NZ617347B2 - Pentylenetetrazole derivatives - Google Patents

Abstract

The disclosure relates to pentylenetetrazole (PTZ) derivates having formula (l) wherein R1 – R10 are independently selected from hydrogen and deuterium, wherein at least one is not hydrogen, or a pharmaceutically salt thereof. The disclosure also relates to pharmaceutical compositions comprising these compounds and their use in the increasing blood flow, heart rate or breathing rate in an individual The disclosure also relates to their use for treating senility, senile confusion, psychoses, psychoneuroses when anxiety and nervous tension were present, cerebral arteriosclerosis, nausea, depression, fatigue, debilitation, mild behavioral disorders, irritability, emotional instability, antisocial attitudes, anxiety, vertigo or incontinence, or symptom thereof, or in improving cognitive function in individuals, for instance, in individuals with Down syndrome and other conditions. Example compounds include: 6,6,8,8,10,10-d6-6,7,8,9-tetrahydro-5Htetrazolo[1,5-a]-azepine se compounds and their use in the increasing blood flow, heart rate or breathing rate in an individual The disclosure also relates to their use for treating senility, senile confusion, psychoses, psychoneuroses when anxiety and nervous tension were present, cerebral arteriosclerosis, nausea, depression, fatigue, debilitation, mild behavioral disorders, irritability, emotional instability, antisocial attitudes, anxiety, vertigo or incontinence, or symptom thereof, or in improving cognitive function in individuals, for instance, in individuals with Down syndrome and other conditions. Example compounds include: 6,6,8,8,10,10-d6-6,7,8,9-tetrahydro-5Htetrazolo[1,5-a]-azepine

Description

PENTYLENETETRAZOLE DERIVATIVES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to US. ional application No. 61/482,533, filed on May 4, 2011, the content of which is incorporated herein by reference in its entirety.

FIELD Isotopically-enriched and/or fluorinated pentylenetetrazole (PTZ) compounds and pharmaceutically acceptable salts thereof are provided. Also provided are pharmaceutical compositions and unit dose forms sing the nds, s of using the compounds for the treatment of certain diseases, and s for the improvement of cognitive impairment in an individual, for instance, in an individual with Down me.

BACKGROUND Since being reported as a possible treatment for schizophrenia in the late 19305, pentylenetetrazole (PTZ) has been used as a therapy for a variety of maladies and conditions involving the central nervous system, such as senile confusion, depression, vertigo, and so forth, as well as being used as a circulatory and respiratory stimulant and cough suppressant. PTZ has the following formula: 6 4 7 5 /N\N3 8 ll N1—N2 PTZ is known by tradenames and synonyms including METRAZOL, CARDIAZOL pentetrazol, among others. In 1982 the US. Food and Drug Administration withdrew its al for marketing of PTZ in the United States and required that evidence be provided for efficacy in support of claims made for PTZ used alone or in combination with other agents. See 47 Federal Register 19208 (May 4, 1982).

PTZ is believed to block or reduce passage of ions through the ion channel associated with type A gamma—aminobutyric acid (GABAA) receptors. GABA is the major tory neurotransmitter in the central s system. GABA, in the absence of PTZ and other channel blockers, GABAA receptor antagonists and/or allosteric modulators, binds to the GABAA receptor leading to receptor channel opening and passage of chloride ions through the channel.

Recent work has shown that administration ofPTZ can lead to improvement in learning and memory. For example, in a transgenic mouse model of Down syndrome, daily doses of PTZ generated ements in learning lasting months after mice were last exposed to PTZ. See, e. g., Fernandez et al., 2007 Nature cience 10(4):411-413; Rueda et al., 2008, Neuroscience Letters 433(1):22-27; and US. Patent Application PublicationNo. 2008/0009475, published January 10, 2008, which disclosures are incorporated herein by reference in their entireties for all purposes.

Peak concentration of PTZ in blood generally occurs within 10 minutes after intravenous (IV) or intraperitoneal (IP) ry. PTZ has high bioavailability after oral dosing (PO), and peak blood levels generally occur within approximately 30 to 60 minutes when PTZ is given . PTZ y crosses the blood brain barrier. It has a relatively short plasma half-life of about 60 minutes. Following administration to mice, rats, dogs, humans and other living systems, PTZ undergoes oxidative metabolism, a key determinant for PTZ's short ife. There are a number of metabolites that have been characterized for PTZ, at least 2 of these are oxidized variants of PTZ and account for over 60% of the eliminated product. These metabolites are 6-hydroxypentetrazole and 8-hydroxypentetrazole. Oxidation is likely carried out by s of the cytochrome P450 superfamily.

Increasing dosing frequency or dosage amounts of PTZ in therapeutic applications to compensate for its relatively short half-life in viva es l consideration in View that its side effects, including seizures and convulsions, are Cmax . PTZ may also cause dose-dependent, significant inhibitory effects on the activity of metabolic enzymes including CYP450 and other s of this superfamily, which could potentially be detrimental when, for instance, PTZ is co-administered with other drugs.

New therapies having a eutic benefit similar or improved to that of PTZ are sought. Included in such sought-after therapies would be, for instance, compounds exhibiting a half-life in viva longer than that for PTZ.

R5, R6, R7, R8, R9, and R10 is not hydrogen. In a particular embodiment, at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is ium. Some embodiments where at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is fluorine, may be claimed in one or more divisional applications. A description of such embodiments is retained herein for completeness. 3 (followed by 3A) 3A wed by 4) [0019A] In related ments, ed is the use of a compound having Formula 1, or pharmaceutically acceptable salt f, in the manufacture of a medicament to increase blood flow, heart rate or ing rate in an individual in need thereof. In other embodiments, provided is the use of a compound having Formula 1, or pharmaceutically able salt thereof, in the cture of a medicament for suppressing coughing in an individual in need thereof. In yet other embodiments, provided is the use of a compound having Formula 1, or pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of senility, senile confusion, psychoses, psychoneuroses when anxiety and nervous tension are present, cerebral arteriosclerosis, nausea, depression, fatigue, debilitation, mild behavioral disorder, irritability, emotional instability, antisocial attitude, 4 (followed by 4A) anxiety, o, incontinence, or symptom thereof. In yet other embodiments, provided is the use of a compound having Formula 1, or pharmaceutically acceptable salt thereof, in the manufacture of a medicament for improving cognitive function in an dual with Down syndrome, phenylketonuria, neurofibromatosis type 1, maple syrup urine disease, Rett syndrome, fetal alcohol syndrome, an autism spectrum er, circadian rhythm disruption, Alzheimer's disease, or dementia. 4A (followed by 5) incontinence, or a symptom f. In certain embodiments, the compound as described herein is for use in improving cognitive function in an individual with Down syndrome, phenylketonuria, neurofibromatosis type 1, maple syrup urine disease, Rett syndrome, fetal alcohol syndrome, an autism spectrum er, circadian rhythm tion, Alzheimer’s disease, or ia.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides a 2H NMR spectrum used in analysis of exemplary compound 1 as described in Example 1 below.

Figure 2 es a 19F NMR um used in analysis of exemplary compound 11 as described in Example 4 below.

Figure 3 provides a 2H NMR spectrum used in analysis of exemplary compound 14 as described in Example 5 below.

DETAILED DESCRIPTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

Unless ise stated, when a position is designated as “H” or “hydrogen,” or when a on in a chemical structure provided herein is implicitly occupied by a hydrogen atom, the position will be understood to have hydrogen at its natural isotopic composition.

Also unless otherwise stated, when a position is designated cally as “D” or “deuterium”, the position is understood to have deuterium at an abundance that is at least 3,340 times greater than the natural abundance ofdeuterium, which is 0.015% (1', e. , at least 50.1% incorporation of deuterium).

The term “deuterium substitution” as used herein refers to the substitution of one or more hydrogen atoms in a molecule with ium atoms.

The terms “isotopically enriched” or “isotopical enrichment” as used herein refer to an atom having an isotopic composition other than the natural isotopic composition of that atom or to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom. For e, in a compound as provided herein, when a position is designated as having 2012/036217 deuterium, it will be understood that the abundance of deuterium at that position is substantially greater than the natural abundance of deuterium, which is about 0.015%. pic enrichment” can be expressed in terms of the percentage of incorporation of an amount of a specific isotope at a given atom in a molecule in the place of the atom’s natural isotopic abundance. For example, deuterium enrichment of 1% at a given position means that 1% of molecules in a given sample contain deuterium at the ed position. The pic ment” of the compounds provided herein can be determined using conventional analytical methods known to one of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.

The term “isotopic enrichment ” as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope within a molecule.

The term “isotopologue” as used herein refers to a s of a specific compound that differs from another species of the given compound only in its isotopic composition, or level of isotopic ment, at one or more positions, e.g., H vs. D.

The term “pharmaceutical composition” refers to a composition that is formulated for pharmaceutical use.

The term “pharmaceutically acceptable” as used herein refers to a component that is compatible with other ingredients of a pharmaceutical composition and is suitable for use in contact with tissues of a subject without undue toxicity, irritation, allergic response, immunogenicity or other cations, commensurate with a reasonable benefit/risk ratio.

As used herein, a aceutically acceptable salt” means any non-toxic salt that, upon administration to a recipient, is capable of ing, either directly or indirectly, a compound of this disclosure. A salt is formed between a basic group of a compound and an acid, or between an acidic group of a compound and a base.

Acids commonly employed to form pharmaceutically acceptable salts include but are not limited to inorganic acids such as en bisulfide, hydrochloric acid, romic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, esulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid.

Bases commonly employed to form pharmaceutically acceptable salts include but are not limited to inorganic bases such as magnesium hydroxide, calcium hydroxide, ium hydroxide, zinc hydroxide, and sodium hydroxide, as well as organic bases such as y, secondary, tertiary, and quaternary, aliphatic and aromatic amines, including but not limited to L-arginine, amine, benzathine, choline, deanol, diethanolamine, lamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethy1amino)-ethanol, ethanolamine, ethylamine, ethylenediamine, pylamine, N—methyl-glucamine, hydrabamine, lH-imidazole, L-lysine, morpholine, 4-(2-hydroxyethyl)-morpholine, amine, dine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, ine, isoquinoline, ary amines, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino(hydroxymethyl)—1,3- propanediol, and hamine.

A pharmaceutically able salt thus includes but is not limited to a sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, ‘pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, late, oxalate, malonate, succinate, te, sebacate, fumarate, maleate, butyne— l,4-dioate, hexyne-l ,6-dioate, benzoate, chlorobenzoate, benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, thalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, B-hydroxybutyrate, glycolate, maleate, te, methanesulfonate, propanesulfonate, naphthalene-1 —sulfonate, naphthalene-Z-sulfonate, and mandelate.

The term “therapeutically effective amount,” as used herein refers to a dosage sufficient to produce a desired result, where the desired result is generally (i) an amelioration or alleviation, if not complete cessation, of one or more symptoms of disease or condition being treated, particularly the cognitive impairment symptoms, 9. g. , memory, learning ability, and the like, or (ii) a measurable improvement in cognitive function as determined on an appropriate assessment test testing some aspect relating to cognition. The term also refers to an amount of a nd that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or clinician.

WO 51343 As used herein, the term “solvate” means a compound that further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, ethanol, methanol, dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces. The term “hydrate” is employed when the solvent is water. Pharmaceutically acceptable solvates and hydrates are xes of a compound with one or more solvent or water les, or 1 to about 100, or 1 to about 10, or 1 to about 2, 3, or 4 solvent or water molecules.

The term “prodrug” as used herein refers to a compound that is a y converted in viva into a compound of Formula I as provided herein (“parent compound”). Prodrugs may have advantages over parent compounds, such as, for e, having better bioavailability or greater lity in pharmaceutical compositions. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic ysis.

The term “stable nds,” as used herein, refers to compounds which possess stability sufficient to allow for their manufacture, and that maintain the integrity ofthe compounds for a sufficient period oftime to be useful for the purposes detailed herein (e.g, formulation into a pharmaceutical composition, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds, treating a disease or condition responsive to therapeutic agents).

A “subject” as used herein means an animal, preferably a mammal, including, for example, mouse, rat, , dog, cat, guinea pig, goat, cow, horse, pig, sheep, monkey, primate, ape, or human. The term “individual” as used herein is when the subject is a human.

The term “disorder” as used herein refers to any abnormal condition of the human or animal body or of its parts that impairs normal oning. A disorder is typically manifested by distinguishing signs and symptoms.

As used herein, ,” “treating” or “treatment” refer to at least an amelioration of the symptoms associated with the disease or condition afflicting the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e. g., symptom, associated with the disease or condition being treated, such as impairment in memory or learning y or mental ion or depression or other cognitive function. As such, treatment also es situations where the disease or condition, or at least symptoms associated therewith, are completely inhibited, e.g. , prevented from happening, or stopped, e. g., terminated, such that the subject no longer suffers from the disease or condition, or at least the symptoms that characterize the disease or condition. It will be understood that where “treat,” “treating” or “treatment” in used in context of treating cognitive impairment, the terms refer to improvement in ion, for example, as can be determined on an appropriate assessment test.

The term “cognitive impairment” as used herein refers to impairment, often but not always from early childhood, of at least one cognitive function, such as a impairment in memory, impairment in learning y, etc.

The term “dosing regimen” as used herein refers to a specified amount of compound administered per time unit and duration of dosing (e.g., 3 times/day for 7 days).

The term “about” as used herein is intended to qualify the numerical values that the term modifies, denoting such a value as variable within a margin of error.

When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term “about” should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account cant figures.

When ranges of values are disclosed, and the notation “from m . . . to n2” or “nl-nz” is used, where n; and n2 are numbers, then unless otherwise specified, this on is intended to include the s themselves and the range between them.

This range may be integral or continuous between and including the end values.

Compounds PTZ derivatives with sed metabolic stability can, in certain embodiments, provide therapeutic benefits over PTZ, for instance, by (a) ing subject compliance by decreasing the number of doses needed to achieve the therapeutic effect of PTZ, (b) decreasing the amount of a dose needed to achieve the therapeutic effect of PTZ and/or reduce the occurrence ofpotential adverse events, (c) ng a more effective drug and/or a safer drug for polypharmacy, whether the polypharmacy be intentional or not, and/or (d) ating inter-patient variability due to rphisms in enzymes that normally metabolize PTZ. Compounds that have one or all of these characteristics compared to PTZ are desirable, and are provided herein.

In one aspect, provided herein is a compound having Formula I: In Formula I, each of R‘, R2, R3, R4, R5, R", R7, R8, R9, and R10 is independently selected from hydrogen, deuterium, and fluorine, wherein at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is not hydrogen.

In certain embodiments, compounds of a I are provided wherein when each of R1, R2, R9, and R10 is hydrogen, at least one of R3, R4, R5, R6, R7, and R8 is not deuterium.

In certain embodiments of the compound of Formula I provided herein, R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from hydrogen and ium.

In some embodiments, R5 and R6 are each deuterium. R1, R2, R3, R4, R7, R8, R9, and R10 are independently selected from hydrogen and deuterium, or R1, R2, R3, R4, R7, R8, R9, and R10 are each hydrogen.

In some embodiments, R9 and R'0 are each deuterium. R1, R2, R3, R4, R5, R6, R7, and R8 are ndently selected from hydrogen and deuterium, or R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen.

In some embodiments, R5, R6, R9, and R10 are each deuterium. R1, R2, R3, R4, R7, and R8 are independently selected from hydrogen and deuterium, or R1, R2, R3, R4, R7, and R8 are each en.

In some embodiments, R1, R2, R5, R6, R9, and R10 are each ium.

R3, R4, R7, and R8 are independently selected from hydrogen and deuterium, or R3, R4, R7, and R8 are each hydrogen.

In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each deuterium.

In certain embodiments, provided herein are compounds having the structure of PTZ in which one or more en are replaced with a deuterium or fluorine. In certain embodiments, a ium isotopologue of PTZ is provided.

Deuterium (referred to as “D” in n formulas herein) is a , non-radioactive isotope of hydrogen. One characteristic of deuterium is that it forms ularly strong bonds with carbon, generally about six to ten times more stable than the ponding hydrogen to carbon bond. General exposure to and oration of deuterium is safe within levels potentially achieved by use of compounds provided herein as therapeutics.

It will be recognized that some variation of natural isotopic abundance occurs in a synthesized compound depending upon the origin of chemical materials used in the synthesis. Thus, typically, any preparation of a compound, e. g., PTZ, will inherently n small amounts of deuterium isotopologues. The concentration of naturally abundant stable hydrogen isotopes, notwithstanding this variation, is small and immaterial as compared to the degree of stable isotopic substitution of compounds provided herein.

A deuterium isotopologue of PTZ as provided herein, can, for example, have a minimum isotopic enrichment factor of at least 3,000 (a deuterium enrichment of 45%) for each designated deuterium in the isotopologue. In other ments, a deuterium isotopologue of PTZ as provided herein has an isotopic enrichment factor for each designated deuterium of at least 3,500 (52.5% deuterium enrichment), at least 4,000 (60% deuterium enrichment), at least 4,500 (67.5% deuterium enrichment), at least 5,000 (75% deuterium enrichment), at least 5,500 (82.5% deuterium enrichment), at least 6,000 (90% deuterium ment), at least 6,333.3 (95% deuterium enrichment), at least 6,466.7 (97% ium enrichment), at least 6,600 (99% deuterium enrichment), or at least 6,633.3 (99.5% deuterium enrichment).

The relative amount of deuterium in the deuterium isotopologues of PTZ provided herein will depend upon a number of s including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorporation of deuterium in the various synthesis steps used to prepare the compound. In certain ments, for a given deuterium isotopologue of PTZ provided herein, the relative amount of other isotopologues will be less than 49.9% of the ologues in toto. In other embodiments, the relative amount of such isotopologues in toto will be less than 47.5%, less than 40%, less than 32.5%, less than 25%, less than 17.5%, less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5%.

In some embodiments, a compound of Formula I provided herein is selected from the group consisting of the ing compounds: DD D D D/N\N D D D /N D /N\N D /N\fi‘ N— :.—'I D N—II D N—N (3) (4) 9 9 9 9 D D D /N\N /N\N /N\ N D / \N D II II II II N—N N—N D N—N N—N (5) (6) (7) and (8) , , , or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of Formula I is selected from the group consisting of the following nds: DD D D /N /N /N\N __II _____II II arld 9 9 9 or pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of Formula I is selected from the group consisting of the following nds, or a pharmaceutically acceptable salt thereof: N N N / \N / \N D / ‘N H II II N—N N—N N—N D D D D D D D D D D 9 9 9 9 N N N ”I D ”I D ”I DD D D N——N N-—N N—N D / \II D D D D D D D N D D D \ D D D D N N N H D / \fi' / \Ii' / If D N N—-—N N--—N N—-—N D 9 5 In other embodiments of the compound of Formula I ed herein, each of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is independently selected from hydrogen and fluorine.

In some embodiments, R5 and R6 are each fluorine. R1, R2, R3, R4, R7, R8, R9, and R10 are independently selected from hydrogen and fluorine, or R1, R2, R3, R4, R7, R8, R9, and R10 are each en.

In some embodiments, R9 and R10 are each fluorine. R], R2, R3, R4, R5, R6, R7, and R8 are independently selected from hydrogen and fluorine, or R‘, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen.

In some embodiments, R5, R6, R9, and R10 are each fluorine. RI , R2, R3, R4, R7, and R8 are ndently selected from hydrogen and fluorine, or R1, R2, R3, R4, R7, and R8 are each hydrogen.

In some embodiments, R‘, R2, R5, R6, R9, and R10 are each fluorine. R3, R4, R7, and R8 are independently selected from hydrogen and fluorine, or R3, R4, R7, and R8 are each hydrogen.

In some ments, R', R2, R3, R4, R5, R6, R7, R8, R9, and R'0 are each fluorine.

In some embodiments, each ole, R2, R9, and R10 is deuterium and each of R5 and R6 is fluorine.

In n embodiments, a compound is provided selected from the group consisting of: F F F F F / \N /N\N F /N\N /N\ H H II F H F N““—N N--—'N F N—N N— F F F F (9) (10) (11) (12) , , 9 ,and N—-N (13 ) or a pharmaceutically acceptable salt thereof.

In certain embodiments, a compound is ed selected from the group consisting of: FF F F ll /NI'll F /N\N /N I'll F —N —N F —lr\|IF —N F FwCEF/fldl'(F10) QM; (11) (12) , , , , F D N—N N—N F D (13) and (14) or a pharmaceutically acceptable salt thereof.

, , In certain embodiments, the compound of Formula I is selected from the group consisting of the following compounds, or a pharmaceutically acceptable salt thereof: F F F N N——” F F F F N F / \Ii' N-—-N F and Without intending to be bound by any ular theory or mechanism, in certain embodiments, it is believed that one or more nds of Formula I provided herein can, when stered to a population of individuals, exhibit decreased inter-individual variation in plasma levels as compared to the individual variation in plasma levels of non-deuterated, non-fluorinated PTZ when administered to a population of individuals in an equivalent dosage unit. In certain embodiments, it is believed that one or more compounds of Formula I provided herein will, when administered to a population of individuals, exhibit average plasma levels greater than the average plasma level of non-deuterated, non-fluorinated PTZ when administered in an equivalent dosage unit to a population of individuals. In yet other embodiments, it is believed that one or more nds of Formula I provided herein will, when administered to a population of individuals, exhibit peak plasma concentrations lower than the peak plasma concentration of non—deuterated, non-fluorinated PTZ when administered in an equivalent dosage unit to a population of individuals.

In certain embodiments, the term “compound,” unless otherwise indicated in the context in which it is used, encompasses its pharmaceutically acceptable salts, solvates (including es), and/or prodrugs.

Synthesis Compounds of Formula I provided herein may be prepared by nce to the known methods for making PTZ. Such methods can be d out utilizing corresponding deuterated and/or fluorinated and optionally, other isotope-containing reagents and/or ediates to synthesize the compounds provided herein, or invoking standard synthetic protocols known in the art for introducing isotopic atoms to a chemical structure. n intermediates can be used with or without purification (e. g. filtration, distillation, sublimation, crystallization, ation, solid phase extraction, and chromatography). For instance, certain intermediates or reagents useful for making PTZ may be replaced with corresponding deuterated or fluorinated intermediates or reagents as may be needed depending on the desired site or sites or deuterium or fluorine incorporation, as exemplified below.

Scheme 1. General Synthetic Route for Preparation of a Compound of Formula I Provided Herein R9 R10 NaN3 R8 R7RaR9 0 N R7 AICI3 R6 / ‘ni R6 g1 R5 N————N R5 R 12 mln, 50° C R4 R1 R4 R3 R3 R2 (A) (I) Scheme 1 shows a general synthetic route useful for preparing nds of Formula I provided herein, including, for example, exemplary compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 (as provided above), as well as other deuterated and/0r fluorinated versions of PTZ. In this , each R is independently selected from H, D, or F, with the proviso that at least one R is D or F.

Alternatively deuterated and/or fluorinated versions of PTZ can be prepared from the appropriately substituted s—caprolactam following the ure described by Lehnhoff and Ugi, 1995, Heterocycles, 40(2): 801—808.

For instance, commercially available substituted cyclohexanones useful as t A for the synthesis of nds of Formula I provided herein, for example, compounds 1, 2, 3, 9, 10, 11 and 12 according to Scheme 1 are, respectively, the following cyclohexanones: O O D D F F D D 9 9 9 O O F F and F An ary synthesis of a reagent A useful for the preparation of compound 4 is described in Lompa-Krzymien and Leitch, 1973, l ofLabelled nds and Radiopharmaceuticals, 9(2): 331—338. An exemplary synthesis of a reagent A useful for the preparation of compound 5 is described in Williams et al., 1964, Monatshefl‘efuer Chemie, 95(1): 166-177. An exemplaly synthesis of a reagent A useful for the preparation of compound 6 is described in Takei et al., 2003, Journal of Organometallic try, 679(1): 32-42. An exemplary synthesis of a reagent A useful for the preparation of compound 7 is described in Wehage and Heesing, 1992, Chem. Ber, 125(1): 209-215. An exemplary sis of a reagent A useful for the preparation of compound 8 is described in Deutsch and Mandelbaum, 1969, Tetrahedron Letters, 10(17): 1351-2. An exemplary synthesis of a reagent A for the preparation of compound 13 is described in Cantacuzene and Atlani, 1970, Tetrahedron, 26(10): 2447— 2468.

The specific approaches and nds shown above are not intended to be ng. The chemical ures in the schemes herein depict variables that are hereby defined surately with chemical group definitions (moieties, atoms, etc.) of the corresponding position in the compound formulae herein, whether identified by the same variable name (i.e., R1, R2, R3, etc.) or not. The suitability of a chemical group in a compound structure for use in the synthesis of another compound is within the knowledge of one of ordinary skill in the art. Additional methods of synthesizing compounds of Formula I provided herein and their tic precursors, including those Within routes not itly shown in schemes herein, are within the means of chemists of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the applicable compounds are known in the art and e, for example, those described in Larock R, Comprehensive Organic Transformations, VCH Publishers (1989); Greene TW et al., Protective Groups in Organic Synthesis, 3rd Ed., John Wiley and Sons (1999); Fieser L et al., Fieser ana’ Fieser's Reagentsfor Organic Synthesis, John Wiley and Sons (1994); and te L, ed., Encyclopedia ofReagentsfor Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.

Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds.

Commercial sources for deuterium isotopically enriched starting materials or reagents include, among others, Icon Services Inc. (Summit, New Jersey USA), dge Isotope tories (Andover, Massachusetts USA) and Sigma- Aldrich Corp. (St. Louis, Missouri USA). Methods of incorporating deuterium in target compounds are extensively documented. See, for instance, Journal ofLabelled Compounds and Radiopharmaceuticals (John Wiley & Sons Ltd), for numerous issues that provided detailed mental descriptions on incorporation of deuterium into bioactive organic molecules.

Pharmaceutical Compositions and Unit Dose Forms In one aspect, provided herein is a pharmaceutical composition comprising a compound of Formula I ed herein or a pharmaceutically acceptable salt, e, or prodrug thereof (collectively ed to below as “the active ingredient”) and a phannaceutically acceptable excipient.

A pharmaceutical composition, as provided herein, is formulated to be ible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g. , intravenous, intradermal, aneous, intramuscular, oral and transdermal al) administration. In a specific ment, the ition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral or topical administration to subjects. In some embodiments, a ceutical composition is formulated in accordance with routine procedures for oral administration to humans. Typically, compositions for enous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.

WO 51343 Excipients are inert substances such as, without limitation, carriers, diluents, fillers, coloring , ng agents, sweeteners, lubricants, solubilizers, suspending agents, binders, vehicles, wetting agents, tablet disintegrating agents and encapsulating material. The choice of ent, to a large extent, depends on factors, such as the particular mode of administration, the effect of the excipient on the solubility and stability of the active ingredient, and the nature of the dosage form.

For example, pharmaceutically acceptable excipients, including carriers, adjuvants, vehicles, and the like, that may be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based nces, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block rs, polyethylene glycol and wool fat.

In making the pharmaceutical compositions, the active ingredient may be mixed with a diluent, or enclosed within a r, which may be in the form of a capsule, sachet, paper, or other container. Consistent with its intended route of administration, the ceutical compositions can be in a solid, semi-solid, or liquid form, for example, in the form of tablets, enteric coated tablets, soft or hard gelatin capsules, depots, pills, powders, lozenges, elixirs, suspensions, emulsions, slurrys, solutions, sterile injectable solutions, sterile packaged powders, suppositories, suspensions, syrups, aerosols, ointments, and the like. In n embodiments, the pharmaceutical compositions contain, for example, up to 0.5%, up to 1%, up to 10%, or up to 25% or more by weight of the active ingredient. ceutical compositions provided herein may be formulated according to conventional pharmaceutical practice (see, e.g., ton: The Science and Practice macy, 2131‘ edition, A.R.

Gennaro, ed. (Lippincott Williams & Wilkins, Phildelphia PA, 2005) and Encyclopedia ofPharmaceutical Technology, Third Edition, J. Swarbrick, editor ma Healthcare USA, Inc., New York, 2006)).

In certain embodiments, the ceutical composition is le for oral, parenteral, or intravenous infusion administration.

Oral administration can e, for instance, buccal, lingual or sublingual administration.

In some embodiments, the pharmaceutical composition is in the form of a tablet or a capsule suitable for oral administration.

Tablets, for instance, can further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation In certain embodiments, the pharmaceutical composition is pyrogen- free. ceutical compositions may be ted in unit dose forms containing a predetermined amount of active ient per unit dose, suitable for administration to a subject. In some embodiments, a unit dose form is provided comprising a compound of the present disclosure and one or more ceutically acceptable exipients. Unit dose forms, as used , refer to ally te units suitable for administration to human and animal ts and packaged individually as is known in the art. Each unit dose contains a predetermined quantity of the active ingredient(s) sufficient to produce the desired therapeutic effect, in association with the pharmaceutical excipients. Examples of unit dose forms include ampoules, syringes, and individually packaged tablets and es. Unit dose forms may be administered in fractions or multiples thereof.

The pharmaceutical composition, including unit dose form, can be in a form such as, for example, a single tablet, pill, capsule, a single solution for intravenous ion, a single drinkable solution, a single patch, and the like. Routes of administration of the unit dose forms include those described above.

The unit dose form can, for example, comprise 0.1 mg to 1 gram of the active ingredient. In certain embodiments, the unit dose form comprises 0.1 mg to 5.0 mg, 0.5 mg to 200 mg, 1 mg to 100 mg, 10 mg to 250 mg, 50 mg to 500 mg, or 100 mg to l g of the active ingredient. In certain embodiments, the unit dose form comprises an amount of the active ingredient consistent with the doses described below for administration to a subject in a method as provided herein.

In certain embodiments, the unit dose form comprises 0.1 mg to 1 g or 0.5 mg to 200 mg of the active ingredient and one or more pharmaceutically acceptable excipients, wherein the unit dose form is le for oral administration to a human.

In other embodiments, the pharmaceutical composition or unit dose form is in the form of a controlled or delayed release formulation. The present invention also provides new formulations and unit dose forms useful in the methods provided below, including controlled or d release and sustained release formulations useful in these methods. In these methods, the effective dose is as bed above, but the dose is only administered once per day, as the sustained release or controlled or delayed e formulation achieves the same therapeutic benefit as more frequent dosing of an immediate-release formulation. Technology for controlled or delayed release and ned release formulations, including those formulated into beads, coated tablets ing osmotically-controlled e s are known in the art, for example, as described in U.S. Patent Nos. 3,062,720; 3,247,066; 4,256,108; 4,160,452; and 4,265,874. In some embodiments, oral compositions can be made, using known technology, which specifically release orally-administered agents in the small or large intestines of a human patient, as described in, for example, Hardy et al., 1987, Alimentary Pharmacology & Therapeutics 1(4) 273—280 or U.S. Pat. No. 4,663,308.

Other lled or delayed release or sustained released formulations may be employed, for instance as described in International Publication No. W0 01/12233, US. Patent Nos 3,773,919 and 4,767,628, and US. Patent Application Publication No. 200300683 84. Such formulations can be used in implants that release an agent over a period of several hours, a day, a few days, a few weeks or several months depending on the polymer, the particle size of the r, and the size of the implant (see, e. g., U.S. Pat. No. 6,620,422). Other sustained release formulations are described in EP 0 467 389 A2, W0 93/241150, U.S. Pat. No. 5,612,052, WO 97/40085, W0 03/075887, W0 01/01964A2, US. Pat. No. 5,922,356, W0 94/155587, W0 02/074247A2, W0 98/25642, US. Pat. Nos. 5,968,895, 6,180,608, U.S. 20030171296, U.S. 20020176841, US. Pat. Nos. 5,672,659, 5,893,985, 122, ,192,741, 5,192,741, 4,668,506, 4,713,244, 5,445,832 4,931,279, 5,980,945, W0 02/058672, W0 9726015, WO 97/04744, and. 0019446.

In certain embodiments, a sustained release or lled or delayed release formulation of the active ient is is delivered during the day, evening or night so that a minimum eutic concentration of the active ingredient in the brain is maintained for a period of time generally greater than 2 or generally greater than 3, generally greater than 4, generally greater than 6, lly greater than 8, or generally greater than 12 hours. 2012/036217 In other embodiments, a pulsatile release formulation of the active ingredient is delivered so that 2, 3 or 4 pulses of the active ient are delivered over a 12 hour cycle. The effective total dose is as described above; the pulsatile release formulation is administered once per day, as the release profile and the mode of release obviates the need for le daily dosing.

Pulsed release technology such as that described in US. Pat. Nos. 4,777,049 and 6,555,136 can thus be used to administer the active agent to a specific location within the gastrointestinal tract. Such systems permit drug delivery at a predetermined time and can be used to deliver the active agent, optionally together with other additives that may alter the local microenvironment to promote agent stability and uptake, directly to the colon, without relying on external conditions other than the presence of water to e in vivo release.

Methods In one aspect, provided herein are methods sing administering a compound of the present disclosure, to a subject to (i) stimulate systemic blood circulation and/or respiration in the subject; (ii) suppress coughing in the t; (iii) treat a disease or condition that the subject has, where the disease or condition is selected from the group consisting of senility, senile confusion, psychoses, psychoneuroses when anxiety and s tension are present, cerebral arteriosclerosis, nausea, sion, fatigue, debilitation, mild behavioral disorder, irritability, cognitive impairment, emotional instability, antisocial attitude, anxiety, vertigo and incontinence; or (iv) improve ion in the subject.

In embodiments of methods for improving cognition, the t can, for example, have a cognitive impairment. In certain embodiments, the cognitive impairment is due to a congenital disorder. Exemplary congenital disorders where ive impairment can be present include Down syndrome, ketonuria, neurofibromatosis type 1, maple syrup urine e, Rett syndrome, and fetal alcohol In some embodiments, the cognitive disorder can have a genetic and/or environmental cause. For instance, in certain embodiments, the method for improving ion comprises administering a compound of the present disclosure to a subject with an autism spectrum disorder.

In yet other embodiments of the methods for improving cognition, the subject can have a cognitive impairment due to, for example, an ed condition. For example, cognitive impairment can be from circadian rhythm tion or a neurodegenerative condition, including Alzheimer's disease and other forms of dementia. ital, acquired and egenerative forms of cognitive impairment reduce the capacity for individuals to store and retrieve memories, learn, communicate and function independently.

In certain embodiments, provided herein are methods of improving cognition in a subject with intellectual disability (mental ation). By intellectual disability is meant a cognitive impairment with a pattern of persistently slow learning of basic motor and language skills during childhood, and a significantly below-normal global intellectual capacity as an adult. One common criterion for diagnosis of intellectual disability is a tested IQ of 70 or below. [001 11] Conditions of interest for treatment, including conditions for improving cognitive function, include Down Syndrome, and other congenital or acquired conditions that impair cognitive on. ed in the ions of interest for treatment are those in which there is impairment, often from early childhood, of at least one cognitive on, such as a impairment in memory, impairment in learning ability, etc. Down syndrome is the most common form of intellectual disability with an incidence rate of about 1 in 700 births and a prevalence of more than 400,000 in the U.S. and just under 6 million worldwide. It is a genetic condition also called Trisomy 21 in which persons with Down syndrome have 3 copies of chromosome 21 rather than 2.

Cognitive ment in Down syndrome is characterized by IQs that range generally from 35 to 70, mental age equivalents of about 7 years of age, and nced deficits in memory and language. [001 12] In certain situations, methods provided herein may result in partial or in a complete removal of a deficit in the cognitive function. For ce, the amount of improvement can be at least about 2 fold, at least about 5 fold or at least about 10 fold as compared to a suitable control, 6. g, an otherwise substantially identical subject not administered a compound as provided herein, that is, a subject having similar a level of cognitive ability that has been administered a placebo, where in certain ments the amount ofimprovement is at least about 1.5 fold, about 2 fold, about 3 fold, about fold, about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold or greater. In certain embodiments, an improvement in cognitive function can be at least about 1% or r. In some ments the improvement can be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 15%, about 20%, about 25%, about %, about 40%, about 50%, about 60%, about 70%, about 80% or greater.

In some embodiments, the improvement is determined by measuring some aspect of cognitive function in a given subject (or population of subjects) before and after administration of a PTZ derivative that is a compound as provided herein. In some embodiments, the aspect of cognitive function can be measured in a given subject (or population of subjects) prior to being stered a PTZ derivative as provided herein, which measurement is then compared to a measurement of the aspect of cognitive function during or upon completion of a closing regimen lasting a period of time (e.g., administrations made over a period of days, weeks, months or even years). In some embodiments, the improvement is determined by ing some aspect of cognitive function in a population of subjects to whom are administered (at least once, or, in other embodiments, a dosing regimen) of a PTZ derivative as provided herein as compared to measurements made in a population of subjects to whom the PTZ derivative as provided herein is not stered.

Assessing treatment efficacy or improvement in cognitive function can be evaluated using any test or protocol known in the art. For instance, the Clinician's Global Impression of Change (CGI/C) has been one of the most commonly used test to assess overall change in al trials. The validity of this type of measure is based on the ability of an experienced ian to detect clinically relevant against l change in a patient’s overall clinical state.

Assessing “improvement in cognitive fimction” can, for example, include a clinical history and/or collection of standardized information. Assessment may e, for instance, intelligence quotient (IQ) testing. It will be understood that an improvement in ive function can refer to any measurable improvement in an aspect of cognition, for example, as determined by performance of a task intended to assess recognition, comprehension, reasoning, remembering, creation of y, on, capacity for nt, learning, etc., or aspect thereof. The cognitive function improvement can be evaluated using any convenient protocol. A variety of ment tests are known in the art. See, e.g., Borkowski et al, “Intellectual Assessment and Intellectual Disability” in Handbook ofIntellectual and pmental Disabilities (eds.

Jacobson et (11., Springer e+Business Media, LLC, New York, 2007), r 14, pages 261-278. [001 16] Assessment tests include, for example, the Diagnostic Adaptive or Scale (DABS); the Wechsler Adult Intelligence Scale (WAIS) including it revisions, WAIS-R and WAIS-III; the Mini-Mental State Examination (MMSE) or “Folstein” test; the Blessed Information-Memory—Concentration Test (BIMC); Fuld Object Memory Evaluation (FOME); the rnia Verbal Learning Test (CVLT) and revised version (CVLT-II); and the like.

The compound provided herein may be administered at once, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or stic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the sional judgment of the person administering or supervising the administration of the formulations. In particular, compounds as provided herein may cause epileptic activity and doses should be well below a dose that will kindle seizures. Orally administered PTZ has, in humans, a ng dose of approximately 20 mg/kg.

Advantageously, the deuterium or fluorine containing analogue of PTZ ed herein may be may be le for administration in r doses, or may be suitable for fewer multiple administrations, to a subject than that for PTZ for a given indication.

Doses of the compounds administered in the methods provided in this disclosure can, for example, be in the range of from 0.005 mg/kg to 10 mg/kg, from 0.001 mg/kg to 0.2 mg/kg, from 0.01 to mg/kg to 2 mg/kg, or from 0.05 to mg/kg to 0.5 mg/kg, where “kg” refers to the subject’s body mass. In certain embodiments, an administered dose is about 10 mg/kg of patient weight, about 5 mg/kg, about 3 mg/kg, about 1 mg/kg, about 0.3 mg/kg, about 0.1 mg/kg, about 0.05 mg/kg, about 0.025 mg/kg, or about 0.01 mg/kg. An effective dose can be in the range of, for example, from 0.01 mg to 1.25 gm per dose, from 1 mg to 250 mg per dose, or from 2.5 mg to 70 mg per dose. The daily dose can be in the range of, for example, 0.1 mg to gm per day, or from 1 mg to 1 gram per day, or from 3 mg to 300 mg per day. In s embodiments, the administered dose is about 1 gm, about 500 mg, about 250 mg, about 200 mg, about 100 mg, about 50 mg, about 25 mg, about 10 mg, about mg, about 1 mg, about 0.5 mg, about 0.25 mg, or about 0.05 mg, which may be administered once, twice, three times or four time per day. In certain embodiments, the dose stered is that as provided above for the unit dose forms.

The dosage regimen with the use of the compounds provided herein is selected by one of ordinary skill in the arts, in view of a variety of factors, including, without limitation, age, weight, sex, and medical ion of the recipient, the severity of the condition to be treated, the route of administration, the level of metabolic and excretory on of the recipient, the dosage form employed, the ular compound and salt thereof employed.

] The compounds provided herein may be administered in a single daily dose, or the total daily dose may be administered in divided doses, two, three, or more times per day. Where delivery is via transdermal forms, administration is continuous.

In accordance with the methods provided, the compounds provided herein can, for example, be administered in a dosing regimen. A dosing regimen can, for example, comprise administering a compound as provided herein to a subject once a day for several days, weeks, months or years. A dosing regimen is usually maintained for at least about two days, at least about one week, at least about two weeks, at least about three weeks, at least about one month, or longer. In some embodiments of the ion, an intermittent dosing regimen is used, i.e., once a month, every other week, every other day, once per week, twice per week, and the like.

In one aspect provided herein are methods for blocking ion flow through the ion channel associated with the GABAA receptor in a cell. Such methods comprise the step of contacting the cell with a compound or ceutical composition provided herein.

In one aspect provided herein are methods for inhibiting GABA activation of ors in the CNS of a subject. Such s comprise the step of administering to the subject a compound or pharmaceutical composition provided herein.

In one aspect provided herein are methods for treating a subject suffering from, or being susceptible to ing from, a disorder that is beneficially d by PTZ. Such methods comprise the step of administering to the subject a therapeutically effective amount of a compound or ceutical composition provided herein.

WO 51343 In one , provided herein are methods of treating a subject suffering from, or being susceptible to suffering from, a disorder that is beneficially d by PTZ wherein the methods comprise the step of administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition provided herein so as to effect: (1) decreased individual variation in plasma levels of the compound or a metabolite f; (2) sed average plasma levels of the compound or decreased average plasma levels of at least one metabolite of the compound per dosage unit; (3) decreased metabolism by at least one cytochrome P450 or monoamine oxidase isoform in the subject; (4) decreased metabolism via at least one polymorphically-expressed cytochrome P450 isoform in the subject; (5) at least one improved sign or symptom of the disorder, in each case of (1)-(5), as ed to PTZ.

In certain embodiments, inter-individual variation in plasma levels of the compound or metabolites thereof is decreased; average plasma levels of the compound are increased; average plasma levels of a lite of the compound are decreased; tion of a rome P450 or monoamine oxidase isoform by the compound is increased; or metabolism of the compound by at least one polymorphically-expressed cytochrome P450 isoform is decreased; by greater than about 5%, r than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, or by greater than about 50% as ed to PTZ.

In one aspect, provided herein are methods for treating a subject suffering from, or being susceptible to suffering from, cognitive impairment wherein the method comprises the step of administering to the subject a derivative of PTZ (a compound as provided ) that upon administration provides a greater plasma exposure level of the derivative of PTZ than the plasma re level of a molar equivalent of PTZ administered in the same dosing regimen and to an equivalent subject. In certain embodiments, the plasma exposure level is at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least about 140%, at least about 145%, or more of the plasma exposure level of PTZ. In some embodiments, the derivative of PTZ has a longer plasma half-live than PTZ as compared to a molar equivalent PTZ composition that is administered using the same dosing regimen. In some embodiments, the derivative ofPTZ has a decreased rate and amount of metabolite production as ed to a molar equivalent PTZ composition that is administered using the same dosing regimen. In some embodiments, the administration of the derivative of PTZ provides both an increase in the plasma exposure level of the derivative of PTZ and a decrease in the plasma exposure level of metabolites as compared to the plasma ofPTZ exposure level of PTZ and PTZ metabolites produced from a molar equivalent administered in the same dosing regimen. Plasma exposure levels of a nd or of metabolites may be ed using the methods described by Li et al., 2005, Rapid Communications in Mass Spectrometry 19: 1943-1950; Jindal et al., 1989, Journal of Chromatography, Biomedical Applications 493(2): 392-7; Schwartz et al., 1966, mical Pharmacology 15(5): 645-55; Mehvar et al.,1987, Drug Metabolism and Disposition 15(2): 250—5; s et al., 1981, Journal ofChromatography, Biomedical Applications 226(1): 175-82; and any references cited therein or any modifications made thereof.

EXAMPLES Example 1: Synthesis of elm-6,7,8,9-Tetrahydro-5H-tetrazolo[1,5- a]azepine (l).

S_ch:c_I_n_¢_2 D D D D NaN D /N~ ~———3—a> ,N D SiCl4, MeCN D N-N' D D D n tetrachloride (0.35 mL, 3 mmol, 1.0 equiv) was added dropwise to a light suspension of all ohexanone (CDN Isotopes, 99.3 atom% D) (0.31 mL, 3 mmol, 1.0 equiv), sodium azide (0.59 g, 9 mmol, 9.0 equiv) and anhydrous acetonitrile (12 mL). The mixture warmed slightly and the suspension became thicker. After the mixture was d at room temperature for 15 hr, an aliquot was quenched into 10% sodium carbonate solution and extracted with dichloromethane. TLC showed a barely detectable amount of compound 1 had been formed. After additional 24 hr, TLC of a quenched aliquot showed that greater amount of compound 1 had been formed but higher Rf components remained. The reaction was continued for a total of 6.5 days to maximize the formation of compound 1. The reaction sion was poured slowly into cold 10% sodium carbonate solution in deuterium oxide (50 mL) and the s mixture extracted with dichloromethane (3 x 25 mL). The combined dichloromethane layers were washed with saturated brine (10 mL), dried over sodium sulfate, filtered and the filtrate concentrated under reduced pressure to give a pale yellow oil that slowly crystallized. The crude product was solved in a minimum volume of dichloromethane and absorbed onto silica gel. The ed al was purified on an IX automated chromatography system g with gradient of 25 to 75% ethyl acetate in heptanes to give a solid. The solid was triturated with es, filtered and dried overnight in a vacuum oven at 25—30 °C to give compound 1 (429 mg) as a white solid.

Compound 1 characterization: Melting point: 59.4-59.5 °C. : >99% by GC analysis on HP—l GC capillary column (30 m x 320 mm x 0.25 pm; hold at 50 °C for 1 min, ramp 20 °C/min to 280 °C, 1 min hold at 280 °C; retention time = 7.51 min).

Mass spectrometry, m/z = 149.1 (M+H+). 1H NMR, 2H NMR and 13C NMR spectra, where each was performed in CDC13, were consistent with product being compound 1.

Figure 1 provides a representative 2H NMR spectrum.

Example 2: Synthesis of 6,6,10,10-d4-6,7,8,9-Tetrahydro-5H— tetrazolo[1,5-a]azepine (2).

Scheme 3 0 D DAbL-DD D NaNa . Q4,:N SmmMeCN N~N Silicon tetra-chloride (0.72 mL, 7.85 mmol, 1.2 equiv) was added dropwise to a suspension of 2,2,6,6-d4-cyclohexanone (Aldrich Isotopes, 98 atom% D) (0.64 g, 6.26 mmol, 1.0 equiv), sodium azide (1.22 g, 18.76 mol, 3 equiv) in anhydrous acetonitrile (24 mL). The mixture was stirred at room temperature over the weekend (60 hr). An aliquot was quenched into 10% sodium carbonate solution in D20 and extracted with EtOAc. TLC indicated compound 2 was generated and a small amount of ng material. The reaction suspension was poured into cold 10% sodium carbonate (36 mL) in deuterium oxide and the aqueous mixture was extracted with ethyl acetate twice (1 x 150 mL and 1 x 50 mL). The combined c layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product as a pale yellow oil (0.8 g), which was solved in a minimum volume of dichloromethane and absorbed onto silica gel (3.4 g). The absorbed material was purified on an ANALOGIX ted chromatography system (SF25-12 g) eluting with a gradient of20 to 80% ethyl acetate in heptanes to give compound 2 (400 mg, 45% yield) as a white solid which was dried in vacuum oven at 25—30 °C.

Compound 2 characterization: Melting point: 59.0—59.1 OC.

Purity: 99.7% by GC analysis on HP-l GC capillary column (30 m x 320 pm x 0.25 pm; split : 20:], hold at 50 °C for 1 min, ramp 20 °C/min to 280 °C, 1 min hold at 280 °C; retention time = 7.49 min). Mass spectrometry, m/z = 143.1 (M+H+). 1H NMR, 13C NMR, COSY NMR and 2H NMR spectra, where each was performed in CDCl3, were tent with product being compound 2.

Example 3: Synthesis of 6,6,8,8,10,10-d6-6,7,8,9-tetrahydro—5H— tetrazolo[1,5-a]-azepine (3).

Scheme 4 O O O D D SiCl4, MeCN N O O D D D D 3-1 3-2 [:5 p-Toluenesulfonylhydrazide (6.0 g, 32 mmol, 1 equiv) was added to a on of 1,4-cyclohexandione monoethyleneketal (SM3) (5.0 g, 32 mmol, 1.0 equiv) in methanol-d (60 mL). The solution became very thick and stirring was continued for 1 hour. Sodium uteride (4 g, 96 mol, 3 equiv) was added slowly portionwise to the reaction. Note: Be cautious during this addition, and leave large amounts of headspace in the reaction vessel. This reaction starts slowly but eventually evolves large amounts of gas with foaming.

After the on of sodium uteride was complete, reaction was refluxed for 1 hour. The reaction was cooled to room temperature and 10% hydrochloric acid was added (100 mL). The reaction was stirred for 10 minutes, and extracted with romethane (3 x 50 mL). The combined organic layers were washed with saturated sodium bicarbonate (5 x 50 mL). The organic layer was dried over sodium sulfate and the reaction was concentrated to ~30 mL under reduced pressure (low vacuum) at ~30°C. This residue was transferred to a Vial and the remaining solvent was removed with a nitrogen stream to give 3-1 as a yellow oil mixed with ~40% p-toluenesulfonyl hydrazide, 3.2 g total mass (~1.3 g product), 40% yield. 4,4-d2—Cyclohexanone (3-1) (1.6 g crude, 6.5 mmol, 1.0 equiv) was suspended in 10% potassium carbonate in deuterium oxide (20 mL) and itrile (0.5 mL). The reaction was stirred for 36 hours at which time NMR indicated the on was complete. The reaction was extracted with romethane (3 x 10 mL).

The combined organic layers were dried over sodium sulfate, filtered and the resulting solution concentrated using a nitrogen stream to give 3-2 as an orange oil (320 mg, 47% yield).

Sodium azide (563 mg, 8.7 mmol, 3 equiv) and silicon tetrachloride (0.375 mL, 3.2 mmol, 1 equiv) was slowly added to a solution of 2,2,4,4,6,6-d6— cyclohexanone (3-2) (300 mg, 2.9 mmol, 1 equiv) in anhydrous acetonitrile (5 mL).

The reaction was stirred at room temperature for ~40 hours, when GC showed complete reaction. The reaction was cooled to 0"C and 10% sodium carbonate in deuterium oxide (3 mL) was added slowly to the reaction. The on was stirred for 30 minutes. The ke suspension was filtered through Celite and the Celite was washed with ethyl e (10 mL). The layers were separated and the ethyl acetate was dried over sodium sulfate. The reaction was filtered, dried over sodium sulfate and concentrated under reduced pressure to give 180 mg of material that was ~90% pure by GC. This material was chromatographed on ANALOGIX automated column chromatography system eluting with gradient of 0 to 100% ethyl acetate in heptanes to give 3 (80 mg, 27% yield) as a white solid.

Compound 3 characterization: Melting point: 58.1-59.3 °C.

Purity: 99.8% by GC analysis on HP—l GC ary column (see Example 1 for details, ; retention time = 7.52 min). Mass spectrometry, m/z = 145.2 (M+H+). 1H NMR, C NMR, 2H NMR and COSY NMR spectra, where each was performed in CDC13, were tent with t being compound 3.

Example 4: Synthesis of 7,7-Difluoro-6,7,8,9—tetrahydro-5H- tetrazolo[1,5 —a] azepine (1 1).

Sc__h____eme5 E 1N HCI NaBH o ——1>[0H N—H>E OBn —-———>- BnO o MeOH BnBr THF THF H20 SM13 13-2 13-3 NaN 14——c clohexadiene —3> BnO ——y——> H0 .,N SiCI4 Pd-0 EtOH ‘N MeCN artin XtalFluor-M F O ’N‘N N~N"N NE13-3HF 00141 1,4—DioxasPiro 4.5 decan-8—ol 13-1 :A cold 0 °C solution of cyclohexanedione monoethylene ketal (SM13) (6.24 g, 40 mmol, 1.0 equiv) in methanol (90 mL) was treated portionwise with sodium borohydride (1.60 g, 42 mmol, 1.05 equiv). The addition was exothermic and H2 was evolved. The reaction temperature was kept below 10 °C on addition of each portion, allowing the mixture to re-cool to 0 °C before the next on. When addition of sodium borohydride was complete, the mixture was stirred at 0 °C for 0.75 hours then stirred 1.5 hours while warming to room temperature. The mixture was concentrated under reduced pressure to remove most of the methanol. The al oily solid was diluted with water (40 mL) and saturated brine (40 mL) — no oil separated. The mixture was ted with sodium chloride and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with saturated brine (25 ml), dried over sodium sulfate, d and the filtrate concentrated under reduced pressure. The residual colorless oil was redissolved in ethyl acetate (15 mL), the solution diluted with heptanes (45 mL) and the turbid on concentrated to give crude compound 13-1 (6.17 g) as a colorless oil. Crude compound 13-1 was used subsequently. 8-(Benzyloxy)—1,4-dioxaspiro[4.Sldecane (13-2): A suspension of 60% sodium hydride in l oil (1.93 g, 48.3 mmol, 1.25 equiv) in THF (50 mL) was cooled in an ter bath. A solution of crude compound 13-1 (6.10 g, 38.6 mmol, 1.0 equiv) in THF (40 mL) was added slowly to maintain at less than 5 °C. The mixture was stirred in an ice-water bath for 20 minutes, allowed to warm to room temperature and stirred lhour. Evolution of H2 was very slow. The mixture was warmed to ~45 °C and held for 1 hour (evolution of H2 had essentially ceased). The tan suspension was cooled to room temperature and benzyl bromide (7.26 g, 5.0 mL, 42.5 mmol, 1.1 equiv) was added dropwise. No noticeable exotherrn occurred on addition, however, after addition was complete the reaction temperature slowly increased from 21 to 25 °C over 0.25 hours. The mixture was stirred at room temperature over a weekend. The mixture was quenched by the very slow addition of saturated ammonium chloride (45 mL). The biphasic suspension was d with water (15 mL) and extracted with a 1 to 1 of ethyl acetate and heptanes (150 mL). The organic phase was washed with saturated brine (2 x 50 mL), dried sodium sulfate, filtered and the filtrate concentrated under reduced volume of pressure. The residual yellow oil was dissolved in a minimum dichloromethane, absorbed onto silica gel and aded on a column of silica gel packed in heptanes. The column was eluted with a gradient ofO to 15% ethyl acetate in heptanes to give 13-2 (9.15 g) as a pale yellow oil. 4—(Benzyloxy)cyclohexanone (13-3): 1N HCl (60 mL) was added to a solution of 13-2 (9.15 g. 36.9 mmol) in THF (90 mL) and the mixture stirred at room temperature for 18 hr. The mixture was concentrated under d pressure to remove most of the THF. The residual aqueous oil was made alkaline by slow on of WmmflwfimflmmMMmmdwmawWMM1m2mmefwwmmmed heptanes. The organic layer was washed with saturated brine (50 mL), dried over sodium sulfate, filtered and the filtrate concentrated under reduced pressure to give crude 13-3 (7.53 g) as a yellow oil. Crude 13-3 was used subsequently. ] 7-(Benzyloxy)-6,7,8,9-tetrahydro-5H-tetrazolo[1,5-a]azepine (13-4): Sodium azide (5.95 g, 91.5 mmol, 3.0 equiv) was added to a solution of crude 13-3 (6.22 g, 20.5 mmol, 1.0 equiv) in acetonitrile (120 mL). Silicon tetrachloride (3.6 mL, .5 mmol, 1.0 equiv) was added dropwise to the sion and the mixture stirred at room temperature with the suspension ng thicker. After ~20 hours, the thick yellow suspension was poured slowly into cold (~5 °C) 10% sodium carbonate (700 mL). The suspension was extracted with ethyl acetate (3 x 200 mL, 1 x 100 mL) — some insolubles remained in the aqueous phase. The combined c layers were washed with saturated brine (2 x 150 mL), dried over sodium sulfate, d and the filtrate trated under reduced re to give a tan solid. TLC (50% ethyl acetate in es, Hanessian’s stain) showed 13-3, some higher Rf impurities and baseline material. The crude product was dissolved in ethyl acetate (250 mL), silica gel (5 g) and charcoal (0.7 g) were added and the solution stirred at 40 °C for 0.25 hr. The mixture was filtered through a CELITE pad, washing the pad with ethyl acetate (400 mL). The filtrate was concentrated under d pressure to give a light tan solid. The crude product was purified on an ANALOGIX automated chromatography system eluting with 0 to 5% methanol in dichloromethane to give 13-4 (4.12 g, ~91% purity by LCMS) as an ite solid. ] 6,7,8,9-Tetrahydro-SH-tetrazolo[1,5-alazepinol (13-5): A mixture of 1,4-cyclohexadiene (10.13 g, 11.8 mL, 126.6 mmol, 7.5 equiv), 20% Pd/C (0.41 g, 50% wet), and 13-4 (4.12 g, 16.88 mmol, 1.0 equiv) in ethanol (150 mL) was refluxed for 21.5 hours. The mixture was cooled to room temperature and filtered through a CELITE pad, washing the pad with ethanol (200 mL). The e (slightly turbid) was concentrated to give a h-white solid. The solid was ved in warm acetone (100 mL), the solution cooled and filtered through a CELITE pad, washing the pad with acetone (100 mL). The filtrate was concentra—ted to give a solid that was triturated with MTBE (50 mL), d and dried for 17 hr in a vacuum oven at 40—45 °C for 17 hours to give 13-5 (2.24 g) as a light gray solid. 8,9-Dihydro-5H—tetrazolo[1,5-a]azepin-7(6H)—one (ll-l): Dess-Martin periodinane (630 mg, 1.52 mmol, 1.3 equiv) was added to a solution of6,7,8,9- tetrahydro-SH-tetrazolo[1,5-a]azepinol (13-5) (200 mg, 1.29 mmol, 1.0 equiv) in THF (3.0 mL) The mixture was stirred overnight. Saturated sodium bicarbonate (20 mL) was added to the reaction and d for 30 minutes. The mixture was extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on AnaLogix automated column chromatography system eluting with gradient of 30 to 100 % ethyl acetate in heptanes to give 11-1 (70 mg, 36 % yield) as a white solid. 7,7-Difluoro-6,7,8,9-tetrahydro-SH-tetrazolo[1,5—alazepine (1 1): Triethylamine trihydrofluoride (0.13 mL, 0.8 mmol, 3.0 equiv) and XTALFLUOR-M reagent (126 mg, 0.52 mmol, 2.0 equiv) were sequentially added to a solution of 8,9-dihydro-5H—tetrazolo[1,5-a]azepin-7(6H)—one (11-1) (40 mg, 0.26 mmol, 1.0 equiv) in dichloromethane (3 mL). The mixture was stirred overnight at room temperature.

Saturated sodium bicarbonate (10 mL) was added to the reaction and stirred for 30 s. The mixture was extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over sodium e, filtered and trated under reduced pressure The residue was chromatographed on ANALOGIX automated column chromatography system eluting with gradient of 10 to 40 % ethyl acetate in heptanes to give 11 (30 mg, 66 % yield) as a white solid.

Compound 11 characterization: Purity: >99 % by GC analysis on HP—SMS GC capillary column (30 m X 250 um x 0.25 pm; hold at 50 °C for 1 min, ramp °C/min to 280 °C, 1 min hold at 280 °C; mobile phase, 5% phenyl methyl siloxane, retention time = 7.37 min). GC-MS, m/z = 174.0 [M]+. IH NMR, COSY NMR, C NMR, and ”P NMR spectra, where each was performed in CDCl3, were consistent with product being compound 11. Figure 2 provides a representative 19F NMR e 5: Synthesis of 6,6,10,10-d4-8,8-Difluoro-6,7,8,9-tetrahydro- 5H-tetrazolo[1,5—a]azepine (14) §_____cheme6 OAPh OAPh NaBH4 :ngzlBnBr CDCI3, base SiCl4/NaN3 _ ’ D D l D D O O SM14 14-1 14-2 14-3 D D DMSO Pd/C oxalylic chloride /N. 4—cyclohexadiene1 NEts BnO 0N 14'4 14-5 14-6 XtalFluor-M N ———> FF ’ N‘N” 1,4-Dioxaspir0[4.5]decanol (14-1): Sodium borohydride (0.8 g, 21 mmol, 1.05 equiv) was added at 0 to 10 °C in portions to a solution of 1,4-dioxaspiro[4.5]decan—8—one (SM14) (3.1 g, 20 mmol, 1.0 equiv) was dissolved in methanol (50 mL). The mixture was stirred at room temperature for 3 h and trated under reduced pressure to remove most of solvent. Water (50 mL) was added and the mixture and stirred for 30 minutes. The mixture was extracted with ethyl acetate (3 x 50 mL)and the combined organic layers were washed with sequentially with 1N HCl (30 mL), water and ted brine . The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give 14-1 (3.0 g, 96% yield) as a colorless oil which was used subsequently . 4-(Benzyloxy)cyclohexanone (14-2): A 60% dispersion of sodium hydride in mineral oil (760 mg, 23 mmol, 1.2 equiv) was added to a solution of 1,4-Dioxaspiro[4.5]decanol (14-1) (3.0 g, 19 mmol, 1.0 equiv) in anhydrous THF (50 mL) at 0 0C. After 6 hours benzyl bromide (2.5 mL, 21.3 mmol, 1.1 equiv) was added to the mixture. The mixture was stirred at room temperature overnight. A 4N HCl solution (30 mL) was added and the reaction was stirred at room temperature for an additional 6 hours. The reaction was neutralized to pH ~7 with 4N sodium hydroxide and ted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was d on an AnaLogix ted column chromatography system eluting with gradient of 0 to 30% ethyl acetate in es to give 14—2 (3.0 g, 77% yield) as a light yellow oil. 4-(Benzyloxy)-2,2,6,6-d4-cyclohexanone (14-3): Triazabicyclodecene (TBD) (200 mg, 1.44 mmol, 0.1 equiv) was added to a solution of 4-(Benzyloxy)cyclohexanone (14-2) (3.0 g, 14.7 mmol, 1.0 equiv) was dissolved in form-d (50 mL). The mixture was stirred at room ature overnight, at which point IH—NMR showed 85 % deuterium incorporation. The mixture was then refluxed for 6 hours, at which point lH-NMR showed ium exchange was te. The reaction was cooled to room temperature and washed sequentially with 1N HCl (20 mL), water, and saturated brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give 14-3 (3.0 g, 98% yield) as a light-yellow oil which was used subsequently. 8-(Benzyloxy)— 6,6,10,10-d4-6,7,8,9-tetrahydro-SH-tetrazolo[1,5- a]azepine (14-4): Sodium azide (2.81 g, 43.2 mmol, 3.0 equiv) and silicon hloride (1.65 mL, 14.4 mmol, 1.0 equiv) were added to a solution of 4-(benzyloxy)-2,2,6,6-d4- cyclohexanone (14—3) (3.0 g, 14.4 mmol, 1.0 equiv) in anhydrous acetonitrile (40 mL).

The reaction was d at room temperature for 48 hours and poured into ice cold saturated sodium bicarbonate (100 mL). The mixture was stirred for 30 minutes and extracted with dichloromethane (3 x 150 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified on ANALOGIX ted column chromatography system eluting with a gradient of 0 to 5% methanol in chloromethane to give 14-4 (2.7 g, 75% yield, 7% proton at positions 6 and 10) as white solid. ,10-d4-6,7,8,9-Tetrahydro-SH-tetrazolo [1,5-a]azepinol (14-5): 7-(Benzyloxy)— 6,6,10,10-d4-6,7,8,9-tetrahydro—5H-tetrazolo[1,5-a]azepine (14-4) (2.7 g, .9 mmol, 1.0 equiv) was dissolved in ethanol (200 mL) followed by the addition of 20 % Pd/C (270 mg, 50% wet) and 1,4-cyclohexadiene (12 mL, 127 mmol, 12 equiv). The e was refluxed overnight, cooled to room temperature, and filtered through a CELITE pad, washing the pad with ethanol (100 mL). The filtrate was concentrated under reduced pressure to give 14-5 (1.55 g, 90% yield) as an off-white solid which was used subsequently. ] 6,6,10,10-d4-8,9-Dihydr0-5H-tetrazolo[1,5-a]azepin-7(6H)-one : Anhydrous DMSO (0.8 mL, 22 mmol, 2.4 equiv) was added to a solution of oxalyl chloride (0.95 mL, 11 mmol, 1.2 equiv) at -78 °C in romethane (20 mL). The mixture was stirred for 0.5 hours at -78 °C followed by the dropwise addition of a solution of 6,6,10,10-d4-6,7,8,9-tetrahydro-5H—tetrazolo[l,5-a]azepinol (14-5) (1.5 g, 9.5 mmol, 1.0 equiv) in dichloromethane (200 mL) maintaining the ature below -65 °C. The reaction was stirred for an additional 2 hours at — 78 °C. Triethylamine (7.7 mL, 55 mmol, 6 equiv) was added and the e was stirred for 30 minutes at -78 °C. After warming to room temperature, water (100 mL) was added and the organic layer was separated, washed with water, saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified on ANALOGIX automated column chromatography system eluting with eluting with a gradient of 0 to 5% methanol in dichloromethane to give 14-6 (800 mg, 55% yield) as white solid. 6,6,10,10-d4-8,8—Difluoro-6,7,8,9—tetrahydro—SH—tetrazolo[1,5- ine (14): Triethylamine hydrofluoride (0.68 mL, 4.2 mmol, 3.0 equiv) and XTALFLUOR-M reagent (680 mg, 2.8 mmol, 2.0 equiv) were added sequentially to a solution of 6,6,10,10-d4-8,9-Dihydro-5H-tetrazolo[1,5-a]azepin-7(6H)-one (14-6) (220 mg, 1.4 mmol, 1.0 equiv) in dichloromethane (10 mL). The mixture was d overnight at room temperature ed by the addition of saturated sodium bicarbonate (30 mL). The mixture d for 30 minutes and extracted with dichloromethane (3 x 50 mL). The combined organic layers was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified on an ANALOGIX ted column chromatography system g with gradient of 10 to 40% ethyl acetate in heptanes to give 14 (200 mg, 80% yield) as a white solid.

Compound 14 characterization: Purity: >99 % by GC analysis on 1 min, ramp HP-SMS GC capillary column (30 m x 250 um x 0.25 pm; hold at 50 °C for °C/min to 280 °C, 1 min hold at 280 °C; mobile phase, 5% phenyl methyl siloxane, retention time = 7.43 min). GC-MS, m/z = 178.11 [M]+. 1H NMR, COSY NMR, 13C NMR, l9F NMR, and 2H NMR spectra, where each was performed in CDC13, were consistent with product being compound 14. Figure 3 provides a representative 2H NMR spectrum.

Example 6: tion of Metabolic Stability in Human Liver Microsomes.

Published accounts have observed that deuterium substitution can have variable and ictable effects on the rate of metabolism of a compound. See, e.g., Blake et al., 1975, J. Pharm. Sci. 64:367-391; Foster, 1985,Adv. Drug Res, 14:1-40; Curr. Opin. r et al., 1999, Can. J. Physiol. Pharmacol. 79-88; Fisher et al., 2006, 34:2871-2876.

Drug Discov. Devel. 9:101-109; Fukuto et al., 1991, J. Med. Chem.

This example demonstrates that metabolic half-lives of ated PTZ ologues differ from each other and from that of PTZ.

Human liver microsomes (Lot# F]M) were obtained from Celsis (Baltimore, MD). Components of the NADPH regenerating system solution A (Lot# 29850) and B (Lot# 28594) were obtained from BD Gentest (Woburn, MA).

Testosterone (Lot# FE111011-01) was purchased from Cerilliant (Round Rock, TX). further solvents and buffers were obtained from commercial sources and used without purification.

Individual test compounds and terone were prepared as a 10 mM buffer stock solution in DMSO. A mixture containing 50 mM potassium phosphate pH 7.4. 3 mM MgClz, 1 mg/mL human liver microsomes and 1 [1M test compound or in a shaking water bath. NADPH testosterone was pre-incubated for 5 min at 37°C rating system (1.3 mM NADP+, 3.3 mM glucosephosphate, 0.4 U/mL glucose- 6-phosphate dehydrogenase, and 3.3 mM magnesium chloride) were prepared by mixing volume of solution A and 1 volume of on B and pre-warmed at 37°C in a shaking water bath. Reactions were initiated by adding NADPH rating system to the incubation mixtures. After 0, 5, 10, 15, 30 and 45 min of incubation, 0.1 mL reaction mixtures were removed from the incubation plate and mixed with 0.15 mL of ice-cold itrile in an appropriate well of a 96-well crash plate. The 96—well crash plate was placed on ice for 15 min, and samples were fuged (2,500 g, 10 min, 4°C) to precipitate protein. The supematants were diluted 1:1 (v/V) with water ning 0.015 uM verapamil (internal standard) in 96-well shallow injection plate, which was sealed and submitted for LC/MS or LC/MS/MS analysis utilitzing a API 150 single quadrupole mas spectrometer.

The residual compound ing (%R) was determined from LC/MS peak areas by comparison to a zero time point. Metabolic half-life (t1 /2) and intrinsic clearance (CLint) values were calculated from the slope of ln( %) plotted vs. time.

As shown in Table 1, under the assay conditions tested, the in vitro t1/2 for Compounds 1, 2, and 4 was increased compared to the tug of PTZ.

Table 1: Metabolic Stability of PTZ Derivatives xx\\\;\ ‘ M x: ._ \ \ \ \\\ iiiiiiiiiiN\ “ “ fPT’Z 521 1.3 — fcomp‘ouml Compoundz The results in Table 1 demonstrate that ives of deuterium PTZ isotopologues are not uniform and differ from each other and from PTZ depending upon the positions of the deuterium atoms within the le.

Without further description, it is believed that one of ry skill in the art can, using the preceding description and the illustrative examples, make and utilize the compounds of the present ion and practice the claimed methods. It should be understood that the foregoing discussion and examples merely present a detailed description of certain embodiments, and are not intended to limit the scope of the disclosure. It will be apparent to those of ordinary skill in the art that s modifications and equivalents can be made without departing from the spirit and scope of the invention.

The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of the disclosure. Modifications of the above-described modes for carrying out the disclosure that are s to persons of skill in the art are intended to be within the scope of the following claims.

All publications, patents, and patent ations cited in this specification are incorporated herein by reference as if each such publication, patent, or patent application were specifically and individually ted to be incorporated herein by reference.

Claims (21)

What we claim is:

1. A compound having Formula I: or a pharmaceutically able salt thereof, wherein: each of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is independently selected from hydrogen, deuterium, and fluorine, wherein at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is deuterium, and wherein when each of R1, R2, R9, and R10 is hydrogen, at least one of R3, R4, R5, R6, R7, and R8 is other than deuterium.

2. The compound of claim 1, or pharmaceutically able salt thereof, n R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from hydrogen and deuterium.

3. The compound of claim 2, or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of , , and .

4. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein the compound is

5. A pharmaceutical composition comprising the compound ing to any one of claims 1-4, or pharmaceutically able salt thereof, and a pharmaceutically acceptable excipient.

6. A unit dose form comprising 0.1 mg to 1 g of the nd of claim 1, or pharmaceutically able salt thereof, and one or more ceutically acceptable excipients, wherein the unit dose form is suitable for oral or intravenous administration to a human.

7. Use of a compound of claim 1, or pharmaceutically acceptable salt thereof, in the manufacture of a medicament to increase blood flow, heart rate or breathing rate in an individual in need thereof.

8. Use of a compound of claim 1, or pharmaceutically acceptable salt thereof, in the manufacture of a ment for suppressing coughing in an individual in need thereof.

9. Use of a compound of claim 1, or pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of senility, senile confusion, psychoses, psychoneuroses when anxiety and nervous tension are present, cerebral arteriosclerosis, nausea, depression, fatigue, debilitation, mild behavioral er, irritability, emotional instability, antisocial attitude, anxiety, vertigo, incontinence, or symptom thereof.

10. Use of a compound of claim 1, or pharmaceutically acceptable salt f, in the manufacture of a medicament for improving cognitive function in an dual with Down syndrome, phenylketonuria, neurofibromatosis type 1, maple syrup urine disease, Rett syndrome, fetal alcohol me, an autism spectrum disorder, circadian rhythm disruption, Alzheimer's disease, or dementia.

11. The nd of claim 1 for use as a circulatory or respiratory stimulant; or for use as a cough suppressant; or for use in treating senility, senile confusion, psychoses, psychoneuroses when anxiety and nervous tension are present, cerebral arteriosclerosis, nausea, depression, fatigue, debilitation, mild oral disorder, irritability, emotional instability, antisocial attitude, anxiety, vertigo, or incontinence, or symptom thereof.

12. The compound of claim 1 for use in improving cognitive on in an individual with Down syndrome, ketonuria, neurofibromatosis type 1, maple syrup urine disease, Rett syndrome, fetal alcohol me, an autism spectrum disorder, circadian rhythm disruption, mer’s disease, or dementia.

13. A compound according to claim 1, substantially as herein described or exemplified.

14. A pharmaceutical composition according to claim 5, substantially as herein described or exemplified.

15. A unit dose form ing to claim 6, substantially as herein described or exemplified.

16. A use according to claim 7, substantially as herein described or exemplified.

17. A use according to claim 8, substantially as herein described or exemplified.

18. A use according to claim 9, substantially as herein described or exemplified.

19. A use according to claim 10, substantially as herein described or exemplified.

20. A nd according to claim 11, substantially as herein described or exemplified.

21. A compound according to claim 12, substantially as herein described or exemplified.