Patent Application
20150038493
The present invention relates to a series of molecules derived from 2,3-diaminopropionic acid (Dap), optionally comprising an 8-hydroxyquinoline (8-HQ) unit, and to the use thereof for scavenging an alpha-oxoaldehyde in particular resulting from glucose degradation and/or for scavenging an alpha, beta-unsaturated aldehyde, in particular resulting from the oxidative degradation of fatty acids. These molecules can also have an application in the cosmetics, agri-food and pharmaceutical fields.
Indeed, these novel compounds have the ability to effectively scavenge α-oxoaldehydes resulting from glucose degradation (for example, glyoxal, methylglyoxal, 3-deoxyglucosone) and/or α,β-unsaturated aldehydes resulting from the oxidative degradation of certain fatty acids (for example, acrolein, malondialdehyde, 4-hydroxynonenal). These aldehydes are partly responsible for irreversible modifications on proteins known under the generic terms AGEs (Advanced Glycation End-products) and ALEs (Advanced Lipid peroxidation End-products). The accumulation of these modifications is, inter alia, closely linked to the development of vascular complications in diabetics, such as atherosclerosis, retinopathy or nephropathy, and of certain neurodegenerative pathological conditions such as Alzheimer's disease.
The therapeutic use of the novel molecules described hereinafter is therefore of particularly advantageous interest for preventing the occurrence of these pathological conditions or even slowing down or treating the development thereof. However, their use in the cosmetics and food fields is also of interest, quite particularly in the treatment or prevention of skin aging.
The reaction for non-enzymatic condensation between sugars and the amino groups of proteins, i.e. a Maillard reaction, results in the formation of “Advanced Glycation End-products”, or AGEs, on proteins. The in vivo occurrence of these irreversible modifications is a lengthy and complex process which has been shown to involve not only sugars, such as glucose, but also some of their degradation products and metabolites, of α-oxoaldehyde type, for instance methylglyoxal (abbreviated as MGO), glyoxal (abbreviated as GO) or 3-deoxyglucosone (abbreviated as 3-DG).
The accumulation of AGEs has two major biological consequences. Firstly, protein crosslinking: this phenomenon is mainly observed on proteins with a long lifetime (collagen, proteins of the crystalline lens, fibronectin, albumin, hemoglobin, etc.) and plays a predominant role in normal aging (loss of physical flexibility of tissues, skin pigmentation) and the occurrence of specific pathological conditions of elderly individuals (cataracts, rheumatic disorders). Secondly, the generation of oxidative stress at the cellular level results in the occurrence of inflammatory and thrombogenic reactions via interaction between AGEs and certain specific receptors (RAGEs). It has thus been possible to demonstrate several intracellular event cascades, initiated by AGE/RAGE interactions, and to directly link them to the development of atherosclerosis and of various microvascular complications (nephropathy, cardiovascular disorders, retinopathy, neuropathy). Persistent hyperglycemic states cause considerable increases in the production of α-oxoaldehydes and in the formation of AGEs; consequently, diabetic individuals are particularly affected by the pathological states mentioned above. The accumulation of AGEs is also involved in the development of certain neurodegenerative diseases such as Alzheimer's disease.
The inhibition of AGE formation and more precisely the in vivo scavenging of the α-oxoaldehydes generated from glucose therefore constitute a therapeutic approach of great interest with regard to the prevention and treatment of the diseases mentioned above. Several compounds have been developed for this purpose since the middle of the 1990s.
Aminoguanidine is one of the most widely studied molecules. In addition to its ability to effectively scavenge MGO and GO, aminoguanidine is a good nitric oxide synthase (abbreviated as NOS) inhibitor and has shown itself to be capable of stopping the development of retinopathy and also of slowing down nephropathic complications in diabetic rats. The development of this molecule has, however, been suspended owing to adverse hepatic and gastrointestinal side effects which occurred during a clinical trial relating to the prevention of the progression of diabetic nephropathy.
Pyridoxamine is also an excellent α-oxoaldehyde scavenger compound which has been found to be capable of reducing the pathological complications usually observed in diabetic rats.
Oxidative stress states, resulting from an imbalance between production of reactive oxygen species (free radicals) and antioxidant cell defenses, are one of the major consequences of diabetes and are, inter alia, responsible for the lipid peroxidation phenomenon. This oxidizing process results in the fragmentation of polyunsaturated fatty acids and in the formation of alpha, beta-unsaturated aldehydes, such as acrolein (abbreviated as ACR), malondialdehyde (abbreviated as MDA) or 4-hydroxy-2-nonenal (abbreviated as 4-HNE), and also of α-oxoaldehydes previously mentioned (abbreviated as MGO, GO). Alpha, beta-unsaturated aldehydes are highly toxic compounds capable of reacting with proteins so as to result in the formation of adducts known under the generic term ALEs (Advanced Lipid peroxidation End-products). Just like AGEs, ALEs induce cell dysfunctions and protein crosslinking phenomena.
Among the alpha, beta-unsaturated aldehydes, acrolein is the compound which exhibits the greatest reactivity toward cysteine, histidine and lysine residues of proteins. The accumulation of the acrolein-derived ALE N-(3-formyl-3,4-dehydropiperidino)lysine (abbreviated as FDP) is thus strongly suspected of contributing to the formation of the abnormalities on Müller glial cells encountered in cases of diabetic retinopathy. High levels of acrolein adducts have also been demonstrated in some neuronal proteins in patients suffering from Alzheimer's disease. Likewise, 4-HNE exhibits an entire range of harmful biological effects, ranging from impairment of genetic expression up to cell apoptosis. Exposure to 4-HNE is implicated in the etiology of numerous diseases associated with oxidative stress, such as atherosclerosis, hepatic ischemia-reperfusion lesions, Alzheimer's disease and Parkinson's disease. Finally, malondialdehyde is known to react with the lysine residues of proteins so as to form dihydropyridine (DHP) derivatives, the presence of which results in phenomena of UV-sensitization of the skin, which contribute to accelerated aging, or even to cancer, of the skin. MDA levels are notably higher in diabetic patients and cause, through crosslinking, deleterious stiffening of collagen in the cardiovascular system. The mutagenic nature of MDA owing to its reactivity toward DNA may also be noted.
One of the therapeutic strategies most widely used for countering the harmful effects of alpha, beta-unsaturated aldehydes consists in using against them nucleophilic molecules capable of diverting them from their biological targets, namely proteins or DNA.
It is therefore of great interest to have novel molecules, devoid of side effects, which make it possible to act on the modifications of ALEs and AGEs by preventing their accumulation responsible for numerous pathological conditions.
In the context of the present invention:
in which:
or of formula (Ib) below:
According to one embodiment, the compound (I) according to the invention of formula (Ia) below:
has an R1 group which may be selected from one of the following moieties:
According to one embodiment, said compound of formula (Ib) below:
has an R1 group which may be selected from one of the following moieties:
According to one embodiment, the compound according to the invention has a number n which is equal to 0 and an atom X which is a nitrogen atom.
According to one embodiment, the compound according to the invention has one or more group(s):
in which:
in which:
in which:
Other advantages may further appear to those skilled in the art on reading the examples below, illustrated by the appended figures, given by way of illustration.
A suspension of (2R)-2,3-diaminopropanoic acid in monohydrochloride form (20.0 g; 142 mmol) in a mixture of dioxane (142 ml) and water (142 ml) is stirred at between 0 and 5° C. Triethylamine (59.0 ml; 426 mmol; 3 eq.) is then added to the medium which becomes homogeneous after a few minutes. Di-tert-butyl dicarbonate (62.0 g; 284 mmol; 2 eq.) is then added, in 4 portions, over the course of 30 min, taking care to keep the temperature below 10° C. The reaction mixture is kept stirring for 16 h at 20° C. The medium is then concentrated by 50%, under reduced pressure, before being taken up with diethyl ether (300 ml) and 1M hydrochloric acid (300 ml). After separation by settling out, the phases are separated and an extraction with diethyl ether (2×300 ml) is carried out on the aqueous phase. The combined organic phases are washed with brine (300 ml), dried over sodium sulfate, filtered and concentrated under vacuum. The residue obtained is dried for several hours under a high vacuum, and triturated, to give the expected compound 1 in the form of a white solid (42.4 g; 139 mmol; 98%).
1H NMR (CDCl3, 300 MHz) δ (ppm): 9.40 (s, 1H); 6.29/5.80 (2s, 1H); 5.50/5.21 (2s, 1H); 4.33 (m, 1H); 3.54 (m, 2H), 1.44 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 173.4; 156.9; 156.3; 80.7; 80.4; 54.7; 42.2; 28.3.
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 eq.) and then 1-hydroxybenzotriazole monohydrate (1.1 eq.) are successively added to a solution of compound 1 (1 eq.) in dichloromethane (10 ml/mmol), stirred at between 0 and 5° C. After stirring for 30 min at the same temperature, the amino derivative 2 (1.0 eq.), chosen according to the compound 3a, b, c, d, e, f, g, h, i, j or k targeted, is added and then the solution is kept stirring for 15 h while allowing the temperature to come back up to 20° C. The reaction mixture is then loaded with silica (1.5 g/mmol) before being concentrated under reduced pressure until a dry fine powder is obtained. A purification by silica gel chromatography is then carried out by depositing the powder obtained directly at the top of the column (eluent: cyclohexane/ethyl acetate, 70/30 to 40/60 or dichloromethane/methanol, 98/2 to 95/5). The expected compound 3a, b, c, d, e, f, g, h, i, j or k is obtained in the form of solid (76-98%).
The amino derivative 2 used to obtain the compound 3a is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.57 (s, 1H); 5.15 (s, 1H); 4.54 (s, 1H); 3.75-3.28 (m, 5H); 3.24 (m, 1H); 2.02-1.78 (m, 4H); 1.41 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.5; 156.0; 155.5; 79.8; 79.4; 52.0; 46.4; 46.0; 42.6; 28.3; 26.0; 24.0.
The amino derivative 2 used to obtain the compound 3b is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.60 (s, 1H); 5.05 (s, 1H); 4.72 (s, 1H); 3.70-3.32 (m, 5H); 3.22 (m, 1H); 1.68-1.49 (m, 6H); 1.42 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.0; 156.1; 155.4; 79.8; 79.4; 50.2; 46.6; 43.2; 28.3; 26.3; 25.4; 24.4.
The amino derivative 2 used to obtain the compound 3c is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.63 (s, 1H); 5.04 (s, 1H); 4.72 (m, 1H); 4.48 (d, J=13.4 Hz, 1H); 3.93 (d, J=13.4 Hz, 1H); 3.37 (m, 1H); 3.20 (m, 1H); 3.04 (m, 1H); 2.58 (m, 1H); 1.64 (m, 3H); 1.42 (s, 18H); 1.30-0.97 (m, 2H); 0.93 (dd, 3J=6.2 Hz, 3J=6.2 Hz, 3H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 167.9; 155.9; 154.9; 79.7; 79.3; 53.3; 53.1; 45.9; 45.7; 43.5; 43.0; 42.7; 42.5; 34.5; 34.4; 33.6; 33.5; 31.0; 30.9; 38.3; 21.6; 21.5.
The amino derivative 2 used to obtain the compound 3d is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.55 (s, 1H); 5.14 (s, 1H); 4.72 (s, 1H); 3.94-3.05 (m, 6H); 2.01-1.27 (m, 26H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 169.9; 155.9; 155.3; 79.7; 79.3; 60.1; 47.7; 46.3; 43.2; 29.0; 28.3; 27.2; 27.1; 26.5.
The amino derivative 2 used to obtain the compound 3e is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.62 (d, 3J=7.9 Hz, 1H); 5.19 (s, 1H); 4.69 (m, 1H); 3.79-3.46 (m, 8H); 3.33 (m, 1H); 3.20 (m, 1H); 1.38 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.7; 155.9; 155.3; 79.8; 79.4; 66.5; 50.0; 45.9; 42.9; 42.4; 28.2.
The amino derivative 2 used to obtain the compound 3f is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.58 (d, 3J=7.7 Hz, 1H); 5.08 (s, 1H); 4.71 (m, 1H); 3.91 (m, 2H); 3.77 (m, 2H); 3.33 (m, 1H); 3.21 (m, 1H); 2.62 (m, 4H); 1.40 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.8; 155.9; 155.3; 79.9; 79.5; 50.1; 48.2; 44.8; 43.0; 28.2; 27.8; 27.2.
The amino derivative 2 used to obtain the compound 3g is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.59 (d, 3J=6.8 Hz, 1H); 5.13 (s, 1H); 4.70 (m, 1H); 3.57 (m, 4H); 3.34 (m, 1H); 3.13 (m, 1H); 2.38 (m, 4H); 2.25 (s, 3H); 1.38 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.4; 155.9; 155.3; 79.8; 79.4; 54.9; 54.4; 50.0; 45.8; 45.3; 43.1; 42.0; 28.3.
The amino derivative 2 used to obtain the compound 3h is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.58 (d, 3J=7.9 Hz, 1H); 5.07 (s, 1H); 4.72 (m, 1H); 3.66 (m, 4H); 3.37 (m, 1H); 3.15 (m, 1H); 2.51 (m, 4H); 2.24 (m, 1H); 1.77 (m, 4H); 1.58 (m, 2H); 1.39 (s, 18H); 1.17 (m, 4H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.1; 155.9; 155.3; 79.7; 79.3; 63.4; 50.0; 49.0; 48.5; 46.0; 43.2; 42.6; 28.8; 28.3; 26.1; 25.7.
The amino derivative 2 used to obtain the compound 3i is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 7.27 (m, 2H); 6.91 (m, 3H); 5.63 (d, 3J=7.4 Hz, 1H); 5.12 (s, 1H); 4.81 (m, 1H); 3.74 (m, 4H); 3.43 (m, 1H); 3.22 (m, 5H); 1.43 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.2; 155.6; 155.0; 150.3; 128.8; 120.1; 116.2; 79.5; 79.1; 49.8; 49.2; 48.8; 45.0; 42.7; 41.7; 27.9.
The amino derivative 2 used to obtain the compound 3j is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.70 (d, 3J=8.1 Hz, 1H); 5.24 (s, 1H); 4.66 (m, 1H); 3.66-3.40 (m, 6H); 3.26 (m, 1H); 3.14 (m, 1H); 2.96 (s, 1H); 2.58-2.28 (m, 6H); 1.33 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.4; 155.9; 155.2; 79.6; 79.2; 59.3; 57.8; 52.8; 52.3; 49.9; 45.2; 42.7; 41.9; 28.1.
The amino derivative 2 used to obtain the compound 3k is:
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.62 (d, 3J=8.1 Hz, 1H); 5.05 (s, 1H); 4.71 (m, 1H); 3.76 (t, 3J=5.7 Hz, 2H); 3.69 (m, 4H); 3.36 (m, 1H); 3.23 (m, 1H); 2.75 (t, 3J=5.7 Hz, 2H); 2.65 (m, 5H); 1.79 (quint, 3J=5.7 Hz, 2H); 1.41 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.3; 156.1; 155.4; 80.0; 79.6; 62.9; 57.5; 52.8; 52.4; 50.3; 44.7; 43.0; 41.4; 28.3; 27.1.
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.27 g; 6.6 mmol; 1.1 eq.) are added to a suspension of compound 1 (1.82 g; 6 mmol) and of 1-hydroxybenzotriazole monohydrate (1.01 g; 6.6 mmol; 1.1 eq.) in acetonitrile (60 ml), stirred at 0° C. After stirring for 15 min at 0° C., this solution is transferred into a dropping funnel and is then added (flow rate=1 ml/min) to a solution of piperazine (5.17 g; 60 mmol; 10 eq.) in acetonitrile (540 ml), at 0° C., with vigorous stirring. The reaction medium is kept stirring for 15 h while allowing the temperature to come back to 20° C. The mixture is then filtered through a sintered glass funnel before being concentrated under reduced pressure. The solid residue obtained is then purified, in three batches, by reverse-phase chromatography (Chromabond Flash RS40 C18ec column) according to the following conditions: elution gradient=10% B from 0 to 5 min, then 10% to 80% B from 5 to 35 min (with A=water and B=acetonitrile), flow rate=40 ml/min, detection=UV at 200 nm. The fractions collected at approximately 15 min are combined and lyophilized to give the expected compound 31 in the form of a white solid (1.68 g; 4.5 mmol; 75%).
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.66 (s, 1H); 5.15 (s, 1H); 4.70 (s, 1H); 3.60-3.44 (m, 4H); 3.34 (m, 1H); 3.18 (m, 1H); 2.90-2.76 (m, 4H); 1.88 (s, 1H); 1.40 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.4; 155.9; 155.4; 79.8; 79.4; 50.0; 46.7; 46.1; 45.6; 43.2; 43.1; 28.2.
MS (ESI+): m/z=373 [M+H]; 395 [M+Na].
Potassium carbonate (207 mg; 1.5 mmol; 1 eq.) and then 1-bromobutane (165 μl; 1.5 mmol; 1 eq.) are successively added to a solution of the compound 3l (558 mg; 1.5 mmol) in acetonitrile (30 ml). The reaction medium is then kept stirring, at reflux, for 24 h, before being filtered through cotton wool and concentrated under reduced pressure. The residue obtained is purified by silica gel chromatography (eluent: dichloromethane/methanol, 95/5) to give the expected compound 3m in the form of a white solid (74%).
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.57 (d, 3J=7.2 Hz, 1H); 5.06 (s, 1H); 4.71 (m, 1H); 3.58 (m, 4H); 3.37 (m, 1H); 3.16 (m, 1H); 2.42 (m, 4H); 2.31 (m, 2H); 1.41 (s, 18H); 1.31 (m, 4H); 0.89 (t, 3J=7.2 Hz, 3H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.3; 155.9; 155.3; 79.8; 79.4; 58.1; 53.2; 52.7; 50.1; 45.5; 43.2; 42.1; 28.8; 28.3; 20.5; 13.9.
MS (ESI+): m/z=429 [M+H]; 451 [M+Na]; 492 [M+MeCN+Na].
The general method A defined above was applied to compound 1, using piperazine (0.5 eq.) as amino derivative 2. The compound 5a is obtained in the form of a white solid (90%).
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.58 (m, 2H); 5.04 (s, 2H); 4.71 (m, 2H); 3.86-3.47 (m, 8H); 3.35 (m, 4H); 1.42 (s, 36H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 168.8; 156.0; 155.3; 80.0; 79.7; 50.6; 50.3; 45.3; 45.0; 43.0; 42.1; 41.6; 28.3.
MS (ESI+): m/z=659 [M+H]; 681 [M+Na].
The general method A was applied to compound 1 using homopiperazine (0.5 eq.) as amino derivative 2. The compound 5b is obtained in the form of a white solid (84%).
1H NMR (CDCl3, 300 MHz) δ (ppm): 5.56 (m, 2H); 5.14 (s, 2H); 4.65 (m, 2H); 3.94-3.26 (m, 12H); 2.21-1.72 (m, 2H); 1.40 (s, 36H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 170.7; 170.5; 156.0; 155.4; 80.0; 79.6; 50.8; 50.5; 45.8; 48.1; 47.5; 47.3; 46.9; 46.4; 45.8; 44.9; 42.9; 42.7; 42.5; 28.3; 26.7.
MS (ESI+): m/z=673 [M+H]; 695 [M+Na].
A 4M solution of hydrochloric acid in dioxane (2.5 ml/mmol; 10 eq.) is added to a solution of the compound 3a, b, c, d, e, f, g, h, i, j, k, l, m, 5a or 5b (1 eq.) in diethyl ether (1.25 ml/mmol). The reaction medium is then kept stirring for 12 h, during which time a precipitate gradually appears. The mixture is then concentrated under reduced pressure and the solid residue obtained is then dissolved in distilled water (5 ml/mmol). This aqueous solution is washed with diethyl ether (3×2.5 ml/mmol), filtered through a 0.45 μm membrane and lyophilized to give the corresponding expected compound, namely 4a, b, c, d, e, f, g, h, i, j, k, l, m, 6a or 6b, in hydrochloride form (85-98%).
1H NMR (D2O, 300 MHz) δ (ppm): 4.72 (m, 1H); 3.77-3.42 (m, 6H); 1.99 (m, 4H).
13C NMR (D2O, 75 MHz) δ (ppm): 163.3; 49.0; 46.7; 46.5; 38.0; 24.9; 23.1.
MS (ESI+): m/z=m/z=158 [M+H]; 180 [M+Na]; 199 [M+MeCN+H]; 221 [M+MeCN+Na]; 337 [2M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 4.93 (m, 1H); 3.84-3.34 (m, 6H); 1.68 (s, 6H).
13C NMR (D2O, 75 MHz) δ (ppm): 163.5; 48.2; 47.2; 44.4; 39.2; 25.9; 24.9; 23.4.
MS (ESI+): m/z=172 [M+H]; 194 [M+Na]; 213 [M+MeCN+H]; 235 [M+MeCN+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 4.95 (m, 1H); 4.32 (m, 1H); 3.87 (m, 1H); 3.55 (m, 2H); 3.29 (m, 1H); 2.85 (m, 1H); 1.79 (m, 3H); 1.19 (m, 2H); 0.96 (m, 3H).
13C NMR (D2O, 75 MHz) δ (ppm): 163.1; 162.9; 47.7; 46.1; 45.7; 43.4; 42.9; 38.8; 38.5; 33.4; 33.1; 32.5; 32.1; 29.6; 29.2; 20.3; 19.9.
MS (ESI+): m/z=186 [M+H]; 208 [M+Na]; 393 [2M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 4.88 (m, 1H); 3.83 (m, 1H); 3.69 (m, 1H); 3.59 (m, 2H); 3.50 (m, 1H); 3.30 (m, 1H); 1.89-1.68 (m, 4H); 1.61 (m, 4H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.5; 47.9; 47.6; 46.7; 38.7; 27.7; 26.1; 25.9; 25.3.
MS (ESI+): m/z=186 [M+H]; 208 [M+Na]; 393 [2M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 4.95 (m, 1H); 3.92-3.46 (m, 10H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.3; 66.0; 65.9; 48.0; 46.0; 43.0; 39.0.
MS (ESI+): m/z=174 [M+H]; 196 [M+Na]; 215 [M+MeCN+H]; 237 [M+MeCN+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 4.96 (dd, 3J=4.1 Hz, 3J=6.1 Hz, 1H); 4.21-4.09 (m, 1H); 4.05-3.94 (m, 1H); 3.89-3.80 (m, 1H); 3.72-3.61 (m, 1H); 3.57 (m, 2H); 2.96-2.66 (m, 4H).
13C NMR (D2O, 75 MHz) δ (ppm): 163.7; 48.1; 47.7; 45.2; 38.5; 26.6; 25.9.
MS (ESI+): m/z=190 [M+H]; 212 [M+Na]; 401 [2M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 5.01 (m, 1H); 4.62 (m, 1H); 4.27 (m, 1H); 3.85-3.53 (m, 5H); 3.41-3.14 (m, 3H); 3.00 (m, 3H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.0; 52.0; 51.9; 51.7; 47.7; 47.5; 42.6; 42.4; 42.3; 42.2; 39.4; 39.2; 38.7; 38.2.
MS (ESI+): m/z=187 [M+H]; 209 [M+Na]; 395 [2M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 4.97 (m, 1H); 4.68 (m, 1H); 4.28 (m, 1H); 3.86-3.51 (m, 5H); 3.46-3.16 (m, 4H); 2.11 (m, 2H); 1.92 (m, 2H); 1.68 (m, 1H); 1.58-1.08 (m, 5H).
13C NMR (D2O, 75 MHz) δ (ppm): 163.9; 65.9; 65.7; 47.6; 47.2; 46.8; 42.4; 39.5; 38.7; 38.3; 26.1; 24.0.
MS (ESI+): m/z=255 [M+H]; 277 [M+Na]; 531 [2M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 7.60 (m, 5H); 5.07 (dd, 3J=3.8 Hz, 3J=6.2 Hz, 1H); 4.32-4.22 (m, 1H); 4.20-4.07 (m, 2H); 4.04-3.93 (m, 1H); 3.91-3.78 (m, 4H); 3.73-3.57 (m, 2H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.1; 140.7; 130.1; 129.5; 120.0; 53.3; 53.2; 47.6; 42.9; 40.0; 38.5.
MS (ESI+): m/z=249 [M+H]; 271 [M+Na]; 519 [2M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 5.01 (m, 1H); 4.24 (m, 1H); 3.98 (m, 2H); 3.90-3.72 (m, 3H); 3.69-3.52 (m, 4H); 3.51-3.18 (m, 4H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.0; 57.7; 54.3; 50.5; 47.6; 42.0; 39.0; 38.4.
MS (ESI+): m/z=217 [M+H]; 239 [M+Na]; 280 [M+MeCN+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 5.00 (m, 1H); 4.26 (m, 1H); 3.87-3.68 (m, 5H); 3.67-3.51 (m, 4H); 3.46-3.12 (m, 4H); 2.02 (m, 2H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.0; 58.0; 54.2; 50.5; 47.6; 42.1; 39.2; 38.4; 25.5.
MS (ESI+): m/z=231 [M+H]; 253 [M+Na]; 294 [M+MeCN+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 5.00 (dd, 3J=3.9 Hz, 3J=6.3 Hz, 1H); 4.17-3.28 (m, 10H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.7; 48.1; 42.8; 42.6; 42.5; 39.5; 39.0.
MS (ESI+): m/z=173 [M+H]; 195 [M+Na]; 214 [M+MeCN+H]; 236 [M+MeCN+Na]; 367 [2M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 5.00 (m, 1H); 4.61 (m, 1H); 4.27 (m, 1H); 3.86-3.51 (m, 5H); 3.40-3.11 (m, 5H); 1.76 (m, 2H); 1.41 (sext, 3J=7.3 Hz, 2H); 0.95 (t, 3J=7.3 Hz, 3H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.4; 164.0; 56.6; 56.2; 50.4; 50.0; 47.7; 47.5; 42.2; 42.1; 39.4; 39.1; 38.7; 38.3; 24.8; 18.7; 12.3.
MS (ESI+): m/z=229 [M+H]; 251 [M+Na].
1H NMR (D2O, 300 MHz) δ (ppm): 4.99 (m, 2H); 3.98 (m, 2H); 3.88-3.44 (m, 10H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.2; 164.0; 47.7; 47.6; 44.5; 43.9; 41.9; 41.4; 38.5; 38.4.
1H NMR (D2O, 300 MHz) δ (ppm): 4.91 (m, 2H); 4.16 (m, 2H); 3.91 (m, 2H); 3.68-3.45 (m, 6H); 3.42-3.09 (m, 2H); 2.18-1.82 (m, 2H).
13C NMR (D2O, 75 MHz) δ (ppm): 165.0; 164.9; 48.3; 48.2; 48.1; 48.0; 47.8; 46.3; 45.9; 45.8; 45.2; 44.1; 38.7; 38.6; 38.4; 27.6; 27.0.
Compound 4a. Calculated for [C7H15N3O]H+ 158.1293. found 158.1301.
Compound 4b. Calculated for [C8H17N3O]H+ 172.1450. found 172.1465.
Compound 4c. Calculated for [C9H19N3O]H+ 186.1606. found 186.1619.
Compound 4d. Calculated for [C9H19N3O]H+ 186.1606. found 186.1602.
Compound 4e. Calculated for [C7H15N3O2]H+ 174.1243. found 174.1259.
Compound 4f. Calculated for [C7H15N3OS]H+ 190.1014. found 190.1023.
Compound 4g. Calculated for [C8H18N4O]H+ 187.1559. found 187.1568.
Compound 4h. Calculated for [C13H26N4O]H+ 255.2185. found 255.2184.
Compound 4i. Calculated for [C13H20N4O]H+ 249.1715. found 249.1719.
Compound 4j. Calculated for [C9H20N4O2]H+ 217.1665. found 217.1657.
Compound 4k. Calculated for [C10H22N4O2]H+ 231.1821. found 231.1808.
Compound 4l. Calculated for [C7H16N4O]H+ 173.1403. found 173.1416.
Compound 4m. Calculated for [C11H24N4O]H+ 229.2028. found 229.2034.
Hydrochloric acid at 37% (10 ml) is added, dropwise, with vigorous stirring, to a suspension of 8-hydroxyquinoline (2.9 g; 20 mmol) in formaldehyde (4 ml). The medium becomes homogeneous and bright yellow and heat is strongly given off during the addition. Hydrochloric acid gas is bubbled through the medium for 1 h and then the stirring is continued for 5 h. The temperature gradually decreases to approximately 25° C. and a fine yellow precipitate appears. The medium is then filtered and the residual yellow solid is washed with hydrochloric acid at 37% (4×5 ml), and then dried under a high vacuum. Compound 7 is obtained in the form of a bright yellow hygroscopic solid (3.15 g; 13.7 mmol; 68%) immediately stored under argon at 4° C.
1H NMR (DMSO-d6, 300 MHz) δ (ppm): 9.24 (dd, 4J=1.2 Hz, 3J=8.7 Hz, 1H); 9.13 (dd, 4J=1.2 Hz, 3J=5.2 Hz, 1H); 8.13 (dd, 3J=5.2 Hz, 3J=8.7 Hz, 1H); 7.87 (d, 3J=8.1 Hz, 1H); 7.54 (d, 3J=8.1 Hz, 1H); 5.32 (s, 2H).
13C NMR (DMSO-d6, 75 MHz) δ (ppm): 149.5; 144.4; 142.9; 132.3; 129.6; 127.8; 124.6; 122.8; 115.2; 43.1.
Molecular sieve 4 Å (6 g; preactivated for 3 h at 300° C.) and then the chlorinated compound 7 (1.38 g; 6 mmol; 1 eq.) are successively added to a solution, under an argon atmosphere, of potassium cyanide (1.98 g; 30 mmol; 5 eq.) in anhydrous dimethylformamide (30 ml). The stirring is maintained at 20° C. for 19 h, during which time the reaction mixture becomes green-yellowish in color. The medium is then filtered through cotton wool before being concentrated under reduced pressure. The residue obtained is taken up in water (20 ml) and this mixture is neutralized, carefully, by adding 1M hydrochloric acid. An extraction with dichloromethane (4×60 ml) is then carried out and the combined organic phases are then washed with brine (20 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The pasty black solid obtained is washed with ether, triturated, and then dried under vacuum to enable the expected compound 8 to be obtained in the form of a light brown solid (974 mg; 5.3 mmol; 88%).
1H NMR (DMSO-d6, 300 MHz) δ (ppm): 9.97 (s, 1H); 8.92 (dd, 4J=1.5 Hz, 3J=4.1 Hz, 1H); 8.45 (dd, 4J=1.5 Hz, 3J=8.6 Hz, 1H); 7.67 (dd, 3J=4.1 Hz, 3J=8.6 Hz, 1H); 7.51 (d, 3J=7.9 Hz, 1H); 7.09 (d, 3J=7.9 Hz, 1H); 4.37 (s, 2H).
13C NMR (DMSO-d6, 75 MHz) δ (ppm): 153.4; 148.2; 138.7; 132.2; 127.9; 126.4; 122.1; 119.1; 117.0; 110.6; 19.3.
MS (ESI+): m/z=185 [M+H].
A suspension of compound 8 (184 mg; 1 mmol) in hydrochloric acid at 37% (5 ml) is brought to reflux for 3 h. The medium is then concentrated under a high vacuum in order to provide the expected compound 9 in the form of a pale yellow solid (240 mg; 1 mmol; 100%).
1H NMR (DMSO-d6, 300 MHz) δ (ppm): 9.11 (m, 2H); 8.07 (dd, 4J=5.2 Hz, 3J=8.6 Hz, 1H); 7.66 (s, 1H); 7.65 (d, 3J=7.9 Hz, 1H); 7.56 (d, 3J=7.9 Hz, 1H); 7.49 (s, 1H); 7.32 (s, 1H); 4.12 (s, 2H).
13C NMR (DMSO-d6, 75 MHz) δ (ppm): 172.2; 147.9; 144.0; 143.3; 131.6; 129.6; 128.7; 123.0; 122.0; 115.3; 36.8.
MS (ESI+): m/z=204 [M+H].
Potassium carbonate (276 mg; 2 mmol; 2 eq.) and then the compound 31 (373 mg; 1 mmol; 1 eq.) are successively added to a suspension of compound 7 (230 mg; 1 mmol) in acetonitrile (10 ml). The reaction medium is kept vigorously stirring at 25° C. for 24 h before being filtered through cotton wool and concentrated under reduced pressure. The residue obtained is purified by reverse-phase chromatography (Chromabond Flash RS15 C18ec column) according to the following conditions: elution gradient=10% to 25% B from 0 to 40 min, 25% to 80% B from 40 to 45 min and then 80% B from 45 to 50 min (with A=water and B=acetonitrile), flow rate=15 ml/min, detection=UV at 200 nm. The fractions collected at approximately 41 min are combined and lyophilized to give the expected compound 10 in the form of a greenish solid (390 mg; 0.74 mmol; 74%).
1H NMR (CDCl3, 300 MHz) δ (ppm): 8.79 (dd, 4J=1.5 Hz, 3J=4.2 Hz, 1H); 8.63 (dd, 4J=1.5 Hz, 3J=8.6 Hz, 1H); 7.46 (dd, 4J=4.2 Hz, 3J=8.6 Hz, 1H); 7.31 (d, 3J=7.8 Hz, 1H); 7.07 (d, 3J=7.8 Hz, 1H); 5.56 (d, 3J=7.7 Hz, 1H); 5.01 (m, 1H); 4.72 (m, 1H); 3.81 (s, 2H); 3.68-3.46 (m, 4H); 3.41 (m, 1H); 3.18 (m, 1H); 2.60-2.34 (m, 4H); 1.42 (s, 18H).
13C NMR (CDCl3, 75 MHz) δ (ppm): 169.6; 156.0; 155.5; 152.0; 147.6; 138.8; 133.9; 129.1; 126.0; 123.7; 121.5; 108.6; 80.0; 79.2; 60.5; 52.9; 52.4; 50.3; 45.6; 43.3; 42.2; 28.3.
MS (ESI+): m/z=530 [M+H]; 552 [M+Na].
The general method B was applied to compound 10 (390 mg; 0.74 mmol) previously synthesized, as compound 3 in said method defined above, to provide compound 11 corresponding to the deprotected compound 10 in the form of a yellow solid (270 mg; 0.57 mmol; 77%).
1H NMR (D2O, 300 MHz) δ (ppm): 9.39 (d, 3J=8.8 Hz, 1H); 9.13 (d, 3J=5.3 Hz, 1H); 8.22 (dd, 3J=8.8 Hz, 3J=5.3 Hz, 1H); 8.04 (d, 3J=8.1 Hz, 1H); 7.56 (d, 3J=8.1 Hz, 1H); 4.99 (m, 3H); 4.18-3.78 (m, 10H).
13C NMR (D2O, 75 MHz) δ (ppm): 164.7; 149.9; 143.4; 142.8; 142.7; 136.6; 129.4; 123.0; 115.8; 115.6; 55.7; 50.9; 50.7; 48.1; 42.6; 39.7; 38.9.
MS (ESI+): m/z=330 [M+H]; 352 [M+Na]; 371 [M+MeCN+H].
Triethylamine (404 μl; 3.6 mmol; 3.6 eq.), the compound 31 (372 mg; 1 mmol; 1 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (230 mg; 1.2 mmol; 1.2 eq.) and then 1-hydroxybenzotriazole monohydrate (184 mg; 1.2 mmol; 1.2 eq.) are successively added to a suspension of compound 9 (240 mg; 1 mmol) in acetonitrile (20 ml). The reaction medium is kept stirring at 20° C. for 24 h, before being filtered through cotton wool, and then concentrated under reduced pressure. The reaction crude is purified by reverse-phase chromatography (Chromabond Flash RS40 C18ec column) according to the following conditions: elution gradient=10% B from 0 to 5 min and then 10% to 50% B from 5 to 35 min (with A=water and B=acetonitrile), flow rate=40 ml/min, detection=UV at 200 nm. The fractions collected at approximately 30 min are combined and lyophilized to give the expected compound 12 in the form of a beige solid (315 mg; 0.56 mmol; 56%).
1H NMR (DMSO-d6, 300 MHz) δ (ppm): 8.79 (d, 3J=4.1 Hz, 1H); 8.38 (d, 3J=8.5 Hz, 1H); 7.48 (dd, 3J=4.1 Hz, 3J=8.5 Hz, 1H); 7.27 (d, 3J=7.8 Hz, 1H); 7.09 (d, 3J=7.8 Hz, 1H); 5.57 (d, 3J=7.3 Hz, 1H); 5.04 (m, 1H); 4.69 (m, 1H); 4.04 (m, 2H); 3.56 (m, 8H); 3.32 (m, 2H); 1.39 (m, 18H).
13C NMR (DMSO-d6, 75 MHz) δ (ppm): 169.5; 168.6; 155.9; 155.3; 151.7; 147.7; 138.6; 132.9; 128.2; 127.4; 121.9; 121.1; 109.2; 79.9; 79.5; 50.4; 45.9; 45.3; 42.9; 42.0; 41.3; 37.5; 28.3.
MS (ESI+): m/z=558 [M+H]; 580 [M+Na].
The general method B was applied to compound 12 (315 mg; 0.56 mmol) previously synthesized, as compound 3 in said method defined above, to provide compound 13, corresponding to the deprotected compound 10, in the form of a yellowish solid (240 mg; 0.51 mmol; 92%).
1H NMR (D2O, 300 MHz) δ (ppm): 8.98 (m, 2H); 8.04 (dd, 3J=5.9 Hz, 3J=8.1 Hz, 1H); 7.55 (d, 3J=7.5 Hz, 1H); 7.35 (d, 3J=7.5 Hz, 1H); 5.01 (m, 1H); 4.36 (s, 2H); 4.10-3.50 (m, 10H).
13C NMR (D2O, 75 MHz) δ (ppm): 171.1; 164.1; 145.9; 143.4; 141.9; 131.6; 128.8; 128.7; 123.1; 121.3; 115.5; 47.7; 44.7; 44.6; 44.4; 44.1; 42.1; 41.9; 41.2; 40.7; 38.5; 35.1.
MS (ESI+): m/z=358 [M+H]; 380 [M+Na].
HRMS (ES+): Compound 13. Calculated for [C18H23N5O3]H+ 358.1879. found 358.1877.
An aqueous solution of glucose (50 mM in phosphate buffer at 100 mM, pH=7.4) is incubated at 37° C. for 14 days in order to “naturally” generate glyoxal (GO) and methylglyoxal (MGO). The compound tested is introduced on the 7th day (final concentration=100 μM). The GO and MGO concentrations of the medium are measured at regular intervals by LC-MS assaying, after derivatization with 2,3-diaminonaphthalene, in order to determine the effect of the compound tested.
The results of the assays are compared with those obtained without the addition of scavenger compound (control) and with those obtained during the addition of a reference compound (aminoguanidine, D-Dap, D-Dap-L-Leu, 8-hydroxyquinoline).
A solution S0 of glucose (50 mM) in phosphate buffer (100 mM, pH=7.4) is prepared and then filtered through a sterile 0.2 μm membrane into a sterile Falcon tube, under a vertical laminar flow filtering hood. 1000 μl of solution S0 are then charged to several sterile capped Eppendorf tubes (1.5 ml) which are then stoppered and placed at 37° C., in the dark, for 14 days (“control” series). The rest of the solution S0 is incubated in the same way, in a Falcon tube. The Eppendorf tubes are then removed, one by one, at regular intervals (approximately 24 h) and are immediately placed at −20° C. while awaiting analysis.
On D=7, an aqueous solution S1 of the compound tested (5 mM) is prepared and then filtered through a sterile 0.2 μm membrane into a sterile Falcon tube, under a vertical laminar flow filtering hood. A mixture of the solution S1 (140 μl) and of the remaining solution S0, incubated for 7 days at 37° C. (6860 μl), is then prepared before being divided up between 6 sterile Eppendorf tubes (6×1000 μl) which are subsequently stoppered and placed at 37° C., in the dark, for 7 days (“test” series). These tubes are removed, one by one, at regular intervals (approximately 24 h) and are immediately placed at −20° C. while awaiting analysis.
On D=14, the tubes of the “control” and “test” series are thawed and are each treated with 100 μl of a solution of 2,3-diaminonaphthalene (10 mM) in order to derivatize the GO and the MGO, respectively in the form of GO-DAN and of MGO-DAN. After homogenization (vortex for 10 sec), the tubes are left to stand for 24 h, at 20° C., in the dark.
The GO-DAN and the MGO-DAN are then assayed in each sample by LC-MS (Shimadzu LCMS-2020), by external calibration carried out with standard solutions of GO-DAN and of MGO-DAN, according to the following conditions. Column: Shim-pack XR-ODS II (75×2 mm, 80 Å), temperature: 40° C., eluent: water/methanol (50/50)+0.1% of formic acid, flow rate: 300 μl/min, analysis time: 10 min, injection volume: 1 μl, detection: ESI+ in SIM mode (m/z=181.1 and 195.1) with the following parameters: interface voltage=4.5 kV, DL voltage=10 V, Q-array DC=0 V, Q-array RF=40 V (for m/z=181.1) or 10 V (for m/z=195.1). The GO-DAN and MGO-DAN compounds are respectively detected at the retention times of 3.9 min and 5.4 min.
The glucose (50 mM), incubated at 37° C., slowly degrades and forms glyoxal (GO) and methylglyoxal (MGO). The concentrations of GO (
The addition of compounds 4a-l or 6a makes it possible to significantly reduce the GO and MGO concentrations in less than 24 h. These compounds are found to be more reactive with respect to MGO than to GO and are on the whole better scavengers than the D-Dap or the D-Dap-L-Leu previously described. In particular, the compounds 4c, 4h and 6a show an activity close to or even greater than that of the reference compound, aminoguanidine.
The use of compounds 11 and 13 in this test also leads to very good results being obtained. Indeed, in addition to a good capacity for scavenging of GO and of MGO, these two compounds have, by virtue of the presence of the 8-hydroxyquinoline unit in their structure, the ability to inhibit the production of GO and of MGO from glucose. Extremely low levels of GO and of MGO are thus observed after 14 days: 3.0 μM (11) and 2.6 μM (13) of GO (compared with 68.4 μM for aminoguanidine) and 0.19 μM (11) and 0.12 μM (13) of MGO (compared with 1.20 μM for aminoguanidine).
The reaction of glucose and of some of its degradation products with proteins generates advanced glycation end-products (AGEs) among which certain species exhibit a specific fluorescence which can be used to demonstrate their presence.
Human albumin at physiological concentration (50 g/l) is incubated with glucose (500 mM in phosphate buffer at 100 mM, pH=7.4) at 37° C. for 20 days in the presence or absence of the compounds tested (50 mM). A measurement of the fluorescence (reading at 440 nm after excitation at 370 nm) is then carried out on each sample. The results are expressed in the form of a fluorescence read (sample)/maximum fluorescence observed (control without scavenger compound, D=20) ratio.
A solution of human albumin (50 g/l) and of glucose (500 mM) in phosphate buffer (100 mM, pH=7.4) is prepared. 1000 μl of the previous solution are then immediately charged to tubes, each containing one of the compounds tested (50 μmol). The resulting solutions are homogenized (vortex for 10 sec) and then immediately filtered through a sterile 0.2 μm membrane into sterile capped Eppendorf tubes (1.5 ml), under a vertical laminar flow filtering hood. The tubes are then stoppered and placed at 37° C., in the dark, for 20 days. After a return to ambient temperature (22° C.), a measurement of the fluorescence (λex=370 nm, λem=440 nm) is carried out on 200 μl of each sample.
The control sample of albumin incubated alone in the glucose solution experiences a multiplication of its fluorescence (and therefore of the amount of AG Es present) by a factor of approximately 7 after 20 days (relative fluorescence: 14% on D=0 and 100% on D=20) (
The presence, in the medium, of known compounds which inhibit AGE formation results in an expected decrease of the fluorescence. Among these reference compounds, aminoguanidine is found to be the most effective (relative fluo.: 3%), far ahead of carnosine (60%) and D-Dap-L-Leu (43%).
The addition of the compounds 4a-l and 6a also makes it possible to maintain low AGE levels (relative fluo.: 11-23%), close to the initial level of the control (14% on D=0). The very large difference in activity between the compound 4e (relative fluo.: 19%) and D-Ala-Morpholine (78%), a derivative of analogous structure which has only one amine function, can be noted, this being evidence of the involvement of the 1,2-diamine unit in the anti-AGE activity of the compounds described here.
Compounds 11 and 13, derived from 8-hydroxyquinoline, are found to be the most effective of the series of molecules tested since they produce the lowest relative fluorescence values observed (respectively 3% and 2%), well below that obtained for 8-hydroxyquinoline (40%) and of the same order of magnitude as that observed for aminoguanidine (3%).
| Number | Date | Country | Kind |
|---|---|---|---|
| 1159061 | Oct 2011 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2012/052268 | 10/5/2012 | WO | 00 |
