OXYSTEROLS AND METHODS OF USE THEREOF

Abstract

Compounds are provided according to Formula (I):

##STR00001##

and pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof; wherein X, Y, R.sup.1, R.sup.2a, R.sup.2b, R.sup.4a, R.sup.4b, R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7, and R.sup.8 are as defined herein. Compounds of the present invention are contemplated useful for the prevention and treatment of a variety of conditions.

Claims

1. A compound of Formula (I): ##STR00032## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, carbocyclyl, or heterocyclyl; each of R.sup.2 and R.sup.2b is independently hydrogen, C.sub.1-C.sub.6 alkyl, halo, cyano, OR.sup.A, or NR.sup.BR.sup.C, or R.sup.2a and R.sup.2b together with the carbon atom to which they are attached form a ring (e.g., a 3-7-membered ring, e.g., a 5-7-membered ring; a ring containing at least one heteroatom, e.g., a nitrogen, oxygen, or sulfur atom); each of R.sup.4a and R.sup.4b is independently absent, hydrogen, C.sub.1-C.sub.6 alkyl, or halo; X is C(R.sup.X).sub.2 or O, wherein R.sup.X is independently hydrogen, halo, or one R.sup.X group and R.sup.5b are joined to form a double bond; Y is OR.sup.Y, wherein R is hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C(O)R.sup.A, C(O)OR.sup.A, C(O)NR.sup.BR.sup.C, or S(O).sub.2R.sup.D; each instance of R.sup.5a and R.sup.5b is independently hydrogen, halo, or C.sub.1-C.sub.6 alkyl; each of R.sup.6a and R.sup.6b is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, or R.sup.6a and R.sup.6b, taken together with the carbon atom to which they are attached, form a ring (e.g., a 3-6-membered ring, e.g. a 4-6-membered ring containing one heteroatom); or R.sup.5a and R.sup.6a, together with the carbon atoms to which they are attached, form a ring (e.g., a 3-6-membered ring, e.g. a 4-6-membered ring containing one heteroatom); and R.sup.7 is absent or hydrogen in the alpha configuration; R.sup.8 is hydrogen, halo, C.sub.1-6alkyl, carbocyclyl, or OR.sup.A; represents a single or double bond, wherein when one is a double bond, the other is a single bond; wherein when the between CR.sup.7 and CR.sup.4aR.sup.4b is a double bond, then one of R.sup.4a or R.sup.4b is absent; and when one of the is a double bond, R.sup.7 is absent; R.sup.A is hydrogen, C.sub.1-C.sub.6 alkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl; each of R.sup.B and R.sup.C is independently hydrogen, C.sub.1-C.sub.6 alkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, or taken together with the atom to which they are attached form a ring (e.g., a 3-7-membered ring, e.g., a 5-7-membered ring; a ring containing at least one heteroatom, e.g., a nitrogen, oxygen, or sulfur atom); and R.sup.D is hydrogen, C.sub.1-C.sub.6 alkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

2. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein: a. R.sup.1 is unsubstituted C.sub.1-3 alkyl; b. R.sup.1 is CH.sub.3, CH.sub.2CH.sub.3, or CH.sub.2CH.sub.2CH.sub.3; c. R.sup.1 is substituted C.sub.1-3 alkyl; or d. R.sup.1 is haloalkyl (e.g., CF.sub.3) or CH.sub.2OCH.sub.3.

3-5. (canceled)

6. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein: a. R.sup.2a or R.sup.2b is hydrogen; or b. R.sup.2a and R.sup.2b are both hydrogen.

7. (canceled)

8. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein R.sup.4a is hydrogen.

9. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein X is CH.sub.2.

10. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein: a. R is substituted or unsubstituted C.sub.1-3 alkyl; or b. R.sup.8 is CH.sub.3.

11. (canceled)

12. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein the compound of Formula (I) is a compound of Formula (II): ##STR00033##

13. The compound of claim 12, or the pharmaceutically acceptable salt thereof, wherein the compound of Formula (II) is a compound of Formula (II-A): ##STR00034##

14. The compound of claim 12, or the pharmaceutically acceptable salt thereof, wherein the compound of Formula (II) is a compound of Formula (II-B): ##STR00035##

15. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein: a R.sup.5a or R.sup.5b is hydrogen; or b. R.sup.5a and R.sup.5b are both hydrogen.

16. (canceled)

17. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein: a. R.sup.6a is a substituted or unsubstituted C.sub.1-3 alkyl; b. R.sup.6a is CH.sub.3 or CH.sub.2CH.sub.3; or c. R.sup.6a is a substituted or unsubstituted C.sub.2-4 alkyl, substituted or unsubstituted C.sub.2-3 alkenyl, substituted or unsubstituted C.sub.2-3 alkynyl, or substituted or unsubstituted carbocyclyl.

18.-19. (canceled)

20. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein: a. R.sup.6b is substituted or unsubstituted C.sub.1-3 alkyl; b. R.sup.6b is CH.sub.3 or CH.sub.2CH.sub.3; c. R.sup.6b is hydrogen; or d. R.sup.6b is CH.sub.3 or CF.sub.3.

21.-23. (canceled)

24. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein: a. R.sup.6a and R.sup.6b are both CH.sub.3; b. R.sup.6a and R.sup.6b, taken together with the atom to which they are attached, form a ring; or c. R.sup.6a and R.sup.6b, taken together with the atom to which they are attached, form a 3-membered ring.

25.-26. (canceled)

27. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein R.sup.1 is hydrogen or C.sub.1-3 alkyl, R.sup.6a is substituted or unsubstituted C.sub.1-3 alkyl, substituted or unsubstituted C.sub.2-3 alkenyl, substituted or unsubstituted C.sub.2-3 alkynyl, or substituted or unsubstituted carbocyclyl, and R.sup.6b is CH.sub.3.

28. The compound of claim 27, or the pharmaceutically acceptable salt thereof, wherein: a. R.sup.6a is selected from the group consisting of substituted or unsubstituted C.sub.1-3 alkyl, unsubstituted C.sub.2-3 alkenyl, unsubstituted C.sub.2-3 alkynyl, or unsubstituted carbocyclyl; or b. R.sup.6a is selected from a substituted or unsubstituted C.sub.1-3 alkyl.

29. (canceled)

30. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein R.sup.1 is CH.sub.3 or CH.sub.2CH.sub.3 and R.sup.6b is CH.sub.3 or CF.sub.3.

31. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein: a. R.sup.Y is substituted or unsubstituted C.sub.1-3 alkyl; b. R.sup.Y is substituted or unsubstituted heterocyclyl; c. R is CH.sub.3; or d. R.sup.Y is CF.sub.3.

32.-34. (canceled)

35. The compound of claim 1, or the pharmaceutically acceptable salt thereof, wherein the compound is: ##STR00036## ##STR00037##

36. A pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier.

37. A method of inducing sedation or anesthesia comprising administering to a subject an effective amount of a compound or pharmaceutically acceptable salt thereof according to claim 1, or pharmaceutical composition thereof.

38. A method for treating or preventing a disorder, comprising administering to a subject in need thereof an effective amount of a compound of or pharmaceutically acceptable salt thereof according to claim 1, or pharmaceutical composition thereof; wherein the disorder is selected from the group consisting of a gastrointestinal (GI) disorder, inflammatory bowel disease (IBD), a structural disorder affecting the GI, an anal disorder, a colon polyp, cancer, diabetes, a sterol synthesis disorder, and colitis.

39.-41. (canceled)

42. A method for treating or preventing a CNS-related condition comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutically acceptable salt thereof according to claim 1, or pharmaceutical composition thereof.

43. The method according to claim 42, wherein the CNS-related condition is an adjustment disorder, anxiety disorder, cognitive disorder, dissociative disorder, eating disorder, mood disorder, schizophrenia or other psychotic disorder, disorder, substance-related disorder, personality disorder, autism spectrum disorders, neurodevelopmental disorder, multiple sclerosis, sterol synthesis disorders, pain, encephalopathy secondary to a medical condition, seizure disorder, stroke, traumatic brain injury, movement disorder, vision impairment, hearing loss, and tinnitus.

44. (canceled)

Description

EXAMPLES

[0152] In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

Example 1. Synthesis of Compound 1

[0153] ##STR00013## ##STR00014##

Synthesis of compound A2. To a solution of reactant A1 (50 g, 127 mmol) in MeOH (500 mL) was added H.sub.2SO.sub.4 (Cat, conc. 5 mL). After heating at reflux for overnight, the solvent was removed under reduced pressure. The residue was diluted with EtOAc (1000 mL), washed by NaHCO.sub.3 (150 mL2), brine (150 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated to give compound A2 (49 g, 94%) as a white solid which was used in the next step without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3), 4.08 (m, 1H), 3.67 (s, 3H), 3.64 (m, 1H), 0.93-0.92 (d, 3H), 0.92 (s, 3H), 0.65 (s, 3H).
Synthesis of compound A3. To a solution of A2 (60 g, 148 mmol) in dry pyridine (400 ml) was added a solution of 4-toluenesulfonyl chloride (62 g, 325 mmol) in dry pyridine (200 ml). After stirring at room temperature for 2 days, ice chips were added gradually to the mixture. The precipitated solid was filtered, then washed with 10% HCl and water to give crude product A3 (100 g, 95%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3), 7.80-7.78 (d, 2H), 7.74-7.72 (d, 2H), 7.35 (t, 4H), 4.81 (m, 1H), 4.32 (m, 1H), 2.47 (s, 6H), 0.90-0.88 (d, 3H), 0.81 (s, 3H), 0.60 (s, 3H).
Synthesis of compound A4. A solution of A3 (6.72 g, 9.4 mmol) and potassium acetate (720 mg, 7.2 mmol) in water (6 mL) and DMF (40 mL) was heated at reflux for overnight. The reaction mixture was poured into ice-cold water and extracted with EtOAc (100 ml3). The combined organic layers were washed with brine (80 mL2), dried over Na.sub.2SO.sub.4 filtered and concentrated. The crude product was purified by column chromatography (silica gel, EA/PE=5:1) to give A4 (1.60 g, 43%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3), 5.36 (t, 1H), 3.67 (s, 3H), 3.53 (m, 1H), 1.00 (s, 3H), 0.93-0.92 (d, 3H), 0.68 (s, 3H).
Synthesis of compound A5. A solution of A4 (1.60 g, 4.1 mmol) in acetic anhydride (40 mL) was heated to 90 C. for overnight. The solvent was removed by reduced pressure, the residue was diluted with saturated NaHCO.sub.3 (50 mL) and stirred for 2 h. The mixture was extracted with EtOAc (50 mL3) and the combined organic layers were washed with brine (60 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was purified by column chromatography (silica gel, EA/PE=1:6) to give A5 (1590 mg, 90%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3), 5.39-5.38 (d, 1H), 4.60 (m, 1H), 3.66 (s, 3H), 2.03 (s, 3H), 1.01 (s, 3H), 0.93-0.92 (d, 3H), 0.68 (s, 3H).
Synthesis of compound A6. To a solution of A5 (200 mg, 0.46 mmol) in 1,4-dioxane (10 mL) was added water (1 mL) and perchloric acid (0.2 mL, 0.78 mmol). The resulting mixture was protected from light and cooled to 10 C. N-Bromosuccinimide (125 mg, 0.70 mmol) was added in one portion. After stirring at 10 C. for 30 min, another portion of N-bromosuccinimide (42 mg, 0.24 mmol) was added. The reaction mixture was stirred until TLC showed no SM. The reaction mixture was quenched with 0.1M of Na.sub.2S.sub.2O.sub.5 solution (40 mL) and extracted with EtOAc (40 mL3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography (PE:EA 10:1, 5:1) to afford A6 (100 mg, 42%) and A6-a (50 mg, 21%) as a white solid. A6 .sup.1H NMR (400 MHz, CDCl.sub.3), 5.09 (m, 1H), 3.98 (s, 1H), 3.67 (s, 3H), 2.06 (s, 3H), 1.36 (s, 3H), 0.94-0.92 (d, 3H), 0.72 (s, 3H); A6-a .sup.1H NMR (400 MHz, CDCl.sub.3), 5.49 (m, 1H), 4.2 (s, 1H), 2.04 (s, 3H), 1.33 (s, 3H), 0.93-0.91 (d, 3H), 0.68 (s, 3H).
Synthesis of compound A7. A solution of Pd(OAc).sub.4 (1.14 g, 3.32 mmol) and 12 (170 mg, 0.67 mmol) in cyclohexane (60 mL) was heated to refluxed for 10 min. Then compound A6 (700 mg, 1.33 mmol) and AIBN (10 mg, 0.08 mmol) were added and the resulting mixture was refluxed for overnight. The reaction mixture was allowed to cool to room temperature, filtered over a plug of celite and washed with EtOAc (100 mL). The organic layer was washed with a solution of 10% sodium metabisulfite (40 mL2), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography (silica gel, EA/PE=1:5) to give A7 (500 mg, 83%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3), 5.21 (m, 1H), 4.07-4.06 (d, 1H), 3.94-3.92 (d, 1H), 3.76-3.74 (d, 1H), 3.67 (s, 3H), 2.04 (s, 3H), 0.92-0.91 (d, 3H), 0.70 (s, 3H).
Synthesis of compound A8. To a solution of A7 (500 mg, 0.95 mmol) in EtOH (40 mL) was added Zn (620 mg, 9.5 mmol), the resulting solution was heated to reflux for 4 h. The reaction mixture was allowed to cool to room temperature, filtered over a plug of celite, washed with EtOAc and concentrated. The residue was purified by column chromatography (silica gel, EA/PE=5:1) to give A8 (310 mg, 72%) as a white solid and A8-a (80 mg, 17%) as a white solid. A8 .sup.1H NMR (400 MHz, CDCl.sub.3), 5.78 (t, 1H), 4.66 (m, 1H), 3.86-3.83 (d, 1H), 3.67 (s, 3H), 3.64-3.61 (d, 1H), 2.05 (s, 3H), 0.94-0.93 (d, 3H), 0.74 (s, 3H). A8-a .sup.1H NMR (400 MHz, CDCl3), 5.75 (m, 1H), 3.84-3.81 (d, 1H), 3.67 (s, 3H), 3.62-3.60 (d, 1H), 0.94-0.92 (d, 3H), 0.74 (s, 3H).
Synthesis of compound 1. To a solution of A8-a (70 mg, 0.17 mmol) in THE (5 mL) was added MeMgBr (2 mL, 1M in THF) dropwise. After stirring at room temperature overnight, the mixture was quenched with water (20 mL) and extracted with EtOAc (15 mL3). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography (silica gel, EA:PE=1:1) to give 1 (20 mg, 30%) as a white solid. .sup.1H NMR (400 MHz, CD.sub.3OD), 5.62-5.61 (d, 1H), 3.85-3.82 (d, 1H), 3.59-3.56 (d, 1H), 1.17 (s, 3H), 3.45 (m, 1H), 1.16 (s, 3H), 0.97-0.96 (d, 3H), 0.78 (s, 3H).

Example 2. Synthesis of Compound 2

[0154] ##STR00015##

Synthesis of compound B2. To a solution of B1 (140 mg, 0.334 mmol) in DCM (5 mL) was added silica gel (100 mg) and PCC (107 mg, 0.5 mmol). The mixture was stirred at 25 C. for 16 hours. TLC (PE:EA=3:1) showed the starting material was consumed completely. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by column chromatography on silica gel (PE:EA=10:1) to give B2 (120 mg, 86.3%) as white solid. LCMS Rt=1.157 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C27H45O3 [M+H].sup.+417, found 399 ([M+H18].sup.+).
Synthesis of compound 2. To a solution of B2 (140 mg, 0.336 mmol) in dry THE (5 mL) at 0 C. under N.sub.2 was added EtMgBr (3 M in diethyl ether, 0.56 mL, 1.67 mmol) dropwise. The mixture was warmed to 25 C. and stirred for 16 hours. LCMS showed the starting material was consumed completely. The reaction mixture was quenched with aqueous NH.sub.4Cl (10 mL), extracted with EtOAc (10 mL*3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by prep. HPLC to give 2 (3 mg, 2%) as white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) =5.57 (d, J=5.02 Hz, 1H), 3.59 (d, J=10.04 Hz, 1H), 3.26-3.32 (m, 4H), 2.47 (d, J=13.05 Hz, 1H), 1.61-2.08 (m, 10H), 1.24-1.49 (m, 9H), 1.05-1.17 (m, 10H), 0.74-1.03 (m, 11H), 0.71 (s, 3H). LCMS Rt=1.239 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI caled. for C.sub.29H.sub.51O.sub.3 [M+H].sup.+447, found 411 ([M+H36].sup.+).

Example 3. Synthesis of Compounds 3 and 4

[0155] ##STR00016##

Synthesis of compound 3. To a solution of C1 (500 mg, 1.15 mmol) and tetraisopropoxytitanium (326 mg, 1.15 mmol) in dry THE (20 mL) under N.sub.2 at 25 C. was added EtMgBr (3 M in diethyl ether, 1.33 mL, 4.02 mmol) dropwise. The mixture was stirred at 25 C. for 16 hours. LCMS showed the starting material was consumed. The reaction mixture was quenched with aqueous NH.sub.4Cl (30 mL), filtered through a pad of celite, and the filtrate was extracted with EtOAc (20 mL*3). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by prep. HPLC to give 3 (211 mg, 42.6%) as white solid and 4 (68 mg, 12.8%) as white solid. 1H NMR (3): (400 MHz, CDCl.sub.3) =5.57 (d, J=5.0 Hz, 1H), 3.59 (d, J=10.0 Hz, 1H), 3.32-3.27 (m, 4H), 2.47 (d, J=12.5 Hz, 1H), 2.10-1.94 (m, 4H), 1.91-1.59 (m, 8H), 1.51-0.83 (m, 20H), 0.78-0.66 (m, 5H), 0.47-0.38 (m, 2H). LCMS (3): Rt=1.142 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.28H.sub.47O.sub.3 [M+H].sup.+ 431, found 453 ([M+Na].sup.+). 1H NMR (4): (400 MHz, CDCl.sub.3) =5.56 (d, J=4.5 Hz, 1H), 5.05-4.93 (m, 1H), 3.59 (d, J=9.5 Hz, 1H), 3.33-3.25 (m, 4H), 2.47 (d, J=12.5 Hz, 1H), 2.36-2.26 (m, 1H), 2.23-2.13 (m, 1H), 2.08-1.73 (m, 8H), 1.70-1.60 (m, 2H), 1.54-1.19 (m, 16H), 1.15 (s, 3H), 1.13-1.00 (m, 4H), 0.96-0.82 (m, 6H), 0.70 (s, 3H). LCMS (4): Rt=1.317 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.30H.sub.53O.sub.3 [M+H].sup.+ 461, found 483 ([M+Na].sup.+).

Example 4. Synthesis of Compound 5

[0156] ##STR00017##

Synthesis of compound D2. To a solution of D1 (20 g, 63.2 mmol) in DME (200 mL) was added KOH (35.4 g, 0.632 mol). The mixture was stirred at 25 C. for 16 hours. TLC (PE:EA=2:1) showed the starting material was remained and the desired compound was observed. The reaction mixture was quenched with ice chips and aqueous citric acid (250 mL), extracted with EtOAc (200 mL*3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography on silica gel (PE:EA=5:1) to give D2 (3 g, 15.0%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) J=5.67-5.56 (m, 1H), 3.66 (d, J=10.0 Hz, 1H), 3.48 (d, J=10.0 Hz, 1H), 3.40-3.28 (m, 4H), 2.91 (dd, J=1.5, 16.6 Hz, 1H), 2.52-2.36 (m, 2H), 2.34-2.28 (m, 1H), 2.23-2.02 (m, 4H), 1.98-1.85 (m, 2H), 1.81-1.72 (m, 1H), 1.69-1.63 (m, 1H), 1.61-1.42 (m, 3H), 1.32-1.19 (m, 2H), 1.09-1.02 (m, 1H), 0.93 (s, 3H).
Synthesis of compound D3. To a stirred solution of D2 (24.8 g, 113 mmol) in toluene (100 mL) was added Me.sub.3Al (2 Min toluene, 28.3 mL, 56.6 mmol) at 0 C. under N.sub.2 dropwise. The resulting solution was stirred for 1 h at 25 C. It was cooled to 70 C. with dry-ice/acetone bath, and a slurry of (8R,9S,10S,13S,14S)-10-(methoxymethyl)-13-methyl-7,8,9,10,11,12,13, 14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-3,17(2H,4H)-dione (6 g, 18.9 mmol) in toluene (150 mL) was added and then stirred for 1 h at 50 to 60 C. MeMgBr in diethyl ether (3M, 18.8 mL, 56.6 mmol) was then added dropwise, while maintaining the temperature during the addition between 50 to 40 C. The reaction mixture was then stirred for 3 h at 50 to 60 C. The mixture was quenched with 10% aqueous citric acid (200 mL), extracted with EtOAc (200 mL*3). The combined organic layers were washed with brine (400 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography on silica gel (PE:EA=8:1) to give D3 (4.5 g, 71.6%) as white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) =5.61 (d, J=5.0 Hz, 1H), 3.65 (d, J=10.0 Hz, 1H), 3.33-3.26 (m, 4H), 2.54-2.39 (m, 2H), 2.17-2.02 (m, 4H), 1.98-1.81 (m, 3H), 1.72-1.61 (m, 3H), 1.57-1.47 (m, 3H), 1.29-1.17 (m, 2H), 1.16 (s, 3H), 1.12-1.04 (m, 1H), 0.99-0.88 (m, 4H). LCMS Rt=1.412 min in 7 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.21H.sub.33O.sub.3 [M+H].sup.+333, found 315 ([M+H18].sup.+).
Synthesis of compound D4. To a solution of bromo(ethyl)triphenylphosphorane (18.3 g, 49.5 mmol) in THE (100 mL) under N.sub.2 was added a solution of t-BuOK (5.55 g, 49.5 mmol) in THE (60 mL). The mixture was becoming orange and stirred for 1 hour. A solution of D3 (3.3 g, 9.92 mmol) in THE (40 mL) was added to this mixture, and the resultant mixture was stirred at 60 C. for additional 16 hours. The reaction mixture was quenched with aqueous NH.sub.4Cl (200 mL), extracted with EtOAC (100 mL*2). The combined organic layers were washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography on silica gel (PE:EA=10:1) to give D4 (2.5 g, 73.3%) as a white solid. 1H NMR (400 MHz, CDCl.sub.3) =5.62-5.54 (m, 1H), 5.16-5.10 (m, 1H), 3.61 (d, J=10.0 Hz, 1H), 3.36-3.26 (m, 4H), 2.53-2.27 (m, 3H), 2.23-1.94 (m, 4H), 1.90-1.81 (m, 1H), 1.69-1.64 (m, 3H), 1.63-1.45 (m, 8H), 1.28-1.19 (m, 1H), 1.16 (s, 3H), 1.11-1.01 (m, 2H), 0.97-0.83 (m, 4H). LCMS Rt=1.506 min in 2 min chromatography, 10-80AB, purity 100%, MS ESI calcd. for C.sub.23H.sub.37O.sub.2 [M+H].sup.+ 345, found 327 ([M+H18].sup.+).
Synthesis of compound D5. To a solution of D4 (1.2 g, 3.48 mmol) and methyl propiolate (874 mg, 10.4 mmol) in dichloromethane (15 mL) under N.sub.2 at 0 C. was added diethylaluminum chloride (0.9 M in toluene, 15.4 mL, 13.9 mmol) dropwise. The resultant mixture was stirred at 25 C. for 16 hours. TLC (PE:EA=3:1) showed the starting material was consumed. The reaction mixture was quenched with aqueous citric acid (100 mL) at 0 C. carefully. The mixture was extracted with dichloromethane (100 mL*3), and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified with the other batch (SAGE-LGY-041) together by column chromatography on silica gel (PE:EA=10:1) to give D5 (3.5 g, 76.4%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) =6.98-6.88 (m, 1H), 5.90-5.72 (m, 1H), 5.57 (d, J=4.0 Hz, 1H), 5.45-5.32 (m, 1H), 3.77-3.69 (m, 3H), 3.61 (d, J=10.0 Hz, 1H), 3.36-3.25 (m, 4H), 3.02 (t, J=6.4 Hz, 1H), 2.47 (d, J=12.4 Hz, 1H), 2.10-1.92 (m, 5H), 1.90-1.59 (m, 2H), 1.23-1.14 (m, 7H), 1.10-0.92 (m, 3H), 0.90-0.81 (m, 5H). LCMS Rt=1.176 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.27H.sub.41O.sub.4 [M+H].sup.+429, found 451 ([M+Na].sup.+).
Synthesis of compound D6. To a solution of D5 (2 g, 4.66 mmol) in EtOAc (50 mL) was added Pd/C (5% on carbon, 0.5 g). The mixture was degassed and purged with H.sub.2 three times, and stirred at 25 C. under H.sub.2 balloon for 2 hours. LCMS showed the starting material was consumed completely. The mixture was filtered through a pad of celite, and the filtrate was concentrated to give D6 (2 g, 99.5%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) =5.56 (d, J=5.0 Hz, 1H), 3.66 (s, 3H), 3.59 (d, J=10.0 Hz, 1H), 3.32-3.26 (m, 4H), 2.46 (d, J=12.5 Hz, 1H), 2.40-2.30 (m, 1H), 2.26-2.17 (m, 1H), 2.08-1.92 (m, 4H), 1.89-1.73 (m, 3H), 1.68-1.59 (m, 2H), 1.54-1.23 (m, 7H), 1.15 (s, 3H), 1.13-0.99 (m, 4H), 0.95-0.83 (m, 5H), 0.70 (s, 3H). LCMS Rt=1.210 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.27H.sub.45O.sub.4 [M+H].sup.+433, found 415 ([M+H18].sup.+).
Synthesis of compound 5. To a solution of D6 (100 mg, 0.231 mmol) in dry THE (10 mL) at 0 C. was added LiAlH.sub.4 (87.2 mg, 2.30 mmol) in portions carefully. The resultant slurry was stirred at 0 C. for 2 hours. TLC (PE:EA=3:1) showed the starting material was consumed. The reaction mixture was quenched with aqueous NH.sub.4Cl (20 mL) at 0 C. dropwise carefully, filtered through a pad of celite, and the filtrate was extracted with EtOAc (10 mL*3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by prep. HPLC to give 5 (32 mg, 34.2%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) J=5.56 (d, J=4.4 Hz, 1H), 3.65-3.56 (m, 3H), 3.34-3.25 (m, 4H), 2.47 (d, J=12.4 Hz, 1H), 2.08-1.94 (m, 4H), 1.87-1.73 (m, 2H), 1.68-1.56 (m, 4H), 1.50-1.21 (m, 9H), 1.17-1.00 (m, 8H), 0.97-0.84 (m, 5H), 0.71 (s, 3H). LCMS Rt=1.074 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI caled. for C.sub.26H.sub.45O.sub.3 [M+H].sup.+405, found 427 ([M+Na]*).

Example 5. Synthesis of Compound 6

[0157] ##STR00018##

Synthesis of compound 6. To a solution of C.sub.1 (100 mg, 0.231 mmol) in dry THE (10 mL) at 0 C. was added MeLi (1.6 M in diethyl ether, 0.72 mL, 25.2 1.15 mmol) dropwise. The mixture was stirred at 0 C. for 2 hours. TLC (PE:EA=3:1) showed the starting material was consumed. The reaction mixture was quenched with aqueous NH.sub.4Cl (20 mL) at 0 C., extracted with EtOAc (10 mL*3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by prep. HPLC to give 6 (47 mg, 47.0%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3)=5.56 (d, J=5.2 Hz, 1H), 3.59 (d, J=9.6 Hz, 1H), 3.34-3.26 (m, 4H), 2.47 (d, J=12.4 Hz, 1H), 2.08-1.94 (m, 4H), 1.87-1.74 (m, 2H), 1.68-1.55 (m, 4H), 1.51-1.25 (m, 9H), 1.19 (s, 6H), 1.17-1.00 (m, 8H), 0.96-0.84 (m, 5H), 0.70 (s, 3H). LCMS t.sub.R=1.177 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.28H.sub.49O.sub.3 [M+H].sup.+433, found 455 ([M+Na].sup.+).

Example 6. Synthesis of Compound 7

[0158] ##STR00019##

Synthesis of compound E2. To a solution of E1 (200 mg, 0.491 mmol) in DCM (10 mL) was added silica gel (200 mg) and PCC (212 mg, 0.982 mmol). The mixture was stirred at 25 C. for 16 hours. TLC (PE:EA=3:1) showed the starting material was consumed. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by silica gel (PE:EA=10:1) to give E2 (100 mg, 50.5%) as colorless oil. LCMS Rt=1.201 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.26H.sub.45O.sub.3 [M+H].sup.+405, found 387 ([M+H18].sup.+).
Synthesis of compound 7. To a solution of E2 (100 mg, 0.247 mmol) and trimethyl(trifluoromethyl)silane (174 mg, 1.23 mmol) in THE (5 mL) was added CsF (3.75 mg, 24.7 mol). The mixture was stirred at 25 C. for 1 hour. TLC (PE:EA=3:1) showed the starting material was consumed. A solution of TBAF (1 M in THF, 1.23 mL, 1.23 mmol) was added to the mixture, and the resulting mixture was stirred at 25 C. for 16 hours. The reaction mixture was concentrated, and the residue was purified by column chromatography on silica gel (PE:EA=10:1) to give 7 (8 mg, 6.83%) as off white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) (=3.92-3.79 (m, 1H), 3.52-3.43 (m, 2H), 3.30 (s, 3H), 2.21-1.91 (m, 4H), 1.89-1.60 (m, 7H), 1.52-1.40 (m, 3H), 1.39-1.18 (m, 9H), 1.16-0.97 (m, 5H), 0.97-0.75 (m, 5H), 0.73-0.62 (m, 4H). LCMS Rt=1.204 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.27H.sub.46F.sub.3O.sub.3 [M+H].sup.+ 475, found 457 ([M+H18].sup.+).

Example 7. Synthesis of Compound 8

[0159] ##STR00020##

Synthesis of compound 8. To a solution of D7 (50 mg, 0.124 mmol) and trimethyl(trifluoromethyl)silane (88.1 mg, 0.62 mmol) in THE (2 mL) was added CsF (1.88 mg, 0.0124 mmol). The mixture was stirred at 25 C. for 1 hour. TLC (PE:EA=3:1) showed the starting material was consumed completely, and HCl (1 M in water, 1.24 mL, 1.24 mmol) was added to the reaction mixture. The resultant mixture was stirred at 25 C. for 16 hours. The desired compound was detected by TLC (PE:EA=3:1). The reaction mixture was neutralized with aqueous sodium bicarbonate (5 mL), extracted with EtOAc (5 mL*3), dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by prep. HPLC to give 8 (5.5 mg, 9.38%) as white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) =5.57 (d, J=4.0 Hz, 1H), 3.94-3.78 (m, 1H), 3.60 (d, J=10.0 Hz, 1H), 3.39-3.23 (m, 4H), 2.47 (d, J=14.1 Hz, 1H), 2.10-1.92 (m, 5H), 1.89-1.69 (m, 4H), 1.46 (br. s., 4H), 1.38-1.04 (m, 12H), 1.03-0.80 (m, 6H), 0.71 (s, 3H). LCMS Rt=1.168 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI caled. for C.sub.27H.sub.44F.sub.3O.sub.3 [M+H].sup.+473, found 455 ([M+H18].sup.+).

Example 8. Synthesis of Compound 9

[0160] ##STR00021##

Synthesis of compound D7. To a solution of 5 (850 mg, 2.10 mmol) in DCM (15 mL) was added PCC (678 mg, 3.15 mmol) and silica gel (1 g). The mixture was stirred at 25 C. for 16 hours. TLC (PE:EA=3:1) showed the starting material was consumed. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by column chromatography on silica gel (PE:EA=8:1) to give D7 (250 mg, 29.5%) as white solid. LCMS Rt=1.089 min in 2 min chromatography, 30-90AB, purity 45.2%, MS ESI calcd. for C.sub.26H.sub.43O.sub.3 [M+H].sup.+403, found 385 ([M+H18].sup.+).
Synthesis of compound 9. To a solution of D7 (200 mg, 0.496 mmol) in dry THE (5 mL) at 0 C. was added MeMgBr (3 M in dimethyl ether, 0.83 mL, 2.48 mmol). The mixture was stirred at 25 C. for 2 hours. TLC (PE:EA=3:1) showed the starting material was consumed. The reaction mixture was quenched with saturated aqueous ammonium chloride (5 mL), extracted with EtOAc (5 mL*3), dried over sodium sulfate, filtered and concentrated to give 9 (190 mg, 91.7%) as white solid. One batch (140 mg) was used directly in the next step, and the other batch (50 mg) was purified by prep. HPLC to give desired compound (3 mg). .sup.1H NMR (400 MHz, CDCl.sub.3) =5.56 (d, J=5.0 Hz, 1H), 3.77-3.71 (m, 1H), 3.59 (d, J=10.0 Hz, 1H), 3.33-3.25 (m, 4H), 2.47 (d, J=13.1 Hz, 1H), 2.10-1.92 (m, 5H), 1.87-1.73 (m, 3H), 1.69-1.58 (m, 3H), 1.49-1.23 (m, 10H), 1.20-1.00 (m, 11H), 0.96-0.81 (m, 6H), 0.71 (s, 3H). LCMS R=1.126 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C %14703 [M+H].sup.+419, found 401 ([M+H18]).

Materials and Methods

[0161] The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization.

[0162] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

[0163] The compounds provided herein may be isolated and purified by known standard procedures. Such procedures include (but are not limited to) recrystallization, column chromatography, HPLC, or supercritical fluid chromatography (SFC). The following schemes are presented with details as to the preparation of representative pyrazoles that have been listed herein. The compounds provided herein may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis. Exemplary chiral columns available for use in the separation/purification of the enantiomers/diastereomers provided herein include, but are not limited to, CHIRALPAK AD-10, CHIRALCEL OB, CHIRALCEL OB-H, CHIRALCEL OD, CHIRALCEL OD-H, CHIRALCEL OF, CHIRALCEL OG, CHIRALCEL OJ and CHIRALCEL OK.

[0164] .sup.1H-NMR reported herein (e.g., for intermediates) may be a partial representation of the full NMR spectrum of a compound, e.g., a compound described herein. For example, the reported .sup.1H NMR may exclude the region between (ppm) of about 1 to about 2.5 ppm.

[0165] Exemplary general method for preparative HPLC: Column: Waters RBridge prep 10 m C18, 19*250 mm. Mobile phase: acetonitrile, water (NH.sub.4HCO.sub.3) (30 L water, 24 g NH.sub.4HCO.sub.3, 30 mL NH.sub.3.H.sub.2O). Flow rate: 25 mL/min

[0166] Exemplary general method for analytical HPLC: Mobile phase: A: water (10 mM NH.sub.4HCO.sub.3), B: acetonitrile Gradient: 5%-95% B in 1.6 or 2 min Flow rate: 1.8 or 2 mL/min; Column: XBridge C18, 4.6*50 mm, 3.5 m at 45 C.

Assay Methods

[0167] Compounds of the present invention can be evaluated using various in vitro and in vivo assays described in the literature; examples of which are described below.

[0168] The following examples are offered to illustrate the biological activity of the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting the scope thereof.

NMDA Potentiation

[0169] NMDA potentiation was assessed using either whole cell patch clamp of mammalian cells which expressed NMDA receptors, or using two-electrode voltage clamp (TEVC) of Xenopus Laevis oocytes expressing NMDA receptors.

Whole-Cell Patch Clamp of Mammalian Cells

[0170] The whole-cell patch-clamp technique was used to investigate the effects of compounds on the NMDA receptor (GRIN1/GRIN2A subunits) expressed in HEK cells. NMDA/Glycine peak and steady-state currents were recorded from stably transfected cells expressing the NMDA receptor and the modulatory effects of the test items on these currents were investigated. Results are shown on Table 1.

[0171] Cells were stably transfected with human GRIN1 (variant NR1-3). These cells were transiently transfected (Lipofectamine) with GRIN2A cDNA and CD8 (pLeu) antigene cDNA. About 24-72 hours following transfection 1 l Dynabeads M-45 CD8 was added to identify successfully transfected cells (Jurman et al., Biotechniques (1994) 17:876-881). Cells were passaged to a confluence of 50-80%. Cells were seeded onto Poly-L-Lysine coated cover slips covered with culture complete medium in a 35 mm culture dish. Confluent clusters of cells are electrically coupled (Pritchett et al., Science (1988), 242:1306-8). Because responses in distant cells are not adequately voltage clamped and because of uncertainties about the extent of coupling (Verdoorn et al., Neuron (1990), 4:919-28), cells were cultivated at a density that enables single cells (without visible connections to neighboring cells) to be measured. Cells were incubated at 37 C. in a humidified atmosphere with 5% CO.sub.2 (rel. humidity about 95%). The cells were continuously maintained in and passaged in sterile culture flasks containing a 1:1 mixture of Dulbecco's modified eagle medium and nutrient mixture F-12 (D-MEM/F-12 1, liquid, with L-Glutamine) supplemented with 9% fetal bovine serum and 0.9% Penicillin/Streptomycin solution. The complete medium was supplemented with 3.0 g/ml Puromycin.

[0172] Whole cell currents were measured with HEKA EPC-10 amplifiers using PatchMaster software. Cell culture dishes for recordings were placed on the dish holder of the microscope and continuously perfused (1 ml/min) with bath solution (NaCl 137 mM, KCl 4 mM, CaCl.sub.2) 1.8 mM, MgCl.sub.2 1 mM, HEPES 10 mM, D-Glucose 10 mM, pH (NaOH) 7.4). All solutions applied to cells including the pipette solution were maintained at room temperature (19 C.-30 C.). After formation of a Gigaohm seal between the patch electrodes and transfected individual HEK 293 cells (pipette resistance range: 2.5 M-6.0 M; seal resistance range:>1 G) the cell membrane across the pipette tip was ruptured to assure electrical access to the cell interior (whole-cell patch-configuration). At this point the bath solution is switched to NAMDA bath solution (NaCl 137 mM, KCl 4 mM, CaCl.sub.2) 2.8 mM, HEPES 10 mM, D-Glucose 10 mM, Cremophore 0.02%, pH (NaOH) 7.4). NMDA inward currents were measured upon application of 30 M NMDA (and 5.0 M Glycine) to patch-clamped cells (2 applications) for 5 s. The cells were voltage clamped at a holding potential of 80 mV. For the analysis of test articles, NMDA receptors were stimulated by 30 M NMDA and 5.0 M Glycine after sequential pre-incubation of increasing concentrations of the test article. Pre-incubation duration was 30 s. Stimulation duration was 5 s Test articles were dissolved in DMSO to form stock solutions of 0.1 mM and 1 mM. Test articles were diluted to 0.1 M and 1 M in NMDA bath solution. Both concentrations of test articles were tested on each cell. The same concentration was applied at least three times or until the steady state current amplitude was reached. Every day one cell was tested with 50 M PREGS (positive control) using the same application protocol to test whether cells were successfully transfected with NMDA receptors.

TABLE-US-00001 TABLE 1 NMDA 1a2A (%) Potentiation Structure 1 M [00022] A [00023] A [00024] A [00025] B [00026] C For Table 1, A indicates >5 to 50%; B: >50%; C indicates not active in the assay.

Whole-Cell Patch Clamp of Mammalian Cells (IWB)

[0173] The whole-cell patch-clamp technique was used to investigate the effects of compounds on NR1/NR2A glutamate receptors expressed in mammalian cells. The results are shown on Table 2.
Test article effects were evaluated in 8-point concentration-response format (4 replicate wells/concentration). All test and control solutions contained 0.3% DMSO and 0.01% Kolliphor EL (C5135, Sigma). The test article formulations were loaded in a 384-well compound plate using an automated liquid handling system (SciClone ALH3000, Caliper LifeScienses). The measurements were perfomed using Ion Works Barracuda platform following this procedure:

Electrophysiological Procedures:

[0174] a) Intracellular solution (mM): 50 mM CsCl, 90 mM CsF, 2 mM MgC.sub.2, 5 mM EGTA, 10 mM HEPES. Adjust to pH 7.2 with CsOH. [0175] b) Extracellular solution, HB-PS (composition in mM): NaCl, 137; KCl, 1.0; CaC.sub.2, 5; HEPES, 10; Glucose, 10; pH adjusted to 7.4 with NaOH (refrigerated until use). [0176] c) Holding potential: 70 mV, potential during agonist/PAM application: 40 mV.
Recording procedure: [0177] a) Extracellular buffer will be loaded into the PPC plate wells (11 L per well). Cell suspension will be pipetted into the wells (9 L per well) of the PPC planar electrode. [0178] b) Whole-cell recording configuration will be established via patch perforation with membrane currents recorded by on-board patch clamp amplifiers. [0179] c) Two recordings (scans) will be performed. First, during pre-application of PAM alone (duration of pre-application5 min) and second, during test articles and agonist (EC.sub.20 L-glutamate and 30 M glycine) co-application to detect positive modulatory effects of the test article.
Test Article Administration: The first pre-application will consist of the addition of 20 L of 2 concentrated test article solution and, second, of 20 L of 1 concentrated test article and agonist at 10 L/s (2 second total application time).

TABLE-US-00002 TABLE 2 GluN2A PCA IWB Ephys % poten- tiation Structure at 3 M [00027] A [00028] B [00029] A [00030] A [00031] A For Table 2, A indicates 10 to 150%, and B indicates potentiation of >150%.

OTHER EMBODIMENTS

[0180] In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0181] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms comprising and containing are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0182] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0183] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.