangeable. .sup.13C-NMR spectrum was recorded at 100.6 or 125.8 MHz, and chemical shifts are expressed in ppm using deuterated solvent signal as internal standard. The purity of final compounds, determined to be >95% by UPLC-MS, and low-resolution mass spectra (LRMS) were recorded on a Waters Acquity H-class equipped with a Photodiode array detector and SQ Detector 2 (UPLC-MS) fitted with a reverse phase column (Aquity UPLC BEH C.sub.18 1.7 m, 2.150 mm). High-resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific Q-Exactive Plus equipped with a Robotic TriVersa NanoMate Advion.
[0540] General Procedures for LCC:
[0541] General procedure 1: Dicyandiamide (2.4 eq.), and the selected diamine (1, eq.) and CuCl.sub.2 (1 eq.) were suspended in a sealed tube in water (0.4 mL/mmol) and stirred for 1 h, then heated at 80 C. for 48 h. The resulting pink mixture was filtrated, and the solid was resuspended in water (10 mL). H.sub.2S, generated from dropwise addition of 37% aqueous (aq.) HCl on FeS (100 mesh powder), was passed into the mixture until it turned black. The black mixture was filtrated, and the filtrate was acidified to pH=5 with an aq. solution (soln.) of 1 M HCl. The solvent was evaporated under reduced pressure. LCC was purified with preparative HPLC (H2O/CH.sub.3CN/Formic acid, 100:0:0.1 to 0:100:0.1) to give the LCC di-formic acid salt as a white powder.
##STR00076##
[0542] LCC-4 with n being 2; LCC-8 with n being 6; LCC-9 with n being 7; LCC-10 with n being 8; and LCC-12 with n being 10.
##STR00077##
[0543] LCC-8 dimethyl with n being 6; LCC-12dimethyl with n being 10.
[0544] LCC-4 di-formic acid salt: (21%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 7.57 (s, 2H, ex), 7.41-6.41 (brs, 12H, ex), 3.06 (brs, 4H), 1.46 (brs, 4H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 159.9, 159.0, 40.7, 26.5 ppm. HRMS (ESI+) m/z: calculated for C.sub.8H.sub.22N.sub.10 [M+2H].sup.2+ 129.1009, found 129.1015.
[0545] LCC-8 di-formic acid salt (11%): .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.68-7.96 (brs, 2H, ex), 8.44 (s, 2H, formate), 7.50-6.76 (brs, 12H, ex), 3.09 (brs, 4H), 1.43 (brs, 4H), 1.34-1.19 (m, 8H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.2 (formate), 160.2, 159.3, 41.3, 29.4, 29.1, 26.7 ppm. HRMS (ESI+) m/z: calculated for C.sub.12H.sub.30N.sub.10 [M+2H].sup.2+ 157.1322, found 157.1328.
[0546] LCC-9 di-formic acid salt (14%): .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.44 (s, 2H, formate), 8.26 (brs, 2H, ex), 7.14 (brs, 12H, ex), 3.06 (t, J=6.2 Hz, 4H), 1.43 (m, 4H), 1.26 (brs, 10H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.0 (formate), 160.2, 159.3, 41.4, 29.4 (2C), 29.2, 26.8 ppm. HRMS (ESI+) m/z calculated for C.sub.13H.sub.32N.sub.10 [M+2H].sup.2+ 164.1400, found 164.1400.
[0547] LCC-10 di-formic acid salt: (13%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.88-7.96 (brs, 2H, ex), 8.47 (s, 2H, formate), 7.43-6.88 (brs, 12H, ex), 3.05 (t, J=6.8 Hz, 4H), 1.43 (m, 4H), 1.34-1.16 (m, 12H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 166.6 (formate), 160.0, 159.0, 41.1, 29.1 (2C), 28.9, 26.5 ppm. HRMS (ESI+) m/z: calculated for C.sub.14H.sub.34N.sub.10 [M+2H].sup.2+ 171.1478, found 171.1485.
[0548] LCC-12 di-formic acid salt: (24%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.80-8.08 (brs, 2H, ex), 8.47 (s, 2H, formate), 7.60-6.78 (brs, 12H, ex), 3.05 (brs, 4H), 1.43 (brs, 4H), 1.32-1.18 (m, 16H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.2 (formate), 160.4, 159.3, 41.3, 29.5 (3C), 29.3, 26.8 ppm. HRMS (ESI+) m/z: calculated for C.sub.16H.sub.38N.sub.10 [M+2H].sup.2+ 185.1635, found 185.1635.
[0549] LCC-12 dimethyl di-formic acid salt: (18%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.47 (s, 2H, formate), 7.70-6.90 (brs, 12H, ex), 3.29 (t, J=7.5 Hz, 4H), 2.90 (s, 6H), 1.48 (m, 4H), 1.32-1.16 (m, 16H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 166.7 (formate), 159.9, 158.7, 49.8, 35.8, 29.5 (2C), 29.3, 27.4, 26.5 ppm. HRMS (ESI+) m/z: calculated for C.sub.18H.sub.42N.sub.10 [M+2H].sup.2+ 199.1791, found 199.1798.
[0550] LCC-8 dimethyl di-formic acid salt: (14%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.44 (s, 2H, formate), 7.62-6.73 (m, 12H, ex), 3.29 (t, J=7.5 Hz, 4H), 2.90 (s, 6H), 1.48 (m, 4H), 1.30-1.17 (m, 8H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 166.4 (formate), 159.8, 158.8, 49.8, 35.9, 29.3, 27.4, 26.4 ppm. HRMS (ESI+) m/z: calculated for C.sub.14H.sub.34N.sub.10 [M+2H].sup.2+ 171.1479, found 171.1484.
[0551] General procedure 2: Selected diamine dihydrochloride (1 eq.) was reacted with sodium di-cyanamide (2 eq.) in butanol (1.4 mL/mmol) at 130 C. overnight. After cooling to rt, the solvent was evaporated under reduced pressure and the crude was washed successively with EtOH and cold water. The bis(cyanoguanidino)alcane di-hydrochloride compound was recrystallized from H.sub.2O/EtOH or from H.sub.2O/ethoxyethanol to afford intermediates as white solids
##STR00078##
[0552] 1,6-Bis(cyanoguanidino)hexane: (66%) NMR is consistent with published procedure (Grberet al. (2013), Angew. Chem. Int. Ed., 52: 4487-4491). Recrystallized from H.sub.2O/EtOH. .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 7.20-6.23 (m, 6H, ex), 3.02 (brs, 4H), 1.40 (brs, 4H), 1.32-1.16 (m, 4H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 161.6, 118.8, 41.1, 29.3, 26.4 ppm.
[0553] 1,8-Bis(cyanoguanidino)octane: (62%) NMR is consistent with published procedure..sup.1 Recrystallized from H.sub.2O/EtOH. .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 7.40-6.14 (m, 6H, ex), 2.93 (m, 4H), 1.31 (m, 4H), 1.23-1.08 (m, 8H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 161.6, 118.9, 41.2, 29.4, 29.2, 26.6 ppm.
[0554] 1,10-Bis(cyanoguanidino)decane: (32%) Recrystallized from H.sub.2O/ethoxyethanol. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 7.66-5.92 (m, 6H, ex), 3.03 (m, 4H), 1.40 (m, 4H), 1.34-1.16 (m, 12H) ppm. .sup.13C-NMR (100.6 MHz, DMSO-d.sub.6) : 161.7, 118.9, 41.2, 29.4 (2C), 29.2, 26.7 ppm.
[0555] 1,12-Bis(cyanoguanidino)dodecane: (44%) Recrystallized from H.sub.2O/ethoxyethanol. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 7.21-6.18 (m, 6H, ex), 3.03 (m, 4H), 1.40 (m, 4H), 1.33-1.14 (m, 16H) ppm. .sup.13C-NMR (100.6 MHz, DMSO-d.sub.6) : 161.6, 118.9, 41.0, 29.4 (3C), 29.2, 26.7 ppm.
[0556] 1,12-Bis(cyanoguanidino)dodecane dimethyl: (86%) No recrystallized. Washing were enough. .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 7.22-6.55 (m, 4H, ex), 3.25 (m, 4H), 2.86 (s, 6H), 1.43 (brs, 4H), 1.38-1.04 (m, 16H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 160.9, 118.9, 49.6, 35.8, 29.4, 29.4, 29.3, 27.3, 26.4 ppm.
[0557] General procedure 3: Bis(cyanoguanidino)alcane di-hydrochloride (1 eq.) and the corresponding primary amine (2 eq.) were mixed together in a sealed tube and heated at 150 C. for 4 to 6 h without solvent. After cooling to rt, the mixture was taken up in ethanol and a large excess of ethyl acetate was added. The white precipitate was filtered off and washed with ethyl acetate to afford LCC-n,m as hydrochloride white salts.
##STR00079##
[0558] LCC-6,2 with n being 4 and m being 1; LCC-6,3 with n being 4 and m being 2; LCC-6,4 with n being 4 and m being 3; LCC-6,5 with n being 4 and m being 4; LCC-8,3 with n being 6 and m being 2; LCC-10,2 with n being 8 and m being 1; LCC-10,3 with n being 8 and m being 2; LCC-10,5 with n being 8 and m being 4; LCC-12,2 with n being 10 and m being 1; LCC-12,3 with n being 10 and m being 2; LCC-12,4 with n being 10 and m being 3; LCC-12,6 with n being 10 and m being 5.
[0559] LCC-6,2 di-hydrochloride salt: (20%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 7.63-6.37 (brs, 12H, ex), 3.16-3.04 (m, 8H), 1.50-1.39 (m, 4H), 1.34-1.26 (m, 4H), 1.06 (t, J=7.2 Hz, 6H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 158.8, 41.3, 36.3, 29.3, 26.5, 15.0 ppm. HRMS (ESI+) m/z: calculated for C.sub.14H.sub.34N.sub.10 [M+2H].sup.2+ 171.1479, found 171.1485.
[0560] LCC-6,3 di-hydrochloride salt: (45%).sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 7.82-6.28 (brs, 12H, ex), 3.16-2.98 (m, 8H), 1.53-1.40 (m, 8H), 1.35-1.25 (m, 4H), 0.87 (t, J=7.5 Hz, 6H) ppm. .sup.13C-NMR (100.6 MHz, DMSO-d.sub.6) : 158.8, 43.2, 41.2, 29.4, 26.5, 22.7, 11.8 ppm. HRMS (ESI+) m/z: calculated for C.sub.16H.sub.38N.sub.10 [M+2H].sup.2+ 185.1635, found 185.1641.
[0561] LCC-6,4 di-hydrochloride salt: (21%).sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 7.90-6.30 (brs, 12H, ex), 3.14-3.04 (m, 8H), 1.53-1.37 (m, 8H), 1.37-1.23 (m, 8H), 0.89 (t, J=7.2 Hz, 6H) ppm. .sup.13C-NMR (100.6 MHz, DMSO-d.sub.6) : 158.8, 41.2, 41.0, 31.5, 29.4, 26.5, 20.0, 14.1 ppm. HRMS (ESI+) m/z: calculated for C.sub.18H.sub.42N.sub.10 [M+2H].sup.2+ 199.1792, found 199.1798.
[0562] LCC-6,5 di-hydrochloride salt: (23%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 7.68-6.47 (m, 12H, ex), 3.08 (brs, 8H), 1.52-1.40 (m, 8H), 1.32-1.23 (m, 12H), 0.88 (t, J=7.1 Hz, 6H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 159.3, 41.2 (2C), 28.9 (3C), 26.5, 22.3, 14.4 ppm. HRMS (ESI+) m/z: calculated for C.sub.20H.sub.46N.sub.10 [M+2H].sup.2+ 213.1948, found 213.1954.
[0563] LCC-8,3 di-hydrochloride salt: (75%).sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 7.70-6.47 (m, 12H, ex), 3.13-3.04 (m, 8H), 1.52-1.39 (m, 8H), 1.33-1.23 (m, 8H), 0.87 (t, J=7.1 Hz, 6H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 159.1, 43.1, 41.3, 29.4, 29.2, 26.7, 22.7, 11.8 ppm. HRMS (ESI+) m/z: calculated for C.sub.18H.sub.42N.sub.10 [M+2H].sup.2+ 199.1792, found 199.1799.
[0564] LCC-10,2 di-formic acid salt. Second purification by preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) to afford a white solid. (46%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.49 (s, 2H, formate), 8.76-7.88 (brs, 4H, ex), 7.33-6.85 (m, 8H, ex), 3.16-2.99 (m, 8H), 1.44 (brs, 4H), 1.25 (brs, 12H), 1.05 (t, J=6.9 Hz, 6H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.4, 159.1, 41.2, 36.1, 29.4 (2C), 29.2, 26.8, 15.1 ppm. HRMS (ESI+) m/z: calculated for C.sub.18H.sub.42N.sub.10 [M+2H].sup.2+ 199.1792, found 199.1793.
[0565] LCC-10,3 di-hydrochloride salt. (75%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 7.82-6.17 (m, 12H, ex), 3.07 (brs, 8H), 1.58-1.37 (m, 8H), 1.26 (brs, 12H), 0.87 (t, J=7.2 Hz, 6H) ppm. .sup.13C-NMR (100.6 MHz, DMSO-d.sub.6) : 159.1, 43.2, 41.4, 29.5 (2C), 29.2, 26.8, 22.8, 11.9 ppm. HRMS (ESI+) m/z: calculated for C.sub.20H.sub.46N.sub.10 [M+2H].sup.2+ 213.1948, found 213.1948.
[0566] LCC-10,5 di-formic acid salt. Second purification by preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 80:20:0.1 to 0:100:0.1) afford a white solid. (35%).sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 8.48-6.55 (m, 12H, ex), 3.17-2.98 (brs, 8H), 1.51-1.35 (m, 8H), 1.35-1.18 (m, 20H), 0.87 (t, J=7.0 Hz, 6H) ppm. .sup.13C-NMR (100.6 MHz, DMSO-d.sub.6) : 167.7 (formate), 159.0, 41.2 (2C), 29.5 (2C), 29.2 (2C), 29.0, 26.8, 22.3, 14.4 ppm. HRMS (ESI+) m/z: calculated for C.sub.24H.sub.54N.sub.10 [M+2H].sup.2+ 241.2261, found 241.2260.
[0567] LCC-12,2 di-formic acid salt. The product was purified by preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 100:0:0.1 to 50:50:0.1) to afford a white solid. (26%).sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 8.50 (s, 2H, formate), 8.80-8.04 (brs, 4H, ex), 7.41-6.85 (m, 8H, ex), 3.18-2.97 (m, 8H), 1.44 (brs, 4H), 1.26 (brs, 16H), 1.05 (t, J=6.9 Hz, 6H) ppm. .sup.13C-NMR (100.6 MHz, DMSO-d.sub.6) : 167.4, 159.0, 41.2, 36.1, 29.4 (3C), 29.2, 26.8, 15.1 ppm. HRMS (ESI+) m/z: calculated for C.sub.20H.sub.46N.sub.10 [M+2H].sup.2+ 213.1948, found 213.1948.
[0568] LCC-12,3 di-formic acid salt. The product was purified by preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) to afford a white solid. (20%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.50 (s, 2H, formate), 8.57-8.18 (brs, 4H, ex), 7.33-6.88 (m, 8H, ex), 3.05 (m, 8H), 1.52-1.38 (brs, 8H), 1.25 (brs, 16H), 0.86 (t, J=7.4 Hz, 6H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.4, 159.1, 43.1, 41.3, 29.5 (3C), 29.2, 26.8, 22.8, 11.8 ppm. HRMS (ESI+) m/z: calculated for C.sub.22H.sub.50N.sub.10 [M+2H].sup.2+ 227.2104, found 227.2105.
[0569] LCC-12,4 Second purification on preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) to afford a white solid. (39%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.49 (s, 2H, formate), 8.73-7.85 (brs, 4H, ex), 7.39-6.93 (brs, 8H, ex), 3.05 (m, 8H), 1.49-1.38 (m, 8H), 1.35-1.20 (brs, 20H), 0.87 (t, J=7.5 Hz, 6H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.4, 159.0, 41.3, 40.9, 31.6, 29.5 (3C), 29.2, 26.8, 20.0, 14.1 ppm. HRMS (ESI+) m/z: calculated for C.sub.24H.sub.54N.sub.10 [M+2H].sup.2+ 241.2261, found 241.2262.
[0570] LCC-12,6 Second purification on preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) afford a white solid. (14%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.48 (s, 2H, formate), 8.30-7.40 (brs, 4H, ex), 7.40-6.70 (brs, 8H, ex), 3.06 (brs, 8H), 1.50-1.37 (m, 8H), 1.35-1.17 (m, 28H), 0.86 (t, J=7.1 Hz, 6H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.5 (formate), 158.8, 41.3 (2C), 31.5, 29.5 (4C), 29.2, 26.8, 26.5, 22.6, 14.3 ppm. HRMS (ESI+) m/z: calculated for C.sub.28H.sub.62N.sub.10 [M+2H].sup.2+ 269.2574, found 269.2572.
[0571] LCC-12,4, alcyne di-formic acid salt. Second purification on preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) afford a white solid. (30%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 9.05-7.60 (brs, 4H, ex), 8.47 (s, 2H, formate), 7.60-6.80 (brs, 8H, ex), 3.21 (t, J=6.2 Hz, 4H), 3.06 (m, 4H), 2.84 (brs, 2H), 2.34 (m, 4H), 1.44 (brs, 4H), 1.25 (brs, 16H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.6 (formate), 159.7, 158.4, 82.6, 72.7 41.3, 40.3, 29.5 (3C), 29.2, 26.8, 19.4 ppm. HRMS (ESI+) m/z: calculated for C.sub.24H.sub.46N.sub.10 [M+2H].sup.2+ 237.1948, found 237.1947.
[0572] LCC-12-dimethyl,1,1 di-formic acid salt. Second purification on preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) afford a white solid. (41%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.53 (s, 2H, formate), 7.31-6.87 (brs, 8H, ex), 3.28 (t, J=7.4 Hz, 4H), 2.92 (s, 12H), 2.90 (s, 6H), 1.48 (m, 4H), 1.33-1.12 (brs, 16H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 165.8 (formate), 158.8, 158.1, 49.8, 37.8 (2C), 35.8, 29.5 (2C), 29.3, 27.4, 26.5 ppm. HRMS (ESI+) m/z: calculated for C.sub.22H.sub.50N.sub.10 [M+2H].sup.2+ 227.2104, found 227.2103.
[0573] LCC-12-dimethyl,2 di-formic acid salt. Second purification on preparative HPLC equipped with C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) afford a white solid. (36%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 9.30-8.67 (brs, 2H, ex), 8.51 (s, 2H, formate), 7.62-6.81 (m, 8H, ex), 3.30 (t, J=7.2 Hz, 4H), 3.08 (m, 4H), 2.91 (s, 6H), 1.49 (m, 4H), 1.33-1.15 (brs, 16H), 1.04 (t, J=7.0 Hz, 6H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.0 (formate), 158.7, 158.4, 49.9, 35.9, 35.8, 29.5 (2C), 29.3, 27.4, 26.5, 15.2 ppm. HRMS (ESI+) m/z: calculated for C.sub.22H.sub.50N.sub.10 [M+2H].sup.2+ 227.2104, found 227.2106.
[0574] Coronaformin-8,8 di-formic, di-ammonium salt. 1,8-Bis(cyanoguanidino)octane (100 mg, 0.36 mmol) and 1,8-diaminooctane dihydrochloride (51.8 mg, 0.36 mmol) were dissolved in DMSO (200 L) followed by addition of aq. HCl (37%, 0.3 mL) and heated at 160 C. overnight. After cooling the resulting brown mixture to rt, the solvent was evaporated under high vacuum. The product was purified by preparative HPLC equipped with a C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) to afford a brown solid. (13%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.43 (s, 2H, formate), 8.40-8.13 (brs, 4H, ex), 7.90-7.03 (brs, 16H, ex), 3.06 (brs, 8H), 1.46 (brs, 8H), 1.28 (brs, 16H). .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 168.0 (formate), 157.6, 41.0, 29.0, 28.9, 26.4 ppm. HRMS (ESI+) m/z: calculated for C.sub.20H.sub.52N.sub.12 [M+2NH.sub.4+2H].sup.4+ 115.1104, found 115.1108.
[0575] Coronaformin-10,10 di-formic, di-ammonium salt. 1,10-Bis(cyanoguanidino)decane (100 mg, 0.33 mmol) and 1,10-diaminodecane dihydrochloride (113 mg, 0.66 mmol) were dissolved in DMSO (200 L) followed by addition of aq. HCl (37%, 2.7 mL) and heated at 160 C. overnight. After cooling the resulting brown mixture to rt, the solvent was evaporated under high vacuum. The product was purified by preparative HPLC equipped with a C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) to afford a brown solid. (24%).sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.42 (s, 2H, formate), 8.34 (brs, 4H, ex), 7.75-7.27 (brs, 16H, ex), 3.05 (m, 8H), 1.45 (brs, 8H), 1.26 (brs, 24H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.9 (formate), 157.8, 41.1, 29.3, 29.0, 28.9, 26.5 ppm. HRMS (ESI+) m/z: calculated for C.sub.24H.sub.60N.sub.12 [M+2NH.sub.4+2H].sup.4+ 129.1260, found 129.1262.
[0576] Coronaformin-12,12 di-formic, di-ammonium salt. 1,12-Bis(cyanoguanidino)dodecane (100 mg, 0.30 mmol) and 1,12-diaminododecane (60 mg, 0.30 mmol) were dissolved in DMSO (200 L) followed by addition of aq. HCl (37%, 2.5 mL) and heated at 160 C. overnight. After cooling the resulting brown mixture to rt, the solvent was evaporated under high vacuum. The product was purified by preparative HPLC equipped with a C18-reverse phase column (H.sub.2O/CH.sub.3CN/formic acid 95:5:0.1 to 0:100:0.1) to afford a light brown solid. (24%).sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 8.41 (s, 2H, formate), 8.21 (brs, 4H, ex), 7.82-7.15 (brs, 16H, ex), 3.05 (q, J=6.3 Hz, 8H), 1.45 (m, 8H), 1.26 (brs, 32H) ppm. .sup.13C-NMR (100.6 MHz, DMSO-d.sub.6) : 167.7 (formate), 157.6, 41.1, 29.4 (2C), 29.1, 28.9, 26.5 ppm. HRMS (ESI+) m/z: calculated for C.sub.28H.sub.68N.sub.12 [M+2NH.sub.4+2H].sup.4+ 143.1417, found 143.1417.
Example 2: Inflammation
[0577] Here, the inventors investigate the molecular mechanisms underlying the regulation of metabolic and epigenetic plasticity of macrophages. They further evaluate the potential of reprogramming cell identity using a novel small molecule towards a less inflammatory signature.
[0578] To study the role of CD44/HA-mediated metal uptake in inflammatory macrophages, the inventors isolated monocytes from human donors and differentiated them using granulocyte-macrophage colony-stimulating factor (GM-CSF) to produce monocyte-derived macrophages (MDM). They then activated MDM (act. MDM) using lipopolysaccharide (LPS) and interferon gamma (IFN) to generate inflammatory macrophages (
[0579] Next, the inventors performed RNA-seq to compare the gene expression signatures of activated MDM with that of macrophages obtained from BALF of severe COVID-19 (sCOVID) patients. These datasets revealed striking similarities between activated MDM and sCOVID macrophages compared to samples from moderate COVID-19 patients and controls (
[0580] The clinically-approved biguanide metformin (Met) has been shown to reduce the production of KG in cancer cells, although with a moderate potency. Interestingly, Met can form bimolecular organometallic complexes with copper. To alleviate the entropic cost inherent to the formation of a bimolecular complex, the inventors synthesized a dimeric small molecule, termed lipophilic copper clamp C.sub.12 (LCC-12), where two biguanides were chemically tethered with a methylene-containing linker (
[0581] Next, the inventors evaluated the transcriptional effect and therapeutic potential of biguanides. LCC-12 treatment led to a different gene expression signature compared to that observed in activated MDM (
[0582] Finally, the inventors evaluated the effect of LCC-12 at 0.3 mg/kg/day, a dose 10 times lower than the maximum tolerated dose (MTD), in another model of sepsis, namely cecal ligation and puncture (CLP). This model is representative of the pathophysiology of subacute polymicrobial abdominal sepsis occurring in humans. LCC-12 delayed death and increased survival rate (
[0583] In conclusion, two of the d-block metals are required for the generation of inflammatory macrophages and blocking the production of KG and acetyl-CoA through mitochondrial copper targeting correlates with therapeutic benefits. This is further supported by the observed improved condition of COVID-19 patients undergoing treatment with Met. Notably, glucose metabolism, which can lead to KG production (
[0584] Materials and Methods
[0585] Antibodies. (WB=Western blot, FC=Flow cytometry, FM=Fluorescence microscopy). CD44 (Abcam, ab189524, WB), CD44-Alexa-Fluor-647 (Novus Biologicals, NB500-481AF647, FC), CD80-AlexaFluor700 (Becton, Dickinson and Company (BD), 561133, FC), CD86-PE/Cy7 (BD, 561128, FC), Cytochrome c (cyt c, Cell Signaling, 12963S, FM), Ferritin (Abcam, ab75973, WB), H3 (Cell Signaling, 9715S, FM), H3K4me3 (Diagenode, C15410003-50, FM), H3K9me2 (Cell Signaling, 4658S, FM), H3K9me3 (Cell Signaling, 13969S, FM), H3K27me3 (Cell Signaling, 9733S, FM), H3K36me2 (Abcam, ab9049, FM), TfR1-APC/Alexa750 (Beckman Coulter, A89313, FC), Transferrin receptor 1 (TfR1, Life Technologies, 13-6800, WB), -Tubulin (Sigma-Aldrich, T5326, WB).
[0586] Cell culture. Peripheral blood samples were collected from distinct healthy donors (Etablissement Franais du Sang). Pan monocytes were isolated by negative magnetic sorting using microbeads according to the manufacturer's instructions (Miltenyi Biotec, 130-096-537), and cultured in RPMI 1640 supplemented with glutamine (Thermo Fisher Scientific, 61870010), 10% fetal bovine serum and exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF, Miltenyi Biotec, 130-093-866, 100 ng/mL) to induce differentiation into macrophages (MDM). At day 5 of differentiation, MDM were treated with lipopolysaccharides (LPS, InvivoGen, tlrl-3pelps, 100 ng/mL, 24 h) and interferon- (IFN, Miltenyi Biotec, 130-096-484, 20 ng/mL, 24 h) to generate activated MDM (act. MDM) and were co-treated with metformin (Met, 1,1-dimethylbiguanid hydrochloride, Alfa Aesar, J63361, 10 mM, 24 h) or LCC-12 (in-house, 10 M, 24 h) as indicated.
[0587] Flow cytometry. Cells were washed with ice-cold 1PBS, incubated with Fc block (Human TruStain FcX, Biolegend, 422302, 1/20) for 15 min, incubated with antibodies for 20 min at 4 C. and washed before analysis using a BD LSRFortessa X-20. Macrophages were analyzed with an antibody panel consisting of antibodies against the following cell surface proteins: CD44, CD80, CD86 and TfR1. The data were analyzed with FlowJo software v. 10.0.00003. Flow cytometry analysis of lysosomal iron content: Lysosomal iron was monitored by incubating cells at 37 C. with 5% CO.sub.2 in medium containing RhoNox-M (in-house, 1 M, 1 h), before flow analysis of fluorescence intensity.
[0588] Fluorescence microscopy. Isolated monocytes were plated on cover slips, differentiated and activated as described in cell culture. For fluorescent detection of HA, Fe.sup.2+ and Cu.sup.2+, live cells were treated with HA-FITC (800 kDa, Carbosynth, YH45321, 0.1 mg/mL, 125 M) and RhoNox-M (Niwa et al., 2014, Org. Biomol. Chem. 12, 6590-6597) (in-house, 1 M) or Lys-Cu (Ren et al., 2015, J. Mater Chem. B 3, 6746-6752) (in-house, 20 M) for 1 h before fixation. Cells were washed three times with 1PBS, fixed with 2% paraformaldehyde in 1PBS for 12 min and then washed three times with 1PBS. After fixation, cells were permeabilized with 0.1% Triton X-100 in 1PBS for 5 min and washed three times with 1PBS. Subsequently, cells were blocked in 2% BSA, 0.2% Tween-20/1PBS (blocking buffer) for 20 min at room temperature. Cells were incubated with the relevant antibody in blocking buffer for 1 h at room temperature, washed three times with 1PBS and were incubated with secondary antibodies for 1 h. Finally, cover slips were washed three times with 1PBS and mounted using VECTASHIELD (Vector Laboratories) containing DAPI. Fluorescence images were acquired using a Deltavision real-time microscope (Applied Precision). 40/1.4NA, 60/1.4NA and 100/1.4NA objectives were used for acquisitions and all images were acquired as z-stacks. Images were deconvoluted with SoftWorx (Ratio conservative15 iterations, Applied Precision) and processed with ImageJ. Histone quantification was performed delineating the nuclei using DAPI fluorescence.
[0589] Bright-field and digital photographs. Bright field images were acquired using a CKX41 microscope (Olympus) and cellSens Entry imaging software (Olympus). Digital images were taken with an iPhone 11 Pro (Apple).
[0590] Click labeling. Activated MDM on coverslips were treated with metforminyn (in-house, 10 M, 3 h) fixed and permeabilized as indicated in fluorescence microscopy. The click reaction cocktail was prepared from a Click-iT EdU Imaging kit (C10337, Life Technologies) according to the manufacturer's protocol. Briefly, mixing 430 L of 1 Click-iT reaction buffer with 20 L of CuSO.sub.4 solution, 1.2 L Alexa Fluor azide, 50 L reaction buffer additive (sodium ascorbate) to reach a final volume of 500 L. Cover-slips were incubated with the click reaction cocktail in the dark at room temperature for 30 min, then washed three times with 1PBS. Immunofluorescence was then performed as described in fluorescence microscopy.
[0591] Western Blotting. Cells were treated as indicated and then washed with 1PBS. Proteins were solubilized in 2 Laemmli buffer containing benzonase (VWR, 70664-3, 1:100), extracts were incubated at 37 C. for 1 h, and quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Protein lysates were resolved by SDS-PAGE electrophoresis (Invitrogen sure-lock system and Nu-PAGE 4-12% Bis-Tris precast gels) and transferred onto nitrocellulose (Amersham Protran 0.45 m) membranes using a Trans-Blot SD semi-dry electrophoretic transfer cell (Bio-rad). Membranes were blocked with 5% non-fat skimmed milk powder in 0.1% Tween-20/1PBS for 1 h. Blots were then probed with the relevant primary antibodies in 5% BSA, 0.1% Tween-20/1PBS at 4 C. overnight with gentle motion. Membranes were washed with 0.1% Tween-20/1PBS three times and incubated with horseradish peroxidase conjugated secondary antibodies (Jackson Laboratories) in 5% non-fat skimmed milk powder, 0.1% Tween-20/1PBS for 1 h at room temperature and washed three times with 0.1% Tween-20/1PBS. Antigens were detected using the SuperSignal West Pico PLUS chemiluminescent detection kits (ThermoFisher Scientific, 34580 and 34096). Signals were recorded using a Fusion Solo S Imaging System (Vilber) and quantified as indicated using ImageJ.
[0592] Inductively coupled plasma mass spectrometry (ICP-MS). HA (Carbosynth, FH45321, 600-1000 kDa, 1 mg/mL) was added together with LPS and IFN and cells were treated for 24 h. Glass vials equipped with Teflon septa were cleaned with nitric acid 65% (VWR, Suprapur, 1.00441.0250), washed with ultrapure water (Sigma-Aldrich, 1012620500) and dried. Cells were harvested followed by two washes with 1PBS. Cells were then counted using an automated cell counter (Entek) and transferred in 200 L 1PBS to the cleaned glass vials, and samples were lyophilized using a freeze dryer (CHRIST, 22080). Samples were subsequently mixed with nitric acid 65% overnight and heated at 80 C. for 2 h. Samples were diluted with ultrapure water to a final concentration of 0.475 N nitric acid and transferred to metal-free centrifuge vials (VWR, 89049-172) for subsequent ICP-MS analysis. Amounts of .sup.56Fe and .sup.63Cu were measured using an Agilent 7900 ICP-QMS in low-resolution mode. Sample introduction was achieved with a micro-nebulizer (MicroMist, 0.2 mL/min) through a Scott spray chamber. Isotopes were measured using a collision-reaction interface with helium gas (5 mL/min) to remove polyatomic interferences. Scandium and indium internal standards were injected after inline mixing with the samples to control the absence of signal drift and matrix effects. A mix of certified standards was measured at concentrations spanning those of the samples to convert count measurements to concentrations in the solution. Uncertainties on sample concentrations were calculated using algebraic propagation of ICP-MS blank and sample counts uncertainties. Values were normalized against cell number.
[0593] siRNA Transfection and CD44 blocking antibody. Human primary monocytes were transfected with Human Monocyte Nucleofector kit (Lonza, VPA-1007) according to the manufacturer's instructions. Briefly, 510.sup.6 monocytes were resuspended into 100 L of nucleofector solution with 200 pmol of ON-TARGETplus CD44 SMARTpool siRNA (Horizon Discovery, L-009999-00-0050) or negative control siRNA (Qiagen, 1027310) before nucleofection with Nucleofector II (Lonza). Cells were then immediately removed and incubated overnight with 5 mL of prewarmed complete RPMI medium (Thermo Fisher Scientific). The following day, CSF-1 was added to the medium. Cells were then treated with anti-human CD44 therapeutic antibody (RG7356, Creative Biolabs, TAB-128CL, 10 g/mL, 24 h) as indicated.
[0594] NMR spectroscopy of HA:copper(II) complex. .sup.1H-NMR spectra were recorded on a 500 MHz Bruker spectrometer at 310 K, and chemical shifts are expressed in ppm using the residual non-deuterated solvent signal as internal standard. Portions of 0.25 mol equiv. of a solution of CuCl.sub.2 in D.sub.2O (8.6 mg in 599 L D.sub.2O) were added to a 2 mM solution of low-molecular-mass HA(LMM Hyal, TCI Chemicals, H1284) in D.sub.2O (1 mg HA in 600 L D.sub.2O) up to 1 mol equiv. into an NMR tube. Then, a drop of trifluoroacetic acid (TFA, Alfa Aesar, A12198) was added. In a separate NMR tube, a drop of TFA was added to a 2 mM solution of LMM-HA in D.sub.2O.
[0595] RNA-seq. RNAs were extracted using the RNeasy mini kit (QIAGEN, 74104). RNA sequencing libraries were prepared from 1 g total RNA using the Illumina TruSeq Stranded mRNA library preparation kit (Illumina, 20020594), which allows strand-specific sequencing. A first step of polyA selection using magnetic beads was performed to allow sequencing of polyadenylated transcripts. After fragmentation, cDNA synthesis was performed and resulting fragments were used for dA-tailing followed by ligation of TruSeq indexed adapters (Illumina, 20020492). Subsequently, polymerase chain reaction amplification was performed to generate the final barcoded cDNA libraries. Sequencing was carried out on a NovaSeq 6000 instrument from Illumina based on a 2100 cycle mode (paired-end reads, 100 bases). Raw sequencing reads were first checked for quality with Fastqc (0.11.8) and trimmed for adapter sequences with the trimGalore (0.6.2) software. Trimmed reads were then aligned on the human hg38 reference genome using the STAR mapper (2.6.1b), up to the generation of a raw count table per gene (GENCODE annotation v29). The bioinformatics pipelines used for these tasks are available online (rawqc v2.1.0: https://github.com/bioinfo-pf-curie/raw-qc, RNA-seq v3.1.4: https://github.com/bioinfo-pf-curie/RNA-seq). The downstream analysis was then restricted to protein-coding genes. Data from (Liao et al., 2020, Nat. Med. 26, 842-844) were converted into bulk by keeping cells annotated as macrophages and then summing the counts for each sample. Counts data from (Pai et al., 2016, PLoS Genet. 12, e1006338) were downloaded from GEO under accession number GSE73502. Raw data from (Fernandes et al., 2016, 7, e00027-16; Gongalves et al., 2020, Nat. Commun. 11, 2282) were downloaded from the NCBI Short Read Archive under records PRJNA528433 and PRJNA290995 and processed as described above. Counts were normalized using TMM normalization from edgeR (v 3.30.3) (Robinson et al., 2010, Bioinformatics 26, 139-140). Differential expression was assessed with the limma voom framework (v 3.44.3) (Ritchie et al., 2015, Nucleic Acids Res. 43, e47). The intra-donor correlation was controlled by using the duplicateCorrelation from limma. Genes with an adjusted p-value<0.05 were called significant. Enrichment analysis from differentially expressed genes has been performed using the enrichGO function from clusterProfilter package v3.16.1.
[0596] -Ketoglutarate (KG) measurements. KG was quantified using a fluorometric assay (Abcam, ab83431) according to the manufacturer's protocol. At least 210.sup.6 cells were treated as indicated and harvested per condition. Floating cells were harvested and adherent cells were washed with 1PBS. Adherent cells were incubated with 1PBS with 10 mM EDTA and then scraped and pooled together with the harvested floating cells. Cells were subsequently washed with ice-cold 1PBS and counted. Then, cells were re-suspended in ice-cold KG buffer (kit component). Cells were centrifuged at 25000 g for 5 min at 4 C. and the supernatant was transferred to clean tubes. Ice-cold perchloric acid (Sigma-Aldrich, 311421-50ML) was added to a final concentration of 1 M and the solution was incubated on ice for 5 min. Cells were centrifuged at 13000 g for 2 min and the supernatant was transferred to a clean tube. Then, vol. of a 2 M solution of KOH was added and the pH adjusted to 7.4 using a 0.1 M aq. solution of KOH. Samples were centrifuged at 13000 g for 15 min and the supernatants collected. KG levels were measured using a standard curve and a control to subtract pyruvate background levels. Fluorescence intensities (ex. 535 nm; em. 590 nm) were recorded using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader, and data were normalized against cell number. Values were derived from the standard curve for each experiment.
[0597] Synthesis. Products were purified on a preparative HPLC Quaternary Gradient 2545 equipped with a Photodiode Array detector (Waters) fitted with a reverse phase column (XBridge Prep C18 5 m OBD 30150 mm). Spectra were run in DMSO-d.sub.6 or Methanol-d.sub.6 at 298 K unless stated otherwise. .sup.1H-NMR spectra were recorded on Bruker spectrometers at 400 or 500 MHz. Chemical shifts are expressed in ppm using the residual non-deuterated solvent signal as internal standard. The following abbreviations are used: ex, exchangeable; s, singlet; d, doublet; t, triplet; brs, broad signal; m, multiplet. .sup.13C-NMR spectrum was recorded at 125.8 MHz, and chemical shifts are expressed in ppm using deuterated solvent signal as internal standard. The purity of final compounds, determined to be >98% by UPLC-MS, and low-resolution mass spectra (LRMS) were recorded on a Waters Acquity H-class equipped with a Photodiode array detector and SQ Detector 2 (UPLC-MS) fitted with a reverse phase column (Aquity UPLC BEH C18 1.7 m, 2.150 mm). High-resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific Q-Exactive Plus equipped with a Robotic TriVersa NanoMate Advion.
[0598] Lipophilic copper clamp (LCC-12): Dicyandiamide (A10451, Alfa Aesar, 500 mg, 5.94 mmol), 1,12-diaminododecane (A04258, Alfa Aesar, 500 mg, 2.50 mmol) and CuCl.sub.2 (22.201-1, Aldrich 249 mg, 1.85 mmol) were suspended in 6 mL water in a sealed tube and stirred for 1 h, then heated at 80 C. for 48 h. The resulting pink mixture was filtrated, and the solid was re-suspended in water (10 mL). H.sub.2S, generated from dropwise addition of 37% aq. HCl (1.00317.100, Supelco) on FeS (100 mesh powder, 17422, Alfa Aesar), was passed into the mixture until it turned black. The black mixture was filtrated, and the filtrate was acidified to pH=5 with a 1M aq. solution of HCl. The solvent was evaporated under reduced pressure. LCC-12 was purified by preparative HPLC (H.sub.2O/CH.sub.3CN/formic acid, 95:5:0.1 to 0:100:0.1) to give the LCC-12 di-formic acid salt as a white powder (280 mg, 24%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.80-8.08 (brs, 2H, ex), 8.47 (s, 2H, formate), 7.60-6.78 (brs, 12H, ex), 3.05 (brs, 4H), 1.43 (brs, 4H), 1.32-1.18 (m, 16H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.2 (formate), 160.4, 159.3, 41.3, 29.5 (3C), 29.3, 26.8 ppm. HRMS (ESI+) m/z: calculated for C.sub.16H.sub.38N.sub.10 [M+2H].sup.2+ 185.1635, found 185.1635. Metforminyn was synthesized as previously reported (S. Mller, A. Versini, F. Sindikubwabo, G. Belthier, S. Niyomchon, J. Pannequin, L. Grimaud, T. Caeque, R. Rodriguez, Metformin reveals a mitochondrial copper addiction of mesenchymal cancer cells. PLoS One 13, e0206764 (2018)). .sup.1H NMR (400 MHz, Methanol-d.sub.6) : 3.60 (t, J=7.0 Hz, 2H), 3.09 (s, 3H), 2.50 (td, J=7.0, 3.0 Hz, 2H), 2.36 (t, J=3.0 Hz, 1H) ppm.
[0599] High-resolution Mass spectrometry of biguanide:copper(II) complexes (HRMS). HRMS solution were prepared and injected without further dilution. Mixtures (1 mL) were prepared in methanol (980 L) with LCC-12-2(HCOOH)/K.sub.2CO.sub.3 aq. soln. (1:1) (10 L, at 10 mM, 1 mM, or 100 M) and CuCl.sub.2 aq. soln. (10 L, at 10 mM, 1 mM, or 100 M) keeping a 1:1 ratio LCC-12/Cu.sup.2+; or in methanol (800 L) with metformin-HCl/K.sub.2CO.sub.3 aq. soln. (2:1) (100 L at 200 mM) and CuCl.sub.2 aq. soln. (100 L at 100 mM); or in methanol (980 L) with metformin-HCl/K.sub.2CO.sub.3 aq. soln. (2:1) (10 L, at 200 mM or 20 mM) and CuCl.sub.2 aq. soln. (10 L, at 100 mM or 10 mM) keeping a 2:1 ratio metformin/Cu.sup.2+.
[0600] Copper-catalyzed oxidation of NADH. The oxidation kinetics of NADH (N4505, Sigma-Aldrich) was followed by measurement of absorbance at 340 nm using a NanoDrop 2000. Measurements were performed at 26 C. A 10 mM sodium phosphate buffer adjusted to pH=7.2 was used as solvent. Each 500 L mixture were prepared with NADH (400 M), imidazole (56750, Sigma-Aldrich, 10 mM), CuSO.sub.4 (451657, Sigma-Aldrich, 4 M), LCC/K.sub.2CO.sub.3 solution (1:1) (4 M or 400 M), metformin/K.sub.2CO.sub.3 solution (2:1) (J63361, Alfa Aesar, 800 M), as indicated, and a H.sub.2O.sub.2 solution (16911, Sigma-Aldrich, 2 mM from an aq. solution 25-35% in H.sub.2O.sub.2) added at the reaction start time. The concentration of NADH was calculated from the measured absorbance at 340 nm and a molar extinction coefficient of 5.510.sup.3 L mol.sup.1 cm.sup.1.
[0601] NADH/NAD.sup.+ measurements in macrophages. NAD.sup.+ and NADH levels were measured using an NAD.sup.+/NADH fluorometric assay (Abcam, ab176723) according to the manufacturer's protocol. In brief, at least 500.000 cells were harvested per condition. Floating cells were harvested and adherent cells were washed with 1PBS. Adherent cells were incubated with 1PBS with 10 mM EDTA and then scraped and pooled together with the harvested floating cells. Cells were subsequently washed with ice-cold 1PBS and counted. Cells were centrifuged at 1500 rpm for 5 min and the supernatant discarded. The pellet was then resuspended in 100 L lysis buffer (kit component) and incubated at 37 C. for 15 min. Standards were prepared according to the manufacturer's protocol. NAD.sup.+ and NADH extraction solutions as well as NAD.sup.+/NADH control solutions (kit components) were added and incubated at 37 C. for 15 min at a volume of 15 L sample to 15 L of the respective buffers. The reactions were stopped using 15 L of respective buffer. Finally, 75 L of NAD.sup.+/NADH reaction mixture (NAD.sup.+/NADH recycling enzyme mixture and sensor buffer, kit components) were added and the resulting mixtures incubated for 1 h at room temperature. Fluorescence intensities (ex. 540 nm; em. 590 nm) were recorded using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader, and data were normalized against cell numbers. Values were derived from the standard curve of each experiment.
[0602] Cytokine measurements. [IL6] and [IL10] were measured in cell culture supernatants using V-Plex validated immunoassay (MSD, Rockville, MD, US). The kit was run according to the manufacturer's protocol and the chemiluminescence signal was measured on a Sector Imager 2400 (MSD).
[0603] Murine model of LPS-induced severe inflammation. Animal work was conducted at Fidelta Ltd according to 2010/63/EU and National legislation regulating the use of laboratory animals in scientific research and for other purposes (Official Gazette 55/13). An Institutional Committee on Animal Research Ethics (CARE-Zg) oversaw that animal-related procedures were not compromising the animal welfare. LPS (Sigma-Aldrich, L2630, 20 mg/kg) was injected intraperitoneally to male BALB/c mice (8 weeks-old). LCC-12 (0.3 mg/kg, IP, n=10) or vehicle (0.9% NaCl, 10 mL/kg, IP, n=10) were injected 2 h prior LPS challenge, then 24 h, 48 h, 72 h and 96 h post challenge. Dexamethasone (10 mg/kg, PO, n=10) was given 1 h prior LPS challenge. Incidence of mortality was monitored every 4 h up to 48 h, then twice daily.
[0604] Murine model of sepsis using cecal ligation and puncture. All animal-related research is conducted in accordance with 2010/63/EU and National legislation regulating the use of laboratory animals in scientific research and for other purposes (Official Gazette 55/13). An Institutional Committee on Animal Research Ethics (CEEA-047) oversees that animal-related procedures are not compromising the animal welfare. 9 weeks-old male BALB/c mice were used for these experiments. Animals were anesthetized by isoflurane (Forene). After abdominal incision, the cecum was ligated, punctured with a gauge needle (25G or 21G), and a small amount of fecal matter was released. After the cecum was returned to the abdomen, the abdominal cavity was closed in two layers and the mice were resuscitated with 30 mL/kg body weight of saline (0.9% NaCl) administered subcutaneously. For the sham group, after abdominal incision, the cecum was manipulated but was neither ligated nor punctured. After the cecum was returned to the abdomen, the abdominal cavity was closed in two layers and the mice were resuscitated with 30 mL/kg body weight of saline administered subcutaneously. LCC-12 (0.3 mg/kg, IP) was administered at 0.3 mg/kg dose 4 h, 24 h, 48 h, 72 h and 96 h following CLP creation. Mortality incidence was monitored every 2 h up to 120 h (except from 10 pm to 6 am) post CLP creation. Dexamethasone was administered intraperitoneally at 1 mg/kg dose 5-minutes prior CLP creation.
[0605] Statistical analyses. All results are presented as mean valuesstandard error of the mean (SEM) unless stated otherwise. PRISM 8 software was used to calculate p-values using a Mann-Whitney test or Kruskal-Wallis test with Dunn's post-test for multiple comparisons as indicated. PRISM 8 software or R programming language was used to generate graphical representations of quantifications unless stated otherwise. Sample sizes (n) are indicated in the figure legends.
[0606] Data and Code Availability
[0607] RNA-seq data are available on the National Center for Biotechnology Information website with accession reference GSE160864 (go to: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE160864; enter token wvqxgwgojxcdboz into the box). Analysis scripts are available at https://github.com/bioinfo-pf-curie/MDMmetals.
Example 3: Biguanides Affect the Activation of Immune Cells
[0608] LCC-12 decreases the inflammatory profile of macrophages as illustrated in Example 2. The inventors equally reported an impact of LCC-12 on other inflammatory cells (
[0609] Materials and Methods
[0610] CD4 Lymphocytes. Peripheral blood samples were collected from healthy donors (Etablissement Franais du Sang). CD4 lymphocytes were isolated by negative magnetic sorting using microbeads according to the manufacturer's instructions (Miltenyi Biotec, 130-096-533) and cultured in RPM11640 supplemented with glutamine and 10% fetal bovine serum. CD4 lymphocytes were activated for 48 h using CD3/CD28 antibodies (2.5 g/mL), in presence of LCC-12 (10 M). The activation status of the lymphocytes was assessed by measuring CD25 and CD69 surface markers by flow cytometry.
[0611] CD8 Lymphocytes. Peripheral blood samples were collected from healthy donors (Etablissement Franais du Sang). CD8 lymphocytes were isolated by negative magnetic sorting using microbeads according to the manufacturer's instructions (Miltenyi Biotec, 130-096-495) and cultured in RPM11640 supplemented with glutamine and 10% fetal bovine serum. CD8 lymphocytes were activated 48 h using CD3/CD28 antibodies (2.5 g/mL), in presence of LCC-12 (10 M). The activation status of the lymphocytes was assessed by measuring CD25 and CD69 surface markers by flow cytometry.
[0612] Neutrophils. Peripheral blood samples were collected from healthy donors (Etablissement Franais du Sang). Then, red cells in whole blood samples were lysed (ebioscience 10RBC lysis buffer, 00-4300-54). The remaining cells were cultured in RPMI 1640 supplemented with glutamine, and 2% human serum, and activated 1 h with LPS (2 g/mL) in presence of LCC-12 (10 M). The granulocytes/neutrophils population was determined by flow cytometry using FSC, SSC and CD15 surface marker. The activation status of the granulocytes was assessed by measuring CD64 and CD66b surface markers by flow cytometry.
[0613] Monocytes. Peripheral blood samples were collected from healthy donors (Etablissement Franais du Sang). Pan monocytes were isolated by negative magnetic sorting using microbeads according to the manufacturer's instructions (Miltenyi Biotec, 130-096-537), and cultured in RPMI 1640 supplemented with glutamine, 10% fetal bovine serum. Monocytes were treated with lipopolysaccharides (LPS, 100 ng/mL, 24 h) to generate activated monocytes and were co-treated with LCC-12 (in-house, 10 M, 24 h). The activation status of the monocytes was assessed by measuring CD25 and CD80 surface markers by flow cytometry.
[0614] Dendritic cells. Peripheral blood samples were collected from healthy donors (Etablissement Franais du Sang). Pan monocytes were isolated by negative magnetic sorting using microbeads according to the manufacturer's instructions (Miltenyi Biotec, 130-096-537), and cultured in RPMI 1640 supplemented with glutamine, 10% fetal bovine serum and treated with granulocyte-macrophage colony-stimulating factor (GM-CSF, 100 ng/mL) and IL-4 (10 ng/mL) to induce differentiation into dendritic cells (DC). At day 5 of differentiation, DC were treated with lipopolysaccharides (LPS, 100 ng/mL, 24 h) to generate activated DC and were co-treated with LCC-12 (in-house, 10 M, 24 h). The activation status of the dendritic cells was assessed by measuring CD40, CD83, CD80 and CD86 surface markers by flow cytometry.
Example 4: Effect of a Series of LCCs on Macrophage Activation and on the Lymphoma Cell Line U937
[0615] The inventors tested a series of LCC molecules on macrophage activation using 10 M or 1 M of compound (as indicated in the table). They also assessed the half maximal inhibitory concentration (IC.sub.50) of cell viability on the lymphoma cell line U-937. Depending on linker length and overall length, there was a varying degree of potency on macrophage activation as well as a range of IC.sub.50 values in the nanomolar to micromolar range. This data highlights the potential of lead structure optimization of this series of biguanides.
TABLE-US-00001 Effect on CD86 macrophage activation marker (Flow cytometry)(All LCCs at IC.sub.50 10 M or 1 M if indicated and Met at (M) 10 mM)(% of activation marker in compared to its level in activated U937 Compound macrophages) cells Metformin 51 10000 59 62 70 56 57 53 53 LCC-7 300 LCC-8 77 200 LCC-9 60 LCC-10 20 LCC-12 25 1 M 2 29 83 26 51 25 18 24 22 20 26 27 LCC-12-15N LCC-8Me 89 150 LCC-12Me 24 6 15 17 LCC-8,3 59 2 39 LCC-10,2 32 1 M 1 29 54 58 LCC-10,3 22 1 M 0.6 38 43 LCC-10,5 0.3 LCC-12,2 0.4 LCC-12,3 0.3 LCC-12,4 1 M 0.3 LCC-12,6 0.4 LCC-12Me, 1 M 1,1 (A) 35 34 LCC-12Me, 1 uM 2 (B) 34 LCC-12-click 0.4 Coronaformin- 3 8,8 Coronaformin- 50 1 10,10 45 Coronaformin- 1 M 0.5 12,12 62 55
[0616] Materials and Methods
[0617] Cell culture. U-937 cells were grown in an incubator equilibrated at 37 C. with 5% CO.sub.2, grown to confluence and split with Trypsin/EDTA (Gibco, TRYPGIB01) once or twice a week according to confluence. U-937 (ATCC, HTB-132, sex: female) were cultured in RPMI 1640 GLUTAMAX (ThermoFisher Scientific, 61870044) supplemented with 10% Fetal Bovine Serum (FBS, Gibco, 10270-106) and Penicillin-Streptomycin mixture (BioWhittaker/Lonza, DE17-602E). U-937 cells and were co-treated with metformin (Met, 1,1-dimethylbiguanid hydrochloride, Alfa Aesar, J63361, 10 mM, 24 h) or different LCC compounds (in-house, 10 M or 1 M, 24 h) as indicated. Peripheral blood samples were collected from distinct healthy donors (Etablissement Franais du Sang). Pan monocytes were isolated by negative magnetic sorting using microbeads according to the manufacturer's instructions (Miltenyi Biotec, 130-096-537), and cultured in RPMI 1640 supplemented with glutamine (Thermo Fisher Scientific, 61870010), 10% fetal bovine serum and exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF, Miltenyi Biotec, 130-093-866, 100 ng/mL) to induce differentiation into macrophages (MDM). At day 5 of differentiation, MDM were treated with lipopolysaccharides (LPS, InvivoGen, tlrl-3pelps, 100 ng/mL, 24 h) and interferon- (IFN, Miltenyi Biotec, 130-096-484, 20 ng/mL, 24 h) to generate activated MDM (act. MDM) and were co-treated with metformin (Met, 1,1-dimethylbiguanid hydrochloride, Alfa Aesar, J63361, 10 mM, 24 h) or different LCC compounds (in-house, 10 M or 1 M, 24 h) as indicated.
[0618] Flow cytometry. Cells were washed with ice-cold 1PBS, incubated with Fc block (Human TruStain FcX, Biolegend, 422302, 1/20) for 15 min, incubated with antibodies for 20 min at 4 C. and washed before analysis using a BD LSRFortessa X-20. Macrophages were analyzed with an antibody panel consisting of antibodies against the following cell surface proteins: CD44, CD80, CD86 and TfR1. The data were analyzed with FlowJo software v. 10.0.00003. Flow cytometry analysis of lysosomal iron content: Lysosomal iron was monitored by incubating cells at 37 C. with 5% CO.sub.2 in medium containing RhoNox-M (in-house, 1 M, 1 h), before flow analysis of fluorescence intensity.
[0619] Cell viability assay (IC.sub.50). Cell viability assay was carried out by plating 1000 cells/well in 96-well plates. Cells were treated for 72 h in a range between to 25 nM and 100 mM using serial dilutions. The inventors followed the manufacturer's protocol. In brief, CellTiter-Blue reagent (G8081, Promega) was added after 72 h treatment and cells were incubated for 3 h before recording fluorescence intensities (.sub.ex. 560/20 nm; .sub.em. 590/10 nm) using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.
Example 5: Effect on Cancer Stem Cells and Cancer Cell Plasticity
[0620] Cancer stem cells (CSC) represent a subpopulation in many cancers and resistance to therapy as well as metastatic dissemination and relapse can be attributed to these cells. Cancer cells can acquire a cell stem cell state without genetic mutations, but rather epigenetic alterations, i.e. cell plasticity. The phenomenon of cell plasticity in cancer has been extensively studied for the epithelial-to-mesenchymal transition (EMT), whereby the mesenchymal state has characteristics attributed to cancer stem cells. Since biguanides affect cell plasticity, the inventors investigated their effect on cancer cell plasticity. Using a well-established model of breast CSCs, namely HMLER CD44.sup.high/CD241.sup.low (Morel et. al, 2008, PlosONE, 3, e2888), the inventors found IC.sub.50-values much lower than metformin depending on the LCC tested (see table). LCC-8, LCC-10 and LCC-12 show an improved IC50, by respectively about 100 fold, about 400 fold and more than 2800 fold.
TABLE-US-00002 IC.sub.50 against HMLER Compound CD44.sup.high/CD24.sup.low Metformin 22.6 LCC-4 22.3 LCC-8 0.240 LCC-10 0.058 LCC-12 0.008 IC.sub.50 are expressed in mM.
[0621] Furthermore, the inventors used the human breast cancer cell line MCF7, the mouse pancreatic cancer cell line FC1242 and the prostate cancer cell line DU-145, where the mesenchymal state of EMT can be induced using TGF- or OSM depending on the cells. LCC-12 showed lower IC.sub.50-values in cells in the mesenchymal state compared to the epithelial counterpart (
[0622] Materials and Methods
[0623] Cell viability assay (IC.sub.50). Cell viability assay was carried out by plating 1000 cells/well in 96-well plates. Cells were treated for 72 h in a range between to 25 nM and 100 mM using serial dilutions. The inventors followed the manufacturer's protocol. In brief, CellTiter-Blue reagent (G8081, Promega) was added after 72 h treatment and cells were incubated for 3 h before recording fluorescence intensities (.sub.ex. 560/20 nm; .sub.em. 590/10 nm) using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.
[0624] Cell culture. MCF7 (ATCC) cells, DU-145 (ATCC) cells and FC1245 cells were cultured in Dulbecco's Modified Eagle Medium GlutaMAX (DMEM, ThermoFisher Scientific, 61965059) supplemented with 10% Fetal Bovine Serum (FBS, Gibco, 10270-106) and Penicillin-Streptomycin mixture (BioWhittaker/Lonza, DE17-602E) unless stated otherwise. Primary lung circulating tumor cells (Celprogen, 36107-34CTC, Lot 219411, sex: female) were grown using stem cell complete media (Celprogen, M36102-29PS) until the third passage. Circulating cancer cells were grown in stem cell ECM T75-flasks (Celprogen, E36102-29-T75) and ECM 6-well plates (Celprogen, E36102-29-6Well). HMLER cells (sex: female) naturally repressing E-cadherin, obtained from human mammary epithelial cells infected with a retrovirus carrying hTERT, SV40 and the oncogenic allele H-rasV12, and HMLER CD44 and TFRC ko clones were cultured in DMEM/F12 (Thermo Fisher Scientific, 31331093) supplemented with 10% FBS (Thermo Fisher Scientific, 10270106), 10 g/mL insulin (Sigma-Aldrich, 10516), 0.5 g/mL hydrocortisone (Sigma-Aldrich, H.sub.0888) and 0.5 g/mL puromycin (Life Technologies, A11138-02), unless stated otherwise. HMLER CD44.sup.high cells were also supplemented with 10 ng/mL EGF (Miltenyi Biotech, 130-097-750).
[0625] Flow cytometry. Cells were washed twice with ice-cold 1PBS and suspended in incubation buffer prior to being analysed by flow cytometry. For each condition, at least 10,000 cells were counted. Data were recorded on a BD Accuri C6 (BD Biosciences) and processed using Cell Quest (BD Biosciences) and FlowJo (FLOWJO, LLC).
[0626] Western Blotting. Cells were treated as indicated and then washed with 1PBS. Proteins were solubilized in 2 Laemmli buffer containing benzonase (VWR, 70664-3, 1:100), extracts were incubated at 37 C. for 1 h, and quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Protein lysates were resolved by SDS-PAGE electrophoresis (Invitrogen sure-lock system and Nu-PAGE 4-12% Bis-Tris precast gels) and transferred onto nitrocellulose (Amersham Protran 0.45 m) membranes using a Trans-Blot SD semi-dry electrophoretic transfer cell (Bio-rad). Membranes were blocked with 5% non-fat skimmed milk powder in 0.1% Tween-20/1PBS for 1 h. Blots were then probed with the relevant primary antibodies in 5% BSA, 0.1% Tween-20/1PBS at 4 C. overnight with gentle motion. Membranes were washed with 0.1% Tween-20/1PBS three times and incubated with horseradish peroxidase conjugated secondary antibodies (Jackson Laboratories) in 5% non-fat skimmed milk powder, 0.1% Tween-20/1PBS for 1 h at room temperature and washed three times with 0.1% Tween-20/1PBS. Antigens were detected using the SuperSignal West Pico PLUS chemiluminescent detection kits (ThermoFisher Scientific, 34580 and 34096). Signals were recorded using a Fusion Solo S Imaging System (Vilber) and quantified as indicated using ImageJ. Antibodies used were: SOD2 (Abcam, ab13534), E-cadherin (Cell Signaling, 3195), -Tubulin (Sigma-Aldrich, T5326), Fibronectin (Sigma-Aldrich, F1141-1MG), SLUG (Cell Signaling, 9585S).
Example 6: Biguanides Show Efficacy on Biopsy-Derived Organoids of Pancreatic Ductal Adenocarcinoma (PDAC)
[0627] The inventors tested LCC-12 on biopsy-derived organoids of PDAC, and found that the molecule had an efficacy in the low micromolar range on a variety of organoids (
[0628] Materials and Methods
[0629] Biopsy-derived pancreatic organoid (BDPO) generation. BDPOs were obtained from endoscopic ultrasound-guided fine-needle aspirations (EUS-FNA) from patients with PDAC included under the PaCaOmics clinical trial (ClinicalTrials.gov: NCT01692873) after approval by the Paoli-Calmettes hospital ethics committee and following patient informed consent. Cultures were established as previously described. Briefly, PDAC biopsies were slightly digested with the Tumor Dissociation Kit (Miltenyi Biotec) at 37 C. for 5 minutes. The pancreatic tissue slurry was transferred into a tissue strainer 100 m and was placed into 12-well plates coated with 150 L GFR matrigel (Corning, Boulogne-Billancourt, France). The samples cultured with Pancreatic Organoid Feeding Media (POFM) consisted of Advanced DMEM/F12 supplemented with 10 mM HEPES (Thermo Fisher Scientifics, Courtaboeuf, France); 1Glutamax (Thermo-Fisher Scientifics); penicillin/streptomycin (Thermo-Fisher Scientifics); 100 ng/mL Animal-Free Recombinant Human FGF10 (Peprotech, Peprotech, Neuilly-Sur-Seine, France); 50 ng/mL Animal-Free Recombinant Human EGF (Peprotech); 100 ng/mL Recombinant Human Noggin (Biotechne, Bio-Techne, Rennes, France); Wnt3a-conditioned medium (30% v/v); RSPO1-conditioned medium (10% v/v); 10 nM human Gastrin 1 (Sigma-Aldrich Lyon, France); 10 mM nicotinamide (Sigma Aldrich); 1.25 mM N-acetylcysteine (Sigma Aldrich); 1B27 (Invitrogen, (Invitrogen, Villebon sur Yvette, France); 500 nM A83-01 (Tocris, Noyal Chtillon sur Seiche, France); 10.5 M Y27632 (Tocris). The plates were incubated at 37 C. in a 5% CO.sub.2 incubator, and the media were changed every 3 or 4 days. For routine passages BDPOs were disaggregated with accutase (Thermo Fisher Scientific) and re-plated as needed.
[0630] Chemograms on BDPO. BDPOs were disaggregated with accutase (Thermo Fisher Scientific), and 1,000 cells/well were plated in two 96-well round bottom ultra-low plates (Corning) with the medium described above. 24 hours later, one plate was used directly for RNA preparation (Time 0 transcriptome) and on the other the medium was supplemented with increasing concentrations of each drug, 72 hours later cell viability was measured with CellTiter-Glo 3D (Promega) reagent quantified using the plate reader Tristar LB941 (Berthold Technologies). Values were normalized and expressed as the percentage of the control (vehicle), which represents 100% of normalized fluorescence. Increasing concentrations of drugs were used. Each experiment was repeated at least twice.
Example 7: Biguanides Target Mitochondria and Mitochondrial Metabolism
[0631] To gain further insights into the mechanism of action (MoA) of LCC-12, the inventors employed nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, which allows for qualitative assessment of specific isotope distributions of elements in a cell. To this end they employed the isotopologue .sup.15N-.sup.13C-LCC-12, which gave rise to a similar NanoSIMS imaging pattern as did .sup.197Au loaded onto an antibody against cytochrome c, suggesting that LCC-12 targets mitochondria (
[0632] Labeling small molecules in cells by means of click chemistry requires a copper(I) catalyst generated in situ by adding copper(II) and ascorbate (Asc) as a reducing agent. Given that the investigation converged towards mitochondrial copper(II) as a mechanistic target of LCC-12, the inventors investigated whether the natural abundance of mitochondrial copper(II) they found in aMDM could allow for click labeling without the need to experimentally add the metal catalyst. The inventors found that fluorescent labeling of the clickable analog of LCC-12 used at a concentration of 100 nM, which is 100-fold lower than the biologically active dose of LCC-12, occurred in the absence of exogenous copper, leading to a fluorescent signal in aMDM that colocalized with mitotracker. Importantly, such a staining was observed only when MDM were activated (
[0633] To delve further into the mechanism at work of biguanides and mitochondrial copper targeting, the inventors investigated the reactions underpinning copper-catalyzed interconversion of NAD(H). In line with the proposed mechanism, they found that mitochondrial superoxide dismutase 2 (SOD2) levels increase during macrophage activation, an enzyme that interconverts superoxide to hydrogen peroxidase (
[0634] Materials and Methods
[0635] Mitochondrial extraction. Mitochondria were isolated using the Qproteome Mitochondria Isolation Kit (Qiagen, 37612) according to the manufacturer's protocol. In brief, cells were washed and centrifuged at 500 g for 10 min and the supernatant was removed. Cells were then washed with a solution of 0.9% NaCl (Sigma-Aldrich, 57653-250G) and resuspended in ice-cold Lysis Buffer and incubated at 4 C. for 10 min. The lysate was then centrifuged at 1000g for 10 min at 4 C. and the supernatant carefully removed. Subsequently, the cell pellet was resuspended in disruption buffer. Complete cell disruption was obtained by using a dounce homogenizer (mitochondria for ICP-MS) or a blunt-ended needle and a syringe (mitochondria for metabolomics). The lysate was then centrifuged at 1000 g for 10 min at 4 C. and the supernatant transferred to a clean tube. The supernatant was then centrifuged at 6000 g for 10 min at 4 C. to obtain pellets containing mitochondria.
[0636] Mitochondrial H.sub.2O.sub.2 content. Mitochondrial H.sub.2O.sub.2 was monitored by incubating cells at 37 C. with 5% CO.sub.2 in medium containing Mito-PY1 (R&D Systems, #4428, 5 M, during the last 24 h), before flow analysis of fluorescence intensity.
[0637] Quantitative metabolomics. In a typical experiment 1.5 million cells were used for total extracts and 15 million cells for mitochondrial extracts. Cells were harvested and the supernatant removed to generate the corresponding cell pellets. Subsequently, pellets were dried and dry pellets were supplemented with 300 l methanol, vortexed 5 min and centrifuged (10 min at 15000 g, 4 C.). Then, the upper phase of the supernatant was split into two parts: 150 L were used for a gas chromatography coupled by mass spectrometry (GC/MS) experiment in microtubes and the remaining 150 L were used for Ultra High Pressure Liquid Chromatography coupled by Mass Spectrometry (UHPLC/MS). For the GC-MS aliquots, supernatants were completely evaporated from the sample. 50 L of methoxyamine (20 mg/mL in pyridine) were added to the dried extracts, then stored at room temperature in the dark for 16 h. The following day, 80 L of MSTF (A-Methyl-N-(trimethylsilyl) trifluoroacetamide) were added and final derivatization occurred at 40 C. for 30 min. Samples were then transferred into vials and directly injected for GC-MS analysis. For the UHPLC-MS aliquots, 150 L were dried in microtubes at 40 C. in a pneumatically-assisted concentrator (Techne DB3, Staffordshire, UK). The dried UHPLC-MS extracts were solubilized with 200 L of MilliQ water. Aliquots for analysis were transferred into LC vials and injected into UHPLC-MS or kept at 80 C. until injection. Widely-targeted analysis of intracellular metabolites gas chromatography (GC) coupled to a triple quadrupole (QQQ) mass spectrometer: GC-MS/MS method was performed on a 7890A gas chromatography (Agilent Technologies, Waldbronn, Germany) coupled to a triple quadrupole 7000C (Agilent Technologies, Waldbronn, Germany) equipped with a High sensitivity electronic impact source (EI) operating in positive mode (Viltard et al., 2019). Peak detection and integration of the analytes were performed using the Agilent Mass Hunter quantitative software (B.07.01). Targeted analysis of nucleotides and cofactors by ion pairing ultra-high performance liquid chromatography (UHPLC) coupled to a Triple Quadrupole (QQQ) mass spectrometer: Targeted analysis was performed on a RRLC 1290 system (Agilent Technologies, Waldbronn, Germany) coupled to a Triple Quadrupole 6470 (Agilent Technologies) equipped with an electrospray source operating in both negative and positive modes. Gas temperature was set to 350 C. with a gas flow of 12 L/min. Capillary voltage was set to 5 kV in positive mode and 4.5 kV in negative mode. 10 L of sample were injected on a Column Zorbax Eclipse XDB-C18 (100 mm2.1 mm particle size 1.8 m) from Agilent technologies, protected by a guard column XDB-C18 (5 mm2.1 mm particle size 1.8 m) and heated at 40 C. by a pelletier oven. The gradient mobile phase consisted of water with 2 mM of dibutylamine acetate concentrate (DBAA) (A) and acetonitrile (B). Flow rate was set to 0.4 mL/min and an initial gradient of 90% phase A and 10% phase B, which was maintained for 3 min. Molecules were then eluted using a gradient from 10% to 95% phase B over 1 min. The column was washed using 95% mobile phase B for 2 minutes and equilibrated using 10% mobile phase B for 1 min and the autosampler was kept at 4 C. Scan mode used was the MRM for biological samples. Peak detection and integration of the analytes were performed using the Agilent Mass Hunter quantitative software (B.10.1). Pseudo-targeted analysis of intracellular metabolites by ultra-high performance liquid chromatography (UHPLC) coupled to a Q-Exactive mass spectrometer. Reversed phase acetonitrile method: The profiling experiment was performed with a Dionex Ultimate 3000 UHPLC system (Thermo Scientific) coupled to a Q-Exactive (Thermo Scientific) equipped with an electrospray source operating in both positive and negative modes and full scan mode from 100 to 1200 m/z. The Q-Exactive parameters were: sheath gas flow rate 55 au, auxiliary gas flow rate 15 au, spray voltage 3.3 kV, capillary temperature 300 C., S-Lens RF level 55 V. The mass spectrometer was calibrated with sodium acetate solution dedicated to low mass calibration. 10 L of sample were injected on a SB-Aq column (100 mm2.1 mm particle size 1.8 am) from Agilent Technologies, protected by a guard column XDB-C18 (5 mm2.1 mm particle size 1.8 am) and heated at 40 C. by a pelletier oven. The gradient mobile phase consisted of water with 0.2% acetic acid (A) and acetonitrile (B). The flow rate was set to 0.3 mL/min. The initial condition was 98% phase A and 2% phase B. Molecules were then eluted using a gradient from 2% to 95% phase B for 22 min. The column was washed using 95% mobile phase B for 2 for min and equilibrated using 2% mobile phase B for 4 min. The autosampler was kept at 4 C. Peak detection and integration were performed using the Thermo Xcalibur quantitative software (2.1) (Viltard et al., 2019, Aging 11, 4783-4800).
[0638] Nanoscale secondary ion mass spectrometry (NanoSIMS). aMDM were grown on coated cover slips and treated with 10 M .sup.15N-.sup.13C-LCC-12 for 3 h. Subsequently, cells were washed twice with 1PBS, once with 0.1 M cacodylate buffer (LFG Distribution, 11653) and then fixed with 2% paraformaldehyde in 0.1 M cacodylate buffer for 20 min. Then, cells were washed three times with 0.1 M cacodylate buffer for 5 min and permeabilized with 0.1% Triton-X in 0.1 M cacodylate buffer for 5 min. Subsequently, cells were washed three times with 0.1 M cacodylate buffer and blocking buffer (2% BSA, 0.1% Tween in 0.1 M cacodylate buffer) was added for 20 min. Primary antibody (1:400) was added for 1 h in blocking buffer. Then, cells were washed three times with 0.1 M cacodylate buffer and the 10 nM gold-nanoparticle-loaded secondary antibody (1:50) was added in blocking buffer for 1 h. Cells were washed three times with 0.1 M cacodylate buffer and treated with 1% OSO.sub.4 (Electron Microscopy Sciences, 19152) in 0.1 M cacodylate buffer for 1 h. Cover slips with samples were washed three times for 10 min with Milli-Q water. Subsequently, cells were dehydrated with sequential EtOH solutions each for 10 min each: 50%, 70%, 290%, 3100% (dried over molecular sieves, Sigma-Aldrich, 69833). Samples were then coated with a 1:1 mixture of resin (Electron Microscopy Sciences, dodecenylsuccinic anhydride, 13710, methyl nadic anhydride, 19000, DMP-30, 13600 and LADD research industries: LX112 resin, 21310) and dry EtOH for 1 h. Then, samples were embedded in pure resin for 1 h. Embedding capsules (Electron Microscopy Sciences, 69910-10) were filled with resin, inverted onto the cover slides and placed in an oven at 56 C. for 24 h. 0.2 am sections were prepared using a Leica Ultracut UCT microtome. Sample sections were deposited onto a clean silicon chip (Institute for Electronic Fundamentals/CNRS and University Paris Sud) and dried upon exposure to air before being introduced into the NanoSIMS-50 ion microprobe (Cameca, Gennevilliers, France). A Cs.sup.+ primary ion was employed to generate negative secondary ion from the sample surface. The probe steps over the image field and the signal of selected secondary ion species were recorded pixel-by-pixel to create 2D images. Image of .sup.12C.sup.14N.sup. was recorded to provide the anatomic structure of the cells, while the one of .sup.31P.sup. highlights the location of cell nucleus. The cellular distribution of .sup.15N-label was imaged by measuring the excess in .sup.12C.sup.15N.sup. to .sup.12C.sup.14N.sup. ratio with respect to the natural abundance level (0.0037), and the one for antibody with gold staining targeting mitochondria was performed by detecting directly .sup.197Au.sup. ion. When detecting .sup.12C.sup.15N.sup. ion, appropriate mass resolution power was required to discriminate abundant .sup.13C.sup.14N.sup. isobaric ions (with an M/M of 4272). For each image recording process, multiframe acquisition mode was applied and hundreds of image planes were recorded. The overall acquisition time corresponding to the .sup.15N image was 12 h and 6 h 30 mins for the .sup.197Au image. During image processing with ImageJ, the successive image planes were properly aligned using TomoJ plugin (Messaoudii et al., 2007, Bioinformatics 8, 288-296), so as to correct the slight primary beam shift during long hours of acquisition. A summed image was then obtained with improved statistics. Further, for the .sup.12C.sup.15N.sup. to .sup.12C.sup.14N.sup. ratio map, an HSI (Hue-Saturation-Intensity) color image was generated using OpenMIMS for display with increased significance (Lechene et al., 2006, J. Biol. 5, 20). The hue corresponds to the absolute .sup.15N/.sup.14N ratio value, and the intensity at a given hue is an index of the statistical reliability.
[0639] Synthesis. Clickable Lipophilic copper clamp 12: Bis-(cyanoguanidino)dodecane (227 mg, 0.60 mmol) and but-3-yne-1-amine hydrochloride (EN300-76524, Enamine, 126 mg, 1.20 mmol) were mixed together in a sealed tube and heated at 150 C. without solvent for 4 h. After cooling to rt, the mixture was taken up in EtOH and a large excess of EtOAc was added slowly. The white precipitate was filtered and purified by preparative HPLC (H.sub.2O/Acetonitrile/formic acid, 95:5:0.1 to 40:60:0.1) to give the clickable LCC-12 di-formic acid salt as a white powder (102 mg, 30%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) =9.05-7.60 (brs, 4H, ex), 8.47 (s, 2H, formate), 7.60-6.80 (m, 8H, ex), 3.29-3.16 (m, 4H), 3.06 (brs, 4H), 2.84 (s, 2H), 2.39-2.29 (m, 4H), 1.44 (brs, 4H), 1.25 (brs, 16H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) =167.6 (formate), 159.7, 158.4, 82.6, 72.7, 41.3, 40.3, 29.5 (3C), 29.2, 26.8, 19.4 ppm. HRMS (ESI+) m/z: calculated for C.sub.24H.sub.46N.sub.10 [M+2H].sup.2+ 237.1948, found 237.1947. Isotopically labelled lipophilic copper clamp: Dicyandiamide .sup.15N and .sup.13C marked (Eurisotop, CNLM-9324-PK, 50 mg, 0.55 mmol), 1,12-diaminododecane (46.8 mg, 0.23 mmol) and CuCl.sub.2 (31.4 mg, 0.23 mmol) were suspended in a sealed tube in water (0.6 mL) and stirred for 1 h, then heated at 80 C. for 48 h. The resulting pink mixture was acidified with an aq. solution of HCl (2 M, 1 mL) until complete dissolution of the precipitate. The mixture was concentrated under reduced pressure and isotopically labeled LCC-12 was purified by preparative HPLC (H.sub.2O/Acetonitrile/formic acid, 95:5:0.1 to 73:27:0.1) to give the isotopically labeled LCC-12 di-formic acid salt as a white powder (35 mg, 32%). .sup.1H-NMR (500 MHz, DMSO-d.sub.6) : 8.60-7.76 (brs, 2H, ex), 8.48 (s, 2H, formate), 7.70-6.50 (brs, 12H, ex), 3.05 (brs, 4H), 1.43 (m, 4H), 1.33-1.18 (m, 16H) ppm. .sup.13C-NMR (125.8 MHz, DMSO-d.sub.6) : 167.0 (formate), 160.2, 159.2, 41.3, 29.5 (3C), 29.2, 26.8 ppm.
[0640] Fluorescence Microscopy
[0641] Isolated monocytes were plated on cover slips, differentiated and activated as described in Cell culture. Cells were washed three times with 1PBS, fixed with 2% paraformaldehyde in 1PBS for 12 min and then washed three times with 1PBS. After fixation, cells were permeabilized with 0.1% Triton X-100 in 1PBS for 5 min and washed three times with 1PBS. Subsequently, cells were blocked in 2% BSA, 0.2% Tween-20/1PBS (blocking buffer) for 20 min at room temperature. Cells were incubated with the relevant antibody in blocking buffer for 1 h at room temperature, washed three times with 1PBS and were incubated with secondary antibodies for 1 h. Finally, cover slips were washed three times with 1PBS and mounted using VECTASHIELD containing DAPI (Vector Laboratories, H-1200-10). Fluorescence images were acquired using a Deltavision real-time microscope (Applied Precision). 40/1.4NA, 60/1.4NA and 100/1.4NA objectives were used for acquisitions and all images were acquired as z-stacks. Images were deconvoluted with SoftWorx (Ratio conservative15 iterations, Applied Precision) and processed with ImageJ.
[0642] Click labeling. aMDM on coverslips were treated with clickable LCC-12 (in-house, 0.1 M, 3 h) in the absence or presence of CCCP (10 M, 3 h) fixed and permeabilized as indicated in fluorescence microscopy. Mitotracker was added to live cells for 45 mins to before fixation. The click reaction cocktail was prepared using the Click-iT EdU Imaging kit (Life Technologies, C10337) according to the manufacturer's protocol. Briefly, we mixed 430 L of 1 Click-iT reaction buffer with 20 L of CuSO.sub.4 solution, 1.2 L Alexa-Fluor-azide, 50 L reaction buffer additive (sodium ascorbate) to reach a final volume of 500 L. Reactions were performed with or without CuSO.sub.4 or ascorbate. Cover-slips were incubated with the click reaction cocktail in the dark at room temperature for 30 min, then washed three times with 1PBS. Immunofluorescence was then performed as described in fluorescence microscopy.