OXYSTEROLS AND METHODS OF USE THEREOF

Abstract

Compounds are provided according to Formula (I) 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.

##STR00001##

Claims

1. A compound of Formula (I): ##STR00034## 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.2a 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; 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.sup.Y 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.5 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; or R.sup.5a and R.sup.6a, together with the carbon atoms to which they are attached, form a ring; 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; and R.sup.D is hydrogen, C.sub.1-C.sub.6 alkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

2. The compound of claim 1, wherein R.sup.1 is unsubstituted C.sub.1-3 alkyl.

3. The compound of claim 2, wherein R.sup.1 is —CH.sub.3, —CH.sub.2CH.sub.3, or —CH.sub.2CH.sub.2CH.sub.3.

4. The compound of claim 1, wherein R.sup.1 is substituted C.sub.1-3 alkyl.

5. The compound of claim 1, wherein R.sup.1 is haloalkyl or —CH.sub.2OCH.sub.3.

6. The compound of claim 1, wherein R.sup.2a or R.sup.2a is hydrogen.

7. The compound of claim 1, wherein R.sup.2a and R.sup.2a is hydrogen.

8. The compound of claim 1, wherein R.sup.4a is hydrogen.

9. The compound of claim 1, wherein X is —CH.sub.2—.

10. The compound of claim 1, wherein R.sup.8 is substituted or unsubstituted C.sub.1-3 alkyl.

11. The compound of claim 10, wherein R.sup.8 is —CH.sub.3.

12. The compound of claim 1, wherein the compound of Formula (I) is a compound of Formula (II): ##STR00035## or a pharmaceutically acceptable salt thereof.

13. The compound of claim 12, wherein the compound of Formula (II) is a compound of Formula (II-A): ##STR00036## or a pharmaceutically acceptable salt thereof.

14. The compound of claim 12, wherein the compound of Formula (II) is a compound of Formula (II-B): ##STR00037## or a pharmaceutically acceptable salt thereof.

15. The compound of claim 1, wherein R.sup.5a or R.sup.5b is hydrogen.

16. The compound of claim 1, wherein R.sup.5a and R.sup.5b are both hydrogen.

17. The compound of claim 1, wherein R.sup.6a is a substituted or unsubstituted C.sub.1-3 alkyl.

18. The compound of claim 17, wherein R.sup.6a is —CH.sub.3 or —CH.sub.2CH.sub.3.

19. The compound of claim 1, wherein 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.

20. The compound of claim 1, wherein R.sup.6b is substituted or unsubstituted C.sub.1-3 alkyl.

21. The compound of claim 20, wherein R.sup.6b is —CH.sub.3 or —CH.sub.2CH.sub.3.

22. The compound of claim 1 wherein R.sup.6b is hydrogen.

23. The compound of claim 20, wherein R.sup.6b is —CH.sub.3 or —CF.sub.3.

24. The compound of claim 1, wherein R.sup.6a and R.sup.6b are both —CH.sub.3.

25. The compound of claim 1, wherein R.sup.6a and R.sup.6b, taken together with the atom to which they are attached, form a ring.

26. The compound of claim 25, wherein the ring is a 3-membered ring.

27. The compound of claim 1, 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, wherein 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.

29. The compound of claim 27, wherein R.sup.6a is selected from a substituted or unsubstituted C.sub.1-3 alkyl.

30. The compound of claim 1, 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, wherein R.sup.Y is substituted or unsubstituted C.sub.1-3 alkyl.

32. The compound of claim 1, wherein R.sup.Y is substituted or unsubstituted heterocyclyl.

33. The compound of claim 31, wherein R.sup.Y is —CH.sub.3.

34. The compound of claim 31, wherein R.sup.Y is —CF.sub.3.

35. The compound of claim 1, wherein the compound is: ##STR00038## ##STR00039## or a pharmaceutically acceptable salt thereof.

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

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

38. A method for treating or preventing a disorder described herein, comprising administering to a subject in need thereof an effective amount of a compound of claim 1, or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof.

39. The method according to claim 38, wherein the disorder is a gastrointestinal (GI) disorder irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), structural disorders affecting the GI, anal disorders, colon polyps, cancer, colitis.

40. The method according to claim 38, wherein the disorder is inflammatory bowel disease.

41. The method according to claim 38, wherein the disorder is cancer, diabetes, or a sterol synthesis disorder.

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 of claim 1, or pharmaceutically acceptable salt thereof, 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, sleep 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. The method according to claim 42, wherein the disorder is sterol synthesis disorder.

Description

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0087] As generally described herein, the present invention provides 19-substituted oxysterols useful for preventing and/or treating a broad range of disorders, including, but not limited to, NMDA-mediated disorders. These compounds are expected to show improved in vivo potency, pharmacokinetic (PK) properties, oral bioavailability, formulatability, stability, and/or safety as compared to other oxysterols.

Compounds

[0088] In one aspect, provided herein are compounds according to Formula (I):

##STR00005##

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.2a 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, halogen, or one R.sup.X group and R.sup.5b are joined to form a double bond; Y is —OR.sup.Y, wherein R.sup.Y 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; and R.sup.D is hydrogen, C.sub.1-C.sub.6 alkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

[0089] In certain embodiments, R.sup.1 is hydrogen. In certain embodiments, R.sup.1 is unsubstituted C.sub.1-3 alkyl. In certain embodiments, R.sup.1 is —CH.sub.3, —CH.sub.2CH.sub.3, or —CH.sub.2CH.sub.2CH.sub.3. In certain embodiments, R.sup.1 is substituted C.sub.1-3 alkyl. In certain embodiments, R.sup.1 is —CF.sub.3 or —CH.sub.2OCH.sub.3.

[0090] In certain embodiments, R.sup.2a is hydrogen. In certain embodiments, R.sup.2b is hydrogen. In certain embodiments, R.sup.2a or R.sup.2b is hydrogen. In certain embodiments, R.sup.2a and R.sup.2b is hydrogen.

[0091] In certain embodiments, R.sup.4a is hydrogen. In certain embodiments, R.sup.4b is hydrogen. In certain embodiments, R.sup.4a or R.sup.4b is hydrogen. In certain embodiments, R.sup.4a and R.sup.4b is hydrogen.

[0092] In certain embodiments, X is —CH.sub.2—.

[0093] In certain embodiments, R.sup.8 is substituted or unsubstituted C.sub.1-3 alkyl. In certain embodiments, R.sup.8 is —CH.sub.3.

[0094] In certain embodiments, the between —CR.sup.7 and —CR.sup.4aR.sup.4b is a double bond, and one of R.sup.4a or R.sup.4b is absent.

[0095] In certain embodiments, is a single bond, and R.sup.7 is hydrogen in the alpha configuration.

[0096] In certain embodiments, the compound of Formula (I) is a compound of Formula (I-A) or (I-B):

##STR00006##

or a pharmaceutically acceptable salts thereof.

[0097] In certain embodiments, the compound of Formula (I) is a compound of Formula (II):

##STR00007##

or a pharmaceutically acceptable salt thereof.

[0098] In certain embodiments, the compound of Formula (II) is a compound of Formula (II-A):

##STR00008##

or a pharmaceutically acceptable salt thereof.

[0099] In certain embodiments, the compound of Formula (II-A) is a compound of Formula (III):

##STR00009##

or a pharmaceutically acceptable salt thereof.

[0100] In certain embodiments, the compound of Formula (II) is a compound of Formula (II-B):

##STR00010##

or a pharmaceutically acceptable salt thereof.

[0101] In certain embodiments, R.sup.5a or R.sup.5b is hydrogen. In certain embodiments, R.sup.5a and R.sup.5b are both hydrogen.

[0102] In certain embodiments, R.sup.6a is a substituted or unsubstituted C.sub.1-3 alkyl (e.g., C.sub.1-3 haloalkyl). In certain embodiments, R.sup.6a is —CH.sub.3 or —CH.sub.2CH.sub.3. In certain embodiments, 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.

[0103] In certain embodiments, R.sup.6b is substituted or unsubstituted C.sub.1-3 alkyl (e.g., C.sub.1-3 haloalkyl). In certain embodiments, R.sup.6b is —CH.sub.3 or —CH.sub.2CH.sub.3. In certain embodiments, R.sup.6b is hydrogen. In certain embodiments, R.sup.6b is —CH.sub.3 or —CF.sub.3.

[0104] In certain embodiments, R.sup.6a or R.sup.6b is hydrogen. In certain embodiments, R.sup.6a and R.sup.6b are both hydrogen.

[0105] In certain embodiments, R.sup.6a is hydrogen and R.sup.6b is substituted or unsubstituted C.sub.1-3 alkyl (e.g., —CH.sub.3, —CH.sub.2CH.sub.3; C.sub.1-3 haloalkyl (e.g., —CF.sub.3)).

[0106] In certain embodiments, R.sup.6a and R.sup.6b are both —CH.sub.3. In certain embodiments, R.sup.6a is —CH.sub.3 and R.sup.6b is —CH.sub.2CH.sub.3. In certain embodiments, R.sup.6a and R.sup.6b, taken together with the atom to which they are attached, form a ring. In certain embodiments, the ring is a 3-membered ring.

[0107] In certain embodiments, R.sup.1 is hydrogen or C.sub.1-3 alkyl, R.sup.6a is substituted or unsubstituted C.sub.1-3 alkyl (e.g., C.sub.1-3 haloalkyl), 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.

[0108] In certain embodiments, R.sup.6a is selected from the group consisting of substituted or unsubstituted C.sub.1-3 alkyl (e.g., C.sub.1-3 haloalkyl), unsubstituted C.sub.2-3 alkenyl, unsubstituted C.sub.2-3 alkynyl, or unsubstituted carbocyclyl. In certain embodiments, R.sup.6a is selected from a substituted or unsubstituted C.sub.1-3 alkyl (e.g., C.sub.1-3 haloalkyl).

[0109] In certain embodiments, 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.

[0110] In certain embodiments, R.sup.Y is substituted or unsubstituted C.sub.1-3 alkyl (e.g., C.sub.1-3 haloalkyl). In certain embodiments, R.sup.Y is substituted or unsubstituted heterocyclyl. In certain embodiments, R.sup.Y is —CH.sub.3 or —CH.sub.2CH.sub.3. In certain embodiments, R.sup.Y is —CF.sub.3.

[0111] In certain embodiments, the compound of Formula (I) is:

##STR00011## ##STR00012##

or a pharmaceutically acceptable salt thereof.

[0112] In one aspect, the present invention provides pharmaceutical compositions comprising a compound as described herein, e.g., a compound of Formula (I), or a pharmaceutically acceptable salt or pharmaceutically acceptable carrier thereof.

[0113] In one aspect, the present invention provides a method of inducing sedation or anesthesia comprising administering to a subject an effective amount of a compound as described herein, e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable carrier thereof.

[0114] In one aspect, provided herein is a method for treating or preventing a disorder described herein, comprising administering to a subject in need thereof an effective amount of a compound as described herein, e.g., a compound of Formula (I), or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof. In some embodiments, the disorder is a NMDA-mediated disorder. In some embodiments, the disorder is a disorder mediated by NMDA, e.g., a disorder which benefits from treatment with a NMDA modulator. In some embodiments, the disorder is cancer. In some embodiments, the disorder is diabetes. In some embodiments, the disorder is a sterol synthesis disorder. In some embodiments, the disorder is a gastrointestinal (GI) disorder e.g., constipation, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn's disease), structural disorders affecting the GI, anal disorders (e.g., hemorrhoids, internal hemorrhoids, external hemorrhoids, anal fissures, perianal abscesses, anal fistula), colon polyps, cancer, colitis. In some embodiments, the disorder is inflammatory bowel disease.

[0115] In one aspect, provided herein is a method for treating or preventing a CNS-related condition comprising administering to a subject in need thereof an effective amount of a compound as described herein, e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable carrier thereof. In some embodiments, the CNS-related condition is an adjustment disorder, anxiety disorder (including obsessive-compulsive disorder, posttraumatic stress disorder, and social phobia), cognitive disorder (including Alzheimer's disease and other forms of dementia), dissociative disorder, eating disorder, mood disorder (including depression (e.g., postpartum depression), bipolar disorder, dysthymic disorder, suicidality), schizophrenia or other psychotic disorder (including schizoaffective disorder), sleep disorder (including insomnia), substance-related disorder, personality disorder (including obsessive-compulsive personality disorder), autism spectrum disorders (including those involving mutations to the Shank group of proteins (e.g., Shank3)), neurodevelopmental disorder (including Rett syndrome, Tuberous Sclerosis complex), multiple sclerosis, sterol synthesis disorders, pain (including acute and chronic pain), encephalopathy secondary to a medical condition (including hepatic encephalopathy and anti-NMDA receptor encephalitis), seizure disorder (including status epilepticus and monogenic forms of epilepsy such as Dravet's disease), stroke, traumatic brain injury, movement disorder (including Huntington's disease and Parkinson's disease), vision impairment, hearing loss, and tinnitus.

Pharmaceutical Compositions

[0116] In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a effective amount of a compound of Formula (I).

[0117] When employed as pharmaceuticals, the compounds provided herein are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.

[0118] In one embodiment, with respect to the pharmaceutical composition, the carrier is a parenteral carrier, oral or topical carrier.

[0119] The present invention also relates to a compound of Formula (I) or pharmaceutical composition thereof for use as a pharmaceutical or a medicament.

[0120] Generally, the compounds provided herein are administered in a therapeutically effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0121] The pharmaceutical compositions provided herein can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. Depending on the intended route of delivery, the compounds provided herein are preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration.

[0122] The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, the compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.

[0123] Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0124] Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. As before, the active compound in such compositions is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.

[0125] Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight. When formulated as a ointment, the active ingredients will typically be combined with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with, for example an oil-in-water cream base. Such transdermal formulations are well-known in the art and generally include additional ingredients to enhance the dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope provided herein.

[0126] The compounds provided herein can also be administered by a transdermal device. Accordingly, transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.

[0127] The above-described components for orally administrable, injectable or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.

[0128] The above-described components for orally administrable, injectable, or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's The Science and Practice of Pharmacy, 21st edition, 2005, Publisher: Lippincott Williams & Wilkins, which is incorporated herein by reference.

[0129] The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

[0130] The present invention also relates to the pharmaceutically acceptable formulations of a compound of Formula (I). In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises a cyclodextrin derivative. The most common cyclodextrins are α-, β- and γ-cyclodextrins consisting of 6, 7 and 8 α-1,4-linked glucose units, respectively, optionally comprising one or more substituents on the linked sugar moieties, which include, but are not limited to, methylated, hydroxyalkylated, acylated, and sulfoalkylether substitution. In certain embodiments, the cyclodextrin is a sulfoalkyl ether β-cyclodextrin, e.g., for example, sulfobutyl ether β-cyclodextrin, also known as Captisol®. See, e.g., U.S. Pat. No. 5,376,645. In certain embodiments, the formulation comprises hexapropyl-β-cyclodextrin. In a more particular embodiment, the formulation comprises hexapropyl-β-cyclodextrin (10-50% in water).

[0131] The present invention also relates to the pharmaceutically acceptable acid addition salt of a compound of Formula (I). The acid which may be used to prepare the pharmaceutically acceptable salt is that which forms a non-toxic acid addition salt, i.e., a salt containing pharmacologically acceptable anions such as the hydrochloride, hydroiodide, hydrobromide, nitrate, sulfate, bisulfate, phosphate, acetate, lactate, citrate, tartrate, succinate, maleate, fumarate, benzoate, para-toluenesulfonate, and the like.

[0132] The following formulation examples illustrate representative pharmaceutical compositions that may be prepared in accordance with this invention. The present invention, however, is not limited to the following pharmaceutical compositions.

Exemplary Formulation 1—Tablets

[0133] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 240-270 mg tablets (80-90 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 2—Capsules

[0134] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a starch diluent in an approximate 1:1 weight ratio. The mixture is filled into 250 mg capsules (125 mg of active compound per capsule).

Exemplary Formulation 3—Liquid

[0135] A compound of Formula (I), or pharmaceutically acceptable salt thereof, (125 mg) may be admixed with sucrose (1.75 g) and xanthan gum (4 mg) and the resultant mixture may be blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water may then be added to produce a total volume of 5 mL.

Exemplary Formulation 4—Tablets

[0136] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active compound) in a tablet press.

Exemplary Formulation 5—Injection

[0137] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/mL.

Exemplary Formulation 6—Tablets

[0138] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 90-150 mg tablets (30-50 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 7-Tablets

[0139] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 30-90 mg tablets (10-30 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 8—Tablets

[0140] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 0.3-30 mg tablets (0.1-10 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 9—Tablets

[0141] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 150-240 mg tablets (50-80 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 10—Tablets

[0142] A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 270-450 mg tablets (90-150 mg of active compound per tablet) in a tablet press.

[0143] Injection dose levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour, all for from about 1 to about 120 hours and especially 24 to 96 hours. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve adequate steady state levels. The maximum total dose is not expected to exceed about 2 g/day for a 40 to 80 kg human patient.

[0144] For the prevention and/or treatment of long-term conditions the regimen for treatment usually stretches over many months or years so oral dosing is preferred for patient convenience and tolerance. With oral dosing, one to five and especially two to four and typically three oral doses per day are representative regimens. Using these dosing patterns, each dose provides from about 0.01 to about 20 mg/kg of the compound provided herein, with preferred doses each providing from about 0.1 to about 10 mg/kg, and especially about 1 to about 5 mg/kg.

[0145] Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses.

[0146] When used to prevent the onset of a CNS-disorder, the compounds provided herein will be administered to a subject at risk for developing the condition, typically on the advice and under the supervision of a physician, at the dosage levels described above. Subjects at risk for developing a particular condition generally include those that have a family history of the condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition.

Methods of Treatment and Use

[0147] Compounds of the present invention, e.g., a compound of Formula (I), and pharmaceutically acceptable salts thereof, as described herein, are generally designed to modulate NMDA function, and therefore to act as oxysterols for the treatment and prevention of, e.g., CNS-related conditions in a subject. In some embodiments, the compounds described herein, e.g., a compound of Formula (I), and pharmaceutically acceptable salts thereof, as described herein, are generally designed to penetrate the blood brain barrier (e.g., designed to be transported across the blood brain barrier). Modulation, as used herein, refers to, for example, the inhibition or potentiation of NMDA receptor function. In certain embodiments, the compound of Formula (I), or pharmaceutically acceptable salt thereof, may act as a negative allosteric modulator (NAM) of NMDA, and inhibit NMDA receptor function. In certain embodiments, the present invention, e.g., a compound of Formula (I), or pharmaceutically acceptable salt thereof, may act as positive allosteric modulators (PAM) of NMDA, and potentiate NMDA receptor function. In certain embodiments, the compound of Formula (I), or pharmaceutically acceptable salt thereof, modulates NMDA function, but does not act as a negative allosteric modulator (NAM) or positive allosteric modulator (PAM) of NMDA.

[0148] In some embodiments, the disorder is cancer. In some embodiments, the disorder is diabetes. In some embodiments, the disorder is a sterol synthesis disorder. In some embodiments, the disorder is a gastrointestinal (GI) disorder, e.g., constipation, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn's disease), structural disorders affecting the GI, anal disorders (e.g., hemorrhoids, internal hemorrhoids, external hemorrhoids, anal fissures, perianal abscesses, anal fistula), colon polyps, cancer, colitis. In some embodiments, the disorder is inflammatory bowel disease.

[0149] Exemplary conditions related to NMDA-modulation includes, but are not limited to, gastrointestinal (GI) disorder, e.g., constipation, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn's disease), structural disorders affecting the GI, anal disorders (e.g., hemorrhoids, internal hemorrhoids, external hemorrhoids, anal fissures, perianal abscesses, anal fistula), colon polyps, cancer, colitis, and CNS conditions, e.g., as described herein.

[0150] Exemplary CNS conditions related to NMDA-modulation include, but are not limited to, adjustment disorders, anxiety disorders (including obsessive-compulsive disorder, posttraumatic stress disorder, social phobia, generalized anxiety disorder), cognitive disorders (including Alzheimer's disease and other forms of dementia), dissociative disorders, eating disorders, mood disorders (including depression (e.g., postpartum depression), bipolar disorder, dysthymic disorder, suicidality), schizophrenia or other psychotic disorders (including schizoaffective disorder), sleep disorders (including insomnia), substance abuse-related disorders, personality disorders (including obsessive-compulsive personality disorder), autism spectrum disorders (including those involving mutations to the Shank group of proteins (e.g., Shank3)), neurodevelopmental disorders (including Rett syndrome), multiple sclerosis, sterol synthesis disorders, pain (including acute and chronic pain), seizure disorders (including status epilepticus and monogenic forms of epilepsy such as Dravet's disease, and Tuberous Sclerosis Complex (TSC)), stroke, traumatic brain injury, movement disorders (including Huntington's disease and Parkinson's disease) and tinnitus. In certain embodiments, the compound of the present invention, e.g., a compound of Formula (I), or pharmaceutically acceptable salt thereof, can be used to induce sedation or anesthesia. In certain embodiments, the compound of Formula (I), or pharmaceutically acceptable salt thereof, is useful in the treatment or prevention of adjustment disorders, anxiety disorders, cognitive disorders, dissociative disorders, eating disorders, mood disorders, schizophrenia or other psychotic disorders, sleep disorders, substance-related disorders, personality disorders, autism spectrum disorders, neurodevelopmental disorders, sterol synthesis disorders, pain, seizure disorders, stroke, traumatic brain injury, movement disorders and vision impairment, hearing loss, and tinnitus.

[0151] In another aspect, provided is a method of treating or preventing brain excitability in a subject susceptible to or afflicted with a condition associated with brain excitability, comprising administering to the subject an effective amount of a compound of the present invention, e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0152] In yet another aspect, the present invention provides a combination of a compound of the present invention, e.g., a compound of Formula (I), or pharmaceutically acceptable salt thereof, and another pharmacologically active agent. The compounds provided herein can be administered as the sole active agent or they can be administered in combination with other agents. Administration in combination can proceed by any technique apparent to those of skill in the art including, for example, separate, sequential, concurrent and alternating administration.

Examples

[0153] 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

[0154] ##STR00013## ##STR00014## ##STR00015##

Synthesis of Compound A2.

[0155] 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 mL×2), brine (150 mL), dried over Na.sub.2SO4, 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), δ 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.

[0156] 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), δ 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.

[0157] 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 ml×3). The combined organic layers were washed with brine (80 mL×2), 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), δ 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.

[0158] 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 mL×3) 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), δ 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.

[0159] 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 mL×3). 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), δ 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), δ 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.

[0160] A solution of Pd(OAc).sub.4 (1.14 g, 3.32 mmol) and I.sub.2 (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 mL×2), 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), δ 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.

[0161] 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), δ 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), δ 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.

[0162] To a solution of A8-a (70 mg, 0.17 mmol) in THF (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 mL×3). 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), δ 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

[0163] ##STR00016##

Synthesis of Compound B2.

[0164] 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+H−18].sup.+).

Synthesis of Compound 2.

[0165] To a solution of B2 (140 mg, 0.336 mmol) in dry THF (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) δ=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 calcd. for C.sub.29H.sub.51O.sub.3[M+H].sup.+ 447, found 411 ([M+H−36].sup.+).

Example 3. Synthesis of Compounds 3 and 4

[0166] ##STR00017##

[0167] Synthesis of compound 3. To a solution of C1 (500 mg, 1.15 mmol) and tetraisopropoxytitanium (326 mg, 1.15 mmol) in dry THF (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. .sup.1H NMR (3): (400 MHz, CDCl.sub.3) δ=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.+). .sup.1H NMR (4): (400 MHz, CDCl.sub.3) δ=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

[0168] ##STR00018## ##STR00019##

Synthesis of Compound D2.

[0169] 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) δ=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.

[0170] To a stirred solution of D2 (24.8 g, 113 mmol) in toluene (100 mL) was added Me.sub.3Al (2 M in 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) δ=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 R.sub.t=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+H−18].sup.+).

Synthesis of Compound D4.

[0171] To a solution of bromo(ethyl)triphenylphosphorane (18.3 g, 49.5 mmol) in THF (100 mL) under N.sub.2 was added a solution of t-BuOK (5.55 g, 49.5 mmol) in THF (60 mL). The mixture was becoming orange and stirred for 1 hour. A solution of D3 (3.3 g, 9.92 mmol) in THF (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. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=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 R.sub.t=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+H−18].sup.+).

Synthesis of Compound D5.

[0172] 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) δ=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 R.sub.t=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.

[0173] 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) δ=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 R.sub.t=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+H−18].sup.+).

Synthesis of Compound 5.

[0174] To a solution of D6 (100 mg, 0.231 mmol) in dry THF (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) δ=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 R.sub.t=1.074 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 427 ([M+Na].sup.+).

Example 5. Synthesis of Compound 6

[0175] ##STR00020##

Synthesis of Compound 6.

[0176] To a solution of C1 (100 mg, 0.231 mmol) in dry THF (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) δ=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

[0177] ##STR00021##

Synthesis of Compound E2.

[0178] 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+H−18].sup.+).

Synthesis of Compound 7.

[0179] To a solution of E2 (100 mg, 0.247 mmol) and trimethyl(trifluoromethyl)silane (174 mg, 1.23 mmol) in THF (5 mL) was added CsF (3.75 mg, 24.7 μmol). 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) δ=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 R.sub.t=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+H−18].sup.+).

Example 7. Synthesis of Compound 8

[0180] ##STR00022##

Synthesis of Compound 8.

[0181] To a solution of D7 (50 mg, 0.124 mmol) and trimethyl(trifluoromethyl)silane (88.1 mg, 0.62 mmol) in THF (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) δ=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 R.sub.t=1.168 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd. for C.sub.27H.sub.44F.sub.3O.sub.3 [M+H].sup.+473, found 455 ([M+H−18]).

Example 8. Synthesis of Compound 9

[0182] ##STR00023##

Synthesis of Compound D7.

[0183] 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 R.sub.t=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+H−18].sup.+).

Synthesis of Compound 9.

[0184] To a solution of D7 (200 mg, 0.496 mmol) in dry THF (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 concentated 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) δ=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.sub.t=1.126 min in 2 min chromatography, 30-90AB, purity 100%, MS ESI calcd, for C.sub.27H.sub.47O.sub.3 [M+H].sup.+ 419, found 401 ([M+H−18].sup.+).

Materials and Methods

[0185] 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.

[0186] 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.

[0187] 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.

[0188] .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 δ (ppm) of about 1 to about 2.5 ppm.

[0189] Exemplary general method for preparative HPLC: Column: Waters RBridge prep 10 μm 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

[0190] 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

[0191] 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.

[0192] 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

[0193] 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

[0194] 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.

[0195] 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 μl 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 μg/ml Puromycin.

[0196] 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 MΩ-6.0 MΩ; seal resistance range:>1 GΩ) 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 “NMDA 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 μM NMDA (and 5.0 μM 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 μM NMDA and 5.0 μM 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 μM 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 μM [00024] A [00025] A [00026] A [00027] B [00028] 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)

[0197] 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 performed using Ion Works Barracuda platform following this procedure:

Electrophysiological Procedures:

[0198] a) Intracellular solution (mM): 50 mM CsCl, 90 mM CsF, 2 mM MgCl.sub.2, 5 mM EGTA, 10 mM HEPES. Adjust to pH 7.2 with CsOH. [0199] b) Extracellular solution, HB-PS (composition in mM): NaCl, 137; KCl, 1.0; CaCl.sub.2, 5; HEPES, 10; Glucose, 10; pH adjusted to 7.4 with NaOH (refrigerated until use). [0200] c) Holding potential: −70 mV, potential during agonist/PAM application: −40 mV.

Recording Procedure:

[0201] a) Extracellular buffer will be loaded into the PPC plate wells (11 μL per well). Cell suspension will be pipetted into the wells (9 μL per well) of the PPC planar electrode. [0202] b) Whole-cell recording configuration will be established via patch perforation with membrane currents recorded by on-board patch clamp amplifiers. [0203] c) Two recordings (scans) will be performed. First, during pre-application of PAM alone (duration of pre-application—5 min) and second, during test articles and agonist (EC.sub.20 L-glutamate and 30 μM 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 μL of 2× concentrated test article solution and, second, of 20 μL of 1× concentrated test article and agonist at 10 μL/s (2 second total application time).

TABLE-US-00002 TABLE 2 GluN2A PCA IWB Ephys % potentiation Structure at 3 μM [00029] A [00030] B [00031] A [00032] A [00033] A For Table 2, “A” indicates 10 to 150%, and “B” indicates potentiation of >150%.

Other Embodiments

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.