[SIZE=+1]Treating renal disease, burns, wounds and spinal cord injury with selective androgen receptor modulators [/SIZE]
<center style="color: rgb(0, 0, 0); font-family: -webkit-standard;">Abstract</center>This invention provides: 1) a method of treating a subject suffering from, or predisposed to a kidney disease or disorder; 2) a method of treating a subject suffering from a wound, or reducing the incidence of, or mitigating the severity of a wound in a subject; 3) a method of treating a subject suffering from a burn, or reducing the incidence of, or mitigating the severity of a burn in a subject, comprising the step of administering to said subject a selective androgen receptor modulator (SARM) compound; 4) a method of treating a subject suffering from a spinal cord injury, by administering to the subject a selective androgen receptor modulator (SARM) and/or an analog, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, prodrug, polymorph, impurity or crystal of said SARM compound, or any combination thereof.
Example 1
Effect of Selective Androgen Receptor Modulators (SARMS) on Subjects with End-Stage Renal Disease
Compounds Tested:
[0290] The following Compounds, which are ligands for the AR with potent binding affinity, exhibiting tissue-selective androgenic and anabolic effects, and oral bioavailability, will be assessed for their effects on end-stage renal disease (ESRD). ##STR50## ##STR51##
[0291] The compounds will be synthesized and characterized, as described for example in U.S. patent application Ser. Nos. 10/277,108, 10/270,732 and 10/371,155, all of which are incorporated herein by reference.
[0292] For example, the process for preparing a SARM compound represented by the structure of formula (I): ##STR52## with substituents as described herein, may comprise the step of coupling an amide of formula (XXII): ##STR53## wherein Z, Y, R.sub.1 and T are as defined above and L is a leaving group, with a compound of formula (XXIII): ##STR54## wherein Q and X are as defined above.
[0293] In one embodiment, the amide of formula XXII is prepared by the following steps: [0294] a) preparing a carboxylic acid of formula XXV by ring opening of a cyclic compound of formula XXIV ##STR55## wherein L, R.sub.1 and T are as defined above, and T.sub.1 is O or NH; and [0295] b) reacting an amine of formula XXVI: ##STR56## wherein Z and Y are as defined above, with the carboxylic acid of formula XXV in the presence of a coupling reagent, to produce the amide of formula XXII. ##STR57##
[0296] In one embodiment, step (a) is carried out in the presence of HBr.
[0297] In one embodiment, whereby compound XXV of step (a) is reacted with a coupling agent prior to step (b).
[0298] In another embodiment, the process for preparing a SARM compound: ##STR58## may comprise the steps of: [0299] a) reacting a ring of formula: ##STR59## wherein L, R.sub.1 are as defined above, and T.sub.1 is O or NH with a compound of: ##STR60## to produce a compound of formula: ##STR61## [0300] b) ring opening of compound produced in a to produce a compound of formula: ##STR62## wherein R.sub.1, T, X and Q are as defined above; and [0301] c) coupling the carboxylic acid of compound in (b) with the amine of formula: ##STR63## wherein Z and Y are as defined above, in the presence of a coupling reagent, to produce the desired SARM compound.
[0302] For example, compound XVI was prepared as follows: ##STR64## (2R)-1-Methacryloylpyrrolidin-2-carboxylic Acid.
[0303] D-Proline, 14.93 g, 0.13 mol) was dissolved in 71 mL of 2 N NaOH and cooled in an ice bath; the resulting alkaline solution was diluted with acetone (71 mL). An acetone solution (71 mL) of metacryloly chloride (13.56 g, 0.13 mol) and 2N NaOH solution (71 mL) were simultaneously added over 40 min to the aqueous solution of D-proline in an ice bath. The pH of the mixture was kept at 10-11.degree. C. during the addition of the metacryloly chloride. After stirring (3 h, room temperature), the mixture was evaporated in vacuo at a temperature at 35-45.degree. C. to remove acetone. The resulting solution was washed with ethyl ether and was acidified to pH 2 with concentrated HCl. The acidic mixture was saturated with NaCl and was extracted with EtOAc (100 mL.times.3). The combined extracts were dried over Na.sub.2SO.sub.4, filtered through Celite, and evaporated in vacuo to give the crude product as a colorless oil. Recrystallization of the oil from ethyl ether and hexanes afforded 16.2 (68%) of the desired compound as colorless crystals: mp 102-103.degree. C. (lit. [214] mp 102.5-103.5.degree. C.); the NMR spectrum of this compound demonstrated the existence of two rotamers of the title compound. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 5.28 (s) and 5.15 (s) for the first rotamer, 5.15 (s) and 5.03 (s) for the second rotamer (totally 2H for both rotamers, vinyl CH.sub.2), 4.48-4.44 for the first rotamer, 4.24-4.20 (m) for the second rotamer (totally 1H for both rotamers, CH at the chiral canter), 3.57-3.38 (m, 2H, CH.sub.2), 2.27-2.12 (1H, CH), 1.97-1.72 (m, 6H, CH.sub.2, CH, Me); .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. for major rotamer 173.3, 169.1, 140.9, 116.4, 58.3, 48.7, 28.9, 24.7, 19.5: for minor rotamer 174.0, 170.0, 141.6, 115.2, 60.3, 45.9, 31.0, 22.3, 19.7; IR (KBr) 3437 (OH), 1737 (C.dbd.O), 1647 (CO, COOH), 1584, 1508, 1459, 1369,1348, 1178 cm.sup.-1; [.alpha.].sub.D.sup.26+80.8.degree. (c=1, MeOH); Anal. Calcd. for C.sub.9H.sub.13NO.sub.3: C 59.00, H 7.15, N 7.65. Found: C 59.13, H 7.19, N 7.61. ##STR65## (3R,8aR)-3-Bromomethyl-3-methyl-tetrahydro-pyrrolo[2,1-c][1,4]oxazine-1,4- -dione.
[0304] A solution of NBS (23.5 g, 0.132 mol) in 100 mL of DMF was added dropwise to a stirred solution of the (methyl-acryloyl)-pyrrolidine (16.1 g, 88 mmol) in 70 mL of DMF under argon at room temperature, and the resulting mixture was stirred 3 days. The solvent was removed in vacuo, and a yellow solid was precipitated. The solid was suspended in water, stirred overnight at room temperature, filtered, and dried to give 18.6 (81%) (smaller weight when dried .about.34%) of the title compound as a yellow solid: mp 152-154.degree. C. (lit. [214] mp 107-109.degree. C. for the S-isomer); .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 4.69 (dd, J=9.6 Hz, J=6.7 Hz, 1H, CH at the chiral center), 4.02 (d, J=11.4 Hz, 1H, CHH.sub.a), 3.86 (d, J=1 1.4 Hz, 1H, CHH.sub.b), 3.53-3.24 (m, 4H, CH.sub.2), 2.30-2.20 (m, 1H, CH), 2.04-1.72 (m, 3H, CH.sub.2 and CH), 1.56 (s, 2H, Me); .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 167.3, 163.1, 83.9, 57.2, 45.4, 37.8, 29.0, 22.9, 21.6; IR (KBr) 3474, 1745 (C.dbd.O), 1687 (C.dbd.O), 1448, 1377, 1360, 1308, 1227, 1159, 1062 cm.sup.-1; [.alpha.].sub.D.sup.26+124.5.degree. (c=1.3, chloroform); Anal. Calcd. for C.sub.9H.sub.12BrNO.sub.3: C 41.24, H 4.61, N 5.34. Found: C 41.46, H 4.64, N 5.32. ##STR66## (2R)-3-Bromo-2-hydroxy-2-methylpropanoic Acid.
[0305] A mixture of bromolactone (18.5 g, 71 mmol) in 300 mL of 24% HBr was heated at reflux for 1 h. The resulting solution was diluted with brine (200 mL), and was extracted with ethyl acetate (100 mL.times.4). The combined extracts were washed with saturated NaHCO.sub.3 (100 mL.times.4). The aqueous solution was acidified with concentrated HCl to pH=1, which, in turn, was extracted with ethyl acetate (100 mL.times.4). The combined organic solution was dried over Na.sub.2SO.sub.4, filtered through Celite, and evaporated in vacuo to dryness. Recrystallization from toluene afforded 10.2 g (86%) of the desired compound as colorless crystals: mp 107-109.degree. C. (lit. [214] mp 109-113.degree. C. for the S-isomer); .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 3.63 (d, J=10.1 Hz, 1H, CHH.sub.a), 3.52 (d, J=10.1 Hz, 1H, CHH.sub.b), 1.35 (s, 3H, Me); IR (KBr) 3434 (OH), 3300-2500 (COOH), 1730 (C.dbd.O), 1449, 1421, 1380, 1292, 1193, 1085 cm.sup.-1; [.alpha.].sub.D.sup.26+10.5.degree. (c=2.6, MeOH); Anal. Calcd. for C.sub.4H.sub.7BrO.sub.3: C 26.25, H 3.86. Found: C 26.28, H 3.75. ##STR67## Synthesis of (2R)-3-Bromo-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylprop- anamide.
[0306] Thionyl chloride (46.02 g, 0.39 mol) was added dropwise to a cooled solution (less than 4.degree. C.) of R-131 (51.13 g, 0.28 mol) in 300 mL of THF under an argon atmosphere. The resulting mixture was stirred for 3 h under the same condition. To this was added Et.sub.3N (39.14 g, 0.39 mol) and stirred for 20 min under the same condition. After 20 min, 5-amino-2-cyanobenzotrifluoride (40.0 g, 0.21 mol), 400 mL of THF were added and then the mixture was allowed to stir overnight at room temperature. The solvent was removed under reduced pressure to give a solid which was treated with 300 mL of H.sub.2O, extracted with EtOAc (2.times.400 mL). The combined organic extracts were washed with saturated NaHCO.sub.3 solution (2.times.300 mL) and brine (300 mL). The organic layer was dried over MgSO.sub.4 and concentrated under reduced pressure to give a solid which was purified from column chromatography using CH.sub.2Cl.sub.2/EtOAc (80:20) to give a solid. This solid was recrystallized from CH.sub.2Cl.sub.2/hexane to give 55.8 g (73.9%) of (2R)-3-Bromo-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylprop- anamide as a light-yellow solid.
[0307] .sup.1H NMR (CDCl.sub.3/TMS) .delta. 1.66 (s, 3H, CH.sub.3), 3.11 (s, 1H, OH), 3.63 (d, J=10.8 Hz, 1H, CH.sub.2), 4.05 (d, J=10.8 Hz, 1H, CH.sub.2), 7.85 (d, J=8.4 Hz, 1H, ArH), 7.99 (dd, J=2.1, 8.4 Hz, 1H, ArH), 8.12 (d, J=2.1 Hz, 1H, ArH), 9.04 (bs, 1H, NH). Calculated Mass: 349.99, [M-H].sup.- 349.0. M.p.: 124-126.degree. C. ##STR68## Synthesis of (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-cyanophenoxy)-2-hydroxy-2-- methylpropanamide.
[0308] A mixture of bromoamide ((2R)-3-Bromo-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpro- panamide, 50 g, 0.14 mol), anhydrous K.sub.2CO.sub.3 (59.04 g, 0.43 mol), 4-cyanophenol (25.44 g, 0.21 mol) in 500 mL of 2-propanol was heated to reflux for 3 h and then concentrated under reduced pressure to give a solid. The resulting residue was treated with 500 mL of H.sub.2O and then extracted with EtOAc (2.times.300 mL). The combined EtOAc extracts were washed with 10% NaOH (4.times.200 mL) and brine. The organic layer was dried over MgSO.sub.4 and then concentrated under reduced pressure to give an oil which was treated with 300 mL of ethanol and an activated carbon. The reaction mixture was heated to reflux for 1 h and then the hot mixture was filtered through Celite. The filtrate was concentrated under reduced pressure to give an oil. This oil was purified by column chromatography using CH.sub.2Cl.sub.2/EtOAc (80:20) to give an oil which was crystallized from CH.sub.2Cl.sub.2/hexane to give 33.2 g (59.9%) of (S)-N-(4-cyano-3-(trifluoromethyl)phenyl)-3-(4-cyanophenoxy)-2-hydroxy-2-- methylpropanamide as a colorless solid (a cotton type).
[0309] .sup.1H NMR (CDCl.sub.3/TMS) .delta. 1.63 (s, 3H, CH.sub.3), 3.35 (s, 1H, OH), 4.07 (d, J=9.04 Hz, 1H, CH), 4.51 (d, J=9.04 Hz, 1H, CH), 6.97-6.99 (m, 2H, ArH), 7.57-7.60 (m, 2H, ArH), 7.81 (d, J=8.55 Hz, 1H, ArH), 7.97 (dd, J=1.95, 8.55 Hz, 1H, ArH), 8.12 (d, J=1.95 Hz, 1H, ArH), 9.13 (bs, 1H, NH). Calculated Mass: 389.10, [M-H].sup.- 388.1. Mp: 92-94.degree. C.
Example 2
Treatment of Subjects with Skin Ulcerations or Burns with SARMs
[0319] In order to determine whether the compounds of this invention are useful in treating skin wounds and/or burns, representative compounds, such as those in Example 1 are prepared as a topical formulation, and in another embodiment, an intravenous formulation. Patients presenting with cutaneous ulcers or burns are assessed.
[0320] In subjects with wounds, the wounds may be debrided and the SARM applied topically, or standard therapy including antiseptics and chemotherapeutics may be administered to the wound site, and the SARM is provided orally or intravenously.
[0321] In subjects with cutaneous ulcers or burns, the SARMs may be applied topically, or intravenously, as described, alone, or in combination with other known therapies, such as antibiotics, growth factors, etc.
[0322] The SARM treatment may be repeated over time, and gross evaluation of the wound or burn site is evaluated, pain is assessed.
[0323] Other parameters evaluated may include determination of the percent body weight loss, and loss of muscle mass. Another group of subjects may comprise those given a high calorie, high protein diets which may include vitamin and mineral supplements.
[0324] Standard animal models of burns and/or wounds may also be evaluated in this context. For example, Sprague-Dawley rats given a standard contact burn (20% TBSA), are evaluated. On day 3, wounds are excised and infected with Pseudomonas aeruginosa and Staphylococcus aureus at 5.0.times.10.sup.5 cfu/ml. The animals are then divided into treatment groups, and treated as described.
Example 3
Treatment of Subjects with Spinal Cord Injuries with SARMs
[0325] Animal models of spinal cord injuries are evaluated as follows: fully adult (approximately 400 grams (g)) female guinea pigs (Hartley Strain) are anesthetized with ketamine/xylyzine by conventional methods (Borgens et al. (2002) J. Exp. Biol. 205, 1-12) prior to surgery, kept warm with heat lamps after surgery, and maintained individually in pens and fed ad libidum. Animals were euthanized at the end of the study, prior to the harvesting of spinal cords for anatomical study by an overdose of the anesthesia (see Borgens et al. (2002) J. Exp. Biol. 205, 1-12).
[0326] A laminectomy procedure exposing the dorsal aspect of the spinal cord was performed on all animals between T9 and T11. A right lateral hemisection is performed, and confirmed to be "complete" (leaving no spared parenchyma) by passing a sharpened pin through the cut tissue. This operation severs the entire right side of the cord from the midline to the far right border of the spinal cord forming a rostral and caudal segment. The entire left side of the spinal cord is left intact. Immediately after transection, a marker device made of surgical stainless steel is inserted into the lesion, as previously described in Borgens et al. (1986) J. Comp. Neurol 250, 168-180 and Borgens and Bohnert (1997) Exp. Neurol. 145, 376-389, which is left in situ for the duration of the study, and removed prior to histological processing. This procedure leaves a hole in the tissue, which accurately marks the exact plane of transaction even in chronic injuries many months old.
[0327] In addition, subjects with spinal cord injury (SCI) may be treated experimentally with SARMs. Human studies have shown that central neurotransmitters may be altered after SCI and thus cause a hypothalamus-pituitary-adrenal axis dysfunction, leading to a decrease in testosterone and other hormone levels.
[0328] Moreover, the effect of SCI or other acute illness or trauma characteristically includes heightened catabolism in conjunction with the lowered anabolic activity resulting in a condition that is prone to loss of lean body tissue. As long as the catabolic process goes uninterrupted, disturbed nutrient utilization will continue. The effects of the loss of lean body mass include the development of wounds and impaired healing mechanisms. Because of poor nutrition and protein combined with immobilization, patients with spinal cord injury are at high risk for bed sores.
[0329] As described in Example 2, SARMs are useful in treating wounds, enhancing muscle mass, and diminishing cachexia, thus SARM treatment in SCI subjects will be evaluated, and its effect on these conditions assessed.
[0330] It will be appreciated by a person skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove.
<center style="color: rgb(0, 0, 0); font-family: -webkit-standard;">Abstract</center>This invention provides: 1) a method of treating a subject suffering from, or predisposed to a kidney disease or disorder; 2) a method of treating a subject suffering from a wound, or reducing the incidence of, or mitigating the severity of a wound in a subject; 3) a method of treating a subject suffering from a burn, or reducing the incidence of, or mitigating the severity of a burn in a subject, comprising the step of administering to said subject a selective androgen receptor modulator (SARM) compound; 4) a method of treating a subject suffering from a spinal cord injury, by administering to the subject a selective androgen receptor modulator (SARM) and/or an analog, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, prodrug, polymorph, impurity or crystal of said SARM compound, or any combination thereof.
Example 1
Effect of Selective Androgen Receptor Modulators (SARMS) on Subjects with End-Stage Renal Disease
Compounds Tested:
[0290] The following Compounds, which are ligands for the AR with potent binding affinity, exhibiting tissue-selective androgenic and anabolic effects, and oral bioavailability, will be assessed for their effects on end-stage renal disease (ESRD). ##STR50## ##STR51##
[0291] The compounds will be synthesized and characterized, as described for example in U.S. patent application Ser. Nos. 10/277,108, 10/270,732 and 10/371,155, all of which are incorporated herein by reference.
[0292] For example, the process for preparing a SARM compound represented by the structure of formula (I): ##STR52## with substituents as described herein, may comprise the step of coupling an amide of formula (XXII): ##STR53## wherein Z, Y, R.sub.1 and T are as defined above and L is a leaving group, with a compound of formula (XXIII): ##STR54## wherein Q and X are as defined above.
[0293] In one embodiment, the amide of formula XXII is prepared by the following steps: [0294] a) preparing a carboxylic acid of formula XXV by ring opening of a cyclic compound of formula XXIV ##STR55## wherein L, R.sub.1 and T are as defined above, and T.sub.1 is O or NH; and [0295] b) reacting an amine of formula XXVI: ##STR56## wherein Z and Y are as defined above, with the carboxylic acid of formula XXV in the presence of a coupling reagent, to produce the amide of formula XXII. ##STR57##
[0296] In one embodiment, step (a) is carried out in the presence of HBr.
[0297] In one embodiment, whereby compound XXV of step (a) is reacted with a coupling agent prior to step (b).
[0298] In another embodiment, the process for preparing a SARM compound: ##STR58## may comprise the steps of: [0299] a) reacting a ring of formula: ##STR59## wherein L, R.sub.1 are as defined above, and T.sub.1 is O or NH with a compound of: ##STR60## to produce a compound of formula: ##STR61## [0300] b) ring opening of compound produced in a to produce a compound of formula: ##STR62## wherein R.sub.1, T, X and Q are as defined above; and [0301] c) coupling the carboxylic acid of compound in (b) with the amine of formula: ##STR63## wherein Z and Y are as defined above, in the presence of a coupling reagent, to produce the desired SARM compound.
[0302] For example, compound XVI was prepared as follows: ##STR64## (2R)-1-Methacryloylpyrrolidin-2-carboxylic Acid.
[0303] D-Proline, 14.93 g, 0.13 mol) was dissolved in 71 mL of 2 N NaOH and cooled in an ice bath; the resulting alkaline solution was diluted with acetone (71 mL). An acetone solution (71 mL) of metacryloly chloride (13.56 g, 0.13 mol) and 2N NaOH solution (71 mL) were simultaneously added over 40 min to the aqueous solution of D-proline in an ice bath. The pH of the mixture was kept at 10-11.degree. C. during the addition of the metacryloly chloride. After stirring (3 h, room temperature), the mixture was evaporated in vacuo at a temperature at 35-45.degree. C. to remove acetone. The resulting solution was washed with ethyl ether and was acidified to pH 2 with concentrated HCl. The acidic mixture was saturated with NaCl and was extracted with EtOAc (100 mL.times.3). The combined extracts were dried over Na.sub.2SO.sub.4, filtered through Celite, and evaporated in vacuo to give the crude product as a colorless oil. Recrystallization of the oil from ethyl ether and hexanes afforded 16.2 (68%) of the desired compound as colorless crystals: mp 102-103.degree. C. (lit. [214] mp 102.5-103.5.degree. C.); the NMR spectrum of this compound demonstrated the existence of two rotamers of the title compound. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 5.28 (s) and 5.15 (s) for the first rotamer, 5.15 (s) and 5.03 (s) for the second rotamer (totally 2H for both rotamers, vinyl CH.sub.2), 4.48-4.44 for the first rotamer, 4.24-4.20 (m) for the second rotamer (totally 1H for both rotamers, CH at the chiral canter), 3.57-3.38 (m, 2H, CH.sub.2), 2.27-2.12 (1H, CH), 1.97-1.72 (m, 6H, CH.sub.2, CH, Me); .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. for major rotamer 173.3, 169.1, 140.9, 116.4, 58.3, 48.7, 28.9, 24.7, 19.5: for minor rotamer 174.0, 170.0, 141.6, 115.2, 60.3, 45.9, 31.0, 22.3, 19.7; IR (KBr) 3437 (OH), 1737 (C.dbd.O), 1647 (CO, COOH), 1584, 1508, 1459, 1369,1348, 1178 cm.sup.-1; [.alpha.].sub.D.sup.26+80.8.degree. (c=1, MeOH); Anal. Calcd. for C.sub.9H.sub.13NO.sub.3: C 59.00, H 7.15, N 7.65. Found: C 59.13, H 7.19, N 7.61. ##STR65## (3R,8aR)-3-Bromomethyl-3-methyl-tetrahydro-pyrrolo[2,1-c][1,4]oxazine-1,4- -dione.
[0304] A solution of NBS (23.5 g, 0.132 mol) in 100 mL of DMF was added dropwise to a stirred solution of the (methyl-acryloyl)-pyrrolidine (16.1 g, 88 mmol) in 70 mL of DMF under argon at room temperature, and the resulting mixture was stirred 3 days. The solvent was removed in vacuo, and a yellow solid was precipitated. The solid was suspended in water, stirred overnight at room temperature, filtered, and dried to give 18.6 (81%) (smaller weight when dried .about.34%) of the title compound as a yellow solid: mp 152-154.degree. C. (lit. [214] mp 107-109.degree. C. for the S-isomer); .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 4.69 (dd, J=9.6 Hz, J=6.7 Hz, 1H, CH at the chiral center), 4.02 (d, J=11.4 Hz, 1H, CHH.sub.a), 3.86 (d, J=1 1.4 Hz, 1H, CHH.sub.b), 3.53-3.24 (m, 4H, CH.sub.2), 2.30-2.20 (m, 1H, CH), 2.04-1.72 (m, 3H, CH.sub.2 and CH), 1.56 (s, 2H, Me); .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 167.3, 163.1, 83.9, 57.2, 45.4, 37.8, 29.0, 22.9, 21.6; IR (KBr) 3474, 1745 (C.dbd.O), 1687 (C.dbd.O), 1448, 1377, 1360, 1308, 1227, 1159, 1062 cm.sup.-1; [.alpha.].sub.D.sup.26+124.5.degree. (c=1.3, chloroform); Anal. Calcd. for C.sub.9H.sub.12BrNO.sub.3: C 41.24, H 4.61, N 5.34. Found: C 41.46, H 4.64, N 5.32. ##STR66## (2R)-3-Bromo-2-hydroxy-2-methylpropanoic Acid.
[0305] A mixture of bromolactone (18.5 g, 71 mmol) in 300 mL of 24% HBr was heated at reflux for 1 h. The resulting solution was diluted with brine (200 mL), and was extracted with ethyl acetate (100 mL.times.4). The combined extracts were washed with saturated NaHCO.sub.3 (100 mL.times.4). The aqueous solution was acidified with concentrated HCl to pH=1, which, in turn, was extracted with ethyl acetate (100 mL.times.4). The combined organic solution was dried over Na.sub.2SO.sub.4, filtered through Celite, and evaporated in vacuo to dryness. Recrystallization from toluene afforded 10.2 g (86%) of the desired compound as colorless crystals: mp 107-109.degree. C. (lit. [214] mp 109-113.degree. C. for the S-isomer); .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 3.63 (d, J=10.1 Hz, 1H, CHH.sub.a), 3.52 (d, J=10.1 Hz, 1H, CHH.sub.b), 1.35 (s, 3H, Me); IR (KBr) 3434 (OH), 3300-2500 (COOH), 1730 (C.dbd.O), 1449, 1421, 1380, 1292, 1193, 1085 cm.sup.-1; [.alpha.].sub.D.sup.26+10.5.degree. (c=2.6, MeOH); Anal. Calcd. for C.sub.4H.sub.7BrO.sub.3: C 26.25, H 3.86. Found: C 26.28, H 3.75. ##STR67## Synthesis of (2R)-3-Bromo-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylprop- anamide.
[0306] Thionyl chloride (46.02 g, 0.39 mol) was added dropwise to a cooled solution (less than 4.degree. C.) of R-131 (51.13 g, 0.28 mol) in 300 mL of THF under an argon atmosphere. The resulting mixture was stirred for 3 h under the same condition. To this was added Et.sub.3N (39.14 g, 0.39 mol) and stirred for 20 min under the same condition. After 20 min, 5-amino-2-cyanobenzotrifluoride (40.0 g, 0.21 mol), 400 mL of THF were added and then the mixture was allowed to stir overnight at room temperature. The solvent was removed under reduced pressure to give a solid which was treated with 300 mL of H.sub.2O, extracted with EtOAc (2.times.400 mL). The combined organic extracts were washed with saturated NaHCO.sub.3 solution (2.times.300 mL) and brine (300 mL). The organic layer was dried over MgSO.sub.4 and concentrated under reduced pressure to give a solid which was purified from column chromatography using CH.sub.2Cl.sub.2/EtOAc (80:20) to give a solid. This solid was recrystallized from CH.sub.2Cl.sub.2/hexane to give 55.8 g (73.9%) of (2R)-3-Bromo-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylprop- anamide as a light-yellow solid.
[0307] .sup.1H NMR (CDCl.sub.3/TMS) .delta. 1.66 (s, 3H, CH.sub.3), 3.11 (s, 1H, OH), 3.63 (d, J=10.8 Hz, 1H, CH.sub.2), 4.05 (d, J=10.8 Hz, 1H, CH.sub.2), 7.85 (d, J=8.4 Hz, 1H, ArH), 7.99 (dd, J=2.1, 8.4 Hz, 1H, ArH), 8.12 (d, J=2.1 Hz, 1H, ArH), 9.04 (bs, 1H, NH). Calculated Mass: 349.99, [M-H].sup.- 349.0. M.p.: 124-126.degree. C. ##STR68## Synthesis of (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-cyanophenoxy)-2-hydroxy-2-- methylpropanamide.
[0308] A mixture of bromoamide ((2R)-3-Bromo-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpro- panamide, 50 g, 0.14 mol), anhydrous K.sub.2CO.sub.3 (59.04 g, 0.43 mol), 4-cyanophenol (25.44 g, 0.21 mol) in 500 mL of 2-propanol was heated to reflux for 3 h and then concentrated under reduced pressure to give a solid. The resulting residue was treated with 500 mL of H.sub.2O and then extracted with EtOAc (2.times.300 mL). The combined EtOAc extracts were washed with 10% NaOH (4.times.200 mL) and brine. The organic layer was dried over MgSO.sub.4 and then concentrated under reduced pressure to give an oil which was treated with 300 mL of ethanol and an activated carbon. The reaction mixture was heated to reflux for 1 h and then the hot mixture was filtered through Celite. The filtrate was concentrated under reduced pressure to give an oil. This oil was purified by column chromatography using CH.sub.2Cl.sub.2/EtOAc (80:20) to give an oil which was crystallized from CH.sub.2Cl.sub.2/hexane to give 33.2 g (59.9%) of (S)-N-(4-cyano-3-(trifluoromethyl)phenyl)-3-(4-cyanophenoxy)-2-hydroxy-2-- methylpropanamide as a colorless solid (a cotton type).
[0309] .sup.1H NMR (CDCl.sub.3/TMS) .delta. 1.63 (s, 3H, CH.sub.3), 3.35 (s, 1H, OH), 4.07 (d, J=9.04 Hz, 1H, CH), 4.51 (d, J=9.04 Hz, 1H, CH), 6.97-6.99 (m, 2H, ArH), 7.57-7.60 (m, 2H, ArH), 7.81 (d, J=8.55 Hz, 1H, ArH), 7.97 (dd, J=1.95, 8.55 Hz, 1H, ArH), 8.12 (d, J=1.95 Hz, 1H, ArH), 9.13 (bs, 1H, NH). Calculated Mass: 389.10, [M-H].sup.- 388.1. Mp: 92-94.degree. C.
Example 2
Treatment of Subjects with Skin Ulcerations or Burns with SARMs
[0319] In order to determine whether the compounds of this invention are useful in treating skin wounds and/or burns, representative compounds, such as those in Example 1 are prepared as a topical formulation, and in another embodiment, an intravenous formulation. Patients presenting with cutaneous ulcers or burns are assessed.
[0320] In subjects with wounds, the wounds may be debrided and the SARM applied topically, or standard therapy including antiseptics and chemotherapeutics may be administered to the wound site, and the SARM is provided orally or intravenously.
[0321] In subjects with cutaneous ulcers or burns, the SARMs may be applied topically, or intravenously, as described, alone, or in combination with other known therapies, such as antibiotics, growth factors, etc.
[0322] The SARM treatment may be repeated over time, and gross evaluation of the wound or burn site is evaluated, pain is assessed.
[0323] Other parameters evaluated may include determination of the percent body weight loss, and loss of muscle mass. Another group of subjects may comprise those given a high calorie, high protein diets which may include vitamin and mineral supplements.
[0324] Standard animal models of burns and/or wounds may also be evaluated in this context. For example, Sprague-Dawley rats given a standard contact burn (20% TBSA), are evaluated. On day 3, wounds are excised and infected with Pseudomonas aeruginosa and Staphylococcus aureus at 5.0.times.10.sup.5 cfu/ml. The animals are then divided into treatment groups, and treated as described.
Example 3
Treatment of Subjects with Spinal Cord Injuries with SARMs
[0325] Animal models of spinal cord injuries are evaluated as follows: fully adult (approximately 400 grams (g)) female guinea pigs (Hartley Strain) are anesthetized with ketamine/xylyzine by conventional methods (Borgens et al. (2002) J. Exp. Biol. 205, 1-12) prior to surgery, kept warm with heat lamps after surgery, and maintained individually in pens and fed ad libidum. Animals were euthanized at the end of the study, prior to the harvesting of spinal cords for anatomical study by an overdose of the anesthesia (see Borgens et al. (2002) J. Exp. Biol. 205, 1-12).
[0326] A laminectomy procedure exposing the dorsal aspect of the spinal cord was performed on all animals between T9 and T11. A right lateral hemisection is performed, and confirmed to be "complete" (leaving no spared parenchyma) by passing a sharpened pin through the cut tissue. This operation severs the entire right side of the cord from the midline to the far right border of the spinal cord forming a rostral and caudal segment. The entire left side of the spinal cord is left intact. Immediately after transection, a marker device made of surgical stainless steel is inserted into the lesion, as previously described in Borgens et al. (1986) J. Comp. Neurol 250, 168-180 and Borgens and Bohnert (1997) Exp. Neurol. 145, 376-389, which is left in situ for the duration of the study, and removed prior to histological processing. This procedure leaves a hole in the tissue, which accurately marks the exact plane of transaction even in chronic injuries many months old.
[0327] In addition, subjects with spinal cord injury (SCI) may be treated experimentally with SARMs. Human studies have shown that central neurotransmitters may be altered after SCI and thus cause a hypothalamus-pituitary-adrenal axis dysfunction, leading to a decrease in testosterone and other hormone levels.
[0328] Moreover, the effect of SCI or other acute illness or trauma characteristically includes heightened catabolism in conjunction with the lowered anabolic activity resulting in a condition that is prone to loss of lean body tissue. As long as the catabolic process goes uninterrupted, disturbed nutrient utilization will continue. The effects of the loss of lean body mass include the development of wounds and impaired healing mechanisms. Because of poor nutrition and protein combined with immobilization, patients with spinal cord injury are at high risk for bed sores.
[0329] As described in Example 2, SARMs are useful in treating wounds, enhancing muscle mass, and diminishing cachexia, thus SARM treatment in SCI subjects will be evaluated, and its effect on these conditions assessed.
[0330] It will be appreciated by a person skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove.
