Patent Application
20230382868
The benefit of priority to Luxembourg Patent Application No. LU501919 filed Apr. 25, 2022 is hereby claimed and the disclosure is incorporated herein by reference in its entirety.
The present invention relates to the fields of medicinal chemistry and medicine, and quinolin-2-yl nitrones as pharmacologically active compounds. These compounds can be in the form of a mixture of enantiomers or in the form of pure enantiomers, in the form of pharmaceutically acceptable salts, as hydrates or solvates thereof. The compounds of the present invention are useful for the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive deterioration of memory and learning ability due to a variety of pathological changes in the central nervous system (CNS) (Scheltens, P.; Strooper, B. D.; Kivipelto, M.; Holstege, H.; Chételat, G.; Teunissen, C. E.; Cummings, J.; Flier, W. M. van der. The Lancet 2021, 397, 1577-1590). The literature review shows that inhibition of cholinesterases (ChEs), namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), monoamine oxidases (MAO-A/B), and β-secretase in addition to modulation of monoaminergic receptors remains the focus of AD-related small molecule drug design (do Carmo Carreiras, M.; Ismaili, L.; Marco-Contelles, J. Bioorg. Med. Chem. Lett. 2020, 30, 126880; Kucwaj-Brysz, K.; Baltrukevich, H.; Czarnota, K.; Handzlik, J. Bioorg. Med. Chem. Lett. 2021, 49, 128275; N. M. Moussa-Pacha, S. M. Abdin, H. A. Omar, H. Alniss, T. H. Al-Tel. Med. Res. Rev. 2020, 40, 339-384; Lalut, J.; Karila, D.; Dallemagne, P.; Rochais, C. Future Med. Chem. 2017, 9, 781-795.) Selective loss of cholinergic neurons leads to a decrease in acetylcholine (ACh) levels in specific brain regions that mediate cognition (Hampel, H.; Mesulam, M.-M.; Cuello, A. C.; Farlow, M. R.; Giacobini, E.; Grossberg, G. T.; Khachaturian, A. S.; Vergallo, A.; Cavedo, E.; Snyder, P. J.; Khachaturian, Z. S. Brain 2018, 141, 1917-1933). Therefore, the inhibition of AChE or BChE increases ACh levels at the synaptic cleft and restores cholinergic neurotransmission (Wang, H.; Zhang, H. ACS Chem. Neurosci. 2019, 10, 852-862). On the other hand, MAOs catalyze the oxidation of amines that act as neurotransmitters, releasing H2O2 and consequently reactive oxygen and nitrogen species (Jones, D. N.; Raghanti, M. A. J. Chem. Neuroanat. 2021, 114, 101957). Inhibiting MAO imparts potent neuroprotective effects by decreasing oxidative stress, and restores impaired synaptic plasticity, memory and learning in mouse model of AD via control of tonic GABA levels (Manzoor, S.; Hoda, N. Eur. J. Med. Chem. 2020, 206, 112787; Youdim, M. B. H. J. Neural Transm. 2018, 125, 1719-1733; Jo, S.; Yarishkin, O.; Hwang, Y. J.; Chun, Y. E.; Park, M.; Woo, D. H.; Bae, J. Y.; Kim, T.; Lee, J.; Chun, H.; Park, H. J.; Lee, D. Y.; Hong, J.; Kim, H. Y.; Oh, S.-J.; Park, S. J.; Lee, H.; Yoon, B.-E.; Kim, Y.; Jeong, Y.; Shim, I.; Bae, Y. C.; Cho, J.; Kowall, N. W.; Ryu, H.; Hwang, E.; Kim, D.; Lee, C. J. Nat. Med. 2014, 20, 886-896; Cho, H.-U.; Kim, S.; Sim, J.; Yang, S.; An, H.; Nam, M.-H.; Jang, D.-P.; Lee, C. J. Exp. Mol. Med. 2021, 53, 1148-1158).
Parkinson's disease (PD) is a chronic neurodegenerative disease affecting 1% of people over the age of 60. PD is characterized by the progressive death of dopaminergic neurons in the substantia nigra, and the formation of Lewy bodies containing aggregates of α-synuclein (Bloem, B. R.; Okun, M. S.; Klein, C. The Lancet 2021, 397, 2284-2303; Hayes, M. T. Am. J. Med. 2019, 132, 802-807). The deficit in dopaminergic neurotransmission is the basis for the well-known symptoms associated with PD pathology, namely tremor, rigidity, bradykinesia, and postural instability. Approved therapies for PD increase dopamine levels in striatum via selective MAO-B inhibition—for example, rasagiline is prescribed as monotherapy in the early stages of PD and as add-on therapy to levodopa in advanced stages of PD (Armstrong, M. J.; Okun, M. S. JAMA 2020, 323, 548-560). Inhibition of MAO-B is thus a validated approach in PD, while a recently developed reversible MAO-B inhibitor also rescued memory impairment and learning in in APP/PS1 mice model of AD (Park, J.-H.; Ju, Y. H.; Choi, J. W.; Song, H. J.; Jang, B. K.; Woo, J.; Chun, H.; Kim, H. J.; Shin, S. J.; Yarishkin, O.; Jo, S.; Park, M.; Yeon, S. K.; Kim, S.; Kim, J.; Nam, M.-H.; Londhe, A. M.; Kim, J.; Cho, S. J.; Cho, S.; Lee, C.; Hwang, S. Y.; Kim, S. W.; Oh, S.-J.; Cho, J.; Pae, A. N.; Lee, C. J.; Park, K. D. Sci. Adv. 5, eaav0316).
The invention relates to quinolin-2-yl nitrones with the formula I, in the form of pure enantiomers or mixtures of enantiomers, and their pharmaceutically acceptable salts. These compounds can be used as drugs for the treatment of neurodegenerative diseases by inhibiting BChE or MAO-B, either alone or in combination with other beneficial activities, such as antioxidant properties and metal chelation.
Several drugs have been approved for the treatment of neurodegenerative diseases, but they cause serious side effects and have limited efficacy in vivo. Therefore, there is an urgent need for the discovery of new drugs for the treatment of neurodegenerative diseases, especially Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.
The above problem is solved by the present invention based on the surprising finding that quinolin-2-yl nitrones with general formula (I) show pharmacological activities beneficial for the treatment of neurodegenerative diseases.
The present invention provides in a first aspect a compound of general formula I
wherein
According to some embodiments, R1 is a -C1-4 alkyl.
According to some embodiments, R1 is a substituted or unsubstituted phenyl, wherein the phenyl, if substituted, is substituted by one or more (such as one, two or three) substituents independently selected from —H, —F, —Cl, —Br, —I, -Me, -Et, —Pr, -iPr, —OMe, —OEt, —OiPr, —OH, —NO2, —NH2, —CF3, and —OCF3;.
According to some embodiments, R1 is a substituted or unsubstituted benzyl, wherein the benzyl, if substituted, is substituted by one or more (such as one, two or three) substituents independently selected from —H, —F, —Cl, —Br, —I, -Me, -Et, —Pr, -iPr, —OMe, —OEt, —OiPr, —OH, —NO2, —NH2, —CF3, and —OCF3;.
According to some embodiments, R2 is H.
According to some embodiments, R2 is -C1-4 alkyl.
According to some embodiments, R2 is phenyl.
According to some embodiments, R2 is halogen.
According to some embodiments, R2 is OR3.
According to some embodiments, R3 in OR3 is H.
According to some embodiments, R3 in OR3 is C1-4 alkyl, optionally substituted by one or more R4 groups selected from halogen, —OH, -OC1-4 alkyl, —NH2, —NH(C1-4 alkyl) or Cy1.
According to some embodiments, R3 in OR3 is C1-4 alkyl, optionally substituted by Cy1.
According to some embodiments, Cy1 represents a 6-membered ring, saturated, partially unsaturated or aromatic, which contains optionally from 1 to 3, such as 1 or 2, heteroatoms selected among N, O, Se and S; Cy1 may be attached to rest of the molecule through any C or N atom available, and Cy1 is optionally substituted by one or more R5 groups.
According to some embodiments, Cy1 represents a 6-membered ring, saturated, partially unsaturated or aromatic, which contains optionally from 1 to 3 N atoms, such as 1 or 2 N atoms; Cy1 may be attached to rest of the molecule through any C or N atom available, and Cy1 is optionally substituted by one or more R5 groups.
According to some embodiments, R2 NHR3.
According to some embodiments, R3 in —NHR3 is H.
According to some embodiments, R3 in —NHR3 is C1-4 alkyl, optionally substituted by one or more R4 groups selected from halogen, —OH, —OC1-4 alkyl, —NH2, —NH(C1-4 alkyl) or Cy1.
According to some embodiments, R3 in —NHR3 is C1-4 alkyl, optionally substituted by Cy1.
According to some embodiments, Cy1 represents a 6-membered ring, saturated, partially unsaturated or aromatic, which contains optionally from 1 to 3, such as 1 or 2, heteroatoms selected among N, O, Se and S; Cy1 may be attached to rest of the molecule through any C or N atom available, and Cy1 is optionally substituted by one or more R5 groups.
According to some embodiments, Cy1 represents a 6-membered ring, saturated, partially unsaturated or aromatic, which contains optionally from 1 to 3 N atoms, such as 1 or 2 N atoms; Cy1 may be attached to rest of the molecule through any C or N atom available, and Cy1 is optionally substituted by one or more R5 groups.
Non-limiting examples of compounds of the present invention are:
According to some embodiments, the compound is selected from the group consisting of:
A Particularly preferred compound of the present invention is quinolin-2-yl nitrone (Z)-N-benzyl-1-(8-hydroxyquinolin-2-yl)methanimine oxide, which structural formula (formula II) is described below:
(Z)-N-Benzyl-1-(8-hydroxyquinolin-2-yl)methanimine oxide showed striking antioxidant capacity against hydroxyl radicals and thus a remarkable activity for neuroprotection of primary cultured neurons after experimental ischemia, as well as a very potent and selective capacity to inhibit human MAO-B and human BChE, as well as strong bio-metal (Zn, Fe, Cu) chelating properties.
Pharmaceutically acceptable pro-drugs, polymorphs, salts and hydrates of any of the above compounds of formula I are included within the present invention.
As used herein, the term “C1-4 alkyl” means a straight or branched chain non-cyclic hydrocarbon having 1, 2, 3 or 4 carbon atoms. The term “C1-4 alkyl” includes methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl. Preferred examples of “C1-4 alkyl” are methyl and tert-butyl.
As used herein, the term “C2-4 alkynyl” means a straight or branched chain non-cyclic hydrocarbon having 2, 3 or 4 carbon atoms and including at least one carbon-carbon triple bond. The term “C2-4 alkynyl” includes ethynyl, 2-propynyl, 3-propynyl, 2-butynyl and 3-butynyl.
It is noted that any of the compounds mentioned as examples throughout the present invention can be used separately or in combination, particularly as adjuvant therapy administered simultaneously, alternatively or successively with respect to a first-line therapy suitable for the treatment of a neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.
Therefore, in a further aspect the present invention provides a pharmaceutical composition comprising a compound of the present invention, or geometric isomers thereof, and a pharmaceutically acceptable excipient and/or carrier.
In a further aspect the present invention provides a compound or pharmaceutical composition of the present invention for use in medicine.
In a further aspect the present invention provides a compound or pharmaceutical composition of the present invention for use in the treatment of a neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.
In a further aspect the present invention provides a compound or pharmaceutical composition of the present invention for use as adjuvant therapy.
The adjuvant therapy may be administered simultaneously, alternatively or successively with respect to a first-line therapy suitable for the treatment of a neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.
The invention is illustrated, though not limited, by the following examples:
General Synthesis Methods. Reactions were monitored by TLC using precoated silica gel aluminium plates containing a fluorescent indicator (Merck, 5539). Detection was done by UV (254 nm) followed by charring with sulfuric-acetic acid spray, 1% aqueous potassium permanganate solution or 0.5% phosphomolybdic acid in 95% EtOH. Anhydrous Na2SO4 was used to dry organic solutions during work-ups and the removal of solvents was carried out under vacuum with a rotary evaporator. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck). Melting points were determined on a Kofler block and are uncorrected. IR spectra were obtained on a Perkin-Elmer Spectrum One spectrophotometer. 1H NMR spectra were recorded with a Varian VXR-200S spectrometer, using tetramethylsilane as internal standard and 13C NMR spectra were recorded with a Bruker WP-200-SY. All the assignments for protons and carbons were in agreement with 2D COSY, HSQC, HMBC, and 1D NOESY spectra. Values with (*) can be interchanged. The purity of compounds was checked by elemental analyses, conducted on a Carlo Erba EA 1108 apparatus, and confirmed to be ≥95%.
General procedure for the synthesis of nitrones. A solution of the corresponding carbaldehyde (1 mmol), Na2SO4 (3 mmol), AcONa (2 mmol) and the appropriate N-alkylhydroxylamine hydrochloride (1.5 mmol) in EtOH (5 mL) was heated at 90° C. for 2-3 h under MWI. After that time, the solvent was evaporated and the crude mixture was purified on column chromatography using the indicated mixtures of solvents.
8-(3-(Piperidin-1-yl)propoxy)quinoline-2-carbaldehyde (2). A solution of commercial 8-hydroxyquinoline-2-carbaldehyde (1) (103.9 mg, 0.6 mmol) in CHCl3 (3.6 mL)/ water (0.6 mL), K2CO3 (249 mg, 1.8 mmol) and commercial 1-(3-chloropropyl)piperidine (178.32 mg, 0.9 mmol) were added. The mixture was vigorously stirred and heated at 80° C. for 1 d. After that time, the solvent was evaporated under reduced pressure and the crude mixture was purified on column chromatography (DCM/MeOH 7%) to yield compound 2 as a yellow solid (158.7 mg, 89%): mp 44-6° C.; IR (KBr) ν 2932, 1709, 1462, 1323, 1102 cm−1;1H NMR (400 MHz, CDCl3) δ 10.20 (s, 1H), 8.18 (dd, J=8.5, 0.9 Hz, 1H), 7.97 (d, J=8.4 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.37 (dd, J=8.3, 1.1 Hz, 1H), 7.12 (dd, J=7.9, 1.2 Hz, 1H), 4.29 (t, J=6.8 Hz, 2H), 2.62-2.51 (m, 2H), 2.50-2.30 (m, 4H), 2.25-2.12 (m, 2H), 1.55 (p, J=5.6 Hz, 4H), 1.39 (q, J=5.7, 4.3 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 193.9, 155.5, 151.4, 140.1, 137.2, 131.4, 129.8, 119.5, 117.7, 109.9, 67.9, 55.78, 54.6 (2C), 26.33, 25.8 (2C), 24.3. HRMS (ESI_ACN) Calcd. for C18H22N2O2: 298.1682. Found: 298.1690.
(Z)-N-tert-Butyl-1-(8-(3-(piperidin-1-yl)propoxy)quinolin-2-yl)methanimine oxide (3). Following the general method for the synthesis of nitrones, a solution of carbaldehyde 2 (79.35 mg, 0.27 mmol), Na2SO4 (76.68 mg, 0.54 mmol), AcONa (26.57 mg, 0.324 mmol) and N-tert-butylhydroxylamine hydrochloride (40.69 mg, 0.324 mmol) in EtOH (5 mL) was heated at 95° C. for 3 h under MWI. Then, the solvent was evaporated and the crude mixture was purified on column chromatography (DCM/MeOH 7%) to yield compound 3 as a yellow solid (52.3 mg, 52%): mp 135-7° C.; IR (KBr) ν 3493, 2942, 1615, 1261, 1096 cm−1; 1H NMR (400 MHz, CDCl 3) δ 9.27 (d, J=8.8 Hz, 1H), 8.36-8.13 (m, 1H), 7.53-7.41 (m, 2H), 7.16-7.04 (m, 1H), 4.37 (t, J=5.7 Hz, 2H), 3.61 (d, J=11.9 Hz, 2H), 3.43-3.28 (m, 2H), 2.68 (dd, J=15.8, 6.7 Hz, 4H), 2.32 (d, J=13.8 Hz, 2H), 2.00-1.79 (m, 4H), 1.67 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 137.5, 132.1, 129.6, 127.6, 127.5, 121.9, 120.4, 119.1, 110.2, 110.0, 72.5. 66.7, 55.4, 53.5 (2C), 28.4 (3C), 23.9, 22.7 (2C), 22.2. HRMS (ESI_ACN) Calcd. for C22H 31N3O2: 369.24163. Found: 369.24136.
(Z)-N-Benzyl-1-(8-(3-(piperidin-1-yl)propoxy)quinolin-2-yl)methanimine oxide (4). Following the general method for the synthesis of nitrones, a solution of carbaldehyde 2 (79.35 mg, 0.27 mmol), Na2SO4 (76.68 mg, 0.54 mmol), AcONa (26.57 mg, 0.324 mmol) and N-benzylhydroxylamine hydrochloride (51.71 mg, 0.324 mmol) in EtOH (5 mL) was heated at 90° C. for 2 h under MWI. After that time, the solvent was evaporated and the crude mixture was purified on column chromatography (DCM/MeOH 7%) to yield compound 4 as a mixture of Z and E isomers in a 3.5:1 ratio, that we were unable to separate, as a yellow solid (59.7 mg, 55%): mp 108-10° C.; IR (KBr) ν 3420, 2935, 1600, 1455, 1105 cm−1; 1H NMR (400 MHz, CDCl3) [(minor isomer E) 8.17 (d, J=8.8 Hz, 1H, H4), 7.79 (s, 1H, CH═N), 7.74 (dd, J=8.4, 1.1 Hz, 1H, H3), 7.55 (dd, J=7.0, 2.5 Hz, 1 H, H5), 7.09 (br d, J=1.5 Hz, 1H, H7), 5.43 (s, 1H, CH2C6H5), (major isomer Z) 9.15 (d, J=8.8 Hz, 1H, H4), 8.17 (d, J=8.8 Hz, 1H, H3), 8.08 (s, 1H, CH═N), 7.07 (br d, J=7.5 Hz, 1H, H7), 5.15 (s, 1H, CH2C6H5)], 8.28-8.26 (m, 1H, C6H5), 7.54 (br d, J=Hz, 1H, H5), 7.44-7.38 (m, 6H, H6, C6H5), 7.45 (t, J=Hz, 1H, H6), 7.52-7.37 (m, 4H, C6H5), 4.31 (t, J=Hz., 2H, OCH2), 3.15-2.65 (m, 6H), 2.43-2.41 (m, 2H), 1.98-1.70 (m, 4H), 1.65-1.51 (m, 2H). HRMS (ESI_ACN) Calcd. for C25H29N3O2: 403.22598. Found: 403.22528.
1-(3-Chloropropyl)-4-(prop-2-yn-1-yl)piperazine (5). To solution of commercial 1-(3-chloropropyl)piperazine dihydrochloride (702 mg, 3 mmol, 1 equiv), TEA (0.84 mL, 6 mmol, 2 equiv) in dry CH2Cl2 (5 mL), cooled at 0° C., propargyl bromide (0.81 mL, 9 mmol, 3 equiv) was added over 30 min under Ar. The mixture was stirred at rt for 24 h and then treated with a saturated sodium bicarbonate solution (10 mL). The organic layer was separated and washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue purified by column chromatography through silica gel (CH2Cl2/MeOH 1%-2%), to yield 5 (312 mg, 52%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 3.53 (t, J=6.6 Hz, 2H), 3.23 (d, J=2.5 Hz, 2H), 2.54-2.39 (m, 10H), 2.18 (t, J=2.5 Hz, 1H), 1.88 (p, J=6.7 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 78.8, 73.2, 55.4, 53.1 (2C), 51.9 (2C), 46.8, 43.2, 29.9.
8-(3-(4-(Prop-2-yn-1-yl)piperazin-1-yl)propoxy)quinoline-2-carbaldehyde (6). A solution of commercial 8-hydroxyquinoline-2-carbaldehyde (1) (91.69 mg, 0.53 mmol) in CHCl3 (5 mL) was added K2CO3 (219.42 mg, 1.59 mmol) and 1-(3-chloropropyl)-4-(prop-2-yn-1-yl)piperazine (5) (138 mg, 0.69 mmol); then, water (1 mL) was added. The mixture was vigorously stirred and heated at 80° C. for 2 d. After that time, the solvent was evaporated under reduced pressure and the crude mixture was purified on column chromatography (DCM/MeOH 4%) to yield compound 6 as a yellow solid (95.5 mg, 53%): mp 99-101° C.; IR (KBr) ν 3128, 2829, 1715, 1462, 1320, 1094 cm−1; 1H NMR (400 MHz, CDCl3) δ 10.20 (s, 1H), 8.20 (d, J=8.4 Hz, 1H), 7.98 (d, J=8.5 Hz, 1H), 7.53 (t, J=8.5 Hz, 1H), 7.39 (d, J=8.5 Hz, 1H), 7.13 (d, J=8.4 Hz, 1H), 4.31 (t, J=6.5 Hz, 2H), 3.24 (s, 2H), 2.76-2.36 (m, 10H), 2.26-2.13 (m, 3H); 13C NMR (101 MHz, CDCl3) δ 193.8, 155.5, 151.4, 140.1, 137.2, 131.4, 129.8, 119.6, 117.8, 110.0, 78.8, 73.2, 67.7, 55.0, 53.0 (2C), 51.8 (2C), 46.8, 26.3. HRMS (ESI_ACN) Calcd. for C20H23N3O2: 337.1790. Found: 337.1790.
(Z)-N-tert-Butyl-1-(8-(3-(4-(prop-2-yn-1-yl)piperazin-1-yl)propoxy)quinolin-2-yl)methanimine oxide (7). Following the general method for the synthesis of nitrones, the reaction of carbaldehyde 6 (69 mg, mmol) with Na2SO4 (57 mg, 0.4 mmol), AcONa (26 mg, 0.32 mmol) and N-tert-butylhydroxylamine hydrochloride (38 mg, 0.3 mmol) in EtOH (5 mL) after 5 min, and column chromatography (DCM/MeOH 3%) yielded compound 7 as a yellow gum (55.7 mg, 66%): IR (KBr) ν 3428, 2819, 1451, 13206, 1155, 1104 cm−1; 1H NMR (400 Hz, CDCl3) δ 9.26 (d, J=8.8 Hz, 1H), 8.19 (d, J=8.8 Hz, 1H), 8.13 (s, 1H), 7.44-7.38 (m, 2H,), 7.08-7.06 (m, 1H), 4.29 (t, J=6.9 Hz, 2H), 3.30-3.28 (m, 2H), 2.63 (s, 10H), 2.32-2.17 (m, 3H), 1.65 (s, 9H); 13C NMR (126 MHz, CDCl3) δ 154.6, 149.8, 140.2, 136.6, 132.6, 129.5, 127.3, 121.7, 119.5, 109.0, 78.7, 73.4, 72.0, 67.7, 55.0, 53.0 (2C), 51.8 (2C), 46.8, 28.3 (3C), 26.3. HRMS (ESI_ACN) Calcd for C24H32N4O2: 408,2525. Found: 408,2525.
(Z)-N-Benzyl-1-(8-(3-(4-(prop-2-yn-1-yl)piperazin-1-yl)propoxy)quinolin-2-yl)methanimine oxide (8). Following the general method for the synthesis of nitrones, a solution of carbaldehyde 6 (95.5 mg, mmol), treated with Na2SO4 (79.52 mg, 0.56 mmol), NaHCO3 (35.28 mg, 0.42 mmol) and N-benzylhydroxylamine hydrochloride (67.03 mg, 0.42 mmol), in THF (5 mL), reacted instantly. After that time, the solvent was evaporated and the crude mixture was purified on column chromatography (DCM/MeOH 3%) to yield compound 8 as a mixture of E and Z isomers in a 1.5:1 ratio, that we were unable to separate, as a yellow gum (92.5 mg, 75%): IR (KBr) ν 3429, 2817, 1457, 1320, 1151, 1104 cm−1; 1H NMR (500 MHz, CDCl3) δ [(major isomer E) 8.17 (d, J=8.5 Hz, 1H, H4), 7.81 (d, J=8.5 Hz, 1H, H3), 7.75 (s, 1H, CH═N), 7.55 (dd, J=7.8, 1.7 Hz, 1H, H5), 7.12 (dd, J=7.7, 1.2 Hz, 1H, H7), 5.44 (s, 1H, CH2C6H5), 3.30 (d, J=2.4 Hz, 2H, CH2C≡CH), (minor isomer Z) 9.18 (d, J=8.7 Hz, 1H, H4), 8.19 (d, J=8.7 Hz, 1H, H3), 8.06 (s, 1H, CH═N), 7.10 (dd, J=7.7, 1.3 Hz, 1H, H7), 5.14 (s, 1H, CH2C6H5), 3.31 (d, J=2.4 Hz, 2H, CH2C≡CH)], 8.26-8.24 (m, 1H, C6H5), 7.45 (t, J=Hz, 1H, H6), 7.52-7.37 [m, 4H, H5 (minor)], C6H5), 4.31 (t, J=Hz, 2H, OCH2), 2.75-2.50 (m, 10H), 2.25-2.24 (m, 1H, CH2C≡CH), 2.24-2.16 (h, J=Hz, 2H, NCH2CH2CH2O). HRMS (ESI_ACN) Calcd for C27H30CIN4O2: 442.2369. Found: 442.2369.
(Z)-1-(8-Hydroxyquinolin-2-yl)-N-methylmethanimine oxide (9). Following the general method for the synthesis of nitrones, the reaction of 8-hydroxyquinoline-2-carbaldehyde (1) (173 mg, 1 mmol, 1 equiv) with Na2SO4 (426 mg, 3 mmol, 3 equiv), AcONa (160 mg, 2 mmol, 1.6 equiv) and N-methylhydroxylamine hydrochloride (239 mg, 1.5 mmol, 1.5 equiv) in EtOH (7 mL) at 90° C. for 10 min, after column chromatography (hexane/AcOEt 9/1) yielded compound 9 as a pale yellow solid (171 mg, 85%): 149-151° C.; 1H NMR (300 MHz, CDCl3) δ 9.16 (d, J=8.8 Hz, 1H), 8.25 (d, J=8.8 Hz, 1H), 8.03 (br s, 1H), 7.86 (s, 1H), 7.51-7.46 (m, 1H), 7.36 (dd, J=7.6, 1.3 Hz, 1H), 7.18 (dd, J=7.6, 1.3 Hz, 1H), 4.01 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 152.1, 147.7, 138.1, 136.8, 136.4, 128.7, 128.6, 121.4, 117.8, 110.1, 55.1. HRMS (ESI_ACN) Calcd. for C11H10N2O2: 202,0742. Found 202,0742.
(Z)-N-tert-butyl-1-(8-hydroxyquinolin-2-yl)methanimine oxide (10). Following the general method for the synthesis of nitrones, the reaction of 8-hydroxyquinoline-2-carbaldehyde (1) (173 mg, 1 mmol, 1 equiv) with Na2SO4 (426 mg, 3 mmol, 3 equiv), AcONa (160 mg, 2 mmol, 1.6 equiv) and N-tert-butylhydroxylamine hydrochloride (188 mg, 1.5 mmol, 1.5 equiv) in EtOH (7 mL) at 90° C. for 5 min, after column chromatography (hexane/AcOEt 9/1), yielded compound 10 as a pale yellow solid (159 mg, 92%): 103-4° C.; 1H NMR (400 MHz, CDCl3) δ 9.16 (d, J=8.8 Hz, 1H), 8.16 (d, J=8.8 Hz, 1H), 8.04 (br s , 1H), 7.97 (s, 1H), 7.41-7.39 (m, 1H), 7.27 (dd, J=7.6, 1.2 Hz, 1H), 7.09 (dd, J=7.6, 1.2 Hz, 1H), 1.62 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 152.0, 148.5, 138.1, 136.7, 131.5, 128.5, 128.4, 121.7, 117.8, 110.0, 72.1, 28.4 (3C). HRMS (ESI_ACN) Calcd. for C14H16N2O2: 244,1212. Found 244,1212.
(Z)-N-Benzyl-1-(8-hydroxyquinolin-2-yl)methanimine oxide (11). Following the general method for the synthesis of nitrones, the reaction of 8-hydroxyquinoline-2-carbaldehyde (1) (173 mg, 1 mmol, 1 equiv), Na2SO4 (426 mg, 3 mmol, 3 equiv.), AcONa (160 mg, 2 mmol, 1.6 equiv.) and N-benzylhydroxylamine hydrochloride (239 mg, 1.5 mmol, 1.5 equiv.) in EtOH (7 mL) was heated at 90° C. during 10 min under mw irradiation. After that time, the solvent was evaporated and the crude mixture was purified on column chromatography (Hexanes/AcOEt 9/1) to yield compound 11 as a pale yellow solid (272 mg, 98%): 110-1° C.; 1H NMR (400 MHz, CDCl3) δ 9.15 (d, J=8.8 Hz, 1H), 8.22 (d, J=8.8 Hz, 1H), 8.00 (br s, 1H), 7.88 (s, 1H), 7.55-7.51(m, 2H), 7.44-7.37 (m, 4H), 7.26 (dd, J=7.6, 1.2 Hz, 1H), 7.08 (dd, J=7.6, 1.2 Hz, 1H), 5.09 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 152.1, 147.7, 138.1, 136.8, 135.3, 132.7, 129.5 (2C), 129.3, 129.2 (2C), 128.7, 128.6, 121.6, 117.8, 110.1, 72.1. HRMS (ESI_ACN) Calcd. for C17H14N2O2: 278,1055. Found 278,1055.
Inhibition of Cholinesterases. The inhibitory potencies of compounds against the ChEs were determined by the method of Ellman (Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88-95). Briefly, compounds were incubated with Ellman's reagent (final concentration, 370 μM) and the ChEs (final concentration, approx. 1 nM or 100 μM hBChE or hAChE, respectively) in 0.1 M sodium phosphate (pH 8.0) for 5 min at 20° C. on 96-well microplates (Brand microplate, pureGrade, F-bottom). Reactions were started adding the substrate (final concentration, 500 μM butyrylthiocholine iodide [BTCI] or acetylthiocholine iodide [ATCI] for hBChE and hAChE, respectively). The final content of DMSO was always 1% (v/v). The increase in absorbance (λ=412 nm) was monitored for 2 min using a microplate reader (Synergy HT, BioTek Instruments, VT, USA). The initial velocities in the presence (vi) and absence (vo) of the compounds were calculated, and the inhibitory potencies were expressed as residual activities (RA=vi/vo). The IC50 values were determined by plotting RA against the applied inhibitor concentrations, with the experimental data fitted to a four-parameter logistic function (GraphPad Prism 9.3, GraphPad Software, San Diego, CA, USA). Donepezil was used as a positive control (Table 1).
Inhibition of Monoamine Oxidases. Recombinant microsomal hMAOs expressed in BTI-TN-5B1-4 cells, HRP (type II, lyophilized powder) and p-tyramine hydrochloride were purchased from Sigma Aldrich (Sigma Aldrich, MO, USA). 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) was synthesized as described previously (von der Eltz, H.; DE; Guder, H.-J.; DE; Muhlegger, K.; DE. U.S. Pat. No. 5,035,998—Hydrolase Substrates. U.S. Pat. No. 5,035,998, Jul. 30, 1991). Briefly, 100 μL of 50 mM sodium phosphate (pH 7.4, 0.05% [v/v] Triton X-114) containing the test compounds, and hMAO-A or hMAO-B were incubated at 37° C. for 15 min in 96-well microplates (Nunc Microwell microplates, Thermo Fisher). After preincubation, the reaction was started by adding Amplex Red (final concentration, 200 μM), HRP (2 U/mL), and p-tyramine (1 mM). The increase in fluorescence intensity (λex=530 nm, λem=590 nm) was monitored at 37° C. for 30 min using a microplate reader (Synergy HT; BioTek Instruments, Inc., VT, USA). DMSO was used for control experiments (1%, v/v). To determine the blank value (b), sodium phosphate buffer replaced the enzyme solution. Initial velocities were calculated from the trends obtained, with each measurement performed in duplicate. The inhibitory potencies are expressed as the RAs according to equation: RA=(vi−b)/(vo−b), where vi is the velocity in the presence of the test compounds, and v0 is the control velocity in the presence of DMSO. IC50 values were determined by plotting the residual MAO activities against the applied inhibitor concentrations, with the experimental data fitted to a Hill four-parameter equation (GraphPad Prism 9.3, GraphPad Software, San Diego, CA, USA). For the reversibility assay, hMAO-B was incubated at 100-fold final concentration with the inhibitors at IC50 concentration at 37° C. (volume, 50 μL). After 15 min, the mixture was diluted 100-fold into the reaction buffer containing Amplex Red, HRP, and p-tyramine hydrochloride. The final concentrations of all reagents and of hMAO-B were the same as described above. Control experiments were performed in the same manner, replacing the inhibitor solution with DMSO (Table 1).
aSEM, standard error of the mean, IC50 values are average of three independent experiments, each performed in triplicate;
bn.a., not active (residual activity - RA at 100 μM ≥50%).
cnonspecific inhibition at the screening concentration (100 μM) due to solubility issues, inhibition disappears upon dilution (10 μM).
dIC50 value do not reflect the true affinity of the compound due to the IC50 value approaching the concentration of the hBChE (approx. 1 nM) in the in vitro assays.
DPPH radical-scavenging potency of compounds 10 and 11. Free-radical scavenging potency was evaluated using the DPPH assay. DPPH (2,2-diphenyl-1-picrylhydrazyl radical) was dissolved in MeOH (150 μL, 140 μM) and added to 150 μL methanol solution of the test sample (screening at 100 μM, serial dilution of compounds for EC50 determination) or methanol (negative control) on 96-well microtiter plates (Brand microplate, pureGrade, F-bottom). The microtiter plate was incubated at room temperature in the dark for 90 min. The absorbance at 517 nm was then determined with a microplate reader (Synergy HT; BioTek Instruments, Inc., VT, USA). The experiments were performed in triplicate, with subtraction of the blank value (compound without DPPH). The percentages of DPPH free radicals were calculated as DPPH free radical (%)=[(A0−A1)/A0]×100, where A0 is the absorbance of the negative control, and A1 is the absorbance of the test sample. The free-radical scavenging potency is expressed as the concentration that scavenged 50% of the DPPH free radicals (EC50)±SEM. Compounds 10 and 11 displayed the free-radical scavenging potency with EC50 values of 119.2±1.2 μM and 126.0±0.1 μM, respectively. Resveratrol and Trolox were used as the positive controls under the same assay conditions.
Metal-chelating properties of 10 and 11. The chelation properties were determined in HEPES buffer (20 mM, 150 mM NaCl, pH 7.4) using a 96-well microplate reader (Synergy HT; BioTek Instruments, Inc., VT, USA). To determine the chelation of the metal ions by the compounds, 30 μM compound solution was treated with equimolar concentrations of CuCl2, ZnCl2, CoCl2, MgCl2, CaCl2, FeCl2, FeCl3, and AlCl3. To prevent oxidation of Fe2+, the solution of FeCl2 was prepared in the presence of 1 mM ascorbic acid. The absorption spectra were recorded after 30 min incubation at room temperature. The chelation was detected by the change in the absorption spectra, which was specific for each metal ion. The Cu2+ binding stoichiometry was resolved by titration of a 30 μM buffered solution of the compounds with additions of CuCl2 buffered stock solution. The absorption of the compounds in the absence and presence of increasing Cu2+ concentrations (0-150 μM) were recorded at the most responsive wavelength after 30 min incubation at room temperature. The absorbance differences in the absence and presence of Cu2+ were plotted against the Cu2+/compound molar ratio. The curves were approximated using data points at the lowest and highest Cu2+/compound ratios, and the intercepts were calculated (Yoe-Jones method) (Bosque-Sendra, J. M.; Almansa-López, E.; Garcia-Campana, M.; Cuadros-Rodriguez, L. Anal. Sci. 2003, 19, 1431-1439).
The absorption spectra of 10 (λabs, max=286 nm) incubated with equimolar quantity of Zn2+, Cu2+, and Al3+ showed characteristic bathochromic shift with new absorption maxima at 309, 310 and 296 nm, respectively (
| Number | Date | Country | Kind |
|---|---|---|---|
| 501919 | Apr 2022 | LU | national |
