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Annulated glycosidase inhibitors

11458140 · 2022-10-04

Assignee

Inventors

Cpc classification

International classification

Abstract

Compounds of formula (I), wherein A, R, W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, W.sup.6, L, Q, R.sup.x and u have the meaning according to the claims, can be employed, inter alia, for the treatment of tauopathies and Alzheimer's disease. ##STR00001##

Claims

1. A compound of formula (I) ##STR00314## wherein R is straight chain or branched alkyl having 1 to 6 carbon atoms, wherein 1 to 5 hydrogen atoms may be replaced by Hal or OH; W.sup.1 and W.sup.2 are, independently, C, CH, or N; W.sup.3, W.sup.4 and W.sup.5 are, independently, C, CH, N, CH.sub.2, NH, S, O, SO, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, CO, ##STR00315## W.sup.6 is CH, N, CH.sub.2, NH, S, O, SO, SO.sub.2, S(O)(NR.sup.3′), or CO; or W.sup.6 is absent; L is a single bond, O, NR.sup.3′, CH.sub.2, OCH.sub.2, CH.sub.2CH.sub.2, CONR.sup.3′, CONR.sup.3′CH.sub.2, NR.sup.3′CO, or NR.sup.3′COCH.sub.2; A is ##STR00316## X is N or CR″′; Y is O, S, SO, or SO.sub.2; R′ and R″ are, independently, H, Hal or straight chain or branched alkyl having 1 to 12 carbon atoms; R″′ and R″″ are, independently, H, Hal, NR.sup.3R.sup.4, CHR.sup.3R.sup.4, OR.sup.3, CN, or a straight chain or branched alkyl having 1 to 12 carbon atoms, wherein 1 to 3 CH.sub.2-groups may be replaced by O, NR.sup.3, S, SO, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, CO, COO, OCO, CONR.sup.3, NR.sup.3CO, ##STR00317## and wherein 1 to 5 hydrogen atoms may be replaced by Hal, NR.sup.3R.sup.4, NO.sub.2, ##STR00318## or R″′ and R″″ are, independently, ##STR00319## R.sup.3 and R.sup.4 are, independently, H or a straight chain or branched alkyl group having 1 to 12 carbon atoms; Q is absent or the group Q(R.sup.x).sub.u is ##STR00320## ##STR00321## Z.sup.1 is S, O, or NR.sup.3; Z.sup.2 and Z.sup.3 are, independently, CR.sup.5 or N; Z.sup.4 is N, CH, CON, or COCH; Z.sup.5 is O, NR.sup.8, CHR.sup.5, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, ##STR00322## Z.sup.6 is CH.sub.2, CO, S(O)(NR.sup.3′), N(SO)R.sup.3′, ##STR00323## Z.sup.7 is C(R.sup.3′).sub.2, S, O, or NR.sup.3′; s is 0 or 1; T is N, CH, or CR.sup.7; R.sup.3′ is H or a straight chain or branched alkyl group having 1 to 12 carbon atoms, wherein 1 to 3 CH.sub.2-groups may be replaced by SO.sub.2, CO, or O, and wherein 1 to 5 hydrogen atoms may be replaced by Hal; R.sup.x, R.sup.5, R.sup.6, R.sup.7 are, independently, H, Hal, CN, NR.sup.3R.sup.4, NO.sub.2, or a straight chain or branched alkyl having 1 to 12 carbon atoms, wherein 1 to 3 CH.sub.2-groups may be replaced by O, NR.sup.3, S, SO, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, CO, COO, OCO, CONR.sup.3, NR.sup.3CO, ##STR00324## and wherein 1 to 5 hydrogen atoms may be replaced by Hal, NR.sup.3R.sup.4, NO.sub.2, OR.sup.3, Het, Ar, Cyc, ##STR00325## or R.sup.x, R.sup.5, R.sup.6, R.sup.7 are Ar, Het, Cyc, ##STR00326## R.sup.8 is H or straight chain or branched alkyl having 1 to 12 carbon atoms, wherein 1 to 3 CH.sub.2-groups may be replaced by SO, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, CO, COO, OCO, CONR.sup.3, NR.sup.3CO, ##STR00327## and further wherein 1 to 5 hydrogen atoms may be replaced by CN, OR.sup.3, SR.sup.3, Hal, NR.sup.3R.sup.4, NO.sub.2, ##STR00328## or R.sup.8 is ##STR00329## Hal is F, Cl, Br, or I; Het is a saturated, unsaturated or aromatic ring, being monocyclic or bicyclic or fused-bicyclic and having 3- to 8-members and containing 1 to 4 heteroatoms selected from N, O and S, which may be substituted by 1 to 3 substituents selected from R.sup.5, Hal, and OR.sup.3; Ar is a 6-membered carbocyclic aromatic ring or a fused or non-fused bicyclic aromatic ring system, which is optionally substituted by 1 to 3 substituents independently selected from R.sup.5, OR.sup.3, and Hal; Cyc is a saturated or an unsaturated carbocyclic ring having from 3 to 8 carbon atoms which is optionally substituted by 1 to 3 substituents independently selected from R.sup.5, Hal, and OH; t and q are, independently, 0, 1, 2 or 3, provided that t+q≥1; u is 1 or 2, or a pharmaceutically usable derivative, solvate, salt, tautomer, enantiomer, racemate, stereoisomer, compound of formula (I) wherein one or more H atoms are replaced by deuterium, or any combination thereof, including mixtures thereof in any ratio.

2. The compound of claim 1, selected from the group consisting of: ##STR00330##

3. A mixture comprising compounds (Ia) and (Ib) according to claim 2, in equal or unequal amounts, wherein: the A groups in (Ia) and (Ib) are identical; the R groups in (Ia) and (Ib) are identical; the L groups in (Ia) and (Ib) are identical; the R.sup.x groups in (Ia) and (Ib) are identical; the W.sup.1 groups in (Ia) and (Ib) are identical; the W.sup.2 groups in (Ia) and (Ib) are identical; the W.sup.3 groups in (Ia) and (Ib) are identical; the W.sup.4 groups in (Ia) and (Ib) are identical; the W.sup.5 groups in (Ia) and (Ib) are identical; the W.sup.6 groups in (Ia) and (Ib) are identical; u in (Ia) and (Ib) are identical; and the Q groups in (Ia) and (Ib) are identical.

4. The compound of claim 1, wherein R is methyl.

5. The compound of claim 1, wherein A is ##STR00331## wherein R′ and R″ have the meaning given in claim 1.

6. The compound of claim 1, wherein the group Q-(R.sup.x).sub.u is H, ##STR00332## ##STR00333## wherein T, Z.sup.5, Z.sup.6, R.sup.6 and R.sup.7 have the meaning given in claim 1.

7. The compound of claim 1, wherein R.sup.x, R.sup.5, R.sup.6, R.sup.7 are independently selected from the group consisting of H, CN, SO.sub.2CH.sub.3, SO.sub.2CH.sub.2CH.sub.3, SO.sub.2CH.sub.2CH.sub.2OH, SO.sub.2CH.sub.2CH.sub.2OCH.sub.3, S(O)(NR.sup.3′)CH.sub.3, S(O)(NR.sup.3′)CH.sub.2CH.sub.3, S(O)(NR.sup.3′)CH.sub.2CH.sub.2OH, S(O)(NR.sup.3′)CH.sub.2CH.sub.2OCH.sub.3, N(SO)R.sup.3′CH.sub.3, N(SO)R.sup.3′CH.sub.2CH.sub.3, N(SO)R.sup.3′CH.sub.2CH.sub.2OH, N(SO)R.sup.3′CH.sub.2CH.sub.2OCH.sub.3, Hal, NR.sup.3R.sup.4, NO.sub.2, phenyl, benzyl, CH.sub.2-pyridyl, O-phenyl, O-pyridyl, O-pyrimidinyl, O-benzyl, 2-, 3- or 4-hydroxy or methoxyphenyl, alkyl, alkoxy (Oalkyl), hydroxyalkylene, alkoxyalkylene, COOH, COOalkyl, CONHalkyl, CONH.sub.2, CON(CH.sub.3).sub.2, NHCOalkyl, NHCOCH.sub.3, NHCOphenyl, NHCOpyridyl, NHCH.sub.2CH.sub.3, NHCH.sub.2CH.sub.2CH.sub.3, NHCOCH.sub.2CH.sub.2OH, CO—N-morpholinyl, CON(CH.sub.3)CH.sub.2CH.sub.2N(CH.sub.3).sub.2, CO-1-piperidinyl, CO-4-hydroxy-1-piperidinyl, CO-1-piperazinyl, CO-4-methyl-1-piperazinyl, CH.sub.2—N-morpholinyl, CH.sub.2N(H)COCH.sub.3, CH.sub.2N(CH.sub.3)COCH.sub.3, CH.sub.2NH.sub.2, NH.sub.2, CH(OH)CH.sub.3, CH(OR.sup.3)CH.sub.3, ##STR00334## wherein t+q is 2 or 3, and Z.sup.7 and R.sup.3′ and R.sup.3 and R.sup.4 have the meaning given in claim 1.

8. The compound of claim 1, wherein L is a single bond.

9. The compound of claim 1, wherein u is 1.

10. The compound of claim 1, selected from the group consisting of: TABLE-US-00005 Example No. Structure  1 embedded image  2 embedded image  3 embedded image  4 embedded image  5 embedded image  6 embedded image  7 embedded image  8 embedded image  9 embedded image  10 embedded image  11 embedded image  12 embedded image  13 embedded image  14 embedded image  15a embedded image  15b embedded image  16a embedded image  16b embedded image  17 embedded image  18 embedded image  19 embedded image  20 embedded image  21 embedded image  22 embedded image  23 embedded image  24 embedded image  25 embedded image  26 embedded image  27 embedded image  28 embedded image  29 embedded image  30 embedded image  31 embedded image  32 embedded image  33 embedded image  34 embedded image  35 embedded image  36 embedded image  37 embedded image  38 embedded image  39 embedded image  40 embedded image  41 embedded image  42 embedded image  43 embedded image  44 embedded image  45 embedded image  46 embedded image  47 embedded image  48 embedded image  49 embedded image  50 embedded image  51 embedded image  52 embedded image  53 embedded image  54 embedded image  55 embedded image  56 embedded image  57 embedded image  58 embedded image  59 embedded image  60 embedded image  61 embedded image  62 embedded image  63 embedded image  64 embedded image  65 embedded image  66 embedded image  67 embedded image  68 embedded image  69 embedded image  70 embedded image  71 embedded image  72 embedded image  73 embedded image  74 embedded image  75 embedded image  76 embedded image  77 embedded image  78 embedded image  79 embedded image  80 embedded image  81 embedded image  82 embedded image  83 embedded image  84 embedded image  85 embedded image  86 embedded image  87 embedded image  88 embedded image  89 embedded image  90 embedded image  91 embedded image  92 embedded image  93 embedded image  94 embedded image  95 embedded image  96 embedded image  97 embedded image  98 embedded image  99 embedded image 100 embedded image 101 embedded image 102 embedded image 103 embedded image 104 embedded image 105 embedded image 106 embedded image 107 embedded image 108 embedded image 109 embedded image 110 embedded image 111 embedded image 112 embedded image 113 embedded image 114 embedded image 115 embedded image 116 embedded image 117 embedded image 118 embedded image 119 embedded image 120 embedded image 121 embedded image 122 embedded image 123 embedded image 124 embedded image 125 embedded image 126 embedded image 127 embedded image 128 embedded image 129 embedded image 130 embedded image 131 embedded image 132 embedded image 133 embedded image 134 embedded image 135 embedded image 136 embedded image 137 embedded image 138 embedded image 139 embedded image 140 embedded image 141 embedded image 142 embedded image 143 embedded image 144 embedded image 145 embedded image 146 embedded image 147 embedded image 148 embedded image 149 embedded image 150 embedded image 151 embedded image 152 embedded image 153 embedded image 154 embedded image 155 embedded image 156 embedded image 157 embedded image 158 embedded image 159 embedded image 160 embedded image 161 embedded image and 162 embedded image or a pharmaceutically usable derivative, solvate, salt, tautomer, enantiomer, racemate, stereoisomer, or any combination thereof, including mixtures thereof in any ratio.

11. A method of treating a neurodegenerative disease, diabetes, or a cardiovascular disease, comprising administering a therapeutically effective amount of a compound of claim 1 to a mammal in need of such treatment.

12. A method of treating a condition selected from the group consisting of one or more tauopathies, Alzheimer's disease, Dementia, Amyotrophic lateral sclerosis, Amyotrophic lateral sclerosis with cognitive impairment, Argyrophilic grain disease, Behavioural variant frontotemporal dementia, Bluit disease, Chronic traumatic encephalopathy, Corticobasal degeneration, Dementia pugilistica, Diffuse neurofibrillary tangles with calcification, Down's syndrome, Familial British dementia, Familial Danish dementia, Frontotemporal dementia with parkinsonism linked to chromosome 17, Frontotemporal lobar degeneration, Ganglioglioma, Gangliocytoma, Gerstmann-Straussler-Scheinker disease, Globular glia tauopathy, Guadeloupean parkinsonism, Hallevorden-Spatz disease, Lead encephalopathy, Lipofuscinosis, Meningioangiomatosis, Multiple system atrophy, Myotonic dystrophy, Niemann-Pick disease, Pallido-ponto-nigral degeneration, Parkinsonism-dementia complex of Guam, Pick's disease, Parkinson's disease dementia, Postencephalitic parkinsonism, Primary progressive aphasia, Prion disease, Fatal Familial Insomnia, Kuru, Progressive supercortical gliosis, Progressive supranuclear palsy, Semantic dementia, Steele-Richardson-Olszewski syndrome, Subacute sclerosing panencephalitis, Tangle-only dementia, Tuberous sclerosis, Huntington's disease, and Parkinson's disease, comprising administering a therapeutically effective amount of a compound of claim 1 to a mammal in need of such treatment.

13. A method of treating a tauopathy, comprising administering a therapeutically effective amount of a compound of claim 1 to a mammal in need of such treatment.

14. A method of inhibiting a glycosidase, comprising contacting a system expressing the glycosidase with a compound of claim 1 under in-vitro conditions such that the glycosidase is inhibited.

15. A pharmaceutical composition comprising an active ingredient that is a compound of claim 1 together with a pharmaceutically tolerable adjuvant and/or excipient.

16. The pharmaceutical composition of claim 15, further comprising one or more additional active ingredients.

17. The compound of claim 1, wherein A is ##STR00499##

18. A compound of formula (I) ##STR00500## wherein R is straight chain or branched alkyl having 1 to 6 carbon atoms, wherein 1 to 5 hydrogen atoms may be replaced by Hal or OH; W.sup.1 and W.sup.2 are, independently, C, CH, or N; W.sup.3, W.sup.4 and W.sup.5 are, independently, C, CH, N, CH.sub.2, NH, S, O, SO, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, CO, ##STR00501## W.sup.6 is CH, N, CH.sub.2, NH, S, O, SO, SO.sub.2, S(O)(NR.sup.3′), or CO; or W.sup.6 is absent; L is a single bond, O, NR.sup.3′, CH.sub.2, OCH.sub.2, CH.sub.2CH.sub.2, CONR.sup.3′, CONR.sup.3′CH.sub.2, NR.sup.3′CO, or NR.sup.3′COCH.sub.2; A is ##STR00502## X is N or CR″′; Y is O, S, SO, or SO.sub.2; R′ and R″ are, independently, H, Hal or straight chain or branched alkyl having 1 to 12 carbon atoms; R″′ and R″″ are, independently, H, Hal, NR.sup.3R.sup.4, CHR.sup.3R.sup.4, OR.sup.3, CN, or a straight chain or branched alkyl having 1 to 12 carbon atoms, wherein 1 to 3 CH.sub.2-groups may be replaced by O, NR.sup.3, S, SO, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, CO, COO, OCO, CONR.sup.3, NR.sup.3CO, ##STR00503## and wherein 1 to 5 hydrogen atoms may be replaced by Hal, NR.sup.3R.sup.4, NO.sub.2, ##STR00504## or R″′ and R″″ are, independently, ##STR00505## R.sup.3 and R.sup.4 are, independently, H or a straight chain or branched alkyl group having 1 to 12 carbon atoms; Q is absent or the group Q(R.sup.x).sub.u is ##STR00506## ##STR00507## Z.sup.1 is S, O, or NR.sup.3; Z.sup.2 and Z.sup.3 are, independently, CR.sup.5 or N; Z.sup.4 is N, CH, CON, or COCH; Z.sup.5 is O, NR.sup.8, CHR.sup.5, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, ##STR00508## Z.sup.6 is CH.sub.2, CO, S(O)(NR.sup.3′), N(SO)R.sup.3′, ##STR00509## Z.sup.7 is C(R.sup.3′).sub.2, S, O, or NR.sup.3′, s is 0 or 1; T is N, CH, or CR.sup.7; R.sup.3′ is H or a straight chain or branched alkyl group having 1 to 12 carbon atoms, wherein 1 to 3 CH.sub.2-groups may be replaced by SO.sub.2, CO, or O, and wherein 1 to 5 hydrogen atoms may be replaced by Hal; R.sup.x, R.sup.5, R.sup.6, R.sup.7 are, independently, H, Hal, CN, NR.sup.3R.sup.4, NO.sub.2, or a straight chain or branched alkyl having 1 to 12 carbon atoms, wherein 1 to 3 CH.sub.2-groups may be replaced by O, NR.sup.3, S, SO, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, CO, COO, OCO, CONR.sup.3, NR.sup.3CO, ##STR00510## and wherein 1 to 5 hydrogen atoms may be replaced by Hal, NR.sup.3R.sup.4, NO.sub.2, OR.sup.3, Het, Ar, Cyc, ##STR00511## or R.sup.x, R.sup.5, R.sup.6, R.sup.7 are Ar, Het, Cyc, ##STR00512## R.sup.8 is H or straight chain or branched alkyl having 1 to 12 carbon atoms, wherein 1 to 3 CH.sub.2-groups may be replaced by SO, SO.sub.2, S(O)(NR.sup.3′), N(SO)R.sup.3′, CO, COO, OCO, CONR.sup.3, NR.sup.3CO, ##STR00513## and further wherein 1 to 5 hydrogen atoms may be replaced by CN, OR.sup.3, SR.sup.3, Hal, NR.sup.3R.sup.4, NO.sub.2, ##STR00514## or R.sup.8 is ##STR00515## Hal is F, Cl, Br, or I; Het is a saturated, unsaturated or aromatic ring, being monocyclic or bicyclic or fused-bicyclic and having 3- to 8-members and containing 1 to 4 heteroatoms selected from N, O and S, which may be substituted by 1 to 3 substituents selected from Hal, and OR.sup.3; Ar is a 6-membered carbocyclic aromatic ring or a fused or non-fused bicyclic aromatic ring system, which is optionally substituted by 1 to 3 substituents independently selected from R.sup.5, OR.sup.3, and Hal; Cyc is a saturated or an unsaturated carbocyclic ring having from 3 to 8 carbon atoms which is optionally substituted by 1 to 3 substituents independently selected from R.sup.5, Hal, and OH; t and q are, independently, 0, 1, 2 or 3, provided that t+q≥1; u is 1 or 2, or a pharmaceutically usable derivative, solvate, salt, tautomer, enantiomer, racemate, stereoisomer, compound of formula (I) wherein one or more H atoms are replaced by deuterium, or any combination thereof, including mixtures thereof in any ratio.

19. A method of treating Alzheimer's disease, comprising administering a therapeutically effective amount of a compound of claim 1 to a mammal in need of such treatment.

20. The method of claim 11, wherein the cardiovascular disease is stroke.

21. The method of claim 12, wherein the Niemann-Pick disease is Niemann-Pick disease type C.

22. The method of claim 12, wherein the Prion disease is selected from the group consisting of Creutzfeldt-Jakob disease, progressive nonfluent aphasia, and variant Creutzfeldt-Jakob disease.

Description

EXPERIMENTAL PART

(1) The compounds according to Formula (I) can be prepared from readily available starting materials by several synthetic approaches, using both solution-phase and solid-phase chemistry protocols or mixed solution and solid phase protocols. Examples of synthetic pathways are described below in the examples. All reported yields are non optimized yields. Unless otherwise stated, compounds of Formula (I) and related formulae obtained as a racemic mixture can be separated to provide an enantiomerically enriched mixture or a pure enantiomer.

(2) The commercially available starting materials used in the following experimental description were purchased from Aldrich, Sigma, ACROS, ABCR, Combi-Blocks, Matrix, Apollo scientific, Alfa Aesar, etc. unless otherwise reported.

(3) The HPLC, MS and NMR data provided in the examples described below are obtained as followed:

(4) .sup.1H NMR analyses were carried out using BRUKER NMR, model AV-II and AV-III 400 MHz FT-NMR. Residual signal of deuterated solvent was used as internal reference. Chemical shifts (δ) are reported in ppm in relative to the residual solvent signal (δ=2.50 for .sup.1H NMR in DMSO-d.sub.6, and 7.26 in CDCl.sub.3). s (singlet), d (doublet), t (triplet), q (quadruplet), br (broad), quint (quintuplet).

(5) LCMS Analysis Condition:

(6) Instrument name: Agilent Technologies 1290 infinity 11.

(7) Method A: Method: A-0.1% TFA in H.sub.2O, B-0.1% TFA in MeCN; flow rate: 2.0 mL/min; column: XBridge C8 (50×4.6 mm, 3.5 μm), +ve mode

(8) Method B: Method: A-10 mM NH.sub.4HCO.sub.3 in H.sub.2O, B-MeCN; flow rate: 1.0 mL/min; column: XBridge C8 (50×4.6 mm, 3.5 μm), +ve mode

(9) HPLC Analysis Condition:

(10) Instrument name: Agilent 1200 Series instruments as followed using % with UV detection (maxplot).

(11) Method A: Method: A-0.1% TFA in H.sub.2O, B-0.1% TFA in MeCN; flow rate: 2.0 mL/min; column: XBridge C8 (50×4.6 mm, 3.5 μm).

(12) Method B: Method: A-10 mM NH.sub.4HCO.sub.3 in H.sub.2O, B-MeCN; flow rate: 1.0 mL/min; column: XBridge C8 (50×4.6 mm, 3.5 μm).

(13) Chiral SFC Analysis Condition:

(14) Instrument name: THAR-SFC 80 and THAR-SFC 200 (analytical) Ratio between CO.sub.2 and co-solvent is ranging between 50:50 and 90:10

(15) Method A: Co-solvent: 20 mM ammonia in IPA; flow: 4 mL/min; column: Lux A1 (250×4.6 mm, 5 μm).

(16) Prep-HPLC Analysis Condition:

(17) Method A: A-0.1% TFA in H.sub.2O, B-MeOH or MeCN; column: Sunfire C8 (19×250 mm, 5 μm) or Sunfire C18 (30×250 mm, 10 μm).

(18) Method B: A-10 mM NH.sub.4HCO.sub.3 in H.sub.2O, B-MeOH or MeCN, Column: Sunfire C8 (19×250 mm, 5 μm) or Sunfire C18 (30×250 mm, 10 μm).

(19) MD Auto-Prep Condition:

(20) The mass directed preparative HPLC purifications were performed with a mass directed autopurification Fractionlynx from Waters.

(21) Method A: 0.1% TFA in H.sub.2O, B-MeOH or MeCN, Column: Symmetry C8 (300×19 mm, 7 μm).

(22) Chiral Preparative SFC Analysis Condition:

(23) Instrument name: THAR-SFC 80, THAR-SFC 200 and Pic SFC 10-150 Ratio between CO.sub.2 and co-solvent is ranging between 50:50 and 90:10

(24) Method A: Co-solvent: 20 mM Ammonia in IPA FLOW: 5 mL/min; COLUMN: Lux A1 (250×30) mm, 5 μm

(25) Method B: Co-solvent: 20 mM MeOH FLOW: 5 mL/min; COLUMN: Lux A1 (250×30) mm, 5 μm

(26) The microwave chemistry was performed on a single mode microwave reactor Initiator™ Sixty from Biotage.

(27) General flash chromatography conditions used for the purification of intermediates or compounds of Formula I: silica gel 230-400 mesh; gradients used as eluent: 10 to 80% EtOAc in petroleum ether or 1 to 15% MeOH in DCM

Intermediate 1: 5-(1-Chloroethyl)benzo[d][1,3]dioxole

(28) ##STR00221##

Step 1: 1-(Benzo[d][1,3]dioxol-5-yl)ethan-1-ol

(29) ##STR00222##

(30) To a stirred solution of 3,4-methylenedioxy acetophenone (50.0 g, 0.31 mol, Alfa aesar) in dry methanol (1000 mL), NaBH.sub.4 (13.83 g, 0.37 mol, Loba chemie) was added slowly at 0° C. and the reaction mixture was stirred at RT for 1 h. Completion of the reaction was monitored by TLC, then the reaction mixture was concentrated under vacuum. The resulting material was dissolved in EtOAc, the organic layer was washed with water (100 mL), brine solution (100 mL) and dried over anhydrous Na.sub.2SO.sub.4. The organic layer was evaporated under vacuum and the resulting crude material was forwarded to the next step without any further purification. Yield: 98% (50.0 g, colorless liquid). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 6.89 (s, 1H), 6.89-6.75 (m, 2H), 5.95 (s, 2H), 4.81 (t, J=8.0 Hz, 1H), 1.46 (d, J=8.0 Hz, 3H). LCMS: (Method B) 149.0 (M−H.sub.2O+H); Rt. 2.5 min, 98.6% (Max). HPLC: (Method A) RT 2.4 min, 99.5% (Max).

Step 2: 5-(1-Chloroethyl)benzo[d][1,3]dioxole

(31) ##STR00223##

(32) To a stirred solution of 1-(benzo[d][1,3]dioxol-5-yl)ethan-1-ol (50.0 g, 0.3 mol) in DCM (400 mL), thionyl chloride was added slowly at 0° C. and continued at RT for 2 h. After completion of the reaction, the reaction mixture was concentrated under vacuum and co-distilled with DCM (100 mL). The resulting crude material was forwarded to the next step as such without further purification. Yield: 64% (35.0 g, brown liquid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.06 (d, J=4.0 Hz, 1H), 6.93 (d, J=8.0 Hz. 1H), 6.86 (d, J=8.0 Hz, 1H), 6.01 (s, 2H), 2.49 (q, J=8.9 Hz, 1H), 1.74 (d, J=8.9 Hz, 3H). LCMS: (Method B) 149.0 (M−Cl+H); Rt. 3.7 min, 80.2% (Max).

Intermediate 2: 6-(1-chloroethyl)-2,3-dihydrobenzofuran

(33) ##STR00224##

Step 1: 2-(2,5-dibromophenoxy)ethan-1-ol

(34) ##STR00225##

(35) To a stirred solution of 1,4-dibromo-2-fluorobenzene (Combi-Blocks, 1000 g, 3.94 mol) in ethylene glycol (5100 mL), NMP (500 mL) was added at RT under nitrogen atmosphere. Then KO.sup.tBu (1547 g, 1.38 mol) was added in portions over 45 min at 5° C. and the resulting mixture was heated at 90° C. for 16 h. Completion of the reaction was monitored by HPLC (Method A), the reaction mixture was then cooled to RT, diluted with water (2000 mL) and stirred for 15 min. The resulting solid was filtered and washed with ethylene glycol (2×300 mL). Water (16000 mL) was added to the filtrate, cooled to 10° C. and stirred for 1 h at the same temperature to precipitate out the whole solid. The obtained solid was filtered and washed with water (2×1000 mL), pet ether (3×1000 mL) and dried under vacuum. This solid was co-distilled with toluene (3×500 mL) to afford the title compound. Yield: 78% (910 g, white solid). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.41 (d, J=8.0 Hz, 1H), 7.06-7.00 (m, 2H), 4.14 (t, J=4.0 Hz, 2H), 4.01 (q, J=3.6 Hz, 2H). LCMS: (Method A) 296.0 (M+H), Rt. 3.9 min, 98.2% (Max). HPLC: (Method A) Rt. 3.7 min, 99.5% (Max).

Step 2: 1,4-dibromo-2-(2-bromoethoxy)benzene

(36) ##STR00226##

(37) To a stirred solution of 2-(2,5-dibromophenoxy)ethan-1-ol (910.0 g, 3.07 mol) in toluene (6370 mL), PBr.sub.3 (Aldrich, 145 mL, 1.54 mol) was added under nitrogen atmosphere at 0° C. over 15 min. The resulting mixture was heated at 90° C. for 4 h and then cooled to 0° C. PBr.sub.3 (13.57 mL, 142.92 mmol) was added followed by the slow addition of water (20 mL) and heating was continued at 90° C. for 3 h. Completion of the reaction was monitored by TLC, the reaction mixture was then cooled to 10° C. and quenched with 1N NaOH solution (2200 mL). The milky solid layer, formed immediately after quenching, was filtered through celite pad. The organic layer was separated, washed with water (1820 mL), brine solution (1820 mL) and dried over anhydrous Na.sub.2SO.sub.4. It was then evaporated at 45° C. under vacuum. The resulting crude material was dissolved in EtOAc (3185 mL), the organic layer was washed with water (1820 mL), brine solution (1820 mL) and dried over anhydrous Na.sub.2SO.sub.4. The organic layer was evaporated at 40° C. under reduced pressure to afford the title compound. Yield: 86% (946 g, white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.54 (d, J=8.4 Hz, 1H), 7.36 (d, J=1.6 Hz, 1H), 7.13-7.10 (m, 1H), 4.45 (t, J=1.2 Hz, 2H), 3.82 (t, J=1.6 Hz, 2H). HPLC: (Method A) Rt. 4.7 min, 93.0% (Max).

Step 3: 2,3-dihydrobenzofuran-6-carbaldehyde

(38) ##STR00227##

(39) To a stirred solution of 1,4-dibromo-2-(2-bromoethoxy)benzene (946 g, 2.64 mol) in dry THF (9.5 L) under nitrogen atmosphere, n-butyl lithium (1812 mL, 2.89 mol, 1.6 M in hexane) was added slowly over 30 min at −78° C. and continued for 1 h at the same temperature. A second batch of n-butyl lithium (1812 mL, 2.89 mol, 1.6 M in hexane) was added slowly over 30 min at −78° C. and stirring was continued for another 1 h. Then DMF (408 mL, 5.27 mol) was added slowly at same temperature and the mixture was stirred for 45 min. After completion of the reaction (monitored by TLC), the reaction mixture was warmed to 10° C., quenched with the addition of sat.NH.sub.4Cl solution (3784 mL) and the aqueous layer was extracted with EtOAc (2×2800 mL). The combined organic layer was washed with water (2838 mL), brine solution (2838 mL), dried over anhydrous Na.sub.2SO.sub.4 and evaporated at 40° C. under reduced pressure to afford the title compound. Yield: 96% crude (404 g, pale brown gummy solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.90 (s, 1H), 7.45 (dd, J=5.2, 1.2 Hz, 2H), 7.19 (s, 1H), 4.60 (t, J=8.7 Hz, 2H), 3.27 (t, J=8.7 Hz, 2H). HPLC: (Method A) Rt. 2.9 min, 84.3% (Max).

Step 4: 1-(2,3-dihydrobenzofuran-6-yl)ethan-1-ol

(40) ##STR00228##

(41) To a stirred solution of 2,3-dihydrobenzofuran-6-carbaldehyde (404 g, 2.73 mol) in dry THF (4040 mL) under nitrogen atmosphere, methyl magnesium chloride solution (1820 mL, 5.45 mol, 3 M in THF) was added slowly over 30 min at 0° C. and stirred for 2 h at RT. Completion of the reaction was monitored by TLC, the reaction mixture was then quenched by using sat. NH.sub.4Cl solution (1616 mL) and extracted with EtOAc (2×2828 mL). The combined organic layer was washed with water (1616 mL), brine solution (1616 mL), dried over Na.sub.2SO.sub.4 and evaporated at 45° C. under reduced pressure. The resulting crude material was purified by flash chromatography (Silica gel: 60-120 mesh, eluent: 18% EtOAc in pet ether) to afford the title compound. Yield: 46% (210 g, pale brown gummy solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.12 (d, J=7.2 Hz, 1H), 6.77 (dd, J=7.6, 0.8 Hz, 1H), 6.72 (s, 1H), 5.05 (d, J=4.4 Hz, 1H), 4.66-4.60 (m, 1H), 4.48 (t, J=8.4 Hz, 2H), 3.12 (t, J=8.4 Hz, 2H), 1.28 (t, J=6.8 Hz, 3H). LCMS: (Method A) 147.0 (M+H) (alkene), Rt. 2.7 min, 90.7% (Max). HPLC: (Method A) Rt. 2.6 min, 91.7% (Max).

Step 5: 6-(1-chloroethyl)-2,3-dihydrobenzofuran

(42) ##STR00229##

(43) To a stirred solution of 1-(2,3-dihydrobenzofuran-6-yl)ethan-1-ol (200 g, 1.22 mmol) in DCM (1600 mL) at 0° C., oxalyl chloride (155 mL, 3.66 mmol) and catalytic amount of DMF (2 mL) were added and the reaction mixture was stirred at RT for 16 h. Then the reaction mixture was concentrated under vacuum and co-distilled with dry DCM (3×500 mL) to afford the title compound. Yield: 97% (crude) (220 g, pale brown gummy solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.32 (d, J=7.6 Hz, 1H), 6.92 (d, J=9.6 Hz, 2H), 5.28 (q, J=13.2 Hz, 1H), 4.52 (t, J=8.4 Hz, 2H), 3.15 (t, J=8.8 Hz, 2H), 1.75 (d, J=8.4 Hz, 3H). LCMS: (Method A) 147.2 (M+H-Chloro), Rt. 4.2 min, 77.2% (Max).

Intermediate 3: tert-butyl (E)-3-((dimethylamino)methylene)-4-oxopiperidine-1-carboxylate

(44) ##STR00230##

(45) To a stirred solution of tert-butyl 4-oxopiperidine-1-carboxylate (10 g, 50.18 mmol), DMF-DMA (73.5 mL, 552 mmol) was added and the reaction mixture was heated at 105° C. for 1 h. Completion of the reaction was monitored by TLC, then the reaction mixture was evaporated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 20-30% EtOAc in hexane) to afford the title compound. Yield: 24% (3 g, brown gummy solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.30 (s, 1H), 4.46 (s, 2H), 3.47 (t, J=8.0 Hz, 2H), 3.06 (s, 6H), 2.26 (t, J=8.8 Hz, 2H), 1.42 (s, 9H). LCMS: (Method B) 255.0 (M+H), Rt. 4.2 min, 77.4% (Max).

Intermediate 4: 1-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine hydrochloride

(46) ##STR00231##

Step 1: tert-butyl 1-phenyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate and tert-butyl 2-phenyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate

(47) ##STR00232##

(48) To a solution of intermediate 3 (1.5 g, 5.90 mmol) in ethanol (15 mL), phenyl hydrazine (0.76 g, 7.08 mmol) was added under nitrogen atmosphere and the reaction mixture was heated at 85° C. for 8 h. Completion of the reaction was monitored by TLC, then the reaction mixture was concentrated under vacuum and the resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 20-30% EtOAc in pet ether) to afford the mixture of regioisomers. The resulting mixture was then separated by Prep HPLC (Method B) and the structure of each regioisomer was confirmed by NOESY experiments.

(49) Isomer 1: Yield: 11% (190 mg, brown solid). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.52-7.48 (m, 5H), 7.38-7.34 (m, 1H), 4.54 (s, 2H), 3.78-3.62 (m, 2H), 2.87 (t, J=5.6 Hz, 2H), 1.53 (s, 9H). LCMS: (Method A) 300.3 (M+H), Rt. 4.7 min, 97.1% (Max).

(50) Isomer 2: Yield: 26% (450 mg, brown solid). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.71 (s, 1H), 7.66-7.64 (m, 2H), 7.47-7.43 (m, 2H), 7.31-7.26 (m, 1H), 4.58 (s, 2H), 3.85-3.72 (m, 2H), 2.91-2.88 (m, 2H), 1.52 (s, 9H). LCMS: (Method A) 300.3 (M+H), Rt. 4.8 min, 99.0% (Max).

Step 2: 1-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine, hydrochloride

(51) ##STR00233##

(52) To a stirred solution of tert-butyl 1-phenyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (Isomer 1) (190 mg, 0.63 mmol) in dry 1,4-dioxane (3 mL) at 0° C., HCl solution in dioxane (2 mL, 4M) was added dropwise and the reaction mixture was stirred at RT for 6 h. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under vacuum and the resulting crude material was triturated with diethyl ether (5 mL), filtered under nitrogen atmosphere to afford the title compound. Yield: 94% (0.14 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.39-9.15 (m, 2H), 7.68 (s, 1H), 7.57-7.53 (m, 4H), 7.45-7.41 (m, 1H), 4.21-4.19 (m, 2H), 3.38-3.35 (m, 2H), 3.11 (t, J=6.0 Hz, 2H). LCMS: (Method A) 200.0 (M+H), Rt. 1.7 min, 99.7% (Max).

Intermediate 5: 2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine hydrochloride

(53) ##STR00234##

(54) To a stirred solution of tert-butyl 2-phenyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (see synthesis of Intermediate 4, step 1, Isomer 2) (450 mg, 1.50 mmol) in dry 1,4-dioxane (4 mL) at 0° C., HCl solution in dioxane (2 mL, 4M) was added dropwise and the reaction mixture was stirred at RT for 6 h. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under vacuum. The resulting crude material was triturated with diethyl ether (8 mL) and filtered under nitrogen atmosphere to afford the title compound. Yield: 88% (0.31 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.52-9.50 (m, 2H), 8.40 (s, 1H), 7.80-7.77 (m, 2H), 7.51-7.48 (m, 2H), 7.33-7.29 (m, 1H), 4.23 (t, J=4.4 Hz, 2H), 3.44-3.42 (m, 2H), 3.00 (t, J=6.4 Hz, 2H). LCMS: (Method A) 200.2 (M+H), Rt. 2.0 min, 97.8% (Max).

Example 1: N-(6-(1-(benzo[d][1,3]dioxol-5-yl)ethyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)acetamide

(55) ##STR00235##

Step 1: t-Butyl 2-amino-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

(56) ##STR00236##

(57) To a stirred solution of intermediate 3 (2.5 g, 9.84 mmol) in ethanol (62 mL), KOAc (3.7 g, 88.58 mmol) and guanidine hydrochloride (4.3 g, 44.29 mmol) were added at RT and the reaction mixture was stirred at 80° C. for 48 h. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under vacuum and the resulting mixture was dissolved in 10% methanol in DCM. The organic layer was washed with saturated NaHCO.sub.3 (30 mL), brine solution (30 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by recrystallization with diethyl ether to afford the title compound. Yield: 29% (600 mg, white solid). .sup.1H NMR (300 MHz, DMSO-d.sub.6): δ 8.03 (s, 1H), 6.41 (s, 2H), 4.03 (s, 2H), 3.57-3.53 (t, J=5.9 Hz, 2H), 2.71 (m, 2H), 1.46 (s, 9H). LCMS: (Method B) 251.2 (M+H), Rt. 4.1 min, 98.2% (Max).

Step 2: tert-butyl 2-acetamido-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

(58) ##STR00237##

(59) To a stirred solution of t-Butyl 2-amino-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (0.6 g, 2.4 mmol) in dry pyridine (4 mL), acetic anhydride (0.3 mL, 2.88 mmol) was added at RT and the reaction mixture was stirred at 80° C. for 3 h. Completion of the reaction was monitored by TLC, then the reaction mixture was concentrated under vacuum and the resulting mixture was dissolved in 5% methanol in DCM. The organic layer was washed with HCl (0.5 N, 30 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 50-60% EtOAc in hexane) to afford the title compound. Yield: 71% (500 mg, off white solid). LCMS: (Method B) 293.1 (M+H), Rt. 3.9 min, 81.4% (Max).

Step 3: N-(5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)acetamide hydrochloride

(60) ##STR00238##

(61) To a stirred solution of tert-butyl 2-acetamido-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (0.5 g, 1.71 mmol) in dry 1,4-dioxane (1 mL), HCl solution in dioxane (4 mL, 4M) was added dropwise at RT and the reaction mixture was stirred for 1 h. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under vacuum and the resulting crude material was washed with diethyl ether to afford the title compound. Yield: 99% (320 mg, yellow solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.63 (s, 1H), 9.70 (m, 2H), 4.25 (s, 2H), 3.44 (m, 2H), 3.01 (t, J=6.3 Hz, 2H), 2.15 (s, 3H).

Step 4: N-(6-(1-(Benzo[d][1,3]dioxol-5-yl)ethyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)acetamide

(62) ##STR00239##

(63) To a stirred solution of N-(5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)acetamide hydrochloride (140 mg, 0.7 mmol) in MeCN (5 mL), TEA (1 mL, 7.1 mmol) and intermediate 1 (300 mg, 1.45 mmol) were added at RT and the reaction mixture was heated at 70° C. overnight. Completion of the reaction was monitored by TLC, then the reaction mixture was evaporated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 1-2% methanol in DCM) to afford the title compound. Yield: 9% (26.61 mg, pale yellow solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.37 (s, 1H), 8.33 (s, 1H), 6.93 (d, J=1.2 Hz, 1H), 6.88-6.86 (m, 1H), 6.82-6.80 (m, 1H), 6.00 (d, J=2.8 Hz, 2H), 3.65-3.58 (m, 2H), 3.46-3.41 (m, 1H), 2.77-2.75 (m, 3H), 2.67-2.62 (m, 1H), 2.14 (s, 3H), 1.35 (d, J=6.8 Hz, 3H). LCMS: (Method A) 341.0 (M+H), Rt. 1.8 min, 95.8% (Max). HPLC: (Method A) Rt. 1.8 min, 97.2% (Max).

Example 2: 2-(1-(benzo[d][1,3]dioxol-5-yl)ethyl)-1,2,3,4-tetrahydroisoquinoline

(64) ##STR00240##

(65) To a stirred solution of 1,2,3,4-tetrahydro isoquinoline (0.29 g, 2.18 mmol) in MeCN (10 mL), TEA (1 mL, 7.10 mmol) and intermediate 1 (0.5 g, 2.72 mmol) were added at RT and the reaction mixture was heated at 90° C. overnight. Completion of the reaction was monitored by TLC, then the reaction mixture was concentrated under vacuum and resulting mixture was dissolved in DCM (20 mL). The organic layer was washed with water (5 mL), brine solution (10 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by MD Auto Prep (method A) to afford the title compound. Yield: 2% (17.64 mg, colorless gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.11-7.07 (m, 3H), 7.05-7.01 (m, 1H), 7.00-6.99 (m, 1H), 6.92-6.85 (m, 1H), 6.81-6.80 (m, 1H), 5.99 (d, J=2.0 Hz, 2H), 3.68-3.64 (m, 1H), 3.54-3.45 (m, 2H), 2.74-2.66 (m, 2H), 2.65-2.64 (m, 1H), 2.51-2.50 (m, 1H), 1.35 (d, J=6.4 Hz, 3H). LCMS: (Method A) 282.1 (M+H), Rt. 2.8 min, 98.6% (Max). HPLC: (Method A) Rt. 2.9 min, 98.7% (Max).

Example 3: N-(5-Phenethyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)acetamide

(66) ##STR00241##

Step 1: t-Butyl 2-amino-6,7-dihydrothiazolo[5,4-c]pyridine-5(4-carboxylate

(67) ##STR00242##

(68) To a solution of t-butyl 3-bromo-4-oxopiperidine-1-carboxylate (1.0 g, 3.59 mmol) in isopropanol (10 mL), thiourea (0.33 g, 4.30 mmol) was added and the reaction mixture was refluxed at 90° C. for 1 h. Completion of the reaction was monitored by TLC. The reaction mixture was then concentrated under vacuum and the resulting crude material was washed with diethyl ether to afford the title compound. Yield: 99% (0.9 g, white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.05 (bs, 2H), 4.32 (m, 2H), 3.61-3.58 (m, 2H), 2.50 (s, 2H), 1.41 (s, 9H). LCMS: (Method A) 256.0 (M+H), Rt. 2.5 min, 64.8% (Max).

Step 2: t-Butyl 2-acetamido-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate

(69) ##STR00243##

(70) To a stirred solution of t-Butyl 2-amino-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (0.9 g, 3.52 mmol) in TEA (5 mL), acetic anhydride (0.43 mL, 4.58 mmol) was added at 0° C. and the reaction mixture was stirred at RT overnight. Completion of the reaction was monitored by TLC, the reaction mixture was concentrated under vacuum and the resulting mixture was dissolved in EtOAc (20 mL). The organic layer was washed with brine solution (5 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was used in the next step as such without any further purification. Yield: 82% (0.85 g, pale yellow liquid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.9 (bs, 1H), 4.46 (m, 2H), 3.63-3.44 (m, 2H), 2.62-2.49 (m, 2H), 2.21 (s, 3H), 1.41 (s, 9H). LCMS: (Method A) 298.0 (M+H), Rt. 3.4 min, 94.5% (Max).

Step 3: N-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)acetamide hydrochloride

(71) ##STR00244##

(72) To a stirred solution of t-Butyl 2-acetamido-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (0.35 g, 3.36 mmol) in dry 1,4-dioxane, HCl solution in dioxane (4 M, 7 mL) was added dropwise at RT and the reaction mixture was stirred for 1 h. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under vacuum and the resulting crude material was triturated with diethyl ether to afford the title compound. Yield: 99% (500 mg, off white solid).

Step 4: N-(5-(1-(benzo[d][1,3]dioxol-5-yl)ethyl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)acetamide

(73) ##STR00245##

(74) To a stirred solution of N-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)acetamide hydrochloride (0.3 g, 1.52 mmol) in MeCN (3 mL), TEA (0.65 mL, 4.56 mmol) and intermediate 1 (0.28 g, 1.52 mmol) were added at RT and the reaction mixture was stirred at 80° C. overnight. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under vacuum and the resulting crude material was dissolved in EtOAc (20 mL). The organic layer was washed with water (10 mL), brine solution (10 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The crude resulting material was purified by flash chromatography (Biotage Isolera, eluent: 1-3% methanol in DCM) to afford the title compound. Yield: 3% (2.87 mg, yellow solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.90 (s, 1H), 6.92 (d, J=1.4 Hz, 1H), 6.85 (d, J=8.0 Hz, 1H), 6.80 (d, J=8.0 Hz, 1H), 5.99 (d, J=2.0 Hz, 2H), 3.67-3.34 (m, 3H), 2.92-2.89 (m, 1H), 2.59-2.50 (m, 3H), 2.10 (s, 3H), 1.30 (d, J=6.8 Hz, 3H). LCMS: (Method B) 346.0 (M+H), Rt. 4.8 min, 96.9% (Max). HPLC: (Method A) Rt. 2.3 min, 97.8% (Max).

Example 4: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(75) ##STR00246##

Step 1: 4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(76) ##STR00247##

(77) To a stirred solution of intermediate 3 (3 g, 11.8 mmol) in dry ethanol (15 mL), hydrazine hydrochloride (0.47 g, 9.4 mmol) was added and the reaction mixture was heated at 85° C. in sealed tube for 4 h. It was evaporated under vacuum and HCl in methanol (20 mL, 4M) was added dropwise at 0° C. under nitrogen atmosphere. The mixture was stirred for 12 h at RT. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated under vacuum and the resulting crude material was used in the next step as such without any further purification. Yield: 69% (1.0 g, yellow solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.28 (s, 2H), 7.55 (s, 1H), 4.11 (s, 2H), 3.51-3.40 (m, 2H), 2.89 (t, J=6.0 Hz, 2H). LCMS: (Method B) 124.0 (M+H), Rt. 0.7 min, 99.3% (Max).

Step 3: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(78) ##STR00248##

(79) To a stirred solution of 4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine (0.1 g, 0.8 mmol) in DMF (10 mL), TEA (0.35 mL, 2.4 mmol) and intermediate 2 (0.221 g, 1.2 mmol) were added at 0° C. and the reaction mixture was stirred at 90° C. overnight. Completion of the reaction was monitored by TLC. The reaction mixture was evaporated at 50° C. under vacuum. The resulting mixture was dissolved in DCM (10 mL) and the organic layer was washed with water (5 mL), brine solution (5 mL) and dried over anhydrous Na.sub.2SO.sub.4. The organic layer was evaporated under vacuum and the resulting crude material was purified by flash chromatography (8% methanol in DCM). The resulting material was further purified by prep HPLC (Method B). After evaporation of the solvents, the material was triturated with DCM (5 mL) to afford the title compound. Yield: 6% (12 mg, white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 12.28 (s, 1H), 7.34 (s, 1H), 7.14 (d, J=7.6 Hz, 1H), 6.78 (d, J=7.6 Hz, 1H), 6.73 (s, 1H), 4.49 (t, J=8.8 Hz, 2H), 3.52 (t, J=6.4 Hz, 2H), 3.13 (t, J=8.8 Hz, 2H), 2.67-2.66 (m, 1H), 2.50-2.49 (m, 4H), 1.30 (d, J=6.4 Hz, 3H). LCMS: (Method A) 270.2 (M+H), Rt. 2.0, 98.7% (Max). HPLC: (Method A) Rt. 2.4 min, 98.9% (Max).

Example 5: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(4-fluorophenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(80) ##STR00249##

Step 1: tert-butyl-1-(4-fluorophenyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate and tert-butyl-2-(4-fluorophenyl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (regioisomers)

(81) ##STR00250##

(82) To a stirred solution of intermediate 3 (1.5 g, 5.90 mmol) in ethanol (15 mL), 4-fluoro phenyl hydrazine hydrochloride (1.15 g, 7.08 mmol) and TEA (1.6 mL, 11.8 mmol) were added under nitrogen atmosphere and the reaction mixture was heated at 85° C. for 8 h. Completion of the reaction was monitored by TLC. The reaction mixture was then concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 15-20% EtOAc in pet ether) to afford the title compound as a mixture of regioisomers. The obtained material was separated by Prep HPLC (Method B).

(83) Isomer 1: Yield: 16% (300 mg, brown gummy oil). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.52 (s, 1H), 7.48-7.45 (m, 2H), 7.17 (t, J=8.8 Hz, 2H), 4.54-4.51 (m, 2H), 3.72-3.70 (m, 2H), 2.82 (t, J=4.8 Hz, 2H), 1.52 (s, 9H). LCMS: (Method A) 318.3 (M+H), Rt. 4.8 min, 91.2% (Max).

(84) Isomer 2: Yield: 17% (320 mg, brown solid). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.64-7.58 (m, 3H), 7.14 (t, J=12.0 Hz, 2H), 4.59-4.57 (m, 2H), 3.78-3.76 (m, 2H), 2.89-2.87 (m, 2H), 1.51 (s, 9H). LCMS: (Method A) 318.3 (M+H), Rt. 4.9 min, 99.9% (Max).

Step 3: 1-(4-fluorophenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine hydrochloride

(85) ##STR00251##

(86) To a stirred solution of tert-butyl-1-(4-fluorophenyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (isomer 1) (300 mg, 0.94 mmol) in dry 1,4-dioxane (3 mL) at 0° C., HCl solution in dioxane (1 mL, 4M) was added dropwise and the reaction mixture was stirred at RT for 6 h. Completion of the reaction was monitored by TLC; the reaction mixture was then concentrated under vacuum. The resulting crude material was triturated with diethyl ether (5 mL) and filtered under nitrogen atmosphere to afford the title compound. Yield: 92% (0.22 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.36-9.35 (m, 2H), 7.67 (s, 1H), 7.62-7.58 (m, 2H), 7.39 (t, J=11.6 Hz, 2H), 4.18-4.20 (m, 2H), 3.39-3.37 (m, 2H), 3.07 (t, J=7.2 Hz, 2H). LCMS: (Method A) 218.2 (M+H), Rt. 1.8 min, 93.5% (Max).

Step 4: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(4-fluorophenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(87) ##STR00252##

(88) To a stirred solution of 1-(4-fluorophenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine hydrochloride (220 mg, 0.86 mmol) in dry MeCN (2 mL), intermediate 2 (190 mg, 1.04 mmol) and TEA (0.35 mL, 2.60 mmol) were added and the reaction mixture was heated at 90° C. for 6 h. Completion of the reaction was monitored by TLC, then the reaction mixture was concentrated under under vacuum. To the resulting mixture, water (5 mL) was added and the aqueous layer was extracted with DCM (3×5 mL). The combined organic layer was washed with brine solution (5 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 30% EtOAc in pet ether) to afford the title compound. Its structure has been confirmed by NOESY. Yield: 2.5% (7.9 mg, yellow gummy liquid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.63-7.59 (m, 2H), 7.47 (s, 1H), 7.34-7.30 (m, 2H), 7.17 (d, J=7.6 Hz, 1H), 6.82 (d, J=7.2 Hz, 1H), 6.78 (s, 1H), 4.52 (t, J=8.4 Hz, 2H), 3.61-3.57 (m, 2H), 3.41-3.39 (m, 1H), 3.15 (t, J=8.8 Hz, 2H), 2.81-2.72 (m, 3H), 2.58-2.52 (m, 1H), 1.35 (d, J=6.4 Hz, 3H). LCMS: (Method A) 364.2 (M+H), Rt. 3.2 min, 98.3% (Max). HPLC: (Method A) Rt. 3.2 min, 97.9% (Max).

Example 6: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(4-fluorophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(89) ##STR00253##

Step 1: 2-(4-fluorophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine hydrochloride

(90) ##STR00254##

(91) To a stirred solution of tert-butyl-2-(4-fluorophenyl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (synthesis described in example 5, step 1, isomer 2) (320 mg, 1.01 mmol) in dry 1,4-dioxane (3 mL) at 0° C., HCl solution in dioxane (4M, 1 mL) was added and the reaction mixture was stirred at RT for 6 h. Completion of the reaction was monitored by TLC; then the reaction mixture was concentrated under vacuum. The resulting crude material was triturated with diethyl ether (5 mL), filtered under nitrogen atmosphere to afford the title compound. Yield: 90% (0.23 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.27 (s, 1H), 8.40 (s, 1H), 7.79 (d, J=10.0 Hz, 2H), 7.50 (t, J=10.4 Hz, 2H), 7.31 (t, J=9.6 Hz, 1H), 4.25 (s, 2H), 3.46-3.43 (m, 2H), 2.99 (t, J=8.4 Hz, 2H). LCMS: (Method A) 218.2 (M+H), Rt. 2.1 min, 96.1% (Max).

Step 2: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(4-fluorophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(92) ##STR00255##

(93) To a stirred solution of 2-(4-fluorophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine hydrochloride (270 mg, 1.06 mmol) in dry DMF (3 mL), intermediate 2 (194 mg, 1.06 mmol) and TEA (0.32 g, 3.20 mmol) were added and the reaction mixture was heated at 90° C. for 6 h. Completion of the reaction was monitored by TLC, then the reaction mixture was concentrated under vacuum. To the resulting mixture, water (2 mL) was added and the aqueous layer was extracted with DCM (3×4 mL). The combined organic layer was washed with brine solution (4 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 1-2% methanol in DCM) and the obtained material was further purified by Prep HPLC (Method B) to afford the title compound. Yield: 6% (22 mg, brown gummy solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.12 (s, 1H), 7.77-7.73 (m, 2H), 7.32-7.27 (m, 2H), 7.17 (d, J=7.6 Hz, 1H), 6.82 (d, J=7.6 Hz, 1H), 6.77 (s, 1H), 4.51 (t, J=8.4 Hz, 2H), 3.61-3.56 (m, 2H), 3.42-3.39 (m, 1H), 3.15 (t, J=8.8 Hz, 2H), 2.80-2.72 (m, 1H), 2.68-2.66 (m, 3H), 1.35 (d, J=6.4 Hz, 3H). LCMS: (Method A) 364.2 (M+H), Rt. 3.3 min, 98.7% (Max). HPLC: (Method A) Rt. 3.0 min, 99.2% (Max).

Example 7: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(94) ##STR00256##

(95) To a stirred solution of intermediate 4 (140 mg, 0.59 mmol) in dry MeCN, intermediate 2 (130 mg, 0.71 mmol) and TEA (0.24 mL, 1.78 mmol) were added and the reaction mixture was heated at 90° C. overnight. Completion of the reaction was monitored by TLC; then the reaction mixture was concentrated under vacuum. To the resulting mixture, water (2 mL) was added and was extracted with DCM (3×3 mL). The combined organic layer was washed with brine solution (3 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 35% EtOAc in pet ether) to afford the title compound. Yield: 13% (25 mg, brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.59-7.57 (m, 2H), 7.51-7.47 (m, 3H), 7.36-7.34 (m, 1H), 7.18-7.16 (m, 1H), 6.84-6.82 (m, 1H), 6.78 (s, 1H), 4.52 (t, J=8.8 Hz, 2H), 3.61-3.57 (m, 2H), 3.41-3.39 (m, 1H), 3.15 (t, J=8.4 Hz, 2H), 2.83-2.71 (m, 3H), 2.58-2.55 (m, 1H), 1.35 (d, J=6.4 Hz, 3H). LCMS: (Method A) 346.2 (M+H), Rt. 3.0 min, 95.7% (Max). HPLC: (Method A) Rt. 3.1 min, 95.6% (Max).

Example 8: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(96) ##STR00257##

(97) To a stirred solution of 2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine hydrochloride (Intermediate 5, 310 mg, 1.31 mmol) in dry MeCN, intermediate 2 (480 mg, 2.63 mmol) and TEA (0.54 mL, 3.95 mmol) were added and the reaction mixture was heated at 90° C. overnight. Completion of the reaction was monitored by TLC; then the reaction mixture was concentrated under vacuum. To the resulting mixture, water (3 mL) was added and was extracted with DCM (3×5 mL). The combined organic layer was washed with brine solution (5 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 35% EtOAc in pet ether) to afford the title compound. Yield: 14% (66 mg, brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.13 (s, 1H), 7.71 (d, J=8.0 Hz, 2H), 7.45-7.42 (m, 2H), 7.24-7.15 (m, 2H), 6.82-6.76 (m, 2H), 4.51 (t, J=3.2 Hz, 2H), 3.61-3.56 (m, 2H), 3.42-3.38 (m, 1H), 3.17-3.12 (m, 2H), 2.76-2.65 (m, 4H), 1.35-1.33 (m, 3H). LCMS: (Method A) 346.3 (M+H), Rt. 3.2 min, 96.9% (Max). HPLC: (Method A) Rt. 3.2 min, 98.7% (Max).

Example 9: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(98) ##STR00258##

Step 1: 2-hydrazinylpyridine

(99) ##STR00259##

(100) To a stirred solution of 2-chloropyridine (3 g, 26.42 mmol) in ethanol (30 mL), hydrazine hydrate (10.56 mL, 211.3 mmol) was added and the reaction mixture was refluxed at 80° C. for 20 h. Completion of the reaction was monitored by TLC; the reaction mixture was then evaporated under vacuum. The resulting crude material was basified with sodium hydroxide (5 mL, 1N) and the aqueous layer was extracted with DCM (2×50 mL). The combined organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was used as such to the next step without any further purification. Yield: 93% (2.6 g, colourless liquid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.97 (t, J=9.2 Hz, 1H), 7.41 (t, J=6.8 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 6.68 (d, J=8.9 Hz, 1H), 6.53 (t, J=6.4 Hz, 1H), 4.08 (s, 2H). LCMS: (Method A) 110.1 (M+H), Rt. 0.6 min, 85.3% (Max).

Step 2: tert-butyl 1-(pyridin-2-yl)-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate and tert-butyl 2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate

(101) ##STR00260##

(102) To a stirred solution of intermediate 3 (3 g, 11.81 mmol) in DMF (20 mL), TEA (4.92 mL, 35.4 mmol) and 2-hydrazinylpyridine (1.54 g, 14.17 mmol) were added and the reaction mixture was heated at 80° C. for 8 h. Completion of the reaction was monitored by TLC; the reaction mixture was then evaporated under vacuum. To the resulting mixture, water (5 mL) was added and was extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine solution (20 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 30% EtOAc in hexane). The pyrazole regioisomers mixture was separated by prep-HPLC (method A) to afford tert-butyl 1-(pyridin-2-yl)-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (Fraction 1) and tert-butyl 2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (Fraction 2).

(103) Fraction 1: Yield: 23% (800 mg, brown gummy oil). LCMS: (Method A) 301.2 (M+H), Rt. 4.5 min, 84.2% (Max). HPLC: (Method A), Rt. 4.0 min, 82.2% (Max).

(104) Fraction 2: Yield: 34% (1.2 g, brown gummy oil). LCMS: (Method A) 301.2 (M+H), Rt. 2.4 min, 65.4% (Max). HPLC: (Method A), Rt. 2.4 min, 68.0% (Max).

Step 3: 1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine

(105) ##STR00261##

(106) To a stirred solution of tert-butyl 1-(pyridin-2-yl)-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (Fraction 1, 800 mg, 26.66 mmol) in dry 1,4-dioxane (10 mL), HCl solution in dioxane (13.33 mL, 4M) was added dropwise at 0° C. and the reaction mixture was stirred at RT for 3 h. Completion of the reaction was monitored by TLC; the reaction mixture was then evaporated under vacuum. The resulting crude material was triturated with EtOAc, hexane and diethyl ether to afford the title compound. Yield: 84% (450 mg, off white solid). LCMS: (Method B) 201.2 (M+H), Rt. 3.4 min, 96.0% (Max). HPLC: (Method B), Rt. 3.6 min, 91.5% (Max).

Step 4: 6-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine

(107) ##STR00262##

(108) To a stirred solution of 1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine (414 mg, 1.12 mmol) in MeCN (10 mL), TEA (0.4 mL, 2.80 mmol) and intermediate 2 (170 mg, 0.93 mmol) were added at RT and the reaction mixture was heated at 80° C. overnight. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum. To the resulting mixture, water (5 mL) was added and was extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine solution (20 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 50% EtOAc in hexane) to afford the title compound. Yield: 12% (97.49 mg, brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d): δ 8.41 (d, J=4.0 Hz, 1H), 7.93 (t, J=7.2 Hz, 1H), 7.86 (d, J=8.4 Hz, 1H), 7.54 (s, 1H), 7.29 (t, J=7.2 Hz, 1H), 7.17 (d, J=7.6 Hz, 1H), 6.82 (d, J=7.2 Hz, 1H), 4.51 (t, J=8.8 Hz, 2H), 3.60-3.58 (m, 1H), 3.56-3.52 (m, 1H), 3.40-3.33 (m, 1H), 3.17-3.13 (m, 5H), 2.76-2.73 (m, 1H), 2.67-2.63 (m, 1H), 1.3 (d, J=6.4 Hz, 3H). LCMS: (Method A) 347.2 (M+H), Rt. 2.7 min, 91.8% (Max). HPLC: (Method A), Rt. 3.0 min, 93.1% (Max).

Example 10: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(109) ##STR00263##

Step 1: tert-butyl 2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate

(110) ##STR00264##

(111) The title compound was synthesized according the protocol described for Example 9, steps 1 and 2. The pyrazole regioisomers mixture was separated by prep-HPLC (method A) to afford the title compound as Fraction 2. Yield: 34% (1.2 g, brown gummy oil). LCMS: (Method A) 301.2 (M+H), Rt. 2.4 min, 65.4% (Max). HPLC: (Method A), Rt. 2.4 min, 68.0% (Max).

Step 2: 2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine

(112) ##STR00265##

(113) To a stirred solution of tert-butyl 2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (1.2 g, 4.0 mmol) in dry 1,4-dioxane (10 mL) at 0° C., HCl solution in dioxane (10 mL, 40 mmol, 4M) was added dropwise and the reaction mixture was stirred at RT for 3 h. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum. The resulting crude material was triturated with EtOAc, hexane and diethyl ether to afford the title compound which was forwarded to the next step as such without any further purification. Yield: 88% (700 mg, yellow solid). LCMS: (Method B) 201.2 (M+H), Rt. 3.4 min, 86.7% (Max). HPLC: (Method B), Rt. 3.2 min, 67.9% (Max).

Step 3: 6-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine

(114) ##STR00266##

(115) To a stirred solution of 1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine hydrochloride (652 mg, 3.26 mmol) in MeCN (10 mL), intermediate 2 (495 mg, 2.71 mmol) followed by TEA (1.52 mL, 10.8 mmol) were added and the reaction mixture was heated at 80° C. overnight. Completion of the reaction was monitored by TLC; the reaction mixture was then evaporated under vacuum. To the resulting mixture, water (5 mL) was added and was extracted with EtOAc (2×25 mL). The combined organic layer was washed with brine solution (25 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by prep. HPLC (method B) to afford the title compound. Yield: 16% (130.75 mg, brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.40 (d, J=6.8 Hz, 1H), 8.30 (s, 1H), 7.91 (t, J=9.2 Hz, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.27 (t, J=5.2 Hz, 1H), 7.16 (d, J=7.6 Hz, 1H), 6.81 (d, J=8.8 Hz, 1H), 6.76 (s, 1H), 4.51 (t, J=8.8 Hz, 2H), 3.63-3.61 (m, 3H), 3.44-3.35 (m, 2H), 3.16-3.12 (m, 2H), 2.69-2.66 (m, 2H), 1.34 (d, J=6.8 Hz, 3H). LCMS: (Method A) 347.2 (M+H), Rt. 2.6 min, 99.5% (Max). HPLC: (Method A), Rt. 2.9 min, 99.5% (Max).

Example 11: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(4-fluorophenyl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine

(116) ##STR00267##

Step 1: 2-(4-fluorophenyl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine

(117) ##STR00268##

(118) To the stirred solution of 4-fluorobenzothioamide (0.38 g, 2.45 mmol) in isopropyl alcohol (10 mL), tert-butyl 3-bromo-4-oxopiperidine-1-carboxylate (0.68 g, 2.45 mmol) was added at RT and the reaction mixture was stirred at 90° C. overnight. Completion of the reaction was monitored by TLC. The reaction mixture was then evaporated under vacuum and was basified using saturated Na.sub.2CO.sub.3 (20 mL). The aqueous layer was extracted with EtOAc (2×50 mL) and the combined organic layer was washed with water (15 mL), brine solution (15 mL) and dried over anhydrous Na.sub.2SO.sub.4. The organic layer was evaporated under vacuum to afford the title compound that was used in the next step as such without any further purification. Yield: 65% (0.5 g, brown solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.95-7.91 (m, 2H), 7.35-7.30 (m, 2H), 3.94 (d, J=1.6 Hz, 2H), 3.04-3.01 (m, 2H), 2.74 (t, J=4.0 Hz, 2H), 1.34 (s, 9H). LCMS: (Method A) 335.3 (M+H), Rt. 2.4 min, 84.3% (Max).

Step 2: 2-(4-fluorophenyl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine hydrochloride

(119) ##STR00269##

(120) To the stirred solution of tert-butyl 2-(4-fluorophenyl)-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (0.5 g, 1.4 mmol) in dry 1,4-dioxane (5 mL), HCl solution in dioxane (4 mL, 4M) was added dropwise at RT and the reaction mixture was stirred for 4 h. Completion of the reaction was monitored by TLC; the reaction mixture was then concentrated under vacuum. The resulting crude material was triturated with EtOAc (2×5 mL) to afford the title compound. Yield: 65% (0.4 g, brown solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.95-7.91 (m, 3H), 7.35-7.30 (m, 2H), 3.94 (d, J=1.6 Hz, 2H), 3.04-3.01 (m, 2H), 2.74 (t, J=4.0 Hz, 2H). LCMS: (Method A) 235.3 (M+H), Rt. 2.4 min, 84.3% (Max).

Step 3: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(4-fluorophenyl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine

(121) ##STR00270##

(122) To the stirred solution of 2-(4-fluorophenyl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine hydrochloride (0.5 g, 2.17 mmol) in DMF (10 mL), TEA (1.2 mL, 8.68 mmol) and intermediate 2 (0.59 g, 3.25 mmol) were added at RT and the reaction mixture was heated at 60° C. overnight. Completion of the reaction was monitored by TLC. The reaction mixture was then quenched with water and was extracted with EtOAc (2×25 mL). The combined organic layer was washed with water (5 mL), brine solution (5 mL), dried over anhydrous Na.sub.2SO.sub.4 and evaporated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 40% EtOAc in pet ether) to afford the title compound. Yield: 5% (0.04 g, pale yellow solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.93-7.90 (m, 2H), 7.34-7.30 (m, 2H), 7.18 (d, J=7.6 Hz, 1H), 6.83 (s, 1H), 6.81-6.77 (m, 1H), 4.52 (t, J=8.8 Hz, 2H), 3.84-3.80 (m, 1H), 3.65-3.51 (m, 2H), 3.16 (t, J=8.8 Hz, 2H), 2.88-2.76 (m, 2H), 2.72-2.70 (m, 2H), 1.36 (d, J=6.8 Hz, 3H). LCMS: (Method A) 381.2 (M+H), Rt. 3.5 min, 92.9% (Max). HPLC: (Method A) Rt. 3.2 min, 94.1% (Max).

Example 12 and Example 13: (R)-5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine and (S)-5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(123) ##STR00271##

(124) The two enantiomers of Example 8 were separated by SFC (Method A: mobile phase: 20 mM ammonia in IPA, column: Lux A1). The first eluting product was associated to Example 12 and the second eluting compound was associated to Example 13.

(125) Analysis of first eluting fraction (Example 12): Yield: 4% (10 mg, yellow gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.14 (s, 1H), 7.72 (d, J=8.4 Hz, 2H), 7.46-7.42 (m, 2H), 7.23 (t, J=7.2 Hz, 1H), 7.16 (d, J=7.2 Hz, 1H), 6.81 (d, J=7.6 Hz, 1H), 6.76 (s, 1H), 4.51 (t, J=8.4 Hz, 2H), 3.58 (t, J=5.6 Hz, 2H), 3.42-3.39 (m, 1H), 3.14 (t, J=8.8 Hz, 2H), 2.76-2.74 (m, 2H), 2.67 (d, J=7.6 Hz, 2H), 1.34 (d, J=6.4 Hz, 3H). LCMS: (Method A) 346.3 (M+H), Rt. 3.1 min, 97.4% (Max). HPLC: (Method A) Rt. 3.1 min, 97.3% (Max). Chiral SFC: (Method A) Rt. 8.98 min, 99.54% (Max).

(126) Analysis of second eluting fraction (Example 13): Yield: 3% (7 mg, brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.14 (s, 1H), 7.72 (d, J=8.4 Hz, 2H), 7.46-7.42 (m, 2H), 7.23 (t, J=7.2 Hz, 1H), 7.16 (d, J=7.2 Hz, 1H), 6.81 (d, J=7.6 Hz, 1H), 6.76 (s, 1H), 4.51 (t, J=8.0 Hz, 2H), 3.58 (t, J=4.8 Hz, 2H), 3.42-3.39 (m, 1H), 3.14 (t, J=8.8 Hz, 2H), 2.76-2.74 (m, 2H), 2.67 (d, J=7.6 Hz, 2H), 1.34 (d, J=6.4 Hz, 3H). LCMS: (Method A) 346.3 (M+H), Rt. 3.1 min, 97.2% (Max). HPLC: (Method A) Rt. 3.1 min, 97.7% (Max). Chiral SFC: (Method A) Rt. 10.0 min, 98.5% (Max).

Example 14: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(pyrimidin-5-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(127) ##STR00272##

Step 1: 5-hydrazinylpyrimidine hydrochloride

(128) ##STR00273##

(129) To a stirred solution of pyrimidine-5-boronic acid (5 g, 40.3 mmol) in dry methanol (160 mL), di-tert-butyl azodicarboxylate (DTAD, 9.3 g, 40.3 mmol) and copper(II) diacetate monohydrate (0.26 g, 1.32 mmol) were added and the reaction mixture was heated at 60° C. for 1 h. Completion of the reaction was monitored by TLC. The reaction mixture was then concentrated under high vacuum and the resulting mixture was dissolved in diethyl ether (50 mL). The organic layer was washed with saturated NaHCO.sub.3 (50 mL), water (50 mL), brine solution (50 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting material was dissolved in 1,4-dioxane (50 mL), HCl solution in dioxane (20 mL, 4M) was added dropwise at 0° C. and the reaction mixture was stirred overnight. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under vacuum and the resulting mixture was triturated with EtOAc (50 mL), filtered under nitrogen to afford the title compound. Yield: 51% (2.2 g, dark green solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.62-10.54 (m, 1H), 8.78 (s, 1H), 8.52-7.81 (m, 2H), 8.58 (s, 2H). LCMS: (Method B) 111.2 (M+H), Rt. 0.8 min, 68.4% (Max).

Step 2: tert-butyl 1-(pyrimidin-5-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate

(130) ##STR00274##

(131) To a solution of intermediate 3 (1.5 g, 5.90 mmol) in ethanol (15 mL), 5-hydrazinylpyrimidine hydrochloride (0.86 g, 5.90 mmol) and TEA (1.8 mL, 17.7 mmol) were added under nitrogen atmosphere and the reaction mixture was heated to 85° C. for 8 h. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under vacuum and the resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 70% EtOAc in pet ether) to afford the title compound. Yield: 70% (1.2 g, brown gummy liquid). LCMS: (Method A) 302.0 (M+H), Rt. 2.3 min, 56.5% (Max).

Step 3: 1-(pyrimidin-5-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine hydrochloride

(132) ##STR00275##

(133) To a stirred solution of tert-butyl 1-(pyrimidin-5-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (1.2 g, 3.83 mmol) in dry 1,4-dioxane (12 mL) at 0° C., HCl solution in dioxane (2.4 mL, 4M) was added dropwise and the reaction mixture was stirred at RT overnight. Completion of the reaction was monitored by TLC; the reaction mixture was then concentrated under vacuum. The resulting crude material was triturated with diethyl ether (15 mL) and filtered under nitrogen atmosphere to afford the title compound. Yield: 43% (0.41 g, brown solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.71-9.46 (m, 2H), 9.23 (s, 1H), 9.11 (s, 1H), 7.82 (s, 1H), 7.56 (s, 1H), 4.18-4.09 (m, 2H), 3.40-3.33 (m, 2H), 3.24-3.21 (m, 1H), 2.91-2.88 (m, 1H).

Step 4: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(pyrimidin-5-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(134) ##STR00276##

(135) To a stirred solution of 1-(2,3-dihydrobenzofuran-6-yl)ethan-1-ol (Intermediate 2, steps 1 to 4; 0.4 g, 2.43 mmol) in dry DCM (10 mL) at 0° C., thionyl chloride (0.26 mL, 3.65 mmol) was added and the reaction mixture was stirred at RT for 2 h. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated under vacuum and the resulting mixture was suspended in dry DMF (3 mL). 1-(Pyrimidin-5-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine hydrochloride (578 mg, 2.43 mmol) and TEA (0.74 g, 7.31 mmol) were added at RT and the reaction mixture was heated at 90° C. for 6 h. Completion of the reaction was monitored by TLC; the reaction mixture was then concentrated under vacuum. To the resulting mixture, water (4 mL) was added and was extracted with DCM (3×4 mL). The combined organic layer was washed with brine solution (10 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 55% EtOAc in pet ether) to afford the title compound. Yield: 4% (6.5 mg, pale brown solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.16 (s, 1H), 9.10 (s, 2H), 7.64 (s, 1H), 7.17 (d, J=7.6 Hz, 1H), 6.82 (d, J=7.2 Hz, 1H), 6.78 (s, 1H), 4.52 (t, J=8.4 Hz, 2H), 3.64-3.57 (m, 2H), 3.37-3.34 (m, 1H), 3.15 (t, J=8.4 Hz, 2H), 2.95-2.89 (m, 2H), 2.77-2.72 (m, 1H), 2.68-2.63 (m, 1H), 1.36 (d, J=6.8 Hz, 3H). LCMS: (Method A) 348.0 (M+H), Rt. 2.2 min, 93.4% (Max). HPLC: (Method A) Rt. 2.2 min, 93.6% (Max).

Example 15: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine or 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(136) ##STR00277##

Step 1: tert-butyl 2-(tetrahydro-2H-pyran-4-yl)hydrazine-1-carboxylate

(137) ##STR00278##

(138) To the stirred solution of tetrahydro-4H-pyran-4-one (2 g, 9.33 mmol), tert-butoxycarbonyl hydrazide (1.8 g, 13.99 mmol) in AcOH:MeOH (1:1, 10 mL), NaCNBH.sub.3 (0.52 g, 8.4 mmol) was added portion wise at 0° C. and the reaction mixture was stirred at RT for 30 min. Completion of the reaction was monitored by TLC; the reaction mixture was then concentrated under vacuum. The resulting mixture was suspended with 10% NaHCO.sub.3 (20 mL) and was extracted with DCM (2×15 mL). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum to afford the title compound. Yield: 99% (crude) (2 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.20 (s, 1H), 4.34 (s, 1H), 3.82-3.81 (m, 2H), 3.29-3.26 (m, 2H), 2.89-2.88 (m, 1H), 1.63 (t, J=2.0 Hz, 2H), 1.38 (s, 9H), 1.24 (t, J=4.0 Hz, 2H). LCMS: (Method A) 117.2 (M-Boc), Rt. 1.2 min, 99.7%.

Step 2: (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride

(139) ##STR00279##

(140) To the stirred solution of tert-butyl 2-(tetrahydro-2H-pyran-4-yl)hydrazine-1-carboxylate (2 g, 9.25 mmol) in dry 1,4-dioxane (6 mL), HCl solution in dioxane (10 mL, 4M) was added and the reaction mixture was stirred at RT for 2 h. Completion of reaction was monitored by TLC; the reaction mixture was then evaporated under vacuum. The resulting crude material was triturated with EtOAc twice and dried under vacuum to afford the title compound which was used in the next step as such without any further purification. Yield: 99% (crude) (2.3 g, off white solid). LCMS: (Method A) 117.2 (M-Boc), Rt. 0.6 min, 99.6%

Step 3: tert-butyl 1-(tetrahydro-2H-pyran-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate or tert-butyl 2-(tetrahydro-2H-pyran-4-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate

(141) ##STR00280##

(142) To the mixture of (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride (2.3 g, 12.16 mmol) and intermediate 3 (3.08 g, 12.16 mmol) in EtOH (23 mL), K.sub.2CO.sub.3 (8.4 g, 60.82 mmol) was added at RT and the reaction mixture was heated at 80° C. for 6 h. Completion of reaction was monitored by TLC. The reaction mixture was concentrated under vacuum. Water (10 mL) was added and was extracted with EtOAc (2×25 mL). The combined organic layer was washed with brine solution (10 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 35% EtOAc in pet ether) to afford the title compound. Yield: 14%, (0.51 g, yellow gummy oil). LCMS: (Method A) 308.3 (M+H), Rt. 4.3 min, 96.4%.

Step 4: 1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine hydrochloride or 2-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine hydrochloride

(143) ##STR00281##

(144) To the stirred solution of tert-butyl 1-(tetrahydro-2H-pyran-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate or tert-butyl 2-(tetrahydro-2H-pyran-4-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (0.5 g, 1.63 mmol) in dry 1,4-dioxane (2 mL), HCl solution in dioxane (5 mL, 4M) was added dropwise and the reaction mixture was stirred at RT for 1 h. Completion of reaction was monitored by TLC; the reaction mixture was then evaporated under vacuum. The resulting crude material was triturated with EtOAc twice and dried under vacuum to afford the title compound. Yield: 79%, (0.31 g, off white solid). .sup.1H NMR (300 MHz, DMSO-d.sub.6): δ 7.38 (s, 1H), 4.40-4.38 (m, 1H), 4.06-4.05 (m, 2H), 3.97-3.96 (m, 2H), 3.49-3.46 (m, 2H), 3.38-3.33 (m, 2H), 3.00 (t, J=6.0 Hz, 2H), 2.05-2.04 (m, 2H), 1.78-1.77 (m, 2H). LCMS: (Method A) 208.0 (M-Boc+H), Rt. 2.6 min, 96.3%.

Step 5: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine or 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(145) ##STR00282##

(146) To a stirred solution of 1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine hydrochloride or 2-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine hydrochloride (0.3 g, 1.2 mmol) in DMF, TEA (0.51 mL, 3.7 mmol) followed by intermediate 2 (0.34 g, 1.8 mmol) were added at RT and the reaction mixture was heated at 75° C. overnight. Completion of reaction was monitored by TLC. The reaction mixture was concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 2% methanol in DCM). The resulting product was further purified by Prep-HPLC (Method B) to afford the title compound. Yield: 8% (32 mg, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.16-7.14 (m, 2H), 6.43 (d, J=8.4 vHz, 1H), 6.74 (s, 1H), 4.50 (t, J=8.8 Hz, 2H), 4.22-4.16 (m, 1H), 3.95-3.93 (m, 2H), 3.54-3.53 (m, 4H), 3.28-3.24 (m, 1H), 3.14 (t, J=8.8 Hz, 2H), 2.72-2.71 (m, 4H), 2.01-1.97 (m, 2H), 1.77-1.74 (m, 2H), 1.31 (d, J=6.8 Hz, 3H). LCMS: (Method A) 354.0 (M+H), Rt. 2.4 min, 99.6% (Max). HPLC: (Method A) Rt 2.4 min, 99.2% (Max).

Example 16: 4-(5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)tetrahydro-2H-thiopyran 1,1-dioxide or 4-(5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)tetrahydro-2H-thiopyran 1,1-dioxide

(147) ##STR00283##

Step 1: tert-butyl ((tetrahydro-4H-thiopyran-4-ylidene)amino)glycinate

(148) ##STR00284##

(149) To a stirred solution of tetrahydro-4H-thiopyran-4-one (5 g, 43.09 mmol) in methanol (50 mL), tert-butyl hydrazinecarboxylate (6.20 g, 46.97 mmol) was added at RT and the reaction mixture was stirred overnight at same temperature. Completion of the reaction was monitored by TLC. The reaction mixture was evaporated under vacuum. To the resulting mixture, water (5 mL) was added and was extracted with EtOAc (2×25 mL). The combined organic layer was washed with brine solution (20 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was used in the next step as such without any further purification. Yield: 96% (9.5 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.68 (s, 1H), 2.75 (t, J=8.1 Hz, 2H), 2.72 (t, J=7.5 Hz, 2H), 2.67 (t, J=6.0 Hz, 2H), 2.65 (t, J=7.4 Hz, 2H), 1.43 (s, 9H). LCMS: (Method A) 175.1 (M-t-Bu+H), Rt. 2.1 min, 35.3% (Max).

Step 2: tert-butyl 2-(tetrahydro-2H-thiopyran-4-yl)hydrazine-1-carboxylate

(150) ##STR00285##

(151) To a stirred solution of step 1 tert-butyl 2-(tetrahydro-4H-thiopyran-4-ylidene)hydrazine-1-carboxylate (9.5 g, 41.02 mmol) in acetic acid (50 mL), NaCNBH.sub.3 (2.85 g, 45.3 mmol) was added at 0° C. and the reaction mixture was stirred for 2 h at RT. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum and to the resulting mixture water (15 mL) was added. The aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layer was dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was used as such in the next step without any further purification. Yield: 83% (7.8 g, colourless liquid). LCMS: (Method A) 177.1 (M-t-Bu+H), Rt. 1.8 min, 96.4% (Max).

Step 3: (tetrahydro-2H-thiopyran-4-yl)hydrazine hydrochloride

(152) ##STR00286##

(153) To a stirred solution of tert-butyl 2-(tetrahydro-2H-thiopyran-4-yl)hydrazine-1-carboxylate (7.8 g, 33.62 mmol) in dry 1,4-dioxane (30 mL), HCl solution in dioxane (33.5 mL, 134 mmol, 4M) was added dropwise at 0° C. and the reaction mixture was stirred at RT for 3 h. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum. The resulting crude material was triturated with EtOAc, hexane and diethyl ether to afford the title compound which was used in the next step without any further purification. Yield: 91% (4 g, off white solid). LCMS: (Method A) 133.3 (M-t-Bu+H), Rt. 0.7 min, 99.5% (Max).

Step 4: tert-butyl 1-(tetrahydro-2H-thiopyran-4-yl)-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate or tert-butyl 2-(tetrahydro-2H-thiopyran-4-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate

(154) ##STR00287##

(155) To a stirred solution of intermediate 3 (3 g, 11.8 mmol) in DMF (20 mL), TEA (4.97 mL, 35.4 mmol) and (tetrahydro-2H-thiopyran-4-yl)hydrazine hydrochloride (1.87 g, 14.19 mmol) were added at RT and the reaction mixture was heated at 80° C. for 8 h. Completion of the reaction was monitored by TLC. The reaction mixture was evaporated under vacuum. To the resulting mixture, water (5 mL) was added and was extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine solution (20 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 60-70% EtOAc in hexane) to afford the title compound. Yield: 85% (3.2 g, yellow gummy oil). LCMS: (Method B) 324.2 (M+H), Rt. 5.8 min, 87.4% (Max). HPLC: (Method B), Rt. 4.9 min, 80.1% (Max).

Step 5: tert-butyl 1-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate or tert-butyl 2-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate

(156) ##STR00288##

(157) To a stirred solution of tert-butyl 1-(tetrahydro-2H-thiopyran-4-yl)-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate or tert-butyl 2-(tetrahydro-2H-thiopyran-4-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (3.1 g, 9.58 mmol) in DCM (30 mL), m-CPBA (4.13 g, 23.95 mmol) was added at 0° C. batchwise and the reaction mixture was stirred at RT for 3 h. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum. To the resulting mixture, water (5 mL) was added and was extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine solution (20 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 80-90% EtOAc in hexane) to afford the title compound. Yield: 83% (2.8 g, yellow gummy oil). LCMS: (Method A) 356.1 (M+H), Rt. 2.4 min, 46.2% (Max).

Step 6: 4-(4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridin-1-yl)tetrahydro-2H-thiopyran 1,1-dioxide hydrochloride or 4-(4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-2-yl)tetrahydro-2H-thiopyran 1,1-dioxide hydrochloride

(158) ##STR00289##

(159) To a stirred solution of tert-butyl 1-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate or tert-butyl 2-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (2.7 g, 7.59 mmol) in dry 1,4-dioxane (20 mL) at 0° C., HCl solution in dioxane (13.5 mL, 30.36 mmol, 4M) was added dropwise and the reaction mixture was stirred at RT for 3 h. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum and the resulting crude material was triturated with EtOAc, hexane and diethyl ether to afford the title compound. Yield: 94% (1.8 g, yellow solid). LCMS: (Method B) 256.1 (M+H), Rt. 0.7 min, 49.9% (Max).

Step 7: 4-(5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)tetrahydro-2H-thiopyran 1,1-dioxide or 4-(5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)tetrahydro-2H-thiopyran 1,1-dioxide

(160) ##STR00290##

(161) To a stirred solution of 4-(4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridin-1-yl)tetrahydro-2H-thiopyran 1,1-dioxide hydrochloride or 4-(4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-2-yl)tetrahydro-2H-thiopyran 1,1-dioxide hydrochloride (200 mg, 0.78 mmol) in DMF (10 mL), intermediate 2 (171 mg, 0.93 mmol) followed by TEA (0.3 mL, 2.34 mmol) were added at RT and the reaction mixture was heated at 80° C. overnight. Completion of the reaction was monitored by TLC. The reaction mixture was then evaporated under vacuum. To the resulting mixture, water (5 mL) was added and the aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine solution (20 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by prep.HPLC (method B) to afford the title compound. Yield: 12% (34.76 mg, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.21 (s, 1H), 7.15 (d, J=7.4 Hz, 1H), 6.79 (d, J=7.2 Hz, 1H), 6.74 (s, 1H), 4.52-4.45 (m, 3H), 3.55-3.49 (m, 2H), 3.41-3.35 (m, 2H), 3.28-3.12 (m, 5H), 2.72-2.60 (m, 4H), 2.42-2.37 (m, 2H), 2.16-2.13 (m, 2H), 1.31 (d, J=4.4 Hz, 3H). LCMS: (Method A) 402.0 (M+H), Rt. 2.3 min, 97.6% (Max). HPLC: (Method A), Rt. 2.3 min, 97.9% (Max).

Example 17: 7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(4-(methylsulfonyl)phenyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo 1,5-a]pyrazine

(162) ##STR00291##

Step 1: [1,2,4]triazolo[1,5-a]pyrazin-2-amine

(163) ##STR00292##

(164) To a solution of 2-amino pyrazine (15.0 g, 157.7 mmol) in dry 1,4-dioxane (150 mL), 1-ethoxycarbonylisothiocyanate (20.7 g, 173.5 mmol) was added at 0° C. and the reaction mixture was stirred at RT for 2 h. After the completion of reaction (monitored by TLC), the reaction mixture was concentrated under vacuum and the resulting material was dissolved in dry methanol (150 mL). To the stirred solution, hydroxylamine hydrochloride (32.87 g, 473.1 mmol) and TEA (106 mL, 788.5 mmol) were added and the reaction mixture was stirred for 2 h at RT, then heated at 80° C. for 3 h. Completion of reaction was monitored by TLC and the reaction mixture was then concentrated under vacuum. To the resulting mixture, water (100 mL) was added and the aqueous layer was extracted with DCM (3×100 mL). The combined organic layer was washed with brine solution (100 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum to afford the title compound. Yield: 71% (15 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.84 (s, 1H), 8.70 (d, J=4.3 Hz, 1H), 7.99 (d, J=4.3 Hz, 1H), 6.48 (s, 2H). LCMS: (Method B) 136.2 (M+H), Rt. 1.14 min, 99.5% (Max).

Step 2: 2-bromo-[1,2,4]triazolo[1,5-a]pyrazine

(165) ##STR00293##

(166) To a solution of tert-butyl nitrite (25.1 g, 244.17 mmol) in dry MeCN (150 mL), copper (II) bromide (37.12 g, 166.47 mmol) was added and heated to 60° C. for 30 min. The reaction mixture was then cooled to RT and stirred for 5 min. Then [1,2,4]triazolo[1,5-a]pyrazin-2-amine (15 g, 110.98 mmol) was added portionwise and the reaction mixture was stirred for 1 h at 60° C. After completion of the reaction (monitored by TLC), the reaction mixture was filtered through celite and washed with DCM (2×100 mL). The combined organic layer was washed with saturated NaHCO.sub.3 (1×100 mL), brine solution (100 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 45% EtOAc in pet ether) to afford the title compound. Yield: 10% (2.1 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.40 (s, 1H), 9.11 (d, J=6.0 Hz, 1H), 8.34 (d, J=6.0 Hz, 1H).

Step 3: 2-bromo-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine

(167) ##STR00294##

(168) To a stirred solution of 2-bromo-[1,2,4]triazolo[1,5-a]pyrazine (2.1 g, 10.71 mmol) in a mixture of dry THF (3 mL) and methanol (1 mL), lithium borohydride solution (2M, 21.43 mL, 21.4 mmol) was added at 0° C. and the reaction mixture was heated at 50° C. for 30 min. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to 0° C. and quenched with saturated NH.sub.4Cl (20 mL). The aqueous layer was extracted with DCM (2×25 mL), then the combined organic layer was washed with brine solution (50 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum to afford the title product. Yield: 41% (0.86 g, pale brown solid). LCMS: (Method A, ELSD) 203.0 (M+1), Rt. 0.7 min, 99.4% (Max).

Step 4: 2-bromo-7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine

(169) ##STR00295##

(170) To a 0° C. solution of 1-(2,3-dihydrobenzofuran-6-yl)ethan-1-ol (2 g, 12.1 mmol) in dry DCM (20 mL), thionyl chloride (2.72 mL, 24.3 mmol) was added and stirred at RT for 2 h. After the completion of reaction (monitored by TLC), the reaction mixture was concentrated completely and added over a reaction mixture of 2-bromo-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine (0.85 g, 4.20 mmol) and TEA (1.7 mL, 12.6 mmol) in dry MeCN (15 mL). The reaction mixture was heated at 60° C. overnight. Completion of reaction was monitored by TLC, then the reaction mixture was concentrated under vacuum. To the resulting mixture, water (15 mL) was added and the aqueous layer was extracted with DCM (2×15 mL). The combined organic layer was washed with brine solution (15 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 5% methanol in DCM) to afford the title compound. Yield: 6% (0.17 g, brown gummy solid). LCMS: (Method A) 351.0 (M+H), Rt. 2.5 min, 59.3% (Max).

Step 5: 7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(4-(methylsulfonyl)phenyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine

(171) ##STR00296##

(172) To a degassed solution of 2-bromo-7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine (0.17 g, 0.48 mmol) in 1,4-dioxane and water (8:2, 6 mL), 4-methylsulphonylphenylboronic acid pinacol ester (0.21 g, 0.73 mmol), Cs.sub.2CO.sub.3 (0.47 g, 1.46 mmol) and Pd(PPh.sub.3).sub.4 (5.6 mg, 0.05 mmol) were added and the reaction mixture was heated at 80° C. for 6 h. Completion of reaction was monitored by TLC, then the reaction mixture was concentrated under vacuum. To the resulting mixture, water (8 mL) was added and the aqueous layer was extracted with DCM (2×8 mL). The combined organic layer was washed with brine solution (8 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 50-60% EtOAc in pet ether), then the obtained material was further purified by prep HPLC (Method B) to afford the title compound. Yield: 3% (5.5 mg, white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.20 (d, J=8.8 Hz, 2H), 8.01 (d, J=8.4 Hz, 2H), 7.21 (d, J=7.6 Hz, 1H), 6.85 (d, J=7.6 Hz, 1H), 6.80 (s, 1H), 4.53 (t, J=8.4 Hz, 2H), 4.19-4.14 (m, 2H), 3.91-3.87 (m, 1H), 3.74-3.68 (m, 2H), 3.26 (s, 3H), 3.16 (t, J=8.4 Hz, 2H), 3.00-2.90 (m, 2H), 1.40 (d, J=6.8 Hz, 3H). LCMS: (Method A) 425.0 (M+H), Rt. 2.8 min, 99.4% (Max). HPLC: (Method A) Rt. 2.7 min, 99.9% (Max).

Example 18: 7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(pyridin-3-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine

(173) ##STR00297##

(174) To a degassed solution of 2-bromo-7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine (0.08 g, 0.22 mmol, synthesis described in example 17, steps 1 to 4), Cs.sub.2CO.sub.3 (0.224 g, 0.68 mmol) and pyridine 3-boronic acid pinacol ester (0.07 g, 0.34 mmol) in 1,4-dioxane (2 ml) and water (0.08 ml), Pd(PPh.sub.3).sub.4 (0.013 g, 0.011 mmol) was added and the resulting solution was purged with argon for another 2 min. The reaction mixture was heated at 80° C. overnight. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to RT, quenched with water (5 mL) and the aqueous layer was extracted with EtOAc (2×10 mL). The combined organic layer was washed with brine solution (20 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by Prep HPLC (method B) to afford the title compound. Yield: 21% (16.74 mg, brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.13 (s, 1H), 8.61 (d, J=4.8 Hz, 1H), 8.27 (d, J=8.0 Hz, 1H), 7.51-7.50 (m, 1H), 7.21 (d, J=7.2 Hz, 1H), 6.85 (d, J=7.6 Hz, 1H), 6.80 (s, 1H), 4.53 (t, J=8.4 Hz, 2H), 4.15-4.14 (m, 2H), 3.86-3.82 (m, 1H), 3.71-3.70 (m, 2H), 3.16-3.14 (m, 2H), 3.08 (m, 3H), 1.40 (d, J=6.8 Hz, 2H). LCMS: (Method A) 348.2 (M+H), Rt. 2.0 min, 94.7% (Max). HPLC: (Method A): Rt. 2.1 min, 94.3% (Max).

Example 19: 6-((7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)amino)nicotinonitrile

(175) ##STR00298##

Step 1: N-([1,2,4]triazolo[1,5-a]pyrazin-2-yl)acetamide

(176) ##STR00299##

(177) To a stirred solution of [1,2,4]triazolo[1,5-a]pyrazin-2-amine (5 g, 0.04 mol, synthesis described in example 17, step 1) in toluene (100 mL), acetic anhydride (7.55 g, 74 mmol) was added at RT and the reaction mixture was heated to reflux overnight. Completion of the reaction was monitored by TLC, then the reaction mixture was filtered and dried under vacuum to afford the title compound. Yield: 55% (3.6 g, white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.07 (s, 1H), 9.21 (d, J=6.4 Hz, 1H), 9.01-8.99 (m, 1H), 8.20 (d, J=6.0 Hz, 1H), 2.17 (s, 3H). LCMS: (Method A) 178.2 (M+H), Rt. 0.7 min, 98.7% (Max).

Step 2: N-(5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)acetamide

(178) ##STR00300##

(179) To a stirred solution of N-(5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)acetamide (3 g, 16.94 mmol) in ethanol (60 mL), PtO.sub.2 (0.6 g, 2.6 mmol) was added under H.sub.2 ballon pressure at RT and continued for 2 h. Completion of the reaction was monitored by TLC, then the reaction mixture was filtered through celite bed, washed with ethanol (120 mL) and dried under vacuum to afford the title compound. Yield: 75% (2.3 g, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.19 (s, 1H), 3.92 (t, J=7.2 Hz, 2H), 3.84 (s, 2H), 3.11 (t, J=7.2 Hz, 2H), 2.01 (s, 3H). LCMS: (Method A) 181.1 (M+H), Rt. 0.5 min, 99.8% (Max).

Step 3: N-(7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)acetamide

(180) ##STR00301##

(181) To a stirred solution of N-(5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)acetamide (1.7 g, 9.38 mmol) in MeCN (68 mL), intermediate 2 (2.56 g, 14.07 mmol) and TEA (5.68 g, 56.29 mmol) were added at RT and the reaction mixture was heated at 60° C. for 6 h. Completion of the reaction was monitored by TLC, then the reaction mixture was concentrated under vacuum. To the resulting mixture, water (100 mL) was added and the aqueous layer was extracted with DCM (3×50 mL). The combined organic layer was washed with brine solution (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 4% methanol in DCM) to afford the title compound. Yield: 24% (740 mg, off white solid). LCMS: (Method A) 328.2 (M+H), Rt. 1.7 min, 74.3% (Max).

Step 4: 7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-amine

(182) ##STR00302##

(183) To a stirred solution of N-(7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)acetamide (0.74 g, 2.26 mmol) in methanol (14 mL), 6N NaOH (0.36 g, 9.04 mmol) was added and the reaction mixture was stirred at RT overnight. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated under vacuum. The resulting crude material was suspended in water (20 mL) and was neutralized with 2N HCl to adjust pH˜6.0. The aqueous layer was extracted with 20% methanol in DCM (3×50 mL). The combined organic layer was washed with brine solution (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum to afford the title compound. Yield: 56% (360 mg, off white solid). LCMS: (Method A) 286.2 (M+H), Rt. 1.6 min, 64.2% (Max).

Step 5: 6-((7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)amino)nicotinonitrile

(184) ##STR00303##

(185) To a stirred solution of 7-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazin-2-amine (0.18 g, 0.63 mmol) in DMF (14 mL) at 0° C., 60% NaH (0.04 g, 0.95 mmol) was added and stirred for 15 min. Then 6-chloronicotinonitrile (0.13 g, 0.95 mmol) was added and the reaction mixture was heated at 70° C. overnight. Completion of the reaction was monitored by TLC, then the reaction mixture was cooled to RT and quenched with water (10 mL). The obtained solid was filtered, dried under vacuum and the resulting material was neutralized with 2N HCl to adjust pH˜6.0. The resulting crude material was suspended in water (20 mL) and the aqueous layer was extracted with 20% methanol in DCM (3×50 mL). The combined organic layer was washed with brine solution (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The resulting crude material was further purified by Prep HPLC (Method B) to afford the title compound. Yield: 18% (21.08 mg, off white solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.49 (s, 1H), 8.59 (d, J=1.6 Hz, 1H), 8.07 (dd, J=8.8, 1.2 Hz, 1H) 7.90 (d, J=8.8 Hz, 1H), 7.18 (d, J=7.2 Hz, 1H), 6.82 (d, J=7.5 Hz, 1H), 6.78 (s, 1H), 4.54-4.50 (m, 2H), 4.01-3.99 (m, 2H), 3.79-3.75 (m, 1H), 3.67 (q, J=7.5 Hz, 1H), 3.55 (d, J=7.5 Hz, 1H), 3.18-3.15 (m, 2H), 2.93-3.86 (m, 2H), 1.36 (d, J=7.5 Hz, 3H). LCMS: (Method B) 388.1 (M+H), Rt. 2.4 min, 97.3% (Max). HPLC: (Method B): Rt. 2.6 min, 98.9% (Max).

Example 20: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(pyrimidin-2-ylmethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(186) ##STR00304##

Step 1: tert-butyl 2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate

(187) ##STR00305##

(188) To a stirred solution of intermediate 3 (25 g, 98.42 mmol) in EtOH (500 mL), K.sub.2CO.sub.3 (27.16 g, 196.8 mmol) and hydrazinehydrate (39.41 g, 787.40 mmol) were added and the reaction mixture was refluxed at 78° C. overnight. Completion of the reaction was monitored by TLC, then the reaction mixture was evaporated under vacuum. To the resulting mixture, water (200 mL) was added and the aqueous layer was extracted with EtOAc (2×300 mL). The combined organic layer was washed with brine solution (50 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by column chromatography (Biotage Isolera, eluent: 70% EtOAc in hexane). Yield: 77% (17 g, off white Solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 12.50 (s, 1H), 7.42 (s, 1H), 4.36 (s, 2H), 3.58 (t, J=6.4 Hz, 2H), 2.62 (t, J=6.4 Hz, 2H), 1.41 (s, 9H). LCMS: (Method A) 224.0 (M+H), Rt. 1.9 min, 93.4% (Max).

Step 2: tert-butyl 2-(pyrimidin-2-ylmethyl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate or tert-butyl 1-(pyrimidin-2-ylmethyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate

(189) ##STR00306##

(190) To a stirred solution of tert-butyl 2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (1.5 g, 6.72 mmol) in THF (30 mL) at 0° C., NaH (60%) (0.23 g, 10.08 mmol) was added and stirred for 15 min. Then 2-2-(chloromethyl)pyrimidine (0.94 g, 7.39 mmol) was added to the reaction mixture and stirred at RT for 4 h. Completion of the reaction was monitored by TLC, then the reaction mixture was quenched with ice cold water (20 mL) and the aqueous layer was extracted with EtOAc (2×200 mL). The combined organic layer was washed with brine solution (10 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by flash chromatography (Biotage Isolera, eluent: 20% EtOAc in hexane) and the regioisomers were separated by SFC (method B) to afford the title compound. The structure of both isomers has been confirmed by NOESY.

(191) Isomer 1: Yield: 19% (400 mg, brown gummy oil). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.76 (d, J=5.2 Hz, 2H), 7.40 (s, 1H), 7.27 (t, J=8.0 Hz, 1H), 5.60 (s, 2H), 4.53 (s, 2H), 3.73 (s, 2H), 2.84 (t, J=5.6 Hz, 2H), 1.52-1.50 (m, 9H). LCMS: (Method A), 316.3 (M+H), Rt. 2.2 min, 99.1% (Max). HPLC: (Method A), Rt. 3.1 min, 99.8% (Max).

(192) Isomer 2: Yield: 33% (700 mg, brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.77 (d, J=6.4 Hz, 2H), 7.43 (t, J=6.4 Hz, 1H), 7.27 (s, 1H), 5.44 (s, 2H), 4.35 (s, 2H), 3.60 (t, J=7.2 Hz, 2H), 2.66-2.62 (m, 2H), 1.42-1.40 (m, 9H). LCMS: (Method A), 316.3 (M+H), Rt. 2.3 min, 91.5% (Max). HPLC: (Method A), Rt. 3.1 min, 94.3% (Max).

Step 3: 2-(pyrimidin-2-ylmethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(193) ##STR00307##

(194) To a stirred solution of tert-butyl 2-(pyrimidin-2-ylmethyl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (Isomer 1 obtained in step 2, 400 mg, 26.66 mmol) in dry 1, 4-dioxane (10 mL) at 0° C., HCl solution in dioxane (13.33 mL, 4M) was added dropwise and the reaction mixture was stirred at RT for 3 h. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum. The resulting crude material was triturated with EtOAc, hexane and diethyl ether to afford the title compound which was forwarded to the next step as such without any further purification. Yield: 49% (150 mg, pale yellow solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.28 (s, 1H), 8.78 (d, J=1.2 Hz, 2H), 7.77 (s, 1H), 7.46-7.45 (m, 1H), 5.50 (s, 2H), 4.15 (d, J=2.8 Hz, 2H), 3.35 (s, 2H), 2.83 (t, J=6.0 Hz, 2H).

Step 4: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-2-(pyrimidin-2-ylmethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine

(195) ##STR00308##

(196) To a stirred solution of 2-(pyrimidin-2-ylmethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine (150 mg, 0.697 mmol) in MeCN (10 mL), intermediate 2 (253 mg, 1.39 mmol) followed by TEA (0.4 mL, 2.79 mmol) were added and the reaction mixture was heated at 60° C. overnight. Completion of the reaction was monitored by TLC, then the reaction mixture was evaporated under vacuum. To the resulting mixture, water (10 mL) was added and the aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine solution (10 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by Prep HPLC (Method B) to afford the title compound. Yield: 2% (2.86 mg, pale brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.78 (d, J=4.8 Hz, 2H), 7.49 (s, 1H), 7.44 (t, J=4.8 Hz, 1H), 7.16 (d, J=7.6 Hz, 1H), 6.80 (d, J=7.6 Hz, 1H), 6.74 (s, 1H), 5.41 (s, 2H), 4.51 (t, J=8.8 Hz, 2H), 3.55-3.52 (m, 2H), 3.34-3.32 (m, 2H), 3.14 (t, J=8.8 Hz, 2H), 2.71 (s, 1H), 2.62-2.51 (m, 2H), 1.32 (d, J=6.4 Hz, 3H). LCMS: (Method A) 362.2 (M+H), Rt. 2.1 min, 95.2% (Max). HPLC: (Method A) Rt. 2.2 min, 97.1% (Max).

Example 21: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(pyrimidin-2-ylmethyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(197) ##STR00309##

Step 1: 1-(pyrimidin-2-ylmethyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(198) ##STR00310##

(199) To a stirred solution of tert-butyl 2-(pyrimidin-2-ylmethyl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (Isomer 2 obtained in Example 20, step 2, 400 mg, 26.66 mmol, isomer 2) in dry 1, 4-dioxane (10 mL) at 0° C., HCl-dioxane (13.33 mL, 4M) was added dropwise and the reaction mixture was stirred at RT for 3 h. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum. The resulting crude material was triturated with EtOAc, hexane and diethyl ether to afford the title compound which was used in the next step as such without any further purification. Yield: 75% (420 mg, pale yellow solid). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.28 (s, 2H), 8.79 (t, J=5.6 Hz, 1H), 7.48-7.37 (m, 1H), 5.51-5.27 (m, 5H), 4.13 (s, 1H), 3.58-3.57 (m, 2H), 2.97 (d, J=6.4 Hz, 1H). LCMS: (Method A), 216.0 (M+H), Rt. 0.6 min, 97.6% (Max).

Step 2: 5-(1-(2,3-dihydrobenzofuran-6-yl)ethyl)-1-(pyrimidin-2-ylmethyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine

(200) ##STR00311##

(201) To a stirred solution of 1-(pyrimidin-2-ylmethyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine (150 mg, 0.69 mmol) in MeCN (10 mL), intermediate 2 (253 mg, 1.39 mmol) followed by TEA (0.4 mL, 2.79 mmol) were added and the reaction mixture was heated at 60° C. overnight. Completion of the reaction was monitored by TLC, then the reaction mixture was evaporated under vacuum. To the resulting mixture, water (10 mL) was added and the aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine solution (10 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude material was purified by Prep HPLC (Method B) to afford the title compound. Yield: 5% (16.4 mg, light brown gummy oil). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.77 (d, J=5.2 Hz, 2H), 7.43 (t, J=4.8 Hz, 1H), 7.16-7.7.14 (m, 2H), 6.79 (dd, J=7.2, 1.2 Hz, 1H), 6.73 (s, 1H), 5.38 (s, 2H), 4.51 (t, J=8.8 Hz, 2H), 3.56-3.52 (q, J=6.8 Hz, 3H), 3.14 (t, J=8.8 Hz, 2H), 2.60-2.57 (m, 1H), 2.56-2.52 (m, 3H), 1.32 (d, J=6.8 Hz, 3H). LCMS: (Method A) 362.2 (M+H), Rt. 2.1 min, 98.2% (Max). HPLC: (Method A) Rt. 2.2 min, 97.9% (Max).

Example B01: Human O-GlcNAcase Enzyme Inhibition Assay

(202) 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λ.sub.exc 485 nm; (λ.sub.emm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC.sub.50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).

Example B02: Pharmacodynamic Model: Total Protein O-GlcNAcylation Immunoassay (RL2 mAb, Meso Scale Electrochemiluminescence (ECL) Assay)

(203) The test compound was administered orally to C57BL/6J mice. At defined time intervals after compound administration, typically a time ranging between 2 and 48 hours, preferably between 4 and 24 hours, mice were sacrificed by decapitation for blood collection and forebrain dissection. Right brain hemispheres were placed in 2 ml Precellys tubes, snap frozen in dry ice and stored at −80° C. Left hemispheres were placed in 2 ml Eppendorf tubes, snap frozen in dry ice and stored at −80° C. until further processing. Blood samples were collected in Sarstedt tubes containing 35 IU of Heparin and kept at 4° C. After centrifugation for 10 min at 3800×g, 4° C., 50 μL of plasma from each sample was transferred to a 1.5 ml Eppendorf tube and stored at −80° C.

(204) For the preparation of soluble brain protein for the immunoassay the hemispheres were homogenized in ice-cold Cytobuster reagent (71009-Merck Millipore) buffer with protease inhibitor cocktail. After centrifugation for 15 min at 17000×g at 4° C. the supernatants were transferred into polycarbonate tubes (1 ml). The supernatants were cleared by centrifugation for 1 h. at 100000×g, 4° C., and the protein concentrations were determined by using the BCA kit (23227-Pierce, Rockford, Ill.) according to the manufacturer's instructions.

(205) Total Protein O-GlcNAcylation Immunoassay:

(206) Samples were randomised and 120 μg/ml (25 μl/well) of soluble brain protein was directly coated on a Multi-array 96-well high bind plate (L15XB-3 High bind—Meso Scale Discovery) overnight at 4° C. After washing (3× with PBS-T buffer), the plate was blocked with MSD blocker A solution for 1 h. at room temperature (RT) under agitation. After washing (3× with PBS-T buffer), the plate was incubated with 0.1 μg/ml of a mouse monoclonal antibody directed against O-GlcNAc moieties (RL2; MA1-072-Thermo Scientific) for 1 h. at RT under agitation. For the ECL assay, after washing (3× with PBS-T buffer), 1 μg/ml of a SULFO-TAG™ labeled anti-mouse secondary antibody (Meso Scale Discovery) was added and the plate was incubated for 1 h. at RT under agitation and protected from light. After washing (3× with PBS-T buffer), 150 μl/well of 1× Read Buffer T was added to the plates before reading on a Sector Imager 6000 (Meso Scale Discovery).

Example B03: Pharmaceutical Preparations

(207) (A) Injection vials: A solution of 100 g of an active ingredient according to the invention and 5 g of disodium hydrogen phosphate in 3 l of bi-distilled water was adjusted to pH 6.5 using 2 N hydrochloric acid, sterile filtered, transferred into injection vials, lyophilized under sterile conditions and sealed under sterile conditions. Each injection vial contained 5 mg of active ingredient.

(208) (B) Suppositories: A mixture of 20 g of an active ingredient according to the invention was melted with 100 g of soy lecithin and 1400 g of cocoa butter, poured into moulds and allowed to cool. Each suppository contained 20 mg of active ingredient.

(209) (C) Solution: A solution was prepared from 1 g of an active ingredient according to the invention, 9.38 g of NaH.sub.2PO.sub.4.2H.sub.2O, 28.48 g of Na.sub.2HPO.sub.4.12H.sub.2O and 0.1 g of benzalkonium chloride in 940 ml of bi-distilled water. The pH was adjusted to 6.8, and the solution was made up to 1 l and sterilized by irradiation. This solution could be used in the form of eye drops.

(210) (D) Ointment: 500 mg of an active ingredient according to the invention were mixed with 99.5 g of Vaseline under aseptic conditions.

(211) (E) Tablets: A mixture of 1 kg of an active ingredient according to the invention, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate was pressed to give tablets in a conventional manner in such a way that each tablet contained 10 mg of active ingredient.

(212) (F) Coated tablets: Tablets were pressed analogously to EXAMPLE E and subsequently coated in a conventional manner with a coating of sucrose, potato starch, talc, tragacanth and dye.

(213) (G) Capsules: 2 kg of an active ingredient according to the invention were introduced into hard gelatin capsules in a conventional manner in such a way that each capsule contained 20 mg of the active ingredient.

(214) (H) Ampoules: A solution of 1 kg of an active ingredient according to the invention in 60 l of bi-distilled water was sterile filtered, transferred into ampoules, lyophilized under sterile conditions and sealed under sterile conditions. Each ampoule contained 10 mg of active ingredient.

(215) (I) Inhalation spray: 14 g of an active ingredient according to the invention were dissolved in 10 l of isotonic NaCl solution, and the solution was transferred into commercially available spray containers with a pump mechanism. The solution could be sprayed into the mouth or nose. One spray shot (about 0.1 ml) corresponded to a dose of about 0.14 mg.

Example B04: Protein Binding in Mice Plasma Using Rapid Equilibrium Dialysis

(216) Materials

(217) CD1 Mice Plasma: pooled male, K2-EDTA (MSEPLEDTA2, Bioreclammation, USA Phosphate Buffered Saline (1×PBS), pH 7.4, 100 mM (Sigma, Cat No. P4417) RED inserts (Pierce, Cat No. 9006, 8 kDa MWCO) Sample Analysis: LC-MS/MS
Methods

(218) Preparation of DMSO Stock Solution

(219) From 20 mM DMSO stock solutions of reference and test compounds, 1 mM DMSO intermediate working solutions are prepared. From 1 mM intermediate working solutions, 100 μM DMSO working solutions are prepared.

(220) Sample Preparation Procedure:

(221) Selected plasma is brought from −20° C. to 37° C. using water bath before its use. Test solution is prepared by adding the DMSO working solution of the reference or test compound (2 μL; 100 μM) to the selected plasma (198 μL). Spiked plasma (200 μl) is transferred to sample compartment of RED insert placed in the teflon plate. 350 μl of 1×PBS is added in the buffer compartment of RED insert. The teflon plate is covered with sealing mat and agitated at 37° C. for 5 hours at 500 RPM in a Thermomixer. After incubation time, an aliquot of plasma (50 μl) from sample compartment is mixed with blank 1×PBS (50 μl). Similarly, an aliquot of buffer (50 μl) from buffer compartment is mixed with blank plasma (50 μl). Quenching solution (200 μL, acetonitrile containing internal standard tolbutamide (0.5 μg/mL)) is added and the resulting solutions are mixed using a vortex mixer and centrifuged (Eppendorf 5415, 13792 g). Supernatants are analyzed using a Mass Spectrometer. The sample (supernatant fraction, 5 μL) is injected into the LC-MS/MS instrument.

(222) Chromatographic Conditions: LC-MS/MS: API 4000 LC-MS/MS Software: Analyst Version 1.6.1 Column Phenomenex Synergy 30*4.6*5μ Column Oven: 40° C. Mode: ESI Positive Injection volume: 5 μl Flow Rate: 1000 μL/mL Buffer: 0.1% Formic acid in Water Method: Isocratic Method/Gradient Composition: A) 0.1% Formic acid in Water B) 0.1% Formic acid in Methanol

(223) TABLE-US-00003 Mobile Mobile Time (Sec) Flow (μL) Phase A Phase B 0.01 1000 10 90 0.4 1000 10 90 0.8 1000 90 10 1.5 1000 90 10 1.8 1000 10 90 2.5 1000 10 90
Results Calculation

(224) After the concentration of free drug and total drug has been determined by LCMS/MS, percent plasma protein binding can be calculated as follows:

(225) % ⁢ ⁢ fraction ⁢ ⁢ unbound = Drug ⁢ ⁢ concentration ⁢ ⁢ in ⁢ ⁢ buffer ⁢ ⁢ after ⁢ ⁢ 5 ⁢ ⁢ hours Drug ⁢ ⁢ concentration ⁢ ⁢ in ⁢ ⁢ plasma ⁢ ⁢ after ⁢ ⁢ 5 ⁢ ⁢ hours × 100

(226) Following this protocol, % fraction unbound in plasma from different species can be also measured.

Example B05: Determination of In Vitro Intrinsic Clearance (Cl.SUB.int.-In Vitro) with Mouse, Rat and Human Liver Microsomes

(227) In this assay, test compounds are incubated with liver microsomes from mouse, rat and human, and rate of disappearance of drug is determined using LC-MS/MS. Conditions used in the assay are summarized below:

(228) Materials

(229) CD-1 Mice liver microsomes, pooled male (Life Technologies, Cat No. MSMC-PL) (20 mg/ml) SD Rat liver microsomes, pooled male (Life Technologies, Cat No. RTMCL-PL) (20 mg/ml) Human liver microsomes, pooled mixed gender (Life Technologies, Cat No. HMMC-PL) (20 mg/ml) NADPH (SRL Mumbai, Cat No. 99197) Verapamil (Sigma, Cat No. V4629) Atenolol (Sigma, Cat No. A7655) Tolbutamide (Sigma Cat. No. T0891) Assay buffer: 50 mM potassium phosphate buffer, pH 7.4 Test & reference compounds: DMSO stock solutions (10 mM concentration) are prepared and stored at room temperature. An intermediate 1 mM solution of test or reference compounds is prepared by mixing 10 μL of 10 mM DMSO stock with 90 μL of DMSO. The contents are mixed vigorously in a vortex mixer.
Methods

(230) Preparation of Working Solutions of Test and Reference Compounds:

(231) Working solution (100 μM concentration) is prepared by mixing 10 μL of 1 mM DMSO solution of test or reference compounds with 90 μL of assay buffer. The mixture is mixed vigorously in a vortex mixer. This resulting solution is containing 10% of DMSO. For the metabolic stability assay, 10 μL of this 100 μM working solution is added to a final assay volume of 1 mL, yielding final test concentration of 1 μM and DMSO concentration of 0.1%.

(232) Metabolic Stability Assay

(233) Metabolic stability assay is done in a final volume of 1 ml in 50 mM assay buffer, potassium phosphate buffer, pH 7.4. Assay is carried out in duplicates (n=2). A mixture containing 955 μL of assay buffer, 25 μL of liver microsomes and 10 μL of 100 μM test compound solution is pre-incubated for 10 minutes in a water-bath maintained at 37° C. After pre-incubation, reaction is started by adding 10 μL of 100 mM NADPH solution. The solution is mixed and incubated at 37° C. in a water-bath. The final concentration of the different components in the assay is: DMSO 0.1%, test compound 1 μM, liver microsome protein 0.5 mg/ml and NADPH 1 mM.

(234) Aliquots (100 μL) are taken at various time-points (0, 5, 15, 30 and 45 minutes) and quenched with 100 μL of acetonitrile containing tolbutamide (500 ng/mL) as internal standard. Samples are mixed using a vortex mixer and centrifuged at 4000 rpm for 10 minutes (Eppendorf 5810R, 3000 g). The supernatants (5 μL) are transferred to 96 well plates and submitted for LC-MS/MS analysis.

(235) Separate incubations in the same assay mixture, but in the absence of NADPH, are run in parallel as control for compound stability. This control assay is carried out in duplicates (n=2). After pre-incubation, addition of NADPH is omitted and replaced with 10 μL of assay buffer. The final assay volume is 1 mL and aliquots (100 μL) are withdrawn and processed for analysis as described for metabolic stability assay.

(236) LC-MS/MS Conditions (Generic Method) LC-MS/MS: API Sciex 4000 with Nexera™ UHPLC Software: Analyst Version 1.6.1 Column: Phenomenex kinetex C18 50×3.0 mm, 2.6μ Column Oven: 40° C. Mode: ESI Positive Injection volume: 5 μl Flow Rate: 1000 μL/mL Buffer: 0.1% Formic acid in Water Method: Isocratic Method/Gradient Composition: A) 0.1% Formic acid in Water B) 0.1% Formic acid in Methanol

(237) TABLE-US-00004 Mobile Mobile Time (Sec) Flow (μL) Phase A Phase B 0.01 1000 10 90 0.4 1000 10 90 1 1000 90 10 1.5 1000 90 10 1.8 1000 10 90 3 1000 10 90
Results Calculation

(238) From LC-MS/MS data, amount of drug remaining at different time points was determined (% PCR). The logarithm of % PCR was plotted against time to get the slope value. From the slope value, in vitro T.sub.1/2 was determined. In vitro intrinsic clearance (Cl.sub.int) was calculated using the following formulae:

(239) CL int = 0.693 In ⁢ ⁢ vitro ⁢ ⁢ t 1 / 2 × Volume ⁢ ⁢ of ⁢ ⁢ incubation mg ⁢ ⁢ of ⁢ ⁢ microsomal ⁢ ⁢ protein In ⁢ ⁢ vitro ⁢ ⁢ t 1 / 2 = 0.693 K el

(240) Where K.sub.el is Elimination Constant (Slope)

(241) Methods for treating the diseases mentioned in this specification, such as tauopathy, by administering one or more of the compounds of the present invention to a patient in need thereof are also object of this invention.

(242) If chemical bonds in the structures above are drawn as follows: custom character or custom character
they indicate a defined, i.e. R or S, stereochemistry at at least one of the atoms to which they are attached to.

(243) This is exemplified below, wherein the structure

(244) ##STR00312##
is representing only one of the two possible enantiomers,

(245) ##STR00313##
i.e. a single individual chemical structure as opposed to a mixture of enantiomers.