EA039381B1 - Benzodiazepine derivatives, compositions, and methods for treating cognitive impairment - Google Patents

Abstract

This invention relates to benzodiazepine derivatives of formulae II, III and IV, pharmaceutical compositions comprising therapeutically effective amounts of these benzodiazepine derivatives and methods of using these derivatives or compositions in treating cognitive impairment associated with central nervous system (CNS) disorders.

Description

This application claims priority to U.S. Provisional Applications 61/919390, filed December 2013, 61/919394, filed December 20, 2013, and 62/075743, filed November 5, 2014. Each of the prior applications is incorporated herein by reference in its entirety.

The invention was made with the support of US Government Grant No. U01 AG041140 issued by the US Government to the National Institutes of Health (NIH) and, in particular, its division the National Institute on Aging (NIA). The US government has the rights to this invention.

Field of invention

The invention relates to compounds, compositions and methods for the treatment of a cognitive impairment associated with disorders of the central nervous system (CNS) in a patient in need of treatment or at risk of developing said cognitive impairment.

State of the art

Cognitive abilities may decline as a normal consequence of aging or as a result of a disorder of the central nervous system.

For example, a significant population of older people experience cognitive decline that is greater than typical with normal aging. This age-related cognitive loss is characterized by a clinically progressive loss of memory, cognition, reasoning, and judgment. Mild cognitive impairment (MCI), age-related memory impairment (AAMI), age-related cognitive decline (ARCD) or similar clinical groups can be named among those associated with such age-related cognitive loss. According to some estimates, there are more than 16 million people with AAMI in the US alone (Barker et al., 1995), and MCI is estimated to affect 5.5-7 million in the US by age 65 (Plassman et al., 2008) .

Cognitive impairment is also associated with other central nervous system (CNS) disorders such as dementia, Alzheimer's disease (AD), prodromal AD, post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder (particularly mania), amyotrophic lateral sclerosis (ALS ), cognitive impairment associated with cancer therapy, mental retardation, Parkinson's disease (PD), autism spectrum disorders, fragile X syndrome, Rett syndrome, compulsive behavior and drug addiction.

Therefore, there is a need for effective treatment of cognitive impairment associated with disorders of the central nervous system (CNS) and improvement of cognitive function in patients diagnosed with, for example, age-related cognitive impairment, MCI causing memory impairment MCI, AAMI, ARCD, dementia, AD , prodromal AD, PTSD, schizophrenia or bipolar disorder (particularly mania), amyotrophic lateral sclerosis (ALS), cancer therapy-related cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorder, fragile X syndrome, syndrome Retta, compulsive behavior and drug dependence and similar disorders of the central nervous system (CNS) with cognitive impairment, or for which there is a risk of developing them.

GABAA receptors (GABAA R) are pentameric constructs composed of a number of different subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ) that form a Cl-permeable channel that is gated by the neurotransmitter γ-aminobutyric acid (GABA). Various pharmacological effects, including anxiety disorders, epilepsy, insomnia, pre-anesthetic sedation, and muscle relaxation, are mediated by different GABA subtypes. Various studies have demonstrated that reduced GABA signaling is associated with various central nervous system disorders with cognitive impairment. In particular, a5-containing GABA R, which is relatively rare in the mammalian brain, plays a role in altering learning and memory. Previous studies have demonstrated a decrease in hippocampal expression of the α5 subunit of the GABA receptor in rats with age-related cognitive decline [see. international patent publication WO 2007/019312). These results suggest that upregulation of α5-containing GABA R function may be effective in the treatment of cognitive impairment associated with these central nervous system disorders.

Thus, there is a need for positive allosteric modulators of α5-containing GABA R that can be used in therapeutic formulations for the treatment of cognitive impairment associated with these disorders of the central nervous system.

The essence of the invention

The present invention meets the above need in the form of a compound of formula II

- 1 039381

or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer, or Z-isomer thereof, or a combination thereof, wherein m is an integer selected from 0-1;

R ¹ is absent or selected from halogen, (C1-C ₁₂ )-aliphatic radical-, -O-(C1-C ₁₂ )aliphatic radical, -OCF3 or -CF ₃ ;

R ² is selected from halogen, -R, -C(O)OR, C(O)R, -(CH ₂ ) _1-3 -OR, -(CH ₂ ) _1-3 R, -(CH ₂ ) _{1- 3} N(R) ₂ or C(O)N(R) ₂ ;

R ³ is selected from halogen, -R, -C(O)OR, -CN, -C(O)R, -C(O)NH _2> -<CH ₂ )i. ₃ -OR or -SO2R;

R ⁴ , R ⁵ and R ⁶ are H;

each R is independently selected from

Η-, (C ₁ -C ₁₂ )αliphatic radical-, (Cb-Cu)aryl, (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical,

3-10 membered heterocyclyl or

5-10 membered heteroaryl; and where the specified heterocyclyl contains 1-4 heteroatoms, independently selected from N or O, and the specified heteroaryl contains 1-4 heteroatoms, independently selected from N or O;

where in each case R is independently substituted with 0-5 R';

or if two R groups are attached to the same atom, the two R groups together with the atom to which they are attached may form a 3-10 membered aromatic or non-aromatic ring containing 0-1 heteroatoms independently selected from N or O, wherein: the ring is optionally substituted with 0-5 R' where in each occurrence R' is independently selected from halogen, -R'', -OR'' , -CN or -CF ₃ ;

where in each case R'' is independently selected from H, -(C ₁ -C ₆ )alkyl, (C ₆ -C ₁₀ )aryl or (C ₆ -C ₁₀ )aryl-(C1C ₁₂ )aliphatic radical.

Some embodiments of this application relate to a compound of formula III

or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer or a combination thereof, wherein m is an integer selected from 0-1;

R ¹ is selected from halogen, (C ₁ -C ₁₂ )aliphatic, -O-(C ₁ -C ₁₂ )aliphatic and -OCF3;

R ² is -(CH2)i- ₃ R or -C(O)OR;

R ³ is -C(O)OR;

R4, R ⁵ and R ⁶ are H;

each R is independently selected from

H-, (C1-C ₁₂ )aliphatic radical or (C6-Cu)aryl.

In another aspect, the present invention relates to a compound of formula IV

- 2 039381

IV or to its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which either a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which

R ¹ is selected from halogen, (C ₁ -C ₁₂ )aliphatic-, -O-(C ₁ -C ₁₂ )aliphatic or -OCF3;

R ² is selected from halo, -OR ⁸ , -SR ⁸ , -(CH2)nOR ⁸ , or -(CH ₂ ) _n O(CH ₂ ) _n R ⁸ , where R ² is independently substituted with 05 R';

R ³ is selected from halogen, -R, -OR, -CN, -(C^^R, -(CH2)i-3-OR, C(O)OR, C(O)R or C(O)NH2;

R ⁴ , R ⁵ and R ⁶ are H;

each R ⁸ is independently -(C1-C ₆ )alkyl, -(C ₃ -C ₁₀ )cycloalkyl, (C ₆ -C ₁₀ )aryl, or 5-10 membered heteroaryl, wherein said (C ₆ -C ₁₀ )aryl is optionally fused with (C ₆ -C ₁₀ )aryl and where each R ⁸ is independently substituted with 0-5 R';

m and n are independently integers selected from 0-1;

each R is independently selected from

H-, (C1-C ₁₂ ) aliphatic radical, (C ₆ -C ₁₀ ) aryl-(C ₁ -C ₁₂ ) aliphatic radical,

3-10 membered heterocyclyl or

5-10 membered heteroaryl;

where the specified heterocyclyl contains 1-4 heteroatoms, independently selected from N or O;

where the specified heteroaryl contains 1-4 heteroatoms, independently selected from N or O;

where each R is independently substituted with 0-5 R';

where each R' is independently selected from halogen, -R", -OR'', -CF ₃ or -OCF ₃ ;

where each R'' is independently selected from H, -(C ₁ -C ₆ )alkyl or (C ₆ -C ₁₀ )aryl.

The present invention also relates to pharmaceutical compositions useful in the treatment of cognitive impairment associated with disorders of the Central nervous system (CNS), comprising the compound of formulas II, III or IV or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in a therapeutically effective amount; and an acceptable carrier, adjuvant or vehicle.

In some embodiments, the compounds of Formula II are positive allosteric modulators of the GABAA α5 receptor. In some embodiments, the compounds of formula III are positive allosteric modulators of the GABAA α5 receptor. In some embodiments, the compounds of formula IV are positive allosteric modulators of the GABAA α5 receptor. The compounds of formula II, III or IV can be used to treat the conditions described here, for example, through the activity of positive allosteric modulators of the GABAA α5 receptor.

In another aspect, the invention relates to a method of treating a cognitive impairment associated with a central nervous system disorder in a patient in need of treatment, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer, or Z isomer, or combinations thereof. In some embodiments, the central nervous system disorder is (1) an age-related cognitive impairment;

(2) mild cognitive impairment (MCI) or amnesic mild cognitive impairment (AMCI);

(3) dementia or Alzheimer's disease;

(4) schizophrenia or bipolar disorder;

(5) amyotrophic lateral sclerosis (ALS);

(6) post-traumatic stress disorder (PTSD);

(7) nervous system disorder associated with cancer therapy;

(8) mental retardation;

(9) Parkinson's disease (PD);

- 3 039381 (10) autism;

(11) compulsive behavior or (12) drug addiction.

Detailed description of the figures

Fig. 1 is a graph depicting the effects of methyl 3,5-diphenylpyridazine-4-carboxylate administration on spatial memory retention of ten age-related (AI) rats in the radial eight-arm maze (RAM) test. Black bars refer to rats treated with vehicle alone; open bars refer to rats treated with methyl 3,5diphenylpyridazine-4-carboxylate at various doses; the shaded bar refers to rats treated with the combination of TB21007 and methyl 3,5-diphenylpyridazine-4-carboxylate.

Fig. 2 is a graph showing the effect of methyl 3,5-diphenylpyridazine-4-carboxylate (introduced intravenously) on Ro154513 binding in the hippocampus and cerebellum. Methyl 3,5diphenylpyridazine-4-carboxylate blocked Ro154513 binding in the hippocampus but had no effect on Ro15413 binding in the cerebellum.

Fig. 3 is a graph showing dose-dependent occupancy of the α5 GABAA receptor by intravenous methyl 3,5-diphenylpyridazine-4-carboxylate, with receptor occupancy determined either by the ratio between exposure of the hippocampus (an area of high density of α5 GABA receptors) to RO 15-4513 exposure and cerebellar exposure ( an area of low density of GABAA α5 receptors) to RO 15-4513, or using GABAA α5 selective compound L-655708 (10 mg/kg, iv) to determine complete filling.

Fig. 4 is a graph showing exposure-fill relationships for methyl 3,5-diphenylpyridazine-4-carboxylate in the hippocampus. Methyl 3,5-diphenylpyridazine-4-carboxylate occupies approximately 32% of the GABAA α5 receptors at exposures that are behaviorally active in aging rats.

Fig. 5 is a graph depicting the effect of ethyl 3-methoxy-7-methyl-9H-benzo[!]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4] diazepine-10-carboxylate on the retention of spatial memory in ten rats with age-related changes (AI) in the test of the radial eight-armed maze (RAM). Fig. 5 shows the effect of ethyl 3-methoxy-7-methyl-9H-benzo[G]imidazo[1,5-a][1,2,4]triazolo[4,3d][1,4]diazepine-10-carboxylate on the retention of spatial memory of ten rats with age-related changes (AI) in the RAM test, where the vehicle control was tested 3 times, and various doses of ethyl-3-methoxy-7-methyl-9H-benzo[f]imidazo[1.5 -a][1,2,4]triazolo[4,3-d][1,4]diazepine-10-carboxylate were double checked; in fig. 5, black bars refer to rats treated with vehicle alone and open bars refer to rats treated with ethyl 3-methoxy-7-methyl-9Hbenzo[f]imidazo[1,5-a][1,2,4] triazolo[4,3-d][1,4]diazepine-10-carboxylate in various doses.

Fig. 6 is a graph showing the effect of ethyl 3-methoxy-7-methyl-9H-benzo[G]imidazo[1,5a][1,2,4]triazolo[4,3-d][1,4]diazepine- 10-carboxylate (introduced intravenously) on Ro154513 binding in the hippocampus and cerebellum. Ethyl 3-methoxy-7-methyl-9H-benzo[G]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4]diazepine-10-carboxylate blocked Ro154513 binding in the hippocampus but had no effect on Ro15413 binding in the cerebellum.

Fig. 7 is a graph showing dose-dependent occupancy of the α5 GABAA receptor ethyl-3methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1, 4]diazepine-10-carboxylate administered intravenously, calculated from the ratio between the exposure of the hippocampus (area of high density of the GABA α5 receptor) to RO 15-4513 and the exposure of the cerebellum (area of low density of the GABA α5 receptor) to RO 15-4513 to determine the total filling.

Fig. 8(A)-(C) are graphs showing the effect of 6,6-dimethyl-3-(3-hydroxypropyl)thio1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4(5H) -one versus vehicle dimethyl sulfoxide (DMSO) in aging-impaired rats using a behavioral task in the Morris water maze. Fig. 8(A) shows the rescue waiting time (i.e., the average time in rats in seconds it took to find the hidden platform in the water pool) during training in 6,6-dimethyl-3-(3-hydroxypropyl)thio-treated rats. -1 -(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one, and rats treated with DMSO vehicle; fig. 8(B) shows the amount of time spent in the target ring and opposite ring by rats treated with 6,6-dimethyl-3-(3-hydroxypropyl)thio-1(thiazol-2-yl)-6,7-dihydro-2- benzothiophen-4(5H)-one, and rats treated with DMSO vehicle; fig. 8(C) shows the number of crossings in the target ring and opposite ring in rats treated with 6,6dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4 (5H)-one, and rats treated with DMSO vehicle.

Detailed description of the invention

Definitions

Unless otherwise indicated, scientific and technical terms used in this application should have the meanings commonly understood by a person skilled in the art. Generally, the nomenclature and methods used in connection with chemistry, cell and tissue culture, molecular biology, cell biology and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry, described here are known and commonly used in the art.

Methods and technologies according to the present invention are usually carried out, unless otherwise indicated, according to standard methods known in the art and as described in various general and more specialized references cited and discussed throughout the description. See, for example, Principles of Neural Science, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, Intuitive Biostatistics, Oxford University Press, Inc. (1995); Lodish et al., Molecular Cell Biology, 4th ed., W. H. Freeman & Co., New York (2000); Griffiths et al., Introduction to Genetic Analysis, 7th ed., W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., Developmental Biology, 6th ed., Sinauer Associates, Inc., Sunderland, MA (2000).

Chemical terms used herein are used according to standard usage in the art, as illustrated by The McGraw-Hill Dictionary of Chemical Terms, Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

All publications, patents and published patent applications mentioned in this application are specifically incorporated into the present description by reference. In case of conflict, the present description, including its partial definitions, shall prevail.

Throughout the description, the word includes, or variations such as includes or including, implies the inclusion of a specified integer (or components) or a group of integers (or components), but not the exclusion of any other integer (or components) or a group of integers (or components). ).

Singular forms also include plural forms, unless the context clearly indicates otherwise.

The term including is used to mean including but not limited to. Including and including, but not limited to, are used interchangeably.

The term agent is used herein to refer to a chemical compound (such as an organic or inorganic compound (including, as such, a compound of the present invention), a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof, as well as humanized , chimeric and human antibodies and monoclonal antibodies, a protein or part thereof, for example, a peptide, lipid, carbohydrate) or an extract obtained from biological materials such as bacteria, plants, fungi or animal cells or tissues (in particular, mammals). Agents include, for example, agents that are known with respect to their structure and those that are not known with respect to their structure. The α5-GABAA receptor agonist activity of such agents may make them suitable for use as therapeutic agents in the methods and compositions of the invention.

Patient, person, or person are used interchangeably and refer to a person or animal. These terms include mammals such as humans, primates, livestock (including cattle, pigs, etc.), domestic animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats) .

Cognitive function or cognitive status refers to any higher-order intellectual brain process or brain state, as appropriate, involved in learning and/or memory, including, but not limited to, attention, information gathering, information processing, working memory, short-term memory, long-term memory. , anterograde memory, retrograde memory, memory retrieval, differential learning, decision making, inhibitory response control, attentional shift, delayed reinforcement of learning, skill rework, temporal integration of voluntary behavior, expression of interest in environment and self-help, processing speed, reasoning and problem solving and social cognition.

In humans, cognitive function can be measured, for example and without limitation, by assessing change on a global clinical impression scale (CIBIC-plus scale); Mini Mental State Assessment (MMSE); using the neuropsychiatric questionnaire (NPI); clinical dementia rating scale (CDR); the Cambridge Automated Neuropsychological Test Battery (CANTAB); Sandoz Geriatric Clinical Assessment Scale (SCAG), Buschke Selective Memory Test (Buschke & Fuld, 1974); an additional criterion for associations of verbal pairs; additional criterion of logical memory; an additional criterion for visual reproduction of the revised Wechsler Memory Scale (WMS-R) (Wechsler, 1997); a Benton visual retention test or a 3-alternative explicit forced choice task or a consensus neuropsychological test with a MATRICS battery. See Folstein et al., J Psychiatric Res 12: 189-98, (1975); Robbins et al., Dementia 5: 266-81, (1994); Rey, L'examen clinique en psychologie, (1964); Kluger et al., J Geriatr Psychiatry Neurol 12:168-79, (1999); Marquis et al., 2002 and Masur et al., 1994. See also Buchanan, R.W., Keefe, R.S.E., Umbricht, D., Green, M.F., Laughren, T., and Marder, S.R. (2011), The FDA-NIMH-MATRICS guidelines for clinical trial design of cognitive-enhancing drugs: what do we know 5 years later? Schizophr. Bull. 37, 1209-1217.

In animal model systems, cognition can be measured by a variety of standard methods known in the art, including using the Morris Water Maze (MWM), the Barnes Ring Maze, the Elevated Radial Eight-Arm Maze, the T Maze, or any other mazes in which animals use spatial information. Cognitive function can be assessed by skill rework, extradimensional shift, conditional differential learning, and reward expectation assessment. Other tests known in the art may also be used to assess cognitive function, such as novel object recognition and scent recognition tasks.

Cognitive function can also be measured using imaging techniques such as positron emission tomography (PET), functional magnetic resonance imaging (fMRI), single photon emission computed tomography (SPECT), or any other imaging technique that measures brain function. In animals, cognitive function can also be measured by electrophysiological methods.

Promotion of cognitive function refers to the effect on reduced cognitive function in such a way that it more closely resembles the function of a normal patient without impairment. Cognitive function may be promoted to any detectable degree, but in humans preferably sufficiently promoted to enable the impaired patient to perform the daily activities necessary for a normal life at a level of proficiency as close as possible to that of a normal, unimpaired patient or a normal, unimpaired patient. age.

In some cases, the promotion of cognitive function in a patient with age-related cognitive impairment refers to the effect on reduced cognitive function so that it more closely resembles the function in a normal patient without age-matched impairments or the function of a young adult patient. This patient's cognition may be promoted to any detectable degree, but preferably sufficiently promoted in a human to allow the impaired patient to perform the daily activities necessary for a normal life at a level of proficiency as close as possible to that of a normal, unimpaired patient or a young adult patient. , or a normal patient with no age-appropriate impairments.

Preservation of cognitive function refers to the effect on normal or impaired cognitive function so that it does not decline or fall below that observed in the patient relative to the first presentation or diagnosis, or to delay such decline.

Improving cognitive function includes promoting cognitive function and/or maintaining cognitive function in a patient.

Cognitive impairment refers to cognitive function in patients that is not as strong as expected in a normal patient without impairment. In some cases, cognitive function is reduced by about 5%, about 10%, about 30% or more compared to the cognitive function expected in a normal patient without impairment. In some cases, cognitive impairment in patients with age-related cognitive impairment refers to cognitive function in patients that is not as strong as expected in a normal patient without impairment of the corresponding age or in a young adult patient (i.e., patients with an average score for a given age). age in a cognitive test).

Age-related cognitive impairment refers to the deterioration of cognitive function in patients at an age at which their cognitive function is not as strong as expected in a normal patient without age-related impairment or as expected in young adult patients. In some cases, cognitive function is reduced by about 5%, about 10%, about 30% or more compared to the cognitive function expected in a normal age-matched patient. In some cases, cognitive function is at the same level as expected in a normal patient of the corresponding age, but reduced by about 5%, about 10%, about 30%, about 50% or more compared to the cognitive function expected in young adult patient. Age-related cognitive decline may be associated with mild cognitive impairment (MCI) (including memory-impairing MCI and non-memory-impairing MCI), age-related memory impairment (AAMI), and age-related cognitive decline (ARCD).

AD-associated or AD-related cognitive impairment or in AD refers to cognitive function in patients that is not as strong as expected in patients in whom AD has not been diagnosed using conventional techniques and standards.

Mild Cognitive Impairment, or MCI, refers to a condition characterized by isolated memory impairment unaccompanied by other cognitive pathologies and with relatively normal functional abilities. One set of MCI clinical performance criteria defines the following characteristics:

(1) memory complaints (as reported by the patient, caregiver, or clinician), (2) normal activities of daily living (ADLs),

- 6 039381 (3) normal general cognitive function, (4) abnormal memory for age (defined as being more than

1.5 standard deviations below the mean for age) and (5) no indicators of dementia (as defined by the DSM-IV guidelines). Petersen et al., Srch. Neurol. 56: 303-308 (1999); Petersen, Mild cognitive impairment: Aging to Alzheimer's Disease. Oxford University Press, N.Y. (2003). Cognitive impairment in patients with MCI may involve any area of cognition or mental activity, including memory, language, association, attention, perception, problem solving, executive function, and visuospatial skills. See, for example, Winbald et al., J. Intern. Med. 256: 240-240, 2004; Meguro, Acta. Neurol. Taiwan. 15:55-57, 2008; Ellison et al., CNS Spectr. 13:66-72, 2008, Petersen, Semin. Neurol. 27:22-31, 2007. MCI is further subdivided into memory-impairing MCI (aMCI) and non-memory-impairing MCI, characterized in particular by impairment (or lack) of memory. MCI is defined as aMCI if memory is impaired for a given age and educational level of the patient. If, on the other hand, the patient's memory is intact for a given age and education, but other, non-memory cognitive areas such as language, executive function, or visuo-spatial skills are impaired, MCI is defined as non-memory-impairing MCI. Both aMCI and non-memory damaging MCI can be further subdivided into single or multiple region MCI. aMCI with a single area refers to a state in which memory, but not other cognitive areas, is impaired. aMCI with multiple areas refers to a state in which memory and at least one other cognitive area are impaired. A non-memory-impairing MCI is one with a single area or multiple areas, depending on whether more than one non-memory cognitive area is impaired. See, for example, Peterson and Negash, CNS Spectr. 13:45-53, 2008.

The diagnosis of MCI usually entails an objective assessment of cognitive impairment, which can be collected using well-known neuropsychological tests, including the Mini Mental Status Assessment Scale (MMSE), the Cambridge Automated Neuropsychological Test Battery (CANTAB), and individual tests such as the Ray test for auditory learning. (AVLT), the Revised Wechsler Memory Scale (WMS-R) Supplemental Visual Reproduction Test, and the New York University (NYU) Paragraph Memory Test. See Folstein et al. , J Psychiatric Res 12: 189-98 (1975); Robbins et al., Dementia 5: 266-81 (1994); Kluger et al., J Geriatric Psychiatry Neurol 12:168-79 (1999).

Age-related memory impairment (AAMI) refers to memory decline due to aging. A patient is considered to have AAMI if he or she is at least 50 years of age and he or she meets all of the following criteria: a) the patient has experienced a decline in memory performance, b) the patient performs worse on standard memory tests compared to younger adults c) all other apparent causes of memory decline other than normal aging have been excluded (in other words, memory decline cannot be attributed to other causes such as recent heart attack or traumatic brain injury, depression, adverse reactions to treatment, Alzheimer's disease, and etc.).

Age-related cognitive decline (ARCD) refers to the declines in memory and cognitive abilities that are a normal consequence of human aging (eg, Craik & Salthouse, 1992). This is also true in virtually all mammalian species. Age-related memory decline refers to older people with objective memory declines relative to their younger years, but whose cognitive functioning is normal relative to their peers (Crook et al., 1986). Age-appropriate memory loss is a less pejorative stigma, emphasizing that these are normal changes (Crook, 1993; Larrabee, 1996) that are not pathophysiological (Smith et al., 1991) and rarely progress to overt dementia (Youngjohn & Crook, 1993) . DSM-IV (1994) encoded the diagnostic classification of ARCD.

Dementia refers to a condition characterized by a severe cognitive impairment that interferes with the normal activities of daily life. Patients with dementia also show other symptoms such as decreased judgment, personality changes, disorientation, confusion, behavioral changes, speech problems, and motor impairments. There are different types of dementias such as Alzheimer's disease (AD), vascular dementia, Lewy body dementia, and frontotemporal dementia.

Alzheimer's disease (AD) is characterized by memory deficits in its early phase. Later symptoms include impaired judgment, disorientation, confusion, behavior changes, trouble speaking, and motor deficits. Histologically, AD is characterized by beta-amyloid plaques and tau protein tangles.

Vascular dementia is caused by strokes. The symptoms overlap with those of AD, but without the focus on memory impairment.

Dementia with Lewy bodies is characterized by abnormal deposits of alpha-synuclein that form internal neurons in the brain. The cognitive impairment may be similar to AD, including memory impairment and changes in behavior and judgment.

Frontotemporal dementia is characterized by gliosis, neuronal loss, superficial spongiform degeneration in the frontal cortex and/or anterior temporal lobes, and Pick's bodies. Symptoms include personality and behavioral changes, including a decrease in social skills and language expression/understanding.

Post-traumatic stress disorder (PTSD) refers to an anxiety disorder characterized by an immediate or delayed response to a catastrophic event, characterized by re-experiencing the trauma, mental numbing or avoidance of trauma-related stimuli, and increased arousal. Re-experiencing phenomena include invading memories, flashbacks, nightmares, and psychological or physiological exhaustion in response to traumatic reminders. Such reactions are worrisome and can have a significant impact, both chronic and acute, on the patient's quality of life and physical and emotional health. PTSD is also associated with reduced cognitive performance, and older adults with PTSD show a greater decline in cognitive performance relative to control patients.

Schizophrenia refers to a chronic, severe disorder characterized by a spectrum of psychopathologies, including positive symptoms such as aberrant or distorted mental representations (eg, hallucinations, delusions), negative symptoms characterized by decreased motivation and adaptive goal-directed action (eg, anhedonia, affective dullness, abulia ), and cognitive impairment. Although it has been suggested that pathologies in the brain underlie the full range of psychopathologies in schizophrenia, currently available antipsychotics are largely ineffective in treating cognitive impairment in patients.

Bipolar disorder or BP or, manic depressive disorder or manic depressive disorder refers to a chronic psychological/mood disorder that can be characterized by significant mood changes, including periods of depression and euphoric manic periods. VR can be diagnosed by a physician based on a life and medical history, consultation, and physical examinations. The term mania or manic periods, or other variants, refers to periods in which an individual exhibits some or all of the following characteristics: racing of ideas, rapid speech, increased levels of activity and arousal, and inflated self-esteem, euphoria, inability to reason, insomnia , reduced concentration and aggression.

Amyotrophic Lateral Sclerosis, also known as ALS, refers to a progressive, fatal neurodegenerative disease characterized by the degeneration of motor neurons, the nerve cells in the central nervous system that control voluntary muscle contractions. ALS is also characterized by neuronal degeneration in the entorhinal cortex and hippocampus, memory deficits, and neuronal hyperexcitability in various areas of the brain such as the cortex.

Cancer therapy-associated cognitive impairment refers to the deterioration in cognitive function that develops in patients undergoing cancer treatment with therapies such as chemotherapy and radiation. Cytotoxicity and other adverse brain side effects of cancer therapies result in cognitive impairment in functions such as memory, learning and attention.

Parkinson's disease (PD) is a neurological disorder characterized by a decrease in voluntary movements. The affected patient exhibits reduced motor activity and slower voluntary movements compared to the normal individual. The patient has a characteristic mask-like face, a tendency to haste when walking, a forward-leaning posture, and generalized muscle weakness. There is typical rigidity in the form of a lead pipe of passive movements. Another important characteristic of this disease is the tremor of the extremities, observed at rest and decreasing with movement.

Autism, within the meaning of the invention, refers to an autism spectrum disorder characterized by a neurodevelopmental disorder resulting in reduced social interaction and communication due to restricted and repetitive behaviour. Autism Spectrum Disorder refers to a group of developmental disorders including autism; Asperger's syndrome; pervasive developmental disorder not otherwise defined (PDD-NOS or atypical autism); Rett syndrome; and disintegrative disorder of childhood.

Mental retardation is a generalized disorder characterized by significantly reduced cognitive function and deficits in adaptive behaviors. Oligophrenia is often defined as an intelligence quotient (IQ) of less than 70. Congenital causes are among the many underlying causes of mental retardation. Dysfunction in neural communication is also considered one of the root causes of mental retardation (Myrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214).

In some cases, mental retardation includes, but is not limited to, Down syndrome, velocardiofacial syndrome, fetal alcohol syndrome, fragile X syndrome, Klinefelter syndrome, neurofibromatosis, congenital hypothyroidism, Williams syndrome, phenylketonuria (PKU), Smith-Lemli-Opitz syndrome, Prader-Willi syndrome, Phelan-McDermid syndrome, Mowat-Wilson syndrome, kiliopathy, Lowe's syndrome and sidereal-type fragile X chromosome syndrome. Down syndrome is a disorder involving a combination of birth defects, including some degree of mental retardation, facial features and often heart defects, increased infections, vision and hearing problems, and other health problems. Fragile X syndrome is a common form of hereditary mental retardation, occurring in 1 in 4,000 men and 1 in 8,000 women. This syndrome is also characterized by developmental delay, hyperactivity, attention deficit disorder, and autistic behavior. There is no effective treatment for fragile X syndrome.

Obsessive-compulsive disorder (OCD) is a mental condition typically characterized by invading, repetitive unwanted thoughts (obsessions) ending in compulsive behaviors and mental acts that the person feels compelled to commit (compulsion). Current epidemiological data indicate that OCD is the fourth most common psychiatric disorder in the US. Some studies suggest that the prevalence of OCD is between one and three percent, despite the fact that the prevalence of clinically recognized OCD is much lower, suggesting that in many people this disorder cannot be diagnosed. Patients with OCD are often diagnosed by a psychologist, psychiatrist, or psychoanalyst according to the diagnostic criteria defined in the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Revised (DSM-IV-TR) (2000), which includes characteristics of obsession and compulsions.

Drug addiction (eg drug addiction, alcoholism) is a mental disorder. Addiction does not occur instantly after contact with the substance of abuse. Rather, it involves multiple, complex neural adaptations evolving with varying dynamics ranging from hours to days to months (Kauer J. A. Nat. Rev. Neurosci. 2007, 8, 844-858). The path to addiction usually begins with the voluntary use of one or more controlled substances, such as narcotics, barbiturates, methamphetamines, alcohol, nicotine, and any number of other such controlled substances. Over time, with increasing use of the controlled substance(s), the ability to voluntarily abstain from the controlled substance(s) decreases due to the effect of long-term use on brain function, and thus behavior. Also, addiction is usually characterized by a compulsive craving for seeking and using a substance that persists even in the face of negative consequences. Cravings may represent changes in the patient's underlying neurobiology, which likely need to be addressed in a meaningful way if recovery is to be achieved. Drug dependence is also characterized in many cases by withdrawal, which for some substances is life-threatening (e.g. alcohol, barbiturates) and for others can lead to significant morbidity (which may include nausea, vomiting, fever, dizziness and profuse sweating) , exhaustion and reduced ability to recover, for example, alcoholism, also known as alcohol dependence, is one type of such drug dependence. Alcoholism is primarily characterized by four symptoms, including cravings, loss of control, physical dependence, and tolerance. These symptoms may also characterize dependence on other controlled substances. Cravings for alcohol, as well as other controlled substances, are often as strong as the need for food or water. Thus, an alcoholic may continue to drink despite serious family, health, and/or legal consequences.

Treating a condition or patient refers to taking steps to obtain beneficial or desirable outcomes, including clinical outcomes. Useful or desirable clinical results include, but are not limited to, preventing or slowing the progression of a disease or disorder, or alleviating, alleviating, or slowing the progression of one or more symptoms of a cognitive impairment associated with disorders of the central nervous system, such as age-related cognitive impairment, mild cognitive impairment. (MCI), memory impairment MCI (aMCI), age-related memory impairment (AAMI), age-related cognitive decline (ARCD), dementia, Alzheimer's disease (AD), prodromal AD, post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis (ALS), cancer therapy-associated cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorders, fragile X syndrome, Rett syndrome, compulsive behavior and drug addiction. In some embodiments, the treatment includes preventing or slowing the progression of a central nervous system disorder (such as described herein). In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with the central nervous system disorder. In some embodiments, the symptom being treated is a cognitive impairment or a cognitive disorder. Treatment of age-related cognitive impairment further includes slowing the transformation of age-related cognitive impairment (including, but not limited to, MCI, ARCD, and AAMI) into dementia (eg, AD).

Treatment of cognitive impairment refers to taking steps to improve cognitive

- 9 039381 function in a patient with a cognitive impairment, so that the patient's performance in one or more cognitive tests is improved to any detectable degree or further decline is prevented. Preferably, the cognitive function of this patient, after treatment for cognitive impairment, more closely resembles that of a normal patient without impairment. Treatment of a human cognitive impairment may improve cognitive function to any detectable degree, but preferably improves enough to allow the impaired patient to perform activities of daily living at the same level of proficiency as a normal patient without impairment. In some cases, the treatment of a cognitive impairment refers to steps taken to improve cognitive function in a patient with a cognitive impairment so that the patient's performance on one or more cognitive tests is improved to any detectable degree or further decline is prevented. Preferably, the cognitive function of this patient, after treatment for cognitive impairment, more closely resembles that of a normal patient without impairment. In some cases, the treatment of a cognitive impairment in a patient suffering from age-related cognitive impairment refers to taking steps to improve the patient's cognitive function so that the patient's cognitive function, after treatment of the cognitive impairment, more closely resembles that of a normal patient without age-appropriate impairment or that of a young adult. patient.

Administration of a substance, compound, or agent to a patient may be by one of a variety of methods known to those skilled in the art. for example, the compound or agent may be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, through the eye, sublingually, orally (by swallowing), intranasally (by inhalation), intraspinally, intracerebral, and transdermally (by absorption, e.g., through the cutaneous duct ). The compound or agent may also suitably be administered by rechargeable or biodegradable polymeric devices or other devices, such as patches and pumps, or formulations providing extended, sustained or controlled release of the compound or agent. The introduction can also be carried out, for example, once, repeatedly and/or over one or more long periods. In some aspects, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug, for example, within the scope of the invention, a doctor directing a patient to self-administer a drug or to take a drug with the help of others and/or who provides a prescription for a drug to a patient is an administration of a drug to the patient.

Suitable routes for administering a substance, compound, or agent to a patient also depend on, for example, the age of the patient, whether the patient is active or inactive at the time of administration, whether the patient has cognitive impairment at the time of administration, the degree of impairment, and the chemical and biological properties of the compound or agent (e.g., , solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, the compound or agent is administered to a patient orally, eg, by ingestion, or intravenously, eg, by injection. In some embodiments, the orally administered compound or agent is in a sustained release or sustained release formulation, or is administered using such a sustained or sustained release device.

Within the scope of the invention, an α5-containing GABA receptor agonist, an α5-containing GABA R agonist, or an α5 GABA receptor agonist and other variations within the scope of the invention refers to a compound that improves the function of an α5-containing GABA receptor (GABA R), i.e., a compound that increases GABA-dependent Cl ^- currents. In some embodiments, an α5 GABA R agonist within the meaning of the invention refers to a positive allosteric modulator that potentiates GABA activity. Suitable α5-GABA receptor agonists suitable for use in the present invention include α5-GABA receptor agonists of all formulas and specific α5-GABA receptor agonists described herein and their hydrates, solvates, polymorphs, salts (e.g., pharmaceutically acceptable salts ), isomers (eg, stereoisomers, E/Z isomers, and tautomers), and combinations thereof.

An antipsychotic, an antipsychotic agent, an antipsychotic, or an antipsychotic compound refers to (1) a typical or atypical antipsychotic; (2) an agent selected from dopaminergic agents, glutamatergic agents, NMDA receptor positive allosteric modulators, glycine reuptake inhibitors, glutamate reuptake inhibitor, metabotropic glutamate receptor agonists (mGluRs), or positive allosteric modulators (PAMs) (e.g., mGluR ² /Agonists) 3 or PAMs), glutamate receptor glur5 positive allosteric modulators (PAMs), muscarinic M1 acetylcholine receptor (mAChR) positive allosteric modulators (PAMs), histamine H3 receptor antagonists, AMPA/kainate receptor antagonists, ampakines (CX-516), glutathione prodrugs, noradrenergic agents, serotonin receptor modulators, cholinergic agents, cannabinoid CB1 antagonists, neurokinin 3 antagonists, neurotensin agonists, MAO B inhibitors, PDE10 inhibitors, nNOS inhibitors, neurosteroids and neurotrophic factors, alpha-7 agonists or allosterically positive x modulators (PAMs) PAMs, serotonin 2C agonists; and/or (3) an agent that can be used in the treatment of one or more of the signs or symptoms of schizophrenia or bipolar disorder (particularly mania).

Exemplary antipsychotics, within the scope of the invention, refer to standard antipsychotics having antipsychotic effects as well as movement-related side effects that are associated with disturbances in the nigrostrial dopamine system. These extrapyramidal side effects (EPS) include Parkinsonism, akathisia, tardive dyskinesia, and dystonia. See Baldessarini and Tarazi in Goodman & Gilman's The Pharmacological Basis of Therapeutics 10 Edition, 2001, pp. 485-520.

Atypical antipsychotics, as used herein, refer to antipsychotics that have antipsychotic effects with minimal or no EPS and include, but are not limited to, aripiprazole, asenapine, clozapine, iloperidone, olanzapine, lurasidone, paliperidone, quetiapine, risperidone, and ziprasidone. Atypical antipsychotics differ from standard antipsychotics in their pharmacological profiles. While standard antipsychotics are characterized predominantly by blockade of the dopamine D ₂ receptor, atypical antipsychotics show multiple receptor antagonistic effects, including the serotonin 5HT _a and 5HT _C receptors, and varying degrees of receptor affinity. Atypical antipsychotics are commonly referred to as serotonin/dopamine antagonists, reflecting the influential hypothesis that a greater affinity for the 5HT2 receptor than for the D2 receptor underlies the atypical action of antipsychotics or second generation antipsychotics. However, atypical antipsychotics often show side effects including, but not limited to, weight gain, diabetes (eg, type II diabetes mellitus), hyperlipidemia, QTc prolongation, myocarditis, genital side effects, extrapyramidal side effects, and cataracts. Thus, atypical antipsychotics do not represent a homogeneous class given their differences in terms of both clinical symptom relief and their potential to induce side effects such as those mentioned above. Further, the common side effects of atypical antipsychotics, as described above, often limit the antipsychotic doses that can be used for these agents.

Memantine is chemically known as 3,5-dimethyladamantan-1-amine or 3,5dimethyltricyclo[3,3,1,13,7]decan-1-amine, which is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist with moderate affinity. Proprietary names for memantine include Axura® and Akatinol® (Merz), Namenda® (Forest Laboratories), Ebixa® and Abixa® (Lundbeck) and Memox® (Unipharm). Memantine has been approved in the US for the treatment of moderate to severe Alzheimer's disease (AD) at doses up to 28 mg/day. Derivatives or analogues of memantine, including compounds structurally or chemically similar to memantine, can also be used in the present invention. Such memantine derivatives or analogues include, but are not limited to, the compounds disclosed in US Pat. Nos. 3,391,142; 4122193; 4273774 and 5061703; publications of US patent applications US 20040087658, US 20050113458, US 20060205822, US 20090081259, US 20090124659 and US 20100227852; publication of patent application EP 2260839A2; EP 1682109B1 and patent application WO 2005079779, all of which are incorporated herein by reference. Memantine, within the scope of the present invention, includes memantine and its derivatives and analogs, as well as its hydrates, polymorphs, prodrugs, salts and solvates. Memantine, within the scope of the invention, also includes a composition comprising memantine, or a derivative or analog thereof, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, or prodrug, wherein the composition optionally further comprises at least one additional therapeutic agent (such as a therapeutic agent, suitable for the treatment of central nervous system disorder or cognitive impairment associated with it). In some embodiments, a memantine composition suitable for use in the present invention comprises memantine and a second therapeutic agent that is donepezil (under the trademark Aricept).

An acetylcholinesterase inhibitor, or ACHE-I, as used herein, refers to an agent that inhibits the ability of the cholinesterase enzyme to break down the neurotransmitter acetylcholine, thereby increasing the concentration and duration of action of acetylcholine, primarily at brain synapses or neuromuscular junctions. AChE-Is that are suitable for use in this application may include, for example, subcategories of (i) reversible non-competitive inhibitors or reversible competitive inhibitors, (ii) irreversible, and (iii) quasi-irreversible inhibitors.

The term co-administration, within the meaning of the invention, means that an α5-GABAa receptor agonist (e.g., a positive allosteric modulator of the a5-GABA receptor) _and a second therapeutic agent (e.g., neuroleptic, memantine, or AChE-I), or pharmaceutically acceptable salts, hydrates, solvates, or polymorphs are administered at time intervals of no more than about 15 minutes, and in some embodiments, no more than about 10 minutes. When drugs are administered simultaneously, an α5- _GABAa receptor agonist (e.g., a positive allosteric modulator of the α5-containing receptor

- 11 039381

ΓΑΜΚ _α ) and a second therapeutic agent (eg, neuroleptic, memantine, or AChE-I), or salts, hydrates, solvates, or polymorphs thereof, may be present in the same dosage (eg, a unit dosage form comprising both an a5- α5-GABA receptor agonist (eg, a positive allosteric modulator of the α5-GABA receptor) and a second therapeutic agent (eg, antipsychotic, memantine, or AChE-I), or in discrete dosages (eg, an α5-GABA receptor agonist (eg, positive allosteric a5-containing GABA receptor modulator) or a salt, hydrate, solvate, or polymorph thereof is contained in one dosage form, and a second therapeutic agent (e.g., neuroleptic, memantine, or AChE-I), or a salt, hydrate, solvate, or polymorph thereof, is contained in another dosage form).

The term sequential administration within the meaning of the invention means that an α5-containing GABA receptor agonist (for example, a positive allosteric modulator of the α5-containing GABA receptor) and a second therapeutic agent (for example, an antipsychotic, memantine or AChE-I), or their pharmaceutically acceptable salts, hydrates, solvates , polymorphs, are administered at greater than about 15 minutes, and in some embodiments, greater than about 1 hour or up to 12-24 hours apart _. ), or a second therapeutic agent (eg, antipsychotic, memantine, or AChE-I) may be administered first. An α5-GABA _A receptor agonist (eg, a positive allosteric modulator of the α5-GABA _A receptor) and a second therapeutic agent (eg, antipsychotic, memantine, or AChE-I), or salts, hydrates, diluents, or polymorphs thereof, for sequential administration may contained in discrete dosage forms, if necessary contained in the same container or package.

A therapeutically effective amount of a drug or agent is an amount of a drug or agent that, when administered to a patient, will have the intended therapeutic effect, such as improving cognitive function in a patient, such as a patient having a cognitive impairment associated with a disorder of the central nervous system. The full therapeutic effect does not necessarily occur with the introduction of a single dose and may occur only after a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount required for a patient will depend on, for example, body size, health and age of the patient, nature and extent of cognitive impairment or other symptoms of a central nervous system disorder (such as age-related cognitive impairment, mild cognitive impairment (MCI), dementia, Alzheimer's disease (AD), prodromal AD, post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, ALS, cancer therapy-related cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorders, fragile X syndrome, Rett syndrome , compulsive behavior and drug addiction) and the therapy or combination of therapies chosen for administration and route of administration. One skilled in the art can readily determine the effective amount for a given situation by routine experimentation.

The term aliphatic within the meaning of the invention refers to straight or branched alkyl, alkenyl or alkynyl. It is understood that the alkenyl or alkynyl must have at least two carbon atoms in the aliphatic chain. Aliphatic groups typically contain from 1 (or 2) to 12 carbon atoms, for example from 1 (or 2) to 4 carbon atoms.

The term aryl in the context of the invention refers to a monocyclic or bicyclic carbocyclic aromatic ring system. Aryl within the scope of the invention includes (C ₆ -C ₁₂ )aryl. For example, aryl within the scope of the invention may be a C ₆ -C ₁₀ monocyclic or C ₈ -C ₁₂ bicyclic carbocyclic aromatic ring system. In some embodiments, the implementation of the aryl, within the scope of the invention may be (C ₆ -C ₁₀ )aryl. Phenyl (or Ph) is an example of a monocyclic aromatic ring system. Bicyclic aromatic ring systems include those in which both rings are aromatic, such as naphthyl, and systems in which only one of the two rings is aromatic, such as tetralin.

The term heterocyclic as used herein refers to a monocyclic or bicyclic non-aromatic ring system having 1 to 4 heteroatoms or groups of heteroatoms in each ring selected from O, N, NH, S, SO or SO2 in a chemically stable order. Heterocyclic within the scope of the invention includes a 3-12-membered heterocyclyl having from 1 to 4 heteroatoms independently selected from O, N, NH, S, SO or SO ₂ . For example, heterocyclic within the scope of the invention may be a 3-10 membered monocyclic or 8-12 membered bicyclic non-aromatic ring system having 1 to 4 heteroatoms or groups of heteroatoms in each ring selected from O, N, NH, S, SO, or SO ₂ in a chemically stable order. In some embodiments, a heterocyclic within the scope of the invention may be a 3-10 membered heterocyclyl having 1 to 4 heteroatoms independently selected from O, N, NH, S, SO, or SO2. In the heterocyclyl variant, which is a bicyclic non-aromatic ring system, one or both rings may contain these heteroatoms or groups of heteroatoms. In another bicyclic variant of hetero-cyclyl, one of the two rings may be aromatic. In yet another embodiment of the heterocyclic ring system, the non-aromatic heterocyclic ring may optionally be fused to an aromatic carbocycle.

Examples of heterocyclic rings include

3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one, 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4- morpholino, 2thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl, 2piperidinyl, 3-piperidinyl, 1-pyrazolinyl 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5- imidazolidinyl, indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiolane, benzodithian and 1,3dihydroimidazol-2-one.

The term heteroaryl in the context of the invention refers to a monocyclic or bicyclic aromatic ring system having from 1 to 4 heteroatoms or groups of heteroatoms in each ring selected from O, N, NH or S in a chemically stable order. Heteroaryl within the scope of the invention includes 5-12 membered heteroaryl having 1 to 4 heteroatoms independently selected from O, N, NH, or S. In some embodiments, heteroaryl within the scope of the invention may be 5-10 membered heteroaryl having 1 to 4 heteroatoms independently selected from O, N, NH, or S. For example, a heteroaryl within the scope of the invention may be a 5-10 membered monocyclic or 8-12 membered bicyclic aromatic ring system having 1 to 4 heteroatoms or groups of heteroatoms selected from O, N, NH, or S on one or both rings in a chemically stable order. In such a heteroaryl embodiment, which is a bicyclic aromatic ring system, both rings are aromatic and one or both rings may contain the indicated heteroatoms or groups of heteroatoms.

Examples of heteroaryl rings include 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5- oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g. 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl , 5-thiazolyl, tetrazolyl (for example, 5-tetrazolyl), triazolyl (for example, 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, benzofuryl, benzothiophenyl, indolyl (for example, 2-indolyl), pyrazolyl (for example, 2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3triazolyl, 1,2,3-thiadiazolyl, 1,3,4 -thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl, 1,3,5triazinyl, quinolinyl (e.g. 2-quinolinyl, 3-quinolinyl, 4-quinolinyl) and isoquinolinyl (e.g. 1-isoquinolinyl, 3-isoquinolinyl or 4-isoquinolinyl).

The term cycloalkyl or cycloalkenyl refers to a monocyclic or fused or bridged bicyclic carbocyclic ring system that is not aromatic. For example, cycloalkyl or cycloalkenyl within the scope of the invention may be a C ₃ -C ₁₀ monocyclic or fused or bridged C ₈ -C ₁₂ bicyclic carbocyclic ring system that is not aromatic. Cycloalkenyl rings have one or more unsaturated units. Preferred cycloalkyl or cycloalkenyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, norbornyl, adamantyl and decalinyl.

Within the scope of the invention, the designations for the carbon atom may have a designated integer and any intermediate integer. For example, the number of carbon atoms in a (C1-C ₄ )alkyl group is 1, 2, 3, or 4. It should be understood that these designations refer to the total number of atoms in the respective group. For example, in (C ₃ -C ₁₀ )heterocyclyl, the total number of carbon atoms and heteroatoms is 3 (as in aziridine), 4, 5, 6 (as in morpholine), 7, 8, 9 or 10.

A pharmaceutically acceptable salt within the meaning of the invention refers to an agent or compound of the invention which is a therapeutically active, non-toxic salt form of the compounds with base and acid. The acid addition salt form of a compound which in its free form is a base can be obtained by treating said free base form with a suitable acid such as an inorganic acid, for example a hydrohalic acid such as hydrochloric or hydrobromic, sulfuric, nitric, phosphoric, etc. P.; or an organic acid such as, for example, acetic, hydroxyacetic, propionic, lactic, pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclic, salicylic, p-aminosalicylic , pamovoy, etc. See, for example, WO 01/062726.

Compounds containing acidic protons can be converted to their therapeutically active, non-toxic base addition salt form, eg metal or amine salts, by treatment with suitable organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali metal and alkaline earth metal salts, for example, lithium, sodium, potassium, magnesium, calcium salts, and the like, salts with organic bases, for example, Nmethyl -D-glucamine, hydrabamine salts and salts with amino acids such as, for example, arginine, lysine, and the like. On the other hand, said salt forms can be converted to free forms by treatment with a suitable base or acid.

This application covers all isomers of the compounds of formulas II-IV.

An isomer within the scope of the invention includes optical isomers (such as stereoisomers, eg enantiomers and diastereoisomers), Z (zusammen) or E (entgegen) isomers and tautomers. Many compounds that can be used in the methods and compositions of the invention have at least one stereogenic center in their structure. This stereogenic center may be present in the R or S configuration, with the R and S designations being used in accordance with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The invention also concerns all stereoisomeric forms, such as enantiomeric and diastereomeric forms of the compounds, or mixtures thereof (including all possible mixtures of stereoisomers). See, for example, WO 01/062726. In addition, some compounds containing alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each case, the invention includes both a mixture and individual isomers. A plurality of substituents on the piperidinyl or azepanyl ring may also be in cis or trans with respect to each other relative to the plane of the piperidinyl or azepanyl ring. Some compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulas described herein, are included within the scope of the present invention. With respect to the methods and compositions of the present invention, reference to a compound or compounds is intended to refer to that compound in each of its possible isomeric forms and mixtures thereof, unless a particular isomeric form is specifically mentioned. See, for example, WO 01/062726.

The compounds of the invention improve the function of α5-containing GABA _a R, i. e. they are α5-containing GABA R agonists (eg, positive allosteric modulators of the α5-containing GABA R receptor) and are able to increase GABA-dependent Cl ^- current.

The invention also relates to pharmaceutical compositions comprising one or more compounds of the invention together with a pharmaceutically acceptable carrier or excipient. In some embodiments, the implementation of the pharmaceutical compositions according to this application may additionally contain a second therapeutic agent, such as an antipsychotic, memantine or AChE-I.

The invention also relates to methods for treating cognitive impairment associated with said disorders of the central nervous system that are sensitive to positive allosteric modulators of the a5-containing GABA receptor, for example, age-related cognitive impairment, mild cognitive impairment (MCI), memory impairment MCI (aMCI), age-related memory impairment (AAMI), age-related cognitive decline (ARCD), dementia, Alzheimer's disease (AD), prodromal AD, post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis (ALS), cognitive impairment associated with therapy for cancer, mental retardation, Parkinson's disease (PD), autism spectrum disorders, fragile X syndrome, Rett syndrome, compulsive behavior and drug addiction. In some embodiments, the method is a method of treating age-related cognitive impairment, mild cognitive impairment (MCI), memory impairment MCI (aMCI), age-related memory impairment (AAMI), age-related cognitive decline (ARCD), dementia, Alzheimer's disease (AD), prodromal AD, post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis (ALS), cancer therapy-related cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorders, fragile X syndrome, Rett syndrome, compulsive behavior and drug addiction. In some embodiments, the implementation of the treatment includes preventing or slowing down the development of disorders of the central nervous system, as described here (such as those described here). In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with the central nervous system disorder. In some embodiments, the symptom being treated is a cognitive impairment or a cognitive disorder.

Various central nervous system disorders with cognitive impairment (eg, age-related cognitive impairment, mild cognitive impairment (MCI), MCI memory impairment (aMCI), age-related memory impairment (AAMI), age-related cognitive decline (ARCD), dementia, Alzheimer's disease ( AD), prodromal AD, post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis (ALS), cancer therapy-related cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorders, fragile X syndrome, Rett syndrome, compulsive behavior and drug addiction) can have many etiologies. However, the symptom of cognitive impairment in each of the aforementioned disorders may have overlapping causes. Thus, a composition or method of treatment that treats a cognitive impairment in one disorder

- 14 039381 of the central nervous system, can also treat cognitive impairment in others.

Benzodiazepine derivatives

The present invention relates to a compound of formula II

or to its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which m is an integer selected from 0-1;

R ¹ is absent or selected from halogen, (C1-C ₁₂ )aliphatic radical-, -O-(C1C ₁₂ )aliphatic radical, -OCF3 or -CF ₃ ;

R ² is selected from halogen, -R, -C(O)OR, C(O)R, -(CH2)1.3-OR, -(CH2)1-3R, -(CHDzXfb or C(O)N( R)2;

R ³ is selected from halogen, -R, -C(O)OR, -CN, -C(O)R, -C(O)NH2, -(CH2)1. ₃ -OR or -SO2R;

R ⁴ , R ⁵ and R ⁶ are H;

each R is independently selected from

H-, (C ₁ -C ₁₂ ) aliphatic radical, (Cb-Cu) aryl, (C ₆ -C ₁₀ ) aryl-(C ₁ -C ₁₂ ) aliphatic radical,

3-10 membered heterocyclyl or

5-10 membered heteroaryl; and where the specified heterocyclyl contains 1-4 heteroatoms, independently selected from N or O, and the specified heteroaryl contains 1-4 heteroatoms, independently selected from N or O;

where in each case R is independently substituted with 0-5 R';

or if two R groups are attached to the same atom, the two R groups together with the atom to which they are attached may form a 3-10 membered aromatic or non-aromatic ring containing 0-1 heteroatoms independently selected from N or O, wherein said ring optionally substituted with 0-5 R', where in each occurrence R' is independently selected from halogen, -R'', -OR'', -CN or -CF ₃ ;

where in each case R'' is independently selected from H, -(C1-C ₆ )alkyl, (C ₆ -C ₁₀ )aryl or (C ₆ -C ₁₀ )aryl-(C1C ₁₂ )aliphatic radical.

Some embodiments relate to a compound of formula III

or to its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which m is an integer selected from 0-1;

R ¹ is selected from halogen, (C1-C ₁₂ )aliphatic radical-, -O-(C1-C12)aliphatic radical and -OCF3;

R2 is -(CH2) _1-3 R or -C(O)OR;

R ³ is -C(O)OR;

R ⁴ , R ⁵ and R ⁶ are H;

each R is independently selected from

H-, (C1-C ₁₂ )aliphatic radical or (Cb-Cu)aryl.

Some embodiments relate to a compound of formula IV

- 15 039381

IV or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer, or Z-isomer thereof, or a combination thereof, wherein

R ¹ is selected from: halo, (C ₁ -C ₁₂ )aliphatic-, -O-(C ₁ -C ₁₂ )aliphatic or -OCF3;

R ² is selected from halo, -OR ⁸ , -SR ⁸ , -(CH2)nOR ⁸ , or -(CH2) _n O(CH ₂ ) _n R ⁸ , where R ² is independently substituted with 0-5 R';

R ³ is selected from halogen, -R, -OR, -CN, -(CH^R -(CH2)i-3-OR, C(O)OR, C(O)R or C(O)NH2;

R4, R ⁵ and R ⁶ are H;

each R ⁸ is independently -(O ₁ -C ₆ )alkyl, -(C ₃ -C ₁₀ )cycloalkyl, (C ₆ -C ₁₀ )aryl, or 5-10 membered heteroaryl, wherein said (C ₆ -C ₁₀ )aryl is optional fused with (C ₆ -C ₁₀ )aryl and where each R ⁸ is independently substituted with 0-5 R';

m and n are independently integers selected from 0-1;

each R is independently selected from

H-, (C1-C ₁₂ ) aliphatic radical, (C ₆ -C 1 ₀ ) aryl-(C 1-C1 ₂ ) aliphatic radical,

3-10 membered heterocyclyl or

5-10 membered heteroaryl;

where the specified heterocyclyl contains 1-4 heteroatoms, independently selected from N or O;

where the specified heteroaryl contains 1-4 heteroatoms, independently selected from N or O;

where each R is independently substituted with 0-5 R';

where each R' is independently selected from halogen, -R'', -OR'', -CF ₃ or -OCF ₃ ;

where each R'' is independently selected from H, -(C1-C ₆ )alkyl or (C ₆ -C ₁₀ )aryl.

In some embodiments, in a compound of formula II, R ² is selected from:

(1) a group consisting of halogen, -R, -(CH2) _1-3 R, -(CH ₂ ) _1-3 -OR, -CF ₃ or -C(O)OR, each R being independently substituted with 0- 5R';

(2) a group consisting of -H, -(C1-C ₆ )alkyl, -CH ₂ -O((C ₁ -C ₆ )alkyl), -CF ₃ , -C(O)O((C ₁ - C ₆ )alkyl), 310-membered heterocyclyl, (C ₆ -C ₁₀ )aryl-, 5-10-membered heteroaryl or (C ₆ -C ₁₀ )aryl-(C ₁ -C ₁₂ )aliphatic radical, and the specified alkyl , aryl or heteroaryl is independently substituted with 0-5 R';

(3) a group consisting of -H, -Me, -Et, propyl, isopropyl, butyl, tert-butyl, CF ₃ , -C(O)OMe, -C(O)OEt, -CH ₂ OMe, -CH ₂ OEt, -CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ -morpholine, -CH ₂ -pyridine or -CH ₂ Ph, said R ² being substituted with 0-3 R';

(4) -Me substituted with 0-3 R' independently selected from -R'', OR'' or -CF3, R'' being independently selected from H, -(C ₁ -C ₆ )alkyl, (C ₆ -C ₁₀ )aryl or (C ₆ -C ₁₀ )aryl-(C ₁ -C ₆ )alkyl;

(5) a group consisting of -CH ₂ Ph, -CH ₂ CH ₂ Ph, -Ph, -CH ₂ OPh, CH ₂ CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ morpholine or -CH ₂ -pyridine, wherein said Ph, pyrrolidine, pyridine or morpholine are substituted with 05 R';

(6) a group consisting of -C^Ph, -C^C^Ph, -Ph, -C^OPh, C^C^OPh, -CH2-pyrrolidine, -CH2morpholine or -CH2-pyridine, said Ph, pyrrolidine, pyridine or morpholine is substituted with 05 R', wherein said -Ph, pyrrolidine, pyridine or morpholine is substituted with 0-5 R' independently selected from -F, -Cl, -CN, -Me, -Et, -OMe or - OEt; and (7) a group consisting of -Me, -C(O)OEt, -CH ₂ Ph, -CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ -pyridine, or -CH 2 -morpholine, said -Ph, pyrrolidine, pyridine or morpholine is substituted with 0-3 R' independently selected from -F, -Me or -OMe.

In some embodiments, in a compound of formula II, III, or IV, R ¹ is independently selected from:

(1) a group consisting of halogen, -(C1-C ₁₂ )aliphatic radical- and -O(C1-C ₁₂ )aliphatic radical;

(2) a group consisting of halogen, -(C1-C ₆ )alkyl and -O((C1-C ₆ )alkyl); or (3) a group consisting of -F, -Cl, -Br, -OMe and -OEt.

In some embodiments, at least one R ¹ is (1) -O(C1C ₁₂ )aliphatic; (2) -O((C1-C ₆ )alkyl) or (3) -OMe.

In some embodiments, in a compound of formula II, R ³ is selected from:

- 16 039381 (1) a group consisting of halogen, -R, -CN, -CF ₃ , -SO2R, -C(O)R and -C(O)OR, each R being independently substituted with 0-5 R';

(2) a group consisting of -H, -F, -Br, -Cl, -(C ₁ -C6) alkyl, -CN, -CHCH, -CF3, -SO ₂ ((C ₁ -C ₆ ) alkyl) , -C(O)NH2, -C(O)((C1-C6)alkyl), -SO2(C6-C1o)aryl, -C(O)O((C _g C6)alkyl), -(C2- C6) alkenyl, -(C2C ₆ ) alkynyl, -(C ₆ -C ₁₀ ) aryl and 5-10 membered heteroaryl-, said alkyl, alkenyl, alkynyl, aryl or heteroaryl being independently substituted with 0-5 R'; or (3) a group consisting of -H, -C(O)OMe, -C(O)Et, -C(O)NH2, C(O)OEt, -C(O)OCH2 (tert-butyl), -C(O)OCH ₂ CF ₃ , -C(O)O (isopropyl), -CHF2, -CN, -C=CH, -SO ₂ Me, -SO2Et, -SO ₂ Ph(Me), -CF ₃ , -Me, -Et, -Br, -Cl, -CH ₂ Ph,

where R ⁹ is selected from -H, -Me, -Et, -CF ₃ , isopropyl or -tert-butyl. In some embodiments, in a compound of formula II, R ³ is:

(1) -C(O)OMe or -C(O)OEt;

or (2) \ where R ⁹ is selected from -H, -Me, -Et, -CF ₃ , isopropyl or -tert-butyl.

In some embodiments, in the compound of formula II, m = 0-1;

each R ¹ is independently selected from halogen, -H, -(C1-C ₆ )alkyl, O((C1-C ₆ )alkyl), CF3 or OCF3;

R ² is selected from -(C1-C ₆ )alkyl, -C(O)O((C1-C ₆ )alkyl), (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical or (C ₆ -C ₁₀ )aryl-O-(C1-C ₁₂ )aliphatic radical, wherein said alkyl, aryl or heteroaryl is independently substituted with 0-5 R';

R ³ is selected from -(C1-C ₆ )alkyl, -SO2 (C ₁ -C ₆ )alkyl or -C(O)O((C ₁ -C ₆ )alkyl), said alkyl being independently substituted with 0-5 R '; and

R ⁴ , R ⁵ and R ⁶ are -H.

In some embodiments, in the compound of formula II, m = 0 or 1;

when m is 1, at least one R ¹ is halogen or —O((C1-C ₆ )alkyl);

R ² is selected from -(C1-C ₆ )alkyl, (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical or (C ₆ -C ₁₀ )aryl-O-(C1C ₁₂ )aliphatic radical, wherein said aryl is independently substituted with 0-5 R' independently selected from -F, -Me or -OMe;

R ³ is -C(O)O((C1-C ₆ )alkyl);

R ⁴ and R ⁵ are both -H and

R ⁶ is -H.

In some embodiments, in the compound of formula II, m = 0-1;

each R ¹ is independently selected from halogen, -H, -(C1-C ₆ )alkyl, O((C1-C ₆ )alkyl), CF3 or OCF3, said R ¹ being independently substituted with 0-5 R';

R ² is selected from -(C1-C ₆ )alkyl, -C(O)O((C1-C ₆ )alkyl), (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical, (C ₆ -C ₁₀ )aryl-O-(C1-C ₁₂ )aliphatic radical, -(CH ₂ ) _1-3 N((C ₆ -C ₁₀ )aryl) ₂ , -(CH ₂ ) _1-3 -(5- 10-membered heteroaryl), -(CH ₂ ) _1-3 -O-(5-10-membered heteroaryl), - (CH ₂ ) _1-3 -(3-10-membered heterocyclyl), and R ² is independently substituted with 0 -5R';

R ³ is selected from -(C ₁ -C ₆ )alkyl, -C=CH, -CN, halogen, -SO ₂ ((C ₆ -C ₁₀ )aryl), -SO ₂ ((C ₁ -C ₆ )alkyl ), -C(O)NH ₂ , -C(O)O((C ₁ -C ₆ )alkyl), -C(O)((C ₁ -C ₆ )alkyl), -(C ₆ -C ₁₀ )aryl or 5-10-membered heteroaryl, wherein R ³ is independently substituted with 0-5 R'; and

R ⁴ , R ⁵ and R ⁶ is -H.

In some embodiments, in the compound of formula II, m = 0 or 1;

when m is 1, at least one R ¹ is halogen or —O((C ₁ -C ₆ )alkyl);

R ² is selected from -(C ₁ -C ₆ )alkyl, (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical, (C ₆ -C ₁₀ )aryl-O-(C1C ₁₂ )aliphatic radical, ((CH ₂ ) _1-3 -(5-10 membered heteroaryl) or -(CH ₂ ) _1-3 -(3-10 membered hetero- 17 039381 cyclyl), wherein R ² is independently substituted with 0-3 R';

R ³ is halogen, -CN, -C CH, -C(O)NH ₂ , -(C ₁ -C ₆ )alkyl, -C(O)((C1-C ₆ )alkyl), -C(O) O ((C1C6) alkyl), -SO2 (Ph (Me)),

wherein said alkyl or Ph is independently substituted with 0-3 R', and wherein R ⁹ is selected from -H, -Me, -Et, -CF ₃ , isopropyl, -OMe or -tert-butyl; R ⁴ , R ⁵ and R ⁶ are -H. In some embodiments, in a compound of formula II

R ³ is , where R ⁹ is selected from -H, -Me, -Et, -CF3, isopropyl or -tert-butyl. In some embodiments, in a compound of formula III, m is 0 or 1, and when m is 1, R ¹ is -O((C1-C ₆ )alkyl);

R ² is selected from -(CH ₂ ) _1-3 -(C _g C ₆ )alkyl or -(CH ₂ ) _1-3 -(C ₆ -C ₁₀ )aryl;

R ³ is -C(O)O((C1-C ₆ )alkyl) and each of R ⁴ and R ⁵ is -H; and

R ⁶ is -H.

In some embodiments, in a compound of formula II, m is 0 or 1, and when m is 1, R ¹ is -O((C1-C ₆ )alkyl);

R ² is -OR ⁸ , where R ⁸ is (C ₆ -C ₁₀ )aryl substituted with 0-3 halogens;

R ³ is -C(O)O((C ₁ -C ₆ )alkyl);

R ⁴ , R ⁵ and R ⁶ are both -H.

In some embodiments, in a compound of formula IV, m = 0 or 1;

R ² is OR ⁸ , -(CH2)nOR ⁸ or -(CH2) _n O(CH ₂ ) _n R ⁸ , where n = 1, and where R ⁸ is -(C1C ₆ )alkyl, (C ₆ -C ₁₀ )aryl or 5-10-membered heteroaryl, wherein R ⁸ is independently substituted with 0-3 R';

R ³ is halogen, -H, -CN, -(C1-C ₆ )alkyl, -C(O)((C1-C ₆ )alkyl), -C(O)O((C1-C ₆ )alkyl) ,

Moreover, the specified alkyl is independently substituted with 0-3 R';

R ⁹ is selected from -Me, -Et, isopropyl or -CF ₃ ;

R ⁴ and R ⁵ are both -H and

R ⁶ is -H.

Examples of some compounds according to the present application include

- 18 039381

Compound Structure one ^С ° ^2В N^ ^CH = 2 X^CO ₂ Et 'N^CH, 3 w CM co o t o o ζΑ^^Λ ² Cz Zz 4 V />-CO ₂ Et N-# ΓΎ ) A H ₃ CO N /--' 5 Q k ⁰ ΊΑ ° pj k 6 Λ L0 /A θ (I J GP 7 ν ^Γ Α ^C ° ^2Et eight Г g — CO ₂ Et ΛΑ ) О M e O N<=/ nine τ /b ΠΊ ten I 0 _b πθτχ GP

- 19 039381

eleven H ₃ co ^x ++- _N ^( 0 OCH ₃ 12 ~ t sh o C o t 44 g /Y-co ₂ E1 .. N-% __/ £P O ^F y-ir ^N Y 45 -Π δ<ΧΧ> ΊΠ£ (1 1 ΠΊ 46 -π η 1-, ο /A ° 47 Γ Ν—CO ₂ Et ___/ ^C| CO O ^N V 48 CD Oh h oh O

- 20 039381

49 jX> O MeO N— ^/ N- / 'X fifty CD O 1 o Z^\ O 1 N> X ^w 51 g />-CO ₂ Et xxf F MeO N—' ⁿ 'n ^/ 52 V /%CO ₂ Et Xd) Ο-c, MeO ^Ν Ά / 53 g CO ₂ Et MeO n X\ / 54 b Μ Μ ^ζ γΧΧγ ^ζ 1 ο ° and ο Χί^ 55 Γ X-CO ₂ Et jCO MeO^^^y-N_o-U \

- 21 039381

56 ,Ν Г />-CO ₂ Et _.CO 101 V />-CO ₂ Et ίΓΎ ) GA 102 ^V-Me ⁿ n 103 C ^N />-CO ₂ Et £O MeO^^y^N__ N o- 104 G / U-CO ₂ Et xx> MeO ^ ^ ^ N N G-O - b 105 r N—CO ₂ Et jctf MeO^^X-'N NA~O ^N λ-\ \Γ~OMe 106 r \-CO ₂ Et GO ΜθΟ' ^ζ ^^\ _Γ 'Ν Ν ^z ^~ ^{Br ν} 'Ν

- 22 039381

107 Π z ^ζ k o o o 1 ^m 0 108 \ >—CO ₂ Et ΓΎ ) CHA νΛ-^c. 109 b ^Z ^44ϊγ ^Z H A* 110 -n h Z ? λ ^ζ 111 sh 0=^ IN II O About H 112 nT° ^{l F} V ^N v 113 from° ^{t F} V- ^N V~

- 23 039381

114 T 0^0 -n 115 z^ T^^T z ' o z 116 z about 1 XT/ QLL 117 ^NH2NV _{_} 118 about About b ό ό 119 i _o Q TD X ¹ o <D 120 v. 9 AT V o <D 2

- 24 039381

121 ^qT nv 122 cW MeO^yN_y-Z \ 123 CD Λ ό 124 0 χ-χ w about <D 125 126 I am 127 XW'? yW ^N \ ^N V

- 25 039381

128 J-Λ p o o 2 129 χχΓ MeO^^XN ©Me Ν'ν ⁷ 130 Ρ-Ν MeO^dV^N_OMe 131 MeO'^^'^-N ©Me 132 QF/ W < ? / ^z 133 P V? /° 134 Q ^ζ ^ρΡΡγ ^ζ %

- 26 039381

135 ххТ ^F V- ^N v 136 oT ^F U- ^N V 137 G N-CO ₂ Et And > __OPh 138 ^.N PN Λ N^\ ίΎ ) ^oPh NV ⁷ 139 0 MPzP 140 b A ^z ^/ 141 PN N-z\\zZ N

- 27 039381

142 f Z Z about CD 2 143 °x ox i ζ'γ^^^'ζ ^Z Z-z 144 -n ,Γ ^Ζ FX V 145 ^F V ^N V 146 V 147 1J / ^1N'N ^ ^V 148 ZhI ^F z)__ ^/O ^C^ N ^/7 A // ⁿ -N

- 28 039381

149 Ν'Ο ^F ν”Λ- ^N N 150 b ζζ z-l ζ ^ζ λ° ο GO 151 χ ^Ν /, ⁿ o ^F £5- 152 nJ\^ ^{nf Ν} ν 153 οΟ ^Λ-_ ^Ν ν 154 z and b 155 Ν% ^f Ο ^Ν ν

-29039381

156 F'OPh 157 χχ Γ F __OPh 158 Ν - ^ F^^^pN_^OPh 159 . ⁼ ο ζ ? 3 ^-6 ο τι 160 .Ν Ν-Ο ^F Ν^Λ- ^ν 'Ν 161 α ο ^Λ ^Ν ν 162 MeCT'^X^-'N__/OPh ^Ν 4ί

- 30 039381

- 31 039381

170 5 f O St 5° 171 Ν-0 II 'MeO^X^y-N_y>Ph NV ⁷ 172 CD Λ ή Π 173 i If o f s 174 xx Χχ V o f 2 175 XX V o f 2 176 XX XX 'V o f 2 177 -5 X X/If o f 2 178 f ,one 179 __ Ν'Ο XX/ ^Ν Λ NX/ ίΎ ) ¹ MeO^^^yN Ο^ΧΧ Ν ~~ ^/ f X ν F

and a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof.

Any embodiment described herein is also intended to represent unlabeled forms as well as isotopically labeled forms of compounds unless otherwise noted. Isotopically labeled compounds have the structures represented by the formulas given here, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that may be included in the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ² H, ³ H, ⁿ C, ¹³ s, ¹⁴ s, ¹⁵ n, ¹⁸ f , ^31p , ^32P , ^35S , ^36Cl , ^125I , respectively. The invention includes various isotopically labeled compounds such as

- 32 039381 is defined here, for example, those in which radioactive isotopes are present, such as ³ H, ¹³ C and ¹⁴ C. Such isotopically labeled compounds can be used in metabolic studies (preferably with ¹⁴ C), reaction kinetics studies (with , for example, ² H or ³ H), detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution tests, or in radiation treatment of patients. In particular, ¹⁸ F or a labeled compound may be particularly preferred for SPECT or PET studies. The isotopically labeled compounds of the invention and their prodrugs can generally be prepared by following the procedures disclosed in the Schemes or in the Examples and Preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

Any of the individual embodiments described herein may define Formula II, III, or IV alone or be combined to form a preferred embodiment of this invention.

General synthesis methodology

Compounds according to the invention can be obtained in general by methods known to the expert. Schemes 1-10 below show general synthetic routes for the preparation of compounds of formulas IIV. Other equivalent schemes, which will be apparent to one of ordinary skill in organic chemistry, may alternatively be used to synthesize various molecular moieties, as illustrated by the general schemes below.

Scheme 1. General synthesis of the compound of formula II

Scheme 2. General synthesis of a compound of formula III in which X, Y, Z, V and W form a pyrazole ring

Ry

NO ₂ 1- diazotization ^ EYU ^ (r'j4 T _______T --- ¹³¹ _{2. restored} ^ ^ G ^ H ₂ abra:

imidazole formation ------------------------------------------]

Scheme 3. General synthesis of the compound of formula II

- 33 039381

Scheme 4. General synthesis of compounds of formula II to provide divergent functionalization on the triazolo ring formed by X, Y, Z, V and W

Scheme 5. General synthesis of the compound of formula II

Scheme 6. General synthesis of the compound of formula II

Scheme 7. General synthesis of the compound of formula IV, in which X, Y, Z, V and W form a substituted

1,2,4-triazole ring

R^is -OR _S1 -5R _S .

ag

-iCHjJ^iR-jRw

Scheme 8. General synthesis of the compound of formula II

- 34 039381

Scheme 9. General synthesis of the compound of formula II

Scheme 10 General synthesis of a compound of formula II or IV wherein X, Y, Z, V and W form a substituted triazole ring such as a 1,2,3-triazole ring or a 1,2,4-triazole ring and an upper imidazole substituted with a 1,2,4-oxadiazole ring as illustrated in 10(a) and 10(b).

10(b)

As one skilled in the art will appreciate, compounds of formulas II-IV with variables other than those depicted above can be prepared by changing the chemicals or synthetic routes.

Pharmaceutical Compositions and Routes of Administration

The present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of formulas I-IV, or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers or combinations thereof.

The basic nitrogen-containing groups present in the compounds of the invention may be quaternized with agents such as lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, arylalkyl halides such as benzyl and phenethyl bromides and others. In this way, water- or fat-soluble or dispersible products are obtained.

It should be understood that the compounds and agents used in the compositions of the invention should preferably readily cross the blood-brain barrier when administered peripherally. Compounds that cannot cross the blood-brain barrier can, however, be effectively administered directly to the central nervous system, for example, by intraventricular or other neurocompatible routes.

In some embodiments of this invention, the a5-containing GABA R positive allosteric modulator is formulated with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchange materials, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents 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 silicon oxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol , sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and wool grease. In other embodiments, no media is used. For example, an a5-containing GABA agonist

R (eg, a positive allosteric modulator of the a5-containing GABA receptor) may be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). An α5-containing GABA R agonist (eg, a positive allosteric modulator of the α5-containing GABA receptor) may be formulated for administration by any convenient route for use in medicine.

In some embodiments, the therapeutic methods of the invention include administering a composition of a compound or agent topically, systemically, or topically. For example, therapeutic compositions of the compounds or agents of the invention may be formulated for administration, for example, by injection (eg, intravenously, subcutaneously, or intramuscularly), inhalation, or insufflation (orally or nasally), or by oral, buccal, sublingual, transdermal, nasal, or parenteral route. introduction. Compositions of the compounds or agents described herein may be formulated as part of an implant or device, or formulated for slow or sustained release. When administered parenterally, the therapeutic composition of the compounds or agents for use in this invention is preferably in a pyrogen-free, physiologically acceptable form. Methods and formulations are generally available from Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA.

In some embodiments, pharmaceutical compositions suitable for parenteral administration may include a positive allosteric modulator of α5-containing GABA R in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted. in sterile injectable solutions or dispersions immediately prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending agents or thickeners. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures of these substances, vegetable oils such as olive oil , and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants.

The composition comprising a positive allosteric modulator of α5-containing GABA R may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be provided by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable dosage form can be provided by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

In some embodiments of the invention, compositions comprising a positive allosteric modulator of a5-containing GABA R can be administered orally, for example, in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth) , powders, granules or in the form of a solution or suspension in an aqueous or non-aqueous liquid, or in the form of an oil-in-water emulsion or a water-in-oil liquid emulsion, or in the form of an elixir or syrup, or in the form of lozenges (using an inert base such as gelatin and glycerol or sucrose and gum arabic) and the like, which contain a predetermined amount of a positive allosteric modulator of a5-containing GABA R as an active ingredient.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more compositions containing a positive allosteric modulator of a5-containing GABA R can be mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and/or any of the following: (1) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or gum arabic; (3) humectants such as glycerin; (4) disintegrants such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution inhibitors such as paraffin; (6) absorption accelerators such as quaternary ammonium bases; (7) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) dyes. In the case of capsules, tablets and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions of this type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols, and the like.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the positive allosteric modulator of a5-containing GABA R, liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol (ethanol), isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 butylene glycol, oils (particularly cottonseed, peanut, corn, wheat germ, olive, castor and sesame oils), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters and sorbitan and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, coloring agents, flavoring agents and preservatives.

Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

As described here, the compounds, agents and their compositions can be entered for slow, controlled or sustained release. The term sustained release is widely known in the pharmaceutical sciences and is used here in relation to the controlled release of an active compound or agent from a dosage form into a medium over (over or during) a long period of time, for example, greater than or equal to one hour. The sustained release dosage form releases the drug in a substantially constant stream over a long period of time, or a substantially constant amount of drug is gradually released over a long period of time. The term extended release as used herein includes the terms controlled release, extended release, sustained release, delayed release, or slow release as these terms are used in the pharmaceutical sciences. In some embodiments, the extended release dosage form is administered in the form of a patch or pump.

One skilled in the art, such as a physician, will readily be able to determine the amount of α5-containing GABA R positive allosteric modulator(s) required to treat a patient using the compositions and methods of the invention. It is understood that the mode of administration will be determined for the individual, taking into account, for example, various factors that alter the effect of the positive allosteric modulator of a5-containing GABA R, the severity or stage of the disease, the route of administration, and characteristics unique to the individual, such as age, body weight, size body and degree of cognitive impairment.

It is known in the art that normalization to surface area is an adequate method for extrapolating doses between species. To calculate the human equivalent dose (HED) based on the dose used in the treatment of age-related cognitive impairment in rats, the formula HED (mg/kg) = rat dose (mg/kg) x 0.16 can be used (see Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers, December 2002, Center for Biologies Evaluation and Research). For example, using this formula, a dosage of 10 mg/kg in rats is equivalent to 1.6 mg/kg in humans. This conversion is based on the more general formula HED=animal dose in mg/kgx (animal body weight in kg/human body weight in kg) ^0, .

In some embodiments of the invention, the dose of the positive allosteric modulator of a5-containing GABA R is from 0.0001 to 100 mg/kg/day (which, given a typical human patient's body weight of 70 kg, is from 0.007 to 7000 mg/day) .

In some embodiments, the administration interval is once every 12 or 24 hours. Administration at less frequent intervals, such as once every 6 hours, may also be used.

When administered in the form of a slow or extended release implant, device or formulation, the α5-containing GABA R positive allosteric modulator may be administered once or at one or more times intermittently throughout the life of the patient as needed. Other administration intervals that are intermediate or shorter than these administration intervals for clinical applications may also be used and may be determined by those skilled in the art based on the methods of the invention.

The desired time of administration can be determined by the skilled artisan through routine experimentation. For example, a positive allosteric modulator of a5-containing GABA R can be administered over a period of 1-4 weeks, 1-3 months, 3-6 months, 6-12 months, 1-2 years, or more, up to a lifetime. patient.

In addition to being a positive allosteric modulator of α5-containing GABA _a R, the compositions of the invention may also include other therapeutically useful agents. These other therapeutically useful agents may be administered in a single formulation, simultaneously or sequentially with a positive allosteric modulator of α5-containing GABA R, in accordance with the methods of the invention.

The skilled artisan will appreciate that the compositions described herein may be adapted and modified according to the intended use, and that the compositions described herein may be used in other suitable applications. For example, the compositions according to this application may further include a second therapeutic agent. Such other additions and modifications do not go beyond the scope of the present description.

Pharmaceutical compositions with antipsychotics

The compounds or compositions of this application may be used in combination with an antipsychotic in the treatment of cognitive impairment associated with schizophrenia or bipolar disorder in a patient suffering from or at risk of said schizophrenia or bipolar disorder (eg mania). An antipsychotic or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof that can be used in the methods and compositions of the invention includes both typical and atypical antipsychotics. In some embodiments, the compounds or compositions of the present invention may be used to treat one or more positive and/or negative symptoms as well as cognitive impairment associated with schizophrenia. In some embodiments, the compounds or compositions of the present invention may be used to treat one or more symptoms as well as a cognitive impairment associated with bipolar disorder (particularly mania). In some embodiments of this invention, the compounds or compositions of the invention prevent or slow the progression of cognitive impairment associated with schizophrenia or bipolar disorder (in particular mania) in said patient.

In some embodiments, atypical antipsychotics suitable for use in the present invention include In some embodiments, an antipsychotic suitable for use in the present invention is selected from aripiprazole (Bristol-Myers Squibb), olanzapine (Lilly) and ziprasidone (Pfizer) and their pharmaceutically acceptable salts, hydrates, solvates and polymorphs.

Pharmaceutical compositions with memantine

The compounds or compositions of this application may be used in combination with memantine, or a derivative or analogue thereof, in the treatment of a central nervous system (CNS) disorder-related cognitive impairment in a patient suffering from or at risk of developing it, including, without limitation, those who have or at risk for age-related cognitive impairment, mild cognitive impairment (MCI), memory-impairing MCI, age-related memory impairment (AAMI), age-related cognitive decline (ARCD), dementia, Alzheimer's disease (AD), prodromal AD, post-traumatic stress disorder (PTSD) ), schizophrenia or bipolar disorder, amyotrophic lateral sclerosis (ALS), and cognitive impairment associated with cancer therapy.

Memantine, also chemically known as 3,5-dimethyladamantan-1-amine or 3,5-dimethyltricyclo[3,3,1,13,7]decan-1-amine, is a noncompetitive N-methyl-Daspartate (NMDA) receptor antagonist. with moderate affinity. Proprietary names for memantine include: Axura® and Akatinol® (Merz), Namenda® (Forest Laboratories), Ebixa® and Abixa® (Lundbeck) and Memox® (Unipharm). Memantine is currently available in the US and in over 42 countries worldwide. It has been approved for the treatment of moderate to severe Alzheimer's disease (AD) in the US at doses up to 28 mg/day. Memantine and some of its derivatives and analogues, which can be used in the present invention, are disclosed in US Patents 3391142; 4122193; 4273774 and 5061703, which are all included in the present description by reference. Other derivatives or analogs of memantine that may be used in the present invention include, but are not limited to, the compounds disclosed in US Patent Application Publications US 20040087658, US 20050113458, US 20060205822, US 20090081259, US 20090124659 and US 20100227852; Publications of the application for EP patent EP 2260839A2; EP Patent EP 1682109B1 and Patent Application Publication WO 2005079779, all of which are incorporated herein by reference. Memantine, within the scope of the present invention, includes memantine and its derivatives and analogs, as well as its hydrates, polymorphs, prodrugs, salts and solvates. Memantine, within the scope of the invention, also includes a composition comprising memantine, or a derivative or analog thereof, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, or prodrug, wherein the composition optionally further comprises at least one additional therapeutic agent (such as a therapeutic agent, suitable for the treatment of central nervous system disorder or associated cognitive impairment). In some variants

- 38 039381 composition of memantine suitable for use in the present invention includes memantine and a second therapeutic agent, which is donepezil (under the trademark

Aricept).

In other embodiments, the a5-containing GABA positive allosteric receptor modulator and memantine (or memantine derivative/analog), or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, or prodrugs thereof, are administered simultaneously or sequentially, either in a single formulation or in separate formulations. formulations packaged together. In other embodiments, the a5-containing GABA positive allosteric receptor modulator and memantine (or memantine derivative/analogue), or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, or prodrugs thereof, are administered by different routes. Within the scope of the invention, the combination includes administration in any of these formulations or by any of these routes of administration.

Pharmaceutical compositions with acetylcholine esterase inhibitors (AChE-Is)

Compounds or compositions according to this application can be used in combination with an acetylcholine esterase inhibitor in the treatment of cognitive impairment associated with disorders of the central nervous system (CNS) in a patient suffering from or at risk of developing it, including, without limitation, having or at risk of developing age-related cognitive impairment, mild cognitive impairment (MCI), memory-impairing MCI, age-related memory impairment (AAMI), age-related cognitive decline (ARCD), dementia, Alzheimer's disease (AD), prodromal AD, post-traumatic stress disorder (PTSD), schizophrenia or bipolar disorder, amyotrophic lateral sclerosis (ALS), and cognitive impairment associated with cancer therapy.

In some embodiments, AChE-Is suitable for use in combination with the compounds and compositions of this application include: Donepezil (aricept), galantamine (razadyne), or rivastigmine (Exelon).

In other embodiments, the a5-containing GABA and AChE-I positive allosteric receptor modulator, or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, or prodrugs thereof, are administered simultaneously or sequentially, either in a single formulation or in separate formulations packaged together. In other embodiments, the a5-containing GABA and AChE-I positive allosteric receptor modulator, or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, or prodrugs thereof, are administered by various routes. Within the scope of the invention, the combination includes administration in any of these formulations or by any of these routes of administration.

Ways to assess cognitive impairment

Animal models serve as an important resource for developing and evaluating treatments for cognitive impairment associated with disorders of the central nervous system. The features that characterize cognitive impairment in animal models tend to extend to human cognitive impairment. Efficacy in such animal models is thus expected to be predictive of efficacy in humans. The degree of cognitive impairment in an animal model for a disorder of the central nervous system and the effectiveness of a method of treating said disorder of the central nervous system can be tested and validated using a variety of cognitive tests.

The behavioral task using the radial eight-arm maze (RAM) is one example of a cognitive test that specifically tests spatial memory (Chappell et al. Neuropharmacology 37: 481-487, 1998). The RAM apparatus consists of, for example, eight equally spaced arms. A labyrinth sleeve extends from each face of the central platform. There is a feeder at the distal end of each sleeve. Food is used as a reward. Blocks can be positioned to prevent entry into any of the arms. Numerous additional labyrinth elements surrounding the apparatus can also be used. After the habituation and training phases, patients' spatial memory can be tested in RAM under control or test compound treatment conditions. As part of the test, patients are pretreated prior to testing with vehicle as a control or one of the test compound dosage ranges. At the beginning of each test, part of the sleeves of the eight-arm labyrinth is blocked. Patients are allowed to receive food in open sleeves, to which access is permitted during this initial informational phase of the trial. Patients are then removed from the maze for a delay period, e.g., 60 second delay, 15 minute delay, one hour delay, two hour delay, six hour delay, 24 hour delay, or longer) between the information phase and the subsequent retention test, during which the barriers on the labyrinth is removed, thus providing access to all eight sleeves. After a delay period, patients are placed back on the central platform (with the previously blocked sleeve barriers removed) and allowed to receive the remaining nutritional rewards during this phase of the trial, which is a retention test. Identity and configuration of blocked sleeves

- 39 039381 vary from test to test. The number of errors that patients make during the retention test phase is tracked. An error in the trial occurs if patients enter a sleeve from which food was already taken in the preliminary phase of the trial, or if, in the second phase, they return to a sleeve that has already been visited. Fewer errors indicate better spatial memory. The number of errors made by subjects in different treatment regimens with test compounds can then be compared in relation to the effectiveness of the test compound in the treatment of cognitive impairment associated with disorders of the central nervous system.

Another cognitive test that can be used to evaluate the effects of a test compound on cognitive impairment in an animal model of central nervous system disorder is the Morris water maze. The water maze is a pool surrounded by a new set of structures relative to the maze. The water maze training protocol can be based on a modified water maze task that has been shown to be hippocampal dependent (de Hoz et al., Eur. J. Neurosci., 22: 745-54, 2005; Steele and Morris, Hippocampus 9:118-36, 1999). The patient is trained to locate a submerged rescue platform hidden under the surface of the pool. During the training trial, the patient is released into the maze (pool) from random starting positions around the perimeter of the pool. The starting position varies from test to test. If the patient does not locate the rescue platform within the given time, the experimenter guides and places the patient on the platform to "teach" the location of the platform. After a delay period after the last training trial, a retention test in the absence of a rescue platform is performed to assess spatial memory. The patient's level of preference for the location of the rescue platform (now missing), as measured, for example, by the time spent by the mouse at that location, or by the number of times the mouse crossed that location, indicates better spatial memory, ie, treatment of cognitive impairment. The location preference of the rescue platform under different treatment conditions can then be compared with respect to the effectiveness of the test compound in the treatment of cognitive impairment associated with disorders of the central nervous system.

There are various tests known in the art for assessing cognitive function in humans, for example, and without limitation, assessment of change in the overall clinical impression scale (CIBIC-plus scale); Mini Mental Health Assessment (MMSE); neuropsychiatric questionnaire (NPI); clinical dementia rating scale (CDR); Cambridge Automated Neuropsychological Test Battery (CANTAB); Sandoz Geriatric Clinical Assessment Scale (SCAG), Buschke Selective Memory Test (Buschke & Fuld, 1974); additional criterion of associations of verbal pairs; additional criterion of logical memory; additional criterion for visual reproduction of the revised Wechsler Memory Scale (WMS-R) (Wechsler, 1997); Benton visual retention test or 3-alternative explicit forced choice task or neuropsychological consensus test with MATRICS battery. See Folstein et al., J Psychiatric Res 12: 189-98, (1975); Robbins et al., Dementia 5: 266-81, (1994); Rey, L'examen clinique en psychologie, (1964); Kluger et al., J Geriatr Psychiatry Neurol 12: 168-79, (1999); Marquis et al., 2002 and Masur et al., 1994. See also Buchanan, R.W., Keefe, R.S.E., Umbricht, D., Green, M.F., Laughren, T., and Marder, S.R. (2011), The FDA-NIMH-MATRICS guidelines for clinical trial design of cognitive-enhancing drugs: what do we know 5 years later? Schizophr. Bull. 37, 1209-1217. Another example of a cognitive test in humans is an explicit forced choice task with 3 alternatives. In this test, patients are presented with color photographs of ordinary objects, consisting of a mixture of three types of image pairs: similar pairs, identical pairs, and unrelated sheets. The second of a pair of similar objects is referred to as a decoy. These pairs of images are completely randomized and presented individually as series of images. Patients are asked to make a judgment as to whether the observed objects are new, old, or similar. A response similar to the decoy presentation indicates successful retrieval by the patient. In contrast, rating a honeypot as old or new indicates that proper memory retrieval did not occur.

In addition to assessing cognitive performance, the progression of age-related cognitive impairment and dementia, as well as the transformation of age-related cognitive impairment into dementia, can be tested by assessing surrogate changes in the patient's brain. Surrogate changes include, without limitation, changes in regional brain volumes, perforator pathway degradation, and changes seen in brain function by resting fMRI (R-fMRI) and fluorodeoxyglucose positron emission tomography (FDG-PET). Examples of regional brain volumes that can be used to control the progression of age-related cognitive impairment and dementia include a reduction in hippocampal volume and a reduction in entorhinal cortex volume or thickness. These volumes can be measured in a patient, for example, using MRI. Aisen et al., Alzheimer's & Dementia 6: 239-246 (2010). Perforant pathway degradation has been shown to be associated with aging as well as cognitive decline. For example, older adults with severe perforator tract degradation tend to perform worse on hippocampus-dependent memory tests. Perforant pathway degradation can be monitored in patients with diffusion tensor imaging.

- 40 039381 Ultra High Definition (DTI) applications. Yassa et al., PNAS 107: 12687-12691 (2010). Resting fMRI (R-fMRI) involves imaging the brain during rest and recording spontaneous low-frequency (<0.1 Hz) large amplitude fluctuations in the fMRI signal temporally correlated across functionally related regions. Bivariate functional connectivity analyses, independent component analyses, and/or frequency signal analyzes are used to uncover functional connectivity between brain regions, especially those areas whose connectivity increases or decreases with age, as well as the degree of cognitive impairment and/or dementia. FDG-PET uses FDG uptake as a measure of regional metabolic activity in the brain. Decreased FDG uptake in areas such as the posterior cingulate cortex, temporoparietal cortex, and prefrontal association cortex has been shown to be associated with the degree of cognitive decline and dementia. Aisen et al., Alzheimer's & Dementia 6:239-246 (2010), Herholz et al., Neurolmage 17: 302-316 (2002).

Age-related cognitive impairment

The invention relates to methods and compositions for the treatment of age-related cognitive impairment or the risk of this impairment using a positive allosteric modulator of the a5-containing GABA _a receptor (i.e., a compound of the invention), such as selected from the compounds or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers or combinations as described here. In some embodiments, the implementation of the treatment includes preventing or slowing the progression of age-related cognitive impairment. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with age-related cognitive impairment. In some embodiments, the treatment of age-related cognitive impairment includes slowing the transformation of age-related cognitive impairment (including, but not limited to, MCI, ARCD, and AAMI) into dementia (eg, AD). The methods and compositions may be used in human patients in clinical applications in the treatment of age-related cognitive decline in conditions such as MCI, ARCD and AAMI or at risk of these conditions. The composition dosage and administration interval for the method are as described herein, which are safe and effective in these applications. In some embodiments, the invention relates to a method of maintaining or improving cognitive function in a patient with age-related cognitive impairment, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof.

In some embodiments, a patient who is receiving treatment with the methods and compositions of the invention exhibits or is at risk of age-related cognitive impairment. In some embodiments, age-related cognitive impairment includes, without limitation, age-related memory impairment (AAMI), mild cognitive impairment (MCI), and age-related cognitive decline (ARCD).

Animal models serve as an important resource for developing and evaluating treatments for such age-related cognitive decline. The features that characterize age-related cognitive impairment in animal models tend to extend to age-related cognitive impairment in humans. Efficacy in such animal models is thus expected to be predictive of efficacy in humans.

Various animal models of age-related cognitive impairment are known in the art. For example, extensive behavioral characterization identifies a natural form of cognitive impairment in a crossed strain of aged Long-Evans rats (Charles River Laboratories; Gallagher et al., Behav. Neurosci. 107:618-626, (1993)). In behavioral assessment with the Morris water maze (MWM), rats learn and remember the location of a rescue platform driven by the pattern of spatial stimuli surrounding the maze. The cognitive basis of performance is tested in test trials by measuring the spatial preferences of the animal in finding the location of the rescue platform. Age-related rats in the study population have no difficulty swimming to a platform in line of sight, but age-related deterioration is found when the platform is camouflaged, requiring the use of spatial information. Performance in the case of individual age rats of the crossed Long-Evans strain varies to a large extent. For example, some of these rats perform tasks on a par with young adult rats. However, approximately 40-50% is outside the performance range for young rats. This variability among aged rats reflects significant individual differences. Thus, in an age population, some animals show cognitive decline and are defined as having age-related deterioration (AI), and other animals do not show deterioration and are defined as having no age-related deterioration (AU). See, for example, Colombo et al., Proc. Natl. Acad. sci. 94: 14195-14199, (1997); Gallagher and Burwell, Neurobiol. Aging 10: 691-708, (1989); Gallagher et al. behavior. neurosci. 107:618-626, (1993); Rapp and Gallagher, Proc. Natl. Acad. Sci. 93: 9926-9930, (1996); Nicolle et al., Neuroscience 74: 741-756, (1996); Nicolle et al., J. Neurosci. 19: 9604-9610, (1999); International Patent

- 41 039381

Publication WO2007/019312 and International Patent Publication WO 2004/048551. Such an animal model of age-related cognitive impairment can be used to test the effectiveness of the methods and compositions of the invention in the treatment of age-related cognitive impairment.

The effectiveness of the methods and compositions of the invention in the treatment of age-related cognitive impairment can be assessed using a variety of cognitive tests, including the Morris water maze and the radial eight-arm maze, as discussed here.

Dementia

The invention also relates to methods and compositions for the treatment of dementia using a positive allosteric modulator of the α5-containing GABA receptor, such as selected from compounds or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations thereof, as described herein. In some embodiments, the treatment includes preventing or slowing the progression of dementia. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with dementia. In some embodiments, the symptom being treated is a cognitive impairment. In some embodiments, the invention relates to a method of maintaining or improving cognitive function in a patient with dementia, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination. In some embodiments, the dementia is Alzheimer's disease (AD), vascular dementia, dementia with Lewy bodies, or frontotemporal dementia. The methods and compositions may be used in human patients in clinical applications in the treatment of dementia. The composition dosage and administration interval for the method are as described herein, which are safe and effective in these applications.

Animal models serve as an important resource for developing and evaluating treatments for dementia. The features that characterize dementia in animal models tend to extend to dementia in humans. Thus, efficacy in such animal models is expected to be predictive of efficacy in humans. Various animal models of dementia are known in the art, such as PDAPP, Tg2576, APP23, TgCRND8, J20, hPS2 Tg, and APP+PS1 transgenic mice. Sankaranarayanan, Curr. top. Medicinal Chem. 6:609-627, 2006; Kobayashi et al. Genes Brain Behav. 4: 173196. 2005; Ashe and Zahns, Neuron. 66: 631-45, 2010. Such animal models of dementia can be used to test the effectiveness of the methods and compositions of the invention in the treatment of dementia.

The effectiveness of the methods and compositions of the invention in treating dementia or dementia-related cognitive impairment can be assessed in animal models of dementia, as well as in humans with dementia, using a variety of cognitive tests known in the art, as discussed here.

Post Traumatic Stress Disorder

The invention also relates to methods and compositions for the treatment of post-traumatic stress disorder (PTSD) using a positive allosteric modulator of the a5-containing GABA receptor, such as selected from the compounds or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations, as described herein . In some embodiments, the treatment includes preventing or slowing the progression of PTSD. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with PTSD. In some embodiments, the symptom being treated is a cognitive impairment. In some embodiments, the invention relates to a method of maintaining or improving cognitive function in a patient with PTSD, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof. The methods and compositions may be used in human patients in clinical applications in the treatment of PTSD. The composition dosage and dosage range for the method are as described herein, which are safe and effective in these applications.

Patients with PTSD (and, to a lesser extent, traumatized patients without PTSD) have smaller hippocampal volumes (Woon et al., Prog. Neuro-Psychopharm. & Biological Psych. 34, 11811188; Wang et al., Arch. Gen. Psychiatry 67:296-303, 2010). PTSD is also associated with reduced cognitive performance. Older adults with PTSD show greater declines in cognitive performance relative to controls (Yehuda et al., Bio. Psych. 60: 714-721, 2006) and are more likely to develop dementia (Yaffe et al., Arch. Gen. Psych. 678: 608-613, 2010).

Animal models serve as an important resource for developing and evaluating treatments for PTSD. The features that characterize PTSD in animal models generally extend to human PTSD. Thus, efficacy in such animal models is expected to be predictive of efficacy in humans. Various animal models of PTSD are known in the art.

One PTSD rat model is Time Dependent Sensitization (TDS).

- 42 039381

TDS involves exposing an animal to a highly stressful event, followed by a situational reminder of the previous stressor. The following is an example of TDS. Rats are placed in a fixative, then placed in a pool and allowed to swim for, for example, 20 minutes. Thereafter, each rat is immediately injected with a gaseous anesthetic until unconscious, and finally dried. Animals are left alone for many days, for example one week. The rats are then subjected to a re-stress procedure consisting of an initial stressful event, such as a swimming pool procedure (Liberzon et al., Psychoneuroendocrinology 22: 443-453, 1997; Harvery et al., Psychopharmacology 175:494-502, 2004). TDS results in an improvement in the acoustic startle reflex (ASR) in the rat, which is comparable to the hypertrophied acoustic startle response that is a prominent symptom of PTSD (Khan and Liberzon, Psychopharmacology 172: 225-229, 2004). Such animal models of PTSD can be used to test the effectiveness of the methods and compositions of the invention in the treatment of PTSD.

The effectiveness of the methods and compositions of the invention in treating PTSD or PTSD-related cognitive impairment can also be assessed in animal models of PTSD, as well as in humans with PTSD, using a variety of cognitive tests known in the art, as discussed here.

Schizophrenia and Bipolar Disorder

The invention further relates to methods and compositions for the treatment of schizophrenia or bipolar disorder (particularly mania) using a positive allosteric modulator of the α5-containing GABAα receptor, such _as selected from the compounds or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations as described here. In some embodiments, the implementation of the treatment includes preventing or slowing the development of schizophrenia or bipolar disorder (in particular, mania). Schizophrenia is characterized by a broad spectrum of psychopathology, including positive symptoms such as aberrant or distorted mental representations (eg, hallucinations, delusions) or dopamine dysregulated symptoms (eg, hyperdopaminergic responses, hyperdopaminergic behavioral responses, dopaminergic hyperactivity or hypermotor activity or psychosis), negative symptoms characterized by a decrease in motivation and adaptive goal-directed actions (eg, anhedonia, affective dullness, abulia), and cognitive impairment. In some embodiments, treatment includes alleviating, alleviating, or slowing the progression of one or more positive and/or negative symptoms, as well as cognitive impairment associated with schizophrenia. Further, there are many other psychiatric disorders, such as schizotypal and schizoaffective disorder, other acute and chronic psychoses, and bipolar disorder (particularly mania), which have overlapping symptomatologies with schizophrenia. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms, as well as cognitive impairment associated with bipolar disorder (in particular, mania). In some embodiments, it relates to a method of maintaining or improving cognitive function in a patient with schizophrenia or bipolar disorder, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof. The methods and compositions may be used in human patients in clinical applications in the treatment of schizophrenia or bipolar disorder (particularly mania). The composition dosage and dosage range for the method are as described herein, which are safe and effective in these applications.

Cognitive impairment is associated with schizophrenia. They precede the onset of psychosis and are present in healthy relatives. The cognitive impairment associated with schizophrenia is a good predictor of functional outcome and is a basic function of the disorder. Cognitive features in schizophrenia reflect dysfunction in networks of the frontal cortex and hippocampus. Patients with schizophrenia also exhibit hippocampal pathologies such as shrinkage of the hippocampus, shrinkage of neurons, and dysfunctional hyperactivity. Imbalances in excitation and inhibition in these brain regions have also been documented in patients with schizophrenia, suggesting that drugs that target inhibitory mechanisms may be of therapeutic value. See, for example, Guidotti et al., Psychopharmacology 180: 191-205, 2005; Zierhut, Psych. Res. Neuroimag. 183:187-194, 2010; Wood et al., NeuroImage 52:62-63, 2010; Vinkers et al., Expert Opin. Investig. Drugs 19:1217-1233, 2009; Young et al. Pharmacol. Ther. 122:150-202, 2009.

Animal models serve as an important resource for developing and evaluating treatments for schizophrenia. The features that characterize schizophrenia in animal models generally extend to schizophrenia in humans. Thus, efficacy in such animal models is expected to be predictive of efficacy in humans. Various animal models of schizophrenia are known in the art.

One animal model of schizophrenia is long-term treatment with methionine. processed

Methionine-treated mice show deficient expression of GAD67 in the frontal cortex and hippocampus, similar to those described in the brains of postmortem patients with schizophrenia. They also exhibit fear suppression and social interaction deficits (Tremonlizzo et al., PNAS, 99: 17095-17100, 2002). Another animal model for schizophrenia is the treatment of rats with methyloxymethanol acetate (MAM). Pregnant female rats were administered MAM (20 mg/kg, ip) on day 17 of pregnancy. MAM treatment stimulates the process of pathological development, resulting in schizophrenia-like phenotypes in offspring, including anatomical changes, behavioral deficits, and altered neural processing. More specifically, MAM-treated rats show reduced density of parvalbumin-positive GABAergic interneurons in portions of the prefrontal cortex and hippocampus. In behavioral tests, MAM-treated rats show reduced latent inhibition. Latent inhibition is a behavioral phenomenon in which the ability to recognize a stimulus to which there was a previous exposure with any consequences is reduced. This tendency to ignore previously positive stimuli and reduce bonding with such stimuli is thought to prevent sensory overload. Low latent inhibition is a sign of psychosis. Latent inhibition can be tested in rats as follows. Rats are divided into two groups. One group is preliminarily exposed to sound in a plurality of tests. The other group is not exposed to sound. Both groups are then subjected to an auditory fear rooting procedure in which the same sound is presented simultaneously with a noxious stimulus, such as an electric shock to the paw. Subsequently, both groups are given a sound and a change in the locomotor activity of the rats is observed during the sound. After the fear is fixed, the rats respond to the sound by a strong weakening of motor activity. However, the group that was exposed to sound before the period of development of the conditioned reflex shows a strong latent inhibition: the suppression of motor activity in response to the sound is reduced. MAM-treated rats, in contrast, show impaired latent inhibition. Those. exposure to sound prior to fear fixation has no significant effect in suppressing fear conditioning, (see Lodge et al., J. Neurosci., 29:23442354, 2009). Such animal models of schizophrenia can be used to test the effectiveness of the methods and compositions of the invention in the treatment of schizophrenia or bipolar disorder (particularly mania).

MAM-treated rats exhibit significantly increased motor responses (or aberrant motor activity) to low dose D-amphetamine administration. MAM-treated rats also show significantly more spontaneously firing ventral tegmental dopamine (DA) neurons. These results are thought to be a consequence of excessive hippocampal activity, since in MAM-treated rats, deactivation of the ventral hippocampus (vHipp) (e.g., intra-vHipp administration of the sodium channel blocker, tetrodotoxin (TTX) to MAM rats) completely reversed the increased activity of the DA and also normalized increased amphetamine-induced motor behavior. The correlation of hippocampal dysfunction and hyperresponsiveness of the DA system is thought to underlie the enhanced amphetamine response in MAM-treated animals and psychosis in schizophrenic patients. See Lodge D. J. et al. Neurobiology of Disease (2007), 27(42), 11424-11430. The use of MAM-treated rats in the study described above may be suitable for use in testing the efficacy of the methods and compositions of the present invention in the treatment of schizophrenia or bipolar disorder (particularly mania). For example, the methods and compositions of the invention can be evaluated using MAM-treated animals for their effects on central hippocampal (vHipp) regulation, increased activity of the DA neuron population, and hyperactive locomotor response to amphetamine in MAM-treated animals.

In MAM-treated rats, hippocampal dysfunction (HDC) leads to systemic dopamine hyperactivity. A benzodiazepine-positive allosteric modulator (PAM) selective for the a5 subunit of the GABA _a receptor, SH-053-2'FR-CH ₃ , was tested for its effects on hippocampal performance (HPC). The effect of SH-053-2'FR-CH ₃ on a hyperactive locomotor response to amphetamine in MAM-treated animals was also investigated. PAM _α5 GABA AR reduces the number of spontaneously active DA neurons in the ventral tegmental area (VTA) of MAM rats to levels seen in saline treated rats (control group) both systemically and by direct infusion into the ventral HPC. In addition, HPC neurons in both saline-treated and MAM-treated animals show reduced cortical responses after treatment with PAM α5 GABA^. In addition, the increased locomotor response to amphetamine observed in MAM-treated rats is reduced after treatment with PAM α5 GABA^. See Gill K. M et al. Neuropsychopharmacology (2011), 1-9. The use of MAM-treated rats in the study described above may be suitable for use in the present invention to test the effectiveness of the methods and compositions of the invention in the treatment of schizophrenia or bipolar disorder (particularly mania). For example, the methods and compositions of the invention can be evaluated using MAM-treated animals for their effects on hippocampal performance (HPC) and hyperactive locomotor response to amphetamine in MAM-treated animals.

Administration of MAM to pregnant rats on embryonic day 15 (E15) severely impairs spatial memory, or the ability to learn the spatial location of the four points on the radial eight-armed maze, in the offspring. In addition, MAM-treated rats on embryonic day 17 (E17) can reach the performance levels of control rats in the initial stages of learning, but are unable to process and retrieve spatial information when a delay of 30 min is introduced, indicating a significant deterioration in working memory. See Gourevitch R. et al. (2004). behavior. Pharmacol, 15, 287-292. Such animal models of schizophrenia can be used to test the effectiveness of the methods and compositions of the invention in the treatment of schizophrenia or bipolar disorder (particularly mania).

Apomorphine-induced climbing (AIC) and stereotyped (AIS) behavior in mice is another animal model that can be used in this invention. The agents are administered to mice at the desired dose level (eg, intraperitoneally). Then, for example, 30 minutes later, experimental mice are challenged with apomorphine (eg, 1 mg/kg s.c.). Five minutes after the injection of apomorphine, the snorting-licking-gnawing syndrome (stereotypical behavior) and climbing behavior induced by apomorphine were assessed and recorded for each animal. Readings may be repeated every 5 minutes during a 30 minute testing session. The scores for each animal are summarized for the entire 30 minute testing session for each syndrome (stereotypic and climbing behavior). If the effect reaches at least 50% inhibition and the ID ₅₀ value (95% confidence interval) is calculated using a nonlinear least squares calculation with inverse prediction. Mean climbing and stereotype scores can be expressed as a percentage of control values observed in vehicle-treated (eg, saline-treated) apomorphine-treated mice. See Grauer SM et al. Psychopharmacology (2009) 204, 37-48. This mouse model can be used to test the effectiveness of the methods and compositions of the invention in the treatment of schizophrenia or bipolar disorder (particularly mania).

In another known preclinical model of schizophrenia, rats are chronically exposed to ketamine, a non-competitive K-methyl-O-aspartate (NMDA) receptor antagonist, resulting in positive and negative psychotic symptoms and cognitive impairment. Male Long-Evans rats are injected intraperitoneally with ketamine (30 mg/kg, twice a day) for two weeks during puberty (2 months). Rats are subjected to behavioral tests when they reach adulthood (approximately 4-5 months old) for behavioral symptoms in response to exposure to ketamine and for the effectiveness of treatment to alleviate these symptoms. See, for example, Enomoto et al. Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 668-675.

The effectiveness of the methods and compositions of the invention in the treatment of schizophrenia or cognitive impairment associated with it can also be evaluated in animal models of schizophrenia or bipolar disorder (in particular mania), as well as in patients with schizophrenia, using a variety of cognitive tests known in the art. the art, as discussed here.

Amyotrophic Lateral Sclerosis (ALS)

The invention further relates to methods and compositions for treating ALS with a positive allosteric α5-GABA receptor modulator, such as selected from the compounds, or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations thereof, as described herein. In some embodiments, the implementation of the treatment includes preventing or slowing the progression of ALS. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with ALS. In some embodiments, the symptom being treated is a cognitive impairment. In some embodiments, the invention relates to a method of maintaining or improving cognitive function in a patient with ALS, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof. The methods and compositions may be used in human patients in clinical applications in the treatment of ALS. The composition dosage and dosage range for the method are as described herein, which are safe and effective in these applications.

In addition to motor neuron degeneration, ALS is characterized by neuronal degeneration in the entorhinal cortex and hippocampus, memory deficits, and neuronal hyperexcitability in various brain areas such as the cortex.

The effectiveness of the methods and compositions of the invention in treating ALS or ALS-related cognitive impairment can also be assessed in animal models of ALS, as well as in humans with ALS, using a variety of cognitive tests known in the art, as discussed here.

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Cognitive Impairment Associated with Cancer Therapy

The invention further relates to methods and compositions for the treatment of cognitive impairment associated with cancer therapy, using a positive allosteric modulator of the a5-containing GABA receptor, such as selected from compounds or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations thereof, as described here. In some embodiments, the implementation of the treatment includes preventing or slowing the progression of cognitive impairment associated with cancer therapy. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with a cognitive impairment associated with cancer therapy. In some embodiments, it relates to a method of maintaining or improving cognitive function in a patient with a cognitive impairment associated with cancer therapy, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combinations. The methods and compositions may be used in human patients in clinical applications in the treatment of cognitive impairment associated with cancer therapy. The composition dosage and dosage range for the method are as described herein, which are safe and effective in these applications.

Therapies used in the treatment of cancer, including chemotherapy, radiation, or combinations thereof, can cause cognitive impairment in patients with respect to functions such as memory, learning, and attention. Cytotoxicity and other adverse brain side effects of cancer therapy are the basis for this form of cognitive impairment, which can persist for decades. (Dietrich et al., Oncologist 13:1285-95, 2008; Soussain et al., Lancet 374:1639-51, 2009).

Cognitive impairment after cancer therapy reflects dysfunction in frontal cortex and hippocampal pathways that are important for normal cognition. In animal models, exposure to chemotherapy or radiation has a negative impact on performance in tests of cognition specifically dependent on these brain systems, especially the hippocampus (Kim et al., J. Radiat. Res. 49:517-526, 2008; Yang et al. al., Neurobiol Learning and Mem 93:487-494, 2010). Thus, drugs targeting these cortical and hippocampal systems may be neuroprotective in patients receiving cancer treatment and be effective in treating symptoms of cognitive impairment that may last longer than interventions used as cancer therapies.

Animal models serve as an important resource for developing and evaluating treatments for cognitive impairment associated with cancer therapy. Features characterizing cancer therapy-associated cognitive impairment in animal models can generally be extrapolated to cancer therapy-associated cognitive impairment in humans. Thus, efficacy in such animal models is expected to be predictive of efficacy in humans. Various animal models of cognitive impairment associated with cancer therapy are known in the art.

Examples of animal models of cognitive impairment associated with cancer therapy include treatment of animals with anti-blastoma agents such as cyclophosphamide (CYP) or using radiation such as ⁶⁰ Co gamma rays. (Kim et al., J. Radiat. Res. 49:517-526, 2008; Yang et al., Neurobiol. Learning and Mem. 93:487-494, 2010). The cognitive function of animal models of cancer therapy-associated cognitive impairment can then be tested using cognitive tests to test the effectiveness of the methods and compositions of the invention in treating cancer therapy-associated cognitive impairment. The effectiveness of the methods and compositions of the invention in treating cancer therapy-related cognitive impairment, as well as human patients with cancer therapy-related cognitive impairment, using a variety of cognitive tests known in the art, as discussed here.

Parkinson's disease (PD)

Parkinson's disease (PD) is a neurological disorder characterized by a decrease in voluntary movements. A patient suffering from this disease shows a reduction in motor activity and slower voluntary movements compared to a healthy person. The patient has a characteristic mask face, a tendency to rush when walking, a bowed posture, and general muscle weakness. There is a typical rigidity of passive movements like a lead pipe. Another important sign of the disease is the tremor of the limbs, observed at rest and decreasing during movement.

Parkinson's disease, whose etiology is unknown, belongs to a group of the most common movement disorders called parkinsonism, affecting approximately one person in a thousand. These other disorders, grouped under the name parkinsonism, may result from viral infection, syphilis, arteriosclerosis and trauma, and exposure to toxic chemicals and drugs. However, it is believed that the abnormal loss of synaptic stability can lead to the destruction of neural circuits and to brain diseases. Whether it is the result of genetics, drug use, the aging process, viral infections, or various other causes, dysfunction in neural communication is the underlying cause for many neurological diseases such as PD (Myrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214).

Regardless of the cause of the disease, the main pathological feature is the degeneration of dopaminergic cells in the basal ganglia, especially in the substantia nigra. Due to the premature death of dopamine-containing neurons in the substantia nigra, the largest structure of the basal ganglia, the striatum, has reduced input from the substantia nigra, resulting in reduced dopamine release. Understanding the underlying pathology led to the introduction of the first successful treatment that could alleviate Parkinson's disease. Virtually all approaches to disease therapy are based on dopamine replacement. Drugs currently used in treatment may be converted to dopamine after crossing the blood-brain barrier, or they may increase dopamine synthesis and decrease its breakdown. Unfortunately, this does not help the main pathological event, the degeneration of cells in the substantia nigra. The disease continues to progress and often after a certain period of time dopamine replacement treatment loses its effectiveness.

The invention relates to methods and compositions for the treatment of PD using a positive allosteric modulator of the α5-containing GABA receptor, such as selected from the compounds or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations, as described here. In some embodiments, the implementation of the treatment includes preventing or slowing the progression of PD. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with PD. In some embodiments, the symptom being treated is a cognitive impairment. For example, the methods and compositions of the present disclosure may be used to alleviate motor/cognitive impairment symptomatic of Parkinson's disease. In addition, the methods and compositions of the present disclosure may be used to treat memory impairment symptomatic of Parkinson's disease. In some embodiments, it relates to a method of maintaining or improving cognitive function in a patient with PD, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof.

There are many animal models for PD. Examples of an animal model for PD include the reserpine model, the methamphetamine model, the 6-hydroxydopamine (6-OHDA) model, the 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) model, the paraquat (PQ)-maneb model, rotenone model, 3nitrotyrosine model and genetic models using transgenic mice. Transgenic models include mice that express α-synuclein, express human mutant forms of α-synuclein, or mice that express LRKK2 mutations. See a review of these models by Ranjita B. et al. (Ranjita B. et al. BioEssays 2002, 24, 308-318). Additional information regarding these animal models is available from Jackson Laboratories (see also http://research.jax.org/grs/parkinsons.html) as well as numerous publications disclosing the use of these validated models.

The effectiveness of the methods and compositions of the invention in treating PD or PD-related cognitive impairment can be assessed in any of the aforementioned animal models of PD, as well as in humans with PD, using a variety of cognitive tests known in the art, as discussed here. .

Autism

Autism is a neurodevelopmental disorder characterized by dysfunction in three main behavioral dimensions: repetitive behavior, social deficits, and cognitive impairment. The area of repetitive behavior includes compulsive behaviors, atypical attachment to objects, strict adherence to regimens or rituals, and repetitive motor patterns such as stereotyping and self-stimulatory behavior. The area of social deficit includes deficits in reciprocal social interactions, lack of eye contact, reduced ability to carry on a conversation, and reduced daily interaction skills. Cognitive disorders may include language pathologies. Autism is a debilitating neurological disorder that affects thousands of Americans and includes many subtypes with various putative causes and few documented improving treatments. Autism spectrum disorders may occur at birth or may have a later onset, such as two or three years of age. There are no clear biological markers for autism. The diagnosis of this disorder is made by looking at the extent to which the child fits into a behavioral syndrome characterized by poor communication skills, differences in social and cognitive abilities, and maladaptive behavioral patterns. Dysfunction in neural communication is considered one of the root causes of autism (Myrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214). Recent studies have shown that there is a deficiency of GABA _a α5 in autism spectrum disorder (ASD), which confirms the need for further research into the system.

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GABA in this disorder (Mendez MA, et al. Neuropharmacology. 2013, 68: 195-201).

The invention also relates to methods and compositions for the treatment of autism using a positive allosteric modulator of the α5-containing GABA receptor, such as selected from the compounds or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations as described herein. In some embodiments, the treatment includes preventing or slowing the progression of autism. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with autism. In some embodiments, the symptom being treated is a cognitive impairment or a cognitive disorder. For example, the methods and compositions of the present disclosure may be used to alleviate motor/cognitive disorders symptomatic of autism. In some embodiments, it relates to a method of maintaining or improving cognitive function in a patient with autism, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof.

The valproic acid (VPA) rat model of autism, which uses in vitro electrophysiological methods developed by Rodier et al. (Rodier, P. M. et al. Reprod. Toxicol. 1997, 11, 417-422), is one of the most comprehensive stroke-based animal models of autism based on the observation that pregnant women who were treated with VPA in the 1960s 1990s, during the limited time window of embryogenesis, had a much higher risk of having an autistic child than the healthy population. The offspring of pregnant VPA-treated rats exhibit several anatomical and behavioral symptoms typical of autism, such as decreased number of cerebellar Purkinje neurons, impaired social interaction, repetitive behavior, and other symptoms of autism, including enhanced memory processing of fear-fading events. See Rinaldi T. et al. Frontiers in Neural Circuits, 2008, 2, 1-7. Another mouse model, the BTBR T+tf/J (BTBR) mice, an established model with strong behavioral phenotypes related to the three diagnostic behavioral symptoms of autism - unusual social interactions, reduced communication, and repetitive behavior - was used to investigate the efficacy of a selective negative allosteric mGluR5 receptor modulator. , GRN-529. See, for example, Silverman J. L. et al. Sci Transl. Med. 2012, 4, 131. The efficacy of the methods and compositions of the invention in the treatment of autism or autism-related cognitive impairment can be evaluated in the VPA rat model or the BTBR T+tf/J (BTBR) mouse model, as well as in humans. with autism using a variety of cognitive tests known in the art, as discussed here.

Mental retardation

Mental retardation is a generalized disorder characterized by significantly reduced cognitive function and deficits in adaptive behaviors. Mental retardation is often defined as an Intelligence Quotient (IQ) score of less than 70.

Congenital causes are among the many underlying causes of mental retardation. Dysfunction in neural communication is also considered one of the root causes of mental retardation (Myrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214).

In some cases, mental retardation includes, but is not limited to, Down syndrome, velocardiofacial syndrome, fetal alcohol syndrome, fragile X syndrome, Klinefelter syndrome, neurofibromatosis, congenital hypothyroidism, Williams syndrome, phenylketonuria (PKU), Smith-Lemli-Opitz syndrome , Prader-Willi syndrome, Phelan-McDermid syndrome, Mowat-Wilson syndrome, ciliopathy, Lowe's syndrome and sidereal-type fragile X syndrome. Down syndrome is a disorder involving a combination of birth defects, including some degree of mental retardation, facial features and often heart defects, increased infections, vision and hearing problems, and other health problems. Fragile X Syndrome is a common form of hereditary mental retardation, occurring at a rate of 1 in 4,000 in men and 1 in 8,000 in women. This syndrome is also characterized by developmental delay, hyperactivity, attention deficit disorder, and autistic-like behavior. There is no effective treatment for fragile X syndrome.

The present invention contemplates the treatment of mild mental retardation, moderate mental retardation, severe mental retardation, profound mental retardation, and mental retardation of unspecified severity. Such mental retardation may be, but not necessarily, associated with chromosomal changes (eg, Down's syndrome due to trisomy 21), heredity, pregnancy and perinatal problems, and other severe mental disorders. This invention relates to methods and compositions for the treatment of mental retardation using a positive allosteric modulator of the a5-containing GABA receptor, such as selected from the compounds or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations, as described herein. In some embodiments, the treatment

- 48 039381 this includes preventing or slowing down the development of mental retardation. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with mental retardation. In some embodiments, the symptom being treated is a cognitive impairment/impairment. For example, the methods and compositions of the present disclosure may be used to alleviate motor/cognitive impairments symptomatic of mental retardation. In some embodiments, the invention relates to a method of maintaining or improving cognitive function in a patient with mental retardation, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof.

Several animal models have been developed for mental retardation. For example, a knockout mouse model has been developed for fragile X syndrome. Fragile X Syndrome is a common form of mental retardation caused by the absence of the FMR1 protein, FMRP. Two FMRP homologues, FXR1P and FXR2P, have been identified. FXR2P is highly expressed in the brain and testis like FMRP. FxR2 and FmR1 knockout mice and FmR1/FxR2 double knockout mice are considered to be useful models for mental retardation such as fragile X syndrome. See, Bontekoe C. J. M. et al. Hum. Mol. Genet. 2002, 11(5): 487-498. The effectiveness of the methods and compositions of the invention in treating mental retardation or cognitive impairment/impairment associated with mental retardation can be evaluated in these mouse models and other animal models designed for mental retardation, as well as in humans with intellectual disability, using a variety of cognitive tests known in the art, as discussed here.

Compulsive behavior (obsessive-compulsive disorder)

Obsessive-compulsive disorder (OCD) is a mental condition typically characterized by invading, repetitive unwanted thoughts (obsessions) leading to compulsive behaviors and mental actions that the person feels compelled to perform (compulsion). Current epidemiological data indicate that OCD is the fourth most common psychiatric disorder in the US. Some researchers suggest that the prevalence of OCD is between one and three percent, despite the fact that the prevalence of clinically recognized OCD is much lower, suggesting that in many people this disorder cannot be diagnosed. Patients with OCD are often diagnosed by a psychologist, psychiatrist, or psychoanalyst according to the diagnostic criteria described in the Diagnostic and Statistical Manual of Mental Disorders, 4th edition text revision (DSM-IV-TR) (2000), including characteristics of obsessions and compulsions. Characteristics of obsessions include: (1) recurrent and persistent thoughts, impulses, or images that are experienced as intrusive and that cause marked restlessness or anxiety; (2) these thoughts, impulses, or images are not just excessive worry about real issues; and (3) the person tries to ignore or suppress such thoughts, impulses, or images, or neutralize them with some other thought or action. The person recognizes that intrusive thoughts, impulses, or images are a product of his or her own mind and are not based on reality. Characteristics of compulsions include:

(1) repetitive behavior or repetitive mental actions that the person feels obligated to perform in response to the obsession, or according to rules that must be strictly observed;

(2) behavior or mental actions aimed at preventing or alleviating anxiety or preventing some terrible event or situation; however, these behaviors or mental actions are not actually related to the problem, or they are excessive.

Individuals with OCD tend to perform tasks (or compulsions) to seek relief from their obsessional anxiety. Repetitive behaviors such as washing hands, counting, checking, or cleaning are often performed with the hope of preventing obsessive thoughts or that it will make them go away. Performing these rituals, however, provides only temporary relief. Individuals with OCD may also be diagnosed with a range of other psychiatric disorders, such as generalized anxiety disorder, anorexia, panic attack, or schizophrenia.

Dysfunction in neural communication is considered one of the root causes for obsessive disorder (Myrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214). Research suggests that OCD may be related to abnormal levels of a neurotransmitter called serotonin. Primary treatment for OCD consists of behavioral therapy, cognitive psychotherapy, and medications. Treatments include serotonin reuptake inhibitors (SRIs) such as paroxetine (Seroxat™, Paxil®, Xetanor™, ParoMerck™, Rexetin™), sertraline (Zoloft®, Stimuloton™), fluoxetine (Prozac®, Bioxetin™) , escitalopram (Lexapro®) and fluvoxamine (Luvox®), as well as tricyclic antidepressants such as clomipramine (Anafranil®). Benzodiazepines are also used in the treatment. As many as 40-60% of patients, however, do not adequately respond to SRI therapy, and an even larger proportion of patients do not experience complete remission of their symptoms.

The invention relates to methods and compositions for the treatment of OCD using an α5-containing GABAα receptor agonist (e.g., a positive allosteric modulator of the α5-containing GABA receptor), such _as selected from the compounds or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers, or combinations as described here. In some embodiments, the treatment includes preventing or slowing the progression of OCD. In some embodiments, the treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with OCD. In some embodiments, the symptom being treated is a cognitive impairment or a cognitive disorder. For example, the methods and compositions of the present disclosure may be used to treat cognitive impairment in OCD and/or improve cognitive function in patients with OCD. In some embodiments, the invention relates to a method of maintaining or improving cognitive function in an OCD patient, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof.

A quinpyrole sensitive rat model has been developed for OCD. Compulsive testing behavior in quinpyrole sensitive rats is interrupted, which is a characteristic of OCD compulsions.

In addition, the schedule-induced polydipsia (SIP) rodent model for obsessive-compulsive disorder was used to evaluate the effects of a novel 5-HT2C receptor agonist, WAY163909. See, for example, Rosenzweig-Lipson S. et al. Psychopharmacology (Berl) 2007, 192, 159-70. The effectiveness of the methods and compositions of the invention in treating OCD, or the cognitive impairment or disorders associated with OCD, can be assessed in the animal models described above and other animal models developed for OCD, as well as in humans with OCD, using a variety of cognitive tests, known in the art, as discussed here.

Drug addiction

Drug addiction (eg drug addiction, alcoholism) is a mental disorder. Addiction does not occur immediately after exposure to the substance being abused. Rather, it involves multiple, complex neural adaptations evolving with varying dynamics ranging from hours to days to months (Kauer J. A. Nat. Rev. Neurosci. 2007, 8, 844-858) . The path to addiction usually begins with the voluntary use of one or more controlled substances, such as narcotics, barbiturates, methamphetamines, alcohol, nicotine, and any number of other such controlled substances. Over time, with increasing use of the controlled substance(s), the ability to voluntarily abstain from the controlled substance(s) decreases due to the effect of long-term use on brain function, and thus behavior. Also, addiction is usually characterized by a compulsive craving for seeking and using the substance, which persists even in the face of negative consequences. The cravings may represent changes in the patient's underlying neurobiology, which likely need to be addressed in a meaningful way if recovery is to be achieved. Drug dependence is also characterized in many cases by withdrawal, which for some substances is life-threatening (e.g. alcohol, barbiturates) and for others can lead to significant morbidity (which may include nausea, vomiting, fever, dizziness and profuse sweating) , exhaustion and reduced ability to recover, for example, alcoholism, also known as alcohol dependence, is one type of such drug dependence. Alcoholism is primarily characterized by four symptoms, including cravings, loss of control, physical dependence, and tolerance. These symptoms may also characterize dependence on other controlled substances. Cravings for alcohol, as well as other controlled substances, are often as strong as the need for food or water. Thus, an alcoholic may continue to drink despite serious family, health, and/or legal consequences.

Recent work investigating the effects of alcohol, central stimulant and opiate abuse on the central nervous system (CNS) has demonstrated a variety of adverse mental health effects, including substance-induced impairments in cognition. See, Nyberg F. Cognitive Impairments in Drug Addicts, Chapter 9. In several laboratories and clinics, cases of significant damage to brain function due to the use of these drugs have been observed. Among the adverse effects of abused drugs on the brain are those that contribute to accelerated aging. An observation that has received particular attention in recent years is that chronic addicts show marked deterioration in areas of the brain associated with executive function and memory function. Marked neuroadaptation induced by addictive substances such as alcohol, central stimulants and opiates results in reduced neurogenesis in the subgranular zone (SGZ) of the hippocampus.

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Indeed, it has been suggested that reduced neurogenesis in the adult SGZ may alter hippocampal function in a way that promotes relapse and persistence of addictive behavior. This also raises the possibility that reduced neurogenesis may contribute to the cognitive impairment caused by these abused substances.

The invention relates to methods and compositions for the treatment of drug dependence using a positive allosteric modulator of the α5-containing GABA receptor, such as selected from the compounds or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, isomers or combinations, as described here. In some embodiments, the implementation of the treatment includes preventing or slowing the progression of drug dependence. In some embodiments, the implementation of the treatment includes alleviating, alleviating or slowing the progression of one or more symptoms associated with drug dependence. In some embodiments, the symptom being treated is a cognitive impairment. For example, the methods and compositions of the present disclosure may be used to treat cognitive impairment and/or improve cognitive function in drug dependent patients. In some embodiments, the invention relates to a method of maintaining or improving cognitive function in a drug dependent patient, comprising the step of administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof.

Several animal models have been developed to study drug addiction. For example, models of genetically selected alcohol-preferring Marchigian Sardinian (msP) alcohol rats have been developed to study the neurobiology of chronic alcoholism. See, Ciccocioppo R. et al. Substance addiction Biology 2006, 11, 339-355. The effectiveness of the methods and compositions of the invention in treating drug addiction or drug addiction-related cognitive impairment can also be assessed in animal models of drug addiction, as well as in human drug addiction, using a variety of cognitive tests known in the art, such as discussed here.

Research Area Criteria (RDoC)

The invention also relates to methods and compositions for the treatment of deterioration in neurological disorders and neuropsychiatric conditions using a positive allosteric modulator of α5-containing GABA R or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer or combination thereof, as described here. In some embodiments, treatment includes alleviating, alleviating, or slowing the progression of one or more symptoms associated with such deterioration. In another aspect, the invention relates to methods and compositions for maintaining or improving cognitive function in a patient using a compound of the invention, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or combination thereof.

It is expected that the RDoC criteria will increase clinical criteria such as DSM and ICD for the diagnosis of disease and disorders affecting the nervous system (see, for example, Am. J. Psychiatry 167:7 (2010)). The RDoC is intended to provide classification based on discoveries in genomics and neuroscience, as well as clinical observations. High expression of α5-containing GABA receptors in specific neural pathways in the nervous system may be therapeutic targets for neural pathway dysfunction identified by RDoC.

α5 GABA subunit binding assays and positive allosteric receptor modulator activity

The affinity of test compounds for the GABA receptor, including the α5 GABA subunit, can be determined using receptor binding assays that are known in the art. See, for example, US Pat. No. 7,642,267 and US Pat. No. 6,743,789, which are incorporated herein by reference.

The activity of test compounds as positive allosteric modulators of α5-containing GABAA R can be tested by electrophysiological methods known in the art. See, for example, US Pat. No. 7,642,267 and Guidotti et al., Psychopharmacology 180: 191205, 2005. Positive allosteric modulator activity can be tested, for example, by testing for GABA-induced chloride ion conductance of GABA receptors comprising the α5 GABA subunit. Cells expressing such receptors may be exposed to an effective amount of a compound of the invention. Such cells can be brought into contact in vivo with the compounds of the invention through contact with a body fluid containing the compound, for example through contact with cerebrospinal fluid. In vitro tests can be performed by contacting cells with a compound of the invention in the presence of GABA. Increased GABA-induced chloride ion conductance in cells expressing GABA receptors, including the α5 GABA subunit, in the presence of a test compound is indicative of positive allosteric modulator activity of said compound. Such changes in conductivity

- 51 039381 can be detected, for example, using a potential clamp test performed on oocytes

Xenopus injected with mRNA of the GABA _a receptor subunit (including RNA of the α5 subunit

GABA _A ), HEK 293 cells transfected with plasmids encoding receptor subunits

GABAA, or in vivo, ex vivo or cultured neurons.

In summary, the invention may be represented by the following embodiments.

Embodiment 1: Compound of formula II

or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which m is an integer selected from 0-1;

R ¹ is absent or selected from halogen, (C1-C ₁₂ )aliphatic radical-, -O-(C1C ₁₂ )aliphatic radical, -OCF ₃ , -CF ₃ ;

R ² is selected from: halogen, -R, -C(O)OR, C(O)R, -(C(R) ₂ ) _1-3 -OR, -(CR) ₁ . ₃ R, -(C(R) ₂ ) _1-3 NR ₂ , C(O)N(R) ₂ ;

R ³ is selected from halogen, -R, -C (O) OR, -CN, -C(O)R, -C(O)NH ₂ , -(C(R) ₂ ) _1-3 -OR, -SO2R ;

R ⁴ , R ⁵ and R ⁶ are H;

each R is independently selected from

H-, (C ₁ -C ₁₂ )aliphatic radical-, (C6-C10)aryl, (C ₆ -Ci ₀ )aryl-(C 1-C ₁₂ )aliphatic radical,

3-10 membered heterocyclyl,

5-10-membered heteroaryl and where the specified heterocyclyl contains 1-4 heteroatoms, independently selected from N and O, and the specified heteroaryl contains 1-4 heteroatoms, independently selected from N and O;

where in each occurrence R is independently substituted with 0-5 R';

or if two R groups are attached to the same atom, the two R groups together with the atom to which they are attached may form a 3-10 membered aromatic or non-aromatic ring containing 0-1 heteroatoms independently selected from N and O, wherein said ring optionally substituted with 0-5 R' where in each occurrence R' is independently selected from halogen, -R", -OR'', -CN and -CF ₃ ;

where in each case R'' is independently selected from H, -(C1-C ₆ )alkyl, (C ₆ -C ₁₀ )aryl and (C ₆ -C ₁₀ )aryl-(C1C ₁₂ )aliphatic radical.

Embodiment 2: Compound of Formula III

or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which m is an integer selected from 0-1;

R ¹ is selected from halogen, (C ₁ -C ₁₂ )aliphatic-, -O-(C ₁ -C ₁₂ )aliphatic and -OCF3;

R ² is -(CR^R and -C(O)OR;

R ³ is -C(O)OR;

R ⁴ , R ⁵ and R ⁶ are H;

each R is independently selected from

H-, (C1-C ₁₂ ) aliphatic radical and (C ₆ -C ₁₀ ) aryl.

Embodiment 3: Compound of Formula IV

- 52 039381

IV or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer, or Z-isomer thereof, or a combination thereof, wherein

R ¹ is selected from halogen, (C1-C ₁₂ )aliphatic radical, -O-(C ₁ -C ₁₂ )aliphatic radical and -OCF3;

R ² is selected from halo, -OR ⁸ , -SR ⁸ , -(CH2)nOR ⁸ , -(CH ₂ ) _n O(CH ₂ ) _n R ⁸ , where R ² is independently substituted with 0-5 R';

R ³ is selected from halogen, -R, -OR, -CN, -(C (R) ₂ )i_3R, -(C (R^-OR, C(O)OR, C(O)R, C(O) NH2;

R ⁴ , R ⁵ and R ⁶ are H;

each R ⁸ is independently -(C1-C ₆ )alkyl, -(C ₃ -C ₁₀ )cycloalkyl, (C ₆ -C ₁₀ )aryl, or 5-10 membered heteroaryl, wherein said (C ₆ -C ₁₀ )aryl is optionally fused with (C ₆ -C ₁₀ )aryl, and where each R ⁸ is independently substituted with 0-5 R';

m and n are independently integers selected from 0-1;

each R is independently selected from

H-, (C1-C ₁₂ ) aliphatic radical, (C ₆ -C ₁₀ ) aryl-(C ₁ -C ₁₂ ) aliphatic radical,

3-10 membered heterocyclyl,

5-10 membered heteroaryl;

where the specified heterocyclyl contains 1-4 heteroatoms, independently selected from N and O;

where the specified heteroaryl contains 1-4 heteroatoms, independently selected from N and O;

where each R is independently substituted with 0-5 R';

where each R' is independently selected from halogen, -R'', -OR'', -CF ₃ , -OCF ₃ ;

where each R'' is independently selected from H, -(C ₁ -C ₆ )alkyl and (C ₆ -C ₁₀ )aryl.

Embodiment 4: The compound of Embodiment 1 wherein R ² is selected from:

(1) a group consisting of halogen, -R, -(C(R)2) _1-3 R, -(C(R)2) _1-3 -OR, -CF ₃ and -C(O)OR, wherein each R is independently substituted with 0-5 R';

(2) a group consisting of -H, -(C1-C ₆ )alkyl, -CH ₂ -O((C ₁ -C ₆ )alkyl), -(C((C ₁ -C ₆ )alkyl) ₂ ) _1-3 -O ((C ₁ C ₆ ) alkyl), -OH, -CF ₃ , -OCF ₃ , (C ₃ -C ₁₀ ) cycloalkyl, -C (O) O ((C ₁ -C ₆ ) alkyl ), 3-10-membered heterocyclyl, (C ₆ -C ₁₀ ) aryl, 5-10-membered heteroaryl, (C ₆ -C ₁₀ ) aryl-(C ₁ -C ₁₂ ) aliphatic radical, (C ₆ -C ₁₀ )aryl(C1-C ₁₂ )aliphatic radical-O-, said R ² being independently substituted with 0-5 R';

(3) a group consisting of -H, -Me, -Et, propyl, isopropyl, butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, CF ₃ , -C(O)OMe, -C(O)OEt , -CH ₂ OMe, -CH ₂ OEt, -CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ -morpholine, -CH ₂ -pyridine, -CH ₂ Ph, said R ² being substituted with 0-3 R';

(4) -Me substituted with 0-3 R' independently selected from -R'', OR'', oxo, -CH2OR, -CH ₂ NR ₂ , -C(O)N(R)2, -C ( O) OR, -CF3, -OCF3 and -N(R)2, wherein R'' is independently selected from H, -(C ₁ -C ₆ )alkyl, (C ₆ -C ₁₀ )aryl and (C ₆ -C ₁₀ ) aryl-(C ₁ -C ₆ ) alkyl;

(5) a group consisting of -CH2Ph, -CH2CH2Ph, -Ph, -CH2OPh, CH2CH2OPh, -CH2-pyrrolidine, -CH2morpholine, _-CH2 -pyridine and _-CH2Ph , said Ph, pyrrolidine, pyridine or morpholine being substituted 0-5R';

(6) a group consisting of -CH ₂ Ph, -CH ₂ CH ₂ Ph, -Ph, -CH ₂ OPh, CH ₂ CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ morpholine, -CH ₂ -pyridine, and -CH ₂ Ph wherein said Ph, pyrrolidine, pyridine or morpholine is substituted with 0-5 R', wherein said -Ph, pyrrolidine, pyridine or morpholine is substituted with 0-5 R' independently selected from -F, -Cl, -CN , -Me, -Et, -OMe and -OEt; and (7) a group consisting of -Me, -C(O)OEt, -CH ₂ Ph, -CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ -pyridine, or -CH ₂ -morpholine, said -Ph , pyrrolidine, pyridine or morpholine is substituted with 0-3 R' independently selected from -F, -Me and -OMe.

Embodiment 5: A compound according to any one of Embodiments 1-4, wherein each R ¹ is independently selected from:

(1) a group consisting of halogen, -R, -OR;

(2) a group consisting of halogen, -H, -(C1-C ₆ )alkyl, -O((C ₁ -C ₆ )alkyl) or (3) a group consisting of -H, -F, -Cl, -Br, -OMe, -OEt.

Embodiment 6: The compound according to Embodiment 5, wherein at least one R ¹ is (1) -OR; (2) -O((C ₁ -C ₆ )alkyl) or (3) -OMe.

Embodiment 7: Compound according to Embodiment 1 in which R ³ is selected

- 53 039381 from:

(1) a group consisting of halogen, -R, -CN, -CF ₃ , -SO2R, -C(O)N(R)2, -C(O)R and -C(O)OR, each R independently substituted with 0-5 R';

(2) a group consisting of -H, -F, -Br, -Cl, - (C1-C ₆ )alkyl, -CN, -C-CH, -CF ₃ , -SO ₂ ((C ₁ -C ₆ )alkyl), -C(O)N((C ₁ -C ₆ )alkyl) ₂ , -C(O)NH ₂ , -C(O)((C1-C ₆ )alkyl), -SO ₂ (C ₆ -C ₁₀ ) aryl) -C (O) O ((C ₁ -C ₆ ) alkyl), - (C ₂ C ₆ ) alkenyl, - (C ₂ -C ₆ ) alkynyl, - (C ₆ -C ₁₀ )aryl, 5-10 membered heteroaryl-, wherein said alkyl, alkenyl, alkynyl, aryl or heteroaryl is independently substituted with 0-5 R'; or (3) a group consisting of -H, -C(O)OMe, -C(O)Et, -C(O)NMe ₂ , -C(O)NH2, -C(O)OEt, -C( O) DOS ₂

-CN, -C-CH, -SO ₂ Me, -SO2Et, (tert-butyl), -C(O)OCH ₂ CF ₃ , -C(O)O(isopropylα), -C(O)NEt ₂ , _-CHF2 ,

-SO ₂ Ph(Me), -CF ₃ , -CHF2, -Me, -Et, -Br, -Cl, -CH ₂ Ph,

where R ⁹ is selected from -H, -Me, -Et, -CF ₃ , isopropyl, -tert-butyl and cyclopropyl.

Embodiment 8: The compound according to Embodiment 7, wherein R ³ is:

(1) -C(O)OMe or -C(O)OEt or o-LA (2) ^V , where R ⁹ is selected from -H, -Me, -Et, -CF3, isopropyl, -tert-butyl and cyclopropyl.

Embodiment 9: The compound according to Embodiment 1, wherein m=0-1;

each R ¹ is independently selected from halo, -H, -(C1-C6)alkyl, -OH, -O((C1- _C6 )alkyl), CF3, _OCF3 , said alkyl being independently substituted with 0-5 R';

R2 is selected from -(C1-C ₆ )alkyl, -C(O)O((C1-C ₆ )alkyl), (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical, (C ₆ - C ₁₀ )aryl-O-(C1-C ₁₂ )aliphatic radical, -(5-10 membered heteroaryl)-(C1-C ₁₂ )aliphatic radical, wherein said alkyl, aryl or heteroaryl is independently substituted with 0-5 R';

R ³ is selected from -(C1-C ₆ )alkyl, -SO ₂ ((C ₁ -C ₆ )alkyl), -C(O)N((C ₁ -C ₆ )alkyl) ₂ and -C(O) O((C ₁ -C ₆ )alkyl), said alkyl being independently substituted with 0-5 R';

R ⁴ , R ⁵ and R ⁶ are -H.

Embodiment 10: The connection according to Embodiment 9, wherein m=0 or 1;

when m is 1, at least one R ¹ is halogen or —O((C ₁ -C ₆ )alkyl);

R ² is selected from -(C ₁ -C ₆ )alkyl, (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical and (C ₆ -C ₁₀ )-aryl-O-(C1C ₁₂ )aliphatic radical , wherein said aryl is independently substituted with 0-5 R' independently selected from -F, -Me and -OMe;

R ³ is -C(O)O((C1-C ₆ )alkyl);

R ⁴ and R ⁵ are both -H and

R ⁶ is -H.

Embodiment 11: The compound according to Embodiment 1, wherein m=0-1;

each R ¹ is independently selected from halogen, -H, -(C1-C ₆ )alkyl, O((C ₁ -C ₆ )alkyl), CF3, OCF3, said R ¹ being independently substituted with 0-5 R';

R ² is selected from

-(C ₁ -C ₆ )alkylα, -C(O)O((C1-C ₆ )alkyl), (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical, (C ₆ -C ₁₀ )aryl-O-(C1-C ₁₂ )aliphatic radical, (C ₆ -C ₁₀ )aryl-N(R'')-(C ₁ -C ₁₂ )aliphatic radical, (5-10-membered heteroaryl)-( C1-C ₁₂ )aliphatic radical, (5-10-membered heteroaryl)-O-(C1-C ₁₂ )aliphatic radical,

- 54 039381 (3-10-membered heterocyclyl)-(C1-C ₁₂ )aliphatic radical, wherein R ² is independently substituted with 0-5 R';

R ³ is selected from

-(C1-C6)alkyl, -C=CH, -CN, halogen, -SO2((C6-C1o)aryl), ^((e^alkyl), -C(O)N((C1C ₆ )alkyl) ₂ , -C(O)NH2, -C(O)O( (C1-C ₆ )alkyl), -C(O)((C1-C ₆ )alkyl), -(C ₆ -C ₁₀ )aryl, 5-10 membered heteroaryl, wherein R ³ is independently substituted with 0-5 R';

R ⁴ , R ⁵ and R ⁶ is -H.

Embodiment 12: The compound according to Embodiment 11, wherein m=0 or 1;

when m is 1, at least one R ¹ is halogen or —O((C1-C ₆ )alkyl);

R ² is selected from

-(C1-C ₆ ) alkyl, (C ₆ -C ₁₀ ) aryl-(C ₁ -C ₁₂ ) aliphatic radical, (C ₆ -C ₁₀ ) aryl-O-(C 1-C ₁₂ ) aliphatic radical, (5 -10-membered heteroaryl)-(C1-C ₁₂ )aliphatic radical and (3-10-membered heterocyclyl)-(C1-C ₁₂ )aliphatic radical, wherein R ² is independently substituted with 0-3 R';

R ³ is halogen, -CN, -C=CH. -C(O)NH2, -(C1-C ₆ )alkyl, -C(O) ((C1-C ₆ )alkyl), -C(O)O((C _G C ₆ )alkyl), -SO ₂ (Ph(Me))

moreover, R ³ is independently substituted with 0-3 R' and moreover, R ⁹ is selected from -H, -Me, -Et, -CF ₃ , isopropyl, -OMe, -tert-butyl and cyclopropyl;

R ⁴ , R ⁵ and R ⁶ are -H.

Embodiment 13: The compound according to Embodiment 12, wherein R ³ is

wherein R ⁹ is selected from -H, -Me, -Et, -CF ₃ , isopropyl and -tert-butyl.

Embodiment 14: A compound according to Embodiment 2 wherein m is 0, 1 or 2, and when m is 1 or 2, at least one R ¹ is -O((C1-C ₆ )alkyl);

R ² is selected from -(C1-C ₆ )alkyl and (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical;

R ³ is -C(O)O((C1-C ₆ )alkyl);

R ⁴ and R ⁵ are both -H and

R ⁶ is -H.

Embodiment 15: The compound according to Embodiment 1 wherein m is 0 or 1, and when m is 1, R ¹ is -O((C ₁ -C ₆ )alkyl);

R ² is OR ⁸ , where R ⁸ is (C ₆ -C ₁₀ )aryl substituted with 0-3 halogens;

R ³ is -C(O)O((C1-C ₆ )alkyl);

R ⁴ , R ⁵ and R ⁶ are both -H.

Embodiment 16: The compound according to Embodiment 3, wherein m=0 or 1;

R ² is OR ⁸ , -(CH2)nOR ⁸ , -(CH2) _n O(CH ₂ ) _n R ⁸ where n=1 and where R ⁸ is -(C1C ₆ )alkyl, (C ₆ -C ₁₀ )aryl or 5-10-membered heteroaryl, wherein R ⁸ is independently substituted with 0-3 R';

R ³ is halogen, -H, -CN, -(C1-C ₆ )alkyl, -C(O)((C ₁ -C ₆ )alkyl), -C(O)O((C ₁ -C ₆ ) alkyl),

Moreover, the specified alkyl is independently substituted with 0-3 R';

R ⁹ is selected from -Me, -Et, isopropyl and -CF3;

R ⁴ and R ⁵ are both -H and

R ⁶ is -H.

- 55 039381

Embodiment 17: A compound selected from

Compound Structure one 2 ^Οθ0 ₂ Εί ου ^F o- 3 X o o then with m ΠΊ 4 ΞΕ O X ^Ζ \ΑΑ/ ^Ζ γο ° (1 J GP 5 Q ^z Χ^.χΧ ^z AND J ΠΊ 6 γ ^ζ γΟΟγ ^ζ 1 o I J m 7 F Lr ^C ° ^2I ΜθθΟΟ _Ν Υ eight 1 e-CO ₂ Et ΧΧ/ ^Ν Ά ΛΑ / A MeO NY\ nine ΞΓ ъ οΧϊ and ten I o X <vo th and

- 56 039381

eleven χ- H3CSH^M''^^^ 0 OCH ₃ 12 Q- ^c °a ^E < ^^-C From H ₃ c, 44 b Ρ ^ζ ρ ^Ζ /ΡΡ/ ^Ζ I o ΊΑ ° (/ 1 CH 45 b ^Z XxX7y ^Z k / V o / A ° (/ / ch 46 to ^Ζ χΡΡ \u003d γ ^Ζ to _ O / A ° (/ ( ch -p 47 β ^Ζ Ρ^Ρ ^Ζ k /K ° ΊΑ ° (/ 1 Ch 48 CD O c ^ζ γΡΡγ ^ζ c o R w

- 57 039381

49 ^N ,CQ o MeO ^N \\ ^N ~~/ N' / ''N fifty ^N ψ* N' ' ' N 51 ^N jccTJp MeO ^X N \\/ ^N —' ⁿ 'n ^z 52 G>-CO ₂ Et j fy. MeO ^N / 'AH 53 d)> — CO ₂ Et JC^C 3 MeO ^N / 54 ъ z ^z I o Y> ° XJ and ο Χί^ 55 r />-co ₂ Et ΌΠ MeO^^^yN ο—Ζ \ Ν./- ⁷ V/

- 58 039381

56 ,Ν G /> -CO ₂ Et AL 101 r >—CO ₂ Et /χ,Ν·/ n7=/ 102 Me^^NA ^V-Me ⁿ n 103 GC CO ₂ Et ΌΟ MeO^^^yN__ N o- 104 Г/-CO ₂ Et MeO^^V'N Ν ^ζ ^~° T 105 r /Y-co ₂ Et jX^ MeO^^XN N 7~O ^N X \_V-OMe 106 r /-CO ₂ Et MeO' ^z ^X _f 'N N ^z ^~ ^{Br n} 'N

- 59 039381

- 60 039381

114 AAO-d Al ^y 'N''N 115 I 7A z /A^Al ^z ' o z 116 -n A ^z z—a zAA/z' o z 117 Al nh ₂ ^N A 118 CD A 1% 119 5.9 1.....1 A o <D 120 _Vo 0 Al A o <D 2

- 61 039381

121 0 122 ΧΓ MeO^yN_y-Z \ ^ν ίΤ ^W 123 CD O o ^z ό 124 About Xu Τ about CD 125 a ^r P° £3- 126 ^? 4°x0 127 ΑΓΆ ^N V

- 62 039381

128 CD O : .. ; ; 129 kV-Br MeSU^''\^-M_oMe ^Ν Ά 130 P'N IX%' ^Y MeO^^N^-N_oMe 131 .cd __/©Me 132 ^a _? l ^N N 133 CkI. ^N V 134 Q 1<

- 63 039381

135 yau. ^N Y 136 X m , )C f/Z-co^t 137 L Yf ^yN QPh II l____/ ^N V 138 f| II F^ _O Ph 139 f| /A II F^ ^{N'N Z} 140 f| Αχ /Λ ' /AA •\N--0/ N-n II χ-y ^N V /yak 141 0 X ^N -b ^ ^ν Ά "l_ Ν I / I ^ν - _Ν

- 64 039381

A 142 A MeO' H J / X^VN ^N A A 143 t| x° A II X\ ^JAN v N=\ / \__ 144 t| LA w II F^- ^N V // ^N ? 145 t| II F^ ^N N 146 A < ^N Λ _Х CL· j ^N V // ^N- ° 147 i| \ζ ^ν Α ¹ JI F^ XX^N one \ °\X^ ^Ν 'Ν ^/λ ~ L 148 i| y ^N A II F^ one \ ^N v

- 65 039381

149 -π 150 ο %-Ζ Ζ-Ζ LL 151 b ^ζ ί-ό 152 ' A'Vn ΓΎ ) 153 αΧ° ^Ν ν 154 b ^Ζ γ^\^γ ^Ζ ο 155 b F

- 66 039381

156 F^^jy^PPh 157 Λ-CN .co F __yph 158 /χ,ΝΧ ίΎ ) __yPh 159 '//2. X" F __y Ph 160 rV/l/ ^F y_ ^Nv 161 Xr* ^OQN v 162 y ^N \_y ^N ~ ^o MeO^^yN QPh NA— ^{7 N} 'N

- 67 039381

163 MeO^/yN O~^7^\ 1l 164 4D jO / MeO-^^^yN_OMe ^N 4i 165 XX V o ω 166 ιχΑ o MeC/XyN_ 167 P~N ΜθΟ'^Χ^Ν-χ^ ^N V 168 S CD O i z ^z Ό z^Z .ύ ⁷ 169 iJvV MeO^X%yN_mind

- 68 039381

or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer, or Z-isomer thereof, or a combination thereof.

Embodiment 18: A pharmaceutical composition useful in the treatment of a cognitive impairment associated with a central nervous system (CNS) disorder, comprising a therapeutically effective amount of a compound according to any one of claims 1 to 17, or a pharmaceutically acceptable amount thereof

- 69 039381 salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in a therapeutically effective amount; and an acceptable carrier, adjuvant or vehicle.

Embodiment 19: The pharmaceutical composition according to Embodiment 18, said composition further comprising a second therapeutic agent, wherein the second therapeutic agent is selected from an antipsychotic or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof; an N-methyl-D-aspartate (NMDA) receptor antagonist or polymorph thereof; or an acetylcholine esterase inhibitor (AChE-I) or a pharmaceutically acceptable salt or polymorph thereof.

Embodiment 20: The pharmaceutical composition according to Embodiment 19 wherein the neuroleptic is selected from aripiprazole, olanzapine and ziprasidone and their pharmaceutically acceptable salts, hydrates, solvates and polymorphs.

Embodiment 21: The pharmaceutical composition according to Embodiment 19, wherein the N-methyl-D-aspartate (NMDA) receptor antagonist is memantine, a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

Embodiment 22: The pharmaceutical composition according to Embodiment 19 wherein the acetylcholine esterase inhibitor (AChE-I) is selected from Donepezil, Galanthamine and Rivastigmine and their pharmaceutically acceptable salts, hydrates, solvates and polymorphs.

Embodiment 23: A method of treating a cognitive impairment associated with a central nervous system (CNS) disorder in a patient, comprising the step of administering a therapeutically effective amount of a compound according to any of Embodiments 1-17 or a pharmaceutical composition according to any of Embodiments 18-22.

Embodiment 24: The method according to Embodiment 23 wherein the central nervous system disorder is

1) age-related cognitive impairment, in particular mild cognitive impairment (MCI), in particular amnesic mild cognitive impairment (AMCI);

(2) dementia, in particular Alzheimer's disease;

(3) schizophrenia or bipolar disorder;

(4) amyotrophic lateral sclerosis (ALS);

(5) post-traumatic stress disorder (PTSD);

(6) associated with cancer therapy;

(7) mental retardation;

(8) Parkinson's disease (PD);

(9) autism;

(10) compulsive behavior or (11) drug addiction.

The skilled artisan will appreciate that the methods described herein may be adapted and modified according to the intended use, and that the methods described herein may be used in other suitable applications, and that such other additions and modifications do not depart from the scope of the present invention.

This invention will be better understood on the basis of the following examples. However, one skilled in the art will appreciate that the particular methods and results discussed are merely illustrative of the invention, as described more fully in the embodiments that follow.

Example 1 Synthesis of Compound 1

- 70 039381

Scheme 11.

A solution of NaNO2 (2.05 g, 29.7 mmol) in H2O ( 19 ml). The internal temperature was maintained below 10°C. After the addition, the mixture was stirred at room temperature for 1 hour. The diazonium salt was collected by filtration and used in the next step. A solution of NaN ₃ (1.93 g, 29.6 mmol) in H2O (7 mL) was added dropwise to the diazonium salt in the crystallization dish with rapid stirring at room temperature. After gas evolution ceased (3 h), the solution was filtered. The collected solid was recrystallized from MeOH to give 4.342 g (75% yield over 2 steps) of product 13 as a yellow solid. Et ₃ N (1.67 ml, 12 mmol). After stirring the mixture at room temperature for 60 hours, the initial suspension turned into a clear yellow solution. The solution was concentrated in vacuo and the residue was purified by chromatography (24 g RediSep silica gel column, 10% to 40% EtOAc in hexanes) to give 2.905 g triazole 14 as a yellow solid. MS: [M+1]=379.

The above triazole 14 (2.95 g, 7.66 mmol) in EtOH (50 ml) with Pd/C (10 wt%, 407 mg, 0.38 mmol) was stirred under H ₂ (balloon) for 24 h This mixture was filtered through Celite. The filtrate was concentrated and the residue was purified by chromatography (24 g RediSep silica gel column, 10% to 50% EtOAc in hexanes) to give 2.453 g of aniline 15 as a white solid. (70% yield over two steps.) MS: [M+1]=349.

Compound 15 (2.45 g, 7.03 mmol) and a catalytic amount of p-TsOH·H ₂ O (24 mg) in p-xylene (30 ml) were heated at 140° C. in an oil bath overnight. The mixture was cooled and filtered. The solid was washed with cold EtOAc. After drying it gave 1.88 g (88% yield) of lactam 16. MS: [M+1]=303.

To a suspension of lactam ester 16 (837 mg, 2.77 mmol) in THF (20 ml) at room temperature was added LiBH ₄ (2M in THF, 1.39 ml, 2.78 mmol). After the mixture was stirred at room temperature for 60 h, additional LiBH ₄ (2M in THF, 0.28 mL, 0.56 mmol) was added and stirred at room temperature for an additional 24 h. EtOAc/EtOH ( 10 ml/10 ml) and the mixture was concentrated in vacuo. The residue was taken up in EtOAc/CH2Cl2/MeOH and free silica gel was added. After evaporating the volatile solvents, the solid was loaded onto a 24 g RediSep silica gel column. Chromatography (solvent A: EtOAc, solvent B: 10:1 v/v CH ₂ Cl ₂ /MeOH; gradient elution: A to B) gave 540 mg (75% yield) of alcohol 17 as a white solid colors. MS: [M+1]=261.

A solution _{of PPh 3 (} 255 mg, 0.97 mmol) in DMF ( 1 ml) in 20 min. After adding TLC showed that the reaction was complete. Water was added to suppress the reaction and the mixture was extracted three times with EtOAc. The combined extracts were washed successively with H ₂ O, brine and dried over Na2SO4. Filtration and concentration gave the crude product. Chromatography (12 g RediSep silica gel column, CH ₂ Cl ₂ to 30% EtOAc in CH ₂ Cl ₂ ) gave 439.2 mg of a mixture of bromide 18 ([M+1]=324) and Ph ₃ PO. The above mixture (439 mg) in EtOAc/EtOH (8 ml/8 ml) with Pd/C (10 wt%, 200 mg, 0.19 mmol) was stirred under H2 (balloon) for 2 h, then filtered through Celite . The filtrate was concentrated and the residue was purified by chromatography (12 g RediSep silica gel column, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexanes, solvent B: EtOAc; gradient elution: A to B) to give 99 mg (you

- 71 039381 stroke -80% over 2 steps) of product 19 in the form of a white solid. MS: [M+1]=245.

In a separate flask, 1,2,3-triazole (55.3 mg, 0.80 mmol) in CH3CN (1 mL) at 0°C was treated with iPr ₂ NEt (146 μL, 0.84 mmol) followed by POCl ₃ ( 23 µl, 0.25 mmol). The solution was stirred at 0° C. for 2 hours. Lactam 19 was added in one batch, and the resulting suspension was heated at 80° C. in an oil bath for 20 hours. Water was added to suppress the reaction. This mixture was extracted with EtOAc three times. The combined extracts were washed with brine and dried over Na ₂ SO ₄ . Filtration and concentration gave 48.8 mg of crude product 20 which was used directly in the next step. A solution of KO-t-Bu (37.2 mg, 0.33 mmol) in DMF (0.5 ml) was cooled to -50°C. Ethyl isocyanoacetate (40 μl, 0.36 mmol) was added dropwise. The mixture was stirred at -50°C for 1 hour. The above crude product 20 in DMF (1 ml) was added dropwise. The mixture was allowed to warm to 10° C. and stirred at 10° C. for 1 h. Saturated aqueous NH ₄ Cl solution was added and it was extracted with EtOAc three times. The combined extracts were washed successively with water, brine and dried over Na ₂ SO ₄ . Filtration and concentration gave the crude product.

Chromatography (12 g RediSep silica gel column, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexanes, solvent B: EtOAc; gradient elution: 20% to 80% B in A) to give 15 mg ( 21% yield over 2 steps) Compound 1 (Example 1) in the form of an off-white solid. MS: [M+1]=340.

1H-NMR (500 MHz, CDCl3) δ: 7.74 (s, 1H), 7.63 (d, 1H, J=3 Hz), 7.51 (d, 1H, J=8.5 Hz), 7.14 (dd, 1H, J=3.0, 8.5 Hz), 4.44 (q, 2H, J=7.0 Hz), 3.95 (s, 3H), 2.44 (s , 3H), 1.45 (t, 3H, J=7.0 Hz).

Example 2 Synthesis of Compound 2

The compound of example 2 was synthesized by a synthesis similar to that described in example 1 using 5-fluoro-2-nitroaniline as starting material to obtain compound 2 in the form of a light brown solid: MS: [M+1]=328.

1H-NMR (500 MHz, CDCl ₃ ) δ: 7.90 (bd dd, 1H, J=2.5, 8.5 Hz), 7.77 (s, 1H), 7.62 (bd dd , 1H, J=5.0, 9.0 Hz), 7.35 (m, 1H), 4.45 (q, 2H, J=7.0 Hz), 2.45 (s, 3H), 1 .45 (t, 3H, J=7.0 Hz).

Example 3 Synthesis of Compound 3

The compound of example 3 was synthesized by a synthesis similar to that described in example 1 using 2-nitroaniline as a starting material to obtain compound 3 in the form of a light yellow solid: MS: [M+1]=310;

1H-NMR (500 MHz, CDCl ₃ ) δ: 8.161 (bd d, 1H, J=8.5 Hz), 7.81 (s, 1H), 7.66 (m, 3H), 4.45 ( q, 2H, J=7.0 Hz), 2.45 (s, 3H), 1.46 (t, 3H, J=7.0 Hz).

Example 4 Synthesis of Compound 110

The acetamide oxime was azeotropically treated three times in toluene prior to use. To a suspension of acetamide oxime (30 mg, 0.4 mmol) in THF (1 ml) was added NaH 60% in oil dispersion (16 mg, 0.4 mmol). The suspension was stirred at room temperature for 15 minutes. Ester compound 2 (65 mg, 0.2 mmol) was added. The vial containing the ester was rinsed with THF (1 ml) which was added to the reaction mixture. The resulting brown suspension was stirred at room temperature for 30 minutes, then heated at 70° C. for 2:30. The suspension was quenched with MeOH. The solvent was evaporated and the crude oil was purified by chromatography (4 g RediSep silica gel column, eluting with 70% EtOAc in Hexanes) to give 28 mg (41% yield) of product. MS: [M+1]=338.

¹ H-NMR (CDCl ₃ ) δ 7.92 (1H, dd, J=2.5, 8.5 Hz), 7.90 (1H, s), 7.67 (1H, dd, J=4, 5, 9.5 Hz), 7.38(1H, m), 2.51(3H, s), 2.46(3H, s).

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The compound was similarly prepared from compound 1 to give compound 167: MS:

[M+1]=350.

¹ H-NMR (CDC^) δ 7.87 (1H, s), 7.65 (1H, d, J=3 Hz), 7.55 (1H, d, J=9 Hz), 7.17 ( 1Н, dd, J=2.5, 9 Hz),

3.96 (3H, s), 2.5 (3H, s), 2.45 (3H, s).

Scheme 12.

Example 5 Synthesis of Compound 167

Example 6 Synthesis of Compound 4

Et ₃ N (0.7 ml, 5 mmol) was added to a solution of compound 17, obtained as in example 1 (260 mg), in DMSO (4 ml) and CH ₂ Cl ₂ (6 ml), then Py SO ₃ ( 398 mg, 2.5 mmol). This mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into water and extracted with EtOAc three times. The combined extracts were washed successively with H ₂ O, brine and dried over Na ₂ SO ₄ . Filtration and concentration gave 198.5 mg of crude aldehyde 21 which was used without further purification. PhMgBr (1M in THF, 1.54 ml, 1.54 mmol) was added dropwise to a suspension of aldehyde 21 (198.5 mg, 0.77 mmol) in THF (10 ml) at 0°C. This mixture was stirred at 0° C. for 30 min, saturated aqueous NH4Cl was added and the mixture was extracted with EtOAc three times.

The combined extracts were washed with brine and dried over Na ₂ SO ₄ . Filtration and concentration gave 252.9 mg of benzyl alcohol 22 as a brown foamy solid. This material was used in the next step without further purification. To a solution of the above crude alcohol 22 in CH ₂ Cl ₂ (8 ml) with Et ₃ SiH (0.60 ml, 3.76 mmol) was added TFA (0.64 ml, 8.27 mmol). The reaction solution was stirred at room temperature for 4 hours. After concentration, the residue was purified by chromatography (12 g RediSep silica gel column, 20 to 80%, EtOAc in hexanes) to give 34.1 mg (12% yield over four steps) of the reduced product 23 as a white foamy solid. MS: [M+1]=321.

In a separate flask, a solution of 1,2,4-triazole (27 mg, 0.39 mmol) in CH3CN (0.5 ml) at 0°C was treated with i-Pr2NEt (72 μl, 0.41 mmol) followed by POCl3 (11 µl, 0.12 mmol). The mixture was stirred at 0° C. for 2 hours. The lactam material 23 (32.2 mg, 0.1 mmol, t) was added in one batch to the reaction mixture and the mixture was heated at 80° C. in an oil bath for 20 hours. cooled to room temperature and a cream colored solid precipitate was observed. Water (0.5 ml) was added and stirred at room temperature for 5 minutes. The precipitated solid was collected by filtration and washed with 0.5 ml of water, followed by drying under high vacuum to give 15.8 mg (42% yield) of adduct 24 as a fluffy off-white solid. MS: [M+1]=372. A solution of KO-t-Bu (9.5 mg, 85 μmol) in DMF (0.5 ml) was cooled to -50°C. Ethyl isocyanoacetate (10.4 μl, 95 μmol) was added dropwise. The resulting mixture was stirred at -50° C. for 1 hour. Triazole amidine 24 (15.8 mg, 42 μmol, solid) was added in one batch. The stirred mixture was allowed to warm to 10°C over 1 hour and kept at 10°C for 1 hour. Saturated aqueous NH ₄ Cl was added and extracted with EtOAc three times. The combined extracts were washed successively with H ₂ O, brine and dried over Na 2 SO 4 . Filtration and concentration gave the crude product. Chromatography (4 g RediSep silica column. Solvent A: 1:1 v/v CH ₂ Cl ₂ /hexanes, Solvent B:

EtOAc; gradient elution: A to 50% B in A) gave 16.8 mg (95% yield) of Example 6 as a white solid. MS: [M+1]=416.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.74 (s, 1H), 7.63 (d, 1H, J=3.0 Hz), 7.50 (d, 1H, J=9, 0 Hz), 7.30 (bd d, 2H, J=7.0 Hz), 7.29 (bd 2H, 7.5 Hz), 7.20 (m, 1H), 7.13 (dd, 1H, J=2.5, 9.0 Hz), 4.41 (q, 2H, J=7.5 Hz), 4.17 (s, 2H), 3.95 (s, 3H) , 1.43 (t, 3H, 7.5 Hz).

Example 7 Synthesis of Compound 5

The compound of example 7 was synthesized by a synthesis similar to that described in example 6 using 2-nitroaniline as starting material to obtain compound 5 in the form of a brown solid: MS: [M+1]=386.

1H-NMR (500 MHz, CDCl3) δ: 8.16 (broad d, 1H, J=7.0 Hz), 7.81 (s, 1H), 7.60-7.68 (m, 3H) , 7.34 (br d, 2H, J=8.0 Hz), 7.29 (br d, 2H, J=7.0 Hz), 7.20 (m, 1H), 4.42 ( q, 2H, J=7.0 Hz), 4.18 (s, 2H), 1.44 (t, 3H, J=7.0 Hz).

Example 8 Synthesis of Compound 6

The compound of example 8 was synthesized by a synthesis similar to that described in example 6 using 5-fluoro-2-nitroaniline as a starting material to obtain compound 8 in the form of a brown solid: MS: [M+1]=404.

1H-NMR (500 MHz, CDCl ₃ ) δ: 7.90 (dd, 1H, J=3.5, 8.5 Hz), 7.77 (s, 1H), 7.61 (dd, 1H, J =5.0, 10.5 Hz), 7.28-7.37 (m, 5H), 7.21 (m, 1H), 4.43 (q, 2H, J=7.0 Hz), 4 .17 (s, 2H), 1.44 (t, 3H, J=7.0 Hz).

Example 9 Synthesis of Compound 44

The compound of example 9 was synthesized by a synthesis similar to that described in example 6 using 5-fluoro-2-nitroaniline as a starting material to obtain the compound of example 9 in the form of a brownish solid: MS: [M+1]=418.

1H-NMR (500 MHz, CDCl3) δ: 7.89 (broad d, 1H, J=9.5 Hz), 7.76 (s, 1H), 7.60 (dd, 1H, J=5, 5, 10.0 Hz), 7.35 (br t, 1H, J=6.0 Hz), 7.22 (br d, 2H, J=8.5 Hz), 7.09 (br d , 2H, J=7.5 Hz), 4.43 (q, 2H, J=7.5 Hz), 4.12 (s, 2H), 2.30 (s, 3H), 1.44 (t , 3H, J=7.5 Hz).

Example 10 Synthesis of Compound 45

The compound of Example 10 was synthesized by a synthesis similar to that described in Example 6 using 5-fluoro-2-nitroaniline as starting material to give the compound of Example 10 as a brownish solid: MS: [M+1]=438.

1H-NMR (500 MHz, CDCl3) δ: 7.90 (dd, 1H, J=3.0, 8.0 Hz), 7.77 (s, 1H), 7.61 (dd, 1H, J= 5.0, 9.0 Hz), 7.36 (m, 1H), 7.25 (broad s, 4H), 4.42 (q, 2H, J=7.0 Hz), 4.14 ( s, 2H), 1.44 (t, 3H, J=7.0 Hz).

Example 11 Synthesis of Compound 46

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The compound of example 11 was synthesized by a synthesis similar to that described in example 6 using 5-fluoro-2-nitroaniline as a starting material to obtain the compound of example 11 in the form of a yellowish solid: MS: [M+1]=422.

D-NMR (500 MHz, CDCl ₃ ) δ: 7.90 (dd, 1H, J=3.0, 8.5 Hz), 7.77 (s, 1H), 7.61 (dd, 1H, J =5.0, 9.0 Hz), 7.36 (m, 1H), 7.28 (m, 2H), 6.96 (m, 2H), 4.42 (q, 2H, J=7, 5 Hz), 4.14 (s, 2H), 1.44 (t, 3H, J=7.0 Hz).

Example 12 Synthesis of Compound 47

The compound of example 12 was synthesized by a synthesis similar to that described in example 6 using 2-nitroaniline as a starting material to obtain the compound of example 12 in the form of a yellowish solid: MS: [M+1]=420.

'H-NMR (500 MHz, CDCl ₃ ) δ: 8.16 (broad d, 1H, J=7.0 Hz), 7.80 (s, 1H), 7.64 (m, 3H), 7 .25 (m, 4H), 4.41 (q, 2H, J=7.0 Hz), 4.14 (s, 2H), 1.44 (t, 3H, J=8.0 Hz).

Example 13 Synthesis of Compound 109

Acetamide oxime (50 mg, 0.67 mmol) was azeotropically treated with toluene 3 times. THF (5 ml) was added followed by NaH 60% in oil dispersion (25 mg, 0.62 mmol). The suspension was stirred at room temperature for 30 min, 2 ml of this suspension was added to ester compound 6 (40 mg, 0.099 mmol) and the final solution was heated at 70° C. for 3 h. The solution was quenched with water. The solution was extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (12 g RediSep silica gel column. Elution with 50% EtOAc in Hexanes) gave 6 mg (20% yield) of Compound 109 as a yellow solid. MS: [M+1]=414).

NMR (CDCl ₃ ) δ 7.93 (1H, dd, J=3, 8.5 Hz), 7.89 (1H, s), 7.65 (1H, dd, J=5.5.9 Hz), 7.38(1H, m), 7.23(5H, m), 4.2(2H, s), 2.50(3H, s).

Example 14 Synthesis of Compound 7

To a stirred mixture of 5-methoxy-2-nitroaniline (5 g, 29.7 mmol) in HCl (conc. 12.9 ml) at 0°C was added NaNO ₂ solution (2.05 g, 29.7 mmol) dropwise. in H ₂ O (8 ml). The internal temperature was maintained below 5°C. After the addition, the mixture was allowed to warm to room temperature over 1 h. The mixture was cooled to 0°C and a solution of SnCl ₂ -2H ₂ O (20.13 g, 89.2 mmol) in HCl (conc. 13 ml) was added slowly over drops. After the addition, this mixture was stirred at room temperature for 2 hours. The resulting yellow solid was collected by filtration and washed with cold (0°C) 6N. HCl. Drying in a vacuum oven gave 3.245 g (50% yield) of a brown solid aryl hydrazine 25. MS: [M+H ₂ O+Na]=224. In a separate flask, a mixture of diethyl 1,3-acetone dicarboxylate (2.426 g, 12 mmol) and diethoxymethyl acetate (1.946 g, 12 mmol) was heated under microwave irradiation at 100°C for 1 h. The reaction mixture was concentrated in vacuo and the residual volatile component co-evaporated with toluene (5 ml) in vacuo to give condensation product 26, which was used directly in the next step.

- 75 039381

Scheme 13

Product 26 above was dissolved in EtOH (30 ml). Molecular sieves (4 A, 2 g) and hydrazine hydrochloride 25 (2.19 g, 10 mmol) were added. The suspension was stirred at room temperature for 24 hours. The mixture was filtered through Celite and the solid was washed with EtOAc (10 mlx3). The filtrate was concentrated. The residue was purified by chromatography (40 g RediSep silica gel column, 10 to 40% EtOAc in hexanes) to give 2.091 g of pyrrole 27, which was used without further purification in the next step. MS: [M+1]=378.

The above nitro group at 27 (2.09 g, 5.5 mmol) was reduced in EtOH (40 ml) with Pd/C (10 wt%, 295 mg, 0.28 mmol) under H ₂ (balloon) for 18 h The mixture was filtered through Celite. The filtrate was concentrated and the residue was purified by chromatography (24 g RediSep silica gel column, hexanes to 50% EtOAc in hexanes) to give 1.127 g of non-cyclized product 28 as a viscous yellow oil ([M+1]=348), plus 154 mg of cyclized product 29 as a gray solid (MS: [M+1]=302). Non-cyclized aniline 28 (1.127 g, 3.2 mmol) in p-xylene (20 ml) was treated with a catalytic amount of p-TsOH-H ₂ O (15 mg) at 140° C. in an oil bath for 20 hours. The reaction mixture was cooled, concentrated and the residue was triturated with cold (0°C) EtOAc. Filtration gave 559 mg of the lactam product 29 as a yellow solid. The total weight of the combined lactam 29 product is 713 mg (24% over 3 steps). MS: [M+1]=302.

Dibal-H (1M in hexane, 6.60 ml, 6.60 mmol) was added to a suspension of ester 29 (566 mg, 1.88 mmol) in CH ₂ Cl ₂ (35 ml) at -78°C.

The suspension was stirred for 10 min at -78°C. The cold bath was removed and the mixture was stirred for 20 minutes while the temperature was raised to room temperature. At this point, TLC showed -80% completion of the reaction. The mixture was cooled to -78° C. and additional Dibal-H (1M in hexane, 1.0 ml, 1.0 mmol) was added. After stirring at -78° C. for 30 minutes, LCMS indicated completion of the reaction. The reaction was stopped by adding an aqueous Rochelle salt solution (20%) followed by EtOAc. The mixture was vigorously stirred at room temperature until it became a clear two layer mixture. The layers were separated and the aqueous layer was extracted with EtOAc three times. The combined organic phase was washed with brine and dried over Na ₂ SO ₄ . Filtration and concentration gave 480 mg of crude alcohol 30 as a yellowish solid. MS: [M+1]=260.

To a solution of alcohol 30 (200 mg, 0.77 mmol) and CBr ₄ (640 mg, 1.93 mmol) in DMF (8 ml) was added slowly over 30 min a solution of PPh ₃ (486 mg, 1.85 mmol) in DMF (2 ml) . After the addition, this mixture was stirred at room temperature for 30 minutes. Water was added to suppress the reaction and the mixture was extracted with EtOAc three times. The combined extracts were washed successively with H ₂ O, brine and dried over Na ₂ SO ₄ . Filtration and concentration gave the crude product. Chromatography (12 g RediSep silica gel column, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexanes, solvent B: EtOAc; gradient elution: 10 to 40% B in A) gave 221 mg bromide 31 and Ph ₃ PO.

The above mixture in EtOAc/EtOH (8 ml/8 ml) with Pd/C (10 wt%, 200 mg, 0.19 mmol) was stirred under H ₂ (balloon) for 1 h. This mixture was filtered through Celite. The filtrate was concentrated and the residue was purified by chromatography (12 g RediSep silica gel column, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexanes, solvent B: EtOAc; gradient elution: 10 to 40% B in A) to obtain 146 mg of a mixture of reduction product 32 ([M+1]=244) and Pn ₃ RO.

In a separate flask, 1,2,4-triazole (81 mg, 1.17 mmol) in CH3CN (1 ml) at 0°C was treated with i-Pr ₂ NEt (214 μl, 1.23 mmol) followed by POCl ₃ ( 34 µl, 0.36 mmol). The solution was stirred at 0°C for 2 hours.

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Lactam 32 (~60% pure LCMS) was added in one batch and the resulting slurry was heated at 80° C. in an oil bath for 18 hours. Water was added to quench the reaction. This mixture was extracted with EtOAc three times. The combined extracts were washed successively with H ₂ O, brine and dried over Na ₂ SO4. Filtration and concentration gave 126.6 mg of crude product 33 in the form of a yellow goo, which was used directly in the next reaction. MS: [M+1]=295. A solution of KOt-Bu (97 mg, 0.86 mmol) in DMF (1 ml) was cooled to -50°C. Ethyl isocyanoacetate (104 μl, 0.95 mmol) was added dropwise. The mixture was stirred at -50° C. for 1 hour. The above crude product 33 in DMF (1.5 ml) was added dropwise. The mixture was allowed to warm to 10° C. and stirred at 10° C. for 1 h. Saturated aqueous NH ₄ Cl solution was added and extracted with EtOAc three times. The combined extracts were washed successively with water, brine and dried over Na ₂ SO ₄ . Filtration and concentration gave the crude product. Chromatography (12 g RediSep silica gel column, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexanes, solvent B: EtOAc; gradient elution: 10 to 40% B in A) to give 22 mg of solid white, which was further purified by preparative TLC (developing using 1:1 A/B) to give 12.8 mg of Compound of the final product 7 (Example 14) in the form of a white solid. MS: [M+1]=339.

1H-NMR (500 MHz, CDCI3) δ: 7.70 (s, 1H), 7.56 (s, 1H), 7.50 (d, 1H, J=3.0 Hz), 7.43 (d , 1H, J=8.5 Hz), 7.00 (dd, 1H, J=2.5, 9.5 Hz), 5.29 (broad s, 1H), 4.44 (q, 2H, J=7.0 Hz), 3.92 (s, 3H), 3.55 (broad s, 1H), 2.17 (s, 3H), 1.45 (t, 3H, J=7.0 Hz).

Example 15 Synthesis of Compound 8

Scheme 14.

connection 8

To a solution of alcohol 30 (261 mg, 1.0 mmol), which was obtained in example 14, in DMSO (4 ml) and CH ₂ Cl ₂ (6 ml) was added Et ₃ N (0.7 ml, 5 mmol), then Py SO ₃ (398 mg, 2.5 mmol). This mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into water and extracted with EtOAc three times. The combined extracts were washed successively with H ₂ O, brine and dried over Na ₂ SO ₄ . Filtration and concentration gave 226 mg of crude aldehyde 34 as a yellow solid. This material was used in the next step without purification. MS: [M+1]=258.

PhMgBr (1 M in THF, 1.58 ml, 1.58 mmol) was added dropwise to a suspension of crude aldehyde 34 (202 mg, 0.79 mmol) in THF (10 ml) at 0°C. This mixture was stirred at 0° C. for 30 min, a saturated aqueous solution of NH ₄ Cl was added and the mixture was extracted with EtOAc three times. The combined extracts were washed with brine and dried over Na ₂ SO ₄ . Filtration and concentration gave 275 mg of crude product 35 as a yellow foamy solid, which was used in the next step without purification.

To a solution of the above crude alcohol 35 in CH ₂ Cl ₂ (10 ml) with Et3SiH (0.66 ml, 4.10 mmol) was added TFA (0.70 ml, 9.02 mmol). The reaction solution was stirred at room temperature for 1 hour. After concentration, the residue was purified by chromatography (24 g RediSep silica gel column, 10% to 50%, EtOAc in hexanes) to give 187.8 mg (59% yield over three steps) of product 36 as a gray solid. MS: [M+1]=320.

In a separate flask, a solution of 1,2,4-triazole (127 mg, 1.83 mmol) in CH3CN (1.6 ml) at 0°C was treated with i-Pr ₂ NEt (336 μl, 1.93 mmol) followed by POC13 (53 µl, 0.56 mmol). The mixture was stirred at 0° C. for 2 hours. Lactam 36 (150 mg, 0.47 mmol, solid) was added in one batch to the reaction mixture and the mixture was heated at 80° C. in an oil bath for 18 hours. The mixture was cooled to room temperature and a solid precipitate was observed. Water (2.1 ml) was added and stirred at room temperature for 10 minutes. Filtration, washing the solid with 2 ml of water followed by drying under high vacuum gave 118.8 mg (69% yield) of triazole amidine 37 as a fluffy off-white solid. MS: [M+1]=371. A solution of KO-t-Bu (72 mg, 0.64 mmol) in DMF (2 ml) was cooled to

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-50°C. Ethyl isocyanoacetate (77 µl, 0.71 molecular weight) was added dropwise. The resulting mixture was stirred at -50° C. for 1 hour. Triazole amidine 37 (118.8 mg, 42 μmol, solid) was added in one part. The stirred mixture was allowed to warm to 10°C over 1 hour and kept at 10°C for 1 hour. Saturated aqueous NH ₄ Cl was added and extracted with EtOAc three times. The combined extracts were washed successively with H2O, brine and dried over Na ₂ SO ₄ . Filtration, concentration, then chromatography (12 g RediSep silica gel column, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexanes, solvent B: EtOAc; gradient elution: A to 40% B in A) gave 125.1 mg (94% yield) Compound 8 as white solid. MS: [M+1]=415.

1H-NMR (500 MHz; CDCl ₃ ) δ: 7.72 (s, 1H), 7.54 (s, 1H), 7.51 (br s, 1H), 7.44 (br d, 1H , J=9.5 Hz), 7.29 (broad d, 2H, J=7.5 Hz), 7.20 (m, 3H), 7.01 (broad d, 1H, J=7, 5 Hz), 5.30 (br s, 1H), 4.38 (q, 2H, J=7.0 Hz), 3.92 (br s, 5H), 3.54 (br s, 1H), 1.41 (t, 3H, J=7.0 Hz).

Example 16 Synthesis of Compound 9

Scheme 15.

r. = main, x = main, connection 11

r. = x = main, compound 12

r. = hx=f: connection 107

LiOH (1.09 g, 45.5 mmol) was added to an active solution of ester 16 (prepared in Example 1) (2.75 g, 9.10 mmol) in THF (24 ml) and water (20 ml) at room temperature temperature. MeOH (4 ml) was added and stirring was continued for 2 hours at room temperature, after which LCMS showed complete consumption of the ester. After concentration in vacuo, the reaction mixture was acidified to pH 3-4 by adding 2N. HCl (20 ml). After 20 minutes of stirring, the reaction mixture was cooled to 0° C., the precipitated solid was collected by filtration, washed with 3-4 ml of water and dried to give 1.59 g (64%) of the corresponding acid 38 as a grayish solid. MS: [M+1]=275. To acid 38 (1.59 g, 5.8 mmol) suspended and stirred in DCM (30 mL) was added EDC (5.6 g, 29.2 mmol), benzyl alcohol (2.5 g, 23.2 mmol) and DMAP (3.54 g, 29.2 mmol). After 3 days of stirring at room temperature, the reaction mixture was concentrated in vacuo. Water (80 ml) was added to the suspension followed by diethyl ether (40 ml) and the mixture was stirred vigorously for 40 minutes, after which the suspension turned into a precipitate which was collected by aspiration filtration.

The solid was washed with water and a small amount of diethyl ether and dried to give 1.65 g (78%) of benzyl ester 39 as a white solid. MS:

[M+1]=365.

Compound 1,2,4-triazole (1.22 g, 17.7 mmol) in CH3CN (15 ml) at 0°C was treated with i-Pr ₂ NEt (3.24 ml, 18.6 mmol) followed by POCl3 ( 0.507 ml, 5.44 mmol). The solution was stirred at 0° C. for 2 hours. Benzyl ester 39 (1.65 g, 4.53 mmol) was added in one part and the resulting suspension was heated at 80° C. in an oil bath for 18 hours. LCMS showed 5 -10% remaining original lactam. In a separate flask, 1,2,4-triazole (307 mg, total 4.9 eq.) in CH3CN (3.8 ml) was treated with i-Pr2NEt (0.82 ml, total 5.1 eq.) and POCl ₃ ( 0.127 ml; total 1.5 eq.) at 0° C. for 2 hours. The resulting clear solution was transferred to the above reaction mixture. After 2 h of heating at 80°C, the reaction mixture was cooled to room temperature, water was added slowly to suppress the reaction (10

- 78 039381 min). After cooling in an ice bath, the solids formed were collected by filtration, washed with water (5 ml) and dried to give 1.61 g (86%) of product 40 as a yellowish solid. MS:

[M+1]=416.

A solution of KO-t-Bu (0.739 g, 6.59 mmol) in DMF (11 ml) was cooled to -50°C. Ethyl isocyanoacetate (0.810 mL, 7.00 mmol) was added dropwise. The mixture was stirred at -50° C. for 1 hour. The above-mentioned triazole intermediate 40 (1.61 g, 3.87 mmol) was added. The mixture was stirred at -50° C. for 30 min and slowly warmed to room temperature over 4-5 h. Saturated aqueous NH ₄ Cl (10 ml) was added followed by EtOAc (10 ml). The mixture was sonicated to destroy the solid, then stirred vigorously for 30 minutes. The precipitate was collected by filtration, washed with water, Et2O and dried to give the crude product as a white solid. The filtrate was partitioned between water and EtOAc; the aqueous layer was separated and extracted with EtOAc twice; the combined EtOAc layer was washed with brine and dried over MgSO ₄ . Filtration and removal of solvent gave a solid residue combined with the solid obtained above for chromatographic purification using a 24 g RediSep silica gel column and gradient elution from 0.5 to 5% MeOH in DCM to give 1.78 g (100% ) imidazole 41 in the form of a white solid. MS: [M+1]=460. Benzyl ester 41 (1.78 g, 3.87 mmol) was subjected to hydrolysis (hydrogen balloon) in the presence of a catalytic amount of 10% Pd on activated carbon in a mixture of solvents THF (40 ml), MeOH (20 ml) and EtOAc (20 ml ) within 20 hours. LCMS showed the disappearance of the starting material without a trace. The solid catalyst was removed by Celite filtration and rinsed repeatedly with sufficient 30% MeOH in DCM until nearly all products were recovered (TLC monitoring). The filtrate containing the product was concentrated in vacuo to give 1.22 g (85%) of the acid product 42 as a yellowish solid. MS:

[M+1]=370.

To acid 42 (1.22 g, 3.30 mmol) suspended and stirred in THF (25 mL) was added dropwise a borane-dimethylsulfide complex (2M THF; 19 mL, 38 mmol) at 0°C. The ice bath was removed and the reaction mixture was stirred at room temperature for 16 hours. After cooling in an ice bath, the reaction was carefully stopped with MeOH (20 ml) and then stirred at room temperature overnight. Solvents were removed in vacuo. MeOH was added and removed in vacuo two more times. Purification by ISCO (24 g RediSep column) using a gradient of 1-8% MeOH in DCM gave 0.625 g (53%) of alcohol product 43 as a white solid.

MS: [M+1]=356.

Diisopropyl azodicarboxylate (48.3 mg, 0.233 mmol) was added dropwise to an active solution of alcohol 43 (37.5 mg, 0.106 mmol), phenol (14.9 mg, 0.158 mmol) and Ph ₃ P (55.6 mg, 0.212 mmol) in anhydrous THF (0.8 ml) at 0°C. The ice bath was removed and stirring was continued at room temperature for 16 hours. LCMS showed no trace of starting alcohol. The reaction mixture was divided between sat. _NaHCO3 and EtOAc. The organic layer was separated and washed with water, brine and dried over MgSO ₄ . The desired product was isolated from the reaction mixture by two consecutive preparative TLCs (4% MeOH in DCM and hexanes/EtOAc/MeOH=47.5/47.5/5, v/v/v) to give 5.3 mg (12 %) of the product, which is Compound 9 in the form of a white solid. MS: [M+1]=432.

1H-NMR (500 MHz, CDCl3) δ: 7.77 (s, 1H), 7.63 (d, 1H, J=3.5 Hz), 7.53 (d, 1H, J=9.0 Hz ), 7.31 (m, 2H), 7.17 (dd, 1H, J=3.0, 8.5 Hz), 7.08 (d, 2H, J=7.0 Hz), 6.99 (t, 1H, J=6.5 Hz), 5.30 (s, 2H) , 4.40 (q, 2H, J=7.0 Hz), 3.96 (s, 3H), 1.38 (t, 3H, J=7.0 Hz).

Example 17 Synthesis of Compound 10

The compound of example 17 was synthesized by a synthesis similar to that described in example 16, using 4-fluorophenol in the final stage to obtain Compound 10 (4.9 mg) in the form of a white solid: MS: [M+1]=450.

1H-NMR (500 MHz, CDCl ₃ ) δ: 7.76 (s, 1H), 7.64 (d, 1H, J=3.5 Hz), 7.53 (d, 1H, J=8.0 Hz), 7.17 (dd, 1H, J=2.5, 8.0 Hz), 7.01 (m, 4H), 5.26 (s, 2H), 4.40 (q, 2H, J =7.0 Hz), 3.96 (s, 3H), 1.40 (t, 3H, J=7.0 Hz).

- 79 039381

Example 18 Synthesis of Compound 11

The compound of Example 18 was synthesized by a synthesis similar to that described in example 16, using 3-methoxyphenol in the final step to obtain Compound 11 (6.1 mg) in the form of a white solid: MS: [M+1]=462.

¹ H-NMR (500 MHz, CDCl3) δ: 7.76 (s, 1H), 7.63 (d, 1H, J=2.5 Hz), 7.53 (d, 1H, J=9.0 Hz), 7.15-7.22 (m, 2H), 6.67 (m, 2H), 6.55 (bd dd, 1H, J=2.5, 8.0 Hz), 5.28 (s, 2H), 4.39 (q, 2H, J=7.0 Hz), 3.96 (s, 3H), 3.81 (s, 3H), 1.39 (t, 3H, J= 7.0 Hz).

Example 19 Synthesis of Compound 12

The compound of example 19 was synthesized by a synthesis similar to that described in example 16, using 2,4-dimethylphenol in the final stage to obtain Compound 12 (3.1 mg) in the form of a white solid: MS: [M+1]=460.

1H-NMR (500 MHz, CDCl3) δ: 7.76 (s, 1H), 7.65 (d, 1H, J=3.0 Hz), 7.53 (d, 1H, J=9.0 Hz ), 7.17 (dd, 1H, J=2.5, 8.5 Hz), 6.98 (m, 3H), 5.26 (s, 2H), 4.37 (q, 2H, J= 7.0 Hz), 3.96 (s, 3H), 2.26 (s, 3H), 2.20 (s, 3H), 1.36 (t, 3H, J=7.0 Hz).

Example 20 Synthesis of Compound 107

Phenol (30 mg, 0.32 mmol) was added to a solution of alcohol 43 where X=F (obtained in a manner identical to the example where X=OCH ₃ ) (60 mg, 0.17 mmol) in THF (0.8 ml) , triphenylphosphine (84 mg, 0.32 mmol). The reaction mixture was stirred at room temperature for 15 minutes. It was then cooled with an ice bath and DIAD (64 μl, 0.32 mmol) in THF (0.2 ml) was added slowly. The ice bath was removed and the reaction mixture was stirred at room temperature for 18 hours. LCMS indicated the presence of a small amount of starting material. Phenol (10 mg), triphenylphosphine (28 mg) and DIAD (21 μl) were added to the reaction mixture and stirred for another 1 h. The solvent was evaporated and the crude material was purified by chromatography (RediSep 12 g silica gel column. Elution solvent: EtOAc) and preparative TLC (eluting solvent: 5% MeOH/47.5% EtOAc/47.5% Hexanes) to give 11.4 mg (16% yield) of Compound 107. [M+1]=421).

1H-NMR (CDCl ₃ ) δ 7.92 (1H, dd, J=3.5, 8.5 Hz), 7.80 (1H, s), 7.63 (1H, dd, J=5, 10 Hz), 7.38 (1H, m), 7.31 (2H, t, J=8.5 Hz), 7.07 (2H, d, J=8.5 Hz), 7.00 (1H, t, J=8.5 Hz), 5.3 (2H, s), 4.39 (2H, q, J=7 Hz), 1.38 (3H, t, J=7 Hz).

Example 21 Synthesis of Compound 111

To a suspension of alcohol 43 (X=Me) (160 mg, 0.47 mmol) in acetonitrile (9 ml) was added POBr ₃ (405 mg, 1.41 mmol). The reaction mixture was heated at 80° C. for 5 hours. The reaction mixture was cooled with an ice bath and sat. aqueous solution of NaHCO ₃ . The final solution was extracted with DCM (3x). The combined organic phases were washed with brine and dried over MgSO ₄ . The solvent was concentrated to give the desired product, 166 mg, 88% yield, [M+1]=403).

To a suspension of the above alkyl bromide derivative (30 mg, 0.075 mmol) in deoxygenated DME (2.7 mL) was added 3-pyridineboronic acid (14 mg, 0.11 mmol) and 2M Na ₂ CO ₃

- 80 039381 (0.22 ml, 0.44 mmol). The suspension was stirred at room temperature for 5 min, then PdCl ₂ (PPh ₃ ) ₂ (10 mg, 0.015 mmol) was added. The suspension was heated in MW at 85° C. for 1 h. The reaction mixture was cooled and diluted with water and extracted with EtOAc (twice). The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration gave a crude product which was purified with 2 preparative TLCs (eluting system: 3% MeOH in DCM) to give 5.3 mg (18% yield) of Compound 111. MS: [M+1]=401.

¹ H-NMR (CDCl ₃ ) δ 8.66 (1H, br s), 8.48 (1H, br s), 7.96 (1H, s), 7.79 (1H, s), 7 .66 (1H, d, J=8 Hz), 7.50 (1H, d, J=8 Hz), 7.43 (1H, d, J=7 Hz), 7.23 (1H, m), 4.42 (2H, q, J=7 Hz), 4.18 (2H, s), 2.54 (3H, s), 1.44 (3H, t, J=7 Hz).

Example 22 Synthesis of Compound 48

Scheme 16.

compound 51

Triethylamine (0.394 ml, 2.82 mmol) and pyridine-sulfur trioxide complex (225 mg, 1.41 mmol). After 3 hours of stirring, the reaction mixture was quenched with water (5 ml) and extracted with ethyl acetate three times. The combined organic solution was washed with water, brine and dried over MgSO ₄ . The aldehyde product 57 was isolated by flash chromatography on an ISCO column (4 g RediSep column) using an elution gradient of 0.5 to 8% MeOH in DCM. 84.4 mg (46%) was obtained as a yellowish foamy solid. MS: [M+1]=354.

Pyrrolidine (5.5 µl, 0.0658 mmol) was added to an active solution of aldehyde 57 (15.5 mg, 0.0439 mmol) in 1,2-dichloroethane (0.3 ml) at room temperature. After 2 min stirring, the solution became clear and NaBH(OAc) ₃ (14.4 mg) was added. The reaction mixture was stirred for 4 hours and quenched with saturated NaHCO3 and extracted with ethyl acetate three times. The combined organic layer was washed with water, brine and dried over Na ₂ SO ₄ . Preparative TLC with 10% MeOH in DCM gave 13.1 mg (73%) of the desired Compound 48 as a clear filmy solid. MS: [M+1]=409.

1H-NMR (500 MHz, CDCl ₃ ) δ: 7.74 (s, 1H), 7.62 (d, 1H, J=3.0 Hz), 7.51 (d, 1H, J=9.0 Hz), 7.14 (dd, 1H, J=3.5, 9.0 Hz), 4.42 (q, 2H, J=6.5 Hz), 3.94 (s, 3H), 3, 87 (br s, 2H), 2.65 (br s, 4H), 1.79 (br s, 4H), 1.44 (t, 3H, J=7.0 Hz).

Example 23 Synthesis of Compound 49

The compound of example 23 was synthesized by a synthesis similar to that described in example 22, using morpholine in the final stage to obtain the compound of example 23 in the form of a transparent film-like solid: MS: [M+1]=425.

1H-NMR (500 MHz, CDCl ₃ ) δ: 7.75 (s, 1H), 7.63 (d, 1H, J=3.0 Hz), 7.52 (d, 1H, J=9.5 Hz), 7.15 (dd, 1H, J=3.0, 9.0 Hz), 4.42 (q, 2H, J=7.5 Hz), 3.95 (s, 3H), 3, 76 (br s, 2H), 3.71 (br s, 4H), 2.57 (br s, 4H), 1.44 (t, 3H, J=8.0 Hz).

Example 24 Synthesis of Compound 50

The compound of example 24 was synthesized by a synthesis similar to that described in example 22, using diethylamine in the final stage to obtain the compound of example 24 in the form

- 81 039381 transparent film-like solid: MS: [M+1]=411.

1H-NMR (500 MHz, CDCl3) δ: 7.74 (s, 1H), 7.64 (broad d, 1H, J=3.0 Hz), 7.51 (d, 1H, J=9, 0 Hz), 7.15 (dd, 1H, J=2.5, 9.0 Hz), 4.43 (q, 2H, J=6.5 Hz), 3.96 (s, 3H) , 3 .86 (br s, 2H), 2.64 (br s, 4H), 1.44 (t, 3H, J=8.5 Hz), 1.15 (br s, 6H).

Example 25 Synthesis of Compound 51

The compound of example 25 was synthesized by a synthesis similar to that described in example 22, using methylbenzylamine in the final stage to obtain the compound of example 25 in the form of a transparent film-like solid: MS: [M+1]=459.

¹ H-NMR (500 MHz, CPS13) δ: 7.75 (s, 1H), 7.63 (d, 1H, J=3.0 Hz), 7.51 (d, 1H, J=8.5 Hz), 7.36 (bd d, 2H, J=8.0 Hz), 7.30 (m, 2H), 7.23 (m, 1H), 7.15 (dd, 1H, J=3 .0, 9.0 Hz), 4.38 (q, 2H, J=7.5 Hz), 3.95 (s, 3H), 3.85 (broad s, 2H), 3.63 (b s, 2H), 2.25 (s, 3H), 1.41 (t, 3H, J=7.0 Hz).

Example 26 Synthesis of Compound 170

Iobutyramidoxime (41.8 mg, 0.41 mmol) and ester 48 (27.9 mg, 0.0683 mmol) in a round bottom flask were azeotropically treated in toluene on a Rotavap several times, suspended in anhydrous THF (0.6 mL) and then cooled to 0°C. NaH (60% oil suspension; 10.9 mg, 0.273 mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 20 minutes, after which it was heated at 70° C. for 6 hours and cooled. Water (4 ml) was added and the mixture was extracted with EtOAc three times. The combined organic solution was washed with brine and dried over MgSO ₄ . Rev. TLC with 10% MeOH in EtOAc gave 10.4 mg (34%) of the desired Compound 170 as a clear filmy solid. MS: [M+1]=447.

Example 27 Synthesis of Compound 52

Scheme 17.

Starting alcohol 43 (160 mg, 0.45 mmol) was treated with phosphorus oxide tribromide (400 mg, 1.4 mmol) in acetonitrile (10 ml) at 80°C for 5 h. Then the reaction mixture was cooled to 0°C, quenched sat. NaHCO ₃ and extracted with dichloromethane twice. The combined dichloromethane solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent in vacuo gave 173.3 mg (92%) of the bromide as a yellowish foamy solid. MS: [M+1]=418.

To a suspension of bromide (55 mg, 0.131 mmol) in dimethoxyethane (2 ml; degassed) was added 2M Na ₂ CO ₃ (0.39 ml, 0.78 mmol) and 3-chlorophenylboronic acid (42.2 mg, 0.27 mmol ). The reaction mixture was stirred at room temperature for 2 min, then Pd(PPh ₃ ) ₄ (75 mg, 0.065 mmol) was added and the suspension was heated at 85° C. in an oil bath for 90 min. After cooling, the reaction mixture was diluted with EtOAc and washed with brine. The aqueous layer was separated and extracted with EtOAc three times. All organic layers were combined and dried over Na ₂ SO ₄ then filtered and the solvent was removed in vacuo. The product was isolated by successive purifications with preparative TLC using 20% hexanes in EtOAc followed by 5% MeOH in DCM. The product in an amount of 9.6 mg (Compound 52) was obtained in the form of a brownish solid. MS: [M+1]=450.

1H-NMR (500 MHz, CDCl ₃ ) δ: 7.75 (s, 1H), 7.64 (d, 1H, J=3.0 Hz), 7.51 (d, 1H, J=9.5 Hz), 7.31 (broad. s,

1H), 7.23 (br s, 1H), 7.17 (m, 3H), 4.43 (q, 2H, J=7.0 Hz), 4.15 (s, 2H), 3, 96 (s, 3H), 1.44 (t, 3H, J=8.0 Hz).

Example 28 Synthesis of Compound 53

The compound of example 28 was synthesized by a synthesis similar to that described in example 27, using 3-cyanophenylboronic acid in the final stage to obtain the compound of example 28 in the form of a brownish solid: MS: [M+1]=441.

1H-NMR (500 MHz, CDCl3) δ: 7.75 (s, 1H), 7.66 (broad s, 1H), 7.64 (d, 1H, J=3.0 Hz), 7.61 (broad d, 1H, J=7.5 Hz), 7.39 (t, 1H, J=7.5 Hz), 7.16 (dd, 1H, J=3.5, 9.5 Hz) , 4.45 (q, 2H, J=7.0H), 4.20 (s, 2H), 3.96 (s, 3H), 1.45 (t, 3H, J=7.0 Hz).

Example 29 Synthesis of Compound 54

The compound of example 29 was synthesized by a synthesis similar to that described in example 27, starting from alcohol, where R ¹ =methyl, and using 2-chlorophenylboronic acid in the final stage to obtain the compound of example 29 in the form of a brownish solid: MS: [M+1 ]=434.

Example 30 Synthesis of Compound 101

The compound of example 30 was synthesized by a synthesis similar to that described in example 27, starting from an alcohol, where R ¹ =methyl, and using in the final stage phenylboronic acid to obtain the compound of example 30 in the form of a brownish solid product, which was purified by chromatography (column with 4 g silica gel RediSep. Elution solvent: EtOAc), then preparative TLC (elution system: 40% DCM/40% Hexanes/17% EtOAc/3% MeOH) to give 5.9 mg (31% yield) of Compound 101. MS: [M +1]=402.

1H-NMR (CDCl ₃ ) δ 7.96 (1H, s), 7.77 (1H, s), 7.55 (1H, m), 7.47 (1H, m), 7.32 (5H, m), 4.41 (2H, q, J=7 Hz), 4.17 (2H, s), 2.53 (3H, s), 1.43 (3H, t, J=7 Hz).

Example 31 Synthesis of Compound 102

To a suspension of bromide in EtOAc (2 ml) and MeOH (2 ml) was added activated 10% Pd/C (5 mg). The suspension was stirred under a hydrogen atmosphere for 48 hours. The solution was filtered through celite. The filtrate was concentrated and purified by chromatography (4 g RediSep silica gel column. Elution solvent: EtOAc) to give 15.9 mg (33%) of the desired Compound 102. MS: [M+1]=324.

¹ H-NMR (CDCl ₃ ) δ 7.96 (1H, s), 7.78 (1H, s), 7.49 (1H, d, J=9 Hz), 7.42 (1H, d, J = 8 Hz), 4.43 (2H, q, J=7.5 Hz), 2.53 (3H, s), 2.44 (3H, s), 1.45 (3H, t, J=7 .5 Hz).

Example 32 Synthesis of Compound 108

MeO

108

To a suspension of the bromide derivative, where R1=OMe, (18 mg; 0.043 mmol) in deoxygenated DME (2 ml) was added 2-chlorophenylboronic acid (10 mg, 0.065 mmol) and 2M Na ₂ CO ₃ solution (0.13 ml,

- 83 039381

0.26 mmol). The suspension was stirred at room temperature for 15 min, then PdCl ₂ dppf (7 mg, 0.009 mmol) was added. The suspension was heated in an oil bath at 85° C. for 1 hour. The reaction mixture was diluted with water and extracted with EtOAc (twice). The combined extracts were washed with brine and dried over Na2SO4. Filtration and concentration gave a crude product which was purified by PrepTLC (eluting system: 5% MeOH/47.5% Hex/47.5% EtOAc) to give 3.5 mg (18% yield) of Compound 108. MS: [M+1]= 451.

¹ H-NMR (CDCl3) δ 7.77 (1H, s), 7.63 (1H, d, J=3 Hz), 7.52 (1H, d, J=11.5 Hz), 7.36 (1H, m), 7.31 (1H, m), 7.18 (2H, m), 7.14 (1H, dd, J=3.9 Hz), 4.38 (2H, q, J= 7 Hz), 4.27 (2H, s), 3.94 (3H, s), 1.41 (3H, t, J=7 Hz).

Scheme 18a.

Scheme 18b.

R' = CH ₂ OFh: compound 124

Example 33 Synthesis of Compound 55

To a solution of compound 58 (6.6 g, 33.5 mmol) in dichloromethane (100 ml) were added DIPEA (8.65 g, 67 mmol), HOBt (5.4 g, 36.85 mmol) and EDCI (9. 6 g, 50.3 mmol). After approximately 15 minutes of stirring, a solution of 2,4-dimethoxybenzylamine (5.6 g, 33.5 mmol) in dichloromethane (50 ml) was added dropwise under nitrogen to the homogeneous reaction mixture. The resulting mixture was stirred under nitrogen at room temperature for 16 hours. The reaction mixture was washed successively with 1N HCl. NaOH (100 ml), water (100 ml) and saline (100 ml). The organic phase was then dried over Na ₂ SO ₄ and evaporated to give crude solid 59 which was crystallized from diethyl ether. Filtration and suction drying in open air gave the pure product as an off-white solid 9.8 g (96%) (MS: [M+1]=347).

To a solution of compound 59 (9.8 g, 28.3 mmol) in MeOH/EtOAc (1:1, 100 ml) was added 10% wet Pd-C (1.8 g, 10% mmol). After three successive vacuum treatments and rinsing with nitrogen, the heterogeneous reaction mixture was hydrogenated using a balloon at atmospheric pressure until hydrogen absorption ceased, for approximately 4 hours. The reaction mixture was filtered through a pad of celite and evaporated to give the pure desired product 60 as a brown oil 8.63 g (96%), (MS: [M+1=317]). This product was used directly in the next step.

To a solution of compound 60 (8.63 g, 27.3 mmol) in dichloromethane (100 ml) was added triethylamine (5.5 g, 54.6 mmol). The mixture was cooled with an ice bath and treated with bromoacetyl chloride (5.2 g, 32.76 mmol) under nitrogen. The ice bath was removed and the mixture was left to stir for 18 hours. The reaction mixture was washed successively with saturated NaHCO ₃ (100 ml), water (100 ml) and brine (100 ml). The organic phase was then dried over Na ₂ SO ₄ and evaporated to give crude solid 61. The crude product was crystallized from methanol, filtered and dried to give pure product as a brown solid 10.3 g (87%),

[MS: 439].

To a solution of compound 61 (10 g, 22.9 mmol) in DMF (1000 ml) was added K2CO3 (4.8 g, 45.8 mmol). The mixture was heated at 50° C. for 24 hours. LCMS showed complete conversion to the desired product. The mixture was cooled to room temperature and the inorganic solid was filtered off. The solvent was removed under high vacuum. The resulting crude product 62 was crystallized from methanol, filtered and dried to give the pure product as a brown solid 6.4 g (78%) (MS: [M+1]=357).

To compound 62 (4.46 g 12.52 mmol) dissolved in 2.5:1 THF/DMF (50 mL) was added t-BuOK (97%, 1.88 g, 16.28 mmol). The mixture was heated to 25°C, and after stirring for 30 min was cooled again to -20°C. After diethyl chlorophosphate (2.35 ml, 16.28 mmol) was added dropwise, the mixture was stirred for 3 h with heating at -20 to 25°C. The reaction mixture was re-cooled to 0° C. and ethyl isocyanoacetate (1.92 ml, 17.53 mmol) was added thereto. After cooling to -78° C., t-BuOK (97%, 1.88 g, 16.28 mmol) was added and stirred at room temperature for 5 h. Progress was checked by LC/MS. The reaction was stopped by adding 1:1 saturated NaHCO ₃ /H ₂ O (140 ml), the precipitate was filtered off, washed with H ₂ O and air dried overnight to give 4.81 g (85%) of the imidazole derivative 63 in the form of a yellow solid (MS:

[M+1]=452).

To Compound 63 (4.81 g, 10.65 mmol) in dichloromethane (35 ml) at 0°C was added trifluoroacetic acid (35 ml), then trifluoromethanesulfonic acid (1.9 ml, 21.31 mmol) dropwise. The mixture was warmed to room temperature, stirred for 2 h, then concentrated to give a residue dissolved in dichloromethane (120 ml). The crude solution was divided between chilled saturated NaHCO ₃ and dichloromethane. The organic extracts were combined, dried (MgSO ₄ ), filtered and concentrated to give 3.2 g (99%) of unprotected product 64 (brown solid) of sufficient purity to be used in the next step (MS: [M+ 1]=302).

POCl ₃ (52.7 mg, 0.344 mmol). The reaction mixture was then heated at 135° C. for 2 hours. After cooling to room temperature, phenoxy acetic acid hydrazide (228.4 mg, 1.36 mmol) was added in situ to imidochloride 65 followed by DIPEA (90 μl). The reaction mixture was stirred at room temperature for 30 min, then heated at 100° C. for 90 min. The reaction mixture was cooled, saturated NaHCO ₃ (aqueous solution) was added and extracted with ethyl acetate three times; the combined organic layer was washed with brine and dried over MgSO4. After filtration and concentration, Compound 55 was isolated by flash chromatography on an ISCO column (4 g RediSep column, 1 to 10% MeOH in DCM as gradient elution) as a white solid, mass: 8.6 mg . MS: [M+1]=432.

1H-NMR (500 MHz, CDCl ,) δ: 7.81 (s, 1H), 7.71 (d, 1H, J=3.5 Hz), 7.52 (d, 1H, J=9, 0 Hz), 7.32 (m, 2H), 7.21 (dd, 1H, J=2.5, 8.5 Hz), 7.11 (d, 2H, J=8.5 Hz), 7 .02 (m, 1H) , 5.44 (s, 2H) , 4.38 (q, 2H, J=7.5 Hz), 3.94 (s, 3H), 1.39 (t, 3H, J=7.0 Hz).

- 85 039381

Example 34 Synthesis of Compound 56

The compound of example 34 was synthesized by a synthesis similar to that described in example 33, using 4-fluorophenoxyacetic acid hydrazide in the final step to obtain the compound of example 34 in the form of a yellowish solid: MS: [M+1]=450.

'H-NMR (500 MHz, CDCl ₃ ) δ: 7.82 (s, 1H), 7.73 (d, 1H, J=3.5 Hz), 7.53 (d, 1H, J=10, 0 Hz), 7.22 (dd, 1H, J=3.5, 9.0 Hz), 7.08-6.99 (m, 4H), 5.41 (s, 2H), 4.41 ( q, 2H, J=7.0 Hz), 3.95 (s, 3H), 1.42 (t, 3H, J=6.5 Hz).

Example 35 Synthesis of Compound 103

103

The compound of example 35 was synthesized by a synthesis similar to that described in example 33, using 2-methoxyacetic acid hydrazide in the final step to obtain the compound of example 35 in the form of a yellowish solid: MS: [M+1]=370.

Example 36 Synthesis of Compound 118

118

Acetamide oxime (8.4 mg, 0.108 mmol) was azeotropically treated in toluene three times on Rotavap, then suspended in THF (1.0 ml). NaH (60% mineral suspension; 3.3 mg, 0.081 mmol) was added and the mixture was stirred at room temperature for 10 minutes. Then ester 55 (23.2 mg, 0.054 mmol) was added. After 40 min stirring at room temperature, the reaction mixture was heated at 70°C for 4 h. After cooling, cold water (5 ml) was added to the reaction mixture and ppts were collected by filtration, washed with water and dried to give 9.7 mg (41%) the desired product in the form of a yellowish solid. MS: [M+1]=442.

Example 37 Synthesis of Compound 128

The compound of example 37 was synthesized by a synthesis similar to that described in example 36 above, using the ether Compound 103 in the final step to obtain the compound of example 37 in the form of a brownish solid: MS: [M+1]=380.

Example 38 Synthesis of Compound 130

The compound of example 38 was synthesized by a synthesis similar to that described in example 36, starting from the ester Compound 103 and by condensation with isobutyramidoxime to give the compound of example 38 in the form of a yellowish solid: MS: [M+1]=408.

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Example 39 Synthesis of Compound 119

To carboxylic acid (13.9 mg, 0.0345 mmol; prepared by hydrolysis of precursor ester 55 with LiOH) stirred in DCM (0.2 mL) was added neopentyl alcohol (30.4 mg, 0.345 mmol), DMAP ( 4.2 mg, 0.0345 mmol) and EDC (20 mg, 0.104 mmol). After five hours of stirring, the reaction mixture was diluted with EtOAc, washed with sat. NH ₄ Cl, sat. NaHCO ₃ , brine and dried over MgSO ₄ . Chromatography on silica gel using a gradient of 0 to 8% MeOH in EtOAc gave 11.7 mg (72%) of the desired Compound 119 as a yellowish solid. MS: [M+1]=474.

Example 40 Synthesis of Compound 120

The compound of example 40 was synthesized by a synthesis similar to that described in example 39 above, using 2-propanol in the final step to obtain the compound of example 40 in the form of a yellowish solid: MS: [M+1]=446.

Example 41 Synthesis of Compound 129

Compound 103 (Scheme 18a) (bb, 1 mg, 0.179 mmol) was hydrolyzed in a solvent system

THF/water/MeOH (total 1.8 ml, ratio 6/5/1) by treating with LiOH (21.4 mg, 0.895 mmol) at room temperature for 2 h. DIL.HCl was added to oxidize (pH ~ 3) reaction mixture. The precipitate was collected by filtration, washed with water and dried to give 49.0 mg (80%) of the acid as a brownish solid.

The acid thus obtained was stirred in DMF (0.7 ml) at 0°C. NaHCO ₃ (48.1 mg, 0.572 mmol) was added followed by N-bromosuccinamide (96.7 mg, 0.543 mmol). After stirring overnight, the reaction mixture was diluted with EtOAc and washed with sat. NaHCO ₃ solution. The aqueous layer was separated and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated. The bromide product was obtained by silica gel column chromatography eluting with a gradient of 0 to 13% MeOH in EtOAc as a white solid (Compound 129). Weight: 28.6 mg (53%). MS: [M+1]=377.

Example 42 Synthesis of Compound 131

Compound 129 (22.6 mg, 0.060 mmol) was hydrogenated in 10% Pd-C in EtOAc (1 ml) and MeOH (1 ml) for 16 hours. Filtration through Celite and removal of the solvent gave 14.9 mg (84%) debrominated Compound 131 in the form of a slightly yellowish solid. MS: [M+1]=298.

Example 43 Synthesis of Compound 122

Meo

'Ν

122

Phenoxy analogue (Scheme 18a, R1=OPh) of acid 66 (20.4 mg, 0.0506 mmol) was suspended and stirred in DCM (0.5 ml) at room temperature. Carbonyldiimidazole (16.4 mg, 0.101 mmol) was added. After 2 hours of stirring, the resulting suspension was cooled to 0° C. and ammonia (30 μl) was added dropwise. After 20 minutes of stirring, the ice bath was removed and the reaction was allowed to continue at room temperature for 1 hour. The reaction mixture was concentrated by removing DCM in vacuo. Water (3 ml) was added and the precipitate was collected by filtration, washed with water and dried to give 16.2 mg of crude primary amide which was used without further purification.

Primary amide (16.2 mg, 0.0402 mmol) was treated with POCl ₃ (46.2 mg, 0.302 mmol) in 1,4-dioxane (0.5 ml) at 95° C. overnight.

The reaction mixture was then quenched with sat. NaHCO ₃ (5 ml), cooled to 0°C and the precipitate was collected by aspiration filtration, washed with water and dried to give 13.6 mg (88%) of the nitrile as a brownish solid, Compound 122. MS: [M+1] =385.

Example 44 Synthesis of compound 123 xLA MeO'^^y-N ο-f Λ

123

To acid 66 (15.8 mg, 0.0392 mmol) while stirring in THF (0.15 ml) and DCM (0.15 ml) was added N,O-dimethylhydroxylamine HCl (4.6 mg, 0.047 mmol) and the hydrate N-hydroxylbenzotriazole (6.0 mg). Then EDC (11.3 mg, 0.0588 mmol) and triethylamine (11.9 mg, 0.118 mmol) were added and the reaction mixture was stirred at room temperature for 12 h, diluted with EtOAc, washed with sat. NH ₄ Cl, brine and dried over MgSO ₄ . Filtration and removal of the solvent in vacuo gave 14.4 mg (82%) Weinreb amide which was used without further purification.

Ethyl magnesium bromide etherate (3M; 0.323 ml) was added to Weinreb amide (14.4 mg, 0.0323 mmol) while stirring in THF (0.3 ml) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 14 h, quenched with sat. NH ₄ Cl, extracted with EtOAc three times; the combined organic layer was washed with brine and dried over MgSO4. Filtration and removal of the solvent gave a crude ketone product which was purified by prep. TLC using 8% MeOH in EtOAc. Weight: 4.6 mg (34%) Compound 123. MS: [M+1]=416.

Example 45 Synthesis of Compound 124

124

Weinreb amide (18.0 mg, 0.0403 mmol) described above was treated with DIBAL (1 M THF; 0.363 ml) at -78°C for 1 h, then also at -78°C, quenched with Rochelle's salt solution (20 %) during the night. The aqueous solution was extracted with EtOAc three times; the combined organic layer was washed with saline and dried over MgSO ₄ . Filtration and removal of the solvent in vacuo gave 13.7 mg of crude aldehyde which was used without further purification.

Crude aldehyde (13.7 mg) in DCM (0.7 ml) at room temperature was treated with deoxofluorite (54.8 mg, 0.248 mmol) for 16 hours. The reaction mixture was quenched with sat. NaHCO ₃ (5 ml) for 20 min, extracted with EtOAc three times; the combined organic layer was washed with brine and dried over MgSO4. Filtration and solvent removal followed by purification prep. TLC using 10% MeOH in EtOAc gave 7.5 mg (52%) of the desired difluoride Compound 124 as a yellowish solid. MS: [M+1]=410.

Example 46 Synthesis of Compound 142

142

Weinreb amide (8.8 mg, 0.0197 mmol) described above in THF (0.15 ml) at 0° C. treated with phenylmagnesium bromide (1 M THF; 0.54 ml) for 2.5 h, quenched with sat. . NH4Cl, extracted with EtOAc twice; the combined organic layer was washed with saline and dried over MgSO ₄ . Filtration and removal of the solvent gave the crude ketone, which was used without further purification. The ketone in THF (0.5 ml) was treated with NaBH ₄ (6 mg) at room temperature for 2 h, then

- 88 039381 quenched sat. NH4C1, extracted with EtOAc three times; the combined organic layer was washed with brine and dried over MgSO4. Filtration and removal of the solvent gave crude alcohol which was used without further purification. The alcohol thus obtained in DCM (1.4 ml) was treated with triethylsilane (86.4 mg, 0.75 mmol) and trifluoroacetic acid (171.0 mg, 1.5 mmol) at 40° C. overnight, then concentrated in vacuum, diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . Filtration and removal of solvent gave crude benzyl which was purified by silica gel column chromatography using 0 to 12% MeOH in EtOAc as eluent; 3.6 mg of Compound 142 was obtained in the form of a yellowish solid. MS: [M+1]=450.

Scheme 19.

Compound 104

Example 47 Synthesis of Compound 106

106

To lactam 64 (185.7 mg, 0.616 mmol) in chlorobenzene (5 ml) was added N,N-dimethyl-p-toluidine (333.3 mg, 2.465 mmol) and phosphorus oxychloride (188.9 mg, 1.232 mmol). The reaction mixture was heated at 135° C. for 2 h, cooled to room temperature and formyl hydrazide (296.0 mg, 4.93 mmol) was added followed by diisopropylethylamine (238.8 mg, 1.85 mmol). After 30 min stirring at room temperature, the reaction mixture was heated at 100°C for 1 h, cooled and sat. NaHCO ₃ (15 ml), extracted with EtOAc twice; the combined organic layer was washed with brine and dried over MgSO4. Filtration and removal of solvent gave a crude triazole product which was purified by silica gel column chromatography using 0 to 15% MeOH in EtOAc as eluent, 35.9 mg (18%) was obtained as a brownish solid. MS: [M+1]=326.

The triazole prepared above in DCM (1 ml) was treated with N-bromosuccinamide (37.6 mg, 0.21 mmol) at 0°C. The reaction mixture was allowed to slowly warm to room temperature and left at room temperature overnight, diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . Filtration and removal of solvent gave crude bromide which was purified by silica gel column chromatography using 0 to 10% MeOH in an EtOAc gradient; 22.9 mg (51%) of Compound 106 was obtained as an off-white solid. [MS]: 406.

Example 48 Synthesis of Compound 104

Phenol (20.3 mg, 0.216 mmol), bromide substrate from example 47 (29.1 mg, 0.0719 mmol), Cs ₂ CO ₃ (117.0 mg, 0.360 mmol), diethyl-1, 3-acetone dicarboxylate (14.5 mg, 0.0719 mmol) and DMF (0.5 ml). The vessel was flushed with nitrogen gas. CuI (6.8 mg, 0.036 mmol) was added and the mixture was stirred at room temperature for 5 minutes, after which it was heated at 140° C. under MW irradiation conditions for 60 minutes. The reaction mixture was diluted with EtOAc, washed with water; the aqueous layer was separated and extracted with EtOAc twice; the combined organic solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent gave a crude ether product which was purified by prep. TLC using 5% MeOH in DCM; 6.6 mg of Compound 104 was obtained in the form of a yellowish solid. MS: [M+1]=418.

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Example 49 Synthesis of Compound 105

The compound of example 49 was synthesized by a synthesis similar to that described in example 48 above, using 3-methoxyphenol instead of phenol, to obtain the compound of example 49 in the form of a yellowish foamy solid: MS: [M+1]=448.

Scheme 20.

Example 50 Synthesis of Compound 112

To a solution of Compound 2 (160 mg, 0.49 mmol) in THF (6 ml), water (5 ml) and MeOH (1 ml) was added LiOH (59 mg, 2.45 mmol). The solution was stirred at room temperature for 3 hours. The solution was concentrated and the crude material was acidified with 1N. HC1 to pH 3-4. No solid was observed. EtOAc was added and the organic phase was extracted (3x). The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration gave 112 mg (77% yield) of the desired carboxylic acid product in the form of an orange solid MS: [M+1]=300.

To a suspension of acid (30 mg, 0.1 mmol) in dichloroethane (0.2 ml) was added thionyl chloride (0.4 ml; 5 mmol) and DMF (20 μl). The final solution was heated at 70° C. for 1 hour. Another 0.2 ml of thionyl chloride was added and the solution was heated for another 30 minutes. The solvent was removed. The crude material was dried in vacuo.

The crude acid chloride (0.1 mmol) was suspended in isopropanol and stirred at room temperature for 18 h.

The solvent was evaporated and the crude material was purified by chromatography (4 g RediSep silica gel column, eluting with 10% MeOH in DCM) to give 8.6 mg (25% yield) of Compound 112 [M+1]=342).

¹ H-NMR (CDCl ₃ ) δ 7.90 (1H, d, J=9 Hz), 7.79 (1H, br s), 7.63 (1H, br s), 7.36 (1H , br s), 3.48 (1H, m), 2.45 (3H, s), 1.43 (6H, d, J=6.5 Hz).

Example 51 Synthesis of Compound 113

The crude acid chloride prepared above (0.066 mmol) was suspended in dichloroethane (1 ml) and 2,2-dimethyl-1-propanol (300 mg, 3.4 mmol) was added. The solution was stirred at room temperature for 18 hours. No product was formed. To the solution obtained above were added DMAP (5 mg, 0.004 mmol) and DCC (15 mg, 0.073 mmol). The solution was stirred at room temperature for 2 hours. The reaction mixture was directly subjected to preparative TLC (eluting system: 75 EtOAc in Hexanes) to give 7.2 mg (30% yield) of Compound 113. MS: [M+1]=370 .

¹ H-NMR (CDCl ₃ ) δ 7.91 (1H, dd, J=3.9 Hz), 7.79 (1H, s), 7.61 (1H, dd, J=4.5.9 Hz ), 7.35(1H, m), 4.11(2H, s), 2.44(3H, s), 1.07(9H, s).

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Example 52 Synthesis of Compound 114

The crude acid chloride prepared above (0.066 mmol) was suspended in dichloroethane (1 ml) and 2,2,2-trifluoroethanol (0.1 ml, 1.4 mmol) was added followed by triethylamine (0.6 ml, 4.3 mmol). The solution was stirred at room temperature for 2:30. The solvent was evaporated and the crude material was purified by chromatography (4 g RediSep silica gel column, eluting with EtOAc), then purified by preparative TLC (elution system: 70% EtOAc in Hexanes) to give 8.1 mg (32% yield) of Compound 114 [M +1]=382).

'H-NMR (CDCI3) δ 7.91 (1H, dd, J=3.5, 9.5 Hz), 7.83 (1H, s), 7.63 (1H, dd, J=4.5 , 9.5 Hz), 7.35(1H, m), 4.77(2H, m), 2.43(3H, s).

Example 53 Synthesis of Compound 136

To a solution of the acid prepared in Example 50 (100 mg, 0.33 mmol) in DMF (1.5 ml) cooled in an ice bath was added NaHCO ₃ (111 mg, 1.32 mmol) followed by NBS (117 mg, 0.66 mmol). The solution was stirred at room temperature for 14 hours. The reaction mixture was diluted with water and extracted with EtOAc (5x). The combined extracts were washed with brine (2x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (4 g RediSep silica gel column, eluting with EtOAc) to give 93 mg (85% yield) Compound 136 [M+1]=334).

'H-NMR (CDCI3) δ 7.87 (1H, dd, J=2.5, 8.5 Hz), 7.72 (1H, s), 7.56 (1H, dd, J=6, 10 Hz), 7.33 (1H, m), 2.44 (3H, s).

Example 54 Synthesis of Compound 139

General combination procedure:

To a solution of Compound 136 (20 mg, 0.061 mmol) in degassed DME (0.9 ml) and water (0.1 ml) was added phenylboronic acid (11 mg, 0.092 mmol), cesium carbonate (80 mg, 0.24 mmol) and PdCl ₂ dppf (5 mg, 0.066 mmol). The suspension was heated at 80°C for one hour. The reaction mixture was diluted with water, extracted with EtOAc (3x). The combined extracts were washed with brine (2x) and dried over MgSO ₄ . Filtration and concentration gave a crude product which was purified by preparative TLC (eluting system: 3% MeOH in EtOAc).

Compound 139 was obtained using phenylboronic acid. Received 10.8 mg (54% yield) of the product. MS: [M+1]=332.

'H-NMR (CDCl ₃ ) δ 7.87 (1H, dd, J=3.5, 9.5 Hz), 7.85 (1H, s), 7.63 (3H, m), 7.50 (2H, t, J=6.5Hz), 7.35(2H, m), 2.41(3H, s).

Example 55 Synthesis of Compound 140

Compound 140 was also obtained using 3-pyridineboronic acid. Received 8.9 mg (27% yield) of the product. MS: [M+1]=333.

'H-NMR (CDCI3) δ 8.86 (1H, s), 8.63 (1H, d, J=5 Hz), 8.01 (1H, m), 7.90 (2H, m), 7 .64(1H, dd, J=5.5, 9Hz), 7.44(1H, m), 7.36(1H, m), 2.39(3H, s).

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Example 56 Synthesis of Compound 152

Compound 152 was prepared using 1-methylpyrazole-4-boronic acid, HCl. 12.5 mg (63% yield) of product was obtained. MS: [M+1]=336.

1H-NMR (CDCl ₃ + MeOD ₄ ) δ 9.04 (1H, br s), 7.99 (1H, br s), 7.75 (2H, m), 7.41 (2H, m) , 3.95 (3H, s), 2.32 (3H, s).

Example 57 Synthesis of Compound 154

Compound 154 was prepared using 2-methylpyridine-4-boronic acid pinacol ester. 7.1 mg (34% yield) of product was obtained. MS: [M+1]=347.

¹ H-NMR (CDCl ₃ ) δ 8.6 (1H, d, J=6 Hz), 7.89 (1H, dd, J=3.5, 8.5 Hz), 7.87 (1H, s ), 7.64 (1H, dd, J=5.5, 9 Hz), 7.48 (1H, s), 7.36 (2H, m), 2.64 (3H, s), 2.41 (3H, s).

Scheme 21.

Connected 153

Example 58 Synthesis of Compound 117

117

In a 100 ml round bottom flask, lactam ester 16' (2 g, 7.35 mmol; which was prepared in a similar manner as 16 described in Scheme 11) was dissolved in 60 ml of anhydrous THF. The solution was stirred at room temperature under a nitrogen atmosphere. LiBH ₄ (2M in THF, 4 ml, 8 mmol) was added slowly. The reaction mixture was stirred under nitrogen for 18 hours. Additional LiBH ₄ (2 M in THF, 2 ml, 4 mmol) was added slowly. The reaction mixture was stirred for an additional 24 hours. A mixture of EtOAc/EtOH (20 ml/20 ml) was added to the reaction mixture and the mixture was concentrated. The residue was taken up in MeOH and silica gel was added. After evaporating the volatile solvents, the solid was loaded onto a 40 g RediSep silica gel column. The desired product was eluted with a 5:1 v/v mixture. CH ₂ Cl ₂ /MeOH. The alcohol was obtained as a white solid (1.14 g, 67% yield). MS: [M+1]=231.

Alcohol (1.14 g, 4.96 mmol) was suspended in 16 ml of 33% HBr in AcOH and heated at 80° C. for 18 hours. The solution was cooled with an ice bath and diluted with EtOAc. A white solid was observed. Saturation was added slowly. aqueous solution of NaHCO ₃ . Large amounts of EtOAc and MeOH were used to dissolve the solid. The organic phase was extracted (3x) and the combined organic phases were washed with brine, dried over MgSO ₄ . Filtration and concentration gave a crude product which was used in the next step without further purification. MS: [M+1]=293.

MeOH (200 ml) and THF (50 ml), wet 10% Pd/C (250 mg) were added to a solution of the alkyl bromide derivative (4.96 mmol) in EtOAc (50 ml) and the resulting suspension was stirred under hydrogen atmosphere for 7 days. The suspension was filtered through Celite and the final solution was concentrated and co-evaporated with toluene. The crude product was used in the next step without further purification.

To a solution of 1,2,4-triazole (2.7 g, 39.7 mmol) in anhydrous CH3CN (20 ml) at 0°C was added iPr ₂ NEt (7.6 ml, 43.6 mmol). Once all the triazole had dissolved, POCl ₃ (1.11 ml, 11.9 mmol) was added. The mixture was stirred at 0° C. for 2 hours. The solution was transferred to a flask containing lactam (4.96 mmol). The final solution was heated in an oil bath at 80° C. for 16 hours. The viscous mixture was cooled with an ice bath and the solvent was evaporated. Dilute with EtOAc and add water. This mixture was extracted with EtOAc five times. The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration gave the crude product, which was used directly in the next reaction. MS: [M+1]=266.

A solution of KOtBu (1.11 g, 9.92 mmol) in DMF (10 ml) was cooled to -50° C. under nitrogen. Ethyl isocyanoacetate (1.2 ml, 10.9 mmol) was added slowly. The mixture was stirred at -60°C to -40°C for 1 hour. The above crude 1,2,4-triazolo intermediate from step 4 (4.96 mmol) in DMF (5 ml) was added slowly. The mixture was allowed to warm to room temperature over 16 hours. Saturated aqueous NH ₄ Cl was added and extracted with EtOAc three times. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (24 g RediSep silica gel column, eluting with 70% EtOAc in Hexanes) yielded 296 mg (20% yield over 4 steps) of product. MS: [M+1]=310.

To a solution of the ether derivative (260 mg, 0.84 mmol) in THF (6 ml), water (5 ml) and MeOH (1 ml) was added LiOH (117 mg, 4.85 mmol). The solution was stirred at room temperature for 3 hours. The solution was concentrated and the crude material was acidified with 1N. HCl to pH 3-4. The solid was collected by repeated filtrations to give 178 mg (75% yield) of the desired product. MS: [M+1]=282.

To a suspension of the acid (80 mg, 0.28 mmol) in THF (2 ml) was added CDI (50 mg, 0.31 mmol). The suspension was heated at 65°C for 3 hours LCMS showed that the reaction was incomplete. Added additional amount of CDI (10 mg) and the solution was heated for another hour. The solution was cooled to room temperature and a solution of NH ₄ OH (1 ml) was added. The solution was stirred for one hour. The solid was collected by filtration to give 33 mg (42%) of Compound 117 as the desired product in the form of a white solid. MS: [M+1]=281.

¹ H-NMR (MeOD ₄ ) δ 8.1 (1H, s), 7.9 (1H, s), 7.73 (3H, m), 7.07 (2H, s), 2.40 (3H , s).

Example 59 Synthesis of Compound 115

115

To a suspension of Compound 117 (8 mg, 0.029 mmol) and triethylamine (8 μl; 0.058 mmol) in THF (1 ml) was added trifluoroacetic anhydride (8 μl; 0.058 mmol). The reaction mixture was stirred at room temperature for 16 hours. LCMS showed only 30% conversion. Additional trifluoroacetic anhydride (30 μl) and triethylamine (30 μl) were added. The solution became clear and was stirred for another hour. The reaction mixture was quenched with MeOH. The solvent was evaporated and the crude material was purified by preparative TLC (eluting system: 70% EtOAc in Hexanes) to give 6.6 mg (83%) of Compound 115. MS: [M+1]=263.

¹ H-NMR (CDCl ₃ ) δ 8.17 (1H, d, J=7 Hz), 7.88 (1H, s), 7.67 (3H, m), 2.46 (3H, s).

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Example 60 Synthesis of Compound 127

To a suspension of Compound 115 (16 mg, 0.06 mmol) in EtOH (0.8 mL) and water (0.2 mL) were added hydroxylamine hydrochloride (6 mg, 0.09 mmol) and potassium carbonate (12 mg, 0.09 mmol). 09 mmol). The suspension was heated at 80° C. for 16 hours. The solution was diluted with EtOAc and washed with water. The aqueous layer was separated and extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over MgSO4. Filtration and concentration gave 12.2 mg (67% yield) of the desired product. MS: [M+1]=296.

A suspension of oxime (10 mg, 0.034 mmol) in acetic anhydride (0.5 ml) was heated at 110°C for 1 hour. The solution was then heated at 130°C for 1 hour. Finally, the temperature was raised to 140°C and heated for another 2 h. The reaction mixture was cooled, and EtOH (1 ml) was added to the reaction mixture and heated for 16 h at 80°C. The solvent was evaporated and the crude material was purified by preparative TLC (eluting system: EtOAc) to give 6.1 mg (56% yield) of the desired Compound 127. MS: [M+1]=320).

1H-NMR (CDCl ₃ ) δ 8.16 (1H, m), 7.92 (1H, s), 7.65 (3H, m), 2.68 (3H, s), 2.46 (3H, s).

Example 61 Synthesis of Compound 133

To a solution of isobutyric acid (19 μl, 0.2 mmol) in THF (0.5 ml) was added CDI (10 mg, 0.062 mmol). The solution was stirred at room temperature for 2 hours. The solution was then transferred to an ampoule containing the oxime derivative described above (12 mg, 0.041 mmol) and heated at 70° C. for 2 hours. LCMS indicated that the reaction was incomplete. Another batch of reagent (isobutyric acid and CDI) was prepared and added to the reaction mixture and heated at 70° C. for another 1 hour. LCMS indicated that all of the starting material had been consumed. The solvent was evaporated and the crude material was suspended in isobutyric acid (1 ml) and heated at 130°C for 1 hour. The solvent was evaporated and the crude material was purified by preparative TLC (eluting system: 70% EtOAc in Hexanes) to give 6.7 mg (71%) of the desired Compound 133. MS: [M+1]=348.

1H-NMR (CDClj) δ 8.16 (1H, m), 7.92 (1H, s), 7.65 (3H, m), 3.32 (1H, m), 2.46 (3H, s ), 1.5 (6H, d, J=7 Hz).

Example 62 Synthesis of Compound 126

The acetamide oxime was azeotropically treated three times in toluene prior to use. To a suspension of acetamide oxime (24 mg, 0.32 mmol) in THF (1 ml) was added 60% NaH in oil dispersion (13 mg, 0.32 mmol). The suspension was stirred at room temperature for 15 minutes. Compound 3 (50 mg, 0.16 mmol) was added. The vial containing the ester was rinsed with DMF (1 ml) which was added to the reaction mixture. The resulting brown slurry was stirred at room temperature for 30 min, then heated at 70° C. for 2 h. The slurry was quenched with water and the solution was kept in the refrigerator overnight. The solid was collected by multiple filtrations to give 16 mg (31% yield) of Compound 126. MS: [M+1]=320.

1H-NMR (CDCl ₃ ) δ 8.18 (1H, m), 7.94 (1H, s), 7.67 (3H, m), 2.51 (3H, s), 2.46 (3H, s).

Example 63 Synthesis of Compound 125

To a suspension of carboxylic acid obtained from Compound 3 (30 mg, 0.11 mmol), N,O

- 94 039381 dimethylhydroxylamine hydrochloride (13 mg, 0.13 mmol), 1-hydroxybenzotriazole hydrate (17 mg, 0.11 mmol) and triethylamine (46 µl, 0.33 mmol) in THF (0.3 ml) and DCM ( 0.3 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (32 mg, 0.17 mmol). The solution was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with EtOAc (3x). The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration gave 31.2 mg (88% yield) of an orange solid which was used in the next step without further purification. MS: [M+1]=325.

To a solution of the above Weinreb amide derivative (31.2 mg, 0.093 mmol) in THF (0.5 ml) cooled at -78°C was added a solution of 3 M ethylmagnesium bromide (0.31 ml, 0.93 mmol). The reaction mixture was stirred at a temperature below -10°C for 60 minutes. The mixture was then quenched with saturated ammonium chloride solution and extracted with EtOAc (2x). The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (4 g RediSep silica gel column, eluting with 80% EtOAc in hexanes) to give 11.1 mg (41% yield) Compound 125. MS: [M+1]=294.

1H-NMR (CDClj) δ 8.15 (1H, m), 7.76 (1H, s), 7.65 (3H, m), 3.08 (2H, q, J=7 Hz), 2, 44 (3H, s), 1.22 (3H, t, J=7 Hz).

Example 64 Synthesis of Compound 132

132

To a solution of isobutyronitrile (2.6 ml; 29 mmol) in EtOH (30 ml) and water (10 ml) were added hydroxylamine hydrochloride (2.01 g, 29 mmol) and potassium carbonate (4 g, 29 mmol). The resulting suspension was heated at 80° C. for 16 hours. The solvent was removed in vacuo. The residue was co-evaporated with toluene. The crude material was washed with EtOH and filtered to remove sodium chloride. The filtrate was evaporated, co-evaporated with toluene several times and dried to give 2 g (69%) of Nhydroxybutyramidine. To a suspension of N-hydroxybutyramidine (47 mg, 0.46 mmol) in THF (1 ml) was added 60% NaH in oil dispersion (18 mg, 0.46 mmol). The suspension was stirred at room temperature for 30 minutes. Compound 3 (47 mg, 0.15 mmol) in THF (1 ml) was added. The resulting suspension was stirred at room temperature for 30 minutes, then at 70° C. for 2 hours. After one hour, only 50% conversion was observed. No change was observed after another hour. Additional reagent (N-hydroxybutyramidine and NaH) as described above was prepared and added to the reaction mixture which was heated for another 40 minutes. At this point in time, LCMS indicated that the reaction was complete. The suspension was quenched with water. Some MeOH was added to dissolve completely and the solution was extracted with EtOAc (3x). The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (4 g RediSep silica gel column, eluting with EtOAc) to give 20 mg (38% yield) of Compound 132. MS: [M+1]=348.

¹ H-NMR (CDCl ₃ ) δ 8.18 (1H, d, J=8 Hz), 7.93 (1H, s), 7.69 (3H, m), 3.22 (1H, m), 2.46 (3H, s), 1.43 (6H, d, J=9.5Hz)

Example 65 Synthesis of Compound 161

To a solution of the acid obtained from Compound 3 (90 mg, 0.32 mmol) in DMF (2 ml) cooled in an ice bath was added NaHCO ₃ (108 mg, 1.28 mmol) followed by NBS (114 mg, 0 .64 mmol). The solution was stirred at room temperature for 18 hours. The reaction mixture was diluted with water and extracted with EtOAc (3x). The combined extracts were washed with brine (2x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (4 g RediSep silica gel column, eluting with EtOAc) to give 54 mg (53% yield) of product. MS: [M+1]=316.

To a solution of the bromide derivative (30 mg, 0.1 mmol) in dioxane (1 ml) and triethylamine (1 ml) was added TMS-acetylene (71 μl, 0.5 mmol), CuI (2 mg, 0.01 mmol) and PdCl ₂ (PPh ₃ ) ₂ (7 mg, 0.01 mmol). The solution was heated at 110° C. for 6 hours. Additional Pd catalyst (7 mg) and TMS-acetylene (0.2 ml) were added and the reaction mixture was heated for another 12 hours. At this time, LCMS showed approximately 80% transformation. Additional Pd catalyst (7 mg) and TMS-acetylene (0.2 ml) were added and the reaction mixture was heated for another 12 hours. LCMS showed complete conversion. The reaction mixture was then diluted with water and extracted with EtOAc (3x). United

- 95 039381 the extracts were washed with brine (2x) and dried over MgSO4. Filtration and concentration gave the crude product. Chromatography (4 g RediSep silica gel column, eluting with 70% EtOAc in

Hexane) to give 23 mg (69% yield) of the product. MS: [M+1]=334.

To a solution of the alkyne derivative (23 mg, 0.069 mmol) in MeOH (0.6 ml) and H ₂ O (0.2 ml) was added

KOH (4 mg, 0.076 mmol) at 0°C. The solution was allowed to warm to room temperature over 16 h. The reaction mixture was diluted with saturated aqueous ammonium chloride and extracted

EtOAc (2x). The combined extracts were washed with brine (2x) and dried over MgSO ₄ . Filtration and concentration gave a crude product which was purified by preparative TLC (eluting system: 80% EtOAc in hexanes) to give 8.1 mg (45% yield) of Compound 161. MS: [M+1]=262.

1H-NMR (CDCl ₃ ) δ 8.13 (1H, m), 7.76 (1H, s), 7.62 (3H, m), 4.09 (2H, br s), 3.28 ( 1H, s), 2.44 (3H, s).

Example 66 Synthesis of Compound 146

To a solution of 3-amino-2-methylacrolein (65 mg, 0.76 mmol) in anhydrous THF (2 ml) was added 60% NaH in oil dispersion (30 mg, 0.76 mmol). The suspension was stirred at room temperature for 15 minutes. Compound 115 (50 mg, 0.19 mmol) was added and the reaction mixture was heated at

65° C. for 3 hours. The reaction mixture was cooled with an ice bath and water was added. The reaction mixture was kept in the refrigerator overnight. The solid was collected by filtration to give 27.5 mg (44% yield) of white solid Compound 146. MS: [M+1]=330.

¹ H-NMR (CDCl ₃ ) δ 8.66 (2H, s), 8.15 (1H, m), 7.89 (1H, s), 7.65 (3H, m), 2.44 (3H , s), 2.36 (3H, s).

Example 67 Synthesis of Compound 153

153

To a suspension of the acid obtained from Compound 3 (30 mg, 0.11 mmol) in dichloroethane (0.2 ml) was added thionyl chloride (1 ml; 13.8 mmol) and DMF (20 μl). The final solution was heated at 70° C. for 1 hour. The solvent was removed. The crude material was dried in vacuo. The crude material was suspended in isopropanol (2 ml) and stirred at room temperature for 16 h. The solvent was evaporated, co-evaporated with methanol, and the crude material was purified by preparative TLC (eluting system: EtOAc) to give 7.2 mg (21% exit) Compounds 153. MS: [M+1]=324.

¹ H NMR (CDCl ₃ ) δ 8.15 (1H, d, J=8 Hz), 7.81 (1H, s), 7.64 (3H, m), 5.32 (1H, q, J= 7 Hz), 2.45 (3H, s), 1.43 (6H, d, J=7 Hz).

Scheme 22.

- 96 039381

Example 68 Synthesis of Compound 116

An alternative route to nitrile-substituted imidazole derivatives has also been made. For example, Compound 116 was prepared from the imino derivative as shown in Scheme 22. A solution of isocyanoacetonitrile (206 mg, 3.12 mmol) in DMF (7 ml) was cooled to -50° C. under nitrogen. KOtBu (320 mg, 2.85 mmol) was added. The mixture was stirred at -50°C for 1 hour. The imino derivative (obtained in a manner identical to that used for the preparation of the imino derivative shown in Scheme 21 above) (350 mg, 1.24 mmol) was added slowly at -50°C. The mixture was allowed to warm to room temperature over 16 hours. Saturated aqueous NH ₄ Cl was added and extracted with EtOAc three times. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (12 g RediSep silica gel column, eluting with 70% EtOAc in Hexanes) to give 230 mg (70% yield) of Compound 116. MS: [M+1]=281.

¹ H-NMR (CDCl ₃ ) δ 7.92 (1H, dd, J=3, 8.5 Hz), 7.81 (1H, s), 7.61 (1H, dd, J=4.5, 9 Hz), 7.38 (1H, m), 2.47 (3H, s).

Example 69 Synthesis of Compound 145

To a suspension of the cyanide derivative of Compound 116 (50 mg, 0.18 mmol) in EtOH (1.6 ml) and water (0.4 ml) were added hydroxylamine hydrochloride (17 mg, 0.24 mmol) and potassium carbonate (28 mg, 0.2 mmol).

The suspension was heated at 80°C for 30 min, then cooled to room temperature. The precipitated solid was collected by filtration to give 37.8 mg (68% yield) of the desired amino oxime product, [M+1]=314.

A suspension of amide oxime (10 mg, 0.032 mmol) in acetic anhydride (0.5 ml) was heated at 140°C for 4 h. The reaction mixture was cooled and EtOH (1 ml) was added to the reaction mixture and heated for 16 h at 80°C. The solvent was evaporated and the crude material was purified by preparative TLC (eluting system: EtOAc) to give 6.6 mg (61% yield) of the desired Compound 145. MS: [M+1]=338.

1H-NMR (CDCl ₃ ) δ 7.91 (1H, dd, J=3.5, 8.5 Hz), 7.89 (1H, s), 7.65 (1H, dd, J=5.5 , 10 Hz), 7.35(1H, m), 2.69(3H, s), 2.45(3H, s).

Example 70 Synthesis of Compound 149

To a solution of isobutyric acid (30 μl, 0.32 mmol) in THF (0.5 ml) was added CDI (16 mg, 0.096 mmol). The solution was stirred at room temperature for 2 hours. The above amide oxime derivative (10 mg, 0.032 mmol) was added and the reaction mixture was heated at 70° C. for 45 minutes. The solvent was evaporated and the crude material was suspended in isobutyric acid (1 ml) and heated at 130° C. for 3 h. %) of the desired Compound 149. MS: [M+1]=366.

1H-NMR (CDCl ₃ ) δ 7.90 (1H, dd, J=3.5.9 Hz), 7.89 (1H, s), 7.66 (1H, dd, J=4.5.8 .5 Hz), 7.36 (1H, m), 3.32 (1H, q, J=6.5 Hz), 2.46 (3H, s), 1.49 (6H, d, J=8 Hz).

Example 71 Synthesis of Compound 150

A suspension of the above amide oxime (10 mg, 0.032 mmol) in trifluoroacetic anhydride (0.5 ml) was refluxed for 10 minutes. The solvent was evaporated and the crude material was purified by preparative TLC (eluting system: 80% EtOAc in Hexanes) to give 11.8 mg (94%)

- 97 039381 of the desired Compound 150. MS: [M+1]=392.

1H-NMR (CDCl ₃ ) δ 7.92 (2H, m), 7.69 (1H, dd, J=5.5, 9.5 Hz), 7.39 (1H, m), 2.45 ( 3H, s).

Example 72 Synthesis of Compound 151

To a solution of formic acid (12 μl, 0.32 mmol) in THF (0.5 ml) was added CDI (16 mg, 0.096 mmol). The solution was stirred at room temperature for 2 hours. The above amide oxime derivative (10 mg, 0.032 mmol) was added and the reaction mixture was heated at 70° C. for 45 minutes. The solvent was evaporated and the crude material was suspended in formic acid (1 ml) and heated at 60°C for 3 hours. The solvent was evaporated and the crude material was purified by preparative TLC (eluting system: 80% EtOAc in Hexanes) to give 2.1 mg ( 20%) of the desired Compound 151. MS: [M+1]=324.

1H-NMR (CDCl ₃ ) δ 8.83 (1H, s), 7.92 (1H, dd, J=3.5, 8 Hz), 7.91 (1H, s), 7.65 (1H, dd, J=4.5, 9Hz), 7.37(1H, m), 2.45(3H, s).

Example 73 Synthesis of Compound 155

To a solution of propionic acid (22 μl, 0.29 mmol) in THF (0.5 ml) was added CDI (14 mg, 0.087 mmol). The solution was stirred at room temperature for 1 hour. The above amide oxime derivative (10 mg, 0.032 mmol) in THF (0.5 ml) was added and the reaction mixture was heated at 70° C. for 90 minutes. The solvent was evaporated and the crude material was suspended in propionic acid (1 ml) and heated at 130°C for 1 h. The solvent was evaporated and the crude material was purified by preparative TLC (eluting system: 80% EtOAc in Hexanes) to give 9.4 mg ( 94%) of the desired Compound 155. MS: [M+1]=352.

Ή-NMR (CDCl3) δ 7.91 (1H, dd, J=2, 8.5 Hz), 7.88 (1H, s), 7.65 (1H, dd, J=6, 9.5 Hz ), 7.36 (1H, m), 3.01 (2H, q, J=8.5 Hz), 2.46 (3H, s), 1.48 (3H, t, J=8.5 Hz ).

Example 74 Synthesis of Compound 160

To a solution of pivalic acid (30 mg, 0.29 mmol) in THF (0.5 ml) was added CDI (14 mg, 0.087 mmol). The solution was stirred at room temperature for 1 hour. The above amide oxime derivative (10 mg, 0.032 mmol) in THF (0.5 ml) was added and the reaction mixture was heated at 70° C. for 90 minutes. The solvent was evaporated and the crude material was suspended in acetic acid (1 ml) and heated under reflux for 3 h. The solvent was evaporated and the crude material was purified by preparative TLC (eluting system: 80% EtOAc in Hexanes) to give 7.4 mg (67 %) of the desired Compound 160. MS: [M+1]=380.

Ή-NMR (CDCl ₃ ) δ 7.90 (1H, dd, J=2.7.9 Hz), 7.88 (1H, s), 7.65 (1H, dd, J=4.5.9 Hz), 7.35(1H, m), 2.47(3H, s), 1.53(9H, s).

Example 75 Synthesis of Compound 143

A solution of KOtBu (40 mg, 0.36 mmol) in DMF (3 ml) was cooled to -50° C. under nitrogen. P-toluenesulfonylmethyl isocyanide (76 mg, 0.39 mmol) was added. The mixture was stirred at -50°C for 1 h. The imino derivative from Scheme 22 (50 mg, 0.18 mmol) was added and the mixture was allowed to warm to room temperature over 16 h. Saturated aqueous NH ₄ Cl was added and extracted with EtOAc five times . The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (4 g RediSep silica gel column, 98 039381 eluting with 70% EtOAc in Hexanes) followed by preparative TLC (eluting system: 30% EtOAc in

DCM) to give 22.2 mg (30% yield) of white solid Compound 143. MS: [M+1]=410.

¹ H-NMR (CDCl ₃ ) δ 7.91 (2H, d, J=8 Hz), 7.87 (1H, dd, J=2.5, 8.5 Hz), 7.74 (1H, s ), 7.65 (1H, dd, J=5.5, 9 Hz), 7.34 (3H, m), 2.50 (3H, s), 2.42 (3H, s).

Example 76 Synthesis of Compound 144

To 3-ethoxymetacrolein (100 mg, 0.88 mmol) was added 7N. ammonia in methanol (1.3 ml, 8.8 mmol). The solution was stirred at room temperature for 16 hours. The solvent was evaporated and the crude yellow solid corresponding to 3-amino-2-methyl-acrolein was used in the next step without further purification.

To a solution of 3-amino-2-methylacrolein (7 mg, 0.087 mmol) in anhydrous THF (1 ml) was added 60% NaH in oil dispersion (6 mg, 0.16 mmol). The suspension was stirred at room temperature for 15 minutes. A cyanide derivative (22 mg, 0.079 mmol) in THF (1 ml) was added and the reaction mixture was heated at 65°C for 1 h. 20 mg) in THF (1 ml) and added to the reaction mixture, which was heated at 65°C for another hour. LCMS indicated completion of the reaction. The reaction mixture was quenched with methanol. The solvent was evaporated. The crude material was suspended in water and the solid was collected by filtration to give 5.2 mg (19% yield) of a light red solid Compound 144. MS: [M+1]=348.

¹ H-NMR (CDCl ₃ ) δ 8.67 (2H, s), 7.90 (1H, d, J=9.5 Hz), 7.85 (1H, s), 7.65 (1H, dd , J=4.5, 9Hz), 7.34(1H, m), 2.44(3H, s), 2.36(3H, s).

Scheme 23.

YL.EYUN R^F Compound 135

n-n. Compound 134

Example 77 Synthesis of Compound 121

121

To a solution of 1,2,4-triazole (2.03 g, 29.4 mmol) in anhydrous CH3CN (20 ml) at 0°C was added iPr ₂ NEt (5.6 ml, 32.4 mmol). Once all the triazole had dissolved, POCl ₃ (0.82 ml, 8.8 mmol) and compound 16' (1 g, 3.68 mmol) were added. The mixture was stirred at 0° C. for 2 hours. The final solution was heated in an oil bath at 80° C. for 16 hours. The mixture was cooled with an ice bath, diluted with EtOAc and water was added. This mixture was extracted with EtOAc three times. The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration gave 1.05 g (88% yield) of an orange solid which was used directly in the next step. MS: [M+1]=324.

A solution of KOtBu (696 mg, 6.2 mmol) in DMF (15 ml) was cooled to -50° C. under nitrogen. Ethyl isocyanoacetate (0.75 ml, 6.8 mmol) was added slowly. The mixture was stirred at -50° C. for 1 h. The above crude product from Step 1 (1 g, 3.1 mmol) was added and the mixture was allowed to warm to room temperature over 18 h. Saturated aqueous NH ₄ Cl was added and extracted EtOAc eight times. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (column with 24 g silica gel

- 99 039381

RediSep, eluting with 70% EtOAc in Hexanes) to give 950 mg (83% yield) of product. MS:

[M+1]=368.

To a solution of the diester (200 mg, 0.54 mmol) in anhydrous THF (4 ml), stirred at room temperature under nitrogen atmosphere, was added LiBH ₄ (2M in THF, 0.66 ml, 1.3 mmol). The reaction mixture was stirred under nitrogen atmosphere for 24 h. EtOAc/EtOH (3 ml/3 ml) was added to the reaction mixture and the mixture was concentrated. The residue was taken up in MeOH and silica gel was added. After evaporating the volatile solvents, the solid was loaded onto a 4 g RediSep silica gel column. The desired product was eluted with a 10:1 v/v mixture. CH ₂ Cl ₂ /MeOH. The diol was obtained as a solid (60 mg, 39% yield).

MS: [M+1]=284.

Diol (60 mg, 0.21 mmol) was suspended in 5 ml of 33% HBr in AcOH and heated at 80° C. for 18 hours. The solution was cooled with an ice bath and diluted with EtOAc. Slowly, saturated aqueous NaHCO ₃ was added. The solution was extracted with EtOAc (3x) and the combined organic phases were washed with brine, dried over MgSO ₄ . Filtration and concentration gave a crude product which was used in the next step without further purification. MS: [M+1]=408.

To a solution of the dialkyl bromide derivative (0.21 mmol) in EtOAc (10 ml) and MeOH (10 ml) was added wet 10% Pd/C (catalytic amount) and the resulting suspension was stirred under a hydrogen atmosphere for 60 h. filtered through Celite and the final solution was concentrated. The crude product was purified by multiple preparative TLC (eluting system: MeOH 3% in EtOAc) to give 6.2 mg (12% yield over 2 steps) of the desired Compound 121. MS: [M+1]=252.

¹ H-NMR (CDCl ₃ ) δ 8.09 (1H, m), 7.74 (1H, s), 7.56 (3H, m), 7.90 (2H, m), 2.42 (3H , s), 2.29 (3H, s).

Example 78 Synthesis of Compound 135

135

Compound 135 was synthesized analogously to Compound 121 as follows:

To a solution of 1,2,4-triazole (952 mg, 13.8 mmol) in anhydrous CH3CN (20 ml) at 0°C was added iPr ₂ NEt (2.6 ml, 15.2 mmol). Once all the triazole had dissolved, POCl ₃ (0.45 ml, 4.8 mmol) and lactam ester (1 g, 3.45 mmol) were added. The mixture was stirred at 0° C. for 2 hours. The final solution was heated in an oil bath at 80° C. for 16 hours. The mixture was cooled with an ice bath, diluted with EtOAc and water was added. This mixture was extracted with EtOAc three times. The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration gave 1.03 g (87% yield) of an orange solid which was used directly in the next step. MS: [M+1]=342.

A solution of KOtBu (658 mg, 5.9 mmol) in DMF (15 ml) was cooled to -50° C. under nitrogen. Ethyl isocyanoacetate (0.71 ml, 6.5 mmol) was added slowly. The mixture was stirred at -50° C. for 1 h. The above crude product from step 1 (1 g, 2.9 mmol) was added and the mixture was allowed to warm to room temperature over 18 h. Saturated aqueous NH ₄ Cl was added and extracted with EtOAc eight times. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (24 g RediSep silica gel column, eluting with 70% EtOAc in Hexanes) gave 1.02 g (90% yield) of product. MS: [M+1]=386.

To a solution of the diester (600 mg, 1.56 mmol) in anhydrous THF (8 mL) stirred at room temperature under nitrogen was added LiBH4 (2M in THF, 3.1 mL, 6.24 mmol). The reaction mixture was stirred under nitrogen atmosphere for 24 h. EtOAc/EtOH (10 ml/10 ml) was added to the reaction mixture and the mixture was concentrated. The residue was taken up in MeOH and silica gel was added. After evaporating the volatile solvents, the solid was loaded onto a 12 g RediSep silica gel column. The desired product was eluted with a 10:1 v/v mixture. CH ₂ Cl ₂ /MeOH. The diol was obtained as a solid (187 mg, 40% yield). MS: [M+1]=302.

Diol (80 mg, 0.27 mmol) was suspended in 7 ml of 33% HBr in AcOH and heated at 80° C. for 48 hours. The solution was cooled with an ice bath and diluted with EtOAc. A saturated aqueous solution of NaHCO ₃ was added slowly. The solution was extracted (3x) and the combined organic phases were washed with brine, dried over MgSO ₄ . Filtration, concentration and co-evaporation with toluene gave 100 mg (88% yield) of a beige solid which was used in the next step without further purification. MS: [M+1]=426.

To a solution of the dialkyl bromide derivative (70 mg, 0.16 mmol) in EtOAc (10 ml) and MeOH (10 ml) was added 10% Pd/C (catalytic amount) and the resulting suspension was stirred under hydrogen atmosphere for 48 hours. The suspension was filtered through Celite and the final solution was concentrated. The crude product was purified by multiple preparative TLC (elution system 1: 75% EtOAc in Hexanes; elution system 2: 5% MeOH in EtOAc; elution system 3: EtOAc) to give 4.1 mg (10% yield) desired Compound 135. MS: [M+1]=270.

'h-NMR (CDCl ₃ ) δ 7.84 (1H, dd, J=2.5, 9 Hz), 7.70 (1H, s), 7.54 (1H, dd, J=5.8 Hz ), 7.30 (1H, m), 2.42 (3H, s), 2.28 (3H, s).

Example 79 Synthesis of Compound 134

To a suspension of the dialkyl bromide derivative described in Scheme 23, R=H, (30 mg, 0.074 mmol) in EtOH (1 ml) and heated at 80°C was added a fresh solution of 2M NaOEt (75 μl, 0.15 mmol). The solution was heated for 10 minutes. The solvent was evaporated. The crude material was suspended in EtOAc and filtered. The filtrate was concentrated and purified by preparative TLC (eluting system: EtOAc) to give 3.1 mg (12% yield) of the desired Compound 134. MS: [M+1]=340.

Scheme 24.

Example 80 Synthesis of Compound 137

To a solution of 5-fluoro-2-nitrobenzoic acid (6.6 g, 35.66 mmol) in dichloromethane (100 ml) was added DIPEA (9.22 g, 71.3 mmol), HOBt (6.0 g, 39. 2 mmol) and EDCI (10.2 g, 53.5 mmol). After approximately 15 minutes of stirring, a solution of 2,4-dimethoxybenzylamine (5.96 g, 35.66 mmol) in dichloromethane (50 ml) was added dropwise under nitrogen atmosphere to the reaction mixture. The resulting mixture was stirred under nitrogen at room temperature for 16 hours. The reaction mixture was washed successively with 1N HCl. HCl (100 ml), sat. NaHCO ₃ (100 ml) and brine (100 ml). The organic phase was then dried over MgSO ₄ . Filtration and removal of the solvent in vacuo gave a yellowish solid, wt: 9.3 g (78%). MS: [M+1]=335.

Powder was added to the nitrobenzene analog (9.3 g, 27.8 mmol) suspended and stirred in a mixture of solvents HOAc/THF/MeOH/H ₂ O (25/100/50/25 ml) at room temperature.

Zinc. The mixture was heated to 70°C for 20 h, cooled and filtered. The solid was washed with THF and the combined filtrate was concentrated in vacuo. To the resulting suspension was slowly and carefully added sat. NaHCO ₃ , avoiding excessive foaming, until a pH of 7 to 8 was reached. The mixture was extracted with EtOAc (3x); the combined organic layer was washed with saline and dried over MgSO ₄ . Filtration and removal of the solvent gave the crude amine product as a dark brown sticky paste, wt: 8.7 g.

To a solution of the aniline above (8.7 g) in dichloromethane (150 ml) was added triethylamine (3.37 g, 33.4 mmol). The mixture was cooled with an ice bath and treated with bromoacetyl chloride (4.81 g, 30.6 mmol) under nitrogen. The ice bath was removed and the mixture was left to stir for 72 hours. The reaction mixture was concentrated in vacuo, the resulting suspension was treated with Et ₂ O (100 ml) and water (100 ml). The precipitated product was collected by filtration and dried to give 5.6 g of the product as a brown solid. The Et ₂ O layer was separated from the aqueous layer and diluted with DCM (50 ml), washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent gave 5.3 g of additional product in the form of a brown foamy solid. Total weight: 11 g (100%).

To a solution of bromide (11 g) in DMF (550 ml) was added K ₂ CO ₃ (7.1 g, 51.7 mmol). The mixture was heated at 50° C. for 48 hours. The mixture was cooled to room temperature and the inorganic solid was filtered off. The filtrate was concentrated in vacuo, treated with water/MeOH (60/10 ml), extracted with DCM (3x); the combined organic layer was washed with saline and dried over MgSO ₄ . Filtration and removal of the solvent followed by silica gel column chromatography using 5-50% EtOAc in DCM gave 3.2 g (36%) of the 7-membered lactam as a brownish solid. MS: [M+1]=345.

To lactam (1.32 g, 3.83 mmol) dissolved and stirred in THF (20 ml) and DMF (3 ml) was added t-BuOK (0.645 g, 5.75 mmol) at -20°C. After 30 minutes of stirring at -20°C, diethyl chlorophosphate (1.19 ml, 6.89 mmol) was added dropwise and the mixture was stirred for 3 hours while heating at -20 to 20°C. The reaction mixture was cooled to -78°C and ethyl isocyanoacetate (0.791 mL, 6.89 mmol) was added thereto, followed by t-BuOK (0.645 g, 5.75 mmol) and stirring continued overnight, while as the temperature reached room temperature. The reaction mixture was quenched with saturated NH4Cl, extracted with EtOAc (2x); the combined organic solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent gave a crude product which was purified by silica gel column chromatography using 15-100% EtOAc in DCM, wt: 0.861 g (47%), as a brown solid. MS: [M+1]=440.

To the imidazole ester above (861 mg) in dichloromethane (5 ml) at 0° C. was added trifluoroacetic acid (5 ml) followed by trifluoromethanesulfonic acid (0.345 ml). The mixture was warmed to room temperature, stirred for 3 h, then concentrated to give a residue which was dissolved in dichloromethane (50 ml) to which sat. NaHCO ₃ (50 ml) followed by stirring for 20 minutes. The pH value of the upper aqueous layer at the check was basic, and it was separated, extracted with DCM (3x); the combined DCM solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent gave 0.58 g (100%) lactam in the form of a yellowish solid. MS: [M+1]=290.

POCl ₃ (133 .0 mg, 0.867 mmol). The reaction mixture was then heated at 135° C. for 2 hours. After cooling to room temperature, phenoxyacetic acid hydrazide (189.0 mg, 1.08 mmol) was added followed by DIPEA (0.455 ml). The reaction mixture was stirred at room temperature for 30 min, then heated at 100° C. for 60 min. The reaction mixture was cooled, saturated NH ₄ Cl (aqueous solution) was added and extracted with ethyl acetate three times; the combined organic layer was washed with brine and dried over MgSO4. After filtration and concentration, the product was isolated by flash chromatography on an ISCO column using 0 to 10% MeOH in EtOAc, wt: 116.7 mg (36%) Compound 137 as a yellowish filmy solid. MS: [M+1]=420.

Example 81 Synthesis of Compound 156

156

An ethyl ester representing Compound 137 (244.2 mg, 0.582 mmol) in a THF/water/MeOH solvent system (total 6.0 ml, ratio 6/5/1) was treated with LiOH (69.7 mg, 2 .91 mmol) at room temperature for 4 h, concentrated in vacuo, acidified to pH~3 and the precipitate was collected by filtration. After washing with water and drying, 179.3 mg (79%) of the acid was obtained in the form of a yellowish solid. MS: [M+1]=392.

To this acid (10.8 mg, 0.0276 mmol) while stirring in DCM (0.1 ml) at room temperature was added EDCI (21.3 mg, 0.11 mmol), DMAP (6.7 mg, 0. 0552 mmol) and isopropyl alcohol (13.2 mg, 0.221 mmol). After 12 hours the reaction mixture was diluted with EtOAc, washed with sat. _NaHCO3 ; the aqueous layer was separated and extracted with EtOAc, the combined organic layer was washed with brine and dried over MgSO4. Filtration and purification prep. TLC concentrate using 10%

MeOH in EtOAc gave 8.7 mg (73%) of the isopropyl ester of Compound 156 as a yellowish foamy solid. MS: [M+1]=434.

Example 82 Synthesis of Compound 138

138

Acetamide oxime (10.7 mg, 0.144 mmol) was azeotroped four times in toluene and added to ethyl ether, Compound 137 (9.5 mg, 0.0226 mmol). THF (0.3 ml) was added followed by a 60% oily suspension of NaH (4.5 mg, 0.112 mmol). The reaction mixture was stirred at room temperature for 30 min, then heated at 70°C for 2 h, cooled to room temperature and the solvent was removed in vacuo, water (1.5 ml) was added to suppress the reaction, stirred for 20 min and cooled to 4°C. The precipitate was collected by filtration, washed with water and dried to give 5.2 mg (59%) of oxadiazole Compound 138 as a light yellow solid. MS: [M+1]=430.

Example 83 Synthesis of Compound 141

141

The compound of example 83 was synthesized by a synthesis similar to that described in example 82 using isobutyramidoxime instead of acetamide oxime to give the compound of example 83 in the form of a yellowish solid: MS: [M+1]=458.

Example 84 Synthesis of Compound 157

157

To the acid prepared in Example 81 above (60.2 mg, 0.154 mmol) was added carbonyldiimidazole (49.9 mg, 0.308 mmol) while stirring in DCM (0.7 mL) at room temperature. The mixture was stirred for 40 minutes, then cooled to 0° C. and ammonia (0.112 ml) was added, warmed to room temperature with stirring continued overnight. The reaction mixture was concentrated, water (8 ml) was added and stirred well for 30 minutes. The resulting precipitate was collected by filtration, washed with water and dried to give 51.1 mg (85%) of the primary amide as a brownish solid. MS: [M+1]=391.

Amide (51.1 mg), above, was treated with POCl ₃ (200.8 mg, 1.31 mmol) in 1,4-dioxane (0.9 ml) at 90° C. for 14 h. After cooling to room temperature the reaction mixture was carefully quenched with sat. NaHCO3 (5 ml) and stirred for 20 min. The precipitate was collected by filtration, washed with water and dried to give 40.9 mg (85%) of nitrile Compound 157 as a brownish solid. MS: [M+1]=373.

Example 85 Synthesis of Compound 147

147

To nitrile (45.8 mg, 0.123 mmol) in a round bottom flask were added hydroxylamine hydrochloride (14.5 mg, 0.209 mmol), K ₂ CO ₃ (22.3 mg, 0.161 mmol), ethanol (0.6 ml) and water (0.15 ml). The reaction mixture was heated at 80°C for 30 min, cooled and concentrated in vacuo. The resulting suspension was treated with water (1.5 ml), sonicated for better mixing and stirred at room temperature for 1 h, after which it was cooled to 4°C. The resulting precipitate was collected by filtration, washed with cold water (1 ml) and dried to give 40.8 mg (82%) of the adduct as an off-white solid. MS: [M+1]=406.

Isobutyric acid (31.4 mg, 0.582 mmol) was treated with carbonyldiimidazole (28.4 mg, 0.175 mmol) in THF (0.5 mL) for 2 h, N-hydroxycarboxamide adduct (11.8 mg, 0.0291 mmol) was added ),

- 103 039381 and the reaction mixture was stirred at room temperature for 30 minutes. Added additional amount of isobutyric acid (0.5 ml) and the reaction mixture was heated at 110°C for 16 h, cooled, sat. NaHCO ₃ (8 ml) and extracted with EtOAc (3x); the combined organic layer was washed with brine and dried over MgSO4. Rev. TLC (5% MeOH in EtOAc) of the concentrated filtrate gave 11.2 mg (84%) of oxadiazole Compound 147 as a white solid. MS: [M+1]=458.

Example 86 Synthesis of Compound 148

148

The compound of example 86 was synthesized by a synthesis similar to that described in example 85, using acetic acid instead of isobutyric acid to obtain the compound of example 86 in the form of a white solid: MS: [M+1]=430.

Example 87 Synthesis of Compound 158

158

The compound of example 87 was synthesized by a synthesis similar to that described in example 85, using propionic acid instead of isobutyric acid to obtain the compound of example 87 in the form of a white solid: MS: [M+1]=444.

Example 88 Synthesis of Compound 159

159

Trifluoroacetic anhydride (196.9 mg, 0.938 mmol) was added at room temperature to the N-hydroxycarboxamide adduct (19.0 mg, 0.0469 mmol) suspended and stirred in THF (0.2 ml). After 30 min stirring, the reaction mixture was heated to 70°C for 1 h, cooled to room temperature and diluted with EtOAc (10 ml), to which sat. NaHCO ₃ and stirred for 30 min. The aqueous layer was separated and extracted with EtOAc (1x); the combined organic layer was washed with saline and dried over MgSO ₄ . Filtration and removal of the solvent gave a paste to which was added nBuOH (5 ml) and HOAc (0.5 ml). This mixture was heated at 115° C. for 16 h, cooled and concentrated in vacuo, diluted with EtOAc, washed with sat. NaHCO3, brine and dried over MgSO ₄ . Rev. TLC (5% MeOH in EtOAc) of the concentrated filtrate gave 11.5 mg (51%) of the desired trifluoromethyl oxadiazole analog Compound 159 as a yellowish solid. MS: [M+1]=484.

Scheme 25.

Example 89 Synthesis of Compound 162

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tBuOK (240.2 mg) was added to lactam 62 (503.4 mg, 1.42 mmol) while stirring in THF (2.9 ml) and DMF (0.8 ml) at -20°C. After 30 min stirring, diethyl chlorophosphate (377.7 mg, 2.12 mmol) was added dropwise and the reaction mixture was slowly heated to 8°C over 3 h, then cooled to -20°C, 2.26 ml (2 .26 mmol) oxadiazole isocyanate (see JMC, 1996, 39, 170; prepared as 1M THF solution). The reaction mixture was further cooled to -78° C., tBuOK (238.4 mg) was added and the reaction mixture was slowly warmed to room temperature overnight. Saturation was added. NH ₄ Cl (5 ml) and the mixture was extracted with EtOAc (2x), washed with brine and dried over MgSO ₄ . After filtration and concentration, the product was isolated by silica gel column chromatography using an elution gradient of 0 to 10% MeOH in EtOAc to give the imidazole product 246.0 mg as a yellowish solid. MS: [M+1]=462.

Imidazole (246.0 mg, 0.533 mmol) obtained above was stirred in DCM (3 ml). Trifluoroacetic acid (3 ml) was added followed by trifluoromethylsulfonic acid (160.0 mg, 1.07 mmol). After 3 h stirring, the reaction mixture was diluted with DCM (20 ml), washed with sat. _NaHCO3 ; the aqueous layer was separated and extracted with DCM (2x); the combined DCM solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent in vacuo gave 208.7 mg of crude lactam product in the form of a yellowish flaky solid. [M+1]=312.

Phosphorus oxychloride (29.9 mg, 0.195 mmol) was added to a solution of the above lactam (22.5 mg, 0.0723 mmol) and N,N-dimethyl-p-toluidine (51.8 mg, 0.383 mmol) stirred in chlorobenzene (0.45 ml) under nitrogen atmosphere. The reaction mixture was heated at 135°C for 3 h, then cooled to room temperature. Diisopropylethylamine (75.7 mg, 0.586 mmol) and phenoxyacetic hydrazide (50.1 mg, 0.302 mmol) were added and the reaction mixture was heated at 100° C. for 14 h, cooled to room temperature and partitioned between sat. _NH4Cl and EtOAc. The aqueous layer was separated and extracted with EtOAc; the combined EtOAc solution was washed with brine and dried over MgSO ₄ . After filtration and concentration, Compound 162 was isolated by silica gel column chromatography using an elution gradient of 0 to 10% MeOH in EtOAc as a yellowish solid. Weight: 11.8 mg (37%). MS: [M+1]=442.

Example 90 Synthesis of Compound 163

The compound of example 90 was synthesized by a synthesis similar to that described in example 89 using 4-fluorophenoxyacetic hydrazide instead of phenoxyacetic hydrazide to give the compound of example 90 in the form of a yellowish solid: MS: [M+1]=460.

Example 91 Synthesis of Compound 164

The compound of example 91 was synthesized by a synthesis similar to that described in example 89 using methoxyacetic hydrazide instead of phenoxyacetic hydrazide to give the compound of example 91 in the form of a yellowish solid: MS: [M+1]=380.

Example 92 Synthesis of Compound 165

Preparation of benzyloxyacetic hydrazide: carbonyldiimidazole (1.52 g, 9.39 mmol) was added to benzyloxyacetic acid (1.2 g, 7.22 mmol) with stirring in THF (60 ml) at 0°C. The ice bath was removed and stirring was continued for 1 hour. The resulting cloudy solution was added to hydrazine (0.927 g, 28.9 mmol) with stirring in THF (40 ml) at room temperature. After 16 h, the reaction mixture was concentrated to a suspension, to which water (120 ml) was added, extracted with DCM (3x); the combined DCM solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent gave 0.908 g (70%) of the hydrazide as a clear viscous oil. This oil was azeotropically treated in toluene several times prior to use.

The compound of example 92 was synthesized by a synthesis similar to that described in example 89, using benzyloxyacetic hydrazide instead of phenoxyacetic hydrazide to obtain the compound of example 92 in the form of a yellowish solid: MS: [M+1]=456.

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Example 93 Synthesis of Compound 166

Compound 165, supra (58.5 mg, 0.128 mmol), was treated with 10% PdoM-C (catalytic) in EtOAc (4 ml) and MeOH (4 ml) under hydrogen atmosphere for 2 h. The catalyst was removed by filtration through Celite . Conc. was added to the filtrate. HCl (0.89 ml) and the mixture was stirred at room temperature for 16 h. An excess of Na ₂ CO ₃ (aqueous solution) was added and the solution was extracted with EtOAc (2x); the combined organic solution was washed with brine and dried over MgSO ₄ . Rev. TLC of the concentrated filtrate using 15% MeOH in EtOAc gave 14.9 mg of primary amide ([M+1]=417) as a yellowish solid. This primary amide was treated with phosphorus oxychloride (54.9 mg, 0.358 mmol) in 1,4-dioxane (1 mL) at 90° C. for 14 h. After cooling, the reaction mixture was diluted with EtOAc, washed with sat. _NaHCO3 ; the aqueous layer was separated and extracted with EtOAc (1 x), the combined organic solution was washed with saline and dried over MgSO ₄ . Rev. TLC of the concentrated filtrate using 5% MeOH in EtOAc gave 5.2 mg of the desired nitrile Compound 166 as white needles. [M+1]=3 99.

Scheme 26.

Example 94 Synthesis of Compound 169

To lactam 62 (2.23 g, 6.24 mmol) while stirring in THF (10 ml) and DMF (3 ml) at -20°C was added tBuOK (1.05 g, 9.36 mmol). After 30 minutes of stirring, diethyl chlorophosphate (1.66 g, 9.36 mmol) was added dropwise and the reaction mixture was slowly heated to 8-10°C over 3 hours, after which it was cooled to -20°C. 10.0 ml (10.0 mmol) oxadiazole isocyanate (see JMC, 1996, 39, 170; prepared as 1M THF solution) was added. The reaction mixture was further cooled to -78° C., tBuOK (1.05 g, 9.36 mmol) was added and the reaction mixture was slowly warmed to room temperature overnight. Saturation was added. NH ₄ Cl (20 ml), and the mixture was extracted with EtOAc (3x), washed with saline and dried over MgSO ₄ . After filtration and concentration, the product was isolated by silica gel column chromatography using a gradient elution of 10-100% EtOAc in DCM to give 1.07 g (35%) imidazole as a yellowish foamy solid. MS: [M+1]=490.

Imidazole (1.07 g, 2.18 mmol) obtained above was stirred in DCM (11 ml). Trifluoroacetic acid (11 ml) was added followed by trifluoromethylsulfonic acid (0.656 g, 4.37 mmol). After 4 h stirring, the reaction mixture was concentrated in vacuo, diluted with DCM (50 ml), washed with sat. _NaHCO3 ; the aqueous layer was separated and extracted with DCM (2x); the combined DCM solution was washed with brine and dried over MgSO4. Filtration and removal of the solvent in vacuo gave 0.872 g of crude lactam as a brownish solid. [M+1]=340.

Phosphorus oxychloride (51.0 mg, 0.333 mmol) was added to a solution of the above lactam (45.0 mg, 0.133 mmol) and N,N-dimethyl-p-toluidine (89.6 mg, 0.663 mmol) stirred in chlorobenzene ( 0.60 ml) under a nitrogen atmosphere. The reaction mixture was heated at 135°C for 3 h, then cooled to room temperature. Diisopropylethylamine (137.5 mg, 1.06 mmol) and methoxyacetic hydrazide (83.1 mg, 0.798 mmol) were added and the reaction mixture was heated at 100° C. for 4 h, cooled to room temperature, diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . After filtration and concentration, Compound 169 was isolated by silica gel column chromatography using an elution gradient of 0 to 13% MeOH in EtOAc as a brownish solid. Weight: 14.3 mg (26%). MS: [M+1]=408.

Example 95 Synthesis of Compound 171

The compound of example 95 was synthesized by a synthesis similar to that described in example 94 using phenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of example 95 in the form of a yellowish solid: MS: [M+1]=470.

Example 96 Synthesis of Compound 172

The compound of example 96 was synthesized by a synthesis similar to that described in example 94 using 4-fluoro-phenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of example 96 in the form of a yellowish solid: MS: [M+1]=488.

Example 97 Synthesis of Compound 173

The compound of example 97 was synthesized by a synthesis similar to that described in example 94 using ethoxyacetic hydrazide instead of methoxyacetic hydrazide to obtain the compound of example 97 in the form of a yellowish solid: MS: [M+1]=422.

Example 98 Synthesis of Compound 174

The compound of example 98 was synthesized by a synthesis similar to that described in example 94 using 2-fluorophenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of example 98 in the form of a yellowish solid: MS: [M+1]=488.

Example 99 Synthesis of Compound 175

The compound of example 99 was synthesized by a synthesis similar to that described in example 94 using 2-chlorophenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of example 99 in the form of a yellowish solid: MS: [M+1]=504.

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Example 100 Synthesis of Compound 176

Preparation of 3-pyridyloxyacetic hydrazide: A solution of ethyl-3-pyridyloxyacetate (0.50 g, 2.76 mmol) and hydrazine (0.31 g, 9.6 mmol) in isopropyl alcohol (35 ml) was heated at 85°C for 30 h, cooled and concentrated in vacuo. The resulting white solid was dissolved in a small amount of sat. NaCl solution and extracted with EtOAc several times. The combined organic solution was dried over MgSO ₄ . Filtration and removal of the solvent gave 177 mg of the desired acetic hydrazide as a white solid. The moisture content of the residual water was removed by azeotropic treatment in toluene.

The compound of example 100 was synthesized by a synthesis similar to that described in example 94 using 3-pyridyloxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of example 100 in the form of a yellowish solid: MS: [M+1]=471.

Example 101 Synthesis of Compound 177

The compound of example 101 was synthesized by a synthesis similar to that described in example 94 using 1-naphthoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of example 101 in the form of an off-white solid: MS: [M+1]=520.

Example 102 Synthesis of Compound 179

The compound of example 102 was synthesized by a synthesis similar to that described in example 94 using 3-fluorophenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of example 102 in the form of a yellowish solid: MS: [M+1]=488.

Example 103 Synthesis of Compound 178

Phosphorus oxychloride (64.8 mg, 0.422 mmol) was added to a solution of oxadiazolyl imidazole lactam (57.5 mg, 0.169 mmol) and N,N-dimethyl-p-toluidine (114.6 mg, 0.847 mmol) stirred in chlorobenzene ( 0.70 ml) under a nitrogen atmosphere. The reaction mixture was heated at 135°C for 3 h, then cooled to room temperature. Diisopropylethylamine (174.7 mg, 1.35 mmol), t-BuOH (0.3 ml) and 2-hydroxyacetic hydrazide (91.3 mg, 1.01 mmol) were added. The reaction mixture was stirred at room temperature for 20 minutes, then heated at 50°C for 1 hour, then heated at 80°C for 1 hour, then finally heated at 100°C overnight. After cooling to room temperature, the reaction mixture was diluted with EtOAc, washed with brine and dried over MgSO ₄ . Silica gel column chromatography of the concentrated filtrate using an elution gradient of 0 to 20% MeOH in EtOAc gave the desired hydroxymethyl triazole as a yellowish solid. Weight: 18.1 mg (27%). MS: [M+1]=394.

To a solution of hydroxymethyl triazole, above (18.1 mg, 0.046 mmol), cyclopentyl bromide (274.0 mg, 1.84 mmol) and HMPA (16.5 mg, 0.092 mmol) with stirring in THF (0.5 ml) NaH (60% suspension; 18.4 mg, 0.46 mmol) was added. After 10 min, the reaction mixture was heated at 100°C for 6 h, cooled, quenched with sat. NaHCO ₃ and extracted with EtOAc (2x), washed with brine and dried over MgSO ₄ . Rev. TLC of the concentrated filtrate using 8% MeOH in EtOAc gave 5.5 mg (26%) of the desired ester Compound 178 as a yellowish solid. [M+1]=462.

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Scheme 27.

Compound 168

Example 104 Synthesis of Compound 168

168

To a suspension of benzyl glycinate hydrochloride (5 g, 24.8 mmol) in DCM (100 ml) were added EDC.HCl (6.2 g, 33.2 mmol) and triethylamine (5.2 ml, 37.2 mmol). The suspension was cooled to -50° C., then formic acid (1.4 ml, 37.2 mmol) in DCM (5 ml) was added. The reaction mixture was stirred at -50°C for 1 h, then at 4°C for 3 h. The solution was diluted with 1N. HCl and extracted with DCM (2x). The combined organic phases were washed with brine and dried over MgSO ₄ . Filtration and concentration gave 3.89 g (81% yield) of formylated glycine in the form of an oil (M+1=194).

To a solution of the formylated glycine derivative (1 g, 5.2 mmol) in DCM (30 ml) was added triethylamine (3.2 ml, 23 mmol). The solution was cooled to -50°C and slowly added POCl ₃ (1.9 ml, 20.8 mmol). The solution was stirred at -50°C for 10 minutes, then stirred at room temperature for 40 minutes. The solution turned light red-brown. This solution was diluted with DCM and 20% sodium carbonate solution (100 ml) was added. The reaction mixture was vigorously stirred for 15 minutes. The organic phase was separated twice and dried over MgSO ₄ . Filtration and concentration to give the desired benzyl isocyanoacetate in quantitative yield, which was used in the next step without further purification.

To a solution of 1,2,4-triazole (914 mg, 13.2 mmol) in anhydrous CH3CN (20 ml) at 0°C was added iPr ₂ NEt (2.5 ml, 14.6 mmol). Once all the triazole had dissolved, POCl ₃ (0.43 ml, 4.6 mmol) was added. The mixture was stirred at 0° C. for 2 hours. The lactam ester 16' (1 g, 3.31 mmol) was added. The final solution was heated in an oil bath at 80° C. for 16 hours. The mixture was cooled with an ice bath. Dilute with EtOAc then add water. The aqueous layer was separated and extracted with EtOAc four times. The combined organic extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration gave a light yellow solid which was used directly in the next step (M+1=354).

A solution of benzyl isocyanate (892 mg, 5.1 mmol) in DMF (10 ml) was cooled to -50° C. under nitrogen. KOtBu (514 mg, 4.6 mmol) was added. The mixture was stirred at -50°C for 1 hour. The triazole derivative prepared above (900 mg, 2.55 mmol) in DMF (5 ml) was added slowly at -50°C. The mixture was allowed to warm to room temperature over 16 hours. Saturated aqueous NH ₄ Cl was added and extracted with EtOAc three times. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration gave the crude product. Chromatography (24 g RediSep silica gel column, eluting with 70% EtOAc in Hexanes) gave 886 mg (76% yield) of product (M+1=460).

To a solution of the benzyl ester derivative (770 mg, 1.68 mmol) in EtOAc (10 mL) and MeOH (30 mL) was added wet Pd/C (60 mg) and the resulting suspension was stirred under hydrogen atmosphere for 48 hours. filtered through Celite, and the final solution was concentrated. The crude debenzylated product (530 mg, 86% yield) was used in the next step without further purification (M+1=370).

To a suspension of the acid (530 mg, 1.44 mmol) in DCM (10 ml) was added CDI (931 mg, 5.75 mmol). The solution was stirred at room temperature for 2 hours. The solution was cooled with an ice bath and NH4OH solution (6 ml) was added. The solution was stirred for 30 minutes and concentrated. The solid was collected by filtration and washed with water to give 422 mg (80%) of the desired product as a brown solid. (M+1=369).

To a suspension of the basic amide derivative (422 mg, 1.15 mmol) in dioxane (10 ml) was added POCl ₃ (160 μl, 1.7 mmol). The suspension was heated at 90° C. for 2 hours. The final solution was cooled with an ice bath and quenched with saturated aqueous NaHCO ₃ . The solid was collected by filtration to give 308 mg (11% yield) of the desired cyanide derivative. (M+1=351).

To a suspension of the cyanide derivative (150 mg, 0.44 mmol) in EtOH (4 ml) and water (1 ml) were added hydroxylamine hydrochloride (40 mg, 0.57 mmol) and potassium carbonate (67 mg, 0.48 mmol). The suspension was stirred at room temperature for 16 hours. LCMS showed approximately 50% conversion. Additional hydroxylamine hydrochloride (40 mg, 0.57 mmol) and potassium carbonate (67 mg, 0.48 mmol) were added and stirred for an additional 24 h. The solution was diluted with EtOAc and washed with water. The combined organic phases were washed with saline, dried over MgSO ₄ . Filtration and concentration gave 145 mg (86% yield) of the desired product. (M+1=384).

To a solution of acetic acid (0.22 ml, 3.8 mmol) in THF (5 ml) was added CDI (123 mg, 0.76 mmol). The solution was stirred at room temperature for 2 h. The solution was then poured into a flask containing the oxime derivative (145 mg, 0.38 mmol) and heated at 70° C. for 1 h. The solvent was evaporated and the crude material was suspended in acetic acid (8 ml) and heated at 130° C. for 1 hour. The solvent was evaporated and the crude material was triturated with water to give 134 mg (86%) of the desired product (M+1=408).

To a suspension of the ether derivative (50 mg, 0.12 mmol) in THF (1 ml) was added lithium aluminum hydride (7 mg, 0.18 mmol). The suspension was stirred at room temperature for 2 hours. LCMS showed approximately 70% conversion along with some other by-products and a small amount of starting material remaining. Additional lithium aluminum hydride (4 mg) was added and the reaction mixture was stirred at room temperature for an additional 30 minutes. The reaction mixture was quenched with 1N. HCl. The solution was extracted with EtOAc (3x). The combined organic phases were washed with saline, dried over MgSO ₄ . Filtration and concentration gave 20 mg (45% yield) of the desired alcohol product. (M+1=366).

To a suspension of alcohol (20 mg, 0.055 mmol) in dioxane (1 ml) was added POBr ₃ (31 mg, 0.11 mmol). The reaction mixture was heated at 110° C. for 1 hour. The reaction mixture was cooled with an ice bath and sat. aqueous solution of NaHCO ₃ . The final solution was extracted with EtOaAc (3x). The combined organic phases were washed with brine and dried over MgSO ₄ . The solvent was concentrated to give 22 mg (96% yield) of the desired product (M+1=428).

To an ampoule containing an alkyl bromide derivative (22 mg, 0.052 mmol) was added 3-fluorophenol (58 mg, 0.52 mmol) in dioxane (1 ml) and potassium carbonate (72 mg, 0.52 mmol). The reaction mixture was heated at 90° C. for 1 hour. The reaction mixture was diluted with sat. aqueous solution of NaHCO ₃ . The final solution was extracted with EtOac (3x).

The combined organic phases were washed with brine and dried over MgSO ₄ . Filtration and concentration gave the crude product. Purification by preparative TLC (eluting system: EtOAc) to give 5 mg (21% yield) of the desired Compound 168 (M+1=460).

1H-NMR (CDCl ₃ ) δ 7.87 (1H, s), 7.65 (1H, d, J=3.5 Hz), 7.57 (1H, d, J=10 Hz), 7.24 (1H, m), 7.19 (1H, dd, J=3.5, 9 Hz), 6.77 (1H, dd, J=2.5, 9.5 Hz), 6.72 (2H, m), 5.26(2H, s), 3.97(3H, s), 2.48(3H, s).

By carrying out reactions similar and analogous to those shown in schemes 1-27, the following compounds were obtained, which are also specifically considered in this application:

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Example 105 Assessing the activity of a positive allosteric modulator of the α5-containing GABA receptor (GABA _A R)

Step 1: Set up clones of GABA _A -R subunits (α5, β3, γ2, α1, α2 and α3) and obtain corresponding cRNAs: Human clones of GABA _A -R subunits α5, β3, γ2, α1, α2 and α3 were obtained from commercial resources ( for example, OriGene, http://www.origene.com and Genescript, http://www.genescript.com). These clones were inserted into pRC, pCDM, pcDNA and pBluescript KSM vectors (for oocyte expression) or other equivalent expression vectors. Standard transfection agents (e.g. FuGene, lipofec

- 113 039381 tamin 2000 or others) are used for transient transfection of host cells.

Stage 2 - Functional Test of GABA _A R subtypes α5β3γ2, α1β3γ2, α2β3γ2 and α3β3γ2 in the Xenopus oocyte expression system: cRNAs encoding subunits α5, β3, γ2, α1, α2 and α3 were transcribed in vitro using the T3 mMESSAGE mMACHINE kit (Ambion) and injected (with respect to α:β:γ=2:2:1 or other optimized conditions) into oocytes recently obtained from Xenopus laevis. After two days of culture, GABA ^- dependent Cl current from oocytes was stimulated using TEVC units (Warner Instruments, Inc., Foster City, CA). GABA, benzodiazepine and diazepam were used as reference compounds to test the system.

Step 3 - Evaluate test compounds for positive allosteric modulator activity on the α5β3γ2 subtype and test for off-target activity on α1-α3 coupled β3γ2 subtypes when a reduction in selectivity EC ₅₀ =5 μM ^is achieved: GABA-dependent Cl current from oocytes was measured in a TEVC setup in the presence of tested compounds. The positive allosteric modulator activity of each test compound was tested in a 5-point dose-response test. Test compounds include some reference compounds (literature EC ₅₀ values for the α5β3γ2 subtype are in the range of 3-10 μM). EC ₅₀ in the α5β3γ2 subtype was obtained for each compound. If the EC ₅₀ in α5β3γ2 is <5 μM, then the EC _{50 of} the other three subtypes (α1β2γ2, α2β3γ2 and α3β3γ2) were further determined individually to test the selectivity of compounds to the α5β3γ2 subtype relative to other subtypes.

Step 4 - Evaluate other test compounds for the α5β3γ2 subtype and check for off-target activity when a decrease in selectivity EC ₅₀ =0.5 μM is achieved: a second batch of test compounds were tested using the same strategy but with a cut-off at a lower EC ₅₀ (0.5 µM). Here, the EC ₅₀ of the α5β3γ2 subtype was also determined for each of the compounds. α1-α3 conjugated β3γ2 subtypes were tested only if the EC ₅₀ for the a5-containing receptor is < 0.5 μM.

Example 106 Evaluation of Compounds for Binding and Activity of a Positive Allosteric Modulator of GABAA α5 Receptors (A) Binding Activity of Test Compounds for GABAAI

Tissue culture and membrane preparation: binding was performed on Ltk cells stably expressing the α1β1γ2, α2β3γ2, α3β3γ2 and α5β3γ2 GABA receptors (supplied by Merck Co., New Jersey, USA). Cells were seeded in 100 mm Petri dishes in DMEM/F12 medium containing 10% serum and antibiotics in 5% CO ₂ and allowed to grow for 1-2 days. GABAAI expression was then induced with dexamethasone as follows: 0.5 μM for 1 day for a5-containing and 2 μM for 3 days for α1, α2 and a3-containing GABAAI. After induction, cells were harvested by scraping into Dulbecco's phosphate buffered saline (DPBS, pH 7.4, Invitrogen, Carlsbad, CA, USA) and centrifuged at 150xg for 10 min. The pellet was washed twice by resuspension and centrifugation. Cell pellets from at least 5 different lots were pooled, suspended in binding test buffer (50 mM KH ₂ PO ₄ ; 1 mM EDTA; 0.2 M KCl, pH 7.4) and membranes prepared by sonication (3-5 times, 30 seconds) using the Branson 150 ultrasonic disintegrator (G.Heinmann, Germany). Protein content was determined using the BCA assay (Bio-Rad Labs, Reinach, Switzerland) with Bovine Serum Albumin (SigmaAldrich, St. Louis, MO, USA) as standard. Aliquots were received and stored at -20°C for further use in binding tests.

Ligand Binding: Compound saturation curves were obtained by incubating membranes with increasing concentrations (0.01-8 nM) of [ ³ H]Rol5-1788 (Flumazepil, 75-85 Ci/mmol, PerkinElmer, MA, USA) measuring non-specific binding in the presence of 10 μM diazepam. Inhibition of the binding of test compounds to [ ³ H]Rol5-1788 was performed at labeled ligand concentrations equal to or lower than the Kd values for α1, α2, α3 and α5-containing GABAAI determined from saturation curves.

All binding tests were performed for 1 hour at 4° C. in test buffer. The total test volume was 0.5 ml containing 0.2 mg/ml of protein for α5 and 0.4 mg/ml for α1, α2 and α3-containing GABAAI membranes. Incubations were terminated by filtration through GF/B filters using a 24-Cell Harvestor (Brandel, Gaithersburg, MD, USA) followed by 3 washes with ice-cold test buffer. The filters were transferred into scintillation ampoules, 5 ml of scintillation liquid was added, vortexed, and stored in the dark. The next day, radioactivity was obtained using a scintillation counter (Beckman Coulter, Brea, CA, USA). All tests were performed in triplicate.

Data Analyzes: Saturation and inhibition curves were generated using GraphPad Prism software (GraphPad Software, Inc., CA, USA). Equilibrium dissociation constants (Ki values) of the unlabeled ligand were determined using the Cheng-Prusoff equation Ki=IC ₅₀ / (1+S/Kd), where IC ₅₀ denotes the concentration of unlabeled ligand that inhibits 50% binding of [ ³ H]ligand, S denotes labeled ligand concentration, and Kd is the equilibrium constant

- 114 039381 dissociation of the radioactive ligand. The log range of compounds (1 nM-10 μM) was used to determine Ki values, presented as Mean ± SD from triplicate tests.

(B) Positive allosteic modulator activity of test compounds on the α5β2γ2 subtype in GABA _A R

Compounds of the present invention were initially tested at 100 nM for their ability to potentiate GABA EC ₂₀ concentration in oocytes containing GABA (α5β2γ2) receptors using a protocol essentially similar to that presented above.

On the day, 1.1 ng/32 nl of GABAA α5β2γ2 cDNA was injected into one oocyte. The test starts on day 2. The cDNA injected into oocytes was a mixture of alpha, beta and gamma, their ratio was 1:1:10 (by weight) and the total weight of the mixed 3 subunits that were injected into one oocyte was 1 ng in volume 32 nl. Injected oocytes can also be tested on day 3. In this case, the amount of cDNA injected into oocytes should be reduced by 20%.

Compounds of the present invention were tested using the following procedures.

Dose-response GABA

one) . 8 oocytes were placed in 8 OpusXpress chambers and infused with Barth's modified saline (MBS) at 3 ml/min. Glass electrodes filled with 3M KCl (0.5-3 mega ohm) were used. The membrane potential of oocytes was fixed at a voltage of -60 mV.

2). The average EC20 GABA obtained in the previous tests was applied five to six times to stabilize the oocytes. Oocytes were washed with MBS for 5-10 min between each GABA application.

3). Lined dose-response GABA to obtain the value of the EU ₂₀ GABA.

Control test (diazepam or methyl 3,5-diphenylpyridazine-4-carboxylate)

1) New oocytes were used for the new test.

2) EC ₂₀ GABA was applied five to six times to stabilize the oocytes. Oocytes were washed with MBS for 5-10 minutes between each GABA application.

3) EU ₂₀ GABA applied to obtain current (1 _GABA ). Oocytes were washed with MBS for 5-10 min.

4) 1 μM diazepam or methyl 3,5-diphenylpyridazine-4-carboxylate was pre-applied for 40 s followed by 1 μM diazepam or methyl 3,5-diphenylpyridazine-4-carboxylate and EC ₂₀ GABA to obtain I _test . I _test was divided into 1 _GABA to obtain potentiation (%).

Analysis of compounds in multiple doses

one) . Repeat steps 1), 2) and 3) above in the control test.

2). The first concentration of test compound was pre-applied for 40 s followed by co-application of test compound at the same concentration and EC ₂₀ GABA to obtain I _test . Divide I _test by 1 _gamma to obtain potentiation (%).

3). All tested oocytes are discarded, new oocytes are used, and steps 1) and 2) above are repeated to test a second concentration of the same compound. Each oocyte is used for only one concentration analysis for a single test compound. The steps are repeated for other test compounds.

In some embodiments, the compounds of this application have a binding affinity (represented by K±) in a5-containing GABAAI less than 200 nM, less than 180 nM, less than 150 nM, or less than 100 nM. In some embodiments, the compounds of this application have a binding affinity (represented by Ki) in a5-containing GABAAI less than 50 nM. In some embodiments, the compounds of this application have a binding affinity (represented by K _i ) in a5-containing GABAAI less than 10 nM.

In some embodiments, the compounds of this application are selective for α5-containing GABAAI over α1-containing GABAAI. In some embodiments, the compounds of this application have greater than 50-fold, greater than 100-fold, greater than 500-fold, or greater than 1000-fold selectivity for α5-containing GABAAI to α1-containing GABAAI .

In some embodiments, the compounds of this application have an EC ₅₀ on α5 containing GABAAI less than 500 nM, less than 100 nM, or less than 50 nM. In some embodiments, the compounds according to this application have an EC ₅₀ on a5-containing GABAAI less than 25 nM.

In some embodiments, the compounds of this application potentiate α5-containing GABAAI by greater than 10%, greater than 25%, greater than 50%, or greater than 75% at a concentration of 100 nM. In some embodiments, the compounds of this application potentiate α5-containing GABAAI by greater than 10%, greater than 25%, greater than 50%, or greater than 75% at a concentration of 1000 nM.

The results of screening in tests of binding and functional activity of PAM are presented below in table. 1 and 2.

Next table. 1 illustrates the Ki ranges of GABA 5α binding to the compounds of the invention:

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Table 1

K values _± GABA 5α binding (nM) <99 nM 100-1000 nM >1000 nM Connections 1, 2, 3, 4, 6, 7.8, 9, 10, 11, 12, 44, 55, 101, 103, 105, 107, 108, 114, 128, 153, 158, 162, 163, 164 , 166, 169, 171, 172, 173, 174, 175, 177, 179, 5, 47, 48, 49, 51, 52, 53, 54, 56, 102, 104, 106, 111, 112, 118, 120 , 126, 127, 130, 133, 137, 145, 147, 148, 149, 155, 156, 157, 160, 165, 168, 178, 45, 46, 109, 122, 129, 132, 150, 151, 159 , 161, 167, 176 Connections 50, 110, 113, 115, 119, 124, 125, 134, 136, 138, 139, 141, 143, 144, 146, 170, Connections 116, 117, 121, 123, 131, 135, 140, 142, 143, 152, 154

Next table. 2 illustrates the ranges of functional potentiation of GABA 5 α associated with the compounds of the invention:

table 2

GABA 5α functional data 20-49% at 100nM >50% at 100nM Connections 1, 2, 9, 11, 48, 45, 55, 109, 110, 111, 118, 120, 126, 127, 128, 130, 132, 137, 147, 148, 153, 155, 158, 162, 163 , 175 Compounds 113, 114, 145, 149, 160, 171, 172, 173, 174, 176, 177, 178, 179

Selected compounds of the invention exhibit >10-fold binding selectivity over GABA 1α, GABA 2 α or GABA 3 α.

Example 107 Effect of methyl 3,5-diphenylpyridazine-4-carboxylate in age-aggravated rats (AI)

Methyl 3,5-diphenylpyridazine-4-carboxylate corresponding to compound number 6 in van Niel et al. J. Med. Chem. 48:6004-6011 (2005), is a selective α5-GABA R agonist. It has an in vitro α5 potency of +27 (EC ₂₀ ). The effect of methyl 3,5-diphenylpyridazine-4-carboxylate in aging-impaired rats was studied using the RAM task. In addition, receptor occupancy with methyl-3,5-diphenylpyridazine-4-carboxylate on the α5-containing GABA receptor was also studied.

(A) Effect of methyl 3,5-diphenylpyridazine-4-carboxylate in aging-disturbed rats using a behavioral task with a radial eight-arm maze (RAM)

The effects of methyl 3,5-diphenylpyridazine-4-carboxylate on in vivo spatial memory retention in age-impaired (AI) rats were assessed in a behavioral radial eight-arm maze (RAM) test using vehicle control and four different dosage levels. methyl 3,5-diphenylpyridazine-4-carboxylate (0.1, 0.3, 1 and 3 mg/kg, IP). The RAM behavioral test was performed on eight AI rats. All five treatment conditions (vehicle and four dosage levels) were tested in all eight rats.

The RAM apparatus used consisted of eight equally spaced arms. An elevated labyrinth arm (7 cm wide ± 75 cm long) extends from each surface of an octagonal central platform (30 cm diameter, 51.5 cm high). The transparent sidewalls of the sleeve are 10 cm high and rotated 65° to form a recess. A cell with food (4 cm in diameter, 2 cm deep) is located at the distal end of each sleeve. Froot Loops™ (Kellogg Company) were used as rewards. Blocks constructed from Plexiglas™ (30 cm high x 12 cm wide) could be placed to prevent penetration into either sleeve. Numerous additional parts of the labyrinth surrounding the apparatus were also provided.

Rats with AI were initially subjected to pre-training testing (Chappell et al. Neuropharmacology 37: 481-487, 1998). Pre-training testing consisted of an habituation phase (4 days), a training phase with a standard task-change pattern (18 days), and another training phase (14 days) in which a brief delay was introduced between the presentation of a subpopulation of sleeves determined by the experimenter (e.g., 5 available arms and 3 blocked arms), and completion of the execution-change task scheme with eight arms (i.e. with all eight available arms).

In the habituation phase, rats were introduced to the maze during an 8-minute procedure for four consecutive days. In each of these procedures, food rewards were distributed in RAM initially on the central platform and in the arms, and then progressively localized only in the arms. After this habituation phase, a standard training protocol was used in which a food ball was placed at the end of each sleeve. The rats underwent one test each day for 18 days. Each daily trial ended when all eight food balls had been received, or when either the choice had been made 16 times or 15 minutes had elapsed. After the completion of this training phase, a second training phase was performed in which the memory requirement was made more difficult by introducing a brief delay during the test. At the beginning of each test, three arms of the eight-arm labyrinth were blocked. The rats were allowed to receive food in five sleeves, which were made available during this initial informational phase of the trial. The rats were then removed from the maze for 60 seconds, during which time the barriers on the maze were removed, thus providing access to all eight arms. The rats were returned back to the central platform and allowed to receive the remaining food rewards during this phase of the retention test. The identity and configuration of the blocked sleeves varied from test to test.

The number of errors made by AI rats during the retention test phase was recorded. A trial error was counted if the rats entered a sleeve from which food had already been removed in the pre-hold phase of the test, or if a rat in the procedure after the delay revisited a sleeve that it had already visited before.

After completion of the pre-training test, rats were subjected to more extended delay intervals, ie, a two-hour delay, between the information phase (presentation with some sleeves blocked) and the retention test (presentation of all sleeves). During the delay interval, the rats were kept outside the maze in the laboratory in their individual cages. Rats with AI were pre-treated once 30-40 min prior to daily trials using the following five conditions: 1) vehicle control - 5% dimethyl sulfoxide, 25% polyethylene glycol 300, and 70% distilled water; 2) 0.1 mg/kg methyl 3,5-diphenylpyridazine-4-carboxylate; 3) 0.3 mg/kg methyl 3,5-diphenylpyridazine-4-carboxylate; 4) 1 mg/kg methyl 3,5-diphenylpyridazine-4-carboxylate; and 5) 3 mg/kg methyl 3,5-diphenylpyridazine-4-carboxylate; by intraperitoneal (i.p). injections. Injections were carried out every other day with intermediate withdrawal days. Each AI rat was treated in all five conditions during the testing period. To balance any potential bias, the effect of the drug was evaluated using an ascending-descending series of doses, ie, the series of doses was first given in ascending order and then repeated in descending order. Therefore, each dose had two definitions.

Parametric statistics (paired t-tests) were used to compare the results of retention tests in AI rats in a two-hour delay version of the RAM test in the context of different doses of methyl 3,5-diphenylpyridazine-4-carboxylate and vehicle control (see Fig. 1). The mean numbers of errors that occurred in the trials were significantly lower for treatment with methyl 3,5diphenylpyridazine-4-carboxylate at 3 mg/kg (mean error ± mean standard deviation (SEM) = 1.31 ± 0.40), than using the vehicle control (mean error±SEM=3.13±0.62). Compared to vehicle control treatment, methyl 3,5-diphenylpyridazine-4-carboxylate significantly improved memory performance at 3 mg/kg (t(7)=4.233, p=0.004).

The therapeutic dose of 3 mg/kg became ineffective when rats with AI were competitively treated with 0.3 mg/kg of TB21007, an inverse agonist of α5-containing GABA _a R. diphenylpyridazine-4-carboxylate (0.3mg/kg TB21007 with 3mg/kg methyl 3,5-diphenylpyridazine-4-carboxylate) was 2.88±1.32 and did not differ from vehicle control (3, 13±1.17 mean errors). Thus, the effect of methyl 3,5-diphenylpyridazine-4-carboxylate on spatial memory is a GABAA α5 receptor dependent effect (see FIG. 1).

(B) Effect of methyl 3,5-diphenylpyridazine-4-carboxylate on occupancy of the α5-containing GABA receptor

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Animals

Adult male Long Evans rats (265-295 g, Charles River, Portage, MI, n=4/group) were used for GABA _A 5a receptor filling studies. Rats were individually housed in ventilated stainless steel racks on a 12:12 light/dark cycle. Food and water were available ad libitum. In additional studies, young or old Long Evans rats (n=2-4/group) were used to evaluate the effect of the compound at behaviorally active doses.

Connections

Ro 15-4513 was used as a radioactive receptor occupancy indicator (RO) for cellular GABA _A 5a receptors in the hippocampus and cerebellum. Ro 15-4513 was chosen as a radiotracer based on its selectivity for GABAa receptors over other alpha subunit-containing GABA receptors and because it has been successfully used in animal and human GABAA5a RO studies [see, e.g., Lingford-Hughes et al. ., J. Cereb. Blood Flow Metab. 22:878-89 (2002); Pym et al., Br. J Pharmacol. 146: 817-825 (2005); and Maeda et al., Synapse 47: 200-208 (2003)). Ro 15-4513 (1 μg/kg) was dissolved in 25% hydroxypropyl beta-cyclodextrin and administered intravenously 20 min prior to RO assessment. Methyl 3,5-diphenylpyridazine-4-carboxylate (0.1-10 mg/kg) was synthesized by Nox Pharmaceuticals (India) and was dissolved in 25% hydroxypropyl beta-cyclodextrin and administered intravenously 15 min prior to radioactive tracer injection. Compounds were administered in a volume of 0.5 ml/kg except for the highest dose of methyl 3,5-diphenylpyridazine-4-carboxylate (10 mg/kg) which was administered in a volume of 1 ml/kg due to solubility limitations.

Tissue preparation and analysis

The rats were sacrificed by turning their necks 20 min after injections of a radioactive tracer. The whole brain was quickly removed and lightly rinsed with sterilized water. Stem blood was collected in EDTA, placed in eppendorf tubes, and kept on wet ice until completion of the study. The hippocampus and cerebellum were analyzed and stored in 1.5 ml eppendorf tubes and placed on wet ice until tissue extraction. Six samples of drug-intact rat cerebral cortex were collected for use in generation of blanks and standard curve samples.

Acetonitrile containing 0.1% formic acid was added to each sample in four times the weight of the tissue sample. For standard curve samples (0.1-30 ng/g), the calculated volume of standard reduced the volume of acetonitrile. The sample was homogenized (FastPrep-24, Lysing Matrix D; 5.5 m/s for 60 seconds or 7-8 W using an acoustic probe dismembrator; Fisher Scientific) and centrifuged for 16 minutes at 14,000 rpm. The supernatant solution (100 µl) was diluted with 300 µl of sterilized water (pH 6.5). This solution was then thoroughly mixed and analyzed by LC/MS/MS for Ro 15-4513 (radiotracer) and methyl 3,5-diphenylpyridazine-4-carboxylate.

For plasma exposure, blood samples were centrifuged at 14,000 rpm for 16 minutes. After centrifugation, 50 µl of supernatant (plasma) from each sample was added to 200 µl of acetonitrile plus 0.1% formic acid. For the standard curve sample (1-1000 ng/mL), the calculated volume of standard reduced the volume of acetonitrile. Samples were sonicated for 5 min in an ultrasonic water bath followed by centrifugation for 30 min at 16,000 rpm. 100 μl of the supernatant was removed from each vial and placed in a new glass vial of the autosampler followed by the addition of 300 μl of sterilized water (pH 6.5). This solution was then thoroughly mixed and analyzed by LC/MS/MS for methyl 3,5-diphenylpyridazine-4-carboxylate.

Receptor uptake was determined by a ratio method that compared uptake in the hippocampus (a region of high density of the GABA5a receptor) with that in the cerebellum (an area of low density of the GABA _A 5a receptor) and an additional higher dose of negative GABA _A 5a allosteric modulator L-655,708 (10 mg /kg, intravenously) to determine complete filling.

Vehicle administration followed by 1 μg/kg intravenous radiotracer, Ro 15-4513 resulted in >5-fold higher levels of Ro 15-4513 in the hippocampus (1.93±0.05 ng/g) compared to the cerebellum ( 0.36±0.02 ng/g). Methyl 3,5-diphenylpyridazine-4-carboxylate (0.01-10 mg/kg, iv) dose-dependently reduced hippocampal Ro 15-4513 binding without affecting cerebellar Ro 15-4513 levels (FIG. 2) in dose of 10 mg/kg, intravenously, showing >90% fill (Fig. 3). Both methods of calculating RO resulted in very similar results with ED ₅₀ values for methyl 3,5-diphenylpyridazine-4-carboxylate of 1.8 mg/kg or 1.1 mg/kg based on the ratio method or using L-755,608 for padding definitions.

Exposure to methyl 3,5-diphenylpyridazine-4-carboxylate was below the Limits of Quantification (BQL) at 0.01 mg/kg, IV, both in plasma and in the hippocampus, but was detectable at low levels in the hippocampus at 0.01 mg/kg. 1 mg/kg IV (see Table 3). Hippocampal exposure was linear as a 10-fold increase in dose from 0.1 to 1 mg/kg IV, resulting in a 12-fold increase in exposure. Increasing the dose from 1 to 10 mg/kg, intravenously, increased exposure only ~5-fold. Plasma exposure increased 12-fold when the dose was increased from 1 to 10 mg/kg, intravenously.

Table 3. % GABA _a receptor occupancy with α5 methyl 3,5-diphenylpyridazine-4-carboxylate (0.01-10 mg/kg, IV). Hippocampal and plasma exposure to methyl 3,5-diphenylpyridazine4-carboxylate in a treatment group in young Long Evans rats

DOSE (mg/kg, IV) %RO (Method L655,708) (SEM) %RO (Relationship method) (SEM) Plasma ng/ml (SEM) Hippocampus ng/g (SEM) 0.01 19.2 (11.1) 15.7 (9.1) BQL BQL 0.1 16.4 (4.9) 13.4 (4.0) BQL 14.6 (3.5) one 38.5 (11.2) 31.5 (9.1) 62.8 (6.1) 180.0 (10.3) ten 110.0 (6.6) 90.2 (5.4) 763.5 (85.7) 947.2 (51.3)

Additional studies were performed in aged Long Evans rats to determine exposure at behaviorally relevant doses in cognition studies. Exposure in young Long Evans rats was also determined to relate to receptor filling studies conducted in young Long Evans rats. Exposure in both young and aged Long Evans rats was relatively similar (Table 4, Fig. 4). A 3-fold increase in dose from 1 to 3 mg/kg, ip, resulted in a more than dose-proportional increase in exposure in both young and aged rats in both the hippocampus and plasma, with increases ranging from 4.5 to 6 ,6 times.

Table 4. Hippocampal and plasma exposure of methyl 3,5-diphenylpyridazine-4-carboxylate in young Long Evans rats in the treatment group

Young Young Age Age Dose (mg/kg, IP) Hippocampus ng/g (SEM) Plasma ng/ml (SEM) Hippocampus ng/g (SEM) Plasma ng/ml (SEM) one 25.9 (1.7) 20.0 (1.4) 38.8 (21.7) 45.2 (29.6) 3 129.1 (22.4) 132.9 (19.5) 177.5 (19.5) 196 (18.2)

In RO studies, exposure to 180 ng/g in the hippocampus (1 mg/kg, IV) represented 32-39% receptor occupancy depending on the method used to determine RO. This exposure is comparable to that observed in aged rats at 3 mg/kg, ip, suggesting that an RO of 30-40% is required for cognitive performance in this model.

These studies demonstrated that methyl 3,5-diphenylpyridazine-4-carboxylate resulted in a dose-dependent increase in α5 GABAA receptor filling. Methyl 3,5-diphenylpyridazine-4-carboxylate also showed good exposure in the brain with brain/plasma ratios >1. Studies further demonstrated that methyl 3,5-diphenylpyridazine-4-carboxylate exerted its cognitive enhancing effects through positive allosteric modulation at _the α5 GABA subtype receptor.

Example 108 Effect of ethyl 3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3d][1,4]diazepine-10- carboxylate in rats with age-related disorders (AI)

Ethyl 3-methoxy-7-methyl-9H-benzo[1]imidazo[1,5-a] [1,2,4]triazolo[4,3-d] [1,4]diazepine-10-carboxylate, corresponding to compound number 49 in Achermann et al. Bioorg. Med. Chem. Lett., 19:5746-5752 (2009), is a selective α5-GABA _a R agonist.

Effect of ethyl 3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4]diazepine-10carboxylate on retention in spatial memory of rats with age-related disorders (AI) in vivo was assessed in a behavioral test using a radial eight-armed maze (RAM), which is essentially similar to the test as described in example 107 (A), using vehicle as control (25% cyclodextrin , with 3-fold testing: at the beginning, in the middle and at the end of the ascending / descending series) and six different dose levels (0.1, 0.3, 1, 3, 10 and 30 mg/kg, with each dose tested twice) ethyl 3-methoxy-7-methyl-9H-benzo[1]imidazo[1,5a][1,2,4]triazolo[4,3-d][1,4]diazenine-10-carboxylate. The same experiment was repeated using the same vehicle as control and doses of ethyl 3-methoxy-7-methyl-9H-benzo[1]imidazo[1,5a][1,2,4]triazolo[4,3-d ][1,4]diazepine-10-carboxylate, where vehicle control was tested 5 times, dose 3mg/kg ethyl-3-methoxy-7-methyl-9H-benzo[1]imidazo[1,5-a ][1,2,4]triazolo[4,3d][1,4]diazepine-10-carboxylate was tested 4 times, and other doses of ethyl 3-methoxy-7-methyl-9Hbenzo[1]imidazo[1, 5-a][1,2,4]triazolo[4,3-d][1,4]diazepine-10-carboxylate were tested twice.

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Parametric statistics (paired t-tests) were used to compare retention test scores in rats with AI in a four-hour version of the RAM retention test in the context of different doses of ethyl-3-methoxy-7-methyl-9H-benzo[t]imidazo[1 ,5-a] [ 1,2,4]triazolo[4,3-d] [ 1,4]diazepine-10carboxylate and vehicle as control (see Fig. 5). Compared with vehicle control treatment, ethyl-3-methoxy-7-methyl-9H-benzo[£]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1 ,4]diazepine-10carboxylate significantly improved memory performance at doses of 3 mg/kg (t(7)=4.13, p=0.004, or t(7)=3.08, p=0.018) and 10 mg/kg ( t (7)=2.82, p=0.026).

Effect of ethyl 3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4]diazepine-10carboxylate on filling The α5-containing GABA receptor was also studied using a procedure that is essentially similar to that described in Example 107(B) (see above). This study demonstrated that ethyl 3-methoxy-7-methyl-9H-benzo[f^imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4]diazepine -10-carboxylate (0.01-10 mg/kg, iv) reduced hippocampal Ro 15-4513 binding without altering cerebellar Ro 15-4513 levels (Figure 6) at 10 mg/kg, iv, demonstrating > 90% coverage (FIG. 7).

Example 109 Effect of 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2benzothiophen-4(5H)-one in rats with age-related disorders using behavioral test in the Morris water maze

6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one corresponding to compound 44 in Chambers et al. J. Med. Chem. 46:2227-2240 (2003), is a selective agonist of α5-containing GABA R.

Effects of 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-b,7-dihydro-2-benzothiophene-4(5Н)one on spatial memory retention in rats with age impairments (AI) in vivo was assessed in a behavioral test in the Morris water maze. The water maze is a pool surrounded by a new set of structures relative to the maze. A water maze training protocol may be based on a modified water maze test that has been shown to be hippocampal dependent (de Hoz et al., Eur. J. Neurosci., 22:745-54, 2005; Steele and Morris, Hippocampus 9:118-36, 1999).

Rats aged with deterioration in cognitive function were introduced unilaterally with a cannula into the lateral ventricle. Stereotaxic coordinates were 1.0 mm posterior to the bregma, 1.5 mm lateral to the midline, and 3.5 mm ventral to the skull surface. After about a week of recovery, the rats were pre-trained in the water maze for 2 days (6 trials per day) to locate a submerged rescue platform hidden below the surface of the pool, with the location of the rescue platform varied from day to day. No intracerebroventricular (ICV) infusions were given during pre-training.

After pre-training, rats received either an ICV infusion of either 100 µg of 6,6-dimethyl-3-(3hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one ( n=6) in 5 μl of DMSO or vehicle representing DMSO (n=5) 40 minutes before training in the water maze and testing. The training consisted of 8 trials per day for 2 days where the hidden rescue platform remained in the same location. The rats were given 60 s to find the location of the platform with an interval of 60 s between tests. The rats were given test analysis (120 s) 24 hours after the end of training when the rescue platform was removed. There were 4 blocks during training, where each block had 4 practice trials.

Rats treated with vehicle and 6,6-dimethyl-3-thio-1-(3-hydroxypropyl)(thiazol-2-yl)-6,7dihydro-2-benzothiophen-4(5H)-one found the rescue platform in one and the same time at the beginning of training (block 1). In this training block, rats treated with vehicle and 6,6-dimethyl-3-(3hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one needed approximately 24 seconds to locate the rescue platform. However, rats treated with 6,6dimethyl-3-thio-1-(3-hydroxypropyl)(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one found the platform more skillfully (t .e., faster) at the end of training (block 4) than rats treated with vehicle alone. In block 4, rats treated with 6,6-dimethyl-3-thio-1-(3-hydroxypropyl)(thiazol-2-yl)-6,7dihydro-2-benzothiophen-4(5H)-one took approximately 9 6 s to locate the rescue platform, while vehicle-treated rats took approximately 19.69 s. These results suggest that 6,6-dimethyl-3-thio-1-(3-hydroxypropyl)(thiazol-2-yl)-6,7dihydro-2-benzothiophene-4(5H)-one improved learning in the water test. maze in rats (see Fig. 8(A)).

During the test run, 24 hours after training, the rescue platform was removed. The rat search/swim pattern was used to measure whether the rats remember where the rescue platform was located during training prior to testing the rats' long-term memory. In this test, the target ring is a defined area 1.5 times the size of the rescue platform located around the area where the platform was located during training prior to the test. The opposite ring is a control region of the same size as the target ring located opposite the target ring in the basin. If the rats had good long-term memory, they would show a tendency to search in the area surrounding the location where the platform was during pre-test learning (ie, in the target ring; not the opposite ring). Ring time is the amount of time, in seconds, that the rat spent in the area of the target or opposite ring. The number (#) of crossings in a ring is the number of times the rat has swum across the region of the target or opposite ring.

The vehicle-treated rats spent the same amount of time in the target ring and the opposite ring, indicating that these rats did not seem to remember where the platform was during pre-test training. In contrast, rats treated with 6,6-dimethyl-3-(3-hydroxypropyl)thio-1(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one spent significantly more time in the target ring and crossed the target ring more often than the time they spent in the opposite ring or the number of times they crossed the opposite ring. These results suggest that 6,6-dimethyl-3-thio-1-(3-hydroxypropyl)(thiazol-2-yl)-6,7-dihydro-2benzothiophene-4(5H)-one improved long-term memory in rats in water maze test (see Figs. 8(B) and 8(C)).

Compounds of the present invention have shown a positive allosteric modulating effect on the α5 GABAA receptor (See, for example, Example 106). These compounds improve the effects of GABA on _the α5 GABA receptor. Therefore, the compounds according to the present invention should have an effect on improving cognitive functions in animals with age-related impairment (such as rats), similar to the effects exerted by other selective α5 GABAA receptor agonists, such as methyl-3,5-diphenylpyridazine-4-carboxylate, ethyl- 3-methoxy-7-methyl-9Hbenzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3H][1,4]diazepine-10-carboxylate and 6,6-dimethyl- 3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (See, for example, Examples 28-30).

CLAIM

Claims (17)

CLAIM (1) -C(O)OMe or -C(O)OEt; or 2) where R ⁹ is selected from -H, -Me, -Et, CF ₃ , isopropyl or -tert-butyl. (1) a group consisting of halo, -R, -CN, -CF ₃ , -SO2R, -C(O)R and -C(O)OR, each R being independently substituted with 0-5 R'; (1) a group consisting of halogen, -(C1-C ₁₂ )aliphatic radical- and -O(C1-C ₁₂ )aliphatic radical; (1) a group consisting of halogen, -R, -(CH ₂ ) _1-3 R, -(CH ₂ ) _1-3 -OR, -CF ₃ or -C(O)OR, each R being independently substituted with 0 -5R'; 1. Compound of formula II II or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which m is an integer selected from 0-1; R ¹ is absent or selected from halogen, (C _g C ₁₂ )aliphatic radical, -O-(C1-C ₁₂ )aliphatic radical, -OCF ₃ or -CF ₃ ; R ² is selected from halogen, -R, -C(O)OR, C(O)R, -(CH ₂ ) _1-3 -OR, -(CH ₂ ) _1-3 R, -(CH ₂ ) _{1- 3} N(R) ₂ or C(O)N(R) ₂ ; R ³ is selected from halogen, -R, -C(O)OR, -CN, -C(O)R, -C(O)NH2, -(CHh^-OR or -SO2R; R ⁴ , R ⁵ and R ⁶ are H; each R is independently selected from H, (C ₁ -C ₁₂ )aliphatic, (C6-Cs)aryl, (C ₆ -C1 ₀ )aryl-(C 1 -C1 ₂ )aliphatic, (2) a group consisting of -H, -F, -Br, -Cl, -(C1-C ₆ )alkyl, -CN, -C=CH, -CF ₃ , -SO ₂ ((C ₁ -C ₆ )alkyl), -C(O)NH2, -C(O)((C1-C6)alkyl), -SO2(C6-C1o)aryl, -C(O)O((C _g C6)alkyl), - (C2-C6) alkenyl, -(C2-C ₆ ) alkynyl, -(C ₆ -C ₁₀ ) aryl and 5-10 membered heteroaryl, said alkyl, alkenyl, alkynyl, aryl or heteroaryl being independently substituted with 0-5 R '; or (3) a group consisting of -H, -C(O)OMe, -C(O)Et, -C(O)NH2, -C(O)OEt, -C(O)OCH2(t-butyl) , -C(O)OCH2CF3, -C(O)O(isopropyl), -CHF2, -CN, -C CH, -SO2Me, -SO2Et, -SO2Ph(Me), -CF3, -Me, -Et, -Br , -Cl, -CH2Ph, where R ⁹ is selected from -H, -Me, -Et, -CF ₃ , isopropyl or -tert-butyl. (2) a group consisting of halogen, -(C1-C ₆ )alkyl and -O((C ₁ -C ₆ )alkyl); or (3) a group consisting of -F, -Cl, -Br, -OMe and -OEt. (2) a group consisting of -H, -(C1-C ₆ )alkyl, -CH ₂ -O((C ₁ -C ₆ )alkyl), -CF ₃ , -C(O)O((C ₁ - C ₆ )alkyl), 310-membered heterocyclyl-, (C ₆ -C ₁₀ )aryl-, 5-10-membered heteroaryl- or (C ₆ -C ₁₀ )aryl-(C1C ₁₂ )aliphatic radical, and the specified alkyl, aryl or heteroaryl is independently substituted with 0-5 R'; 2. Compound of formula III - 121 039381 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which m is an integer selected from 0-1; R ¹ is selected from halogen, (C ₁ -C ₁₂ )aliphatic-, -O-(C ₁ -C ₁₂ )aliphatic and -OCF3; R ² is -(CH ₂ ) ₁ . ₃ R or -C(O)OR; R ³ is -C(O)OR; R ⁴ , R ⁵ and R ⁶ are H; each R is independently selected from H, (C1-C ₁₂ )aliphatic radical or (C6-Csh)aryl. (3) a group consisting of -H, -Me, -Et, propyl, isopropyl, butyl, tert-butyl, CF ₃ , -C(O)OMe, -C(O)OEt, -CH ₂ OMe, -CH ₂ OEt, -CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ -morpholine, -CH ₂ -pyridine or CH ₂ Ph, said R ² being substituted with 0-3 R'; - 122 039381 3-10 membered heterocyclyl or 3. Compound of formula IV IV or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer, or Z-isomer thereof, or a combination thereof, wherein R ¹ is selected from halogen, (C1-C ₁₂ )aliphatic-, -O-(C ₁ -C ₁₂ )aliphatic or -OCF3; R ² is selected from halo, -OR ⁸ , -SR ⁸ , -(CH2)nOR ⁸ , or (CH2) _n O(CH ₂ ) _n R ⁸ , where R ² is independently substituted with 0-5 R'; R ³ is selected from halogen, -R, -OR, -CN, -(CH ₂ ) _1-3 R, -(CH ₂ ) _1-3 -OR, C(O)OR, C(O)R or C( O)NH ₂ ; R ⁴ , R ⁵ and R ⁶ are H; each R ⁸ is independently -(C ₁ -C ₆ )alkyl, -(C ₃ -C ₁₀ )cycloalkyl, (C ₆ -C ₁₀ )aryl or 5-10 membered heteroaryl, wherein said (C ₆ -C ₁₀ )aryl is optional fused with (C ₆ -C ₁₀ )aryl and where each R ⁸ is independently substituted with 0-5 R'; m and n are independently integers selected from 0-1; each R is independently selected from H, (C1-C ₁₂ ) aliphatic radical, (C ₆ -C1 ₀ ) aryl-(C 1-C ₁₂ ) aliphatic radical, 3-10 membered heterocyclyl or (4) -Me substituted with 0-3 R' independently selected from -R'', OR'' or -CF ₃ , wherein R'' is independently selected from H, -(C ₁ -C ₆ )alkyl, (C ₆ -C ₁₀ )aryl or (C ₆ -C ₁₀ )aryl-(C ₁ -C ₆ )alkyl; 4. The compound according to claim 1 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer or Z-isomer, or combination thereof, in which R ² is selected from: 5. A compound according to any one of claims 1-4, or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer, or Z-isomer, or combination thereof, wherein each R ¹ is independently selected from: (5) a group consisting of -CH2Ph, -CH2CH2Ph, -Ph, -CH2OPh, -CH ₂ CH2OPh, -CH ₂ -pyrrolidine, -CH ₂ -morpholine or -CH ₂ -pyridine, said Ph, pyrrolidine, pyridine or morpholine being substituted with 0-5 R'; 5-10 membered heteroaryl; where the specified heterocyclyl contains 1-4 heteroatoms, independently selected from N or O; where the specified heteroaryl contains 1-4 heteroatoms, independently selected from N or O; where each R is independently substituted with 0-5 R'; where each R' is independently selected from halogen, -R", -OR", -CF ₃ or -OCF ₃ ; where each R'' is independently selected from H, -(C ₁ -C ₆ )alkyl or (C ₆ -C ₁₀ )aryl. 5-10-membered heteroaryl and where the specified heterocyclyl contains 1-4 heteroatoms, independently selected from N or O, and the specified heteroaryl contains 1-4 heteroatoms, independently selected from N or O; where in each case R is independently substituted with 0-5 R'; or if two R groups are attached to the same atom, the two R groups together with the atom to which they are attached may form a 3-10 membered aromatic or non-aromatic ring containing 0-1 heteroatoms independently selected from N or O, wherein said ring optionally substituted with 0-5 R', where in each occurrence R' is independently selected from halogen, -R'', -OR'', -CN or -CF ₃ ; where in each case R'' is independently selected from H, -(C1-C ₆ )alkyl, (C ₆ -C ₁₀ )aryl or (C ₆ -C ₁₀ )aryl-(C1C ₁₂ )aliphatic radical. 6. The compound according to claim 5 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer or Z-isomer, or a combination thereof, in which at least one R ¹ denotes (1) -O(C1C ₁₂ )aliphatic radical, which is (2) -O((C1-C ₆ )alkyl) selected from (3) -OMe. (6) a group consisting of -CH ₂ Ph, -CH ₂ CH ₂ Ph, -Ph, -CH ₂ OPh, -CH ₂ CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ -morpholine, or -CH ₂ - pyridine, wherein said Ph, pyrrolidine, pyridine, or morpholine is substituted with 0-5 R', wherein said -Ph, pyrrolidine, pyridine, or morpholine is substituted with 0-5 R', independently selected from -F, -Cl, -CN, -Me , -Et, -OMe, or -OEt; and (7) a group consisting of -Me, -C(O)OEt, -CH ₂ Ph, -CH ₂ OPh, -CH ₂ -pyrrolidine, -CH ₂ -pyridine, or -CH ₂ -morpholine, said -Ph , pyrrolidine, pyridine or morpholine is substituted with 0-3 R' independently selected from -F, -Me or -OMe. 7. The compound according to claim 1 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer or Z-isomer, or a combination thereof, in which R ³ is selected from: 8. The compound according to claim 7, or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer, or Z-isomer, or a combination thereof, wherein R ³ is: 9. The compound according to claim 1, or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer or Z-isomer, or a combination thereof, in which m = 0-1; each R ¹ is independently selected from halogen, -H, -(C1-C ₆ )alkyl, -O((C1-C ₆ )alkyl), CF ₃ or OCF ₃ ; R ² is selected from -(C1-C ₆ )alkyl, -C(O)O((C1-C ₆ )alkyl), (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical or (C ₆ -C ₁₀ )aryl-O-(C1-C ₁₂ )aliphatic radical, wherein said alkyl, aryl or heteroaryl is independently substituted with 0-5 R'; R ³ is selected from -(C1-C ₆ )alkyl, -SO ₂ (C ₁ -C ₆ )alkyl or -C(O)O((C1-C ₆ )alkyl), said al- 123 039381 kyl being independently substituted 0-5R'; and R ⁴ , R ⁵ and R ⁶ are -H. 10. The compound according to claim 9 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer or Z-isomer, or a combination thereof, in which m = 0 or 1; when m is 1, at least one R ¹ is halo or —O((C _g C ₆ )alkyl); R ² is selected from -(C1-C ₆ )alkyl, (C ₆ -C1 ₀ )aryl-(C1-C1 ₂ )aliphatic radical or (C ₆ -C1 ₀ )aryl-O-(C1C1 ₂ )aliphatic radical, wherein said aryl is independently substituted with 0-5 R' independently selected from -F, -Me or -OMe; R ³ is -C(O)O((C1-C ₆ )alkyl); R ⁴ and R ⁵ are both -H; and R ⁶ is -H. 11. The compound according to claim 1 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer or Z-isomer, or a combination thereof, in which m = 0-1; each R ¹ is independently selected from halogen, -H, -(C1-C ₆ )alkyl, -O((C1-C ₆ )alkyl), CF ₃ or OCF ₃ , said R ¹ being independently substituted with 0-5 R'; R ² is selected from -(C1-C ₆ )alkyl, -C(O)O((C1-C ₆ )alkyl), (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical, (C ₆ -C 1 ₀ )aryl-O-(C 1-C1 ₂ )aliphatic radical, -(CH2)i-3N((C6-C _w ) aryl)2, -(CH ₂ ) _1-3 -(5-10-membered heteroaryl), -(CH ₂ ) _1-3 -O-(5-10-membered heteroaryl), -(CH ₂ ) _1-3 -(3-10-membered heterocyclyl), wherein R ² is independently substituted with 0-5 R'; R ³ is selected from -(C1-C6) alkyl, -C CH -CN, halogen, -SO2 ((C6-C1o) aryl), -SO2 ((Aralkyl), -C (O) NH2, -C (O) O ((C ₁ -C ₆ )alkyl), -C(O)((C ₁ -C ₆ )alkyl), -(C ₆ -C ₁₀ )aryl or 5-10 membered heteroaryl, R ³ being independently substituted with 0-5 R ' and R ⁴ , R ⁵ and R ⁶ are -H. 12. The compound according to claim 11 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which m = 0 or 1; when m is 1, at least one R ¹ is halogen or —O((C1-C ₆ )alkyl); R ² is selected from -(C1-C ₆ )alkyl, (C ₆ -C ₁₀ )aryl-(C1-C ₁₂ )aliphatic radical, (C ₆ -C ₁₀ )aryl-O-(C1C ₁₂ )aliphatic radical, ( -(CH ₂ ) _1-3 -(5-10 membered heteroaryl) or -(CH ₂ ) _1-3 -(3-10 membered heterocyclyl), wherein R ² is independently substituted with 0-3 R'; R ³ is halogen, -CN, -C-CH, -C(O)NH2, -(Aralkyl, -C(O)((C1-C6)alkyl), -C(O)O((C1- -CF ₃ , isopropyl, -OMe or -tert-butyl; R ⁴ , R ⁵ and R ⁶ are -H. 13. The compound according to claim 12, or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, E-isomer or Z-isomer, or a combination thereof, in which R ³ is where R ⁹ is selected from -H, -Me, -Et, -CF ₃ , isopropyl or -tert-butyl. 14. A compound according to claim 2, or a pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer, or Z-isomer, or combination thereof, in which m is 0 or 1, and when m is 1, R ¹ is -O((C1- C ₆ )alkyl); R ² is selected from -(CH ₂ ) _1-3 -(C ₁ -C ₆ )alkyl or -(CH ₂ ) _1-3 -(C ₆ -C ₁₀ )aryl; R ³ is -C(O)O( (C1-C ₆ )alkyl); and each of R ⁴ and R ⁵ is -H; and R ⁶ is -H. 15. The compound according to claim 1 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer or Z-isomer, or a combination thereof, in which - 124 039381 m = 0 or 1 and when m is 1, R ¹ is -O((C1-C ₆ )alkyl); R ² is -OR ⁸ , where R ⁸ is (C6-C ₁₀ )aryl substituted with 0-3 halogens; R ³ is -C(O)O((C1-C ₆ )alkyl); R ⁴ , R ⁵ and R ⁶ are -H. 16. The compound according to claim 3 or its pharmaceutically acceptable salt, diastereoisomer, enantiomer, Eisomer or Z-isomer, or a combination thereof, in which m = 0 or 1; R ² is OR ⁸ , -(CH2)nOR ⁸ or -(CH2) _n O(CH ₂ ) _n R ⁸ , where n = 1, and where R ⁸ is -(C _r C ₆ )alkyl, (C ₆ - C ₁₀ )aryl or 5-10 membered heteroaryl, R ⁸ being independently substituted with 0-3 R'; R ³ is halogen, -H, -CN, -(C ₁ -C ₆ )alkyl, -C(O)((C ₁ -C ₆ )alkyl), -C(O)O((C ₁ -C ₆ )alkyl), I ---R ⁹ j ---R ⁹ A OR A wherein said alkyl is independently substituted with 0-3 R'; R ⁹ is selected from -Me, -Et, isopropyl or -CF3; R ⁴ and R ⁵ are both -H and R ⁶ is -H. 17. Connection selected from