This application represents a National Stage application of PCT/DE2010/000001 entitled “Nω-Hydroxy-L-Arginine Derivatives for the Treatment of Diseases” filed Jan. 4, 2010, pending.
1. Field of the Invention
The present invention relates to physically-chemically and pharmacokinetically enhanced Nω-hydroxy-L-arginine (NOHA) derivatives and a method for producing the NOHA derivatives having enhanced physical-chemical and pharmacokinetic properties according to the invention.
2. Discussion of the Prior Art
Nω-hydroxy-L-arginine is the physiologically occurring intermediate of the NO synthase catalysed oxidation L-arginine. NOHA is oxidised in a further step of NO synthase, nitrogen monoxide being released and L-citrulline being formed.
Thus the semi-essential amino acid L-arginine is the natural source for the nitrogen monoxide (NO).
Nitrogen monoxide (NO) is of decisive importance among others for supplying the organs with blood. NO leads indirectly to an increase of the vessels. The extent of the vascular dilation then has different effects in the individual organs. In the heart there is for example an improved circulation. In addition to expanding the vessels, NO also has other properties:
A dysregulation or reduced NO availability is thus related to diverse cardiovascular diseases. A limited NO availability is therefore associated with the so-called endothelial dysfunction—a state of multifactorial genesis—that is associated with high blood pressure, atherosclerosis, arterial thrombosis, coronary heart disease, heart failure, heart attack, hypercholesterolaemia and diabetes.
Old, rigid, atherosclerotically changed vessels can be deformed again with NO. Blood circulation is thus improved and among others high blood pressure can be normalised. For children that are born with serious respiratory dysfunction, the inhalation of NO is even today in use successfully. NO promotes the erection of the penis which has led to the development of drugs against impotence (Viagra®).
Also for fighting tumors it is expected that new treatments can be pursued with NO since NO produced by white blood cells does not only destroy bacteria but also cells.
However, NO has quite a short life. Within a short period it reacts with oxygen molecules to form nitrite (NO2−) and nitrate (NO3−). The short life also explains why NO can only be formed directly at its target.
It is further known that NOHA is a potent inhibitor of arginase I with a Ki value between 30-42 μM.
In smaller in-vivo studies with rats, desired effects for treating the erectile dysfunction, endothelial dysfunction and hypertension could already be demonstrated using an i.v.-NOHA therapy.
Treating diseases associated with endothelial dysfunction, using conventional NO donors has a few disadvantages. In the case of nitrates for example the short therapeutic half time, the low oral bioavailability, partly adverse haemodynamic effects and tolerance effects are to be mentioned in this context. Indeed, a proatherogene effect during long-term treatment using organic nitrates is being discussed recently.
So that a therapy of NO-deficient diseases is possible with few side effects, the physiological situation has to be imitated as best as possible, i.e. NO must only be released for a short time, in the right amounts and of the right location. All these prerequisites are met using NOHA as an NO-donor or, to take into consideration the time factor, with a retarding prodrug of NOHA. In addition to the fact that using this strategy, nitrogen monoxide is only released where it is needed and is formed to an extent that is too small, the fact can be exploited that NOHA presents one of the most potent arginase inhibitors. Specifically an increased arginase activity is discussed as a mechanism for reduced NO availability and thus as a contributing factor in the development of the endothelial dysfunction. Thus a dual mode of action is exploited when using NOHA.
The use of L-arginine and Nω-hydroxy-L-arginine and their simple carboxylic acid esters, and also analogue N-hydroxyguanidine for treating a multiplicity of diseases, have been described and patented (a selection: WO03045369, U.S. Pat. No. 6,277,884, WO0132167, CA02386938).
In practice, use of such compounds is limited by their bad pharmacokinetic profile. The insufficient drug qualities of this substance can be explained above all by the presence of an unsubstituted N-hydroxyguanidine function. In particular, the following effects can be observed for the physical-chemical instability of N-hydroxyguanidines:
N-hydroxyguanidines decompose at room temperature and should be stored at 4-8° C., better −20° C. They are most stable in the form of their salts of strong acids. The following decomposition processes are known:
Metabolic instability of N-hydroxyguanidines:
This leads to a strong limitation of the biological half time as a result of thermal, hydrolytic, oxidative and enzymatic processes. Also a high first-pass effect is to be expected, considering the metabolic instability that was mentioned.
It is therefore the object of the invention to improve the physical-chemical and metabolic stability and thus the pharmacokinetic properties of Nω-hydroxy-L-arginine and their simple carboxylic acid esters as well as analogue N-hydroxyguanidines.
It was surprisingly found that by substituting the hydroxyguanidine function according to the invention thermal and oxidation-stable derivatives of the NOHA were produced for the first time.
The chemical formula of the inventive derivatives is shown below.
The inventive substitutions are marked by X. Both a substitution at the oxygen of the hydroxylic groups X2 leads to surprisingly stable derivatives, as also a substitution at the nitrogen X1 and also a combination of the two inventive substitutions. In a usual way, the carboxylic acid function can be esterified in a conventional manner with an alkyl or aryl radical or be present as an acid (R1). Y can correspond to hydrogen, alkoxycarbonyl or aryloxycarbonyl radical. n can be 2 or 3. The chirality centre m can be R or S configured.
Material and Methods
Exemplary Embodiment 1: O-Alkylised NOHA Derivatives
General regulation in the style of Linton et al. [J. Org. Chem. 2000, 65, 1566]:
7.0 mmol of Nα-(t-butyloxycarbonyl)-L-ornithine-t-butylester are dissolved in 250 mL of dry dichloromethane. The solution is cooled to 0° C. and 14 mL of a 0.5 M solution (in dichloromethane) of benzyloxycarbonylisothiocyanate (7.0 mmol) are added in drops over 30 minutes. The reaction mixture is stirred for two hours, the solution being warmed to room temperature. Using a rotary evaporator, the mixture is concentrated to approximately one third of the original volume in vacuum and washed in each case with 25 mL of 1% HCl, water and saturated NaCl solution. The organic phase is dried over Na2SO4 and removed using the rotary evaporator. The thiourea 1 is mostly already >96% pure (DC) and is purified further using column chromatography.
After column chromatography over silica gel (cyclohexane/ethylacetate, 4:1), a light-yellow oil is obtained that becomes solid in the refrigerator.
Yield: 3.13 g (93%)
TLC: Rf=0.31 (cyclohexane/ethylacetate, 4:1; ninhydrine)
1H-NMR (CDCl3):
δ/ppm=1.44, 1.46 (s, 9H, 2×C(CH3)3), 1.64-1.75 (m, 4H, β,γ-CH2) 3.66 (m, 2H, N—CH2), 4.20 (m, 1H, α-CH), 5.08 (m, 1H, NH), 5.10 (s, 2H, CH2-Cbz), 7.36 (m, 5H, ArH), 8.15 (br s, 1H, NH), 9.65 (br s, 1H, NH).
13C-NMR (CDCl3):
δ/ppm=24.8 (γ-CH2), 28.7, 29.0 (2×C(CH3)3), 30.9 (β-CH2), 45.8 (N—CH2), 54.2 (α-CH), 68.8 (CH2-Cbz), 80.4, 82.8 (2×C(CH3)3), 129.0, 129.4, 129.5 (ArCH), 135.2 (ArC), 153.2 (CO-Cbz), 156.0 (CO-Boc), 172.2 (COOtBu), 179.8 (C═S).
MS (ESI):
m/z=482 [M+H]+, 426 [M−C4H8+H]+, 370 [M−2×C4H8+H]+, 326 [M−2×C4H8−CO2+H]+.
C23H35N3O6S (481.61)
8.0 mmol of Nα-(t-butyloxycarbonyl)-L-ornithine-t-butylester are dissolved in 250 mL of dry dichloromethane. The solution is cooled to 0° C., and 927 mg ethoxycarbonylisothiocyanate (7.0 mmol), dissolved in 15 mL of dry dichloromethane, are added drop-wise over a period of 30 minutes. The reaction mixture is stirred at room temperature for two hours. The mixture is then concentrated using the rotary evaporator to approximately one third of the original volume and washed with in each case 25 mL of 1% HCl, water and saturated NaCl solution. The organic phase is dried over Na2SO4 and concentrated in vacuum. The thiourea 2 at this point is mostly already >than 96% pure (DC). The product is started to be dissolved in the solvent, activated carbon is added to it and it is cleaned by means of flash chromatography over a short silica gel column. A column with about 20 g of silica gel is used, and the eluent is cyclohexane/ethylacetate (4:1).
Yield: 2.76 g of a colourless oil (94%)
TLC: Rf=0.31 (cyclohexane/ethylacetate, 4:1; ninhydrine)
Melting point: 81° C.
1H-NMR (CDCl3):
δ/ppm=1.31 (t, 3H, 3J=7.1 Hz, CH2—CH3), 1.45, 1.47 (2×s, 9H, C(CH3)3, 1.61-1.90 (m, 4H, β,γ-CH2), 3.66 (pseudo q, 2H, N—CH2), 4.22 (q, 2H, 3J=7.1 Hz, CH2—CH3), 5.08 (m, 1H, α-CH), 8.06, 9.70 (2×br s, 1H, NH).
13C-NMR (CDCl3):
δ/ppm=14.9 (CH2—CH3), 24.9 (γ-CH2), 28.7, 29.0 (2×C(CH3)3), 31.0 (β-CH2), 45.8 (N—CH2), 54.3 (α-CH), 63.4 (O—CH2), 82.8 (C(CH3)3), 153.4 (CO-Boc), 156.2 (CO-Boc), 172.5 (COOt/Bu), 180.1 (C═S).
MS (ESI):
m/z=442 [M+Na]+, 420 [M+H]+, 308 [M−2×C4H8+H]+, 264 [M−2×C4H8—CO2+H]+.
C18H33N3O6S (419.54)
0.5 mmol of the thiourea 1 are dissolved in 5 mL of dry dichloromethane and 522 μL DIPEA (3 mmol) and 1.5 mmol of hydroxylammonia hydrochloride is added. The solution is brought to 0° C. for about 30 minutes and 287 mg N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.5 mmol) are added. Unless specified, the mixture is stirred over night at room temperature. The solution is diluted with approximately 10 mL of dichloromethane and washed in, each case with 5 mL of 1% HCl, water and saturated NaCl solution. The organic phase is dried over Na2SO4 and concentrated using the rotary evaporator. The result is mostly oils that are further purified using flash chromatography over, silica gel. The eluents used and the yields that were achieved are specified with the respective substances.
The eluent used is dichloromethane/methanol (99:1).
Yield: 235 mg of a colourless oil (95%)
TLC: Rf=0.30 (dichloromethane/methanol, 99:1; ninhydrine)
1H-NMR (CDCl3):
δ/ppm=1.44, 1.46 (2×s, 9H, C(CH3)3), 1.58-2.02 (m, 4H, β,γ-CH2), 3.11 (m, 2H, N—CH2), 3.66 (s, 3H, O—CH3), 4.18 (m, 1H, α-CH), 5.11 (m, 1H, NH), 5.13 (s, 2H, CH2-Cbz), 6.25 (m, 1H, NH), 7.36 (m, 5H, ArH), 7.91 (br s, 1H, NH).
13C-NMR (CDCl3):
δ/ppm=25.6 (γ-CH2), 28.7, 29.0 (2×C(CH3)3), 30.9 (β-CH2), 41.2 (N—CH2), 54.5 (α-CH), 62.0 (O—CH3), 68.3 (CH2-Cbz), 80.3, 82.5 (2×C(CH3)3), 129.0, 129.3, 129.4 (ArCH), 135.8 (ArC), 148.8 (C═N), 153.6 (CO-Cbz), 156.0 (CO-Boc), 172.5 (COOtBu).
MS (ESI):
m/z=517 [M+Na]+, 495 [M+H]+, 439 [M−C4H8+H]+.
C24H38N4O7 (494.58)
The protected L-arginine 3 (0.4 mmol) is stirred in 8 mL of TFA and 2.4 mL of thioanisol for 30 minutes at room temperature. Then the majority of TFA is distilled off in vacuum, and 5 mL of water and 15 mL of diethylether are added. The organic phase is also extracted twice using 5 mL of water, and the combined aqueous phases are finally washed with 5 mL of diethylether. The aqueous phase is concentrated using the rotary evaporator (at approximately 35° C.) and taken up with little 0.1% of TFA (in Aqua bidest.). Flash chromatography follows over an RP-18 column (eluent: 0.1% of TFA(aq)), ninhydrine-positive fractions being combined. The combined fractions are concentrated to a volume of approximately 10 mL using the rotary evaporator and then lyophilised with freeze drying.
Yield: 171 mg of a colourless oil (99%), Rf=0.56 (i-propanol/water/acetic acid, 6:3:1; ninhydrine)
1H-NMR (D2O):
δ/ppm=1.78 (m, 2H, γ-CH2), 1.99 (m, 2H, β-CH2), 3.31 (t, 2H, 3J=6.8 Hz, N—CH2), 3.75 (s, 3H, O—CH3), 4.06 (t, 3J=6.2 Hz, 1H, α-CH).
13C-NMR (D2O, TPS):
δ/ppm=26.4 (γ-CH2), 29.7 (β-CH2), 43.0 (N—CH2), 55.5 (α-CH), 67.3 (O—CH3), 160.1 (C═N), 174.8 (CO).
HRMS (m/z):
calculated for C7H17N4O3 [M+H]+=205.12952, found: 205.12951.
For the esterification, 238 mg of the free amino acid 3 (0.55 mmol) are dissolved in 5 mL of absolute ethanol in an argon atmosphere. The solution is stirred for 30 minutes at −10° C. before HCl gas is introduced into the solution for approximately 5-10 minutes. The batch is then stirred further for an hour at 0° C. and placed in the refrigerator for 36 hours. The solution is carefully concentrated at room temperature in vacuum and lyophilised. The product thus obtained is a very hygroscopic, amorph solid which liquefies on contact with air.
Yield: 168 mg of a clear oil (99%)
TLC: Rf=0.18 (i-propanol/water/acetic acid, 8:1:1; ninhydrine)
1H-NMR (DMSO-d6):
δ/ppm=1.24 (t, 3J=7.2 Hz, 3H, CH2—CH), 1.47-1.90 (m, 4H, β,γ-CH2), 3.22 (m, 2H, N—CH2), 3.64 (s, 3H, O—CH3), 3.99 (m, 1H, α-CH), 4.21 (q, 3J=7.2 Hz, 2H, CH2—CH3), 8.04 (br s, 2H, NH2), 8.31 (br t, 1H, NH), 8.72 (br s, 3H, NH3+), 11.33 (br s, 1H, NH+).
13C-NMR (DMSO-d6):
δ/ppm=13.9 (CH2—CH3), 24.0 (γ-CH2), 27.0 (β-CH2), 40.0 (N—CH2), 51.4 (α-CH), 61.7 (CH2—CH3), 64.4 (O—CH3), 157.2 (C═N), 169.2 (CO).
HRMS (m/z):
calculated for C9H21N4O3 [M+H]+=233.16082, found: 233.16064.
C9H20N4O3.2.0HCl.0.7H2O (317.82)
210 mg of the thiourea 2 (0.5 mmol) are dissolved in 5 mL of dry dichloromethane and 261 μL of DIPEA (1.5 mmol) and 62.6 mg of methoxylamine hydrochloride (0.75 mmol) are added. The solution is brought to 0° C. for approximately 30 minutes and 143.5 mg of EDCl (0.75 mmol) are added. The mixture is stirred at room temperature over night. The solution is diluted with approximately 10 mL of dichloromethane and washed in each case with 5 mL of 1% HCl, water and saturated NaCl solution. The organic phase is dried over Na2SO4 and concentrated in vacuum. The raw product is further purified by means of column chromatography over silica gel (dichloromethane/methanol, 98:2).
Yield: 203 mg of a colourless oil (94%)
TLC: Rf=0.26 (dichloromethane/methanol, 98:2; ninhydrine)
1H-NMR (CDCl3):
δ/ppm=1.27 (t, 3J=7.1 Hz, 3H, CH2—CH3), 1.43, 1.45 (2×s, 9H, C(CH3)3), 1.56-1.89 (m, 4H, β,γ-CH2), 3.09 (m, 2H, N—CH2), 3.66 (s, 3H, O—CH3), 4.16 (br q, 3J=7.1 Hz, 3H, CH2—CH3, α-CH), 5.10, 6.26 (2×br m, 1H, NH), 7.80 (br s, 1H, NH).
13C-NMR (CDCl3):
δ/ppm=14.9 (CH2—CH3), 25.6 (γ-CH2), 29.7 (β-CH2), 29.0, 30.9 (2×C(CH3)3), 41.2 (N—CH2), 54.5 (α-CH), 62.0 (CH2—CH3), 62.6 (O—CH3), 80.2, 82.5, (2×C(CH3)3), 149.0 (C═N), 153.8 (CO-Eoc), 156.0 (CO-Boc), 172.4 (COOtBu).
MS (ESI ):
m/z=455 [M+Na]+, 433 [M+H]+, 377 [M−C4H8+H]+.
C19H36N4O7 (432.52)
200 mg of the completely protected A′ -methoxy-L-arginine 5 (0.46 mmol) are stirred in 5 mL of TFA at first for 30 minutes at 0° C. and then for three hours at room temperature. TFA is carefully distilled off at the lowest possible temperature in vacuum, and the residue is taken up with a little Aqua bidest. There follows a further purification by means of flash chromatography on an RP-18 column (0.1% of TFA in Aqua bidest.). The ninhydrine-positive fractions are combined, concentrated using the rotary evaporator at approximately 30° C. to a residual volume of approximately 10 mL and lyophilised.
Yield: 225 mg of a colourless oil (97%)
TLC: Rf=0.44 (i-propanol/water/acetic acid, 8:1:1, ninhydrine)
1H-NMR (DMSO-d6):
δ/ppm=1.22 (t, 3J =7.1 Hz, 3H, CH2—CH3), 1.50-1.87 (m, 4H, β,γ-CH2), 3.12 (br t, 2H, N—CH2), 3.62 (s, 3H, O—CH3), 3.90 (m, 1H, α-CH), 4.13 (q, 3J=7.1 Hz, 2H, CH2—CH3), 7.35 (br s, 1H, NH), 8.28 (br s, 3H, NH3+).
13C-NMR (DMSO-d6):
δ/ppm=14.1 (CH2—CH3), 24.6 (γ-CH2), 27.3 (β-CH2), 40.6 (N—CH2), 51.7 (α-CH), 61.6 (CH2—CH3), 62.1 (O—CH3), 149.0 (C═N), 153.5 (CO-Eoc), 170.9 (CO).
HRMS (m/z):
calculated for C10H21N4O5 [M+H]+=277.15065, found: 277.15049.
C10H20N4O5.2.0 CF3COOH.0.4 H2O (513.56)
For the esterification, 200 mg of the free amino acid 6 (0.397 mmol) are dissolved in 5 mL of absolute ethanol in an argon atmosphere. The solution is stirred at −10° C. for 30 minutes, before HCl gas is introduced into the solution for approximately 5-10 minutes. The batch is then stirred further for an hour at 0° C. and placed in the refrigerator for 36 hours. The solution is carefully concentrated in vacuum at room temperature and lyophilised. The product thus obtained is a very hygroscopic, amorph solid that liquefies on contact with air.
Yield: 150 mg of a colourless oil (99%)
1H-NMR (DMSO-d6):
δ/ppm=1.23, 1.25 (2×t, 3J=7.0 Hz, 3H, CH2—CH), 1.55-1.88 (m, 4H, β,γ-CH2), 3.32 (br t, 2H, N—CH2), 3.71 (s, 3H, O—CH3), 3.95 (m, 1H, α-CH), 4.19 (m, 4H, 2×CH2—CH3), 8.80 (br s, 4H, NH3+, NH).
13C-NMR (DMSO-d6):
δ/ppm=13.9, 14.0 (2×CH2—CH3), 24.0 (γ-CH2), 26.9 (β-CH2), 41.1 (N-CH2), 51.4 (α-CH2), 61.7, 62.5 (2×CH2—CH3), 63.9 (O—CH3), 150.7 (C═N), 152.6 (CO-Eoc), 169.2 (COOEt).
HRMS (m/z):
calculated for C12H25N4O5 [M+H]+=305.18195, found: 305.18176.
C12H24N4O5.2.0 HCl.0.6 H2O (388.08)
Exemplary Embodiment 2: O-carboxyalkylated NOHA Derivatives
241 mg of the thiourea 1 (0.5 mmol) are dissolved in 5 mL of dry dichloromethane and 261 μL of DIPEA (0.75 mmol) and 79 mg of aminooxyacetic acid methylester (0.75 mmol) are added. The solution is brought to 0° C. for approximately 30 minutes and 143.5 mg of EDCl (0.75 mmol) are added. The mixture is stirred over night at room temperature. The solution is diluted with approximately 10 mL of dichloromethane and washed in each case with 5 mL of 1% HCl, water and saturated NaCl solution. The organic phase is dried over Na2SO4 and concentrated in vacuum. The raw product is purified further by means of chromatography over silica gel (cyclohexane/ethylacetate, 3:2).
Yield: 254 mg of a colourless oil (92%) TLC: Rf=0.48 (cyclohexane/ethylacetate, 3:2; ninhydrine)
1H-NMR (CDCl3):
δ/ppm=1.44, 1.46 (2×s, 9H, C(CH3)3), 1.50-1.87 (m, 4H, β,γ-CH2), 3.07 (m, 2H, N—CH2), 3.73 (s, 3H, O—CH3), 4.16 (m, 1H, α-CH), 4.41 (s, 2H, O—CH2), 5.08 (m, 1H, NH), 5.15 (s, 2H, CH2-Cbz), 6.37 (br t, 3J=5.3 Hz, 1H, NH), 7.30-7.39 (m, 5H, ArH), 8.23 (br s, 1H, NH).
13C-NMR (CDCl3):
δ/ppm=25.5 (γ-CH2), 28.7 (β-CH2), 29.0, 30.9 (2×C(CH3)3), 41.2 (N—CH2), 52.4 (O—CH3), 54.5 (α-CH), 68.3 (CH2-Cbz), 71.2 (O—CH2), 80.3, 82.5 (2×C(CH3)3), 129.0, 129.28, 129.34 (ArCH), 135.9 (ArC), 150.8 (C═N), 153.7 (CO-Cbz), 156.0 (CO-Boc), 171.7, 172.4 (COOtBu, COOMe).
MS (ESI):
m/z=575 [M+Na]+, 553 [M+H]+, 497 [M−C4H8+H]+.
270 mg of the completely protected Nω-carboxymethoxy-L-arginine 8 (0.489 mmol) are stirred at 50-60° C. for four hours in 5 mL of 6 N HCl. The batch is concentrated in vacuum, approximately 1-2 mL of Aqua bidest. are added, and it is then purified by means of flash chromatography on an RP-18 column (0.1% of TFA in Aqua bidest.). Ninhydrine-positive fractions are combined, concentrated using the rotary evaporator at 30° C. to only a few milliliters and then lyophilised.
Yield: 150 mg of a white, amorphous solid (96%)
TLC: Rf=0.53 (i-propanol/water/acetic acid, 6:3:1; ninhydrine) 1H-NMR (D2O):
δ/ppm=1.76-2.18 (m, 4H, β,γ-CH2), 3.41 (br t, 3J=6.7 Hz, 2H, N—CH2), 4.17 (br t, 3J=6.2 Hz, α-CH), 4.63 (s, 2H, O—CH2).
13C-NMR (D2O, TPS):
δ/ppm=26.3 (γ-CH2), 29.6 (β-CH2), 43.2 (N—CH2), 55.3 (α-CH), 75.5 (O—CH2), 161.0 (C═N), 174.6, 175.3 (2×CO).
MS (ESI):
m/z=249 [M+H]+.
C8H16N4O5.2.0 HCl.0.5 H2O (330.17)
The preparation and working-up take place under the same conditions as described for the analogous compound 8, with the thiourea 2 and aminooxyacetic acid ethylester as starting substances (batch size: 1.0 mmol).
Yield: 500 mg of a colourless oil (99%)
TLC: Rf=0.51 (cyclohexane/ethylacetate, 3:2; ninhydrine)
1H-NMR (CDCl3):
δ/ppm=1.26 (t, 3J=7.1 Hz, 3H, CH2—CH3), 1.27 (t, 3J=7.1 Hz, 3H, CH2-CH), 1.42, 1.44 (2×s, 9H, C(CH3)3), 1.51-1.85 (m, 4H, β,γ-CH2), 3.06 (m, 2H, N—CH2), 4.16 (q, 3J=7.11 Hz, 2H, CH2—CH3), 4.19 (q, 3J=7.17 Hz, 2H, CH2—CH3), 4.39 (s, 2H, O—CH2), 5.08 (m, 1H, NH), 6.40 (br t, 1H, NH), 8.19 (br s, 1H, NH).
13C-NMR (CDCl3):
δ/ppm=14.1, 14.2 (2×CH2—CH3), 24.8 (γ-CH2), 27.9, 28.3 (233 C(CH3)3), 30.2 (β-CH2), 40.5 (N—CH2), 53.7 (α-CH), 60.8, 61.9 (2×CH2—CH3), 70.7 (O—CH2), 79.5, 81.8 (2×C(CH3)3), 150.4 (C═N), 153.2 (CO-Eoc), 155.3 (CO-Boc), 170.7,. 171.7 (COOEt, COOtBu).
MS (ESI):
m/z=527 [M+Na]+, 505 [M+H]+, 449 [M−C4H8]+.
C22H40N4O9 (504.59)
252 mg of the completely protected precursor 10 (0.5 mmol) are stirred in 5 mL of TFA at first at 0° C. for 30 minutes and then at room temperature for three hours. TFA is carefully distilled off at the lowest possible temperature in vacuum, and the residue is taken up with a little Aqua bidest. There follows the further purification by means of flash chromatography on an RP-18 column (0.1% TFA(aq)/methanol step gradient, 5-30%). The ninhydrine-positive fractions are combined, concentrated using the rotary evaporator at approximately 30° C. to a residual volume of approximately 10 mL and lyophilised.
Yield: 277 mg of a clear oil (96%)
TLC: Rf =0.62 (i-propanol/water/acetic acid, 8:1:1; ninhydrine)
The substance is present as a solvent in DMSO-d6 as an isomer mixture in a ratio of approximately 8.6:1.4 (at 300 K, relative to the singulet of CH2 of the ethoxycarbonylmethoxy radical). The shifts specified refer to the main isomer.
1H-NMR (DMSO-d6):
δ/ppm=1.19 (t, 3J=7.10 Hz, 3H, CH2—CH 1.21 (t, 3 J=7.08 Hz, 3H, CH2—CH3), 1.48-1.87 (m, 4H, β,γ-CH2), 3.02 (m, 2H, N—CH2), 3.89 (m, 1H, α-CH), 4.10 (q, 3J=7.04 Hz, 2H, CH2—CH3), 4.12 (q, 3J=7.13 Hz, 2H, CH2—CH3), 4.37 (s, 2H, O—CH2), 6.54 (br s, 1H, NH), 8.24 (br s, 3H, NH3+), 10.64 (br s, COOH).
13C-NMR (DMSO-d6):
δ/ppm=14.0, 14.2 (2×CH2—CH3), 27.4 (CH2), 40.3 (N—CH2), 51.8 (α-CH), 60.2, 61.3 (2×CH2—CH3), 70.3 (O—CH2), 169.9, 171.0 (3×CO).
HRMS (m/z):
calculated for C13H25N4O7 [M+H]+=349.17178, found: 349.17155.
C13H24N4O7.3.2 CF3COOH.1.5 H2O (740.26)
For the esterification, 288 mg of the free amino acid 11 (0.5 mmol) are dissolved in an argon atmosphere in 5 mL of absolute ethanol. The solution is stirred at 31 10° C. for 30 minutes, before HCl gas is introduced into the solution for approximately 5-10 minutes. The batch is then stirred further for an hour at 0° C. and placed in the refrigerator over night. The solution is carefully concentrated in vacuum at room temperature, lyophilised and yields a strongly hygroscopic, amorphous solid that liquefies on contact with air.
Yield: 222 mg of a colourless oil (99%)
TLC: Rf =0.57 (i-propanol/water/acetic acid, 8:1:1; ninhydrine)
1H-NMR (DMSO-d6):
δ/ppm=1.20 (t, 3J=7.16 Hz, 3H, CH2—CH3), 1.23 (t, 3J=7.13 Hz, 3H, CH2—CH3), 1.26 (t, 3J=7.10 Hz, 3H, CH2—CH3), 1.51-1.87 (m, 4H, β,γ-CH2), 3.17 (m, 2H, N—CH2), 3.95 (m, 1H, α-CH), 4.14 (q, 3J=7.12 Hz, 2H, CH2—CH3), 4.15 (q, 3J=7.10 Hz, 2H, CH2—CH3), 4.17-4.25 (m, 2H, CH2—CH3), 4.51 (s, 2H, O—CH2), 7.80 (br s, 1H, NH), 8.55 (br s, 1H, NH), 8.72 (br s, 3H, NH3+).
13C-NMR (DMSO-d6):
δ/ppm=13.9, 14.0, 14.1 (3×CH2—CH3), 24.2 (γ-CH2), 27.2 (β-CH2), 40.8 (N—CH2), 51.5 (α-CH), 60.5, 61.7, 61.9 (3×CH2—CH3), 71.1 (O—CH2), 150.3 (C═N), 153.0 (CO—Eoc), 169.1, 169.3 (2×CO).
HRMS (m/z):
calculated for C15H29N4O7 [M+H]+=377.20308, found: 377.20287.
Exemplary Embodiment 3: O-Glycosidically Conjugated NOHA Derivatives
210 mg of the thiourea 2 (0.5 mmol), 218 mg of 1-aminooxy-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (0.6 mmol) and 104.5 μl of DIPEA (0.6 mmol) are dissolved in 5 mL of dry dichloromethane. The batch is cooled to 0° C., 115 mg of EDCl (0.6 mmol) are added and stirred at room temperature for 48 hours. The solution is diluted with approximately 10 mL of dichloromethane and in each case washed with 5 mL of 1% HCl, water and saturated NaCl solution. The organic phase is dried over Na2SO4 and concentrated in vacuum. The raw product is purified further by means of column chromatography over silica gel (dichloromethane/methanol, 97:3). The combined fractions of the purified product are concentrated in vacuum to form a clear oil. Repeatedly adding and removing dry dichloromethane yields a solid white foam that liquefies on contact with air.
Yield: 262 mg of a colourless oil (70%)
TLC: Rf=0.35 (dichloromethane/methanol, 97:3; ninhydrine)
1H-NMR (CDCl3):
δ/ppm=1.29 (t, 3J=7.1 Hz, 3H, CH2—CH3), 1.43, 1.45 (2×s, 9H, C(CH3)3), 1.52-1.86 (m, 4H, β,γ-CH2), 1.98, 2.02, 2.05, 2.13 (4×COCH3), 3.08 (m, 2H, N—CH2), 3.96 (br t, 3J=6.8 Hz, 1H, 5′-CH), 4.11-4.21 (m, 5H, α-CH, CH2—CH3, 3′-CH, NH), 4.85 (d, 3J=8.3 Hz, 1H, 1′-CH), 5.06 (m, 2H, 6′-CH2), 5.25 (dd, 3J=10.4, 8.3 Hz, 1H, 2′-CH), 5.39 (dd, 3J=3.5, 1.0 Hz, 1H, 4′-CH), 6.47 (br t, 1H, NH), 7.65 (br s, 1H, NH).
MS (ESI):
m/z=772 [M+Na]+, 750 [M+H]+, 707 [M−C2H2O+H]+.
In a Schlenk tube 120 mg (0.16 mmol) of the completely protected and carefully dried precursor 13 are dissolved with argon fumigation in approximately 10 mL of dry diethylether. The solution is stirred for approximately 30 minutes at −15° C., and then HCl gas is introduced carefully with simultaneous argon fumigation for approximately 5 minutes. The reaction mixture is placed over night in the refrigerator and concentrated the following day for working up in vacuum. The white-yellow solid is then taken up with approximately 1-2 mL of 0.5 M NaHCO3 solution and purified by means of flash chromatography over an RP-18 column (flow agent: Aqua bidest./methanol), a step gradient being used for the elution. The start is made with a concentration of 10% methanol, then increased to 25% and finally to 50% methanol. The product-containing fractions are combined, concentrated at 30° C. in vacuum to a residual volume of approximately 50 mL and then lyophilised.
Yield: 74 mg of a fine white powder (75%)
In D2O as solvent, the substance is present as an isomer mixture in a ratio of approximately 6:4 (at 300 K, relative to the triplet of CH3 of the ethoxycarbonyl function). The shifts specified refer to the main isomer.
1H-NMR (D2O, TPS):
δ/ppm=1.28 (t, 3J=7.1 Hz, 3H, CH2—CH3), 1.52-1.97 (m, 4H, β,γ-CH2), 2.02, 2.08, 2.12, 2.23 (4×s, 3H, COCH3), 3.16 (br t, 3J=6.8 Hz, 2H, 6′-CH2), 3.75 (t, 3J=6.3 Hz, 1H, 5′-CH), 4.15-4.30 (m, 5H, CH2—CH3, α-CH, N—CH2), 5.07 (d, 3J=8.0 Hz, 1H, 1′-CH), 5.21-5.33 (m, 2H, 2′,3′-CH), 5.47 (m, 1H, 4′-CH).
13C-NMR (D2O, TPS):
δ/ppm=16.2 (CH2—CH3), 22.7, 22.8, 22.9, (4×COCH3), 26.6 (γ-CH2), 30.6 (β-CH2), 42.7 (N—CH2), 57.2 (α-CH), 64.6 (6′-CH2), 65.8 (CH2—CH3), 70.6, 70.7, 73.4, 74.1 (2′,3′,4′,5′-CH), 104.1 (1′-CH), 155.6 (C═N), 156.8 (CO-Eoc), 175.4, 175.8, 176.2, 177.1 (4×COCH3).
MS (ESIESI):
m/z=615 [M+Na]+, 593 [M+H]+, 551 [M−C2H2O+H]+, 331 [C14H19O9]+.
HRMS (m/z):
calculated for C23H33N4O14Na [M+Na]+=615.21202, found: 615.21164
C23H35N4NaO14 (614.53)
Number | Date | Country | Kind |
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10 2009 004 203 | Jan 2009 | DE | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/DE2010/000001 | 1/4/2010 | WO | 00 | 9/23/2011 |
Publishing Document | Publishing Date | Country | Kind |
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WO2010/078865 | 7/15/2010 | WO | A |
Number | Name | Date | Kind |
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6277884 | De Tejada | Aug 2001 | B1 |
20050158401 | Morris | Jul 2005 | A1 |
Number | Date | Country |
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WO 0132167 | May 2001 | WO |
WO 03045369 | Jun 2003 | WO |
WO 2007005620 | Jan 2007 | WO |
Entry |
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Moali et al., “Óxidations of Nω-Hydroxyarginine Analogues and Various N-Hydroxyguanidines by NO Synthase II: Key Role of Tetrahydrobiopterin in the Reaction Mechanism and Substrate Selectivity”, Chemical Research in Toxicology, vol. 14, pp. 202-210, 2001. |
Labby et al., “Methylated Nω-Hydroxy-L-Arginine Analogous as Mechanistic Probes for the Second Step of the Nitric Oxide Synthase-Catalyzed Reaction”, Biochemistry, 52, pp. 3062-3073, 2013. |
Schade et al., “Efficient Synthesis of Optically Pure Nω-Alkylated L-Arginines”, Synthesis, No. 15, pp. 2391-2397, 2008. |
Schade et al., “Prodrug Design for the Potent Cardiovascular Agent -Hydroxy-L-Arginine (NOHA): Synthetic Approaches and Physicochemical Characterization”, Organic Biomolecular Chemistry, 9, pp. 5249-5259, 2011. |
Number | Date | Country | |
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20120123111 A1 | May 2012 | US |