Patent Grant
7273856
The present invention relates to compounds antagonists of tachykinins in general, in particular of neurokinin A, and to their use in pharmaceutical formulations.
Tachykinins is a family including at least three peptides, known as Substance P, Neurokinin A (NKA) e Neurokinin B (NKB).
The research in the field of the tachykinins antagonist, mainly based on single or multiple substitution of amino acids of the sequence of the peptidic agonists of Substance P and of the other tachykinins, lead to the discovery of nonapeptides containing one or more units of D-triptophan (Regoli et al., Pharmacol. 28,301 (1984)). However, the problems derived from the pharmacological use of high molecular weight peptides (multiple sites of enzymatic hydrolytic attack, poor bioavailability, rapid hepatic and renal excretion) induced to search the minimum peptidic fragment which is still capable of exerting an antagonist activity. These studies lead to the detection of adequately derivatised bicyclic and monocyclic peptides, antagonist of neurokinin A (Patent Applications No. WO 9834949 and No. WO 200129066).
Various compounds have been claimed as selective antagonists of Substance P, for instance in Patent Applications No. Wo 9519966 and No. WO 9845262; but, besides being selective for NK1 receptor, these compounds have structural characteristics which are different from the compounds of the present invention, mainly the lack of a basic amino group.
Among NK1 antagonists, we can also mention those described in Patent Application No. WO 200014109; among these compounds, there is not even one alpha,alpha-disubstituted amino acid, and the basic group, when present, is in positions that are very different from those in the compounds of the invention.
Also in Patent No. EP 394 989 the compounds with NK1 activity do not usually have a basic group and do not exhibit an alpha,alpha-disubstituted-amino acid.
In Biorganic & Med. Chem. (1994), 2 (2), 101–113 (S. Boile et; al.) compounds with NK2 activity are described, which contain an alpha,alpha-disubstituted phenylalanine (Phe), but they do not exhibit the basic characteristics nor they can be associated to the structure described by the general formula (I).
In Patent Application No. WO 9404494 NK1 antagonists are described, which exhibit a disubstituted alpha,alpha-amino acid whose structure do not correspond to the general formula (I), in particular for the presence—among other things—of a —O—CO— group in the place of X1.
It has been surprisingly found that the present non peptidic compounds of general formula (I) as defined hereinafter, show a better behaviour in inhibiting the bonding of tachykinins onto the receptor NK2, and a better in vivo antagonist activity than that showed by the products disclosed in the above cited prior art patents.
The present invention refers therefore to linear compounds of general formula (I) comprising an alfa,alfa-disubstituted amino acid and at least an amino group capable of giving basic characteristics to the compounds
wherein:
possibly substituted by one or more substituents independently chosen from halogen, C1–C6 alkyl possibly substituted by not more than three fluorine atoms (such as a trifluoromethylic group), C1–C6 alkyloxy possibly substituted by not more than three fluorine atoms (such as a trifluoromethoxylic group), —OH, —NHR10, —N(R10)2, —SR10, —CONHR10, —COR10, —COOR10, —R9COOR10, —OR9COOR10, —R9COR10, —R9CONHR10, —NHCOR10, and -nitro, wherein R10 is H or a linear or branched C1–C6 alkyl chain, and R9 is a linear or branched C1–C6 alkylene chain, and
wherein D=O, S, CH2, O—CH2 or N—R7, wherein R7 is selected from the group consisting of H, a linear or branched C1–C6 alkyl chain, and acyl radical R8-CO, wherein R8 is selected from the group consisting of H and linear or branched C1–C6 alkyl chain;
wherein the broken line indicates a possible double bond; n and m can independently be 0, 1 or 2; R13 and R14 are independently selected from the group consisting of H, C1–C6 alkyl chain, or they can be linked to form an aromatic group selected from the group consisting of benzothiophene, indol, pyridine, pyrrol, benzofurane, thiophene, benzene, naphthalene, imidazol, and biphenyl, which can be possibly substituted by one or more substituents independently selected from halogen, C1–C6 alkyl possibly substituted by not more than three fluorine atoms (such as trifluoromethylic group), C1–C6 alkyloxy chain possibly substituted by not more than three fluorine atoms (such as trifluoromethoxylic group), —OH, —NHR10, —N(R10)2, —SR10, —CONHR10, —COR10, —COOR10, —R9COOR10, —OR9COOR10, —R9COR10, —R9CONHR10, —NHCOR10, and -nitro, wherein
Further object of the present invention are the ‘retro-inverted’ compounds of the present formula (1) compounds, i.e. the compounds of general formula (I) in which one or more amidic bonds are inverted.
The presence of an alpha,alpha-disubstituted amino acid and the presence of at least one amino group in R3, which gives to the compounds a strong basicity, can be considered as a peculiar structural characteristic of the products belonging to the general formula (I). Further object of the present invention are the pharmaceutically acceptable salts of the compounds of general formula (I) with organic or inorganic acids selected from the group consisting of hydrochloric acid, sulphuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, oxalic acid, malonic acid, maleic acid, fumaric acid, succinic acid, tartaric acid and citric acid.
Furthermore, object of the present invention are the single enantiomers and diastereoisomers of the compounds of formula (I) or mixtures thereof, originating from the insertion into the structure of formula (I) of chiral residues or groups.
Further object of the present invention are the pharmaceutical formulations comprising the compounds of general formula (I) and the use of said compounds for preparing pharmaceutical formulations for the treatment of diseases in which neurokinin A plays a pathogenetic role.
Preferred compounds of the invention are the compounds of general formula (I), wherein the aminoacidic residue of general formula (III)
is selected from the group consisting of aminoacidic residues of α,α-• disubstituted glycine-type selected from the group consisting of 1-aminocyclohexane-1-carboxylic acid (Ac6c), 1-aminocyclopentane-1-carboxylic acid (Ac5c), 1-aminocyclopent-3-ene-1-carboxylic acid (Ac5c), 1-aminoisobutyrric acid, 1-aminoindane-1-carboxylic acid (1-Aic), 2-aminoindane-2-carboxylic acid (2-Aic), 2-aminotetraline-2-carboxylic acid (2-Atc), 2-methyl-2-ethylglycine, 2-methyl-2-isopropylglycine, 2-methyl-2-n-propylglycine, 2-methyl-2-(2-butyl) glycine, 2-methyl-2-isobutylglycine, 2-methyl-phenylalanine; and the other groups are as defined above.
Preferred compounds according to the present invention are the compounds of formula (I) wherein:
possibly substituted by one or more substituents independently selected from halogen, C1–C6 alkyl possibly substituted by not more than three fluorine atoms (such as trifluoromethylic group), C1–C6 alkyloxy possibly substituted by not more than three fluorine atoms (such as trifluoromethoxylic group), —OH, —NHR10, —N(R10)2, —SR10, —CONHR10, —COR10, —COOR10, —R9COOR10, —OR9COOR10, —R9COR10, —R9CONHR10, —NHCOR10, and -nitro, wherein R10 is H or linear or branched C1–C6 alkyl chain, and R9 is a linear or branched C1–C6 alkylene chain, and
wherein D=O, S or N—R7 wherein R7 is selected from the group consisting of H, linear or branched C1–C6 alkyl chain, acyl radical R8-CO, wherein R8 is selected from the group consisting of H and linear or branched C1–C6 alkyl chain;
is an α,α-disubstituted glycine-type residue selected from the group consisting of 1-aminocyclohexane-1-carboxylic (Ac6c), 1-aminocyclopentane-1-carboxylic acid (Ac5c), 1-aminoindane-1-carboxylic acid (1-Aic), 1-aminocyclopentan-3-ene 1- carboxylic acid (Ac5c), 2-methyl-phenylalanine, 2-methyl-2-ethyl-glicine,
Amongst these compounds particularly preferred are the compounds wherein:
Amongst these compounds more preferred are the present compounds of formula (I) wherein:
Among the terms used for describing the present invention the following are preferred:
The compounds of the present invention have shown an antagonist activity to the action of Substance P, Neurokinin A, and Neurokinin B; and they demonstrated particularly selective against the action of Neurokinin A.
Thus the present compounds of formula (I) can be used for preparing pharmaceutical formulations, possibly comprising pharmaceutically acceptable diluents and excipients commonly used in drug products, useful for the treatment and prevention of diseases in which tachykinins in general, and namely Neurokinin A, are involved as neuromodulators; as an example we can list the following diseases: respiratory pathologies, such as asthma, allergic rhinitis, and chronic obstructive bronchitis; ophthalmic diseases, such as conjunctivitis, skin diseases, such as allergic and contact dermatitis, and psoriasis, intestinal disorders, such as irritable colon, ulcerous colitis and Chron disease, gastric diseases, urinary diseases, such as cystitis and incontinence, erectile dysfunctions, diseases of the nervous central system, such as anxiety, depression and schizophreny, tumoural pathologies, autoimmunitary diseases or diseases related to AIDS, cardiovascular pathologies, neuritis, neuralgia and treatment of pain, in particular visceralgia, inflammatory processes, such as osteoarthritis or rheumatoid arthritis.
The present compounds of formula (I), as defined above, can be prepared according to methods described in literature and well known to the person skilled in the art, such as by reactions of amidic condensation, substitution, addition or reductive amination.
As an example the synthetic paths described in the general guidelines in the following reaction schemes can be followed by making the obvious and suitable changes according to the substituents.
In the following schemes, unless otherwise clearly specified, the substituents are as defined above.
For example, the compounds of general formula (I) may be obtained according to the following Scheme 1 by reacting the activated carboxylic acid derivatives of general formula (IV) with the intermediate compound of general formula (V).
In this case, as an example, X1=CO.
Again, as an example, the compounds of general formula (I) can be obtained, as described in the following Scheme 2, according to the following sequence of reactions:
In this case, as an example, X1═CO and n=0, 1, 2.
The compounds of the present invention can occur in various isomeric forms. In fact, whereas the configuration of the carbon linked to R5 is univocally prefixed by using during the synthesis the specific isomer of the amino acid derivative, frequently the other starting products can be constituted by mixtures of stereo isomers of difficult separation.
Therefore, the compounds of the present invention can be obtained as mixtures of diastereoisomers. These mixtures can be resolved by chromatography. The compounds of formula (I) can however be used as single enantiomers as well as mixtures of isomers.
Some examples of the present compounds and of the preparation method thereof are provided in the following for illustrative and non limiting purposes of the present invention.
1a) to a solution of 1-amino-cyclopentane-1-carboxylic acid (1 g, 7.66 mmol) in 30 ml of anhydrous dichloromethane (DCM) N,O-bis(trimethylsilyl)acetamide (BSA) (3.8 ml, 15.4 mmol) is added under magnetic stirring; after 15 min trimethylchlorosylane (TMSCl) (0.38 ml, 10% of the BSA volume) is added. The amino acid is completely sylanised (the solution at the end of the addition is clear), and after about 2 hours of stirring at room temperature benzo[b]thiophene-2-carbonyl chloride (7.66 mmol) dissolved in 10 ml of DCM is added to the reaction mixture. The reaction is kept for 12 hours under stirring at room temperature.
The solution is concentrated under reduced pressure, then 50 ml of NaHCO3 aq. 1M are added, and is kept under stirring for 30′. Everything is transferred into a separatory funnel, then ethyl acetate (AcOEt) (50 ml) is added; the mixture is shaked and the organic phase is removed.
The aqueous solution is acidified up to pH=1 with HCl 6N and washed with AcOEt (3×50 ml). The organic phases are collected together, transferred in a separatory funnel and washed with H2O and brine up to pH=5–6. The organic phase is dried on anhydrous Na2SO4, then brought to dryness. The isolated residue is crystallised from acetonitrile, thus obtaining 1.4 g (4.84 mmol, yield=63%) of Nα(benzo[b]thiophene-2-carbonyl)-1-aminocyclopentane-1-carboxylic acid.
HPLC (method E): Rt=14.04 min.
1b) To a solution in anhydrous THF (25 ml) of the product coming from Example 1a) (500 mg, 1.73 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (2.08 mmol, 0.402 g) and diisopropylethylamide (DiPEA) (5.2 mmol, 0.89 ml) are added. The solution is kept under stirring for 2 hours at room temperature. The solvent is evaporated, and the residue is dissolved with AcOEt (50 ml); the organic phase is washed with NaHCO3 1M (3×50 ml), HCl 1M (3×50 ml), saturated aqueous solution of sodium chloride (3×50 ml). The organic solution is dried on anhydrous Na2SO4 and then it is brought to dryness. 0.450 g of 2-(benzo[b]thiophen-2-yl)-4-cyclopentyl-1,3-oxazolin-5-one (1.66 mmol, yield=96%) are obtained.
HPLC (method E): Rt=21.86 min.
1c) To a solution in 15 ml of anhydrous THF of tert-butyloxycarbonyl-D-phenylalanine N-hydroxysuccinimmidyl ester (500 mg, 1.38 mmol), N-(3-aminopropyl)-morpholine (1.328 mmol, 0.201 ml) is added. The stirring is maintained for 90 min. The solvent is then evaporated, and the residue dissolved with AcOEt (50 ml); the organic phase is washed with NaHCO3 1M (3×50 ml), HCl 1M (3×50 ml), saturated aqueous solution of sodium chloride (3×50 ml). The solution dried on anhydrous Na2SO4. Following the evaporation of the solvent, 474 mg of (ter-butyloxycarbonyl-D-phenylalanine-N-[3(morpholin-4-yl)propyl]amide) (1.21 mmol, yield=88%) are obtained.
HPLC (method E): Rt=10.35 mm.
1d) The product coming from Example 1c) (0.474 g, 1.21 mmol) is dissolved in 50 ml of a solution of HCl 4N in dioxane. After 30′ the solvent is evaporated, and the residue is dissolved and brought to dryness with toluene and with absolute ethanol. The so obtained solid is transferred into a separatory funnel with 50 ml of NaHCO3 and 50 ml of CHCl3. After shaking, the organic phase is collected and the aqueous phase is washed with other 5 portions of CHCl3. All the organic phases are collected, then transferred into a separatory funnel; one washing is made with a sodium chloride saturated solution. The organic phase is dried on Na2SO4. The solvent is evaporated. 0.291 g (1 mmol, yield=83%) of clear oil of D-phenylalanine-N-[3(morpholin4-yl)propyl]amide hydrochloride.
1e) To a solution in a mixture of 10 ml of anhydrous CH3CN and 1 ml of anhydrous THF of the product coming from Example 1b) (66.5 mg; 0.245 mmol), a solution of the product coming from Example 1d) (72 mg, 0.247 mmol) in 5 ml of anhydrous CH3CN is added. The reaction is allowed to reflux for 48 hours.
After removing the solvent at reduced pressure, the residue is dissolved with AcOEt (50 ml), and the solution is transferred in a separatory funnel, and washed once with a sodium chloride saturated solution. The organic phase is dried on Na2SO4 and the solvent evaporated. 135 mg of the product are obtained, which is then purified by reverse phase chromatography, using a column Hibar Merck LiChrospher 100, RP-18e (5 μm), eluents=A: H2O+0.1% TFA; B: CH3CN+0.1% TFA; elution with a linear gradient from 10% B at 50% B in 40 min, flow 10 ml/min; WV detection (λ=230 nm). The fractions corresponding to the peak of the product have been collected, concentrated to small volume at reduced pressure and lyophilised, yielding 0.124 mmol of Nα[Nα(benzo[b]thiophen-2-ylcarbonyl)-1-aminocyclopentane-1-carbonyl]-D-phenylalanine-N-[3(morpholin-4-yl)propyl]amide (yield=51% after purification).
MS (m/z): 563.3 (MH+). (HPLC (method E): Rt=14.03 min.
2a) To a solution of (1S,2S)-diaminocyclohexane (1.14 g, 10 mmol) in 50 ml of DCM, dibenzyl dicarbonate (2 g ,7 mmol) dissolved in 20 ml of DCM is added drop by drop. When the addition is over, the obtained precipitate is filtered and dried, obtaining 0.55 g of a white solid which is the starting diamine cyclohexane. The organic filtrate (80 ml) is extracted with HCl 1N (3×10 ml) and the aqueous extract are collected, alkalinise to pH=10 and extracted with DCM (3×10 ml). The collected organic extracts are dried on anhydrous Na2SO4, then filtered and brought to dryness. The residue is dissolved in 3 ml of methanol, then ethyl acetate saturated of hydrochloric acid (EtOAc/HCl) (2 ml) is added and, subsequently, ethyl ether (20 ml) yielding a white precipitate in suspension. This precipitate is filtered and dried thus obtaining 1.44 g of (1S,2S)-N-monobenzyloxycarbonyldiaminocyclohexane hydrochloride (yield=50,5%).
HPLC (E): Rt=7.85 min.
HPLC(E): Rt=15.5 min.
The residue is then dissolved in 10 ml of EtOAc and added with 20 ml of EtOAc/HCl; after 1 hour 100 ml of Et2O are added to the mixture, thus obtaining a white precipitate that, after filtration, gives 0.425 g of hydrochloride of (1R,3S)-3-aminocyclopentan-3-carboxylic-N-[(1S,2S-2-N-(benzyloxycarbonyl)aminocyclohexyl]amide acid.
HPLC(E): Rt=8.9 min.
2c) 0.425 g of the product coming from Example 2b) are dissolved in DMF (10 ml) and added with Nα(tert-butyloxycarbonyl)-D-phenylalanine (0.312 g, 1.29 mmol), EDC (0.230 g, 1.29 mmol), HOBt (0.175 g, 1.29 mmol) and DiPEA until an alkaline reaction is achieved. The mixture is kept to react overnight, then 30 ml of DCM are added and the organic phase is washed with KHSO4 5% (3×50 ml), NaHCO3 (3×50 ml), H2O (3×50 ml), and dried on anhydrous Na2SO4. After filtration and removal of the solvent, 531 mg of raw product are obtained from which, after grinding in Et2O, 470 mg of a white solid (yield=83%) are obtained. HPLC(E): Rt=16.06 ml.
The release of the Boc group with EtOAc/HCl and subsequent precipitation with Et2O (70 ml) allows the isolation of 410 mg of hydrochloride of (1R,3S)-Nγ-[D-phenylalanil]-3-aminocyclopentan-3-carboxylic-N-[(1S,2S)-2-N-(benzyloxycarbonyl)aminocyclohexyl) amide acid. HPLC(E)=11.27 min.
2d) Into a 100 ml glass protected with a CaCl2 tube 1.29 g (10 mmol) of 1-aminocyclohexan-1-carboxylic acid are suspended in 20 ml of DCM, and added with 5 ml of N,O-bis-(trimethylsilyl)-acetamide. The reaction mixture is kept under stirring for 2 hours until the amino acid derivative is completely dissolved. To the solution 1.8 g (0.92 mmol) of chloride of benzothiophene-2-carboxylic acid are added drop by drop in 20 ml of DCM, and the mixture is allowed to react for 1 hour at room temperature. The organic solution is then washed with water (5×50 ml), dried on anhydrous Na2SO4, filtered, brought to dryness and grinded with Et2O, thus yielding 2.60 g (yield=90.2%) of a white solid of Nα(benzo[b]thiophen-2-yl-carbonyl)-1-aminocyclohexane-1-carboxylic acid.
HPLC (method E): Rt=17.7 min.
2e) To a solution in anhydrous THF (25 ml) of the product coming from Example 2d) (0.50 g, 1.67 mmol), EDC (2.08 mmol, 0.402 g) and DiPEA (5.2 mmol, 0.890 ml) are added. The solution is maintained under stirring for 2 hours at room temperature. The solvent is then evaporated, the residue is dissolved in AcOEt (50 ml), the organic phase is washed with NaHCO3 1M (3×50 ml), HCl 1M (3×50 ml), sodium chloride saturated aqueous solution (3×50 ml). The organic solution is anhydrified on anhydrous Na2SO4 and then brought to dryness. 0.450 g of 2-(benzo[b]thiophen-2-yl)4-cyclohexyl-oxazol-5-one (1.57 mmol, yield=94%) are obtained. HPLC (method E): Rt=22.1 min.
2f) 0.176 g of the product coming from Example 2c), dissolved in 5 ml of anhydrous CH3CN, are added with 82 mg of the product coming from Example 2d) and with DiPEA until an alkaline reaction is achieved. The mixture is then heated to reflux for 18 hours. The reaction mixture is then diluted with DCM (20 ml) and washed with KHSO4 5% (3×50 ml), NaHCO3 (3×50 ml), H2O (3×50 ml), and dried on anhydrous Na2SO4. The mixture is filtered, brought to dryness and grinded with Et2O, thus obtaining 185 mg of a white solid. HPLC(E): Rt=17.08 min.
The so obtained solid product is dissolved in 12 ml of MeOH and 1 ml of acetic acid. The resulting solution is added with 250 mg of palladium on carbon and insufflated with hydrogen for 2 hours until, under HPLC at the above described conditions, the peak at Rt=17.08 min disappears and a main peak at Rt=11.78 min appears.
After filtration of the catalyst and removal of the solvent, the reaction mixture is then purified in 10 runs of preparative chromatography using a column Vydac Peptide&Protein (250×22 mm), 10μ, eluting with H2O+0,1% TFA (A) and CH3CN+0,1% TFA (B) (gradient 10% to 50% of B in 110 min; flow 12 ml/min.).
The chromatographic fractions which resulted pure from HPLC analysis are collected and evaporated to yield a residue of 70 mg of (1R,3S)-acid-Nγ{Nα[Nα(benzo[b]thiophen-2-yl-carbonyl) -1-aminocyclopentan-1-carboxy]-D-phenylalanil}-3-aminocyclopentan-1-carboxylic-N-[(1S,2S)-2-aminocyclohexyl]amide.
1H-NMR (δ, DMSO-d6): 1.2–1.9 (m, 22H, CH2); 2.6 (m, 1H, CH—CO-3Ac5c); 2.9–3.20 (m, 2H, (β)CH2—D-Phe); 3.85 (m, 1H, CH—NH, 1,2 di-NH2-cyclohexane); 4.2 (m, 1H, CH—NH-3Ac5c); 4.45 (m, 1H, (α)CH—D-Phe)); 7.1–7.25 (m, 5H, Carom—D-Phe); 7.45–7.85 (m, 8H, C(5)H+C(6)H+NHCH-1,2di-NH2-cyclohexane+NHCH-3Ac6c+NH3++NHCH—D-Phe); 7.90–8.02 (m, 2H, C(4)H+C(7)H); 8.3 (s, 1H, C(3)H); 8.9 (s, 1H, NH-1Ac5c). MS-FAB: 644,3 (M+H)+
With analogous methods or by obvious changes for a skilled person, the following products have been prepared:
MS-FAB: 664.32 (M+H)+ HPLC (method E): Rt=10.5 min.
MS-FAB: 641.32 (M+H)+ HPLC (method F): Rt=4.8 ml.
MS-FAB: 628.4 (M+H)+ HPLC (method F): Rt=3.37 min.
MS-FAB: 628.3 (M+H)+ HPLC (method E): Rt=9.25 min.
MS-FAB: 635.2 (M+H)+ HPLC (method E): Rt=12.9 ml.
MS-FAB: 572.3 (M+H)+ HPLC (method E): Rt=12.0 min.
MS-FAB: 686.3 (M+H)+ HPLC (method E): Rt=12.1 min.
MS-FAB: 656.3 (M+H)+ HPLC (method E): Rt=11.9 min.
MS-FAB: 656.3 (M+H)+ HPLC (method E): Rt=11.8 min.
MS-FAB: 692.4 (M+H)+ HPLC (method E): Rt=12.7 min.
1H-NMR (δ, DMSO-d6): 1.2–1.9 (m, 22H, CH2); 2.6 (m, 1H, CH—CO-3Ac5c); 2.90–3.20 (m, 2H, (β)CH2—D-Phe); 2.75 (m, 6H, N(CH3)2); 3.8 (s, 3H, 1-(CH3) indole) 4.2 (m, 1H, CH—NH-3Ac5c); 4.45 (m, 1H, (α)CH—D-Phe); 7.10–7.25 (m, 5H, Carom—D-Phe); 7.45–7.85 (m, 6H, C(5)H+C(6)H+NHCH-1,2di-NH2-cyclohexane+NHCH-3Ac6c+HN(CH3)2++NHCH—D-Phe); 7.90–8.02 (m, 2H, C(4)H+C(7)H); 8.5 (s, 1H, C(3)H); 8.9 (bs, 1H, NH-1Ac5c). MS-FAB: 669.3 (M+H)+ HPLC (method E): Rt=12.2 min.
MS-FAB: 632.2 (M+H)+ HPLC (method F): Rt=5.51 mm.
MS-FAB: 616.3 (M+H)+ HPLC (method F): Rt=5.58 min.
MS-FAB: 678 (M+H)+ HPLC (method E): Rt=12.8 min.
MS-FAB: 655 (M+H)+ HPLC (method E): Rt=12.2 min.
MS-FAB: 642.2 (M+H)+ HPLC (method E): Rt=12.0 mm.
1H-NMR (δ, DMSO-d6): 1.2–1.9 (m, 26H, CH2); 2.3 (m, 1H, CH—CO-3Ac6c); 2.8–3.15 (m, 2H, (β)CH2—D-Phe); 3.55 (m, 1H, CH—NH 1,2 di-NH2-cyclohexane); 4.1 (m, 1H, CH—NH-3Ac6c); 4.35 (m, 1H, (α)CH—D-Phe); 7.05–7.18 (m, 5H, Carom—D-Phe); 7.39–7.45 (m, 2H, C(5)H+C(6)H); 7.59–7.65 (m, 3H, NHCH); 7.89–7.96 (m, 2H, C(4)H+C(7)H); 8.3 (s, 1H, C(3)H); 8.42 (s, 1H, NH-1Ac6c). MS-FAB: 672,45 (M+H)+ HPLC(method E): Rt=12.7 min.
1H-NMR (δ, DMSO-d6): 1.2–1.9 (m, 24H, —CH2—); 2.2 (m, 1H, CH—CO-3Ac6c); 2.7 (m,2H, NH—CH2); 2.8–3.15 (m, 2H, (β)CH2—D-Phe); 3.0 (m, 2H, CH2—NH3+); 4.1 (m, 1H, CH—NH-3Ac6c); 4.35 (m, 1H, (α)CH—D-Phe); 7.05–7.18 (m, 5H, Carom—D-Phe); 7.59–7.65 (m, 3H, NHCH); 7.40–7.55 (m, 5H,C(5)H+C(6)H+NH3+); 7.89–7.96 (m, 2H,C(4)H+C(7)H); 8.3 (s, 1H, C(3)H), 8.42 (s, 1H, NH-1Ac6c). MS-FAB: 660.22 (M+H)+ HPLC (method E): Rt=12.4 min.
1H-NMR (δ, DMSO-d6): 1.2–1.9 (m, 24H, CH2); 2.2 (m, 1H, CH—CO-3Ac5c); 2.8–3.25 (m, 2H, (β)CH2—D-Phe); 4.0 (m, 1H, CH—NH-3Ac5c); 4.25 (m, 1H, CH—NH, 1,2 di-NH2—); 4.5 (m, 1H, (α)CH—D-Phe);); 7.1–7.25 (m, 5H, Carom—D-Phe); 7.45–7.55 (m, 2H, C(5)H+C(6)H); 7.6–7.75 (m, 3H, NHCH); 7.93–8.02 (m, 2H, C(4)H+C(7)H); 8.3 (s, 1H, C(3)H); 8.42 (s, 1H, NH-1Ac6c). MS-FAB: 658.30 (M+H)+ HPLC (method E): Rt=12.5 min.
MS-FAB: 659.20 (M+H)+ HPLC (method E): Rt=11.4 min
MS-FAB: 672.24 (M+H)+ HPLC (method E): Rt=12.8 min.
1H-NMR (δ, DMSO-d6): 1.2–1.9 (m, 24H, CH2); 2.2 (m, 1H, CH—CO-3Ac6c); 2.80–3.20 (m, 2H, (β)CH2—D-Phe); 3.75 (m, 1H, CH—NH 1,2 di-NH2-cyclohexane); 4.2 (m, 1H, CH—NH-3Ac6c); 4.45 (m, 1H, (α)CH—D-Phe); 7.1–7.25 (m, 5H, Carom—D-Phe); 7.40–7.50 (m, 3H, C(5)H+C(6)H+NHCH-3Ac6c); 7.60–7.70 (m, 4H, NHCH-1,2di-NH2-cyclohexane+NH3+); 7.85 (1H, NHCH—D-Phe);7.90–8.02 (m, 2H, C(4)H+C(7)H); 8.25 (s, 1H, C(3)H); 8.9 (s, 1H, NH-1Ac5c). MS-FAB: 658.35 (M+H)+ HPLC(method E): Rt=12.0 min.
MS-FAB: 658.30 (M+H)+ HPLC(method E): Rt=11.9 min.
1H-NMR (δ, DMSO-d6): 1.2–1.9 (m, 22H, CH2); 2.2 (m, 1H, CH—CO-3Ac5c); 2.70–3.25 (m, 2H, (β)CH2—D-Phe); 4.0 (m, 1H, CH—NH-3Ac5c); 4.2 (m, 1H, CH—NH 1,2 di-NH2-cyclohexane CH—NH-3Ac6c); 4.45 (m, 1H, (α)CH—D-Phe)); 7.10–7.25 (m, 5H, Carom—D-Phe); 7.45–7.70 (m, 7H, C(5)H+C(6)H+NHCH-1,2 di-NH2-cyclohexane+NHCH-3Ac6c+NH3+);7.85 (d, 1H, NHCH—D-Phe);• 7.90–8.02 (m, 2H, C(4)H+C(7)H); 8.25 (s, 1H, C(3)H); 8.9 (s, 1H, NH-1Ac5c). MS-FAB: 644.3 (M+H)+ HPLC(method E): Rt=11.8 min.
MS m/z: 561.3 (M+H+). HPLC (method C)Rt=12.16 min.
MS (m/z): 605.4 (MH+). HPLC (method B): Rt=4.57 min.
52a) To a suspension of 1-amino-1-cyclopentancarboxylic acid (19.2 g, 149 mmol) in 500 ml of anhydrous CH2Cl2, is added, under magnetic stirring, N,O-bis(trimethylsilyl)acetamide (BSA) (54 g, 268 mmol, containing 5% of TMSCI). After about 1 hour under stirring at room temperature, to this solution benzo[b]thiophene-2-carbonylchloride (29.2 g, 149 mmol) dissolved in 200 ml of CH2Cl2 is added in about 2 hours; the reaction mixture is kept under stirring for further 3 hours. The solution is then evaporated under reduced pressure, and the residue is treated with 200 ml of aqueous K2CO3 5% for 15 min, then extracted with AcOEt (2×100 ml). The aqueous phase is then acidified with aqueous HCl 37% until complete precipitation of a white solid, which is extracted with AcOEt (2×200 ml) and dried on anhydrous Na2SO4. The organic phase is filtered and evaporated, thus obtaining 1-[(benzo[b]thiophene-2-carbonyl)-amino]-cyclopentanecarboxylic acid (38.7 g, 134 mmol). HPLC (method A): Rt=3.51 min.
With analogous procedures the following intermediate products have also been obtained:
HPLC (method A): Rt=4.08 min.
HPLC (method A): Rt=4.03 min.
HPLC (method A): Rt=3.47 min.
HPLC (method A): Rt=3.74 min.
52b) To a solution in anhydrous THF (500 ml) of the product coming from Example 52a) (16 g, 55 mmol), EDC.HCl (12.7 g, 66 mmol) and diisopropylethylamine (30 ml) are added under magnetic stirring at room temperature. The reaction mixture is kept under stirring for 4 hours, then the solvent is evaporated to dryness, and the residue is dissolved in AcOEt (1000 ml) and washed with NaHCO3 10% (3×300 ml), citric acid 10% (3×300 ml), H2O (3×500 ml), then dried on Na2SO4 and brought to dryness, thus obtaining a whitish solid of 2-benzo[b]thiophen-2-yl-3-oxa-1-azaspiro[4.4]-non-1-en-4-one (13 g, 47.9 mmol).
HPLC (A): Rt=4.97 min.
With analogous procedures the following intermediate products have also been obtained:
HPLC(method A): Rt=5.55 min.
Rt=5.65 min.
Rt=4.97 min.
52c) To a solution in anhydrous DMF (100 ml) of Z-D-phenylalanine (5 g, 16.7 mmol) HOBt (2.34 g, 17.6 mmol) and EDC.HCl (8.73 g, 17.55 mmol) are added under magnetic stirring. After 30 min 3-aminopropanol (1.25 g, 16.72 mmol) dissolved in DMF (60 ml) is added to the solution, and the reaction mixture is kept at room temperature for 12 hours. The mixture is then diluted with AcOEt (200 ml) and extracted with KHSO4 (5% solution, 200 ml) and with NaHCO3 (saturated solution, 200 ml). The organic phase, dried on Na2SO4, filtered and brought to dryness, is then washed with ethyl ether and the white solid in suspension is filtered to give the desired product (5.15 g, 14.5 mmol).
HPLC (method B): Rt=3.48 ml.
The product coming from the previous reaction, is deprotected by hydrogenation (H2, Pd/C 10%) according to a procedure known to any person skilled in the art, to give a white solid of 2(R)-amino-N-(3-hydroxypropyl)-3-phenyl-propionamide (2.91 g, 14.5 mmol), which is used directly in the subsequent reaction.
52d) To the product coming from step 52b) (1 g, 3.7 mmol) dissolved in anhydrous DMF (40 ml), the intermediate described in 52c) (1.11 g, 5.6 mmol) is added, and the reaction is kept at room temperature under magnetic stirring for 24 hours. Then the mixture is diluted with AcOEt (150 ml) and the organic phase is washed with distilled water (3×50 ml) and a saturated solution of NaHCO3 (2×50 ml), then dried on anhydrous Na2SO4 and brought to dryness to give a white solid of benzo[b]thiophene-2-carboxylic acid {1-[1(R)-(3-hydroxypropylcarbamoyl)-2-phenyl-ethylcarbamoyl]-cyclopentyl}-amide (1.51 g, 3 mmol). HPLC (method A): Rt=3.73 min.
52e) The so obtained product (1.2 g, 2.44 mmol), without further purification, is dissolved in AcOEt (30 ml). To this solution, maintained at 0° C. with an ice bath, an aqueous solution of NaBr (0.5 M, 5.35 ml, 2.68 mmol), 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO, 3.4 mg, 0.022 mmol) is added and, drop by drop, aqueous NaClO (1.83 M, 1.55 ml). The biphasic mixture is kept at 0° C. under stirring for further 20 min, then the organic phase is separated and the aqueous phase is extracted with AcOEt (20 ml). The organic fractions are collected and washed with a buffered solution of KI (20 ml), 10% solution of Na2S2O3 (20 ml) and NaHCO3 (5%, 20 ml); they are then dried and evaporated to give a product, which is purified by flash chromatography (EtOAc/hexane, 80/20) to yield 0.82 g of a white solid of 4-[2(R)-({1-[(Benzo[b]thiophene-2-carbonyl)-amino]-cyclopentanecarbonyl}-amino)-3-phenyl-propionylamino]-propanal.
HPLC (method B): Rt=3.99 min.
The so obtained aldehyde (100 mg, 0.203 mmol) is dissolved in methanol (5 ml) and added with 1-methyl-[1,4]diazepan (115 mg, 1.01 mmol) and glacial acetic acid (0.2 ml). After the solution has been stirred for 15 min, Na(CN)BH3 (20 mg, 0.32 mmol) is added to the solution. After 12 hours the solvent is evaporated under reduced pressure and the raw product is dissolved in HCl 1M. The acid extract is washed with AcOEt (10 ml) and brought up to alkaline pH by adding solid NaHCO3, then it is extracted with AcOEt (2×25 ml). The organic phase is evaporated, then purified by means of preparative HPLC (colonna: Combi HT™ (SB C18, 5 μm, 100 Å, 21×50 mm), Method: H2O+0.1% TFA/MeCN+0.1% TFA, 95/5>5/95 in 10 min, flow=40 ml/min, λ=220, 270 nm) to yield benzo[b]thiophene-2-carboxylic acid (1-{1-[3-(4-methyl-[1,4]diazepan-1-yl)-propylcarbamoyl]-2-(R)-phenylethylcarbamoyl}-cyclopentyl)-amide TFA salt (40 mg, 0.067 mmol). MS (m/z): 590.5 (MH+). HPLC (method A): Rt=3.05 min.
With analogous methods the following products have also been prepared:
MS (m/z): 591.3 (MH+). HPLC (method A): Rt=3.64 min.
MS (m/z): 577.3 (MH+). HPLC (method A): Rt=3.31 min.
MS (m/z): 619.3 (MH+). HPLC (method A): Rt=3.68 min.
56a) Boc-D-phenylalanine O-succinimidoester (5 g, 16.56 mmol) is dissolved in CH3CN (60 ml) and added to a solution of β-alanine (2.08 g, 16.56 mmol) and TEA (4.61 ml) in 60 ml of water. After 30 min the organic solvent is evaporated and the aqueous residue, acidified by adding HCl, is extracted with AcOEt (3×100 ml). The organic extract is washed with water, dried and brought to dryness to give a colourless oil (4.19 g, 12.08 mmol). HPLC (B): Rt=3.19 min.
The so obtained product is deprotected under standard conditions (HCl, dioxane) and the so obtained amine (2.9 g, 12 mmol) is used, without flier purification steps, in the reaction with the intermediate product described in Example 52b), in the presence of stoichiometric DIPEA, in the conditions above described, to yield 3-[2(R)-({1-[(Benzo[b]thiophene-2-carbonyl)-amino]-cyclopentanecarbonyl}-amino)-3-phenyl-propionylamino]-propionic acid (5.17 g, 10.2 mmol). HPLC (B): Rt=3.98 min.
56b) The so obtained acid (100 mg, 0.2 mmol) is caused to react with 2,6-cis-dimethylpiperazine by a standard method for the formation of the amidic bond (EDC, HOBt, CH2Cl2). The raw product of the reaction is purified by preparative HPLC (same conditions of Example 52) to yield benzo[b]thiophene-2-carboxylic acid (1-{1(R)-[3-(3,5-cis-dimethyl-piperazin-1-yl)-3-oxo-propylcarbamoyl]-2-phenyl-ethylcarbamoyl}-cyclopentyl)-amide TFA salt (25 mg, 0.035 mmol).
MS (m/z): 604.2 (MH+). HPLC (method A): Rt=3.36 min.
With analogous methods, starting from suitable intermediate products as described in Example 52b) and using the obtained suitable amines when necessary, the following products have been prepared according to methods known to the skilled person:
MS (m/z): 653.3 (MH+). HPLC (method A): Rt=3.40 min.
MS (m/z): 725 (MH+, monoisotopic). HPLC (method A): Rt=3.76 min.
MS (m/z): 731 (MH+, monoisotopic). HPLC (method A): Rt=3.73 min.
MS (m/z): 674.2 (MH+). HPLC (method A): Rt=3.43 min.
MS (m/z): 660.3 (MH+). HPLC (method A): Rt=3.40 min.
MS (m/z): 676.2 (MH+). HPLC (method A): Rt=3.43 ml.
MS (m/z): 644.3 (MH+). HPLC (method A): Rt=3.55 min.
MS (m/z): 660.2 (MH+). HPLC (method A): Rt=3.68 min.
MS (m/z): 615.1 (MH+). HPLC (method A): Rt=3.79 min.
Benzo[b]thiophene-2-carboxylic acid {1-[2-phenyl-1(R)-(1-pyridin-2-ylmethyl-piperidin-4-ylcarbamoyl)-ethylcarbamoyl]-cyclopentyl}-amide TFA salt
66a) Z-D-phenylalanina (3.28 g, 11 mmol) is caused to react with N-Boc-(4-amino)piperidine hydrochloride (2.6 g, 11 mmol) under the usual conditions for peptidic coupling (HOBt, EDC, DIPEA, CH2Cl2) as described in Example 56b). The so obtained adduct (5 g, 10.3 mmol), after a simple extractive processing, is hydrogenated in the presence of Pd/C as the catalyst, so to obtain 4-(2(R)-amino-3-phenyl-propionylamino)-piperidine-1-carboxylic acid tert-butyl ester (3.21 g, 9.27 mmol).
HPLC (method B): Rt=3.22 min.
With analogous procedures the following intermidiates have also been obtained:
4-[(2(R-amino-3-phenyl-propionylamino) methyl]-piperidine-1-carboxylic acid tert-butyl ester
4-[2-(2(R)-amino-3-phenyl-propionylamino)-ethyl]-piperidine-1-carboxylic acid tert-butyl ester (in this case, the N-Boc-4-aminoethyl)piperidine obtained starting from the corresponding alcohol by obvious reaction for a skilled person, has been used).
66b) The compound obtained as in Example 66a) (1.69 g, 4.87 mmol) is reacted with DMF at room temperature for 24 hours with the oxazolinone described in Example 52b) (1.29 g, 4.8 mmol). The so obtained intermediate (2.9 g, 4.7 mmol, HPLC (B): Rt=4.55 min), after a simple extractive working, is deprotected by reaction with TFA in CH2Cl2, to yield benzo[b]thiophene-2-carboxylic acid {1-[2-phenyl-1(R)-(piperidin-4-ylcarbamoyl)-ethylcarbamoyl]-cyclopentyl}-amide TFA salt. MS (m/z): 519.2 (MH+). HPLC (method A): Rt=3.29 mm.
With analogous methods the following compounds have been prepared:
MS (m/z): 533.3 (MH+). HPLC (method A): Rt=3.29 min.
MS (m/z): 547.3 (MH+). HPLC (method A): Rt=3.39 min.
66c) 200 mg (0.31 mmol) of benzo[b]thiophene-2-carboxylic acid {1-[2-phenyl-1(R)-(piperidin-4-ylcarbamoyl)-ethylcarbamoyl]-cyclopentyl}-amide are used for the reaction of reductive amination with pyridine 2-carboxaldehyde (64 mg, 0.6 mmol), to yield, after purification by preparative HPLC according to the method already described in the previous Examples, the desired product benzo[b]thiophene-2-carboxylic acid {1-[2-phenyl-1(R)-(1-pyridin-2-ylmethyl-piperidin-4-ylcarbamoyl)-ethylcarbamoyl]-cyclopentyl}-amide TFA salt (120 mg, 0.16 mmol).
MS (m/z): 610.3 (MH+). HPLC (method A): Rt=3.58 min.
In analogy to what described in Example 66c), the reaction in DMF, at room temperature and for times ranging from 12 to 48 hours, of the intermediate products described in Example 66a) with oxazolinones described in Example 52b), and the reaction of the intermediate products described in Example 66a) with 1-(3-(E)p-Tolyl-acryloylamino)-cyclopentanecarboxylic acid described in Example 52a) under conditions of peptidic coupling, gives, after deprotection of the amino groups, intermediates analogous to those described in Example 66b). Such intermediates are alkylated under conditions which are widely reported in the literature and well known to any person skilled in the art, such as:
With the synthetic procedure described in Example 66 the following products have been prepared:
MS (m/z): 617.3 (MH+). HPLC (method A): Rt=3.44 min.
MS (m/z): 631.3 (MH+). HPLC (method A): Rt=3.57 min.
MS (m/z): 709 (MH+, isotopic pattern of Br). HPLC (method A): Rt=3.82 mm. 1H NMR (400 MHz): (δ, DMSO-d6) 1.12–2.13 (m, 17 H); 2.17–2.30 (m, 1H); 2.74–3.00 (m, 5H), 3.02–3.38 (m, 5H); 3.44–3.57 (m, 2H); 3.80–3.93(m, 2H); 4.39–4.50 (m, 1H); 7.11–7.24 (m, 5H); 7.55–7.66 (m, 2H); 7.85–7.92 (m, 1H); 7.93–7.98 (m, 1H); 8.30 (s, 1H); 8.34–8.38 (m, 1H); 8.97 (s, 1H); 8.78 and 9.01 (2 broad signal, 1H total).
MS (m/z): 575.3 (MH+). HPLC (method A): Rt=3.56 min.
MS (m/z): 631.3 (MH+). HPLC (method A)=Rt=3.51 min. 1H NMR (500 MHz): (δ, DMSO-d6) amongst the others 0.99–1.14 (m, 4H); 2.18–2.29 (m, 1H), 2.65–2.75 (m, 2H); 2.78–3.02 (m, 3H); 3.75–3.85 (m, 2H); 4.41–4.50 (m, 1H); 7.09–7.23 (m, 5H); 7.43–7.52 (m, 3H); 7.86 (d, 1H, J=8.6); 7.94–8.07 (m, 2H); 8.30 (s, 1H); 8.89 (s, 1H).
MS (m/z): 703.3 (MH+, isotopic pattern of Br). HPLC (method A): Rt=3.88 ml. 1H NMR (600 MHz): (δ, DMSO-d6) 0.90–1.11 (m, 4H); 1.18–1.29 (m, 1H); 1.38–1.73 (m, 12H); 1.74–1.82 (m, 1); 1.87–1.91 (m, 2H); 1.92–1.99 (m, 1H); 2.27–2.34 (m, 1H); 2.50–2.56 (m, 2H); 2.81–2.89 (m, 2H); 2.94–3.02 (m, 1H); 3.16–3.28 (m, 3H); 3.75–3.83 (m, 2H); 4.44–4.50 (m, 1H); 7.11–7.21 (m, 5H); 7.46 (t, 1H, J=5.77 Hz); 7.71–7.75 (m, 1H); 7.82(d, 1H, J=8.65 Hz); 7.98–8.04 (m, 3H); 8.29–8.31 (m, 1H); 8.53 (s, 1H); 8.83 (s, 1H).
MS (m/z): 614.4 (MH+). HPLC (method A): Rt=3.46 min.
MS (m/z): 615.4 (MH+). HPLC (method A): Rt=3.56 min. 1H NMR (400 MHz): (δ, DMSO-d6) amongst the others 1.13–1.25 (m, 2H); 2.15–2.23 (m, 1H); 2.34 (s, 3H); 3.80–3.87 (m, 2H); 4.36–4.44 (2 m, 1H total); 6.68 and 6.69 (2 d, 1H total J=15.81 for both); 7.13–7.30 (m, 7H); 7.375 and 7.38 (2 d, 1 H total, J=15.81 for both); 7.45–7.51 (m, 2H); 7.66 and 7.73 (2 t,1 H total, J=5.8 Hz for both); 7.82 and 7.83 (2 d, 1H total, J=8.67 for both); 8.54 and 8.55 (2 s, 1H total); 8.80 and 8.97 (2 broad signal, 1H total).
MS (m/z): 645.5 (MH+). HPLC (method A): Rt=3.63 min.
MS (m/z): 633.4 (MH+, con isotopic pattern of Br). HPLC (method A): Rt=3.86 min.
MS (m/z): 631.4 (MH+). HPLC (method A): Rt=3.53 min.
MS (m/z): 645.3 (MH+). HPLC (method A): Rt=3.61 min.
MS (m/z): 629.3 (MH+). HPLC (method A): Rt=4.11 min.
MS (m/z): 689.3 (MH+, isotopic pattern of Br). HPLC (method A): Rt=3.83 min.
MS (m/z): 633.5 (MH+). HPLC (method A): Rt=3.79 min.
MS (m/z): 647.3 (MH+). HPLC (method A): Rt=3.91 min.
MS (m/z): 613.3 (MH+). HPLC (method A): Rt=3.82 ml.
MS (m/z): 702 (MH+, isotopic pattern of Br). HPLC (method A): Rt=3.55 min.
MS (m/z): 605 (MH+, isotopic pattern of Br). HPLC (method A): Rt=3.71 min.
MS (m/z): 628.4 (MH+). HPLC (method A): Rt=3.60 min.
MS (m/z): 586.3 (MH+). HPLC (method A): Rt=3.60 min.
MS (m/z): 633.3 (MH+). HPLC (method A): Rt=3.50 min.
MS (m/z): 705.3 (MH+, isotopic pattern of Br). HPLC (method A): Rt=3.62 min.
MS (m/z): 633.4 (MH+). HPLC (method A): Rt=3.66 min.
MS (m/z): 651.3 (MH+, isotopic pattern of Cl). HPLC (method A): Rt=3.73 ml.
MS (m/z): 635.3 (MH+, isotopic pattern of Cl). HPLC (method A): Rt=3.70 min.
MS (m/z): 651.3 (MH+, isotopic pattern of Cl). HPLC (method A): Rt=3.98 min.
94a) 1-trityl-2-(R)-benzyl-1,2-ethylendiamine.
To a suspension of 24 g of D-phenylalaninamide in DMF (200 ml) 41.0 g di trityl chloride are added under stirring at room temperature and, after 1 hour, 80 ml of TEA diluted in 100 ml of DMF are also added. Stirring is maintained for 10 hours, then the solvent is evaporated, the residue is dissolved in ethyl acetate and washed with NaCl aq. 10%, citric acid aq. 10% and again with NaCl aq. 10%. After drying and evaporation, the residue is then dissolved in 200 ml of DMF. To th eso obtained solution 2000 ml of distilled water are added drop by drop. A precipitate is obtained, which is first brought to dryness then dissolved in 200 ml of THF and this solution is added, drop by drop, in 20 min at 0° C., under stirring and under nitrogen atmosphere, to a solution of 400 ml of LiAlH4 0.62 M in THF. The mixture is heated and maintained under reflux for 4 hours. After the mixture has been brought to 0° C., 15 ml of distilled water, 15 ml of NaOH aq. 15% and 45 ml of water are added in this order. By filtration of the so obtained precipitate, 1-trityl-2-(R)-benzyl 1,2-ethylendiamine is obtained.
94b) To a solution of 1-trityl-2-(R)-benzyl-1,2-ethylendiamine coming from Example 94a) (21.6 g, 55 mmol) in anhydrous CH2Cl2 (300 ml) and TEA (8.4 ml, 60.5 mmol) maintained at 0° C., 2-nitrobenzensolphonylchloride (12.2 g, 55 mmol) is added in small portions. Once the addition is over, the mixture is kept under stirring at room temperature for 4 hours more, then it is washed with NaCl aq. 15% (150 ml), NaHCO3 aq. 5% (150 ml) and again with NaCl aq. 15% (150 ml). The organic phase dried and evaporated at reduced temperature yield a solid product, which is then dissolved in 150 ml of CH3CN, cooled at 0° C. and treated with an excess of HCl in dioxane. After 2 hours at room temperature the solvent is evaporated to dryness, and the solid residue is dissolved in ethyl ether and filtered to yield a white solid of N-(2(R)-Amino-3-phenyl-propyl)-2-nitro-benzenesulphonamide hydrochloride.
HPLC (method A): Rt=2.70 min.
94c) The oxazolinone described in Example 52b) (407 mg, 1.5 mmol) and the intermediate 94b) described above (670 mg, 1.5 mmol) are dissolved in DMF (8 ml), added with TEA (0.64 ml, 4.5 mmol) and maintained at 80° C. under stirring for 2 hours. The residue obtained from evaporation of the solvent is dissolved with AcOEt (30 ml) and washed with HCl 1M (10 ml), NaHCO3 (5%, 10 ml), and NaCl aq. 15% (10 ml). The so obtained raw product is purified by flash chromatography (AcOEt/hexane 1/1) to give 0.5 g (0.8 mmol) of the intermediate benzo[b]thiophene-2-carboxylic acid {1-[1(R)-benzyl-2-(2-nitro-benzenesulphonylamino)-ethylcarbamoyl]-cyclopentyl}-amide.
HPLC (method B): 4.74 min.
94d) 1.2 g (3.36 mmol, 3 mmol/g of loading) of triphenylphosphinic resin are maintained to swell for 30 min in CH2Cl2 (25 ml) under balancing stirring; to the suspension the intermediate described in Example 94c) (0.5 g, 0.84 mmol), N-Boc-4-hydroxyethyl-piperidine (600 mg, 2.52 mmol) and tert-butyl-diazodicarboxylate (600 mg, 2.52 mmol) are added. After 16 hours under stirring the resin is removed by filtration and the solution is brought to small volume (10 ml). 5 ml of trifluoroacetic acid are added under stirring. After 1 hour the solution is brought to dryness, the residue is dissolved with AcOEt (30 ml) and the solution washed with Na2CO3 aq. 5% (15 ml) e NaCl aq. 15% (10 ml), dried and evaporated Benzo[b]thiophene-2-carboxylic acid (1-{1(R)-benzyl-2-[(2-nitro-benzenesulphonyl)-(2-piperidin-4-yl-ethyl)-amino]-ethylcarbamoyl}-cyclopentyl)-amide is obtained as a foamy solid (553 mg, 0.77 mmol).
HPLC (method A): Rt=4.20 min
95e) The product coming from the previous step (60 mg, 0,084 mmol) is dissolved in CH2Cl2 (2 ml) and to this solution 60 μl of TEA and 13.5 μl of thiophen-2-yl-acethyl chloride are added. The magnetic stirring is maintained for 12 hours, then the solvent is evaporated and the residue, dissolved with AcOEt (10 ml), is washed with Na2CO3 aq. 5% (2×5 ml). The raw product obtained from evaporation of the solvent (60 mg, 0.07 mmol), is dissolved in DMF (3 ml) and treated with diazabicycloundecene (DBU, 32 mg, 0.21 mmol) and 2-mercaptoethanol (17 mg, 0.21 mmol) for 12 hours. The residue obtained from evaporation of the solvent at reduced pressure is washed with ethyl ether and purified by preparative HPLC yielding 10 mg (0.013 mmol) of benzo[b]thiophene-2-carboxylic acid [1-(1(R)-benzyl-2-{2-[1-(thiophen-2-yl-acetyl)-piperidin-4-yl]-ethylamino}-ethylcarbamoyl)-cyclopentyl]-amide TFA salt as white lyophilic product.
MS (m/z): 657.3 (MH+). HPLC (method A): Rt=4.12 min.
[2-(2(R)-Amino-3-phenyl-propionylamino)-ethyl]-carbamic acid tert-butyl ester (200 mg, 0.65 mmol), obtained by coupling of N-Boc-ethylendiamine and Z-D-phenylalanine succinimidoester followed by hydrogenation, is added to a solution of 2-benzo[b]thiophen-2-yl-3-oxa-1-azaspiro[4.4]-non-1-en-4-one (176 mg, 0.65 mmol) in DMF and maintained under magnetic stirring at room temperature for 48 hours. The so obtained product is deprotected (HCl, dioxane), acylated with 1-Boc-amino-1-cyclohexancarboxylic acid (156 mg, 0.65 mmol), by standard activation as described above, then again deprotected (HCl, dioxane). The raw product obtained from extractive work up is purified by flash chromatography (eluent: CHCl3/MeOH 9:1) to give benzo[b]thiophene-2-carboxylic acid [1-(1(R)-{2-[(1-amino-cyclohexanecarbonyl)-amino]ethylcarbamoyl}-2-phenyl-ethylcarbamoyl)-cyclopentyl]-amide (160 mg, 0.27 mmol).
MS (m/z): 604.3 (MH+). HPLC (method A): Rt=3.58 min.
With analogous methods the following products have been prepared:
MS (m/z): 658.3 (MH+). HPLC (method A): Rt=3.62 min.
MS (m/z): 730.2 (MH+, isotopic pattern of Br). HPLC (method A): Rt=4.00 min.
MS (in/z): 716.3 (MH+, isotopic pattern of Br). HPLC (method A): Rt=3.95 min.
MS (m/z): 716.3 (MH+, isotopic pattern of Br). HPLC (method A): Rt=3.97 min.
Analogously to Example 95) 4-[2-2(R)-amino-3-phenyl-propionylamino)-ethyl]-piperazine-1-carboxylic acid tert-butyl ester (obtained starting from Z-D-phenylalanine and N-Boc-4-hydroxyethylpiperazine by reactions and couplings widely described in literature and in part already reported in previous examples) (100 mg, 0.26 mmol) and 2-benzo[b]thiophen-2-yl-3-oxa-1-azaspiro[4.4]-non-1-en-4-one (72 mg, 0.26 mmol) are caused to react in DMF to yield a product which is deprotected and subsequently acylated with thiophen-2-yl-acethyl chloride in CH2Cl2 (as described in Example 94 and purified by flash chromatography using AcOEt as the eluent. 69 mg (0.10 mmol) of benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1(R)-{2-[4-(thiophen-2-yl-acetyl)-piperazin-1-yl]-ethylcarbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide are so obtained.
MS (m/z): 672.3 (MH+). HPLC (method A): Rt=3.87 min.
The compound of the title is obtained by a procedure analogous to that described in Example 100, but carrying out a reductive amination with tetrahydropyranyl 4-carboxyaldehyde in place of an acylation step with thiophen-2-yl-acethyl chloride.
MS (m/z): 646.2 (MH+). HPLC (method A): Rt=3.24 min.
196 mg of 5-chlorobenzofuran-2-carboxylic acid are suspended in 12 ml of anhydrous CH2Cl2, in nitrogen atmosphere. 100 μl of oxalyl chloride and one drop of DMF are then added. The stirring is maintained until the reaction is finished. The solvent is then evaporated and the residue is brought to dryness in a hard vacuum. 87 mg of the so obtained acylic chloride are added to a mixture of 132 mg of 1-amino-cyclopentanecarboxylic acid [1(R)-(3-morpholin-4-yl-propylcarbamoyl)-2-phenyl-ethyl]-amide bis-hydrochloride, 200 μl of TEA and 10 ml of anhydrous CH2Cl2 under magnetic stirring. Once the reaction is over, the solvent is evaporated and the residue is dissolved with a mixture of ethyl acetate and K2CO3 aq. 10%, by vigorously stirring. After separation of the organic phase, washings with basic water, drying on anhydrous Na2SO4, filtration and evaporation of the solvent, 134 mg of a residue are obtained, which are then purified by flash chromatography, eluting with increasing amounts of methanol in ethyl acetate until MeOH/AcOH=1/6 v/v, so to obtain the desired amide.
MS (m/z): 581.3 (MH+). HPLC (method C): Rt=13.14 min.
With analogous procedure the following compounds have been prepared:
MS (m/z): 597.3 (M+H+). HPLC (method C): Rt=13.80 min.
MS m/z: 625.2 (M+H+, monoisotopic). HPLC (method C): Rt=13.41 min.
MS m/z: 597.3 (N+H+). HPLC (method C): Rt=13.58 min.
MS m/z: 593.3 (M+H+). HPLC (method C): Rt=12.62 min.
MS m/z: 597.3 (M+H+). HPLC (method C): Rt=13.79 min.
MS m/z: 641.3 (M+H+, monoisotopic). HPLC (method C): Rt=13.86 min.
MS m/z: 655.2 (M+H+, monoisotopic). HPLC (method C): Rt=14.63 min.
MS m/z: 578.3 (M+H+). HPLC (method C): Rt=13.18 min.
MS m/z: 594.3 (M+H+). HPLC (method C): Rt=14.03 min.
MS m/z: 577.3 (M+H+). HPLC (method C): Rt=13.26 min.
MS m/z: 561.3 (M+H+). HPLC (method C): Rt=13.01 min.
MS m/z: 574.3 (M+H+). HPLC (method D): Rt=13.62 min.
MS m/z: 578.2 (M+H+). HPLC (method D): Rt=8.62 min.
MS m/z: 631.3 (M+H+). HPLC (method C): Rt=14.57 min.
118a) 18.0 g of 4-tetrahydropyrancarboxylic acid are introduced in a 250 ml flask with a calcium chloride tube, and dissolved in 130 ml of CH2Cl2. Under magnetic stirring, 15 ml of oxalyl chloride and 3 drops of DMF are added successively. The solution, that regularly degasses, is kept under magnetic stirring for 16 hours. After evaporation of the solvent, the residue is dried in hard vacuum at room temperature, dissolved in 100 ml of CH2Cl2 and put in a bath of ice and water, maintaining a vigorous magnetic stirring. A solution of 21.75 g of ethyl 4-piperidincarboxylate in 30 ml of CH2Cl2 containing 15.35 g of TEA is added to the mixture by means of a dripping funnel. During the addition, that lasts 3 hours, a clear suspension forms. The reaction mixture is left to stand overnight, then CH2Cl2 is evaporated and the residue is brought to dryness in hard vacuum. The residue is dissolved in 110 ml of ammonia aq. 25%, then methanol is added until complete solubilisation. Methanol is refluxed until the ester disappears (samples are taken, evaporated and analised by 1H-NMR). The solution is brought to small volume and extracted 25 times with 100 ml of chloroform to give, after evaporation of the solvent, 21.4 g of raw diamide.
14.79 g of the so obtained diamide are added in portions to 175 ml of a solution 1M of borane in THF. The addition lasts about 1 hour and it is carried out under a nitrogen flow, so that the temperature does not exceed 35° C. Once the addition is completed, the reaction mixture is heated to reflux and the reflux is maintained for 11 hours. In a bath of ice and water, 130 ml of a solution 4M of HCl in 1,4-dioxane, previously diluted with 100 ml of methanol, are added drop by drop to the solution obtained as above. The reaction mixture is heated to reflux, and the reflux is maintained for 12 hours before cooling at 0–4° C.
By filtration 7.95 g of C-[1-(tetrahydro-pyran-4-ylmethyl)-piperidin-4-yl]-methylamine bis-hydrochloride are recovered. From mother liquor, by dilution to doubled volume with diethyl ether, further 1.85 g of the desired product are recovered.
1H-NMR (200 MHz, DMSO-d6), • (ppm): 1.09–1.33 (m, 2H); 1.51–2.20 (m, 8H); 2.59–3.08 (m, 6H); 3.09–3.58 (m, 4H); 3.76–3.91 (m, 2H); 8.18 (br, 3H); 10.20 (br, 1H).
Analogously, the following amines have also been prepared:
MS (m/z): 227.2 (MH+) 1H NMR (200 MHz): (δ, CDCl3) 0.94 (s, 3H); 1.08–1.69 (m, 9H); 2.10 (s, 2H); 2.10–2.30 (m, 2H); 2.48–2.59 (m, 2H); 2.67–2.83 (m, 2H); 3.47–3.79 (m, 4H). C-[4-Methyl-1-(4-methyl-tetrahydro-pyran-4-ylmethyl)-piperidin-4-yl]-methylamine MS (m/z): 227.3 (MH+)
MS (m/z): 241.2 (MH+)
117b) Staring from the amine obtained in Example 117a), by a peptidic synthesis using Boc, under the operative conditions well known to a skilled person, by reacting the above said amine with Boc-D-phenylalanine O-succinimidoester, deprotecting, reacting with N-Boc 1-amino-1-cyclopentancarboxylic acid and deprotecting, the compound 1-amino-cyclopentanecarboxylic acid (2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethyl)amide is obtained
This compound is caused to react with the chloride of 6-iodonaphthalen-2-carboxylic acid, by a procedure analogous to that described in Example 102), thus obtaining the final product &iodo-naphthalene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide.
MS m/z: 751.3 (M+H+). HPLC (method D): Rt=14.00 min.
Analogously to that described in Example 117), by reaction of the corresponding carboxylic acids activated with 1-amino-cyclopentanecarboxylic acid (2-phenyl-1R-{[1-(tetrahydro-pyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethyl)-amide, the following compounds have been obtained:
MS m/z: 655.2 (M+H+). HPLC (method D): Rt=12.42 min. 1H-NMR (DMSO-d6). δ (amongst the others): 0.94–1.07 (m, 4H); 1.42–1.72 (m, 12H); 1.74–1.81 (m, 1H); 1.89–2.00 (m, 3H); 2.24–2.33 (m, 1H); 2.55–2.63 (m, 2H); 2.82–2.90 (m, 2H); 2.94–3.01 (m, 1H); 3.74–3.83 (m, 2H); 3.91 (s, 3H); 4.42–4.49 (m, 1H); 7.10–7.21 (m, 5H); 7.23–7.27 (m, 1H); 7.38–7.41 (m, 1H); 7.51 (t, J=5.7 Hz, 1H); 7.79 (d, J=8.6, 1H); 7.86–7.97 (m, 3H); 8.45 (s, 1H); 8.69 (s, 1H).
MS m/z: 693.5 (M+H+, monoisotopic). HPLC (method D): Rt=13.18 min. 1H-NMR (DMSO-d6). δ(amongst the others): 0.97–1.12 (m, 4H); 1.39–1.73 (m, 12H); 1.74–1.81 (m, 1H); 1.89–1.96 (in, 1H); 1.97–2.04 (m, 2H); 2.21–2.29 (m, 1H); 2.63–2.71 (m, 2H); 2.79–2.92 (m, 2H); 2.95–3.03 (m, 1H); 3.77–3.85 (m, 2H); 4.40–4.48 (m, 1H); 7.10–7.22 (m, 5H); 7.45 (t, J=5.7 Hz, 1H); 7.54 (dd, J=1.7 and 8.4 Hz, 1H); 7.67 (d, J=0.8 Hz, 1H) 7.79 (d, J 8.4, 1H); 7.85 (d, J=8.6 Hz, 1H); 7.95 (s,br, 1H); 8.85 (s, 1H).
MS m/z: 649.3 (M+H+). HPLC (method D): Rt=13.00 min. 1H-NMR (DMSO-d6). • (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.74–1.81 (m,1H); 1.89–1.96 (m, 1H); 1.97–2.04 (m, 2H); 2.212.29 (m, 1H); 2.63–2.71 (m, 2H); 2.79–2.92 (m, 2H); 2.95–3.03 (m, 1H); 3.77–3.85 (m, 2H); 4.40–4.48 (m, 1H); 7.10–7.22 (m, 5H); 7.42 (dd, J=1.8 and 8.4 Hz, 1H); 7.46 (t, J=5.8 Hz, 1H); 7.66 (d, J=0.9 Hz, 1H) 7.81–7.86 (m, 21H); 8.84 (s, 1H).
MS m/z: 633.5 (M+H+). HPLC (method D): Rt=12.10 min. 1H-NMR (DMSO-d6).• (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.74–1.81 (m, 1H); 1.89–1.96 (m, 1H); 1.97–2.04 (m, 2H); 2.21–2.29 (m, 1H); 2.63–2.71 (m, 2H); 2.78–2.93 (m, 2H); 2.95–3.03 (m, 1H); 3.75–3.85 (m, 2H); 4.40–4.48 (m, 1H); 7.10–7.22 (m, 5H); 7.32–7.37 (m, 1H); 7.46 (t, J=5.8 Hz, 1H); 7.60–7.66 (m, 2H); 7.71 (dd, J=4.1 and 9.0 Hz, 1H) 7.83 (d, J=8.6 Hz, 1H); 8.84 (s, 1H).
MS m/z: 649.5 (M+H+). HPLC (method D): Rt=12.78 min. 1H-NMR (DMSO-d6). • (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.74–1.81 (m, 1H); 1.89–1.96 (m, 1H); 1.97–2.04 (m, 2H); 2.21–2.29 (m, 1H); 2.63–2.71 (m, 2H); 2.78–2.93 (m, 2H); 2.95–3.03 (m, 1H); 3.75–3.85 (m, 2H); 4.40–4.48 (m, 1H); 7.10–7.22 (m, 5H); 7.46 (t, J=5.8 Hz, 1H); 7.50–7.53 (m, 1H); 7.61 (s, br, 1H); 7.71 (d, J=8.8 Hz, 1H) 7.85 (d, J=8.6 Hz, 1H); 7.92 (d, J=2.2, 11H); 8.87 (s, 1H).
MS m/z: 693.5 (M+H+, monoisotopic). HPLC (method D): Rt=13.14 ml. 1H-NMR (DMSO-d6). • (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.74–1.81 (m, 1H); 1.89–1.96 (m, 1H); 1.97–2.04 (m, 2H); 2.21–2.29 (m, 1H); 2.63–2.71 (m, 2H); 2.78–2.93 (m, 2H); 2.95–3.03 (m, 1H); 3.75–3.84 (m, 2H); 4.40–4.48 (m, 1H); 7.11–7.21 (m, 5H); 7.44 (t, J=5.8 Hz, 1H); 7.58–7.69 (m, 3H) 7.85 (d, J=8.6 Hz, 1H); 8.06 (d, J=2.0, 1H); 8.87 (s, 1H).
MS m/z: 671.6 (M+H+). HPLC (method D): Rt=15.16 min.
MS m/z: 629.5 (M+H+). HPLC (method D): Rt=12.69 min.
MS m/z: 629.5 (M+H+). HPLC (method D): Rt=12.66 min. 1H-NMR (DMSO-d6). • (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.74–1.81 (m, 1H); 1.89–1.96 (m, 1H); 1.97–2.04 (m, 2H); 2.21–2.29 (m, 1H); 2.44 (s, 3H) 2.63–2.71 (m, 2H); 2.78–2.93 (m, 2H); 2.95–3.03 (m, 1H); 3.75–3.84 (m, 2H); 4.40–4.48 (m, 1H); 7.11–7.21 (m, 5H); 7.29–7.33 (m, 1H) 7.49 (t, J=5.7 Hz, 1H); 7.53–7.60 (m, 3H); 7.86 (d, J=8.5 Hz, 1H); 8.76 (s, 1H).
MS m/z: 741.5 (M+H+). HPLC (method D): Rt=13.42 min. 1H-NMR (DMSO-d6). • (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.741.81 (m, 1H); 1.89–1.96 (m, 1H); 1.97–2.04 (m, 214); 2.21–2.29 (m, 1H); 2.63–2.71 (m, 2H); 2.78–2.93 (m, 2H); 2.95–3.03 (m, 1H); 3.75–3.84 (m, 2H); 4.40–4.48 (m, 1H); 7.11–7.21 (m, 5H); 7.43 (t, J=5.8 Hz, 1H); 7.53 (d, J=8.7 Hz, 111) 7.58 (s, 1H); 7.76 (dd, J=1.8 and 8.7 Hz, 1H); 7.86 (d, J=8.6 Hz, 1H); 8.22 (d, J=1.7, 11H); 8.88 (s, 1H).
MS m/z: 671.5 (M+H+). HPLC (method D): Rt=14.90 min.
MS m/z: 757.2 (M+H+). HPLC (method D): Rt=13.85 min.
MS m/z: 665.4 (M+H+). HPLC (method D): Rt=13.32 min. 1H-NMR (DMSO-d6). • (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.74–1.81 (m, 1H); 1.87–1.94 (m, 1H); 2.17–2.28 (m, 1H); 2.65–2.71 (m, 2H); 2.79–2.87 (m, 1H); 2.88–2.99 (m, 2H); 3.31–3.20 (m, 1H); 3.76–3.84 (m, 2H); 4.42–4.49 (n, 1H); 7.10–7.21 (m, 5H); 7.44 (t, J=5.8 Hz, 1H); 7.49 (dd, J=2.0 and 8.5, 1H), 7.85 (d, J=8.6 Hz, 1H); 8.00 (d, J=8.5 Hz, 1H); 8.22 (d, J=1.9 Hz, i); 8.28 (s, 1H); 8.93 (s, 1H).
MS m/z: 709.4 (M+H+, monoisotopic). HPLC (method D): Rt=13.12 min.
MS m/z: 757.4 (M+H+). HPLC (method D): Rt=13.38 min.
MS m/z: 699.5 (M+H+). HPLC (method D): Rt=13.88 min.
MS m/z: 645.5 (M+H+). HPLC (method D): Rt=12.83 min 1H-NMR (DMSO-d6). • (amongst the others): 0.98–1.12 (m, 4H); 1.36–1.82 (m, 14H); 1.87–1.95 (m, 1H); 1.95–2.06 (m, 2H); 2.19–2.29 (m, 1H); 2.53 (s, 3H); 2.67–2.74 (m, 2H); 2.80–2.94 (m, 2H); 3.15–3.28 (m, 3H); 3.76–3.83 (m, 2H); 4.41–4.48 (m, 1H); 7.10–7.23 (m, 5H); 7.29–7.33 (m, 1H); 7.37–7.41 (m, 1H); 7.44 (t, J=5.7 Hz, 1H); 7.86–7.83 (m, 1H); 7.86 (d, J=8.6 Hz, 1H); 8.31 (s, 1H); 8.86 (s, 1H).
MS m/z: 661.5 (M+H+). HPLC (method D): Rt=12.32 min.
MS m/z: 699.5 (M+H+). HPLC (method D): Rt=13.31 min.
MS m/z: 665.4 (M+H+). HPLC (method D): Rt=12.99 min.
MS m/z: 645.5 (M+H+). HPLC (method D): Rt=13.03 min. 1H-NMR (DMSO-d6). • (amongst the others): 1.00–1.11 (m, 4H); 1.43–1.80 (m, 13H); 1.87–1.94 (m, 1H); 1.95–2.04 (m, 2H); 2.18–2.28 (m, 1H); 2.44 (s, 3H); 2.66–2.73 (m, 2H); 2.80–2.87 (m, 1H); 2.90–3.00 (m, 2H); 3.14–3.27 (m, 3H); 3.75–3.84 (m, 2H); 4.41–4.50 (m, 1H); 7.10–7.21 (m, 5H); 7.29–7.33 (m, 1H); 7.47 (t, J=5.8 Hz, 1H); 7.75 (s, 1H); 7.84 (d, J=8.6 Hz, 1H); 7.87–7.91 (m, 1H); 8.19 (s, 1H); 8.83 (s, 1H).
MS m/z: 645.5 (M+H+). HPLC (method D): Rt=13.02 min. 1H-NMR (DMSO-d6). • (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.74–1.81 (m, 1H); 1.87–1.94 (m, 1H); 2.17–2.28 (m, 1H); 2.45 (s, 3H); 2.67–2.73 (m, 2H); 2.79–2.87 (m, 1H); 2.88–2.99 (m, 2H); 3.31–3.20 (m, 1H); 3.76–3.84 (m, 2H); 4.42–4.49 (m, 1H); 7.10–7.21 (m, 5H); 7.28 (d, J=8.2 Hz, 1H); 7.47 (t, J=5.6 Hz, 1H); 7.78–7.88 (m, 3H); 8.22 (d, J=1.9 Hz, 1H); 8.22 (s, 1H); 8.80 (s, 1H).
MS m/z: 649.3 (M+H+). HPLC (method D): Rt=12.51 min. 1H-NMR (DMSO-d6). • (amongst the others): 0.97–1.12 (m, 4H); 1.42–1.73 (m, 12H); 1.74–1.81 (m, 1H); 1.87–1.94 (m, 1H); 2.17–2.28 (m, 1H); 2.45 (s, 3H); 2.67–2.73 (m, 2H); 2.79–2.87 (m, 1H); 2.88–3.00 (m, 2H); 3.31–3.20 (m, 1H); 3.76–3.84 (m, 2H); 4.42–4.49 (in, 1H); 7.10–7.21 (m, 5H); 7.34 (m, 1H); 7.47 (t, J=5.8 Hz, 1H); 7.84 (d, J=8.6, 1H); 7.95–7.98 (m, 1H); 8.00–8.04 (m, 1H); 8.27 (s, 1H); 8.89 (s, 1H).
MS m/z: 686.4 (M+H+). HPLC (method D): Rt=8.76 min.
MS m/z: 645.5 (M+H+). HPLC (method D): Rt=11.98 min.
MS m/z: 686.6 (M+H+). HPLC (method D): Rt=8.58 min.
MS m/z: 639.5 (M+H+). HPLC (method D): Rt=13.14 min.
MS m/z: 703.4 (M+H+, monoisotopic). HPLC (method D): Rt=13.53 in.
MS m/z: 625.5 (M+H+). HPLC (method D) Rt=12.29 min. 1H-NMR (DMSO-d6). δ (amongst the others): 0.94–1.07 (m, 4H); 1.43–1.72 (m, 12H); 1.76–1.83 (m, 1H); 1.90–2.00 (m, 3H); 2.25–2.34 (m, 1H); 2.55–2.63 (m, 2H); 2.82–2.90 (m, 2H); 2.94–3.01 (m, 1H); 3.74–3.83 (m, 2H); 3.91 (s, 3H); 4.42–4.49 (m, 1H); 7.10–7.21 (m, 5H); 7.23–7.27 (m, 1H); 7.52 (t, J=5.7 Hz, 1H); 7.58–7.67 (m, 2H); 7.80 (d, J=8.6, 1H); 7.95–8.06 (m, 41); 8.53 (s, 1H); 8.78 (s, 1H).
By analogous procedure to that described in Example 117, the following compounds have moreover been prepared:
MS (m/z): 603.3 (MH+). HPLC (method A): Rt=3.63 min.
MS (m/z): 617.4 (MH+, isotopic pattern of Br). HPLC (method A): Rt=4.01 min.
MS (m/z): 717.4 (MH+, isotopic pattern of Br). HPLC (method A): Rt=4.16 min.
HPLC methods
Mobile phase: A=H2O+0.1% TFA; B=MeCN+0.1% TFA
METHOD A
Column: Zorbax™ SB-18, 3.5 μm, 100 Å(50×4.6 mm)
Gradient: from A/B=9515 to A/B=5/95 in 6.5 min+1 min isocratic
Flow rate: 3 ml/min
λ=220, 270 nm.
METHOD B
Column: Platinum™ RP-18, 3 μm, 100 Å(33×7 mm)
Gradient: from A/B=95/5 to A/B=5/95 in 6.5 min+1 min isocratic
Flow rate: 3 ml/min
λ=220, 270 nm.
METHOD C
Column: Jupiter™ C18, 5 μm (250×4.6 mm)
Gradient: from A/B=85/15 to A/B=5/95 in 20 min
Flow rate: 1 ml/min
λ=210 nm.
METHOD D
Column: Symmetry™ 300 C18, 5 μm (250×4.6 mm)
Gradient: from A/B=85/15 to A/B=5/95 in 20 mil
Flow rate: 1 ml/min
λ=210 mm.
METHOD E
Column: Protein & Peptide Vydac™ C18 (250×4.6 mm)
Gradient: from A/B=80/20 to A/B=20/80 in 25 min+A/B=20/80 for 10 min
Flow rate: 1 ml/min
λ=230 nm.
METHOD F
Column: Inertsil ODS-3 (GL Sciences), 3 μm (50×3 mm)
(250×4.6 mm)
Gradient: from A/B=80/20 to A/B=30/70 in 9 min
Flow rate: 0.8 ml/min
λ=230 1 nm.
List of Abbreviations
In the present description the following abbreviations have been used:
Ac5c, aminocyclopentanecarboxylic;Ac6c, aminocyclohexanecarboxylic; AcOEt, ethyl acetate; Boc, N-tert-butyloxycarbonyl;BSA,N,O-bis(trimethylsilyl)acetamide; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EtOAc, ethyl acetate; HOBt, 1-hydroxybenzotriazole; TEA, triethylamine; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSCI, trimethylsilylchloride; Z, N-benzyloxycarbonyl.
The evaluation of the antagonist activity on NK-2 receptors has been assessed by binding and functional tests according to that already described in literature in relation to NK-2 antagonists.
In particular, the affinity of the present compounds for the human NK-2 receptor has been assessed by a binding test using Chinese hamster ovary (CHO) cell membranes transfected with NK-2 receptor of human ileum together with [125I]NKA (Amersham, aspecific activity 2000 Ci/mmol) radiobinder at the concentration of 100 pM in competition studies.
The test substances have been tested within a concentration range from 0.01 nM to 10 nM. At the end of the incubation time (30 min, 20° C.) the samples have been filtered and radioactivity has been determined using a gamma-counter.
The data shown in the following Table I have been obtained for some compounds of general formula (I) and refer to the affinity values of human NK-2 receptor:
The present compounds of formula (I) can be handled according to the common pharmacopoeial techniques in order to prepare formulations suitable for oral, intranasal, parentheral, sublingual, inhalatory, transdermic, local or rectal use according to the data known in literature for this kind of products; these formulations comprise oral formulations, such as tablets, capsules, powders, granulated preparations, and oral solutions or suspensions, formulations for sublingual administration, for intranasal administration, aerosol formulations, implantations, formulations for subcutaneous, intramuscular, intravenous, intraocular and rectal administration. The effective doses are 0.1 to 50 mg/kg of body weight. For humans the effective dose may preferably range from 0.5 to 4000 mg/day, in particular from 2.5 to 1000 mg according to the age of patients and to the type of treatment.
The treatment is performed by administering to the patient the required amount 1 to 4 times-per-day for periods of time up to 2 weeks or in any case until remission of symptoms; for chronic pathologies, administration can be prolonged for significantly longer periods of time according to the physician judgement
Thanks to their high antagonist activity to tachykinins NK-2 receptor, the present compounds are useful in the treatment of diseases in which Neurokinine A plays a pathogenetic role, and namely in the following pathologies:
| Number | Date | Country | Kind |
|---|---|---|---|
| FI2001A0203 | Oct 2001 | IT | national |
| FI2002A0104 | Jun 2002 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP02/12022 | 10/28/2002 | WO | 00 | 4/29/2004 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO03/037916 | 5/8/2003 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5610176 | Horwell et al. | Mar 1997 | A |
| 5780467 | Dorn et al. | Jul 1998 | A |
| 6262069 | Liebeschuetz et al. | Jul 2001 | B1 |
| 6294547 | Oka et al. | Sep 2001 | B1 |
| 6740682 | Liebeschuetz | May 2004 | B2 |
| 6855715 | Liebeschuetz et al. | Feb 2005 | B1 |
| 6878725 | Liebeschuetz et al. | Apr 2005 | B2 |
| 6900196 | Liebeschuetz et al. | May 2005 | B2 |
| Number | Date | Country |
|---|---|---|
| 0 394 989 | Oct 1990 | EP |
| WO9404494 | Mar 1994 | WO |
| WO 0519966 | Jul 1995 | WO |
| WO9834949 | Aug 1998 | WO |
| WO9845262 | Oct 1998 | WO |
| WO 0014109 | Mar 2000 | WO |
| WO 0129066 | Apr 2001 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20040259930 A1 | Dec 2004 | US |
