EA040488B1 - Benzodiazepine derivatives, compositions and methods of treatment of cognitive impairment - Google Patents

Description

Government Support Statement

This invention was made with government support under grants No. U01 AG041140 and No. UH2NS101856 awarded by the National Institutes of Health (NIH) and, in particular, its division, the National Institute on Aging (NIA), a government agency of the United States of America. The government of the United States of America has certain rights to the invention.

Related Applications

This application claims the benefit and priority of U.S. Provisional Application 62/436,272, filed December 19, 2016, and PCT/US2017/67448, filed December 19, 2017, both of which are incorporated herein by reference in in full.

The field of technology to which the invention belongs

The invention relates to compounds, compositions and methods for the treatment of brain cancer or cognitive impairment associated with brain cancer in a subject in need of such treatment.

State of the art

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

For example, a significant population of older people experience cognitive decline that is greater than that of the normal aging process. This age-related cognitive loss is clinically characterized by a progressive loss of memory, cognition, thinking, and judgment. Mild Cognitive Impairment (MCI), Age-Related Memory Impairment (AML), Age-Related Cognitive Decline (ADFR), or similar clinical groupings are among those associated with such age-related cognitive loss. According to some estimates, there are more than 16 million people with GNP in the US alone (Barker et al., 1995), and MCI is estimated to affect 5.5–7 million in the US over the age of 65 (Plassman et al., 2008). ).

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

Therefore, there is a need for effective treatment of cognitive impairment associated with disorders of the central nervous system (CNS) and improvement of cognitive function in patients diagnosed with, for example, age-related cognitive impairment, MCI, amnesic MCI, GNP, GRFR, dementia, AD , prodromal period of AD, PTSD, schizophrenia or bipolar disorder (particularly mania), amyotrophic lateral sclerosis, cancer therapy-related cognitive impairment, mental retardation, Parkinson's disease, autism spectrum disorders, fragile X syndrome, Rett syndrome, compulsive behavior , drug dependence and similar disorders of the central nervous system with cognitive impairment or with the risk of their development.

GABAA receptors (GABAA-P) are pentameric complexes from a set of different subunits (α1-6, (β1-3, γ1-3, δ, ε, π, θ) that form a Cl-permeable channel that is controlled by the neurotransmitter γ -aminobutyric acid (GABA) Various pharmacological effects, including anxiety disorders, epilepsy, insomnia, pre-anesthetic sedation and muscle relaxation, are mediated by different GABA subtypes.

Various studies have shown that reduced GABA signaling is associated with various CNS disorders with cognitive impairment. In particular, a5-containing GABA-P, which are relatively rare in the mammalian brain, play a role in the modification of learning and memory. Previous studies have demonstrated a decrease in the expression in the hippocampus of the α5 subunit of the GABA receptor in rats with age-related cognitive decline (see international patent publication WO 2007/019312). These results suggest that activation of a5-containing GABA-P function may be effective in the treatment of cognitive impairment associated with these CNS disorders.

Thus, there is a need for positive allosteric modulators of a5-containing GABAA-P that can be used in therapeutic formulations for the treatment of cognitive impairment associated with these disorders of the CNS.

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The essence of the invention

The present invention satisfies the above need by providing the following compounds comprising a 1,5-benzodiazepine skeleton and a 1,2,3-triazole ring:

Compound Structure 344 § CD About m 356 III O 357 o b Hm /=\ S 358 o b 359 360 361 362 363 364 365 C Μ $ 0 ^s >& ν X^ _n V Xgm X ^ζ αΎ> QQQ « Q 4 Q ° ° o o 366 CD About δ X-J

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367 2 CD O 0 F^ FF n 368 2 CD O 0 F^ ^F ^z -y^%V z^p 369 § CD O 0 0 370 2 CD O 0 F^ FF 371 s CD O 0 F ^z ^F ^Z ^F^4/ ^Z ζ^θ 0 q 372 S CD O 0 F ^{z Z} F ^z \^/0/ ^z 9 373 2 CD O 0 F^ FF ^Z \^\^y ^Z z ^z o 0 374 O z / \ o zF/^^Fz 0-Z z-z 0 About 2 375 2 CD O 0 F^ ^Ζ ν^\^γ ^Ζ ^YJ 0 -Π q 382 O 0 F^ ^z \iA^Ay ^z z^ Q 383 2 CD O 0 F^ ^Z ^A^0y ^z 385 o 0 F^ 0F ^z \^\^0y ^z 99 386 O J0 <XX^ q 387 NO^i pr^V ^N 'N

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388 MeO' D ^N ' N N 389 J MeO X , ^χγ 390 J MeO o ^Ο Χ^ ^Ζ zπ ^Νν 391 Ji MeO^ / 01^^^=^01 ΝΝ 392 ji MeO^ C JV _F UL V 393 ( ^{:ι Ν} ν 394 MeO r/f- Ν - / ^ν - _Ν 395 Cl I 396 SG I 397 Jf Cl^ ^ ^Ν χ ΛΛ / ο-^ο° ^yj ^N V 398 or nO^Vf ¹ D ^N 'N ^F \ 399 Q-^O sg ^N'N 400 jf Cl'^ M about 401 cK 4- ^N'N

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402 b 403 CD About i 404 CD ABOUT 405 Q V about CD 2 406 2 CD O .k o 407 2 CD OK 408 2 CD O A 409 sA, _ /=\ J! 1/ome M ν 410 2 CD About Λ π 411 CD OK ίΑ 412 Λ - W ^osg “ MeO^^N/ ⁿ 'n 413 CD ABOUT i and 414 CD ABOUT Y .... O CD 415 h i

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442 b O 443 °M 448 O b i and ° 449 2 CD Oh α ^Ϊ! 450 P ba iiiYW t-Z Z-z b o CD 2 451 CD About b SH 452 m ζΥγΥ Ύζ Z-Z b O 453 Us ¹ 454 y) ^ζ Υ\Υγ ^ζ O)

or their pharmaceutically acceptable salts, enantiomers, diastereoisomers, Z-isomers, E-isomers or combinations thereof, as well as the following compounds containing a 1,4-benzodiazepine skeleton and a 1,2,4-triazole ring:

314 A-L 1J / ο _χ 315 ABOUT Ζν 7 Α Ζ Υ -*\ 316 χΛ ο 317 ·. :. ' Ο

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319 xD o 320 γ o 321 o ΧΪΧ α^γ ^Ν o I 322 o 323 o f /GL J ^F ^.N-% ^c| o^ ^N -N 324 O

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325 γ o 326 ml N-Y 327 γΥ ^with ' Υ/ΥΥ 328 ιγ Υ ο 2 330 ο 331 CY ΥΧ Ϋ ο 332 γ 333 ΥΥΪ^θ jX> Cl 334 335 A/-Π -π 336 Ϋ 337 /^V ^CF3 CI'^Y'N θ _χ 338 % Qω

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339 XT _Οχ ⁿ -n 340 MeO^^A--N 341 CI^^A^n o^/ 342 „oA Cl \ /°^ ^CF3 At 343 CT £ Ύ ) MecA^yN_ 345 χ/A Cl^^yN o _x NV ⁷ 346 HPb 348 . \ ^: 349 £? X ό 350 A-a3 LAV c ^|X ^£ta ^ν Άο^ b 351 vxb 352 ^Aci ^ALAo. b 353 )1 Λ x)

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354 „A/ ^s n />—\ /=\ Af F 355 m N—, AA AA ^with n />—\ /=\ F 384 A-P . N '^A II /Y—. ^Ν ^M \ ^N OMe 421 G'A-l ΓΎ ^Ν ) O ^{F С|} __/OMe 422 ^Γ 'Ή% ΓΥ ^Ν ) SU ^{with |} CI^AA\^.n_/OMe 423 jAA ) /%-° ^CF ₃ οι ^^^ν^νομθ 429 Ab^ __/OMe 430 ayy Cl__OMe 431 aTa Cl^^^yN OMe ⁿ -n 432 ^AAxr __/OMe 433 d °L ? X Ύ ) C|AA\^n_ NA? \ ^Nome 434 ^Ya /4 bA n X Ύ ) ^c| ^AA^-n_NA? \Nome 435 ^N ' ^N ^OMe

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444 O-\ jTjf 'eV ^cl Cl^^yN 4=/ ^ΝOMe 445 θ ¹ ΛΑ / ci^^yN yy ^ΝOMe 446 AuCh) n / _r, 1 447 sh p, I 455 dti cl Ν__mind ⁿ 'n 458 hum 460 -N Pi O^^y-- N o— F^F ^N 'N ^Z 464 % g%L CQ 'U 466 9' rumble 00 ^N 467 Ν θ~-Ί r Uch 1 Jo > °^^/T ^N \ /°~~ _f 0f ^N 'N ^Z 470 ^y<l N y F^i I > fV^T^ ^{0 FN} -N 471 N O-ι fOO^ ⁰ -

or their pharmaceutically acceptable salts, enantiomers, diastereoisomers, Z-isomers, E-isomers, or combinations thereof.

In another aspect of the invention, there is provided a pharmaceutical composition comprising the aforementioned compound or a pharmaceutically acceptable salt, enantiomer, diastereomer, Z-isomer, E-isomer or combination thereof in a therapeutically effective amount and an acceptable carrier, adjuvant or excipient.

In some embodiments, said composition further comprises a second therapeutic agent.

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In another aspect of the invention, there is provided a method of treating a CNS-related cognitive impairment in a subject in need of such treatment, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, Z-isomer, E-isomer or a combination thereof or a pharmaceutical composition containing a compound of the invention. In some embodiments, a CNS disorder with cognitive impairment includes, but is not limited to, age-related cognitive impairment, mild cognitive impairment (MCI), amnestic MCI (aMCI), dementia, Alzheimer's disease (AD), post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis (ALS), cancer therapy-associated cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorders, compulsive behavior, and drug addiction.

In another aspect of the invention, there is provided a method for treating brain cancer or a brain cancer-related cognitive impairment in a subject in need of such treatment, comprising the step of administering a compound or a pharmaceutical composition comprising a compound selected from the following compounds:

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4 I^N-COsEt ΛΑ / O H ₃ CO N\<y— 5 i y-co ₂ Et <P 6 \ CO ₂ Et XQ 0 ^Nv 9 /A* H-CO^-^'N^ d 10 H ₃ CO' ^Z ^ ^ii>4 'N'A ^ 0 F eleven r > —CO ₂ Et ΠΏ HsCO'^^N-^i

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12 I “oh oh s? “ 44 w g p o P-A O j _z Aj-^-vA _z XZ Zz X LL 45 ^“DS! /CO o ^G X 46 LL w H o ChJ L-z z-z X LL 47 O,,-, sh | i) About o I 'Lz Z-z Q 48 ίΎ^Τ n MeO-^^^N'V ^N ~ 49 -- ^N _n Q-co ₂ e, 03 o N- / ^ZN 'N 50 6- ^c ° _g E. ⁿ -n

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51 Г y-CO ₂ Et χΡΡ P MeO-^^^^Np nP "P 52 ,^Ν Г >~CO ₂ Et a/ \ /-X jpQ (0 MeO " N3\ 53 s Ji ‘Ϋ~^Υ 9 * About 2 54 ъ lx V ^s ) aJ i 107 . 9 about about. ° to ζΡ-^Ρζ %Ζ Ζ-ζ Q and. 108 r > —CO ₂ Et ίΎ ) /A MeO'^^-^'^'Np Pl 109 “Π β 0 -μ 110 Ρ-Ν .rrgA Ρ Ν- / 'Ν

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111 f,)-CO ₂ Et xe j ^N Y_ ^N V X-, <TA N=/ 112 Ya' ^1: Y 113 O) °t ^F ^> 4 ^N V 114 YZYf KI z'N 115 z ABOUT , Ύζ z-z 116 1Ί (7 1 about z 117 <\l X °γ ⁷ 121 qT ri ^Nv

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139 b ό 140 About zSfA V-Z Z-z Ll 143 0 F0 fF X° XT° 144 Λ ^Z F z^VFF Fz zF 0 LL 145 0 ^ζ γΑ^Α^ ^ζ V ^z 0° 146 αΓ 149 /F° ^f jl ^N N 150 0F ^? o J ¹

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168 πί ^Meo y_/°~O ^Ν 'Ν \ _f 170 .Ν θ-Ν 3 //—3 JL ABOUT/ \ Ν Ν—. 180 %ο γΤ Y ^o Y _F ^ν 'ν 181 χχχ ηΧΧ “ Y°-Q ^Ν - Υ 182 _me ° kr°x ⁿ 'n 194 ^.Ν __, ^Ν -0 03 η MeO ^ν Ό ΟΟ 195 /% ⁰ ηΧ ^Ν ^ Ν yJCX >4^ 207 Γ y-CN ίΎ ) ΛΛ ΜβΟ' ^χ ^^ ^/4 'Νγ^ /

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208 ΓCN ΓΎ / SU Cl 209 ,: ^N jQ J €% Meo - ^; _N y j- ^N 'N 210 S F O L Sn ² ~Σ 211 GU "ΓΎ ) 212 ΓΎ ) i ^N ff 213 Г* A ^вг Λ A / 1 >---4 ^N W yX 214 SU» ΓΎ ) 1 >----X ^N *Ss / -X ^N /Uxx

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218 r ^N y-= al / O L/ V Ν ' ₀ 219 About 220 2 δ ft and ^to 221 / £l ) O A 222 fAcn OE /^ N - 7 ⁷ R-;/ N 223 _-N G CN _M . ₀ Q^^.°^ 224 G y- ^CN MeO-^^ ⁴ N ^N 'N 225 Г 1-CN ...χ.... ^Ν - ^χ Il Ί > ^{Br N} 'N 226 ft .-CN C' : .-, ^α rL°O ^N V

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227 Г 3 - CN χΑ ^α α°Ό 228 -N r /A ^CN Να £CA ^Ν 'Ν 229 - Cl m η Vz Z-z 1 230 ^F aa ^N IT ) ¹ o F 231 r Vcl, ^Ν νλ о F 247 ^-N A ^Br Ν*κί\ ^N NH / 248 A ^{Br Me0} nA° ^N 0

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250 y™ ^N V 251 F o bb o za ^z 252 .N oTc MeO N—/ 253 About Μβθ^Λ _Ν -ΧΟ 257 Ρ'Ν Μβο-^ΥΥ-Χ Ύ 258 2 (0 Ο Y Λ ^? λ ^Ζ 262 Γ 1-CN ..CO Μθ° nXaS) ^Νί Ν 272 ΥαΙ. 273 Ck°T ίΎ ) MeO'^-^N Ά_/=\ 'VW

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277 oh oh 279 g φ o JI z^A^^z o 1 1 Г ι) O 1 II ^m OJ D' 289 s δ J Jd 290 .cd ⁿ MeO n4 Ό 291 OC^ T^^dd ² VZ ZZ J o F s 292 <HD. ΧΪ ) MeO'^^ ⁴ 'ndd X ^M N 298 PT Lc\X 0—N- / ' ^N N 299 . Y'Ts: । .1-./ N ^ 300 O b cOd y ^z dy

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or its pharmaceutically acceptable salt, enantiomer, diastereoisomer, Z-isomer, E-isomer, or combinations thereof.

In another aspect of the invention, there is provided a method of treating a brain cancer or brain cancer-related cognitive impairment in a subject in need of such treatment, comprising the step of administering a compound or a pharmaceutical composition containing a compound, wherein the compound is selected from:

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Compound Structure X 55 MeO^ at L ^coge ' 56 MeO^ L L ^O CHL 103 V F—CO ₂ Et GL MeSG N Y \ N o- G Y-s° ₂ in .nJ 104 MeO ^x g(l Ut G / U-co ₂ Et . kk 105 X MeO^ Oh 0 oh 106 r /> - ^C0 2Et Meo U/Lvg ⁱⁿ N 118 J MeSG h 0 Χ-Λ -y 119 J MesL 120 £ l rL Ύ U MesL ϊ^</ 122 No. MesL Luu°L 123 JI MeSG l o ό 124 JI MeSG \ ^F l νΌ ^N 'N^ 128 P-N ^/A <\ Λ z^nJ MeSG __/ ^0Me

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129 mg MeO ^^ y-N /OMe 130 Ο-Ν _ oMe 131 A OME 137 Г 0 —-CO ₂ Et jCO F^^y_N_/OPh 138 ^Ν _χ QN ^^nAnA ΛΑ / ___/° ¹ ° ¹¹ 141 O'-N Au-O ⁿ 'N 142 ΛΑ 0 ^N A ,n-/ jTY ) MeO^^VN Ad ⁿ 'N 147 // ^N '° A Ά U 148 . ^N 'O ,^νΑνΑ X j ) ^F /)_/°X0 N ⁷ ' % /7 ⁱⁿ 'N 156 _η ψ°4 F __/ ^OPh NV ⁷ 157 A-cn n 5 F __OPh NV ⁷ 158 .N N'O F__/OPh 159 ^N __^ ^Ν 'Ο X%>/ ^N l ⁿ ^cf ₃ AA / ^F '^]f' ^N \__/ ^OPh 162 ^N N~O jo/A MeO ^^y-N OPh

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163 Λ41 N'b ΓΥ > MeO^^^y-N O-Ai NV /7-F 164 "OU MeO^^ \yN ©Me 165 _λ ^Ν Ό _X NY N ^ Y / CO and MeO ⁴ / VN ^N -N 166 ΑΓζ MeO^^yy 0-^7^ ^N A 169 /Y° xX> / MeO^^y-y_y Me 171 N~O χχΓ ^Ύ MeST^y-y_yPh 172 λ ^Ν Ό Af^Y MeO^^y-y_yy^L ^N 'N ^f 173 N-0 ^.TA'U' MeO^^y-NOEt 174 // ^N ~O .Oj ' ^Y MeO^^y^y_y-_/=y N ^/ V /7 ^N 175 // ^N 'O γγ ^N 0^ ^N ^Υ N Ά /7 N Cl 176 // ^N O .a5 ' ^Y MeO^^yN O^/^y ^N N n 177 // ^N '° „iyAy^ MeO^^YN ⁿ 'N /A w 178 N-0 ,α/ ^Ύ MeO^^yyN ^N \i \ /А N ο—ζ J

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179 Meck ... NO- ^7V /F L.N-# N° vv 183 MeO^ rd vd ^F 184 MeO ^x To II A Ύ°7 185 X F Wow /° C / ⁼ \ / ° W -( ^N to N° 186 d to ^Nk Nγ ome . ABOUT' A° ΛΑζ 187 d to ^Nv 1 OEt 188 X F uu ?=\ Υ'Ύ N-O u <v 189 d Xdk ^Ν ν c N° 190 d with ^N ^Γ -k ^N .to-Z -CN 191 Meo^ >-CN 192 MeO' To H-CN 193 MeO ^x cd c •k -CN 196 MeO ^x θνν b -CN 197 ci^ c ^Nk _^O^z^

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198 HuA Λ ή-D ΓΎ ) CI 199 j Οχ ο ο 200 Ρχχο 201 /Ν Ν'Ο Ο ^r 1 202 ιχΓ CI ^^γ-Ν_ ^ν Ό and 203 Α ^σ XX D ο S 204 Ρ ΧΑ. Ο 205 /Ν ΟΓ ΟΙ^^γ-Ν ^Ν 'ν TJ 206 ΡΝ ^with ι CH /°\ 215 .Ν-Ο jQf ) С1 ''^''ΐί 4__/°\ 216 Ο 217 /Ν /DD 232 Ο ΜθΟ^^γ-Ν_ ^Ν 'ν U 233 L-dc pfO N-ho

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234 YYv Cl 235 χΥ MeO^^-^V'N c< k__/ >ι Ν ^Ν Ύ U 236 γν H3CO^^^y χϊ U 237 _P\ λ IJ / CI -^Υ^Υ^Ν_ 238 ΥκΥ /χΥΥ ГГ CI^TYX^-n 239 ί>Υ Ci __ ^Ν ^ί 240 .Ν γΥ Ν-κιΥ Ν ο— 241 ΥΓ ^•^YrYV^ ^Ν 'Ν Μ 242 b x/ ο ο ^ζ 243 Ργ ίΥΥΧ ^cl ^^iT ^N \_Λ 244 M, FY /° 245 ο Ν γ/' ΟΙ^^Υ-Ν ο^ ^Ν '|/ ^/ 246 χΥ ⁰¹ __/°\

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249 A-A /AC n ίΎ ) ^c| 254 b 255 A : :. ABOUT 256 V5 X about 259 Abb Cl^y _N 260 Ab CI^AA^n 261 x/T ci^bAy, _N o^/ 263 Cl _d ^N A 264 αΥαΊ ψ. .Q Cl ^''y ^N >_ 265 φν..ΰ ^C| __Px 266 αα ^{ό C|} __nN- ^A 267 Ρ-η I clxx C|^%AXy. _{N 0} \ 268 D ΐ X JA Aa ABOUT 270 χς^Ό ^Cl ΑΑΥ 271 Ab ^N ' N ^/

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274 b 275 ρ O t ci 276 q O 278 611 N~# jTY > ^Cl_P ^ 280 F /°0 1 r>-v X 1 ί J > ^N F 281 P~N ίΎ > CI^FX^n o^/ N'N^ T 282 £ D ^F° n-F 283 0H1 JO > ^c|/ FXp_N o^ ^N O 284 py x4z ^N £ Ύ ) ^{1 n} O 285 O 286 oCC C T 287 ^ ⁿ /°x ^pFA , ίΎ > ^c| ^^p_N q, ^N OI 288 hT ^C| ^\-NQ, ^N 'N 1 293 IP, V O 294 ^N Op QpP II/ ^C| '^^]p ^N \ _y °\ o

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295 ΧΎ ) ci __/°\ 296 mr °C ' O 297 Οη γυ / Cl __o ₄ 301 N'N^ 302 MM Pp-N°\ 303 P'N N-M pr / Cl N_P^F ₃ 304 (A-<~1 Pf-p 0 Cr γ ^Ν ,__/O^CH3 ^N 'N^ 305 >>= lYf ^N 'N^ I 306 xt; ⁿ m 1 307 - /=\ [Pf ) F ^N 'N A-CO ₂ E, 308 XX > ^C| _y^SRz 309 Cl N o^CFs 310 _ )=\ ΓΎ ) ^N 'NI 311 -X\jlI> _// ^Np 312 lX> ^C| _y^CFa ^N 'N^

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313 0 Ύ Ύ ) _/O^ CP3 ^Ν 'γΓ 329 оС γ O 427 °х 5 χχ V O 428 ^n< ^CF ₃ ίΓ Ύ ) Cl __ OMe 456 £ XX ' Χ ^ζ 457 / XX γ ο 459 / XX 5 461 / XX V ώ 462 __Ρ'Ν £ Ύ ) M 463 / χχ 465 X ΥΧ % Ζ^ο ' Χ ^ζ 468 / χχ 469 .Ν Ο' _Ν ΟΛΑ Ν 4 Ο—

or its pharmaceutically acceptable salt, enantiomer, diastereoisomer, Z-isomer, E-isomer, or combinations thereof.

Detailed description of the drawings

In FIG. 1 is a graph showing the effect of methyl 3,5-diphenylpyridazine-4-carboxylate administration on spatial memory retention in ten aging-impaired (AD) rats in the Radial Eight-Arm Maze (REL) test. Black bars refer to vehicle-only treated rats; white bars refer to rats treated with methyl 3,5diphenylpyridazine-4-carboxylate at various doses; the shaded bar refers to rats treated with the combination of TB21007 and methyl 3,5-diphenylpyridazine-4-carboxylate.

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In FIG. 2 is a graph showing the effect of methyl 3,5-diphenylpyridazine-4carboxylate (administered intravenously) on Ro 154513 binding in the hippocampus and cerebellum.

Methyl 3,5-diphenylpyridazine-4-carboxylate blocked Ro 154513 binding in the hippocampus but did not affect Ro 15413 binding in the cerebellum.

In FIG. 3 is a graph showing dose-dependent occupancy of α5 GABAA receptors by intravenous methyl-3,5-diphenylpyridazine-4-carboxylate, with receptor occupancy determined by either the ratio between exposure to Ro 15-4513 on the hippocampus (an area with a high density of α5 GABAA receptors) and exposure to Ro 15-4513 on the cerebellum (an area of low density of GABAA α5 receptors), or using the GABAA a5 selective compound L-655708 (10 mg/kg, iv) to determine complete occupation.

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

In FIG. 5 is a graph showing the effect of ethyl 3-methoxy-7-methyl-9Hbenzo[(]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4]diazepin- 10-carboxylate on retention of spatial memory in ten rats with age-related disorders (AD) in the Radial Eight-Arm Maze (REL) test Fig. 5 shows the effect of ethyl-3-methoxy-7-methyl-9H-benzo[f]imidazo[1 ,5a][1,2,4]triazolo[4,3-d][1,4]diazepine-10-carboxylate on the retention of spatial memory in ten rats with age-related disorders (AD) in the RVL test, where the control vehicle was studied 3 times and various doses of ethyl-3-methoxy-7-methyl-9H-benzo[f|imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4]diazepine10- carboxylate was tested twice, in Fig. 5 black bars refer to rats treated with vehicle only and white bars refer to rats treated with ethyl-3-methoxy-7-methyl-9Hbenzo[f]imidazo[1,5-a][1, 2,4]triazolo[4,3-d][1,4]diazepine-10-carboxylate in various doses.

In FIG. 6 is a graph showing the effect of ethyl 3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4] diazepine-10-carboxylate (administered intravenously) on Ro 154513 binding in the hippocampus and cerebellum. Ethyl 3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5a][1,2,4]triazolo[4,3-d][1,4]diazepine-10-carboxylate blocked Ro binding 154513 in the hippocampus but did not affect Ro 15413 binding in the cerebellum.

In FIG. 7 is a graph showing the dose-dependent occupation of α5 GABAA receptors by ethyl-3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3-d][ 1,4]diazepine-10-carboxylate administered intravenously as calculated from the ratio between exposure to Ro 15-4513 on the hippocampus (area with high density of GABA α5 receptors) and exposure to Ro 15-4513 on the cerebellum (area of low density of GABA α5 receptors) ) to determine full occupation.

In FIG. 8A-8C are graphs showing the effect of 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one on compared with vehicle dimethyl sulfoxide (DMSO) in aging-impaired rats using the Morris Water Maze behavioral test. In FIG. 8A shows rescue latency (i.e., the average time in seconds that rats found a hidden platform in a pool of water) during exercise in rats treated with 6,6-dimethyl-3-(3-hydroxypropyl)thio-1- (thiazol-2-yl)-6,7-dihydro-2-benzothiophene4(5H)-one, and vehicle-treated DMSO rats; in fig. 8B shows the amount of time spent in the target annulus and opposite annulus by rats treated with 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2- benzothiophen-4(5H)-one, and rats treated with vehicle DMSO; in fig. 8C shows the number of crossings in the target annulus and opposite annulus in rats treated with 6,6-dimethyl-3-(3hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4( 5H)-one and rats treated with DMSO vehicle.

Detailed description of the invention

Definitions.

Unless otherwise specified in the present description, scientific and technical terms used in this document should have the meanings that are commonly understood by a person skilled in the art. Generally, the nomenclature used in connection with chemical methods, cell and tissue culture, molecular biology, cell and oncological biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described in this document, well known and widely used in the art.

The methods and methods of the present invention are generally carried out, unless otherwise indicated, in accordance with standard methods well known in the art and as described in the various general and more specific references that are cited and discussed throughout this specification. See, for example, Principles of Neural Science, McGraw-Hill Medical, New York, NY (2000); Motulsky, Intuitive Biostatistics, Oxford University Press, Inc. (1995); Lodish et al., Molecular Cell Biology, ^4th ed.,

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W. H. Freeman; Co., New York (2000); Griffiths et al., Introduction to Genetic Analysis, ^7th ed.,

W. H. Freeman; Co., NY (1999); and Gilbert et al., Developmental Biology, ^6th ed., Sinauer Associates, Inc.,

Sunderland, M.A. (2000).

Chemical terms used in this specification are used in accordance with conventional usage in the art, exemplified by The McGraw-Hill Dictionary of Chemical Terms, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

All publications, patents and published patent applications mentioned in this document are expressly incorporated into the present description by reference. In the event of a conflict, the present description, including its specific definitions, will take precedence.

Throughout this specification, the word contains, or variations thereof such as contains or containing, should be understood to mean the inclusion of the specified whole (or components) or group of integers (or components), but not the exclusion of any other whole (or components) or group of integers (or components).

The singular forms include the plural unless the context clearly dictates otherwise.

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

The term agent is used herein to refer to a chemical compound (such as an organic or inorganic compound (including, for example, a compound of the present invention), a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof, as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or part thereof, such as a peptide, lipid, carbohydrate), or an extract obtained from biological materials such as bacteria, plants, fungi, or cells or tissues of animals (especially mammals). Agents include, for example, agents that are known with respect to structure and agents that are unknown with respect to structure. The agonistic activity of α5-containing GABAA receptors of such agents may make them useful as therapeutic agents in the methods and compositions of this invention.

Patient, object, or individual are used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock (including cows, pigs, etc.), domestic animals (eg, dogs, cats, etc.), and rodents (eg, mice and rats).

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

In humans, cognitive function can be measured by, for example, but not limited to, the Global Clinical Impression Scale (CIBIC-plus scale); the brief mental status assessment scale (BSMS); neuropsychiatric questionnaire (NPI); clinical rating scale for dementia (CRSD); Cambridge Automated Neuropsychological Test Battery (CANTAB); the Sandoz Geriatric Clinical Assessment Scale (SCAG), the Buschke Selective Memory Test (Buschke and Fuld, 1974); subtest for verbal pair associations; subtest for logical memory; the Visual Reproduction Subtest of the Revised Wechsler Memory Scale (WMS-R) (Wechsler, 1997); Benton's visual retention test, or a specific task with forced three choice alternatives, or the consensus battery of neuropsychological tests MATRICS. See Folstein et al., J. Psychiatric. Res. 12:189-98, (1975); Robbins et al. Dementia 5:266-81 (1994); Rey, L'examen clinique en psychologie, (1964); Kluger et al., J. Geriatr. Psychiatry. neuroi. 12:168-79, (1999); Marquis et al., 2002 and Masur et al., 1994. See also Buchanan, R.W., Keefe, R.S.E., Umbricht, D., Green, M.F., Laughren, T., and Marder, S.R. (2011), The FDA-NIMH-MATRICS guidelines for clinical trial design of cognitive-enhancing drugs: what do we know 5 years later? Schizophr. Bull. 37, 1209-1217.

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

Cognitive function can also be measured using imaging techniques such as

- 39 040488 positron emission tomography (PET), functional magnetic resonance imaging (fMRI), single photon emission computed tomography (SPECT) or any other imaging modality that measures brain function. In animals, cognitive function can also be measured using electrophysiological methods.

Cognitive stimulation refers to influencing impaired cognitive function so that it more closely resembles that of a normal, unaffected subject. Cognitive function may be stimulated to any detectable degree, but in humans it is preferably sufficiently stimulated to enable the affected subject to perform daily activities of normal life at a level of skill as close as possible to that of a normal, unaffected subject or an age-appropriate, normal, unaffected subject.

In some cases, the stimulation of cognitive function in a subject affected by age-related cognitive impairment refers to the impairment of cognitive function, so that it more closely resembles the function of an age-appropriate, normal, unaffected subject, or the function of a young subject. The subject's cognitive function may be stimulated to any detectable degree, but in humans is preferably sufficiently stimulated to enable the affected subject to perform daily activities of normal life at a level of skill as close as possible to a normal, unaffected subject, or a young subject, or age appropriate. group, normal, unaffected object.

Preservation of cognitive function refers to the effect on normal or impaired cognitive function, so that it does not deteriorate or fall below the level that is observed in the subject at first presentation or diagnosis, or delay such deterioration.

Improving cognitive function includes promoting cognitive function and/or maintaining cognitive function in the subject.

Cognitive impairment refers to cognitive function in subjects that is not as robust as expected in a normal, unaffected subject. In some cases, cognitive function is reduced by about 5%, about 10%, about 30% or more compared to the cognitive function expected in a normal unaffected subject. In some cases, cognitive impairment in subjects affected by age-related cognitive impairment refers to cognitive function in subjects that is not as robust as expected in an age-appropriate, normal, unaffected subject, or function of a young subject (i.e., subjects with average scores for given age in a cognitive test).

Age-related cognitive impairment refers to a cognitive impairment in the elderly in which their cognitive function is not as robust as that expected in an age-appropriate, normal subject, or as expected in a young subject. In some cases, cognitive function is reduced by about 5%, about 10%, about 30% or more compared to the cognitive function expected in an age-appropriate, normal subject. In some cases, cognitive function is as expected in an age-appropriate normal subject, but is reduced by about 5%, about 10%, about 30%, about 50%, or more compared to that expected in a young subject. . Age-related cognitive impairment may be associated with mild cognitive impairment (MCI) (including amnestic MCI and non-amnestic MCI), age-related memory impairment (AML) and age-related cognitive decline (AMCF).

Cognitive impairment associated with or related to AD or in AD refers to cognitive function in subjects that is not as robust as expected in subjects who have not been diagnosed with AD using generally accepted methodologies and standards.

Mild Cognitive Impairment or MCI refers to a condition characterized by a discrete memory impairment not accompanied by other cognitive impairment and relatively normal performance. One set of criteria for the clinical characterization of MCI includes the following characteristics: (1) memory complaint (as reported by the patient, informant, or clinician); (2) normal activities of daily living (ADLs); (3) normal global cognitive function; (4) abnormal memory for age (defined as a score greater than 1.5 standard deviations below the mean for age) and (5) no dementia indicators (as defined in the DSM-IV guidelines). Petersen et al., Srch. Neurol. 56:303-308 (1999); Petersen, Mild cognitive impairment: Aging to Alzheimer's Disease. Cognitive deficits in subjects with MCI may involve any cognitive area or mental process, including memory, language, association, attention, perception, problem solving, executive function, and visuospatial skills. See, for example, Winbald et al., J. Intern. Med. 256:240-240, 2004; Meguro, Acta. Neurol. Taiwan. 15:5557, 2008; Ellison et al., CNS Spectr. 13:66-72, 2008, Petersen, Semin. Neurol. 27:22-31, 2007. MCI is further subdivided into amnestic MCI (aAMN) and non-amnestic MCI characterized, in particular, by impairment (or lack thereof) of memory. MCH is defined as a MCH if memory is found to be impaired, taking into account the subject's age and educational level. If, on the other hand, it is found

- 40 040488 that the subject's memory is intact for age and educational level, but other non-memory cognitive areas such as language, executive function, or visuo-spatial skills are damaged, MCI is defined as non-amnestic MCI. aUKN and non-amnestic UCH can be further divided into single or multi-domain UCH. aUKN-single domain refers to a condition in which memory is impaired but not other cognitive areas. aUKN-multidomain refers to a condition in which memory and at least one other cognitive area are impaired. Non-amnestic MCI is single-domain or multi-domain, depending on whether more than one non-memory cognitive area is damaged. See, for example, Peterson and Negash, CNS Spectr. 13:45-53, 2008.

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

Dementia refers to a condition characterized by a severe cognitive deficit that interferes with normal life activities. Subjects with dementia also exhibit other symptoms such as clouding of consciousness, personality changes, disorientation, mental confusion, behavioral changes, speech problems, and motor defects. There are different types of dementia such as Alzheimer's disease (AD), vascular dementia, Lewy body dementia, and frontotemporal dementia.

Alzheimer's disease is characterized by early memory deficits. Later symptoms include clouding of consciousness, disorientation, confusion, behavioral changes, speech problems, and motor defects. Histologically, AD is characterized by beta-amyloid plaques and tau-protein tangles.

Vascular dementia is caused by strokes. The symptoms are similar to those of AD, but without focusing on memory impairment.

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

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

Post-traumatic stress disorder (PTSD) refers to an anxiety disorder characterized by an immediate or delayed response to a catastrophic event, characterized by re-experiencing the trauma, mental numbness or avoidance of trauma-related stimuli, and increased arousal. Re-experiencing the event includes intrusive memories, flashbacks of the past, nightmares, and psychological or physiological distress in response to the trauma reminder. Such responses are worrisome and can have a significant impact, both chronic and acute, on the patient's quality of life and physical and emotional well-being. PTSD is also associated with impaired cognition, and older people with PTSD show more significant cognitive decline compared to controls.

Schizophrenia refers to a chronic, severe disorder characterized by a spectrum of psychopathology, including positive symptoms such as aberrant or distorted mental representations (eg, hallucinations, delusions), negative symptoms characterized by decreased motivation and adaptive goal-directed activity (eg, anhedonia, affective dullness, abulia ), and cognitive impairment. Although brain abnormalities are thought to underlie the entire spectrum of psychopathology in schizophrenia, currently available antipsychotics are largely ineffective in treating cognitive impairment in patients.

Bipolar disorder, or BD, or manic-depressive disorder, or manic-depressive illness refers to a chronic psychological/affective disorder that can be characterized by significant mood swings, including periods of depression and euphoric manic periods. BD can be diagnosed by a qualified physician based on a personal and medical history, an appointment consultation, and physical examinations. The term mania, or manic periods, or other variations, refers to periods when the patient exhibits some or all of the following characteristics: racing of ideas, rapid speech, increased levels of activity and excitement, as well as inflated self-esteem, euphoria, inability to

- 41 040488 reasoning, insomnia, impaired concentration and aggression.

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

Cancer therapy-associated cognitive impairment refers to cognitive impairment that develops in subjects treated with cancer therapies such as chemotherapy (eg, chemical brain) and radiation. Cytotoxicity and other undesirable brain side effects of cancer therapy lead to cognitive impairment in functions such as memory, learning and attention.

Parkinson's disease (PD) is a neurological disorder characterized by decreased voluntary movement. The affected patient has decreased motor activity and slower voluntary movements compared to a normal person. The patient has a characteristic masking face, a tendency to rush when walking, a flexion posture, and general muscle weakness. There is a characteristic rigidity of passive movements like a lead pipe. Another important feature of the disease is the tremor of the extremities, which occurs at rest and decreases during movement.

As used herein, the term autism refers to an autism spectrum disorder characterized by a neurodevelopmental disorder resulting in impaired social interaction and communication through restricted and repetitive behavior. Autism spectrum disorder refers to a group of developmental disorders that includes autism; Asperger's syndrome; pervasive developmental disorder not otherwise specified (PDD-NOS or atypical autism); Rett syndrome and childhood disintegrative disorder.

Mental retardation is a common disorder characterized by significantly impaired cognitive function and deficits in adaptive behavior. Mental retardation is often defined as an intelligence quotient (IQ) of less than 70. Hereditary causes are among the many underlying causes of mental retardation. Dysfunction in neural connectivity is also considered to be one of the main causes of mental retardation (Myrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214).

In some cases, mental retardation includes, but is not limited to, Down syndrome, velocardiofacial syndrome, fetal alcohol syndrome, fragile X syndrome, Klinefelter syndrome, neurofibromatosis, congenital hypothyroidism, Williams syndrome, phenylketonuria (PKU), Smith-Lemli-Opitz syndrome , Prader-Willi syndrome, Phelan-McDermid syndrome, Mowat-Wilson syndrome, ciliopathy, Lowe's syndrome and X-linked mental retardation, Siderius type.

Down syndrome is a disorder that includes a combination of birth defects, including some degree of mental retardation, facial features and often heart defects, increased susceptibility to infections, vision and hearing problems, and other pathologies.

Fragile X syndrome is a common form of hereditary mental retardation, occurring in 1 in 4,000 men and 1 in 8,000 women. The syndrome is also characterized by mental retardation, hyperactivity, attention deficit disorder, and autistic behavior. There is no effective treatment for fragile X syndrome.

Obsessive-compulsive disorder (OCD) is a mental condition that is most often characterized by obsessive, repetitive, disturbing thoughts (obsessions) leading to compulsive behaviors and conscious actions that the person feels they must perform (compulsiveness). Currently available epidemiological data indicate that OCD is the fourth most common mental disorder in the United States. Some studies suggest that the prevalence of OCD is between one and three percent, although the prevalence of clinically recognized OCD is much lower, suggesting that many people with the disorder may be undiagnosed. Patients with OCD are often diagnosed by a psychologist, psychiatrist, or psychoanalyst according to the diagnostic criteria in the Diagnostic and Statistical Manual of Mental Disorders, 4th Ed. revised (DSM-IV-TR) (2000), which include the characteristics of obsessions and compulsiveness.

Drug addiction (eg drug addiction, alcohol addiction) is a mental disorder. Addiction does not occur instantly when exposed to a substance of abuse. Rather, it involves multiple, complex neural adaptations that develop on varying time courses ranging from hours to days to months (Kauer J.A. Nat. Rev. Neurosci. 2007, 8, 844-858). The path to addiction usually begins with the conscious use of one or more controlled substances, such as narcotics, barbiturates, methamphetamines, alcohol, nicotine, and any of a number of other such controlled substances. With time

- 42 040488 With prolonged use of a controlled substance(s), the conscious ability to abstain from the controlled substance(s) is reduced due to the effect of prolonged use on brain function and therefore behavior. Essentially, addiction is usually characterized by a compulsive craving, seeking, and use of the substance that persists even in the face of negative consequences. Cravings may represent changes in the patient's underlying neurobiology that likely need to be addressed in a significant way if recovery is to be achieved. In many cases, drug dependence is also characterized by withdrawal symptoms that are life-threatening for some substances (e.g., alcohol, barbiturates) and in other cases can lead to significant morbidity (which may include nausea, vomiting, fever, dizziness, and increased sweating) , distress and reduced ability to recover. For example, alcoholism, also known as alcohol addiction, is one such drug addiction. Alcoholism is primarily characterized by four symptoms, which include cravings, loss of control, physical dependence, and addiction. These symptoms can also characterize dependence on other controlled substances. The craving for alcohol, like other controlled substances, is often as strong as the craving for food or water. Thus, an alcoholic may continue to drink despite dire family, health, and/or legal consequences.

Treatment of a condition or patient refers to taking action to obtain beneficial or desired outcomes, including clinical outcomes. Beneficial or desirable clinical outcomes include, but are not limited to, preventing or slowing the progression of a disease or disorder, or lessening, improving, or slowing the progression of one or more symptoms of a cognitive impairment associated with CNS disorders such as age-related cognitive impairment, mild cognitive impairment (MCI) , amnesic MCI (aAMI), age-related memory impairment (AMD), dementia, Alzheimer's disease (AD), post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis (ALS), cancer therapy-associated cognitive impairment, mental retardation , Parkinson's disease (PD), autism spectrum disorders, compulsive behavior and drug addiction. In some embodiments, the treatment includes preventing or slowing the progression of a CNS disorder (such as described herein). In certain embodiments, treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with a given CNS disorder. In certain embodiments, the symptom to be treated is a cognitive impairment or cognitive deficit.

Treatment of age-related cognitive impairment further includes slowing the transformation of age-related cognitive impairment (including, but not limited to, MCI, GRFR, and GNP) into dementia (eg, AD).

Treatment of cognitive impairment refers to taking steps to improve the cognitive function of a subject with a cognitive impairment such that the subject's performance on one or more cognitive tests is improved to any detectable degree or is prevented from further decline. Preferably, the subject's cognitive function after the treatment of cognitive impairment more closely resembles that of a normal, unaffected subject. Treatment of cognitive impairment in humans may improve cognitive function to any detectable degree, but preferably improves sufficiently to allow the affected subject to perform daily activities of normal life with the same level of skill as a normal, unaffected subject. In some instances, treating a cognitive impairment refers to taking steps to improve the cognitive function of a subject with a cognitive impairment such that the subject's performance on one or more cognitive tests is improved to any detectable degree or is prevented from further decline. Preferably, the subject's cognitive function after the treatment of cognitive impairment more closely resembles that of a normal, unaffected subject. In some cases, treating a cognitive impairment in a subject affected by age-related cognitive impairment refers to taking measures to improve the subject's cognitive function so that the subject's cognitive function after treatment for the cognitive impairment more closely resembles that of an age-appropriate, normal, unaffected subject, or that of a young subject.

Administration of a substance, compound, or agent to an object may be accomplished using one of a variety of methods known to those skilled in the art. For example, a compound or agent can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, through the eyes, sublingually, orally (by swallowing), intranasally (by inhalation), intraspinally, intracerebral, and transdermally (by absorption, for example, through skin canal). The compound or agent may also be appropriately administered using rechargeable or biodegradable polymeric devices or other devices such as patches and pumps or formulations that provide sustained, sustained or controlled release of the compound or agent. The introduction can also be carried out, for example, once, multiple times and/or for one or more extended periods. In some aspects, the introduction includes both

- 43 040488 direct administration, including taking a drug without the supervision of a doctor, and indirect administration, including the effect of prescribing a drug. For example, as used herein, a physician who prescribes a drug for self-administration to a patient or has a drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

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

As used herein, an a5-containing GABAA receptor agonist, an a5-containing GABAA-P agonist, or a GABAA α5 receptor agonist and other variants used in the present description refer to a compound that activates the function of an a5-containing GABAA receptor (GABA-P ), i.e. a compound that increases GABA-dependent Cl' channels. In some embodiments, an α5-containing GABA-P agonist as used herein refers to a positive allosteric modulator that potentiates GABA activity. Suitable α5-containing GABA receptor agonists suitable for use in the present invention include α5-containing GABA receptor agonists of all formulae and certain α5-containing GABA receptor agonists described herein and their salts (e.g., pharmaceutically acceptable salts) , isomers (eg stereoisomers, E/Z isomers) and combinations thereof.

An antipsychotic, antipsychotic agent, antipsychotic, or antipsychotic compound refers to (1) a typical or atypical antipsychotic; (2) an agent that is selected from dopaminergic agents, glutamatergic agents, NMDA receptor positive allosteric modulators, glycine reuptake inhibitors, glutamate reuptake inhibitors, agonists, or positive allosteric modulators (PAMs) of metabotropic glutamate receptors (mGluR) (e.g., agonists or PAM mGluR2/3), positive allosteric modulators (PAM) of the glutamate receptor glur5, positive allosteric modulators (PAM) of the M1-muscarinic acetylcholine receptor (mAChR), histamine H3 receptor antagonists, AMPA/kainate receptor antagonists, ampakines (CX-516), glutathione prodrugs, noradrenergic agents, serotonin receptor modulators, cholinergic agents, cannabinoid CB1 antagonists, neurokinin 3 antagonists, neurotensin agonists, MAO B inhibitors, PDE10 inhibitors, nNOS inhibitors, neurosteroids and neurotrophic factors, agonists or positive allosteric moduli tori (PAM) alpha-7, serotonin 2C agonists; and/or (3) an agent that is useful in treating one or more of the signs or symptoms of schizophrenia or bipolar disorder (particularly mania).

As used herein, exemplary antipsychotics refer to standard antipsychotics that produce antipsychotic effects as well as movement-related side effects associated with disturbances in the nigrostriatal dopamine system. These extrapyramidal side effects (EPS) include parkinsonism, akathisia, tardive dyskinesia, and dystonia. See Baldessarini and Tarazi in Goodman & Gilman's The Pharmacological Basis of Therapeutics 10 Edition, 2001, p. 485-520.

As used herein, atypical antipsychotics refer to antipsychotics that produce antipsychotic effects with little or no EPS and include, but are not limited to, aripiprazole, asenapine, clozapine, iloperidone, olanzapine, lurasidone, paliperidone, quetiapine, risperidone, and ziprasidone. Atypical antipsychotics differ from standard antipsychotics in their pharmacological profiles. While standard antipsychotics are characterized primarily by blockade of the dopamine D ₂ receptor, atypical antipsychotics antagonize multiple receptors, including the 5HT _a and 5HT _c serotonin receptors, and varying degrees of receptor affinity. Atypical antipsychotics are commonly referred to as serotonin/dopamine antagonists, reflecting the important hypothesis that a higher affinity for the 5HT ₂ receptor than for the D ₂ receptor underlies the action of the atypical or second-generation antipsychotics. However, atypical antipsychotics often exhibit side effects including, but not limited to, weight gain, diabetes (eg, type II diabetes mellitus), hyperlipidemia, QTc prolongation, myocarditis, sexual side effects, extrapyramidal side effects, and cataracts. Thus, atypical antipsychotics do not represent a homogeneous class given their differences in terms of both clinical symptom relief and their ability to cause side effects such as those listed above. In addition, the common side effects of atypical antipsychotics, as described above, often limit the doses of antipsychotics that can be used with these agents.

Memantine is chemically known as 3,5-dimethyladamantan-1-amine or 3,5-dimethyltricyclo[3.3.1.1 ^3,7 ]decan-1-amine, which is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist with moderate affinity. Proprietary names for memantine include: Aksura® and Akatinol® (Merz), Namenda® (Forest Laboratories), Ebixa® and Abix® (Lundbeck) and Memox® (Unipharm). Memantine is approved for the treatment of moderate to severe Alzheimer's disease (AD) in the US at doses up to 28 mg/day. Derivatives or analogues of memantine, which include compounds that structurally or chemically resemble memantine, are also useful in the present invention. Such memantine derivatives or analogs include, but are not limited to, the compounds disclosed in US Pat. Nos. 3,391,142; 4122193; 4273774 and 5061703; publications of US patent applications US20040087658, US20050113458, US20060205822, US20090081259, US20090124659 and US20100227852; publication of patent application EP 2260839A2; in EP 1682109 B1 and PCT Application Publication WO 2005/079779, all of which are incorporated herein by reference. Used in the present invention, memantine includes memantine and its derivatives and analogs, as well as its hydrates, polymorphs, prodrugs, salts and solvates. As used herein, memantine also includes a composition comprising memantine, or a derivative or analog thereof, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, or prodrug thereof, the composition optionally further comprising at least one additional therapeutic agent (such as a therapeutic agent useful in the treatment of a CNS disorder or associated cognitive impairment). In some embodiments, a memantine composition suitable for use in the present invention comprises memantine and a second therapeutic agent that is donepezil (under the trade name Aricept).

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

As used herein, the term co-administration means that an α5-containing GABA _A receptor agonist (for example, a positive allosteric modulator of the α5-containing GABA receptor) and a second therapeutic agent (for example, an antipsychotic, memantine, or AChE-I) or pharmaceutically acceptable salts, hydrates, solvates, or polymorphs are administered with a time separation of no more than about 15 minutes, and in some embodiments, no more than about 10 minutes. When the drugs are administered simultaneously, an α5-containing GABA _A receptor agonist (eg, a positive allosteric modulator of the α5-containing GABA receptor) and a second therapeutic agent (eg, an antipsychotic, memantine, or AChE-I) or salts, hydrates, solvates thereof or polymorphic modifications may be contained in the same dosage (eg, a unit dosage form containing both an α5-containing GABA receptor agonist (eg, a positive allosteric modulator of the α5-containing GABA receptor) and a second therapeutic agent (eg, an antipsychotic agent, memantine or AChE-I) or in separate dosages (for example, an α5-containing GABAA receptor agonist (for example, a positive allosteric modulator of the α5-containing GABAA receptor) or its salt, hydrate, solvate, or polymorph modification is contained in a single dosage form and a second therapeutic agent (eg, an antipsychotic, memantine, or AChE-I) or its salt, hydrate, solvate or polymorph contained in another dosage form).

As used herein, the term sequential administration means that an α5-containing GABA receptor agonist (for example, a positive allosteric modulator of the α5-containing GABA receptor) and a second therapeutic agent (for example, an antipsychotic agent, memantine or AChE-I) or their pharmaceutically acceptable salts, hydrates, solvates, polymorphs are administered with time separations greater than about 15 minutes and in some embodiments greater than about 1 hour or up to 12-24 hours. Either an α5-containing GABAA receptor agonist (e.g., a positive allosteric modulator of α5 -containing GABA receptor), or a second therapeutic agent (for example, an antipsychotic agent, memantine or AChE-I). α5 GABA receptor agonist (e.g., a positive allosteric modulator of the α 5 GABA receptor) and a second therapeutic agent

- 45 040488 (for example, an antipsychotic agent, memantine or AChE-I) or their salts, hydrates, solvents or polymorphs for sequential administration may be contained in separate dosage forms, optionally contained in the same container or package.

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

The compounds of the present invention also include prodrugs, analogs or derivatives. The term prodrug is recognized in the art and is intended to encompass compounds or agents that, under physiological conditions, are converted into positive allosteric modulators of α5-containing GABA-P. A common way to obtain a prodrug is to select fragments that are hydrolyzed or metabolized under physiological conditions to obtain the desired compound or agent. In other embodiments, the prodrug is converted by enzymatic activity of the host animal into a positive allosteric modulator of the GABAA α5 receptor.

As used herein, a pharmaceutically acceptable salt refers to an agent or compound according to the invention which is a therapeutically active, non-toxic base and acid salt form of the compounds. An acid addition salt form of a compound that occurs in its free base form can be obtained by treating said free base form with an appropriate acid such as an inorganic acid, for example a hydrohalic acid such as hydrochloric or hydrobromic, sulfuric, nitric, phosphoric and similar; or an organic acid such as, for example, acetic, hydroxyacetic, propanoic, lactic, pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclic, salicylic, γ-aminosalicylic , pamovoy and the like. See, for example, WO 01/062726.

Compounds containing acidic protons can be converted to their therapeutically active, non-toxic base addition salt form, such as a metal or amine salt, by treatment with appropriate organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali metal and alkaline earth metal salts, such as lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, such as N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. In turn, these forms of salts can be converted into free forms by treatment with an appropriate base or acid.

As used herein, the term hydrate refers to the combination of water with a compound in which water retains its molecular state as water and is either absorbed, adsorbed, or contained in the crystal lattice of the substrate compound.

As used herein, the term polymorph refers to different crystal forms of the same compound and other solid state molecular forms, including pseudo polymorphs such as hydrates (for example, bound water present in the crystal structure) and solvates (for example, bound solvents with the exception of water) of the same compound. Various crystalline polymorphs have different crystal structures due to different packing of molecules in the lattice. This leads to different crystal symmetry and/or unit cell parameters, which directly affects its physical properties, such as the X-ray diffraction characteristics of crystals or powders. For example, a different polymorph will typically deflect at a different set of angles and will give different values for intensities. Therefore, X-ray powder diffraction can be used to identify different polymorphs or a solid form that contains more than one polymorph in a reproducible and reliable manner. Crystalline polymorphs are of interest to the pharmaceutical industry and especially to those involved in the development of suitable dosage forms. If the polymorphic form is not maintained constant during clinical or stability studies, the exact dosage form used or investigated may not be comparable from one lot to another. It is also desirable to have processes for preparing a compound with a selected polymorph of high purity when the compound is used in clinical studies or in commercial products, since impurities present may cause undesirable toxicological effects. Certain polymorphs may exhibit increased thermodynamic stability or may be more easily manufactured in high purity in large quantities and thus are more suitable for inclusion in pharmaceutical formulations. Certain polymorphs may exhibit other preferred physical properties such as lack of hygroscopicity, improved solubility, and increased dissolution rates due to different lattice energies.

An isomer as used herein includes optical isomers (such as stereoisomers, eg enantiomers and diastereomers), Z (together) or E (opposite) isomers. Many of the compounds useful in the methods and compositions of this invention have at least one stereogenic center in their structure. This stereogenic center may be present in the R- or S-configuration, the indicated R- and S-designation is used in accordance with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The invention also relates to all stereoisomeric forms such as enantiomeric and diastereomeric forms of the compounds or mixtures thereof (including all possible mixtures of stereoisomers). See, for example, WO 01/062726. In addition, certain compounds that contain alkenyl groups may exist as Z (together) or E (opposite) isomers. In each case, the invention includes both a mixture and separate individual isomers. A plurality of substituents on the piperidinyl or azepanyl ring may also be either cis or trans relative to each other with respect to the plane of the piperidinyl or azepanyl ring. With respect to the methods and compositions of the present invention, reference to a compound or compounds is intended to encompass such a compound in each of its possible isomeric forms and mixtures thereof, unless a particular isomeric form is specifically named. See, for example, WO 01/062726.

The compounds of the invention enhance the function of a5-containing GABA-P, ie. they are α5-containing ΓΑΜΚ _α -Ρ agonists (for example, positive allosteric modulators of the α5-containing GABA receptor) and are able to increase GABA-dependent Cl' channels.

The invention further provides pharmaceutical compositions comprising a compound of the invention together with a pharmaceutically acceptable carrier or excipient. In some embodiments, the implementation of the pharmaceutical compositions of this invention may additionally contain a second therapeutic agent, such as an antipsychotic agent, memantine or AChE-I.

The invention further provides methods for treating cognitive impairment associated with said CNS disorders that are susceptible to positive allosteric modulators of the a5-containing GABAA receptor, e.g., age-related cognitive impairment, mild cognitive impairment (MCI), amnesic MCI (aMCI), dementia, Alzheimer's disease (AD), post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis (ALS), cancer therapy-associated cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorders, compulsive behavior and drug addiction. In certain embodiments, the method is a method for treating age-related cognitive impairment, mild cognitive impairment (MCI), amnestic MCI (aMCI), dementia, Alzheimer's disease (AD), post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis ( ALS) associated with cancer therapy of cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorders, compulsive behavior and drug addiction. In certain embodiments, the treatment includes preventing or slowing the progression of a CNS disorder as described herein (such as described herein). In certain embodiments, the treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with a CNS disorder. In certain embodiments, the symptom to be treated is a cognitive impairment or cognitive deficit. In another aspect of the invention, there is provided a method for maintaining or improving cognitive function in a subject in need of such treatment, the method comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, isomer, or combination thereof.

Various CNS disorders with cognitive impairment (eg, age-related cognitive impairment, mild cognitive impairment (MCI), amnestic MCI (aMCI), dementia, Alzheimer's disease (AD), post-traumatic stress disorder (PTSD), schizophrenia, bipolar disorder, amyotrophic lateral sclerosis ( ALS), cancer therapy-related cognitive impairment, mental retardation, Parkinson's disease (PD), autism spectrum disorders, compulsive

- 47 040488 behavior and drug addiction) may have different etiologies. However, the symptom of cognitive impairment in each of the above disorders may have overlapping causes.

Thus, a composition or method of treatment that treats a cognitive impairment in one CNS disorder may also treat a cognitive impairment in another.

Derivatives of benzodiazepines.

The present invention provides the following compounds containing a 1,5-benzodiazepine skeleton and a 1,2,3-triazole ring:

Compound Structure 344 L Z-Z y o o 2 356 '1 J o 357 o h l ^z \AJy ^z /=\ 2 358 o d At 359 o b L ^z XXv ^z R 360 X /°У ^{F C|} ^H ^N V

- 48 040488

- 49 040488

- 50 040488

- 51 040488

- 52 040488

- 53 040488

- 54 040488

or their pharmaceutically acceptable salts, enantiomers, diastereoisomers,

Z-isomers,

E-isomers or combinations thereof.

The present invention also provides the following compounds containing a 1,4-benzodiazepine skeleton and a 1,2,4-triazole ring:

- 55 040488

- 56 040488

- 57 040488

334 O dr about 335 c ¹ ^Υ ^Ν χ_/°^ 336 η joT°^ .^γ ^Ν °\ 337 4 . dij O 338 %ϊ C o 339 Id v 340 oh d-d NJ ΜθΟ^^^γ-Ν_

- 58 040488

- 59 040488

- 60 040488

431 Υ/'Ϋ Cl ^^^NOMe 432 ΥΗ/ __/OMe 433 A-l %\ χχ,ΝΥ 0-4 // X Ύ ) CI^YyN_ n' / \ ^N OMe 434 XV-x /=< /Х/NY 0-4 / ί Ύ ) Ν./ \ ^N OMe 435 Y /=\ Yy ⁿ ~Y°YY ^{f Cl} ^ ^X ^y--N ^ ^N ^OMe 444 N-kg \ ^N OMe 445 „χγΓγ ¹ Ν-κΓλ ^N OMe

- 61 040488

446 cf _ Rx r 447 GU ) Cl N_ρ, ^Ν 'Ν Τ 455 Ly I 458 Pi γ 460 Ρ/VJ 464 ? ΡΡ CQ 466 γΛ % / 467 Ν Ρ-Ο νΡ ιΡ £ Y ) GRGUL _f Pf ^ν ~ν 470 Pϊ 471 pi y

or their pharmaceutically acceptable salts, enantiomers, diastereoisomers, Z-isomers, E-isomers, or combinations thereof.

Any embodiment described herein is also intended to represent unlabeled forms as well as isotopically labeled forms of compounds, unless otherwise indicated. Isotopically labeled compounds have structures depicted by the formulas given in the present description, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that may be included in the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ² H, ³ H, ⁿ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³¹ P, ³² P, ³⁵ S, ³⁶ Cl, ¹²⁵ I, respectively. The invention includes various isotopically labeled compounds as defined herein, for example those in which radioactive isotopes are present such as ³ H, ¹³ C and ¹⁴ C. Such isotopically labeled compounds are useful in metabolism studies (preferably with ¹⁴ C) , reaction kinetics studies (e.g. with ² H or ³ H), detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including analyzes of drug or substrate distribution in tissues or during radioactive treatment of patients. In particular, ¹⁸ F or a labeled compound may be most preferred for PET or SPECT studies. The isotopically labeled compounds of this invention and their prodrugs can generally be prepared using the procedures disclosed in the Schemes or in the examples and preparations described below by substituting a non-isotopically labeled reagent with a readily available isotopically labeled reagent.

General methodology of synthesis.

The compounds of this invention can be obtained usually using methods known to the person skilled in the art. Schemes 1-10 show general synthetic routes for the preparation of compounds of formulas I-IV. Other equivalent schemes, which will be apparent to those skilled in the art of organic chemistry, may alternatively be used to synthesize various molecular moieties, as illustrated by the general schemes below.

Scheme 1

Scheme 2 about R?A-CO ₂ Et no ₂ ekG ^{r4r5 (r1)} XX

1, diazotization of AG __________► ^m

Ing 2.recovery ™ formation of pyrazole

Scheme 3

Scheme 4

- 63 040488

Scheme 5

R

Scheme 6

Scheme 7

OME

Meo

R ² \u003d -OR _a , -SRg, - (CH ^ ORg,

-(CHsIpRe or

Scheme 8

halide, etc.

Scheme 9

- 64 040488

Scheme 10 (1) LiOH; (2)CDI, NHL OH; (3) TFUA

Scheme 10a

Scheme 10c (1) oxalyl chloride. Et ₃ N or HOBT, EDC;

(2) DAST, K ₂ CO ₃

Schemes 10b(a) and 10b(b)

Those skilled in the art will recognize that the compounds of the invention may be prepared using a variety of chemicals or synthetic routes.

Pharmaceutical compositions and methods of administration.

The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant or excipient and a compound of the invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, Z-isomer, E-isomer, or combination thereof, in a therapeutically effective amount.

The basic nitrogen-containing groups present in the compounds of the invention may be quaternized with agents such as lower alkyl halides such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides such as benzyl and phenethyl bromides and others.

- 65 040488 products that are washable or dispersible in water or oil.

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

In some embodiments of the invention, the a5-containing GABA-P positive allosteric modulator is formulated with a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid , potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol sodium carboxymethyl cellulose , polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. In other embodiments, no carrier is used. For example, an α5-containing GABA-P agonist (eg, a positive allosteric modulator of the α5-containing GABA-P receptor) can be administered alone or as a component of a pharmaceutical composition (therapeutic composition). An α5-containing GABA-P agonist (eg, a positive allosteric modulator of the α5-containing GABA-P receptor) may be formulated for administration by any convenient route for use in human medicine.

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

In certain embodiments, pharmaceutical compositions suitable for parenteral administration may comprise a positive allosteric modulator of a5-containing GABA-P in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which may be converted to sterile injectable solutions or dispersions shortly before use, which may contain antioxidants, buffering agents, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. The necessary fluidity can be achieved, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

A composition containing a positive allosteric modulator of a5-containing GABA-P may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of exposure to microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of an injectable pharmaceutical form can be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

In certain embodiments of the invention, compositions containing a positive allosteric modulator of a5-containing GABAA-P can be administered orally, for example, in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum or tragacanth), powders , granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir

- 66 040488 or syrup or lozenges (using an inert base such as gelatin and glycerin or sucrose and acacia) and the like, each containing a predetermined amount of positive allosteric modulator a5-containing GABA-P as an active ingredient.

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

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

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

As described herein, the compounds, agents, and compositions thereof can be administered in a sustained, controlled, or sustained release manner. The term sustained release is widely known in the pharmaceutical field and is used herein to refer to the controlled release of an active compound or agent from a dosage form into the environment over (over or over) an extended period of time, such as greater than or equal to 1 hour. sustained release will release the drug at a substantially constant rate over an extended period of time, or a substantially constant amount of drug will be released gradually over an extended period of time. As used herein, the term extended release includes the terms controlled release, extended release, sustained release, delayed release, or slow release as these terms are used in the pharmaceutical sciences. In some embodiments, the sustained release dosage is administered in the form of a patch or pump.

A person skilled in the art, such as a physician, can readily determine the amount of α5-containing GABA-P positive allosteric modulator(s) required to treat a subject using the compositions and methods of the invention. It is understood that the dosing regimen will be determined for the subject, taking into account, for example, various factors that modify the effect of the a5-containing GABA-P positive allosteric modulator, disease severity or stage, route of administration, and subject-specific characteristics such as age, weight , size and degree of cognitive impairment.

It is well known in the art that body surface area normalization is a suitable method for extrapolating doses between species. To calculate the human equivalent dose (HED) from the dose used to treat age-related cognitive impairment in rats, one can

- 67 040488 use the formula HED (mg/kg) = rat dose (mg/kg) x 0.16 (see Estimating the Safe Starting Dose in

Clinical Trials for Therapeutics in Adult Healthy Volunteers, December 2002, Center for Biologies Evaluation and Research). For example, using this formula, a dose of 10 mg/kg in rats is equivalent to 1.6 mg/kg in humans. This conversion is based on the more general formula HED = animal dose in mg/kg x (animal weight in kg/human weight in kg) ^0.33 .

In certain embodiments, the dose of a5-containing GABA-P positive allosteric modulator is 0.0001 to 100 mg/kg/day (which for a typical 70 kg human is 0.007 to 7000 mg/day).

In certain embodiments, the administration interval is once every 12 or 24 hours. Administration at shorter intervals, such as once every 6 hours, can also be used.

When administered via an implant, device or formulation with a sustained or sustained release, the a5-containing GABA-P positive allosteric modulator may be administered once or one or more times intermittently throughout the life of the patient as needed. Other administration intervals, intermediate or less than these dosage intervals for clinical applications, can also be used and can be determined by a person skilled in the art in accordance with the methods of this invention.

The desired time of administration can be determined by routine experimentation by one of skill in the art. For example, a positive allosteric modulator of a5-containing GABA-P can be administered over a period of 1-4 weeks, 1-3 months, 3-6 months, 6-12 months, 1-2 years, or more, up to the lifespan of the patient.

In addition to being a positive allosteric modulator of α5-containing GABA-P, the compositions of this invention may also include other therapeutically useful agents. These other therapeutically useful agents may be administered in the same formulation simultaneously or sequentially with a5-containing GABA-P positive allosteric modulator in accordance with the methods of the invention.

One of skill in the art will appreciate that the compositions described herein may be adapted and modified according to the present application and that the compositions described herein may be used in other suitable applications. For example, the compositions of this invention may further comprise a second therapeutic agent. Such other additions and modifications do not go beyond the scope of the application for this invention.

Pharmaceutical compositions with antipsychotics.

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

In some embodiments, antipsychotics suitable for use in the present invention are selected from atypical antipsychotics.

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

In some embodiments, antipsychotics suitable for use in the present invention are typical antipsychotics, including, but not limited to, acepromazine, benperidol, bromazepam, bromperidol, chlorpromazine, chlorprothixene, clothiapine, cyamemazine, diazepam, dixirazine, droperidol, flupentixol , fluphenazine, fluspirilene, haloperidol, heptaminol, isopropamide iodide, levomepromazine, levosulpiride, loxapine, melperone, mesoridazine, molindone, oxypertine, oxyprotepin, penfluridol, perazine, periciazine, perphenazine, pimozide, pipamperone, pipothiazine, prochlorperazine, promazine, protipendone, pyridoxine, sulpiride, sultopride, tetrabenazine, thioproperazine, thioridazine, tiapride, thiothixene, trifluperazine,

- 68 040488 triflupromazine, trihexyphenidyl and zuclopenthixol and their pharmaceutically acceptable salts, hydrates, solvates and polymorphs.

In some embodiments, the antipsychotic agent or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof may be selected from compounds that are dopaminergic agents (such as dopamine D1 receptor antagonists or agonists, dopamine D2 receptor antagonists or partial agonists). , dopamine D3 receptor antagonists or partial agonists, dopamine D4 receptor antagonists), glutamatergic agents, positive allosteric modulators of N-methyl-D-aspartate receptors (NMDA), glycine reuptake inhibitors, glutamate reuptake inhibitors, agonists or positive allosteric modulators (PAM) of metabotropic glutamate receptors (mGluRs) (e.g. mGluR2/3 agonists or PAM), positive allosteric modulators (PAM) of glutamate receptor glur5, positive allosteric modulators (PAM) of M1-muscarinic acetylcholine receptor (mAChR), antagonist histamine H3 receptor, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonists, ampakines (CX-516), glutathione prodrugs, noradrenergic agents (such as agonists or antagonists of alpha- 2-adrenergic receptors and catechol-O-methyltransferase (COMT) inhibitors), serotonin receptor modulators (such as 5-HT _2A receptor antagonists, 5-HT _1A receptor partial agonists, 5-HT2C agonists and 5-HT6 antagonists, serotonin 2C agonists) , cholinergic agents (such as alpha-7 nicotinic receptor agonists or PAMs, alpha-4-beta2 nicotinic receptor agonists, nicotinic receptor allosteric modulators and acetylcholinesterase inhibitors, muscarinic receptor agonists and antagonists), cannabinoid CB1 antagonists, neurokinin 3 antagonists, agonists neurotensin, monoamine oxidase (MAO) B inhibitors, PDE10 inhibitors, neuronal nitric oxide synthase (nNOS) inhibitors, neurosteroids and neurotrophic factors.

In some embodiments, an a5-containing GABA receptor positive allosteric modulator as described herein and an antipsychotic agent as described herein, or pharmaceutically acceptable salts, hydrates, solvates, or polymorphs thereof, are administered simultaneously or sequentially or in one or in separate formulations packaged together. In other embodiments, the α5-containing GABA receptor positive allosteric modulator and the antipsychotic agent, or pharmaceutically acceptable salts, hydrates, solvates, or polymorphs thereof, are administered by various routes. Used in the present description, the combination includes the introduction using any of these compositions or routes of administration.

Pharmaceutical compositions with memantine.

The compounds or compositions of the present invention may be used in combination with memantine, or a derivative or analogue thereof, for the treatment of cognitive impairment associated with disorders of the central nervous system (CNS) in a subject in need or at risk of developing, including but not limited to subjects with or at risk of developing age-related cognitive impairment, mild cognitive impairment (AMCI), amnestic MCI, dementia, Alzheimer's disease (AD), post-traumatic stress disorder (PTSD), schizophrenia or bipolar disorder, amyotrophic lateral sclerosis (ALS) and related with cognitive impairment cancer therapy.

Memantine, also chemically known as 3,5-dimethyladamantan-1-amine or 3,5-dimethyltricyclo[3.3.1.1 ^3,7 ]decan-1-amine, is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. with moderate affinity. Proprietary names for memantine include Aksura® and Akatinol® (Merz), Namenda® (Forest Laboratories), Ebixa® and Abix® (Lundbeck) and Memox® (Unipharm). Memantine is currently available in the US and in over 42 countries worldwide. It is approved for the treatment of moderate to severe Alzheimer's disease (AD) in the US at doses up to 28 mg/day. Memantine and some of its derivatives and analogues, which can be used in the present invention, are disclosed in US patent No. 3391142; 4122193; 4273774 and 5061703, all of which are incorporated into the present description by reference. Other memantine derivatives or analogs that are useful in the present invention include, but are not limited to, those compounds disclosed in US Patent Application Publications US20040087658, US20050113458, US20060205822, US20090081259, US20090124659, and US20100227852; publication of patent application EP 2260839 A2; patent EP 1682109 B1 and PCT application publication WO 2005/079779, all of which are incorporated herein by reference. Used in the present invention, memantine includes memantine and its derivatives and analogs, as well as its hydrates, polymorphs, prodrugs, salts and solvates. As used herein, memantine also includes a composition comprising memantine, or a derivative or analog thereof, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, or prodrug thereof, the composition optionally further comprising at least one additional therapeutic agent.

- 69 040488 (such as a therapeutic agent suitable for the treatment of CNS disorders or associated cognitive impairments). In some embodiments, a memantine composition suitable for use in the present invention comprises memantine and a second therapeutic agent that is donepezil (under the trade name Aricept).

In other embodiments, the α5-containing GABA receptor positive allosteric modulator and memantine (or memantine derivative/analogue), or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, or prodrugs thereof, are administered simultaneously or sequentially or in one or separate packaged formulations. together. In other embodiments, the α5-containing GABA receptor positive allosteric modulator and memantine (or memantine derivative/analogue), or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, or prodrugs thereof, are administered by various routes. Used in the present description, the combination includes the introduction using any of these compositions or routes of administration.

Pharmaceutical compositions with acetylcholinesterase inhibitors (AChE-Is).

The compounds or compositions of this invention may be used in combination with an acetylcholinesterase inhibitor to treat central nervous system (CNS)-related cognitive impairment in a subject in need of or at risk of developing, including, but not limited to, subjects having or at risk of developing development of age-related cognitive impairment, mild cognitive impairment (AMCI), amnestic MCI, dementia, Alzheimer's disease (AD), post-traumatic stress disorder (PTSD), schizophrenia or bipolar disorder, amyotrophic lateral sclerosis (ALS), and cancer therapy-associated cognitive impairment.

AChE-Is known to one of skill in the art may fall into the subcategories of (i) reversible non-competitive inhibitors or reversible competitive inhibitors, (ii) irreversible and/or (iii) quasi-reversible inhibitors.

In a particular embodiment, AChE-Is useful in the present invention include those described in PCT applications WO 2014/039920 and WO 2002/032412; patents EP No. 468187; 481429-A and US Pat. Nos. 4,816,456; 4895841; 5041455; 5106856; 5602176; 6677330; 7340299; 7635709; 8058268; 8741808 and 8853219, all of which are incorporated into the present description by reference.

In a certain embodiment, typical AChE-Is that can be used in accordance with this invention include Donepezil (Aricept), Galantamine (Razadin) or Rivastigmine (Exelon).

In other embodiments, the positive allosteric modulator of the a5-containing GABA receptor and AChE-I, or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, or prodrugs thereof, are administered simultaneously or sequentially or in one or separate formulations packaged together. In other embodiments, the positive allosteric modulator of the a5-containing GABAA receptor and AChE-I, or pharmaceutically acceptable salts, hydrates, solvates, polymorphs, or prodrugs thereof, are administered by various routes. Used in the present description, the combination includes the introduction using any of these compositions or routes of administration.

Methods for assessing cognitive impairment.

Animal models provide an important source for the development and evaluation of treatments for cognitive impairment associated with CNS disorders. The hallmarks that characterize cognitive impairment in animal models generally extend to cognitive impairment in humans. Efficacy in such animal models is thus expected to be predictive of efficacy in humans. The degree of cognitive impairment in an animal model for a CNS disorder and the effectiveness of a treatment method for said CNS disorder can be tested and validated using various cognitive tests.

Behavioral Test The Radial Eight Arm Maze (REL) is one example of a cognitive test, specifically a test of spatial memory (Chappell et al. Neuropharmacology, 37:481-487, 1998). The RVL device consists, for example, of eight equally spaced sleeves. The sleeve of the labyrinth is projected from each face of the central platform. The food hole is located at the distal end of each sleeve. Food is used as a reward. Blocks can be positioned to prevent entry into either arm. Numerous additional maze stimuli surrounding the device may also be provided. After the stages of habituation and learning, the spatial memory of objects can be studied in the RWL under control conditions or conditions of treatment with the test compound. As part of the test, objects are pretreated prior to testing with a control vehicle or one of the dosage ranges of the test compound. At the beginning of each challenge, a subset of the arms of the eight-arm maze is blocked. Objects are allowed to receive food from unblocked arms, access to which is allowed during this initial informational phase of the trial. Objects are then removed from the maze during a delay period, e.g. 60 second delay, 15 minute delay, 1 hour delay, 2 hour delay, 6 hour delay, 24 hour delay or more between the informational step and the subsequent retention test, during which the barriers in the maze are removed, thus allowing access to all eight arms. After the grace period has elapsed, the objects are placed back on the central platform (with the barriers to the pre-locked arms removed) and allowed to receive the remaining food rewards during this phase of the trial retention test. The identity and configuration of the blocked sleeves varies depending on the tests. Tracks the number of errors that objects make during the retention test phase. An error occurs during the trial if subjects enter an arm from which food was already taken in the pre-deferral component of the trial, or if they revisit an arm in a post-delay session that they have already visited. Fewer errors will mean better spatial memory. The number of errors made by a test subject under different test compound treatment regimens can then be compared for the efficacy of the test compound in treating CNS-related cognitive impairment.

Another cognitive test that can be used to evaluate the effect of a test compound on CNS cognitive impairment in an animal model is the Morris Water Maze. The water labyrinth is a pool surrounded by a new system of images in relation to the labyrinth. The water maze test protocol can be based on a modified water maze task that has been shown to be hippocampal dependent (de Hoz et al., Eur. J. Neurosci., 22:745-54, 2005; Steele and Morris, Hippocampus, 9:118-36, 1999). The subject is trained to locate a submerged rescue platform hidden below the surface of the pool. During the mock test, an object is released into the labyrinth (pool) from random starting positions around the perimeter of the pool. The starting position varies from test to test. If the subject does not find the rescue platform within the set time, the experimenter guides and places the object on the platform to learn the location of the platform. After a delay period after the last training trial, a retention test in the absence of a rescue platform is performed to assess spatial memory. The object's preference level for the location of the (currently missing) rescue platform, as measured, for example, by the time spent at that location or the number of mouse crossings of that location, indicates better spatial memory, i.e. treatment of cognitive impairment. The preference for the location of the rescue platform under different treatment conditions can then be compared for the effectiveness of the study compound in the treatment of cognitive impairment associated with CNS disorders.

Various tests are known in the art for assessing cognitive function in humans, such as, but not limited to, the Global Clinical Impression Scale (CIBIC-plus scale); brief mental status assessment scale (MSSS); neuropsychiatric questionnaire (NPI); clinical rating scale for dementia (CRSD); Cambridge Automated Neuropsychological Test Battery (CANTAB); Sandoz Geriatric Scale of Clinical Assessment (SCAG), Buschke Selective Memory Test (Buschke and Fuld, 1974); subtest for verbal pair associations; subtest for logical memory; Visual Reproduction Subtest of the Revised Wechsler Memory Scale (WMS-R) (Wechsler, 1997); the Benton Visual Retention Test or the MATRICS Consensus Neuropsychological Test Battery, which includes tests for working memory, processing speed, attention, verbal learning, visual learning, thinking and decision making, and social cognition. See Folstein et al., J. Psychiatric. Res. 12:189-98, (1975); Robbins et al. Dementia 5:266-81 (1994); Rey, L'examen clinique en psychologie, (1964); Kluger et al., J. Geriatr. Psychiatry. neuroi. 12:168-79, (1999); Marquis et al., 2002 and Masur et al., 1994. See also Buchanan, R.W., Keefe, R.S.E., Umbricht, D., Green, M.F., Laughren, T., and Marder, S.R. (2011), The FDA-NIMH-MATRICS guidelines for clinical trial design of cognitive-enhancing drugs: what do we know 5 years later? Schizophr. Bull. 37, 1209-1217. Another example of a cognitive test in humans is a specific task with forced three choice alternatives. In this test, subjects are presented with color photographs of common objects, consisting of a mixture of three types of image pairs: identical pairs, identical pairs, and unbonded films. The second of a pair of identical objects is called a decoy. These image pairs are completely randomized and presented individually as a series of images. Objects are instructed to decide whether the objects they see are new, old, or the same. The same response to the presentation of the decoy stimulus indicates the successful extraction of memory by the object. On the contrary, calling the bait stimulus old or new, one can say that the correct memory retrieval did not occur.

In addition to assessing cognitive performance, the progression of age-related cognitive impairment and dementia can be monitored, as well as the transformation of age-related cognitive impairment into dementia, by assessing surrogate changes in the subject's brain. Surrogate changes include, but are not limited to, changes in the volumes of brain regions, degra

- 71 040488 perforant pathway and changes observed in brain function via resting fMRI (P-fMRI) and fluorodeoxyglucose positron emission tomography (FDG-PET). Examples of volumes of brain regions suitable for monitoring the progression of age-related cognitive impairment and dementia include a decrease in the volume of the hippocampus and a decrease in the volume or thickness of the entorhinal cortex. These volumes can be measured at the object, for example, using MPT. Aisen et al., Alzheimer's; Dementia 6:239-246 (2010). Perforant pathway degradation has been shown to be associated with age as well as cognitive decline. For example, older adults with more severe perforator pathway degradation tend to perform worse on hippocampus-dependent memory tests. Perforant pathway degradation can be monitored in subjects using ultra-high resolution diffusion tensor imaging (DTI). Yassa et al., PNAS, 107:12687-12691 (2010). Resting-state fMRI (R-fMRI) involves imaging the brain at rest and recording spontaneous low-frequency (<0.1 Hz) high-amplitude fluctuations in the fMRI signal that are temporally correlated in all functionally related areas. Functional connectivity correlation analysis, independent component analyzes, and/or signal frequency response analyzes are used to identify functional connectivity between brain regions, in particular those areas whose connectivity increases or decreases with age, as well as the degree of cognitive impairment and/or dementia. FDG-PET uses FDG uptake as a measure of regional metabolic activity in the brain. Reduced FDG uptake in areas such as the posterior cingulate cortex, temporoparietal cortex, and prefrontal association cortex has been shown to be associated with the degree of cognitive decline and dementia. Aisen et al., Alzheimer's; Dementia, 6:239-246 (2010), Herholz et al., NeuroImage, 17:302-316 (2002).

Age-related cognitive impairment.

The invention provides methods and compositions for the treatment of age-related cognitive impairment or its risk of development using a positive allosteric modulator of the a5-containing GABA _A receptor (i.e., a compound of the invention), such as selected from compounds, or pharmaceutically acceptable salts, isomers, or combinations thereof as described in this document. In certain embodiments, the treatment includes preventing or slowing the progression of age-related cognitive impairment. In certain embodiments, the treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with age-related cognitive impairment. In certain embodiments, the treatment of age-related cognitive impairment includes slowing the transformation of age-related cognitive impairment into dementia (eg, AD). The methods and compositions can be used in human patients in clinical applications for the treatment of age-related cognitive impairment in conditions such as MCI or its risk of developing. This document describes that the composition dose and dosage range for the method are safe and effective in these applications. In some embodiments, the invention provides a method of maintaining or improving cognitive function in a subject with age-related cognitive impairment, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, isomer, or a combination thereof.

In some embodiments, the subject being treated with the methods and compositions of this invention exhibits or is at risk of developing an age-related cognitive impairment. In some embodiments, the age-related cognitive impairment includes mild cognitive impairment (MCI).

Animal models provide an important source for developing and evaluating treatments for these age-related cognitive impairments. The hallmarks that characterize age-related cognitive impairment in animal models generally extend to age-related cognitive impairment in humans. Efficacy in such animal models is thus expected to be predictive of efficacy in humans.

Various animal models of age-related cognitive impairment are known in the art. For example, extensive behavioral characterization has identified a naturally occurring form of cognitive impairment in old Long Evans outbred rats (Charles River Laboratories; Gallagher et al., Behav. Neurosci. 107:618-626, (1993)). In a behavioral assessment using the Morris Water Maze (MWM), rats recognize and remember the location of a rescue platform based on the pattern of spatial cues surrounding the maze. The cognitive basis of performance is explored in probe tests using the measurement of the animal's spatial error in search of the location of the rescue platform. Older rats in the study population do not have difficulty swimming to the visible platform, but age-related impairment is determined when the platform is camouflaged, requiring the use of spatial parameter information. Performance for individual older Long Evans outbred rats varies considerably. For example, some of these rats perform tasks on the same level as young objects. However, approximately 40-50% is outside the performance range of young objects. This variability among older rats reflects confirmed individual differences. Ta

- 72 040488 Thus, in the age population, some animals have cognitive impairment and are defined as age-related impairment (AD), and other animals do not have impairment and are not defined as age-related impairment (AD). (See, for example, Colombo et al., Proc. Natl. Acad. Sci. 94:1419514199, (1997); Gallagher and Burwell, Neurobiol. Aging, 10:691-708, (1989); Gallagher et al. Behav Neurosci 107:618-626 (1993) Rapp and Gallagher Proc Natl Acad Sci 93:9926-9930 (1996) Nicolle et al Neuroscience 74:741-756 (1996) ), Nicolle et al., J. Neurosci 19:9604-9610, (1999), International Patent Publication WO 2007/019312 and International Patent Publication WO 2004/048551. compositions of this invention for the treatment of age-related cognitive impairment.

The effectiveness of the methods and compositions of the present invention for the treatment of age-related cognitive impairment can be assessed using various cognitive tests, including the Morris water maze and the radial eight-arm maze, as discussed herein.

Dementia.

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

Animal models are an important source for developing and evaluating treatments for dementia. The hallmarks that characterize dementia in animal models generally extend to dementia in humans. Thus, efficacy in such animal models is expected to be predictive of efficacy in humans. Various animal models of dementia are known in the art, such as PDAPP, Tg2576, APP23, TgCRND8, J20, hPS2 Tg, and APP+PS1 transgenic mice. Sankaranarayanan, Curr. top. Medicinal Chem, 6:609-627, 2006; Kobayashi et al. Genes Brain Behav. 4:173-196. 2005; Ashe and Zahns, Neuron. 66:631-45, 2010. Such animal models of dementia can be used to analyze the effectiveness of the methods and compositions of this invention for the treatment of dementia.

The effectiveness of the methods and compositions of the present invention for the treatment of dementia or dementia-related cognitive impairment can be assessed in animal models of dementia, as well as in humans with dementia, using various cognitive tests known in the art, as discussed herein. description.

Post-traumatic stress disorder.

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

Patients with PTSD (and less trauma-prone non-PTSD patients) have smaller hippocampal volumes (Woon et al., Prog. Neuro-Psychopharm.; Biological Psych. 34, 1181-1188; Wang et al., Arch. Gen. Psychiatry 67:296-303, 2010). PTSD is also associated with impaired cognitive performance. Older patients with PTSD have more cognitive decline than controls (Yehuda et al., Bio. Psych. 60:714-721, 2006) and have more

- 73 040488 the likelihood of developing dementia (Yaffe et al., Arch. Gen. Psych. 678:608-613, 2010).

Animal models provide an important source for the development and evaluation of treatments for PTSD. The hallmarks that characterize PTSD in animal models generally extend to PTSD in humans. Thus, efficacy in such animal models is expected to be predictive of efficacy in humans. Various animal models of PTSD are known in the art.

One model of PTSD in rats is time-dependent sensitization (TDS). TDS involves exposing an animal to a significant stressful event followed by a situational reminder of the previous stressor. Below is an example of TDS. The rats are placed in a restraint, then placed in a swimming tank and allowed to swim for a period of time, eg 20 minutes. Thereafter, each rat is immediately exposed to a gaseous anesthetic until unconscious and finally dried. Animals are kept alone for several days, for example one week. The rats are then exposed to a re-stress session consisting of the original stressor, such as a swimming session in a swim tank (Liberzon et al., Psychoneuroendocrinology, 22:443-453, 1997; Harvery et al., Psychopharmacology, 175:494502, 2004). TDS results in an increase in the acoustic startle response (ASR) in rats, which is comparable to the exaggerated acoustic startle response that is a known symptom of PTSD (Khan and Liberzon, Psychopharmacology, 172:225-229, 2004). Such animal models of PTSD can be used to analyze the effectiveness of the methods and compositions of this invention for the treatment of PTSD.

The effectiveness of the methods and compositions of the present invention for the treatment of PTSD or PTSD-related cognitive impairment can also be assessed in animal models of PTSD, as well as in humans with PTSD, using various cognitive tests known in the art, as discussed herein. description.

Schizophrenia and bipolar disorder.

The invention further provides methods and compositions for the treatment of schizophrenia or bipolar disorder (particularly mania) using a positive allosteric modulator of the a5-containing GABA receptor, such as selected from compounds, or pharmaceutically acceptable salts, isomers, or combinations thereof, as described in this document. In certain embodiments, the implementation of the treatment includes preventing or slowing the progression of schizophrenia or bipolar disorder (in particular, mania). Schizophrenia is characterized by a broad spectrum of psychopathology, including positive symptoms such as aberrant or distorted mental representations (eg, hallucinations, delusions) or symptoms associated with dopamine dysregulation (eg, hyperdopaminergic responses, hyperdopaminergic behavioral responses, dopaminergic hyperactivity, or hyperlocomotor activity, or psychosis), negative symptoms characterized by decreased motivation and adaptive goal-directed activity (eg, anhedonia, affective dullness, abulia), and cognitive impairment. In certain embodiments, treatment includes alleviating, improving, or slowing the progression of one or more positive and/or negative symptoms, as well as cognitive impairment associated with schizophrenia. In addition, there are a number of other mental illnesses, such as schizotypal and schizoaffective disorder, other acute and chronic psychoses, and bipolar disorder (particularly mania), that have similar symptoms to schizophrenia. In some embodiments, the treatment includes alleviating, improving, or slowing the progression of one or more symptoms, as well as the cognitive impairment associated with bipolar disorder (in particular, mania). In some embodiments, the invention provides a method of maintaining or improving cognitive function in a subject with schizophrenia or bipolar disorder, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt, isomer, or combination thereof. The methods and compositions may be used in human patients in clinical applications for the treatment of schizophrenia or bipolar disorder (particularly mania). This document describes that the composition dose and dosage range for the method are safe and effective in these applications.

Cognitive impairment is associated with schizophrenia. They precede the onset of psychosis and are present in unaffected relatives. Cognitive impairment associated with schizophrenia is a good indicator of the functional outcome of the disease and is a key feature of the disorder. Cognitive features in schizophrenia reflect dysfunction in the frontal cortical and hippocampal systems. Patients with schizophrenia also have hippocampal pathologies such as hippocampal volume contractions, neuronal shrinkage, and dysfunctional hyperactivity. An imbalance in excitation and inhibition in these brain regions has also been documented in patients with schizophrenia, suggesting that drugs that target inhibitory mechanisms may be therapeutic. See, for example, Guidotti et al., Psychopharmacology, 180:191-205, 2005; Zierhut, Psych. Res. Neuroimag. 183:187-194, 2010; Wood et al., NeuroImage, 52:62-63, 2010; Vinkers et al., Expert Opin. Investig. Drugs, 19:1217-1233, 2009; Young et al. Pharmacol. Ther. 122:150-202, 2009.

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Animal models provide an important source for the development and evaluation of treatments for schizophrenia. The hallmarks that characterize schizophrenia in animal models generally extend to schizophrenia in humans. Thus, efficacy in such animal models is expected to be predictive of efficacy in humans. Various animal models of schizophrenia are known in the art.

One animal model of schizophrenia is a course treatment with methionine. Methionine-treated mice show underexpression of GAD67 in the frontal cortex and hippocampus, similar to that found in the brains of patients with post-mortem schizophrenia. They also exhibit prepulse inhibition of startle and deficits in social interaction (Tremonlizzo et al., PNAS, 99:17095-17100, 2002). Another animal model of schizophrenia is the treatment of rats with methyloxymethanol acetate (MAM). Pregnant female rats are administered MAM (20 mg/kg, ip) on the 17th day of pregnancy. MAM processing summarizes the pathological development of schizophrenia-like phenotypes in offspring, including anatomical changes, behavioral disturbances, and altered neural processing. More specifically, MAM-treated rats exhibit a reduced density of parvalbumin-positive GABAergic interneurons in portions of the prefrontal cortex and hippocampus. In behavioral tests, MAM-treated rats exhibit reduced latent inhibition. Latent inhibition is a behavioral phenomenon in which there is reduced recognition of a stimulus for which there has been a preliminary exposure with any consequence. This tendency to disregard previously favorable stimuli and to reduce association formation with such stimuli is thought to prevent sensory overload. A low level of latent inhibition indicates psychosis. Latent inhibition can be studied in rats as follows. Rats are divided into two groups. One group is pre-exposed to tone for several trials. The other group is not affected by the tone. Both groups are then subjected to the auditory conditioned freezing method, in which the same tone is presented simultaneously with a painful stimulus, such as an electrical shock to the paw. In the future, both groups are presented with a tone and the change in motor activity in rats is monitored during the presentation of the tone. After conditioned reflex freezing, the rats react to the presentation of the tone with significantly reduced motor activity. However, the group that was exposed to the tone before the conditional period exhibits stable latent inhibition: the suppression of motor activity in response to the presentation of the tone decreases. MAM-treated rats, in contrast, show impaired latent inhibition. Thus, exposure to tone prior to the conditioned freezing method has no significant effect in suppressing conditioned freezing (see Lodge et al., J. Neurosci., 29:2344-2354, 2009). Such animal models of schizophrenia can be used to analyze the effectiveness of the methods and compositions of the invention for the treatment of schizophrenia or bipolar disorder (particularly mania).

MAM-treated rats show a significantly improved locomotor response (or aberrant locomotor activity) to administration of D-amphetamine at low doses. MAM-treated rats also show significantly higher numbers of spontaneously firing dopamine (DA) neurons in the ventral tegmental area. These results are believed to be due to overactive hippocampus because, in MAM-treated rats, inactivation of the ventral hippocampus (vHipp) (e.g., by intra-vHipp administration of a sodium channel blocker, tetrodotoxin (TTX), to MAM rats) reversed the increased activity of the DA population. -neurons, and also normalized the increased motor behavior caused by amphetamine. The correlation of hippocampal dysfunction and hypersensitivity of the DA system is believed to underlie the enhanced amphetamine response in MAM-treated animals and psychosis in schizophrenic patients. See Lodge D.J. et al. Neurobiology of Disease (2007), 27(42), 11424-11430. The use of MAM-treated rats in the above study may be suitable for use in evaluating the effectiveness of the methods and compositions of the present invention for the treatment of schizophrenia or bipolar disorder (particularly mania). For example, the methods and compositions of this invention can be evaluated using MAM-treated animals for their effect on central hippocampal (vHipp) regulation, for increased activity of the DA neuron population, and for the hyperactive motor response to amphetamine in MAM-treated animals.

In MAM-treated rats, hippocampal (HPC) dysfunction leads to hyperactivity of the dopamine system.

A benzodiazepine positive allosteric modulator (PAM) selective for the a5 subunit of the GABA receptor, SH-053-2'FR-CH ₃ , is being investigated for its effect on the hippocampal (HPC) response. Also investigate the effect of SH-053-2'FR-CH ₃ on hyperactive motor response to amphetamine in MAM-treated animals. α5GABA-R PAM reduced the number of spontaneously active DA neurons in the ventral tegmental region (VTA) in MAM rats to levels observed in rats treated with saline (control group), both systemically and

- 75 040488 for direct insertion into the ventral HPC. In addition, HPC neurons in both saline-treated and MAM-treated animals show reduced cortically induced responses after treatment with a5GABA-P PAM. In addition, the enhanced motor response to amphetamine observed in MAM-treated rats is reduced after α5GABA _A -P PAM treatment. See Gill KM et al. Neuropsychopharmacology, (2011), 1-9. The use of MAM-treated rats in the above study may be suitable for use in the present invention in evaluating the effectiveness of the methods and compositions of the invention for the treatment of schizophrenia or bipolar disorder (particularly mania). For example, the methods and compositions of the present invention can be evaluated using MAM-treated animals for their effect on the hippocampal response (HPC) and hyperactive motor response to amphetamine in MAM-treated animals.

Administration of MAM to pregnant rats at day 15 of gestation (E15) severely impairs spatial memory, or the ability to learn the spatial arrangement of four objects in a radial eight-armed maze, in the offspring. In addition, MAM-treated rats at day 17 of gestation (E17) are able to achieve performance levels of control rats in the initial stages of learning, but are unable to process and retrieve spatial information at a 30-minute delay, indicating a significant deterioration in short-term memory. See Gourevitch R. et al. (2004). behavior. Pharmacol, 15, 287-292. Such animal models of schizophrenia can be used to evaluate the effectiveness of the methods and compositions of the invention for the treatment of schizophrenia or bipolar disorder (particularly mania).

Apomorphine-induced climbing (AIC) and stereotypy (AIS) in mice is another animal model used in this invention. The agents are administered to mice at the desired dose level (eg, via intraperitoneal injection). Subsequently, for example, after 30 minutes, apomorphine is administered to experimental mice (for example, 1 mg/kg sc). Five minutes after the injection of apomorphine, sniff-lick-nibble syndrome (stereotypic behavior) and climbing behavior induced by apomorphine were assessed and recorded for each animal. Readings may be repeated every 5 minutes during a 30-minute testing session. The results for each animal were summarized over a 30-minute testing session for each syndrome (stereotypic behavior and climbing). If the effect achieved at least 50% inhibition and the SH ₅₀ value (95% confidence interval) is calculated using a non-linear least squares calculation with inverse prediction, the mean climbing and stereotypy scores can be expressed as a percentage of the control values observed in mice treated with excipient (eg treated with saline) that receive apomorphine. See Grauer SM et al. Psychopharmacology (2009), 204, 37-48. This mouse model can be used to evaluate the effectiveness of the methods and compositions of the invention for the treatment of schizophrenia or bipolar disorder (particularly mania).

In another well-known preclinical model of schizophrenia, rats chronically exposed to ketamine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, reproduce positive and negative psychotic symptoms and cognitive impairment. Male Long Evans rats are treated intraperitoneally with ketamine (30 mg/kg, twice daily) for two weeks at a young age (2 months). Rats are behaviorally examined when they reach adulthood (approximately 4-5 months of age) to identify behavioral symptoms of ketamine exposure and the effectiveness of treatment to reduce these symptoms. See, for example, Enomoto et al. Progress in NeuroPsychopharmacology; Biological Psychiatry, 33 (2009), 668-675.

The effectiveness of the methods and compositions of this invention for the treatment of schizophrenia or associated cognitive impairment can also be assessed in animal models of schizophrenia or bipolar disorder (particularly mania), as well as in humans with schizophrenia, using a variety of cognitive tests known in the art. techniques, as discussed in the present description.

Amyotrophic lateral sclerosis (ALS).

The invention further provides methods and compositions for the treatment of ALS using a positive allosteric modulator of the a5-containing GABAA receptor, such as one selected from compounds, or pharmaceutically acceptable salts, isomers, or combinations thereof, as described herein. In certain embodiments, the treatment includes preventing or slowing the progression of ALS. In certain embodiments, the treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with ALS. In certain embodiments, the symptom being treated is a cognitive impairment. In some embodiments, the invention provides a method of maintaining or improving cognitive function in an ALS subject, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt, isomer, or combination thereof. The methods and compositions may be used in human patients in clinical applications for the treatment of ALS. This document describes that the composition dose and dosage range for the method are safe and effective in these applications.

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

The effectiveness of the methods and compositions of the present invention for the treatment of ALS or ALS-related cognitive impairment can also be assessed in animal models of ALS, as well as in humans with ALS, using various cognitive tests known in the art, as discussed herein. .

Cancer therapy-associated cognitive impairment.

The invention further provides methods and compositions for treating cancer therapy-associated cognitive impairment using a positive allosteric modulator of the a5-containing GABAA receptor, such as selected from compounds, or pharmaceutically acceptable salts, isomers, or combinations thereof, as described herein. The methods and compositions may be used in human patients in clinical applications for the treatment of cancer therapy-associated cognitive impairment. This document describes that the composition dose and dosage range for the method are safe and effective in these applications.

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

Cognitive impairment after cancer therapy reflects dysfunction in the frontal cortical and hippocampal systems, which are essential for normal cognition. In animal models, exposure to either chemotherapy or radiation has an adverse effect on performance on cognition tests, which is clearly dependent on these brain systems, in particular the hippocampus (Kim et al., J. Radiat. Res. 49:517-526, 2008; Yang et al., Neurobiol Learning and Mem 93:487-494, 2010). Thus, drugs targeting these cortical and hippocampal systems may be neuroprotective in patients receiving cancer therapy and effective in treating symptoms of cognitive impairment that may persist after interventions used as cancer therapy.

Animal models provide an important source for developing and evaluating treatments for cancer therapy-associated cognitive impairment. The hallmarks that characterize cancer therapy-associated cognitive impairment in animal models generally extend to cancer therapy-associated cognitive impairment in humans. Thus, efficacy in such animal models is expected to be predictive of efficacy in humans. Various animal models of cancer therapy-associated cognitive impairment are known in the art.

Examples of animal models of cancer therapy-associated cognitive impairment include treatment of animals with anticancer agents such as cyclophosphamide (CYP) or with radiation such as ⁶⁰ Co gamma rays. (Kim et al., J. Radiat. Res. 49:517-526, 2008; Yang et al., Neurobiol. Learning and Mem. 93:487-494, 2010). The cognitive function of animal models of cancer therapy-associated cognitive impairment can then be examined using cognitive tests to evaluate the effectiveness of the methods and compositions of the invention in treating cancer therapy-associated cognitive impairment. The effectiveness of the methods and compositions of the present invention in treating cancer therapy-associated cognitive impairment can also be assessed in animal models of cancer therapy-associated cognitive impairment, as well as in humans with cancer therapy-associated cognitive impairment, using various cognitive tests known in the art. techniques, as discussed in the present description.

Parkinson's disease.

Parkinson's disease (PD) is a neurological disorder characterized by decreased voluntary movement. The affected patient has decreased motor activity and slower voluntary movements compared to a normal person. The patient has a characteristic masking face, a tendency to rush when walking, a flexion posture, and general muscle weakness. There is a characteristic rigidity of passive movements like a lead pipe. Another important feature of the disease is the tremor of the extremities, which occurs at rest and decreases during movement.

Parkinson's disease, whose etiology is unknown, belongs to a group of the most common movement disorders called parkinsonism, which affects approximately one person in a thousand. These other disorders, grouped under the name parkinsonism, may result from viral infection, syphilis, atherosclerosis, and trauma and exposure.

- 77 040488 toxic chemicals and drugs. At the same time, it is believed that improper loss of synaptic stability can lead to disruption of neural circuits and to brain diseases. Despite the result of genetics, drug use, the aging process, viral infections, or various other causes, dysfunction in neuronal communication is believed to be the root cause of many neurological diseases such as PD (Myrrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214).

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

The invention provides methods and compositions for the treatment of PD using a positive allosteric modulator of the α5-containing GABA _A receptor, such as selected from compounds, or pharmaceutically acceptable salts, isomers, or combinations thereof, as described herein. In certain embodiments, treatment includes preventing or slowing the progression of PD. In certain embodiments, the symptom being treated is a cognitive impairment. For example, the disclosure methods and compositions can be used to improve motor/cognitive impairment symptomatic of Parkinson's disease. In addition, the methods and compositions of the disclosure may be useful in the treatment of memory disorders symptomatic of Parkinson's disease. In some embodiments, the invention provides a method of maintaining or improving cognitive function in a subject with PD, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt, isomer, or combination thereof.

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

The effectiveness of the methods and compositions of this invention for the treatment of PD or PD-related cognitive impairment can be assessed in any of the above animal models of PD, as well as in humans with PD, using various cognitive tests known in the art, as discussed in the present description.

Autism.

Autism is a neurodevelopmental disorder characterized by dysfunction in three main behavioral dimensions: repetitive behavior, social deficits, and cognitive deficits. The domain of repetitive behavior includes compulsive behavior, unusual attachment to objects, rigid adherence to routines or rituals, and repetitive movement patterns such as stereotypy and self-stimulatory behavior. Measurements of social deficits include deficits in social interaction, lack of eye contact, reduced ability to carry on conversations, and impaired daily communication skills. Cognitive deficits may include language impairments. Autism is a disabling neurological disorder that affects thousands of Americans and spans a number of subtypes with various putative causes and several documented improving treatments. Autism spectrum disorders may be present at birth or may have a late onset, such as at two or three years of age. There are no clear biological markers of autism. The diagnosis of the disorder is based on the degree to which the child fits the behavioral syndrome, which is characterized by poor communication skills, specificity of social and cognitive abilities, and maladaptive behavior patterns. Dysfunction in neural connectivity is considered one of the main causes of autism (Myrhe van Spronsen and Casper C. Hoogenraad, Curr.

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Neurol. neurosci. Rep. 2010, 10, 207-214). Recent studies have shown that there is a GABA _α α5 deficiency in autism spectrum disorder (ASD), and further research into the GABA system in this disorder is supported (Mendez MA, et al. Neuropharmacology. 2013, 68:195-201).

The invention also provides methods and compositions for the treatment of autism using a positive allosteric modulator of the a5-containing GABAA receptor, such as selected from compounds or pharmaceutically acceptable salts, isomers, or combinations thereof, as described herein. In certain embodiments, the treatment includes preventing or slowing the progression of autism. In certain embodiments, the treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with autism. In certain embodiments, the symptom to be treated is a cognitive impairment or cognitive deficit. For example, disclosure methods and compositions can be used to improve motor/cognitive impairments symptomatic of autism. In some embodiments, a method for maintaining or improving cognitive function in an autistic subject is provided, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, isomer, or combination thereof.

The valproic acid (VPA) model of autism in rats using in vitro electrophysiological methods established by Rodier et al. (Rodier, P.M. et al. Reprod. Toxicol. 1997, 11, 417-422), is one of the most comprehensive reliable animal damage-based models of autism and is based on the observation that pregnant women who received VPA in the 1960s. within a limited time interval of embryogenesis, had a much higher risk of having an autistic child than the normal population. The offspring of pregnant rats exposed to VPA exhibit some of the anatomical and behavioral symptoms typical of autism, such as a reduced number of cerebellar Purkinje neurons, impaired social interaction, repetitive behaviors, as well as other symptoms of autism, including increased fear memory processing. See Rinaldi T. et al. Frontiers in Neural Circuits, 2008, 2, 1-7. Another BTBR T+tf/J (BTBR) mouse model, a valid model with consistent behavioral phenotypes related to the three diagnostic behavioral symptoms of autism - unusual social interactions, impaired communication, and repetitive behavior - was used to investigate the efficacy of a selective negative allosteric mGluR5 receptor modulator. GRN-529. See, for example, Silverman J.L. et al. sci. Transl. Med. 2012, 4, 131. The effectiveness of the methods and compositions of the present invention for the treatment of autism or autism-related cognitive impairment can be evaluated in the VPA-treated rat model of autism, or the BTBR T+tf/J (BTBR) mouse model, also as in people with autism, using various cognitive tests known in the art, as discussed in the present description.

Mental retardation.

Mental retardation is a common disorder characterized by significantly impaired cognitive function and deficits in adaptive behavior. Mental retardation is often defined as an intelligence quotient (IQ) of less than 70. Hereditary causes are among the many underlying causes of mental retardation. Dysfunction in neural connectivity is also considered to be one of the main causes of mental retardation (Myrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214).

In some cases, mental retardation includes, but is not limited to, Down syndrome, velocardiofacial syndrome, fetal alcohol syndrome, fragile X syndrome, Klinefelter syndrome, neurofibromatosis, congenital hypothyroidism, Williams syndrome, phenylketonuria (PKU), Smith-Lemli-Opitz syndrome , Prader-Willi syndrome, Phelan-McDermid syndrome, Mowat-Wilson syndrome, ciliopathy, Lowe's syndrome and X-linked mental retardation, Siderius type.

Down syndrome is a disorder that includes a combination of birth defects, including some degree of mental retardation, facial features and often heart defects, increased susceptibility to infections, vision and hearing problems, and other pathologies.

Fragile X syndrome is a common form of hereditary mental retardation, occurring in 1 in 4,000 men and 1 in 8,000 women. The syndrome is also characterized by mental retardation, hyperactivity, attention deficit disorder, and autistic behavior. There is no effective treatment for fragile X syndrome.

The present invention provides for the treatment of mild mental retardation, moderate mental retardation, severe mental retardation, profound mental retardation and unspecified mental retardation. Such mental retardation may, but need not, be associated with chromosomal changes (eg, Down's syndrome due to trisomy 21), heredity, pregnancy and perinatal problems, and other severe psychiatric disorders. This invention provides methods and compositions for the treatment of mental retardation using a positive allosteric modulator of a5-containing

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A GABA receptor, such as selected from compounds, or pharmaceutically acceptable salts, isomers, or combinations thereof, as described herein. In certain embodiments, the treatment includes preventing or slowing the progression of mental retardation. In certain embodiments, the treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with mental retardation. In certain embodiments, the symptom being treated is a cognitive deficit/impairment. For example, disclosure methods and compositions can be used to improve motor/cognitive impairments symptomatic of mental retardation. In some embodiments, the invention provides a method of maintaining or improving cognitive function in a subject with mental retardation, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt, isomer, or combination thereof.

Several animal models have been developed for mental retardation. For example, a gene knockout mouse model for fragile X syndrome has been developed. Fragile X syndrome is a common form of mental retardation caused by the absence of the FMR1 protein, FMRP. Two FMRP homologues, FXR1P and FXR2P, have been identified. FXR2P shows strong expression in the brain and testicles, like FMRP. Both Fxr2 and Fmr1 knockout mice and Fmr1/Fxr2 double knockout mice are believed to be useful models for mental retardation such as fragile X syndrome. See Bontekoe C.J.M. et al. Hum. Mol. Genet. 2002, 11(5):487498. The effectiveness of the methods and compositions of this invention for the treatment of mental retardation or cognitive deficits/impairment associated with mental retardation can be assessed in these mouse models and other animal models designed for mental retardation, as well as in humans with intellectual disability, using various cognitive tests. , known in the art, as discussed in the present description.

Compulsive behavior (obsessive-compulsive disorder).

Obsessive-compulsive disorder (OCD) is a mental condition that is most often characterized by obsessive, repetitive, disturbing thoughts (obsessions) leading to compulsive behaviors and conscious actions that the person feels they must perform (compulsiveness). Currently available epidemiological data indicate that OCD is the fourth most common mental disorder in the United States. Some studies suggest that the prevalence of OCD is between one and three percent, although the prevalence of clinically recognized OCD is much lower, suggesting that many people with the disorder may be undiagnosed. Patients with OCD are often diagnosed by a psychologist, psychiatrist, or psychoanalyst according to the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition Revised (DSM-IV-TR) (2000) diagnostic criteria, which include characteristics of obsessions and compulsiveness. Characteristics of obsessions include (1) repetitive and persistent thoughts, urges, or images that are perceived as intrusive and cause marked anxiety or distress; (2) the thoughts, impulses, or images are not just excessive worries about real issues; and (3) the person attempts to ignore or suppress such thoughts, urges, or images, or neutralize them through other thoughts or actions. The person becomes aware that intrusive thoughts, urges, or images are a product of his or her own mind and are not based in reality. Characteristics of compulsiveness include (1) repetitive behaviors or conscious actions that the person feels compelled to perform in response to compulsiveness or in accordance with rules that must be strictly enforced; (2) the behavior or conscious actions are aimed at preventing or reducing distress or preventing any fearful event or situation; however, the behavior or conscious actions are not actually related to the problem or are excessive.

People with OCD typically perform tasks (or compulsions) to find relief from obsessive-compulsive anxiety. Repetitive behaviors such as washing hands, counting, checking, or cleaning are often performed with the hope of preventing intrusive thoughts or making them go away. Performing these rituals, however, provides only temporary relief. People with OCD may also be diagnosed with a range of other psychiatric disorders, such as generalized anxiety disorder, anorexia nervosa, panic attack, or schizophrenia.

Dysfunction in neural connectivity is considered one of the main causes of obsessive disorder (Myrhe van Spronsen and Casper C. Hoogenraad, Curr. Neurol. Neurosci. Rep. 2010, 10, 207-214). Research suggests that OCD may be due to abnormal levels of a neurotransmitter called serotonin. Primary treatment for OCD consists of behavioral therapy, cognitive therapy, and medications. Treatment drugs include selective serotonin reuptake inhibitors (SSRIs) such as paroxetine (Seroxat™, Paxil®, Xetanor™, Paromerk™, Rexetine™), sertraline (Zoloft®, Stimuloton™), fluoxetine (Prozac®,

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Bioxetine™), escitalopram (Lexapro®) and fluvoxamine (Luvox®), as well as tricyclic antidepressants such as clomipramine (Anafranil®). Benzodiazepines are also used in the treatment.

However, up to 40-60% of patients fail to respond adequately to SSRI therapy, and an even greater proportion of patients do not experience complete remission of their symptoms.

The invention provides methods and compositions for the treatment of OCD using an α5-containing GABA receptor agonist (e.g., a positive allosteric modulator of the α5-containing GABA receptor), such as selected from compounds, or pharmaceutically acceptable salts, isomers, or combinations thereof, as described in this document. In certain embodiments, the treatment includes preventing or slowing the progression of OCD. In certain embodiments, treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with OCD. In certain embodiments, the symptom to be treated is a cognitive impairment or cognitive deficit. For example, the disclosure methods and compositions can be used to treat cognitive deficits in OCD and/or to improve cognitive function in patients with OCD. In some embodiments, the invention provides a method of maintaining or improving cognitive function in a subject with OCD, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt, isomer, or combination thereof.

For OCD, a quinpyrol-sensitized rat model was developed. The compulsive testing behavior of quinpyrol-sensitized rats was interrupted, which is a qualitative characteristic of OCD compulsiveness. In addition, the rodent model of schedule-induced polydipsia (SIP) of obsessive-compulsive disorder was used to evaluate the effects of the new 5-HT2C receptor agonist WAY-163909. See, for example, Rosenzweig-Lipson S. et al. Psychopharmacology (Berl), 2007, 192, 159-70. The effectiveness of the methods and compositions of this invention for the treatment of OCD, or cognitive impairment, or cognitive impairment associated with OCD, can be assessed in the above animal models and other animal models developed for OCD, as well as in humans with OCD, using various cognitive tests. , known in the art, as discussed in the present description.

Drug addiction.

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

Recent work investigating the effects of alcohol, CNS stimulant and opiate abuse on the central nervous system (CNS) has demonstrated a range of adverse mental health effects, including substance use-induced cognitive impairment. See Nyberg F. Cognitive Impairments in Drug Addicts, Chapter 9. Significant impairment of brain function has been observed in some laboratories and clinics due to these drugs. Among the harmful effects on the brain of substances that cause abuse, there are those that contribute to accelerated aging. An observation that has received particular attention in recent years is that chronic drug users show marked deterioration in areas of the brain associated with executive function.

- 81 040488 function and memory function. Marked neuroadaptation induced by narcotics such as alcohol, CNS stimulants, and opiates includes reduced neurogenesis in the subgranular zone (SGZ) of the hippocampus. Indeed, it has been hypothesized that decreased neurogenesis in adults in HCZ may alter hippocampal function in a way that promotes relapse and persistence of addictive behavior. It also raises the possibility that reduced neurogenesis may contribute to the development of cognitive impairment caused by these drugs.

The invention provides methods and compositions for the treatment of drug dependence using a positive allosteric modulator of the a5-containing GABAA receptor, such as selected from compounds, or pharmaceutically acceptable salts, isomers, or combinations thereof, as described herein. In certain embodiments, the implementation of the treatment includes preventing or slowing the progression of drug dependence. In certain embodiments, treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with drug dependence. In certain embodiments, the symptom being treated is a cognitive impairment. For example, the methods and compositions of the disclosure may be used to treat cognitive impairment and/or to improve cognitive function in drug addicted patients. In some embodiments, the invention provides a method of maintaining or improving cognitive function in a drug addicted subject, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt, isomer, or combination thereof.

Several animal models have been developed to study drug addiction. For example, to study the neurobiology of alcoholism, a model of genetically selected Marchigan-Sardinian alcohol-preferring rats was developed. See Ciccocioppo R. et al. Substance addiction Biology, 2006, 11, 339-355. The effectiveness of the methods and compositions of the present invention for the treatment of drug dependence or drug dependence-related cognitive impairment can also be assessed in animal models of drug dependence, as well as in humans with drug dependence, using various cognitive tests known in the art, such as discussed in the present description.

Brain cancer.

Brain cancer is the growth of abnormal cells in brain tissues, usually associated with the growth of malignant brain tumors. Brain tumors grow and press on nearby areas of the brain, which can prevent that part of the brain from working properly. Brain cancer rarely spreads to other tissues outside the brain. Tumor grade, based on how abnormal cancer cells look under a microscope, can be used to tell the difference between slow-growing and fast-growing tumors. Brain tumors are classified according to the type of cells from which the tumor appears to originate. Diffuse, fibrillar astrocytomas are the most common type of primary brain tumor in adults. These tumors are classified histopathologically into three grades: World Health Organization (WHO) grade II astrocytoma, WHO grade III anaplastic astrocytoma, and WHO grade IV glioblastoma multiforme (GBM). WHO grade II astocytomas are the slowest-growing of the spectrum of diffuse astrocytomas. Astrocytomas show a significant tendency to infiltrate the surrounding brain, complicating a locally controlled trial course of treatment. These invasive abilities are often seen in both low grade and high grade tumors.

Glioblastoma multiforme is the most malignant stage of astrocytoma, with the survival time for most patients being less than 2 years. Histologically, these tumors are characterized by dense cellularity, high rates of proliferation, endothelial proliferation, and focal necrosis. The high proliferative nature of these lesions is likely due to multiple mitogenic effects. One of the hallmarks of MGB is endothelial proliferation. Many angiogenic growth factors and their receptors have been found in the MGB.

There are biological subsets of astrocytomas that may reflect the clinical heterogeneity seen in these tumors. These subsets include brainstem gliomas, which are a form of childhood diffuse, fibrillar astrocytoma that often has a malignant course. MGBs of the brainstem have genetic characteristics in adults, so that younger patients are affected. Pleomorphic xanthoastrocytoma (PCA) is a low-grade superficial astrocytic tumor that predominantly affects young people. Although these tumors have an unusual histological appearance, they are usually slow growing tumors that can be treated surgically. However, some PCAs may return as MGBs. Pilocytic astrocytoma is the most common astrocytic tumor in children, both clinically and histopathologically from

- 82 040488 is different from diffuse, fibrillar astrocytoma, which affects adults.

Pilocytic astrocytomas do not have the same genomic changes as diffuse, fibrillar astrocytomas. Subependymal giant cell astrocytomas (SEGAs) are low-grade periventricular astrocytic tumors that are commonly associated with tuberous sclerosis (TS) and are histologically identical to the so-called suppository grooves that line the ventricles in patients with TS. Like other tumor lesions in TS, they are slow growing and may look more like hamartomas than true neoplasms. Desmoplastic cerebral astrocytoma of infancy (DCAM) and desmoplastic infantile ganglioglioma (DIIGG) are large, superficial, usually cystic, benign astrocytomas that affect children during the first year or two years of life.

Oligodendrogliomas and oligoastrocytomas (mixed gliomas) are diffuse, usually cerebral tumors that are clinically and biologically most closely related to diffuse, fibrillar astrocytomas. However, tumors are much less common than astrocytomas and generally have a better prognosis than diffuse astrocytomas. Oligodendrogliomas and oligoastrocytomas can progress to either WHO grade III anaplastic oligodendroglioma, or WHO grade anaplastic oligoastrocytoma, or WHO grade IV MGB. Thus, the genetic changes that lead to oligodendroglial tumors represent another route to MGD.

Ependymomas are a clinically diverse group of gliomas that range from aggressive intraventricular tumors in children to benign spinal cord tumors in adults. Transitions of ependymoma to MGB are rare. Choroid plexus tumors also represent a diverse group of tumors that predominantly occur in the ventricular system, ranging from aggressive supratentorial intraventricular tumors in children to benign tumors of the cerebellopontine angle in adults. Choroid plexus tumors have occasionally been reported in patients with Li-Fraumeni syndrome and Hippel-Lindau disease (LHL).

Medulloblastomas are highly malignant, primitive tumors that occur in the posterior fossa, predominantly in children. Medulloblastoma is the most common malignant brain tumor in children. The most lethal subtype of medulloblastoma exhibits high expression of the GABA receptor a5 subunit gene and MYC amplification. See, for example, J. Biomed. Nanotechnol. Jun 2016; 12(6):1297-302.

Meningiomas are common intracranial tumors that originate in the lining of the brain and compress the underlying brain. Meningiomas are usually benign, but some atypical meningiomas may recur locally, and some meningiomas are frankly malignant and may involve the brain or metastasize. Atypical and malignant meningiomas are not as common as benign meningiomas. Schwannomas are benign tumors that arise in peripheral nerves. Schwannomas can occur in cranial nerves, particularly in the vestibular part of the eighth cranial nerve (vestibular schwannomas, acoustic neuromas), where they appear as masses of the cerebellopontine angle. Hemangioblastomas are tumors of uncertain origin that consist of endothelial cells, pericytes, and so-called stromal cells. These benign tumors are most often found in the cerebellum and spinal cord of young people. Multiple hemangioblastomas are characteristic of Hippel-Lindau disease (BHL). Hemangiopericytomas (HPCs) are dural tumors that can exhibit locally aggressive behavior and can metastasize. The histogenesis of dural hemangiopericytoma (HPC) has been debated for a long time, with some authors classifying it as a distinct entity and others classifying it as a subtype of meningioma.

The invention provides methods and compositions for the treatment of brain cancer (e.g., brain tumors as described herein) using a positive allosteric modulator of the a5-containing GABA receptor, such as selected from compounds, or pharmaceutically acceptable salts, isomers, or their combinations as described herein. In certain embodiments, the treatment includes preventing or slowing the progression of brain cancer. In certain embodiments, the treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with brain cancer. In certain embodiments, the symptom being treated is a cognitive impairment. For example, the methods and compositions of the disclosure may be used to treat cognitive impairment and/or to improve cognitive function in patients with brain cancer. In some embodiments, the invention provides a method of maintaining or improving cognitive function in a subject with brain cancer, comprising the step of administering to said subject a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, isomer, or combination thereof. In some embodiments, the brain tumor is a medulloblastoma.

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Research Area Criteria (RDoC).

The invention further provides methods and compositions for treating disorders in neurological disorders and neuropsychiatric conditions using a positive allosteric modulator of a5-containing GABA-P, or a pharmaceutically acceptable salt, isomer, or combination thereof, as described herein. In certain embodiments, treatment includes alleviating, improving, or slowing the progression of one or more symptoms associated with such a disorder. In another aspect of the invention, methods and compositions are provided for maintaining or improving cognitive function in a subject in need of such treatment using a compound of the invention, or a pharmaceutically acceptable salt, isomer, or combination thereof.

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

Studies on the binding of the α5-subunit of GABA and the activity of a positive allosteric receptor modulator.

The affinity of test compounds for the GABA receptor containing the GABA α5 subunit can be determined using receptor binding assays known in the art. See, for example, US Pat. Nos. 7,642,267 and 6,743,789, which are incorporated herein by reference.

The activity of test compounds as a positive allosteric modulator of a5-containing GABAA-P can be tested using electrophysiological methods known in the art. See, for example, US Pat. No. 7,642,267 and Guidotti et al., Psychopharmacology, 180:191-205, 2005.

The activity of a positive allosteric modulator can be investigated, for example, using the analysis of GABA-induced conductivity of chloride ions of GABA receptors containing the α5 subunit of GABA. Cells expressing such receptors may be exposed to an effective amount of a compound of the invention. Such cells can interact in vivo with the compounds of the invention by contact with a body fluid containing the compound, for example, by contact with cerebrospinal fluid. In vitro studies can be carried out by reacting cells with a compound of the invention in the presence of GABA. Increased GABA-induced chloride conductance in cells expressing GABA receptors containing the α5 GABA subunits in the presence of the test compound would indicate positive allosteric modulator activity of said compound. Such changes in conductance can be detected, for example, using a voltage clamp assay performed on Xenopus oocytes injected with the GABA receptor subunit mRNA (including the GABA α5 RNA subunit), HEK 293 cells transfected with plasmids encoding the GABA receptor subunits, or in vivo, ex vivo or cultured neurons.

One skilled in the art will appreciate that the methods described herein may be adapted and modified in accordance with the present application, and that the methods described herein may be used in other suitable applications, and that such other additions and modifications will not go beyond the scope of this document.

The present invention can be better understood from the following examples. However, it will be readily apparent to a person skilled in the art that the specific methods and results described are only illustrative of the invention, as described in more detail in the embodiments they follow.

Examples

Example 1 Synthesis of Compound 1

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Scheme 11

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

MS: [M+1] = 379.

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

MS: [M+1] = 349.

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

MS: [M+1] = 303.

To a suspension of lactam ester 16 (837 mg, 2.77 mmol) in THF (20 ml) at room temperature was added LiBH ₄ (2 M in THF, 1.39 ml, 2.78 mmol). The mixture was then stirred at room temperature for 60 h, more LiBH4 (2 M in THF, 0.28 mL, 0.56 mmol) was added and stirred at room temperature for an additional 24 h. EtOAc/EtOH ( 10 ml/10 ml) and concentrated under vacuum. The residue was dissolved in EtOAc/CH ₂ Cl ₂ /MeOH and free silica gel was added. After evaporation of the volatile solvents, the solid was loaded onto a RediSep column with 24 g of silica gel. Chromatography (solvent A: EtOAc, solvent B: 10:1 v/v CH ₂ Cl ₂ /MeOH; gradient eluent: A to B) provided 540 mg (75% yield) of alcohol 17 as a white solid.

MS: [M+1] = 261.

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A solution of PPh ₃ ( ₂₅₅ mg, 0.97 mmol) in DMF ( 1 ml) more than 20 min. After the addition, TLC indicated that the reaction was complete. Water was added to quench the reaction and the mixture was extracted three times with EtOAc. The combined extracts were washed successively with H2O, brine and dried over Na ₂ SO ₄ . Filtration and concentration provided the crude product. Chromatography (RediSep column with 12 g silica gel, CH ₂ Cl ₂ to 30% EtOAc in CH ₂ Cl ₂ ) provided 439.2 mg of a mixture of bromide 18 ([M+1] = 324) and Ph ₃ PO. The above mixture (439 mg) in EtOAc/EtOH (8 mL/8 mL) with Pd/C (10 wt%, 200 mg, 0.19 mmol) was stirred under H2 (balloon) for 2 h, then filtered through heals. The filtrate was concentrated and the residue was purified by chromatography (RediSep column with 12 g of silica gel, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexane, solvent B: EtOAc; gradient eluent: A to B) to give 99 mg (80% yield over two steps) of product 19 as a white solid.

MS: [M+1] = 245.

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

Chromatography (RediSep column with 12 g silica gel, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexane, solvent B: EtOAc; gradient eluent: 20 to 80% B in A) to give 15 mg (yield 21% over two steps) Compound 1 (Example 1) as a white to yellowish or grayish solid.

MS: [M+1] = 340.

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

Example 2 Synthesis of Compound 2:

The compound of Example 2 was synthesized in a similar synthetic route as described for Example 1 using 5-fluoro-2-nitroaniline as starting material to give Compound 2 as a light brown solid.

MS: [M+1] = 328.

¹ H-NMR (500 MHz, CDCl3) δ: 7.90 (br.dd, 1H, J=2.5, 8.5 Hz), 7.77 (s, 1H), 7.62 (br.dd , 1H, J=5.0, 9.0 Hz), 7.35 (m, 1H), 4.45 (q, 2H, J=7.0 Hz), 2.45 (s, 3H), 1 .45 (t, 3H, J=7.0 Hz).

Example 3 Synthesis of Compound 3:

The compound of Example 3 was synthesized in a similar synthetic route as described for Example 1 using 2-nitroaniline as starting material to give Compound 3 as a light yellow solid.

MS: [M+1] = 310;

¹ H-NMR (500 MHz, CDCl3) δ: 8.161 (br. d, 1H, J=8.5 Hz), 7.81 (s, 1H), 7.66 (m, 3H), 4.45 ( k, 2H, J=7.0 Hz), 2.45 (s, 3H), 1.46 (t, 3H, J=7.0 Hz).

- 86 040488

Example 4 Synthesis of Compound 110

Acetamidoxime was azeotropically distilled three times in toluene prior to use. To a suspension of acetamidoxime (30 mg, 0.4 mmol) in THF (1 ml) was added 60% NaH in oil dispersion (16 mg, 0.4 mmol). The suspension was stirred at room temperature for 15 minutes. Ester compound 2 (65 mg, 0.2 mmol) was added. The tube containing the ester was washed with THF (1 ml) which was added to the reaction mixture. The resulting brown suspension was stirred at room temperature for 30 minutes, then heated at 70° C. for 2 hours and 30 minutes. The suspension was quenched with MeOH. The solvent was evaporated and the crude oil was purified by chromatography (4 g silica gel RediSep column, eluted with 70% EtOAc in hexanes) to give 28 mg (41% yield) of the product.

MS: [M+1] = 338;

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

Example 5 Synthesis of Compound 167

The compound was prepared similarly from Compound 1 to give Compound 167.

MS: [M+1] = 350;

¹ H-NMR (CDCl ₃ ) δ: 7.87 (1H, s), 7.65 (1H, d, J=3 Hz), 7.55 (1H, d, J=9 Hz), 7.17 (1H, dd, J=2.5, 9Hz), 3.96(3H, s), 2.5(3H, s), 2.45(3H, s).

Scheme 12

Example 6 Synthesis of Compound 4:

To a solution of compound 17 prepared as in Example 1 (260 mg) in DMSO (4 ml) and CH ₂ Cl ₂ (6 ml) was added Et ₃ N (0.7 ml, 5 mmol) followed by Py-SO ₃ (398 mg, 2.5 mmol). Stirred at room temperature for 1 hour. The reaction mixture was poured into water and extracted three times with EtOAc. The combined extracts were washed successively with H2O, brine and dried over Na ₂ SO ₄ . Filtration and concentration provided 198.5 mg of crude aldehyde 21 which was used without further purification. PhMgBr (1 M in THF, 1.54 ml, 1.54 mmol) was added dropwise to a suspension of aldehyde 21 (198.5 mg, 0.77 mmol) in THF (10 ml) at 0°C. Stirred at 0°C for 30 min. Saturated aqueous NH4Cl was added and extracted three times with EtOAc.

The combined extracts were washed with brine and dried over Na ₂ SO ₄ . Filtration and concentration provided 252.9 mg of benzyl alcohol 22 as a brown foamy solid.

- 87 040488 of a different substance. It was used in the next step without further purification. To a solution of the above crude alcohol 22 in CH ₂ Cl ₂ (8 ml) with Et ₃ SiH (0.60 ml, 3.76 mmol) was added TFA (0.64 ml, 8.27 mmol). The reaction solution was stirred at room temperature for 4 hours. After concentration, the residue was purified by chromatography (RediSep column with 12 g silica gel, 20 to 80% EtOAc in hexanes) to give 34.1 mg (12% yield over four steps) of the reduced product 23 as a white foamy solid.

MS: [M+1] = 321.

In a separate flask, a solution of 1,2,4-triazole (27 mg, 0.39 mmol) in CH3CN (0.5 ml) at 0°C was treated with i-Pr ₂ NEt (72 μl, 0.41 mmol) followed by addition POCl ₃ (11 µl, 0.12 mmol). The mixture was stirred at 0°C for 2 hours. Lactam substance 23 (32.2 mg, 0.1 mmol, solid) was added to the reaction mixture in one portion and heated in an oil bath at 80°C for 20 hours. The mixture was cooled to room temperature and a creamy solid precipitate was observed. Water (0.5 ml) was added and stirred at room temperature for 5 minutes. The solid precipitate was collected by filtration and washed with 0.5 ml of water, followed by drying under high vacuum to give 15.8 mg (42% yield) of adduct 24 as a white to yellowish or grayish friable solid.

MS: [M+1] = 372.

A solution of KO-t-Bu (9.5 mg, 85 μmol) in DMF (0.5 ml) was cooled to -50°C. Ethyl isocyanoacetate (10.4 μl, 95 μmol) was added dropwise. The resulting mixture was stirred at -50° C. for 1 hour. Triazolamidine 24 (15.8 mg, 42 μmol, solid) was added in one portion. The stirred mixture was allowed to warm to 10°C over 1 h and kept at 10°C for 1 h. Saturated aqueous NH4Cl was added and extracted three times with EtOAc. The combined extracts were washed successively with H2O, brine and dried over Na ₂ SO ₄ .

Filtration and concentration provided the crude product. Chromatography (RediSep column with 4 g silica gel. Solvent A: 1:1 v/v CH ₂ Cl ₂ /hexane, solvent B: EtOAc; gradient eluent: A to 50% B in A) provided 16.8 mg (yield 95%) of the compound of Example 6 as a white solid.

MS: [M+1] = 416;

¹ H-NMR (500 MHz, CDCU) δ: 7.74 (s, 1H), 7.63 (d, 1H, J=3.0 Hz), 7.50 (d, 1H, J=9.0 Hz) 7.30 (br.d, 2H, J=7.0 Hz), 7.29 (br.d., 2H, 7.5 Hz), 7.20 (m, 1H), 7.13 (dd , 1H, J=2.5, 9.0 Hz), 4.41 (c, 2H, J=7.5 Hz), 4.17 (s, 2H), 3.95 (s, 3H), 1 .43 (t, 3H, 7.5 Hz).

Example 7 Synthesis of Compound 5:

The compound of Example 7 was synthesized in a similar synthetic route as described for Example 6 using 2-nitroaniline as starting material to give Compound 5 as a brown solid.

MS: [M+1] = 386;

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 8.16 (br. d, 1H, J=7.0 Hz), 7.81 (s, 1H), 7.60-7.68 (m, 3H), 7.34 (br.d, 2H, J=8.0 Hz), 7.29 (br. d, 2H, J=7.0 Hz), 7.20 (m, 1H), 4, 42 (q, 2H, J=7.0 Hz), 4.18 (s, 2H), 1.44 (t, 3H, J=7.0 Hz).

Example 8 Synthesis of Compound 6:

The compound of Example 8 was synthesized in a similar synthetic route as described for Example 6 using 5-fluoro-2-nitroaniline as starting material to give the compound of Example 8 as a brown solid.

MS: [M+1] = 404;

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

Example 9 Synthesis of Compound 44:

- 88 040488

The compound of Example 9 was synthesized in a similar synthetic route as described for Example 6 using 5-fluoro-2-nitroaniline as starting material to give the compound of Example 9 as a brownish solid.

MS: [M+1] = 418;

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

Example 10 Synthesis of Compound 45:

The compound of Example 10 was synthesized in a similar synthetic route as described for Example 6 using 5-fluoro-2-nitroaniline as starting material to give the compound of Example 10 as a brownish solid.

MS: [M+1] = 438;

1H-NMR (500 MHz, CDCI3) δ: 7.90 (dd, 1H, J=3.0, 8.0 Hz), 7.77 (s, 1H), 7.61 (dd, 1H, J= 5.0, 9.0 Hz), 7.36 (m, 1H), 7.25 (br.s, 4H), 4.42 (q, 2H, J=7.0 Hz), 4.14 ( s, 2H), 1.44 (t, 3H, J=7.0 Hz).

Example 11 Synthesis of Compound 46:

The compound of Example 11 was synthesized in a similar synthetic route as described for Example 6 using 5-fluoro-2-nitroaniline as starting material to give the compound of Example 11 as a yellowish solid.

MS: [M+1] = 422;

'H-NMR (500 MHz, CDCl ₃ ) δ: 7.90 (dd, 1H, J=3.0, 8.5 Hz), 7.77 (s, 1H), 7.61 (dd, 1H, J=5.0, 9.0 Hz), 7.36(m, 1H), 7.28(m, 2H), 6.96(m, 2H), 4.42(k, 2H, J=7 .5 Hz), 4.14 (s, 2H), 1.44 (t, 3H, J=7.0 Hz).

Example 12 Synthesis of Compound 47:

The compound of Example 12 was synthesized in a similar synthetic route as described for Example 6 using 2-nitroaniline as starting material to give the compound of Example 12 as a yellowish solid.

MS: [M+1] = 420;

'h-NMR (500 MHz, CDCI3) δ: 8.16 (br. d, 1H, J=7.0 Hz), 7.80 (s, 1H), 7.64 (m, 3H), 7. 25 (m, 4H), 4.41 (q, 2H, J=7.0 Hz), 4.14 (s, 2H), 1.44 (t, 3H, J=8.0 Hz).

Example 13 Synthesis of Compound 109:

Acetamidoxime (50 mg, 0.67 mmol) was azeotropically distilled with toluene 3 times. THF (5 ml) was added followed by 60% NaH in oil dispersion (25 mg, 0.62 mmol). The suspension was stirred at room temperature for 30 minutes. 2 ml of this suspension was added to ester compound 6 (40 mg, 0.099 mmol) and the resulting solution was heated at 70° C. for 3 hours. The solution was quenched with water. The solution was extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over MgSO ₄ . Filtration and concentration provided the crude product. Chromatography (RediSep column with 12 g silica gel. Eluted with 50% EtOAc in hexane) provided 6 mg (20% yield) of Compound 109 as a yellow solid.

MS: [M+1] = 414;

- 89 040488 ¹ H-NMR (CDCl ₃ ) δ: 7.93 (1H, dd, J=3, 8.5 Hz), 7.89 (1H, s), 7.65 (1H, dd, J= 5.5, 9 Hz), 7.38(1H, m), 7.23(5H, m), 4.2(2H, s), 2.50(3H, s).

Example 14 Synthesis of Compound 7:

Meo

A solution of NaNO2 (2.05 g, 29.7 mmol) in H2O (8 ml). The internal temperature was maintained below 5°C. After the addition, the mixture was allowed to warm to room temperature for 1 h. The mixture was cooled to 0°C and a solution of SnCl ₂ -2H ₂ O (20.13 g, 89.2 mmol) in HCl (conc. 13 ml) was slowly added dropwise ). After the addition, it was stirred at room temperature for 2 hours. The resulting yellow solid was collected by filtration and washed with cold (0°C) 6N. HCl. Drying in a vacuum oven gave 3.245 g (50% yield) of a brown solid as arylhydrazine 25.

MS: [M+H ₂ O+Na] = 224.

In a separate flask, a mixture of diethyl 1,3-acetone dicarboxylate (2.426 g, 12 mmol) and diethoxymethyl acetate (1.946 g, 12 mmol) was heated under microwave radiation at 100°C for

h. The reaction mixture was concentrated under vacuum and the residual volatile component was co-distilled off with toluene (5 ml) under vacuum to obtain the condensation product 26, which was used directly in the next stage.

Scheme 13

NaNQ2 HCl (conc.)

H2O

(~50% for 2 stages) ² $

MS (4A)

EUN, ct

ABOUT

100°C, 1h EtOiC pure, microwave radiation

Dibal-N CH _? Ch 74%

M

H?, Pd/C EtOAc/EtOH (-78% over 2 steps)

1) 1,2,4-triazole POCI ₃ , 0Pr ₂ NEt CH ₃ CN

2) CN^CO ₂ Et

ΚΟ-ί-Bu, DMF

Product 26 above was dissolved in EtOH (30 ml). Molecular sieves (4 A, 2 g) and hydrazine hydrochloride 25 (2.19 g, 10 mmol) were added. The suspension was stirred at room temperature for 24 hours. Filtered through celite and the solid was washed with EtOAc (3x10 ml). The filtrate was concentrated. The residue was purified by chromatography (RediSep column with 40 g silica gel, 10 to 40% EtOAc in hexanes) to give 2.091 g of pyrrole 27, which was used without further purification in the next step.

MS: [M+1] = 378.

The above nitro group 27 (2.09 g, 5.5 mmol) was reduced in EtOH (40 mL) with Pd/C (10 wt%, 295 mg, 0.28 mmol) under H2 (balloon) for 18 h. The mixture was filtered through celite. The filtrate was concentrated and the residue was purified by chromatography (RediSep column with 24 g silica gel, hexane to 50% EtOAc in hexane) to give 1.127 g of non-cyclized product 28 as

- 90 040488 yellow sticky oil ([M+1] = 348) plus 154 mg of cyclized product 29 as a gray solid (MS: [M+1] = 302). Non-cyclized aniline 28 (1.127 g, 3.2 mmol) in p-xylene (20 ml) was treated with a catalytic amount of p-TsOHH ₂ O (15 mg) in an oil bath at 140°C for 20 h. The reaction mixture was cooled, concentrated and the residue was triturated with cold (0°C) EtOAc. Filtration provided 559 mg of lactam product 29 as a yellow solid. The total weight of the combined lactam product 29 is 713 mg (24% over three steps).

MS: [M+1] = 302.

To a suspension of ester 29 (566 mg, 1.88 mmol) in CH ₂ Cl ₂ (35 ml) at -78°C was added Dibal-H (1 M in hexane, 6.60 ml, 6.60 mmol). The suspension was stirred for 10 min at -78°C. The cold bath was removed and stirred for 20 minutes until the temperature rose to room temperature. At this point, TLC indicated; 80% completion of the reaction. Cooled to -78°C and added more Dibal-N (1 M in hexane, 1.0 ml, 1.0 mmol). After stirring at -78°C for 30 min, LCMS indicated that the reaction was complete. The reaction was quenched by adding an aqueous solution of Rochelle's salt (20%) followed by the addition of EtOAc. Stir vigorously at room temperature until the mixture became a clear two-layer mixture. The layers were separated and the aqueous layer was extracted three times with EtOAc. The combined organic phase was washed with brine and dried over Na ₂ SO ₄ . Filtration and concentration provided 480 mg of crude alcohol 30 as a slightly yellow solid.

MS: [M+1] = 260.

To a solution of alcohol 30 (200 mg, 0.77 mmol) and CBr ₄ (640 mg, 1.93 mmol) in DMF (8 ml) was slowly added a solution of PPh ₃ (486 mg, 1.85 mmol) in DMF (2 ml ) after 30 min. After the addition, the mixture was stirred at room temperature for 30 minutes. Water was added to quench the reaction and the mixture was extracted three times with EtOAc. The combined extracts were washed successively with H2O, brine and dried over Na ₂ SO ₄ . Filtration and concentration provided the crude product.

Chromatography (RediSep column with 12 g silica gel, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexane, solvent B: EtOAc; gradient eluent: 10 to 40% B in A) provided 221 mg of a mixture of bromide 31 and Ph3PO.

The above mixture in EtOAc/EtOH (8 ml/8 ml) with Pd/C (10 wt%, 200 mg, 0.19 mmol) was stirred under H2 (balloon) for 1 h. It was filtered through celite. The filtrate was concentrated and the residue was purified by chromatography (RediSep column with 12 g silica gel, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexane, solvent B: EtOAc; gradient eluent: 10 to 40% B in A ) to give 146 mg of a mixture of reduced product 32 ([M+1] = 244) and Ph ₃ PO.

In a separate flask, 1,2,4-triazole (81 mg, 1.17 mmol) in CH3CN (1 ml) at 0°C was treated with i-Pr ₂ NEt (214 μl, 1.23 mmol) followed by the addition of POCl ₃ ( 34 µl, 0.36 mmol). The solution was stirred at 0°C for 2 hours. Lactam 32 (purity; 60% by LCMS) was added in one portion and the resulting suspension was heated in an oil bath at 80°C for 18 hours. Water was added to quench the reaction. Extracted three times with EtOAc. The combined extracts were washed successively with H2O, brine and dried over Na ₂ SO ₄ . Filtration and concentration provided 126.6 mg of crude product 33 as a yellow glue, which was used directly in the next reaction.

MS: [M+1] = 295.

A solution of KO-t-Bu (97 mg, 0.86 mmol) in DMF (1 ml) was cooled to -50°C. Ethyl isocyanoacetate (104 μl, 0.95 mmol) was added dropwise. The mixture was stirred at -50°C for 1 hour. The above crude product 33 in DMF (1.5 ml) was added dropwise. The mixture was allowed to warm to 10° C. and stirred at 10° C. for 1 h. Saturated aqueous NH ₄ Cl was added and extracted three times with EtOAc. The combined extracts were washed successively with water, brine and dried over Na ₂ SO ₄ . Filtration and concentration provided the crude product. Chromatography (RediSep column with 12 g silica gel, solvent A: 1:1 v/v CH ₂ Cl ₂ /hexane, solvent B: EtOAc; gradient eluent: 10 to 40% B in A) provided 22 mg of a white solid , which was further purified by preparative TLC (designed with 1:1 A/B) to give 12.8 mg of the final product Compound 7 (Example 14) as a white solid.

MS: [M+1] = 339;

¹ H-NMR (500 MHz, CDCU) δ: 7.70 (s, 1H), 7.56 (s, 1H), 7.50 (d, 1H, J=3.0 Hz), 7.43 ( d, 1H, J=8.5 Hz), 7.00 (dd, 1H, J=2.5, 9.5 Hz), 5.29 (br.s, 1H), 4.44 (c, 2H , J=7.0 Hz), 3.92 (s, 3H), 3.55 (br. s, 1H), 2.17 (s, 3H), 1.45 (t, 3H, J=7, 0 Hz).

Example 15 Synthesis of Compound 8:

- 91 040488

Scheme 14

To a solution of alcohol 30 (261 mg, 1.0 mmol), which was obtained in Example 14, in DMSO (4 ml) and CH ₂ Cl ₂ (6 ml) was added Et ₃ N (0.7 ml, 5 mmol) followed by adding Py-SO ₃ (398 mg, 2.5 mmol). Stirred at room temperature for 1 hour. The reaction mixture was poured into water and extracted three times with EtOAc. The combined extracts were washed successively with H2O, brine and dried over Na ₂ SO ₄ . Filtration and concentration provided 226 mg of crude aldehyde 34 as a yellow solid. It was used in the next step without purification.

MS: [M+1] = 258.

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

To a solution of the above crude alcohol 35 in CH ₂ Cl ₂ (10 ml) with Et ₃ SiH (0.66 ml, 4.10 mmol) was added TFA (0.70 ml, 9.02 mmol). The reaction solution was stirred at room temperature for 1 hour. After concentration, the residue was purified by chromatography (RediSep column with 24 g silica gel, 10 to 50% EtOAc in hexane) to give 187.8 mg (59% yield over three steps) of the product 36 as a gray solid.

MS: [M+1] = 320.

In a separate flask, a solution of 1,2,4-triazole (127 mg, 1.83 mmol) in CH3CN (1.6 ml) at 0°C was treated with i-Pr ₂ NEt (336 μl, 1.93 mmol) followed by POCl ₃ (53 μl, 0.56 mmol). The mixture was stirred at 0°C for 2 hours. Lactam 36 (150 mg, 0.47 mmol, solid) was added to the reaction mixture in one batch and heated in an oil bath at 80°C for 18 hours. The mixture was cooled to room temperature and a solid precipitate was observed. Water (2.1 ml) was added and stirred at room temperature for 10 minutes. Filtration, washing the solid with 2 ml of water followed by drying under high vacuum provided 118.8 mg (69% yield) of triazolamidine 37 as a white with a yellowish or greyish tint, friable solid.

MS: [M+1] = 371.

A solution of KO-t-Bu (72 mg, 0.64 mmol) in DMF (2 ml) was cooled to -50°C. (77 μl, 0.71 mol) was added dropwise. The resulting mixture was stirred at

Ethyl isocyanoacetate

-50°C for 1 hour

Triazolamidine 37 (118.8 mg, 42 µmol, solid) was added in batches. The stirred mixture was allowed to warm to 10° C. over 1 h and kept at 10° C. for 1 h. Saturated aqueous NH ₄ Cl was added and extracted three times with EtOAc. The combined extracts were washed successively with H ₂ O, brine and dried over Na ₂ SO ₄ . Filtration, concentration, then chromatography (RediSep column with 12 g silica gel. Solvent A: 1:1 v/v CH ₂ Cl ₂ /hexane, solvent B: EtOAc; gradient eluent: A to 40% B in A) provided 125.1 mg (94% yield) Compound 8 as a white solid.

MS: [M+1] = 415;

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

Example 16 Synthesis of Compound 9:

- 92 040488

Scheme 15

LiOH (1.09 g, 45.5 mmol) was added to a stirred solution of ester 16 (prepared in Example 1) (2.75 g, 9.10 mmol) in THF (24 ml) and water (20 ml) at room temperature. temperature. MeOH (4 ml) was added and stirring was continued for 2 hours at room temperature, at which point LCMS showed complete consumption of the ester. After concentration under vacuum, the reaction mixture was acidified to pH 3-4 by adding 2N. HCl (20 ml). After 20 minutes of stirring, the reaction mixture was cooled to 0°C, the solid precipitate was collected by filtration, washed with 3-4 ml of water and dried to obtain 1.59 g (64%) of the corresponding acid 38 as a grayish solid.

MS: [M+1] = 275.

To acid 38 (1.59 g, 5.8 mmol) suspended and stirred in DCM (30 ml) was added EDC (5.6 g, 29.2 mmol), benzyl alcohol (2.5 g, 23.2 mmol) and DMAP (3.54 g, 29.2 mmol). After 3 days of stirring at room temperature, the reaction mixture was concentrated under vacuum. Water (80 ml) was added to the suspension, followed by diethyl ether (40 ml), and the mixture was vigorously stirred for 40 minutes, after which the suspension precipitated and was collected by suction filtration. The solid was washed with water and a small amount of diethyl ether and dried to give 1.65 g (78%) of benzyl ether 39 as a white solid.

MS: [M+1] = 365.

1,2,4-triazole compound (1.22 g, 17.7 mmol) in CH3CN (15 mL) at 0°C was treated with i-Pr ₂ NEt (3.24 mL, 18.6 mmol) followed by POCl ₃ (0.507 ml, 5.44 mmol). The solution was stirred at 0°C for 2 hours. Benzyl ether 39 (1.65 g, 4.53 mmol) was added in batches and the resulting suspension was heated in an oil bath at 80°C for 18 hours. LCMS showed 5-10% remaining original lactam. In a separate flask, 1,2,4-triazole (307 mg, total 4.9 eq.) in CH3CN (3.8 ml) was treated with i-Pr ₂ NEt (0.82 ml, total 5.1 eq.) and POCl ₃ (0.127 ml; total 1.5 eq.) at 0°C for 2 hours. The resulting clear solution was transferred to the above reaction mixture. After 2 h heating at 80°C, the reaction mixture was cooled to room temperature, water was slowly added to quench the reaction (10 min). After cooling in an ice bath, the solids formed were collected by filtration, washed with water (5 ml) and dried to give 1.61 g (86%) of product 40 as a light yellow solid.

MS: [M+1] = 416.

A solution of KO-t-Bu (0.739 g, 6.59 mmol) in DMF (11 ml) was cooled to -50°C. Ethyl isocyanoacetate (0.810 mL, 7.00 mmol) was added dropwise. The mixture was stirred at -50° C. for 1 h. The above triazole intermediate 40 (1.61 g, 3.87 mmol) was added. The mixture was stirred at -50°C for 30 min and slowly warmed to room temperature over 4-5 h. Saturated aqueous NH ₄ Cl (10 ml) was added followed by EtOAc (10 ml). The mixture was sonicated to break up solid pieces, then thoroughly mixed for 30 minutes. The precipitate was collected by filtration, washed with water, Et2O and dried to give the crude product as a white solid. The filtrate was partitioned between water and EtOAc; the aqueous layer was separated and extracted twice with EtOAc; the combined EtOAc layer was washed with brine and dried over

- 93 040488

_MgSO4 . Filtration and removal of the solvent provided a solid residue which was combined with the solid obtained above for chromatographic purification using a RediSep column with g silica gel and gradient elution from 0.5 to 5% MeOH in DCM to give 1.78 g (100% ) imidazole 41 as a white solid.

MS: [M+1] = 460.

Benzyl ester 41 (1.78 g, 3.87 mmol) was subjected to hydrogenolysis (hydrogen balloon) in the presence of a catalytic amount of 10% Pd on carbon in a mixture of solvents THF (40 ml), MeOH (20 ml) and EtOAc (20 ml) within 20 hours, LCMS showed complete disappearance of the starting material. The solid catalyst was removed by filtration through celite and washed repeatedly with sufficient 30% MeOH in DCM until nearly all products were recovered (TLC monitoring). The product-containing filtrate was concentrated in vacuo to give 1.22 g (85%) of the acid product 42 as an off-white solid.

MS: [M+1] = 370.

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

MS: [M+1] = 356.

Diisopropyl azodicarboxylate (48.3 mg, 0.233 mmol) was added dropwise to a stirred solution of alcohol 43 (37.5 mg, 0.106 mmol), phenol (14.9 mg, 0.158 mmol) and Ph ₃ P (55.6 mg, 0.212 mmol ) in anhydrous THF (0.8 ml) at 0°C. The ice bath was removed and stirring continued at room temperature for 16 hours. LCMS indicated complete disappearance of the starting alcohol. The reaction mixture was partitioned between sat. _NaHCO3 and EtOAc. The organic layer was separated and washed with water, brine and dried over MgSO4. The desired product was isolated from the reaction mixture by two sequential preparative TLC (4% MeOH in DCM and hexane/EtOAc/MeOH = 47.5/47.5/5, v/v/v) to give 5.3 mg (12%) of the product which is Compound 9 as a white solid.

MS: [M+1] = 432;

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.77 (s, 1H), 7.63 (d, 1H, J=3.5 Hz), 7.53 (d, 1H, J=9, 0 Hz). 7.31 (m, 2H), 7.17 (dd, 1H, J=3.0, 8.5 Hz), 7.08 (d, 2H, J=7.0 Hz), 6.99 (t , 1H, J=6.5 Hz), 5.30 (s, 2H), 4.40 (c, 2H, J=7.0 Hz), 3.96 (s, 3H), 1.38 (t , 3H, J=7.0 Hz).

Example 17 Synthesis of Compound 10:

The compound of Example 17 was synthesized in a similar synthetic route as described for Example 16 using 4-fluorophenol in the final step to give Compound 10 (4.9 mg) as a white solid.

MS: [M+1] = 450;

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

Example 18 Synthesis of Compound 11:

The compound of Example 18 was synthesized in a similar synthetic route as described for Example 16 using 3-methoxyphenol in the final step to give Compound 11 (6.1 mg) as a white solid.

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MS: [M+1] = 462;

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.76 (s, 1H), 7.63 (d, 1H, J=2.5 Hz), 7.53 (d, 1H, J=9, 0 Hz), 7.15-7.22 (m,

2H), 6.67 (m, 2H), 6.55 (br.dd, 1H, J=2.5, 8.0 Hz), 5.28 (s, 2H), 4.39 (q, 2H , J=7.0 Hz), 3.96 (s, 3H), 3.81 (s,

3H), 1.39 (t, 3H, J=7.0 Hz).

Example 19 Synthesis of Compound 12:

The compound of Example 19 was synthesized in a similar synthetic route as described for Example 16 using 2,4-dimethylphenol in the final step to give Compound 12 (3.1 mg) as a white solid.

MS: [M+1] = 460;

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

Example 20 Synthesis of Compound 107:

Phenol ( ₃₀ mg, 0.32 mmol ), triphenylphosphine (84 mg, 0.32 mmol). The reaction mixture was stirred at room temperature for 15 minutes. Then cooled in an ice bath and slowly added DIAD (64 μl, 0.32 mmol) in THF (0.2 ml). The ice bath was removed and the reaction mixture was stirred at room temperature for 18 hours. LCMS still showed some starting material. Phenol (10 mg), triphenylphosphine (28 mg) and DIAD (21 μl) were added to the reaction mixture and stirred for another 1 h. The solvent was evaporated and the crude material was purified by chromatography (RediSep column with 12 g silica gel. Eluent: EtOAc ) and preparative TLC (eluting solvent: 5% MeOH/47.5% EtOAc/47.5% hexane) to give 11.4 mg (16% yield) of Compound 107.

MS: [M+1] = 421);

¹ H-NMR (CDCl3) δ: 7.92 (1H, dd, J=3.5, 8.5 Hz), 7.80 (1H, s), 7.63 (1H, dd, J=5, 10Hz), 7.38(1H, m), 7.31(2H, t, J=8.5Hz), 7.07(2H, d, J=8.5Hz), 7.00(1H , t, J=8.5 Hz), 5.3 (2H, s), 4.39 (2H, k, J=7 Hz), 1.38 (3H, t, J=7 Hz).

Example 21 Synthesis of Compound 111:

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

MS: [M+1] = 403.

To a suspension of the above alkyl bromide derivative (30 mg; 0.075 mmol) in anoxic DME (2.7 ml) was added 3-pyridineboronic acid (14 mg, 0.11 mmol) and 2 M Na ₂ CO ₃ solution (0.22 ml, 0 .44 mmol). The suspension was stirred at room temperature for 5 min, then PdCl ₂ (PPh ₃ ) ₂ (10 mg, 0.015 mmol) was added. The suspension was heated at MW at 85°C for 1 h. The reaction mixture was cooled, diluted with water and extracted with EtOAc (twice). The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration provided the crude product, which was purified using 2 prep. TLC (eluting system: 3%

MeOH in DCM) to give 5.3 mg (18% yield) of Compound 111.

MS: [M+1] = 401;

¹ H-NMR (CDCl ₃ ) δ: 8.66 (1H, br.s), 8.48 (1H, br.s), 7.96 (1H, s), 7.79 (1H, s), 7.66 (1H, d, J=8 Hz), 7.50 (1H, d, J=8 Hz), 7.43 (1H, d, J=7 Hz), 7.23 (1H, m) , 4.42 (2H, q, J=7 Hz), 4.18 (2H, s), 2.54 (3H, s) 1.44 (3H, t, J=7 Hz).

Example 22 Synthesis of Compound 48:

Scheme 16

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

MS: [M+1] = 354.

To a stirred solution of aldehyde 57 (15.5 mg, 0.0439 mmol) in 1,2-dichloroethane (0.3 ml) at room temperature was added pyrrolidine (5.5 μl, 0.0658 mmol). After 2 minutes of stirring, the solution became clear and NaBH(OAc) ₃ (14.4 mg) was added. The reaction mixture was stirred for 4 h, and extinguished with saturated NaHCO ₃ and was extracted three times with ethyl acetate. The combined organic layer was washed with water, brine and dried over Na ₂ SO ₄ . Rev. TLC with 10% MeOH in DCM provided 13.1 mg (73%) of the desired Compound 48 as a clear filmy solid.

MS: [M+1] = 409;

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.74 (s, 1H), 7.62 (d, 1H, J=3.0 Hz), 7.51 (d, 1H, J=9, 0 Hz) 7.14 (dd, 1H, J=3.5, 9.0 Hz), 4.42 (k, 2H, J=6.5 Hz), 3.94 (s, 3H), 3, 87 (br.s, 2H), 2.65 (br.s, 4H), 1.79 (br.s, 4H), 1.44 (t, 3H, J=7.0 Hz).

Example 23 Synthesis of Compound 49:

The compound of Example 23 was synthesized in a similar synthetic route as described for Example 22, using morpholine in the final step to give the compound of Example 23 as a clear, filmy solid.

MS: [M+1] = 425;

1H-NMR (500 MHz, CDCl3) δ: 7.75 (s, 1H), 7.63 (d, 1H, J=3.0 Hz), 7.52 (d, 1H, J=9.5 Hz ), 7.15 (dd, 1H, J=3.0, 9.0 Hz), 4.42 (c, 2H, J=7.5 Hz), 3.95 (s, 3H), 3.76 (br.s, 2H), 3.71 (br.s, 4H), 2.57 (br.s, 4H), 1.44 (t, 3H, J=8.0 Hz).

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Example 24 Synthesis of Compound 50:

The compound of Example 24 was synthesized in a similar synthetic route as described for Example 22 using diethylamine in the final step to give the compound of Example 24 as a clear filmy solid.

MS: [M+1] = 411;

1H-NMR (500 MHz, CDCi ₃ ) δ: 7.74 (s, 1H), 7.64 (br. d, 1H, J=3.0 Hz), 7.51 (d, 1H, J=9 .0 Hz), 7.15 (dd, 1H, J=2.5, 9.0 Hz), 4.43 (k, 2H, J=6.5 Hz), 3.96 (s, 3H), 3.86 (br.s, 2H), 2.64 (br.s, 4H), 1.44 (t, 3H, J=8.5 Hz), 1.15 (br.s, 6H).

Example 25 Synthesis of Compound 51:

The compound of Example 25 was synthesized in a similar synthetic route as described for Example 22 using methylbenzylamine in the final step to give the compound of Example 25 as a clear film-like solid.

MS: [M+1] = 459;

¹ H-NMR (500 MHz, CDCl3) δ: 7.75 (s, 1H), 7.63 (d, 1H, J=3.0 Hz), 7.51 (d, 1H, J=8.5 Hz), 7.36 (br.d, 2H, J=8.0 Hz), 7.30 (m, 2H), 7.23 (m, 1H), 7.15 (dd, 1H, J=3 .0, 9.0 Hz), 4.38 (k, 2H, J=7.5 Hz), 3.95 (s, 3H), 3.85 (br. s, 2H), 3.63 (br. .s, 2H), 2.25 (s, 3H), 1.41 (t, 3H, J=7.0 Hz).

Example 26 Synthesis of Compound 170:

Isobutyramidoxime (41.8 mg, 0.41 mmol) and ester 48 (27.9 mg, 0.0683 mmol) in a round bottom flask were azeotropically distilled in toluene on a rotary evaporator several times, suspended in anhydrous THF (0.6 ml ) and then cooled to 0°C. NaH (60% oily suspension; 10.9 mg, 0.273 mmol) was added. The ice bath was removed, the reaction mixture was stirred at RT for 20 min before heating at 70°C for 6 h and cooled. Water (4 ml) was added and the mixture was extracted three times with EtOAc. The combined organic solution was washed with brine and dried over MgSO ₄ . Rev. TLC with 10% MeOH in EtOAc provided 10.4 mg (34%) of the desired Compound 170 product as a clear filmy solid.

MS: [M+1] = 447.

Example 27 Synthesis of Compound 52:

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Scheme 17

Ri = OMe; R ₂ = m-CI-Ph: compound 52

Ri = OMe; R ₂ = m-CN-Ph: compound 53

Ri = Me; R ₂ = o-CI-Ph: compound 54

Ri = Me; R ₂ = Ph: compound 101

Ri=OMe; o-CI-Ph: compound 108

The starting alcohol 43 (160 mg, 0.45 mmol) was treated with phosphorus oxide tribromide (400 mg, 1.4 mmol) in acetonitrile (10 mL) at 80°C for 5 h. The reaction mixture was then cooled to 0°C, quenched sat. NaHCO ₃ and extracted twice with dichloromethane. The combined dichloromethane solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under vacuum provided 173.3 mg (92%) of the bromide as a yellowish foamy solid.

MS: [M+1] = 418.

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

MS: [M+1] = 450;

'S-NMR (500 MHz, CDCl ₃ ) δ: 7.75 (s, 1H), 7.64 (d, 1H, J=3.0 Hz), 7.51 (d, 1H, J=9, 5 Hz), 7.31 (br.s, 1H), 7.23 (br.s, 1H), 7.17 (m, 3H), 4.43 (k, 2H, J=7.0 Hz) , 4.15 (s, 2H), 3.96 (s, 3H) 1.44 (t, 3H, J=8.0 Hz).

Example 28 Synthesis of Compound 53:

The compound of Example 28 was synthesized in a similar synthetic route as described for Example 27 using 3-cyanophenylboronic acid in the final step to give the compound of Example 28 as a brownish solid.

MS: [M+1] = 441;

¹ H-NMR (500 MHz, CDCl3) δ: 7.75 (s, 1H), 7.66 (br. s, 1H), 7.64 (d, 1H, J=3.0 Hz), 7. 61 (br.d, 1H, J=7.5 Hz), 7.39 (t, 1H, J=7.5 Hz), 7.16 (dd, 1H, J=3.5, 9.5 Hz ), 4.45 (q, 2H, J=7.0H), 4.20 (s, 2H), 3.96 (s, 3H), 1.45 (t, 3H, J=7.0 Hz) .

Example 29 Synthesis of Compound 54:

The compound of Example 29 was synthesized in a similar synthetic route as described for Example 27 starting from an alcohol in which R ₁ = methyl and using 2-chlorophenylboronic acid in the final step to give the compound of Example 29 as a brownish solid.

MS: [M+1] = 434.

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Example 30 Synthesis of Compound 101:

The compound of Example 30 was synthesized in a similar synthetic route as described for Example 27 starting from an alcohol in which R1 = methyl and using phenylboronic acid in the final step to give the compound of Example 30 as a brownish solid which was purified by chromatography (column RediSep with 4 g silica gel Elution solvent: EtOAc), then prep. TLC (eluting system: 40% DCM/40% hexane/17% EtOAc/3% MeOH) gave 5.9 mg (31% yield) of Compound 101.

MS: [M+1] = 402;

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

Example 31 Synthesis of Compound 102:

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

MS: [M+1] = 32 4;

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

Example 32 Synthesis of Compound 108:

To a suspension of a bromide derivative in which R ₁ = OMe (18 mg; 0.043 mmol) in oxygen-free DME (2 ml) was added 2-chlorophenylboronic acid (10 mg, 0.065 mmol) and 2 M Na ₂ CO ₃ solution (0.13 ml, 0.26 mmol). The suspension was stirred at room temperature for 15 min, then PdCl ₂ dppf (7 mg, 0.009 mmol) was added. The suspension was heated in an oil bath at 85°C for 1 h. The reaction mixture was diluted with water and extracted with EtOAc (twice). The combined extracts were washed with brine and dried over Na ₂ SO ₄ . Filtration and concentration provided a crude product which was purified with prep. TLC (eluting system: 5% MeOH/47.5% Hex/47.5% EtOAc) to give 3.5 mg (18% yield) of Compound 108.

MS: [M+1] = 451;

¹ H-NMR (CDCl ₃ ) δ: 7.77 (1H, s), 7.63 (1H, d, J=3 Hz), 7.52 (1H, d, J=11.5 Hz), 7 .36 (1H, m), 7.31 (1H, m), 7.18 (2H, m), 7.14 (1H, dd, J=3.9 Hz), 4.38 (2H, k, J=7 Hz), 4.27(2H, s), 3.94(3H, s), 1.41(3H, t, J=7 Hz).

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Scheme 18a

Diagram 18b

Example 33 Synthesis of Compound 55:

To a solution of compound 58 (6.6 g, 33.5 mmol) in dichloromethane (100 ml) were added DIPEA (8.65 g, 67 mmol), HOBt (5.4 g, 36.85 mmol) and EDCI (9. 6 g, 50.3 mmol). After approximately 15 minutes of stirring, a solution of 2,4-dimethoxybenzylamine (5.6 g, 33.5 mmol) in dichloromethane (50 ml) was added dropwise under nitrogen atmosphere to the homogeneous reaction mixture. The resulting mixture was stirred under nitrogen at room temperature for 16 h. NaOH (100 ml), water (100 ml) and saline (100 ml). The organic phase was then dried over Na ₂ SO ₄ and evaporated to give crude solid 59 which was crystallized from ethyl ether. Filtration and drying with suction in open air provided a white with a yellowish or grayish tint solid pure product of 9.8 g (96%).

MS: [M+1] = 347.

To a solution of compound 59 (9.8 g, 28.3 mmol) in MeOH/EtOAc (1:1, 100 ml) was added 10% wet Pd-C (1.8 g, 10% mmol). After three successive evacuation and nitrogen purges, the heterogeneous reaction mixture was balloon hydrogenated at atmospheric pressure until hydrogen uptake ceased, approximately 4 h. The reaction mixture was filtered through a Celite pad and evaporated to give the pure desired product 60 as a brown oil .63 g (96%) (MS: [M+1=317]). This product was used directly in the next step.

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

MS: 439.

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

MS: [M+1] = 357.

To compound 62 (4.46 g, 12.52 mmol) dissolved in 2.5:1 THF/DMF (50 ml) was added t-BuOK (97%, 1.88 g, 16, 28 mmol). The mixture was heated to 25°C and after stirring for 30 min was again cooled to -20°C. After diethyl chlorophosphate (2.35 ml, 16.28 mmol) was added dropwise, the mixture was stirred for 3 hours while heating from -20 to 25°C. The reaction mixture was re-cooled to 0° C. and ethyl isocyanoacetate (1.92 ml, 17.53 mmol) was added thereto. After subsequent cooling to -78°C, t-BuOK (97%, 1.88 g, 16.28 mmol) was added and stirred at RT for 5 h. Progress was monitored by LC/MS. The reaction was quenched by adding 1:1 saturated NaHCO ₃ /H ₂ O (140 ml), the precipitate was filtered off, washed with H 2 O and air dried overnight to give 4.81 g (85%) of the imidazole derivative 63 as a yellow solid.

MS: [M+1] = 452.

To compound 63 (4.81 g, 10.65 mmol) in dichloromethane (35 ml) at 0°C was added trifluoroacetic acid (35 ml), followed by dropwise addition of trifluoromethanesulfonic acid (1.9 ml, 21.31 mmol). The mixture was heated to RT, stirred for 2 h, then concentrated to give a residue which was dissolved in dichloromethane (120 ml). The crude solution was partitioned between chilled saturated NaHCO ₃ and dichloromethane. The organic extracts were combined, dried (MgSO ₄ ), filtered and concentrated to give 3.2 g (99%) of unprotected product 64 (brown solid) of sufficient purity for the next step.

MS: [M+1] = 302.

POC13 (52.7 mg, 0.344 mmol). The reaction mixture was then heated at 135° C. for 2 hours. After cooling to room temperature, phenoxyacetic acid hydrazide (228.4 mg, 1.36 mmol) was added in situ to imino chloride 65, followed by DIPEA (90 μl). The reaction mixture was stirred at room temperature for 30 min, then heated at 100°C for 90 min. The reaction mixture was cooled, saturated NaHCO ₃ (aq.) was added and extracted three times with ethyl acetate; the combined organic layer was washed with brine and dried over MgSO ₄ . After filtration and concentration, the product as Compound 55 was isolated by ISCO flash column chromatography (4 g RediSep column, 1 to 10% MeOH in DCM as elution gradient) as a white solid, wt: 8.6 mg.

MS: [M+1] = 432;

1H-NMR (500 MHz, CDC13) δ: 7.81 (s, 1H), 7.71 (d, 1H, J=3.5 Hz), 7.52 (d, 1H, J=9.0 Hz ), 7.32 (m, 2H), 7.21 (dd, 1H, J=2.5, 8.5 Hz), 7.11 (d, 2H, J=8.5 Hz), 7.02 (m, 1H), 5.44 (s, 2H), 4.38 (q, 2H, J=7.5 Hz), 3.94 (s, 3H), 1.39 (t, 3H, J= 7.0 Hz).

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Example 34 Synthesis of Compound 56:

The compound of Example 34 was synthesized in a similar synthetic route as described for Example 33 using 4-fluorophenoxyacetic acid hydrazide in the final step to give the compound of Example 34 as a yellowish solid.

MS: [M+1] = 450;

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.82 (s, 1H), 7.73 (d, 1H, J=3.5 Hz), 7.53 (d, 1H, J=10, 0 Hz), 7.22 (dd, 1H, J=3.5, 9.0 Hz), 7.08-6.99 (m, 4H), 5.41 (s, 2H), 4.41 ( k, 2H, J=7.0 Hz), 3.95 (s, 3H), 1.42 (t, 3H, J=6.5 Hz).

Example 35 Synthesis of Compound 103:

103

The compound of Example 35 was synthesized in a similar synthetic route as described for Example 33 using 2-methoxyacetic acid hydrazide in the final step to give the compound of Example 35 as a yellowish solid.

MS: [M+1] = 370.

Example 36 Synthesis of Compound 118:

118

Acetamidoxime (8.4 mg, 0.108 mmol) was azeotropically distilled in toluene three times on a rotary evaporator, then suspended in THF (1.0 ml). NaH (60% mineral slurry; 3.3 mg, 0.081 mmol) was added and the mixture was stirred at RT for 10 minutes. Then ester 55 (23.2 mg, 0.054 mmol) was added. After stirring for 40 min at RT, the reaction mixture was heated at 70°C for 4 h. After cooling, cold water (5 ml) was added to the reaction mixture and the precipitate was collected by filtration, washed with water and dried to give 9.7 mg (41% ) of the desired product as a yellowish solid.

MS: [M+1] = 442.

Example 37 Synthesis of Compound 128:

128

The compound of Example 37 was synthesized in a similar synthetic route as described for Example 36 above, using the ester of Compound 103 in the final step to give the compound of Example 37 as a brownish solid.

MS: [M+1] = 380.

Example 38 Synthesis of Compound 130:

130

The compound of Example 38 was synthesized in a similar synthetic route as described for Example 36 starting from the ester of Compound 103 and condensing with isobutyramidoxime to give the compound of Example 38 as a yellowish solid.

MS: [M+1] = 408.

Example 39 Synthesis of Compound 119:

119

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Neopentyl alcohol (30.4 mg, 0.345 mmol), DMAP (4, 2 mg, 0.0345 mmol) and EDC (20 mg, 0.104 mmol). After 5 hours stirring, the reaction mixture was diluted with EtOAc, washed with sat. NH4Cl , sat. NaHCO ₃ , brine and dried over MgSO ₄ . Chromatography on silica gel using a gradient of 0 to 8% MeOH in EtOAc provided 11.7 mg (72%) of the desired Compound 119 product as a yellowish solid.

MS: [M+1] = 474.

Example 40 Synthesis of Compound 120:

The compound of Example 40 was synthesized in a similar synthetic route as described for Example 39 above, using 2-propyl alcohol in the final step to give the compound of Example 40 as a yellowish solid.

MS: [M+1] = 446.

Example 41 Synthesis of Compound 129:

Compound 103 (Scheme 18a) (66.1 mg, 0.179 mmol) was hydrolyzed in a solvent system

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

The acid thus obtained was stirred in DMF (0.7 ml) at 0°C. NaHCO ₃ (48.1 mg, 0.572 mmol) was added followed by N-bromosuccinamide (96.7 mg, 0.543 mmol). After stirring overnight, the reaction mixture was diluted with EtOAc and washed with sat. NaHCO3. The aqueous layer was separated and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated. The product bromide was obtained by silica gel column chromatography eluting with a gradient of 0 to 13% MeOH EtOAc as a white solid (Compound 129). Weight: 28.6 mg (53%).

MS: [M+1] = 377.

Example 42 Synthesis of Compound 131:

Compound 129 (22.6 mg, 0.060 mmol) was hydrogenated over 10% Pd-C in EtOAc (1 ml) and MeOH (1 ml) for 16 h. the product of Compound 131 as a slightly yellowish solid.

MS: [M+1] = 298.

Example 43 Synthesis of Compound 122:

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

0.0402 mmol) was treated with POCl ₃ (46.2 mg, 0.302 mmol) in 1,4-dioxane (0.5 ml) at 95° C. overnight. The reaction mixture was then quenched with sat. NaHCO ₃ (5 ml), cooled to 0°C and the precipitate was collected by suction filtration, washed with water and dried to give 13.6 mg (88%) of the nitrile as a brownish solid, Compound 122.

MS: [M+1] = 385.

Example 44 Synthesis of Compound 123:

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

Ethyl magnesium bromide etherate (3 M; 0.323 ml) was added to Weinreb's amide (14.4 mg, 0.0323 mmol) while stirring in THF (0.3 ml) at 0°C. The reaction mixture was allowed to warm to RT and stirred for 14 h, quenched with sat. NH ₄ Cl , extracted three times with EtOAc; the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of solvent provided a crude ketone product which was purified with prep. TLC using 8% MeOH in EtOAc.

Mass: 4.6 mg (34%) Compound 123.

MS: [M+1] = 416.

Example 45 Synthesis of Compound 124:

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

The crude aldehyde (13.7 mg) in DCM (0.7 ml) at RT was treated with deoxofluoro (54.8 mg, 0.248 mmol) for 16 h. The reaction was quenched with sat. NaHCO ₃ (5 ml) for 20 min, extracted three times with EtOAc; the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and solvent removal followed by purification with prep. TLC using 10% MeOH in EtOAc provided 7.5 mg (52%) of the desired Compound 124 difluoride as a yellowish solid.

MS: [M+1] = 410.

Example 46 Synthesis of Compound 142:

142

The above Weinreb amide (8.8 mg, 0.0197 mmol) in THF (0.15 ml) at 0° C. was treated with phenylmagnesium bromide (1 M THF; 0.54 ml) for 2.5 h, sat. quenched. NH4Cl, extracted twice with EtOAc; the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent provided the crude ketone, which was used without further purification. The ketone in THF (0.5 ml) was treated with NaBH ₄ (6 mg) at RT for 2 h, then quenched with sat. NH4Cl, extracted three times with EtOAc; the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of solvent provided crude alcohol which was used without further purification. The alcohol thus obtained in DCM (1.4 ml) was treated with triethylsilane (86.4 mg, 0.75 mmol) and trifluoroacetic acid

- 104 040488 (171.0 mg, 1.5 mmol) at 40°C overnight, then concentrated under vacuum, diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . Filtration and removal of solvent provided a crude benzyl product which was purified by silica gel column chromatography using 0 to 12% MeOH in EtOAc as eluent; 3.6 mg of Compound 142 was obtained as a yellowish solid.

MS: [M+1] = 450.

Scheme 19

Example 47 Synthesis of Compound 106:

106

To lactam 64 (185.7 mg, 0.616 mmol) in chlorobenzene (5 ml) was added N,N-dimethyl-p-toluidine (333.3 mg, 2.465 mmol) and phosphorus oxychloride (188.9 mg, 1.232 mmol). The reaction mixture was heated at 135° C. for 2 h, cooled to RT and formyl hydrazide (296.0 mg, 4.93 mmol) was added followed by diisopropylethylamine (238.8 mg, 1.85 mmol). After 30 min stirring at RT, the reaction mixture was heated at 100° C. for 1 h, cooled and sat. NaHCO ₃ (15 ml), extracted twice with EtOAc; the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of solvent provided the crude triazole product, which was purified by silica gel column chromatography using 0-15% MeOH in EtOAc as eluent, 35.9 mg (18%) was obtained as a brownish solid.

MS: [M+1] = 326.

The above triazole in DCM (1 ml) was treated with N-bromosuccinamide (37.6 mg, 0.21 mmol) at 0°C. The reaction mixture was allowed to warm slowly to RT and the process continued at RT overnight, diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO4. Filtration and removal of solvent provided crude bromide, which was purified by silica gel column chromatography using a gradient of 0-10% MeOH in EtOAc; 22.9 mg (51%) of Compound 106 was obtained as a white with a yellowish or grayish tinge of a solid.

MS: [MS]: 406.

Example 48 Synthesis of Compound 104:

Phenol (20.3 mg, 0.216 mmol), bromide substrate from Example 47 (29.1 mg, 0.0719 mmol), Cs ₂ CO ₃ (117.0 mg, 0.360 mmol), diethyl -1,3acetone dicarboxylate (14.5 mg, 0.0719 mmol) and DMF (0.5 ml). The vessel was purged with nitrogen. CuI (6.8 mg, 0.036 mmol) was added and the mixture was stirred at RT for 5 min before heating at 140° C. under MB irradiation for 60 min. The reaction mixture was diluted with EtOAc, washed with water; aq. the layer was separated and extracted twice with EtOAc; the combined organic solution was washed with brine and dried over MgSO ₄ . Filtration and removal of solvent provided a crude ether product which was purified with prep. TLC using 5% MeOH in

DXM; 6.6 mg of Compound 104 was obtained as a yellowish solid.

MS: [M+1] = 418.

Example 49 Synthesis of Compound 105:

105

The compound of Example 49 was synthesized in a similar synthetic route as described for Example 48 above using 3-methoxyphenol instead of phenol to give the compound of Example 49 as a yellowish foamy solid.

MS: [M+1] = 448.

Scheme 20

R = ί-Ρπ compound 112

NBS/ NaHCO ₃ R = CH _g C(CH ₃ ) _e : compound 113 and R = CH ₂ CF ₃ : compound 114

R - Ph: Compound 13S

R = 3-Pyridyl: Compound 140

R = 1-Me-4-Pyrazolyl: Compound 152

R = 2-Me-4-Pyridyl: Compound 154

Example 50 Synthesis of Compound 112:

112

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

MS: [M+1] = 300.

To a suspension of the acid (30 mg, 0.1 mmol) in dichloroethane (0.2 ml) was added thionyl chloride (0.4 ml; 5 mmol) and DMF (20 μl). The resulting solution was heated at 70° C. for 1 hour. An additional 0.2 ml of thionyl chloride was added and the solution was heated for another 30 minutes. The solvent was removed. The crude material was dried under vacuum. The crude acid chloride (0.1 mmol) was suspended in isopropanol and stirred at room temperature for 18 h. The solvent was evaporated and the crude material was purified by chromatography (4 g silica gel RediSep column eluted with 10% MeOH in DCM) to give 8.6 mg (yield 25%) of Compound 112.

MS: [M+1] = 342.

¹ H-NMR (CDCl3) δ: 7.90 (1H, d, J=9 Hz), 7.79 (1H, br.s), 7.63 (1H, br.s), 7.36 (1H , br.s), 3.48 (1H, m), 2.45 (3H, s), 1.43 (6H, d, J=6.5 Hz).

- 106 040488

Example 51 Synthesis of Compound 113:

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

MS: [M+1] = 370;

'H-NMR (CDCI3) δ: 7.91 (1H, dd, J=3.9 Hz), 7.79 (1H, s), 7.61 (1H, dd, J=4.5, 9 Hz ), 7.35(1H, m), 4.11(2H, s), 2.44(3H, s), 1.07(9H, s).

Example 52 Synthesis of Compound 114:

The crude acid chloride (0.066 mmol) obtained above was suspended in dichloroethane (1 ml) and 2,2,2-trifluoroethanol (0.1 ml, 1.4 mmol) followed by the addition of triethylamine (0.6 ml, 4.3 mmol ). The solution was stirred at room temperature for 2 hours 30 minutes. The solvent was evaporated and the crude material was purified by chromatography (4 g silica gel RediSep column eluted with EtOAc), then purified by prep. TLC (eluting system: 70% EtOAc in hexanes) gave 8.1 mg (32% yield) of Compound 114.

MS: [M+1] = 382;

'H-NMR (CDCI3) δ: 7.91 (1H, dd, J=3.5, 9.5 Hz), 7.83 (1H, s), 7.63 (1H, dd, J=4, 5, 9.5 Hz), 7.35(1H, m), 4.77(2H, m) 2.43(3H, s).

Example 53 Synthesis of Compound 136:

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

MS: [M+1] = 334;

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

Example 54 Synthesis of Compound 139:

General coupling procedure: To a solution of Compound 136 (20 mg, 0.061 mmol) in degassed DME (0.9 ml) and water (0.1 ml) was added phenylboronic acid (11 mg, 0.092 mmol), cesium carbonate (80 mg, 0 .24 mmol) and PdCl ₂ dppf (5 mg, 0.066 mmol). The suspension was heated at 80°C for 1 h. The reaction mixture was diluted with water, extracted with EtOAc (3x). The combined extracts were washed with brine (2x) and dried over MgSO ₄ . Filtration and concentration provided a crude product which was purified with prep. TLC (eluting system: 3% MeOH in

- 107 040488

EtOAc).

Compound 139 was obtained using phenylboronic acid. Received 10.8 mg (yield 54%) of the product.

MS: [M+1] = 332;

¹ H-NMR (CDCl3) δ: 7.87 (1H, dd, J=3.5, 9.5 Hz), 7.85 (1H, s), 7.63 (3H, m), 7.50 (2H, t, J=6.5 Hz), 7.35 (2H, m), 2.41 (3H, s).

Example 55 Synthesis of Compound 140:

Compound 140 was prepared in a similar manner using 3-pyridineboronic acid. Received 8.9 mg (yield 27%) of the product.

MS: [M+1] = 333;

¹ H-NMR (CDCl3) δ: 8.86 (1H, s), 8.63 (1H, d, J=5 Hz), 8.01 (1H, m), 7.90 (2H, m), 7.64(1H, dd, J=5.5, 9Hz), 7.44(1H, m), 7.36(1H, m), 2.39(3H, s).

Example 56 Synthesis of Compound 152:

Compound 152 was prepared using 1-methylpyrazole-4-boronic acid, HCl. Received 12.5 mg (yield 63%) of the product.

MS: [M+1] = 336;

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

Example 57 Synthesis of Compound 154:

Compound 154 was prepared using 2-methylpyridine-4-boronic acid pinacol ester. 7.1 mg (yield 34%) of product was obtained.

MS: [M+1] = 347;

1H-NMR (CDCl3) δ: 8.6 (1H, d, J=6 Hz), 7.89 (1H, dd, J=3.5, 8.5 Hz), 7.87 (1H, s) , 7.64 (1H, dd, J=5.5, 9 Hz), 7.48 (1H, s), 7.36 (2H, m), 2.64 (3H, s), 2.41 ( 3H, s).

- 108 040488

Scheme 21

Example 58 Synthesis of Compound 117:

h \u003d SN ₃ ; compound 127 x = i-Fr compound 133

R=SN ₃ connection 126

R-i-Pr: Connection 132

In a 100 ml round bottom flask, the lactam ester 16' (2 g, 7.35 mmol; which was obtained in the same manner as 16 described in Scheme 11) was dissolved in 60 ml of anhydrous THF. The solution was stirred at room temperature under nitrogen atmosphere. LiBH4 (2 M in THF, 4 ml, 8 mmol) was added slowly. The reaction mixture was stirred under nitrogen for 18 h. More LiBH4 (2 M in THF, 2 mL, 4 mmol) was added slowly. The reaction mixture was stirred for another 24 hours. A mixture of EtOAc/EtOH (20 ml/20 ml) was added to the reaction mixture and it was concentrated. The residue was dissolved in MeOH and silica gel was added. After evaporation of the volatile solvents, the solid was loaded onto a RediSep column with 40 g of silica gel. The desired product was eluted 5:1 v/v. CH ₂ Cl ₂ /MeOH. The alcohol was obtained as a white solid (1.14 g, 67% yield).

MS: [M+1] = 231.

Alcohol (1.14 g, 4.96 mmol) was suspended in 16 ml of 33% HBr in AcOH and heated at 80° C. for 18 hours. The solution was cooled in an ice bath and diluted with EtOAc. A white solid was observed. A solution of sat. aq. _NaHC03 . Large amounts of EtOAc and MeOH were used to solubilize the solid. The organic phase was extracted (3x) and the combined organic phases were washed with brine, dried over MgSO ₄ . Filtration and concentration provided a crude product which was used in the next step without further purification.

MS: [M+1] = 293.

- 109 040488

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

To a solution of 1,2,4-triazole (2.7 g, 39.7 mmol) in anhydrous CH ₃ CN (20 ml) at 0°C was added i-Pr ₂ NEt (7.6 ml, 43.6 mmol) . After all the triazole had dissolved, POCl ₃ (1.11 ml, 11.9 mmol) was added. The mixture was stirred at 0° C. for 2 hours. The solution was transferred to a flask containing lactam (4.96 mmol). The resulting solution was heated in an oil bath at 80° C. for 16 hours. The viscous mixture was cooled in an ice bath and the solvent was evaporated. Dilute with EtOAc and add water. Extracted with EtOAc five times. The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration provided the crude product, which was used directly in the next reaction.

MS: [M+1] = 266.

A solution of KOtBu (1.11 g, 9.92 mmol) in DMF (10 ml) was cooled to -50° C. under nitrogen. Ethyl isocyanoacetate (1.2 ml, 10.9 mmol) was added slowly. The mixture was stirred at -60 to -40° C. for 1 hour. The above crude 1,2,4-triazolo intermediate from step 4 (4.96 mmol) in DMF (5 ml) was added slowly. The mixture was allowed to warm to room temperature over 16 hours. Saturated aqueous NH4Cl was added and extracted three times with EtOAc. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration provided the crude product. Chromatography (24 g silica gel RediSep column eluted with 70% EtOAc in hexanes) provided 296 mg (20% yield over 4 steps) of product.

MS: [M+1] = 310.

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

MS: [M+1] = 282.

To a suspension of the acid (80 mg, 0.28 mmol) in THF (2 ml) was added CDI (50 mg, 0.31 mmol). The suspension was heated at 65° C. for 3 hours. LCMS indicated that the reaction was incomplete. More CDI (10 mg) was added and the solution was heated for another 1 h. The solution was cooled to room temperature and a solution of NH ₄ OH (1 ml) was added. The solution was stirred for 1 hour. The solid was collected by filtration to give 33 mg (42%) of Compound 117 as the desired product as a white solid.

MS: [M+1] = 281;

¹ H-NMR (MeOD4) δ: 8.1 (1H, s), 7.9 (1H, s), 7.73 (3H, m), 7.07 (2H, s), 2.40 (3H , With).

Example 59 Synthesis of Compound 115:

115

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

MS: [M+1] = 263;

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

Example 60 Synthesis of Compound 127:

127

To a suspension of Compound 115 (16 mg, 0.06 mmol) in EtOH (0.8 ml) and water (0.2 ml) was added hydroxylamine chloride (6 mg, 0.09 mmol) and potassium carbonate (12 mg, 0.09 mmol). The suspension was heated at 80° C. for 16 hours. The solution was diluted with EtOAc and washed with water. The aqueous layer was separated and extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over MgSO ₄ . Filtration and concentration provided 12.2 mg (67% yield) of the desired product.

MS: [M+1] = 296.

A suspension of oxime (10 mg, 0.034 mmol) in acetic anhydride (0.5 ml) was heated at 110°C for 1 hour. The solution was then heated at 130°C for 1 hour. Finally, the temperature was raised to 140°C and heated another 2 h. The reaction mixture was cooled and EtOH (1 ml) was added to the reaction mixture, which was heated for 16 h at 80°C. The solvent was evaporated and the crude material was purified with prep. TLC (eluting system: EtOAc) afforded 6.1 mg (56% yield) of the desired compound 127.

MS: [M+1] = 320;

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

Example 61 Synthesis of Compound 133:

To a solution of isobutyric acid (19 μl, 0.2 mmol) in THF (0.5 ml) was added CDI (10 mg, 0.062 mmol). The solution was stirred at room temperature for 2 hours. The solution was then transferred to a tube containing the oxime derivative described above (12 mg, 0.041 mmol) and heated at 70° C. for 2 hours. LCMS indicated that the reaction was incomplete. Prepared another portion of the reagent (isobutyric acid and CDI) and added to the reaction mixture, which was heated at 70°C for another 1 hour LCMS showed that all of the starting material was consumed. The solvent was evaporated, the crude material was suspended in isobutyric acid (1 ml) and heated at 130°C for 1 h. The solvent was evaporated and the crude material was purified with prep. TLC (eluting system: 70% EtOAc in hexane) gave 6.7 mg (71%) of the desired compound 133.

MS: [M+1] = 348.

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

Example 62 Synthesis of Compound 126:

Acetamidoxime was azeotropically distilled in toluene three times prior to use. To a suspension of acetamidoxime (24 mg, 0.32 mmol) in THF (1 ml) was added 60% NaH in oil dispersion (13 mg, 0.32 mmol). The suspension was stirred at room temperature for 15 minutes. Compound 3 (50 mg, 0.16 mmol) was added. The tube containing the ester was washed with DMF (1 ml) which was added to the reaction mixture. The resulting brown slurry was stirred at room temperature for 30 min, then heated at 70° C. for 2 h. The slurry was quenched with water and the solution was stored in the refrigerator overnight. The solid was collected by repeated filtrations to give 16 mg (31% yield) of Compound 126.

MS: [M+1] = 320;

1H-NMR (CDCl3) δ: 8.18 (1H, m), 7.94 (1H, s), 7.67 (3H, m), 2.51 (3H, s), 2.46 (3H, With).

Example 63 Synthesis of Compound 125:

To a carboxylic acid suspension prepared from Compound 3 (30 mg, 0.11 mmol), N,O-dimethylhydroxylamine hydrochloride (13 mg, 0.13 mmol), 1-hydroxybenzotriazole hydrate (17 mg, 0.11 mmol) and triethylamine (46 μl, 0.33 mmol) in THF (0.3 ml) and DCM (0.3 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (32 mg, 0.17 mmol). The solution was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated ammonium chloride solution and

- 111 040488 were extracted with EtOAc (3x). The combined extracts were washed with brine and dried over

_MgSO4 . Filtration and concentration provided 31.2 mg (88% yield) of an orange solid which was used in the next step without further purification.

MS: [M+1] = 325.

To a solution of the above Weinreb amide derivative (31.2 mg, 0.093 mmol) in THF (0.5 ml) cooled at -78°C was added a solution of 3 M ethylmagnesium bromide (0.31 ml, 0.93 mmol). The reaction mixture was stirred at a temperature below -10°C for more than 60 minutes. It was then quenched with saturated ammonium chloride solution and extracted with EtOAc (2x). The combined extracts were washed with brine and dried over MgSO4. Filtration and concentration provided the crude product. Chromatography (RediSep column with 4 g silica gel eluted with 80% EtOAc in hexane) to give 11.1 mg (41% yield) of Compound 125.

MS: [M+1] = 294;

¹ H-NMR (CDCl3) δ: 8.15 (1H, m), 7.76 (1H, s), 7.65 (3H, m), 3.08 (2H, q, J=7 Hz), 2.44 (3H, s), 1.22 (3H, t, J=7 Hz).

Example 64 Synthesis of Compound 132:

132

To a solution of isobutyronitrile (2.6 ml; 29 mmol) in EtOH (30 ml) and water (10 ml) were added hydroxylamine hydrochloride (2.01 g, 29 mmol) and potassium carbonate (4 g, 29 mmol). The resulting suspension was heated at 80° C. for 16 hours. The solvent was removed under vacuum. The residue was co-evaporated with toluene. The crude material was washed with EtOH and filtered to remove sodium chloride. The filtrate was evaporated, co-evaporated several times with toluene and dried under vacuum to give 2 g (69%) of N-hydroxybutyramidine.

To a suspension of N-hydroxybutyramidine (47 mg, 0.46 mmol) in THF (1 ml) was added 60% NaH in oil dispersion (18 mg, 0.46 mmol). The suspension was stirred at room temperature for 30 minutes. Compound 3 (47 mg, 0.15 mmol) in THF (1 ml) was added. The resulting suspension was stirred at room temperature for 30 min, then heated at 70° C. for 2 h. After 1 h, only 50% conversion was observed. After another 1 hour, no change was observed. More reagent (N-hydroxybutyramidine and NaH) was prepared as described above and added to the reaction mixture which was heated for another 40 minutes. At this point, LCMS indicated that the reaction was complete. The suspension was quenched with water. Some MeOH was added to dissolve completely and the solution was extracted with EtOAc (3x). The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration provided the crude product. Chromatography (4 g silica gel RediSep column eluted with EtOAc) to give 20 mg (38% yield) of Compound 132.

MS: [M+1] = 348;

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

Example 65 Synthesis of Compound 161:

161

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

MS: [M+1] = 316.

To a solution of the bromide derivative (30 mg, 0.1 mmol) in dioxane (1 ml) and triethylamine (1 ml) was added TMC-acetylene (71 μl, 0.5 mmol), CuI (2 mg, 0.01 mmol) and PdCl ₂ (PPh ₃ ) ₂ (7 mg, 0.01 mmol). The solution was heated at 110° C. for 6 hours. More Pd catalyst (7 mg) and TMC-acetylene (0.2 ml) were added and the reaction mixture was heated for an additional 12 hours. At this point, LCMS indicated approx.

- 112 040488 a positive 80% conversion. More Pd catalyst (7 mg) and TMC-acetylene (0.2 ml) were added and the reaction mixture was heated for an additional 12 hours. LCMS indicated complete conversion. The reaction mixture was then diluted with water and extracted with EtOAc (3x). The combined extracts were washed with brine (2x) and dried over MgSO ₄ . Filtration and concentration provided the crude product. Chromatography (4 g silica gel RediSep column eluted with 70% EtOAc in hexanes) gave 23 mg (69% yield) of product.

MS: [M+1] = 334.

To a solution of the alkyne derivative (23 mg, 0.069 mmol) in MeOH (0.6 ml) and H2O (0.2 ml) was added KOH (4 mg, 0.076 mmol) at 0°C. The solution was allowed to warm to room temperature over 16 hours. The reaction mixture was diluted with saturated aqueous ammonium chloride and extracted with EtOAc (2x). The combined extracts were washed with brine (2x) and dried over MgSO ₄ . Filtration and concentration provided a crude product which was purified with prep. TLC (eluting system: 80% EtOAc in hexane) to give 8.1 mg (45% yield) of Compound 161.

MS: [M+1] = 262;

¹ H-NMR (CDCl3) δ: 8.13 (1H, m), 7.76 (1H, s), 7.62 (3H, m), 4.09 (2H, br.s), 3.28 (1H, s), 2.44 (3H, s).

Example 66 Synthesis of Compound 146:

To a solution of 3-amino-2-methylacrolein (65 mg, 0.76 mmol) in anhydrous THF (2 ml) was added 60% NaH in oil dispersion (30 mg, 0.76 mmol). The suspension was stirred at room temperature for 15 minutes. Compound 115 (50 mg, 0.19 mmol) was added and the reaction mixture was heated at 65° C. for 3 hours. The reaction mixture was cooled in an ice bath and water was added. The reaction mixture was stored in the refrigerator overnight. The solid was collected by filtration to give 27.5 mg (44% yield) of a white solid Compound 146.

MS: [M+1] = 330;

¹ H-NMR (CDCl3) δ: 8.66 (2H, s), 8.15 (1H, m), 7.89 (1H, s), 7.65 (3H, m), 2.44 (3H , s), 2.36 (3H, s).

Example 67 Synthesis of Compound 153:

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

MS: [M+1] = 324.

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

- 113 040488

Scheme 22 about

Trifluoroacetic anhydride

R =CF ₃ : Compound 150 formic acid

CDI propionic acid

CDI pi wali nova ya acid

R to Compound 151

R - No Connection 155

R=g-wee: Compound 160

Example 68 Synthesis of Compound 116:

CDI

116

An alternative route to nitrile-substituted imidazole derivatives has also been realized. As an example, Compound 116 was prepared from the imino derivative as shown in Scheme 22. A solution of isocyanoacetonitrile (206 mg, 3.12 mmol) in DMF (7 ml) was cooled to -50°C under nitrogen atmosphere. KOtBu (320 mg, 2.85 mmol) was added. The mixture was stirred at -50°C for 1 hour. The imino derivative (obtained identically to the imino derivative shown above in Scheme 21) (350 mg, 1.24 mmol) was added slowly at -50°C. The mixture was allowed to warm to room temperature over 16 hours. Saturated aqueous NH ₄ Cl was added and extracted three times with EtOAc. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration provided the crude product. Chromatography (RediSep column with 12 g silica gel eluted with 70% EtOAc in hexanes) to give 230 mg (70% yield) of Compound 116.

MS: [M+1] = 281;

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

Example 69 Synthesis of Compound 145:

145

To a suspension of the cyanide derivative Compound 116 (50 mg, 0.18 mmol) in EtOH (1.6 ml) and water (0.4 ml) were added hydroxylamine hydrochloride (17 mg, 0.24 mmol) and potassium carbonate (28 mg, 0.2 mmol). The suspension was heated at 80°C for 30 min, then cooled to room temperature. The solid precipitate was collected by filtration to give 37.8 mg (68% yield) of the desired amino oxime product.

MS: [M+1] = 314.

A suspension of amidoxime (10 mg, 0.032 mmol) in acetic anhydride (0.5 ml) was heated at 140° C.

- 114 040488 for 4 h. The reaction mixture was cooled and EtOH (1 ml) was added to the reaction mixture, which was heated for 16 h at 80°C. The solvent was evaporated and the crude material was purified with prep. TLC (eluting system: EtOAc) gave 6.6 mg (61% yield) of the desired product Compound 145.

MS: [M+1] = 338.

¹ H-NMR (CDCl ₃ ) δ: 7.91 (1H, dd, J=3.5, 8.5 Hz), 7.89 (1H, s), 7.65 (1H, dd, J=5 .5, 10 Hz), 7.35(1H, m), 2.69(3H, s), 2.45(3H, s).

Example 70 Synthesis of Compound 149:

To a solution of isobutyric acid (30 μl, 0.32 mmol) in THF (0.5 ml) was added CDI (16 mg, 0.096 mmol). The solution was stirred at room temperature for 2 hours. The above amidoxime derivative (10 mg, 0.032 mmol) was added and the reaction mixture was heated at 70° C. for 45 minutes. The solvent was evaporated and the crude material was suspended in isobutyric acid (1 ml) and heated at 130° C. for 3 hours. The solvent was evaporated and the crude material was purified with prep. TLC (eluting system: 80% EtOAc in hexane) to give 10.6 mg (91%) of the desired product Compound 149.

MS: [M+1] = 366;

¹ I-NMR (CDCI3) δ: 7.90 (1H, dd, J=3.5, 9 Hz), 7.89 (1H, s), 7.66 (1H, dd, J=4.5, 8.5 Hz), 7.36 (1H, m), 3.32 (1H, k, J=6.5 Hz), 2.46 (3H, s), 1.49 (6H, d, J= 8 Hz).

Example 71 Synthesis of Compound 150:

A suspension of the above amidoxime (10 mg, 0.032 mmol) in trifluoroacetic anhydride (0.5 ml) was heated at reflux for 10 minutes. The solvent was evaporated and the crude material was purified with prep. TLC (eluting system: 80% EtOAc in hexanes) to give 11.8 mg (94%) of the desired product Compound 150.

MS: [M+1] = 392;

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

Example 72 Synthesis of Compound 151:

To a solution of formic acid (12 μl, 0.32 mmol) in THF (0.5 ml) was added CDI (16 mg, 0.096 mmol). The solution was stirred at room temperature for 2 hours. The above amidoxime derivative (10 mg, 0.032 mmol) was added and the reaction mixture was heated at 70° C. for 45 minutes. The solvent was evaporated, the crude material was suspended in formic acid (1 ml) and heated at 60°C for 3 h. The solvent was evaporated and the crude material was purified with prep. TLC (eluting system: 80% EtOAc in hexane) gave 2.1 mg (20%) of the desired compound 151.

MS: [M+1] = 324;

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

Example 73 Synthesis of Compound 155:

To a solution of propionic acid (22 μl, 0.29 mmol) in THF (0.5 ml) was added CDI (14 mg,

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0.087 mmol). The solution was stirred at room temperature for 1 hour. The above amidoxime derivative (10 mg, 0.032 mmol) in THF (0.5 ml) was added and the reaction mixture was heated at 70° C. for 90 minutes. The solvent was evaporated, the crude material was suspended in propionic acid (1 ml) and heated at 130°C for 1 h. The solvent was evaporated and the crude material was purified with prep. TLC (eluting system: 80% EtOAc in hexane) to give 9.4 mg (94%) of the desired product Compound 155.

MS: [M+1] = 352;

'H-NMR (CDCI3) δ: 7.91 (1H, dd, J=2, 8.5 Hz), 7.88 (1H, s), 7.65 (1H, dd, J=6.9, 5 Hz), 7.36 (1H, m), 3.01 (2H, k, J=8.5 Hz), 2.46 (3H, s), 1.48 (3H, t, J=8, 5 Hz).

Example 74 Synthesis of Compound 160:

To a solution of pivalic acid (30 mg, 0.29 mmol) in THF (0.5 ml) was added CDI (14 mg, 0.087 mmol). The solution was stirred at room temperature for 1 hour. The above amidoxime derivative (10 mg, 0.032 mmol) in THF (0.5 ml) was added and the reaction mixture was heated at 70° C. for 90 minutes. The solvent was evaporated, the crude material was suspended in acetic acid (1 ml) and heated under reflux for 3 hours. The solvent was evaporated and the crude material was purified with prep. TLC (eluting system: 80% EtOAc in hexane) afforded 7.4 mg (67%) of the desired compound 160.

MS: [M+1] = 380;

'H-NMR (CDCI3) δ: 7.90 (1H, dd, J=2.7, 9 Hz), 7.88 (1H, s), 7.65 (1H, dd, J=4.5, 9 Hz), 7.35(1H, m), 2.47(3H, s) 1.53(9H, s).

Example 75 Synthesis of Compound 143:

A solution of KOtBu (40 mg, 0.36 mmol) in DMF (3 ml) was cooled to -50° C. under nitrogen. P-toluenesulfonylmethyl isocyanide (76 mg, 0.39 mmol) was added. The mixture was stirred at -50°C for 1 h. The imino derivative from Scheme 22 (50 mg, 0.18 mmol) was added and the mixture was allowed to warm to room temperature over 16 h. Saturated aqueous NH ₄ Cl was added and extracted five times with EtOAc. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration provided the crude product. Chromatography (4 g silica gel RediSep column eluted with 70% EtOAc in hexanes) followed by prep. TLC (eluting system: 30% EtOAc in DCM) gave 22.2 mg (30% yield) of a white solid Compound 143.

MS: [M+1] = 410.

'H-NMR (CDCI3) δ: 7.91 (2H, d, J=8 Hz), 7.87 (1H, dd, J=2.5, 8.5 Hz), 7.74 (1H, s ), 7.65 (1H, dd, J=5.5, 9 Hz), 7.34 (3H, m), 2.50 (3H, s), 2.42 (3H, s).

Example 76 Synthesis of Compound 144:

To 3-ethoxymetacrolein (100 mg, 0.88 mmol) was added 7N. ammonia in methanol (1.3 ml, 8.8 mmol). The solution was stirred at room temperature for 16 hours. The solvent was evaporated and the crude yellow solid corresponding to 3-amino-2-methylacrolein was used in the next step without further purification. To a solution of 3-amino-2-methylacrolein (7 mg, 0.087 mmol) in anhydrous THF (1 ml) was added 60% NaH in oil dispersion (6 mg, 0.16 mmol). The suspension was stirred at room temperature for 15 minutes. A cyanide derivative (22 mg, 0.079 mmol) in THF (1 ml) was added and the reaction mixture was heated at 65°C for 1 h. As described above, a new batch of reagents was prepared with 3-amino-2-methylacrolein (20 mg) and NaH (20 mg) in THF (1 ml) was added to the reaction mixture, which was heated at 65° C. for another 1 h. LCMS indicated completion of the reaction. The reaction mixture was quenched with methanol. The solvent was evaporated. The crude material was suspended

- 116 040488 in water and the solid was collected by filtration to give 5.2 mg (yield 19%) of a light red solid Compound 144.

MS: [M+1] = 348;

¹ H-NMR (CDCl3) δ: 8.67 (2H, s), 7.90 (1H, d, J=9.5 Hz), 7.85 (1H, s), 7.65 (1H, dd , J=4.5, 9Hz), 7.34(1H, m), 2.44(3H, s), 2.36(3H, s).

Scheme 23

R

Example 77 Synthesis of Compound 121:

134

R = H: Compound 121

R = F: Connection 135

To a solution of 1,2,4-triazole (2.03 g, 29.4 mmol) in anhydrous CH3CN (20 ml) at 0°C was added i-Pr ₂ NEt (5.6 ml, 32.4 mmol). After all the triazole was dissolved, POCl ₃ (0.82 ml, 8.8 mmol) and compound 16' (1 g, 3.68 mmol) were added. The mixture was stirred at 0° C. for 2 hours. The resulting solution was heated in an oil bath at 80° C. for 16 hours. The mixture was cooled in an ice bath, diluted with EtOAc and water was added. Extracted with EtOAc three times. The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration provided 1.05 g (88% yield) of an orange solid which was used directly in the next step.

MS: [M+1] = 324.

A solution of KOtBu (696 mg, 6.2 mmol) in DMF (15 ml) was cooled to -50° C. under nitrogen. Ethyl isocyanoacetate (0.75 ml, 6.8 mmol) was added slowly. The mixture was stirred at -50°C for 1 h. The above crude product from step 1 (1 g, 3.1 mmol) was added and the mixture was allowed to warm to room temperature over 18 h. Saturated aqueous NH4Cl was added and extracted eight times with EtOAc. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration provided the crude product. Chromatography (24 g silica gel RediSep column eluted with 70% EtOAc in hexane) gave 950 mg (83% yield) of product.

MS: [M+1] = 368.

To a solution of the diester (200 mg, 0.54 mmol) in anhydrous THF (4 ml), stirred at room temperature under nitrogen atmosphere, was added LiBH4 (2 M in THF, 0.66 ml, 1.3 mmol). The reaction mixture was stirred under nitrogen atmosphere for 24 hours. A mixture of EtOAc/EtOH (3 ml/3 ml) was added to the reaction mixture and concentrated. The residue was dissolved in MeOH and silica gel was added. After evaporation of the volatile solvents, the solid was loaded onto a RediSep column with 4 g of silica gel. The desired product was eluted 10:1 v/v. _CH2Cl2 /MeOH. The diol was obtained as a solid (60 mg, 39% yield).

MS: [M+1] = 284.

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

- 117 040488

MS: [M+1] = 408.

To a solution of the dialkyl bromide derivative (0.21 mmol) in EtOAc (10 ml) and MeOH (10 ml) was added wet 10% Pd/C (catalytic amount) and the resulting suspension was stirred under a hydrogen atmosphere for 60 h. The suspension was filtered through celite and the resulting solution was concentrated. The crude product was purified by repeated prep. TLC (eluting system: 3% MeOH in EtOAc) gave 6.2 mg (12% yield over 2 steps) of the desired compound 121.

MS: [M+1] = 252.

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

Example 78 Synthesis of Compound 135:

135

Compound 135 was synthesized analogously to Compound 121 as follows: i-Pr ₂ NEt (2.6 ml, 15.2 mmol). After all the triazole was dissolved, POCl ₃ (0.45 ml, 4.8 mmol) and lactam ester (1 g, 3.45 mmol) were added. The mixture was stirred at 0° C. for 2 hours. The resulting solution was heated in an oil bath at 80° C. for 16 hours. The mixture was cooled in an ice bath, diluted with EtOAc and water was added. Extracted with EtOAc three times. The combined extracts were washed with brine and dried over MgSO ₄ . Filtration and concentration provided 1.03 g (87% yield) of an orange solid which was used directly in the next step.

MS: [M+1] = 342.

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

MS: [M+1] = 386.

To a solution of the diester (600 mg, 1.56 mmol) in anhydrous THF (8 ml), stirred at room temperature under nitrogen atmosphere, was added LiBH ₄ (2 M in THF, 3.1 ml, 6.24 mmol). The reaction mixture was stirred under nitrogen atmosphere for 24 hours. A mixture of EtOAc/EtOH (10 ml/10 ml) was added to the reaction mixture and concentrated. The residue was dissolved in MeOH and silica gel was added. After evaporation of the volatile solvents, the solid was loaded onto a RediSep column with 12 g of silica gel. The desired product was eluted 10:1 v/v. CH ₂ Cl ₂ /MeOH. The diol was obtained as a solid (187 mg, 40% yield).

MS: [M+1] = 302.

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

MS: [M+1] = 426.

To a solution of the dialkyl bromide derivative (70 mg, 0.16 mmol) in EtOAc (10 ml) and MeOH (10 ml) was added 10% Pd/C (catalytic amount) and the resulting suspension was stirred under hydrogen atmosphere for 48 hours. The suspension was filtered through celite and the resulting solution was concentrated. The crude product was purified by repeated prep. TLC (elution system 1: 75% EtOAc in hexane; elution system 2: 5% MeOH in EtOAc; elution system 3: EtOAc) to give 4.1 mg (10% yield) of the desired compound 135.

MS: [M+1] = 270.

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

- 118 040488

Example 79 Synthesis of Compound 134:

134

To a suspension of the dialkyl bromide derivative described in Scheme 23, R=H (30 mg, 0.074 mmol) in EtOH (1 ml) and heated at 80°C, was added a freshly prepared solution of 2 M NaOEt (75 μl, 0.15 mmol). The solution was heated for 10 minutes. The solvent was evaporated. The crude material was suspended in EtOAc and filtered. The filtrate was concentrated and purified with prep. TLC (eluting system: EtOAc) yielded 3.1 mg (12% yield) of the desired compound 134.

MS: [M+1] = 340.

Scheme 24

About Me

Meo

R = i-Pr: connection 147

R = Me: compound 148

R = Et: connection 158

R=CF3; connection 15E

Example 80 Synthesis of Compound 137:

137

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

MS: [M+1] = 335.

Zn powder was added to a nitrobenzene analog (9.3 g, 27.8 mmol) suspended and stirred in a mixture of solvents HOAc/THF/MeOH/H ₂ O (25/100/50/25 mL) at RT. The mixture was heated to

- 119 040488

70°C for 20 h, cooled and filtered. The solid was washed with THF and the combined filtrate was concentrated in vacuo. To the resulting suspension was slowly and carefully added sat. NaHCO3 to avoid excessive foaming until the pH reaches 7-8. The mixture was extracted with EtOAc (3x); the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent provided the crude amine product as a dark brown tacky paste, wt: 8.7 g.

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

To a solution of bromide (11 g) in DMF (550 ml) was added K2CO3 (7.1 g, 51.7 mmol). The mixture was heated at 50° C. for 48 hours. The mixture was cooled to room temperature and the inorganic solid was filtered off. The filtrate was concentrated in vacuo, treated with water/MeOH (60/10 ml), extracted with DCM (3x); the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent followed by silica gel column chromatography using 5 to 50% EtOAc in DCM provided 3.2 g (36%) of the 7-membered lactam as a brownish solid.

MS: [M+1] = 345.

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

MS: [M+1] = 440.

Trifluoroacetic acid (5 ml) was added to the above imidazole ester (861 mg) in dichloromethane (5 ml) at 0°C, followed by trifluoromethanesulfonic acid (0.345 ml). The mixture was warmed to RT, stirred for 3 h, then concentrated to give a residue which was dissolved in dichloromethane (50 ml) to which sat. NaHCO ₃ (50 ml) followed by stirring for 20 minutes. upper aq pH layers were checked for basicity and separated, extracted with DCM (3x); the combined DCM solution was washed with brine and dried over MgSO ₄ . Filtration and removal of solvent provided 0.58 g (100%) lactam as a yellowish solid.

MS: [M+1] = 290.

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

MS: [M+1] = 420.

Example 81 Synthesis of Compound 156:

156

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Ethyl ester of Compound 137 (244.2 mg, 0.582 mmol) in THF/water/MeOH solvent system (6.0 ml total, 6/5/1 ratio) was treated with LiOH (69.7 mg, 2.91 mmol) at RT for 4 h, concentrated under vacuum, acidified to pH 3 and the precipitate was collected by filtration. After washing with water and drying, 179.3 mg (79%) of the acid was obtained as a yellowish solid.

MS: [M+1] = 392.

EDCI (21.3 mg, 0.11 mmol), DMAP (6.7 mg, 0.0552 mmol ) and isopropyl alcohol (13.2 mg, 0.221 mmol). After 12 hours the reaction mixture was diluted with EtOAc, washed with sat. _NaHCO3 ; aq. the layer was separated and extracted with EtOAc, the combined organic layer was washed with saline and dried over MgSO ₄ . Filtration and purification with prep. TLC of the concentrate using 10% MeOH in EtOAc provided 8.7 mg (73%) of the isopropyl ester of Compound 156 as a yellowish foamy solid.

MS: [M+1] = 434.

Example 82 Synthesis of Compound 138:

Acetamidoxime (10.7 mg, 0.144 mmol) was azeotropically distilled in toluene four times and added to the ethyl ester of Compound 137 (9.5 mg, 0.0226 mmol). THF (0.3 ml) was added followed by a 60% NaH oil suspension (4.5 mg, 0.112 mmol). The reaction mixture was stirred at RT for 30 min, then heated at 70°C for 2 h, cooled to RT and the solvent was removed under vacuum, water (1.5 ml) was added to quench the reaction, stirred for 20 min and cooled to 4°C. The precipitate was collected by filtration, washed with water and dried to give 5.2 mg (59%) of the oxadiazole product of Compound 138 as a light yellow solid.

MS: [M+1] = 430.

Example 83 Synthesis of Compound 141:

The compound of Example 83 was synthesized in a similar synthetic route as described for Example 82 using isobutyramidoxime instead of acetamidoxime to give the compound of Example 83 as a yellowish solid.

MS: [M+1] = 458.

Example 84 Synthesis of Compound 157:

Carbonyldiimidazole (49.9 mg, 0.308 mmol) was added to the acid prepared in Example 81 above (60.2 mg, 0.154 mmol) while stirring in DCM (0.7 ml) at RT. The mixture was stirred for 40 min, then cooled to 0° C. and ammonia (0.112 ml) was added, warmed to RT while continuing to stir overnight. The reaction mixture was concentrated, water (8 ml) was added and stirred well for 30 minutes. The resulting precipitate was collected by filtration, washed with water and dried to give 51.1 mg (85%) of the primary amide as a brownish solid.

MS: [M+1] = 391.

The above amide (51.1 mg) was treated with POCl ₃ (200.8 mg, 1.31 mmol) in 1,4-dioxane (0.9 ml) at 90°C for 14 h. After cooling to RT, the reaction mixture was carefully quenched satiety. NaHCO ₃ (5 ml), stirred for 20 min. The precipitate was collected by filtration, washed with water and dried to give 40.9 mg (85%) of Compound 157 nitrile product as a brownish solid.

MS: [M+1] = 373.

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Example 85 Synthesis of Compound 147:

147

To nitrile (45.8 mg, 0.123 mmol) in a round bottom flask were added hydroxylamine hydrochloride (14.5 mg, 0.209 mmol), K ₂ CO ₃ (22.3 mg, 0.161 mmol), ethanol (0.6 ml) and water (0.15 ml). The reaction mixture was heated at 80°C for 30 min, cooled and concentrated under vacuum. The resulting suspension was treated with water (1.5 ml), sonicated to facilitate mixing and stirred at RT for 1 h before cooling to 4°C. The resulting precipitate was collected by filtration, washed with cold water (1 ml) and dried to give 40.8 mg (82%) of the adduct as a white with a yellowish or grayish tinge of a solid.

MS: [M+1] = 406.

Isobutyric acid (31.4 mg, 0.582 mmol) was treated with carbonyldiimidazole (28.4 mg, 0.175 mmol) in THF (0.5 ml) for 2 h. N-hydroxycarboxamide adduct (11.8 mg, 0.0291 mmol) was added ) and the reaction mixture was stirred at RT for 30 min. More isobutyric acid (0.5 ml) was added and the reaction mixture was heated at 110° C. for 16 h, cooled, sat. NaHCO ₃ (8 ml) and extracted with EtOAc (3x); the combined organic layer was washed with brine and dried over MgSO ₄ . Rev. TLC (5% MeOH in EtOAc) of the concentrated filtrate provided 11.2 mg (84%) of oxadiazole Compound 147 as a white solid.

MS: [M+1] = 458.

Example 86 Synthesis of Compound 148:

148

The compound of Example 86 was synthesized in a similar synthesis as described for Example 85 using acetic acid instead of isobutyric acid to give the compound of Example 86 as a white solid.

MS: [M+1] = 430.

Example 87 Synthesis of Compound 158:

158

The compound of Example 87 was synthesized in a similar synthetic route as described for Example 85 using propionic acid instead of isobutyric acid to give the compound of Example 87 as a white solid.

MS: [M+1] = 444.

Example 88 Synthesis of Compound 159:

159

Trifluoroacetic anhydride (196.9 mg, 0.938 mmol) was added to the N-hydroxycarboxamide adduct (19.0 mg, 0.0469 mmol) suspended and stirred in THF (0.2 ml) at RT. After 30 minutes of stirring, the reaction mixture was heated to 70°C for 1 h, cooled to RT and diluted with EtOAc (10 ml), to which was added sat. NaHCO ₃ and stirred for 30 min. The aqueous layer was separated and extracted with EtOAc (1x); the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent provided a paste to which n-BuOH (5 ml) and HOAc (0.5 ml) were added. Heated at 115°C for 16 h, cooled and concentrated under vacuum, diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . Rev. TLC (5% MeOH in EtOAc) of the concentrated filtrate provided 11.5 mg (51%) of the desired trifluoromethyl oxadiazole analog Compound 159 as a yellowish solid.

MS: [M+1] = 484.

Scheme 25

Meo

H-CHjOPh: compound 162 compound 166

R= CH ₂ O4-F-Ph: compound 163

R = CH ₂ OCH ₃ : compound 164

R = CH ₅ OCH ₂ Ph: compound 165

Example 89 Synthesis of Compound 162:

162

To lactam 62 (503.4 mg, 1.42 mmol) while stirring in THF (2.9 ml) and DMF (0.8 ml) at -20°C was added tBuOK (240.2 mg). After 30 minutes stirring, diethyl chlorophosphate (377.7 mg, 2.12 mmol) was added dropwise and the reaction mixture was slowly heated to 8°C over 3 hours before cooling to -20°C. 2.26 ml (2.26 mmol) of oxadiazole isocyanate (ref. JMC, 1996, 39, 170; obtained as a 1 M THF solution) was added. The reaction mixture was further cooled to -78°C, tBuOK (238.4 mg) was added and the reaction mixture was slowly heated to RT overnight. Saturation was added. NH4Cl (5 ml) and the mixture was extracted with EtOAc (2x), washed with brine and dried over MgSO ₄ . After filtration and concentration, the product was isolated by silica gel column chromatography using a gradient elution of 0 to 10% MeOH in EtOAc to give 246.0 mg of the imidazole product as a yellowish solid.

MS: [M+1] = 462.

The imidazole obtained above (246.0 mg, 0.533 mmol) was stirred in DCM (3 ml). Trifluoroacetic acid (3 ml) was added followed by trifluoromethylsulfonic acid (160.0 mg, 1.07 mmol). After 3 hours stirring, the reaction mixture was diluted with DCM (20 ml), washed with sat. _NaHCO3 ; aq. the layer was separated and extracted with DCM (2x); the combined DCM solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under vacuum provided 208.7 mg of the crude lactam product as a yellowish flaky solid.

MS: [M+1] = 312.

Phosphorus oxychloride (29.9 mg, 0.195 mmol) was added to a solution of the above lactam (22.5 mg, 0.0723 mmol) and N,N-dimethyl-p-toluidine (51.8 mg, 0.383 mmol) with stirring in chlorobenzene (0.45 ml) under nitrogen atmosphere. The reaction mixture was heated at 135°C for 3 h, then cooled to RT. Diisopropylethylamine (75.7 mg, 0.586 mmol) and phenoxyacetic hydrazide (50.1 mg, 0.302 mmol) were added and the reaction mixture was heated at 100° C. for 14 h, cooled to RT and partitioned between sat. NH4Cl and EtOAc. The aqueous layer was separated and extracted with EtOAc; the combined EtOAc solution was washed with brine and dried over MgSO ₄ . After filtration and concentration, the product of Compound 162 was isolated by silica gel column chromatography using a gradient elution of 0 to 10% MeOH in EtOAc as a yellowish solid. Weight: 11.8 mg (37%).

MS: [M+1] = 442.

Example 90 Synthesis of Compound 163:

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The compound of Example 90 was synthesized in a similar synthetic route as described for Example 89 using 4-fluorophenoxyacetic hydrazide instead of phenoxyacetic hydrazide to give the compound of Example 90 as a yellowish solid.

MS: [M+1] = 460.

Example 91 Synthesis of Compound 164:

164

The compound of Example 91 was synthesized in a similar synthetic route as described for Example 89 using methoxyacetic hydrazide instead of phenoxyacetic hydrazide to give the compound of Example 91 as a yellowish solid.

MS: [M+1] = 380.

Example 92 Synthesis of Compound 165:

165

Preparation of benzyloxyacetic hydrazide: carbonyldiimidazole (1.52 g, 9.39 mmol) was added to benzyloxyacetic acid (1.2 g, 7.22 mmol) with stirring in THF (60 ml) at 0°C. The ice bath was removed and stirring was continued for 1 hour. The resulting cloudy solution was added to hydrazine (0.927 g, 28.9 mmol) with stirring in THF (40 mL) at RT. After 16 h, the reaction mixture was concentrated to a suspension, to which water (120 ml) was added, extracted with DCM (3x); the combined DCM solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent provided 0.908 g (70%) of the hydrazide as a clear viscous oil. It was azeotropically distilled in toluene several times before use. The compound of Example 92 was synthesized in a similar synthetic route as described for Example 89 using benzyloxyacetic hydrazide instead of phenoxyacetic hydrazide to give the compound of Example 92 as a yellowish solid.

MS: [M+1] = 456.

Example 93 Synthesis of Compound 166:

166

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

MS: [M+1] = 399.

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Scheme 26

r ¹ conhnh ₂

DIPEA, PhCI

R ¹ = CH ₂ OCH ₃ : compound 169

R ¹ = CH ₂ OPh: compound 171

R ¹ - CH ₂ O-4-F-Pht compound 172

R ¹ = CH ₂ OEt: compound 173

R ¹ = CH ₂ O-2-F-Pht compound 174

R ¹ = CH ₂ O-2-CI-Ph: compound 175

R ¹ = CH ₂ O-3-Pyr: compound 176

R ¹ = CH ₂ O-1-Naphthyl: compound 177

R ¹ - CH ₂ O-3-F-Ph: compound 179 compound 178 (2135)

Example 94 Synthesis of Compound 169:

169

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

MS: [M+1] = 490.

The imidazole obtained above (1.07 g, 2.18 mmol) was stirred in DCM (11 ml). Trifluoroacetic acid (11 ml) was added followed by trifluoromethylsulfonic acid (0.656 g, 4.37 mmol). After 4 hours stirring, the reaction mixture was concentrated in vacuo, diluted with DCM (50 ml), washed with sat. _NaHCO3 ; aq. the layer was separated and extracted with DCM (2x); the combined DCM solution was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under vacuum provided 0.872 g of the crude lactam product as a brownish solid.

MS: [M+1] = 340.

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

Weight: 14.3 mg (26%).

MS: [M+1] = 408.

Example 95 Synthesis of Compound 171:

The compound of Example 95 was synthesized in a similar synthetic route as described for Example 94 using phenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of Example 95 as a yellowish solid.

MS: [M+1] = 470.

Example 96 Synthesis of Compound 172:

The compound of Example 96 was synthesized in a similar synthetic route as described for Example 94 using 4-fluorophenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of Example 96 as a yellowish solid.

MS: [M+1] = 488.

Example 97 Synthesis of Compound 173:

Example 97 was synthesized in a similar synthesis as described for Example 94 using ethoxyacetic hydrazide instead of methoxyacetic hydrazide to give Example 97 as a yellowish solid.

MS: [M+1] = 422.

Example 98 Synthesis of Compound 174:

The compound of Example 98 was synthesized in a similar synthesis as described for Example 94 using 2-fluorophenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of Example 98 as a yellowish solid.

MS: [M+1] = 488.

Example 99 Synthesis of Compound 175:

175

The compound of Example 99 was synthesized in a similar synthesis as described for Example 94 using 2-chlorophenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of Example 99 as a yellowish solid.

MS: [M+1] = 504.

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Example 100 Synthesis of Compound 176:

Preparation of 3-pyridyloxyacetic hydrazide: a solution of ethyl 3-pyridyloxyacetate (0.50 g, 2.76 mmol) and hydrazine (0.31 g, 9.66 mmol) in isopropyl alcohol (35 ml) was heated at 85°C for 30 h, cooled and concentrated under vacuum. The resulting white solid was dissolved in a small amount of sat. NaCl solution and re-extracted with EtOAc. The combined organic solution was dried over MgSO ₄ . Filtration and removal of the solvent provided 177 mg of the desired acetic hydrazide as a white solid. Residual water moisture was removed by azeotropic distillation in toluene. Example 100 was synthesized in a similar synthesis as described for Example 94 using 3-pyridyloxyacetic hydrazide instead of methoxyacetic hydrazide to give Example 100 as a yellowish solid.

MS: [M+1] = 471.

Example 101 Synthesis of Compound 177:

The compound of Example 101 was synthesized in a similar synthetic route as described for Example 94 using 1-naphthoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of Example 101 as a white to yellowish or grayish solid.

MS: [M+1] = 520.

Example 102 Synthesis of Compound 179:

The compound of Example 102 was synthesized in a similar synthetic route as described for Example 94 using 3-fluorophenoxyacetic hydrazide instead of methoxyacetic hydrazide to give the compound of Example 102 as a yellowish solid.

MS: [M+1] = 488.

Example 103 Synthesis of Compound 178:

Phosphorus oxychloride (64.8 mg, 0.422 mmol) was added to a solution of oxadiazolilimidazollactam (57.5 mg, 0.169 mmol) and N,N-dimethyl-p-toluidine (114.6 mg, 0.847 mmol) with stirring in chlorobenzene (0. 70 ml) under a nitrogen atmosphere. The reaction mixture was heated at 135°C for 3 h, then cooled to RT. Diisopropylethylamine (174.7 mg, 1.35 mmol), t-BuOH (0.3 ml) and 2-hydroxyacetic hydrazide (91.3 mg, 1.01 mmol) were added. The reaction mixture was stirred at RT for 20 min, then heated at 50°C for 1 h followed by heating at 80°C for 1 h, then finally heated at 100°C overnight. After cooling to RT, the reaction mixture was diluted with EtOAc, washed with brine and dried over MgSO ₄ . Silica gel column chromatography of the concentrated filtrate, using a gradient elution of 0 to 20% MeOH in EtOAc, provided the desired hydroxymethyltriazole product as a yellowish solid.

Weight: 18.1 mg (27%).

MS: [M+1] = 394.

To a solution of the above hydroxymethyltriazole (18.1 mg, 0.046 mmol), cyclopentyl bromide (274.0 mg, 1.84 mmol) and HMPTA (16.5 mg, 0.092 mmol) in THF (0.5 ml) was added with stirring

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NaH (60% suspension; 18.4 mg, 0.46 mmol). After 10 min, the reaction mixture was heated at 100°C for 6 h, cooled, quenched with sat. NaHCO ₃ and extracted with EtOAc (2x), washed with brine and dried over MgSO ₄ . Rev. TLC of concentrated filtrate using 8% MeOH in

EtOAc provided 5.5 mg (26%) of the desired ester Compound 178 as a yellowish solid.

MS: [M+1] = 462.

Scheme 27

ABOUT

1-triazole. DIPEA ROS! ₃

2-NCCH _? CO,Bn, KOtBu

> ₂ Et ^m 2

Ν'™

Example 104 Synthesis of Compound 168:

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

M+1=194.

To a solution of the formylated glycine derivative (1 g, 5.2 mmol) in DCM (30 ml) was added triethylamine (3.2 ml, 23 mmol). The solution was cooled to -50°C and slowly added POCl ₃ (1.9 ml, 20.8 mmol). The solution was stirred at -50°C for 10 minutes, then stirred at room temperature for 40 minutes. The solution turned light red-brown. Dilute with DCM and add 20% sodium carbonate solution (100 ml). The reaction mixture was vigorously stirred for 15 min. The organic phase was separated twice and dried over MgSO4. Filtration and concentration provided the desired benzyl isocyanoacetate in quantitative yield, which was used in the next step without further purification.

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

M+1=354.

A solution of benzyl isocyanoacetate (892 mg, 5.1 mmol) in DMF (10 ml) was cooled to -50° C. under atmospheric

- 128 040488 nitrogen. KOtBu (514 mg, 4.6 mmol) was added. The mixture was stirred at -50°C for 1 hour. The triazole derivative obtained above (900 mg, 2.55 mmol) in DMF (5 ml) was added slowly at -50°C. The mixture was allowed to warm to room temperature over 16 hours. Saturated aqueous NH ₄ Cl was added and extracted three times with EtOAc. The combined extracts were washed with brine (3x) and dried over MgSO ₄ . Filtration and concentration provided the crude product. Chromatography (RediSep column with 24 g silica gel eluted with 70% EtOAc in hexanes) to give 886 mg (76% yield) of product (M+1=460).

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

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

To a suspension of the primary amide derivative (422 mg, 1.15 mmol) in dioxane (10 ml) was added POCl ₃ (160 μl, 1.7 mmol). The suspension was heated at 90° C. for 2 hours. The resulting solution was cooled in an ice bath and quenched with saturated aqueous NaHCO ₃ . The solid was collected by filtration to give 308 mg (77% yield) of the desired cyanide derivative (M+1=351).

To a suspension of the cyanide derivative (150 mg, 0.44 mmol) in EtOH (4 ml) and water (1 ml) were added hydroxylamine hydrochloride (40 mg, 0.57 mmol) and potassium carbonate (67 mg, 0.48 mmol). The suspension was stirred at room temperature for 16 hours. LCMS showed approximately 50% conversion. More hydroxylamine hydrochloride (40 mg, 0.57 mmol) and potassium carbonate (67 mg, 0.48 mmol) were added and stirred for another 24 h. The solution was diluted with EtOAc and washed with water. The combined organic phases were washed with brine, dried over MgSO4. Filtration and concentration provided 145 mg (86% yield) of the desired product (M+1=384).

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

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

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

To a tube containing an alkyl bromide derivative (22 mg, 0.052 mmol) was added 3-fluorophenol (58 mg, 0.52 mmol) in dioxane (1 ml) and potassium carbonate (72 mg, 0.52 mmol). The reaction mixture was heated at 90° C. for 1 h. The reaction mixture was diluted with sat. aq. NaHCO ₃ solution. The resulting solution was extracted with EtOAc (3x). The combined organic phases were washed with brine and dried over MgSO ₄ . Filtration and concentration provided the crude product. Cleaning with Prep. TLC (eluting system: EtOAc) provided 5 mg (21% yield) of the desired compound 168 (M+1=460).

¹ H-NMR (CDCl3) δ: 7.87 (1H, s), 7.65 (1H, d, J=3.5 Hz), 7.57 (1H, d, J=10 Hz), 7. 24 (1H, m), 7.19 (1H, dd, J=3.5, 9 Hz), 6.77 (1H, dd, J=2.5, 9.5 Hz), 6.72 (2H , m), 5.26 (2H, s), 3.97 (3H, s), 2.48 (3H, s).

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Synthesis of Compounds 215-313 Scheme 28

Synthesis ^{of Intermediate} A ,9,11,13,15-heptaene-5-carboxylate).

A solution of ethyl bromoacetate (Scheme 28) (10.0 g, 59.87 mmol) in 20.0 ml anhydrous THF was added dropwise to a solution of (2,4-dimethoxybenzyl)amine (10.0 g, 59.81 mmol) and triethylamine (6.06 g, 59.87 mmol) in anhydrous THF (20.0 ml) at 0° C. under nitrogen. The reaction mixture was warmed to room temperature and stirred overnight. Saline solution was added; 100 ml and the reaction mixture was extracted with ethyl acetate (2x100 ml). The combined extracts were dried over anhydrous MgSO ₄ and concentrated under reduced pressure. Purification was carried out using combiFlash chromatography, gradient: 20:80 to 50:50 v/v. ethyl acetate:hexane. 7.6 g (yield 50.2%) of the alkylation product was obtained as a colorless liquid.

m/z calculated for C ₁₃ H ₁₉ NO ₄ [M+H]+: 254; found: 254.1.

The ether (7.5 g, 29.6 mmol) was dissolved in 40.0 ml of methanol. The reaction mixture was cooled and a solution of 2 N aq. NaOH (88.82 mmol, 44.0 ml). The reaction mixture was warmed to room temperature and stirred for 2 h. aq. HCl. Excess water was concentrated under reduced pressure and air flow was directed to obtain a white solid powder. The solid was dissolved in 85:15 v/v DCM:MeOH (100.0 mL) and filtered, the filtrate was evaporated to give 7.1 g of the carboxylic acid as a white powder (hygroscopic).

m/z calculated for CnH ₁₅ NO ₄ [M+Na]+: 248; found: 248.1.

The above compound (7.0 g, 31.08 mmol) and 6.14 g, 31.08 mmol of 5-chloroisatoic anhydride were mixed in 70.0 ml of p-xylene and refluxed at 140° C. for 3 hours The reaction mixture was filtered and the crude product was recrystallized from methanol. 8.5 g of 7-chloro-4-[(2,4-dimethoxyphenyl)methyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2,5-dione was obtained as a white powder ( yield 75.8%).

m/z calculated for C ₁₈ H ₁₇ ClN ₂ O ₄ [M+H]+: 361; found: 361.1.

The above benzodiazepine-2,5-dione (4 g, 11.1 mmol) was dissolved in THF/DMF (57.2/12.7 ml) and cooled at -20°C. Potassium tert-butoxide fine powder (1.9 g, 16.6 mmol) was added and the reaction mixture was stirred at -20° C. for 20.0 min. 3.1 g, 17.7 mmol of diethyl chlorophosphate was added dropwise to the reaction mixture at −20° C. and allowed to warm to 0-5° C. for 3 hours. The reaction mixture was stirred at ambient temperature for 10.0 minutes. To the reaction mixture was added 2.1 g, 18.4 mmol of ethyl isocyanoacetate at -20°C and the reaction mixture was further cooled to -78°C. 1.9 g, 16.6 mmol of finely divided potassium tert-butoxide powder was added at -78° C. and the reaction mixture was stirred overnight while warming slowly to ambient temperature. The reaction mixture was quenched with saturated aq. NH4Cl solution (10 ml), extracted with ethyl acetate (3x20 ml). The combined extracts were dried over anhydrous

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MgSO ₄ and concentrated under reduced pressure. The crude product was recrystallized from ethyl acetate to give 2.2 g of ethyl 12-chloro-8-[(2,4-dimethoxyphenyl)methyl]-9-oxo-2,4,8triazatricyclo[8.4.0.0 ^2.6 ]tetradec-1 (14),3,5,10,12-pentaene-5-carboxylate as a white solid. A second crop was obtained from the mother liquor to give an additional 3.5 g of product (64% yield).

The dimethoxybenzyl protecting group was removed by dissolving the above compound (2.2 g, 4.83 mmol) in DCM (25.0 ml) followed by the addition of 25.0 ml trifluoroacetic acid and 1.45 g, 9.65 mmol trifluoromethanesulfonic acid. The reaction mixture was stirred at room temperature for 90 minutes. The reaction mixture was neutralized with aq. NaHCO ₃ and the precipitate was filtered off, washed with water and dried to give 1.9 g of ethyl 12-chloro-9-oxo-2,4,8-triazatricyclo[8.4.0.0 ^2.6 ]tetradeca-1(14),3, 5,10,12-pentaene-5-carboxylate as a solid.

m/z calculated for C ₁₄ H ₁₂ ClN ₃ O ₃ [M+H]+: 306; found: 306.1.

In the first stage, the above ethyl 12-chloro-9-oxo-2,4,8-triazatricyclo[8.4.0.0 ^2,6 ]tetradeca1(14),3,5,10,12-pentaene-5-carboxylate (1, 9 g, 6.21 mmol) was dissolved in 25.0 ml chlorobenzene followed by the addition of 2.52 g, 18.64 mmol 4,N,N-trimethylaniline, 1.42 g, 9.32 mmol POCl ₃ and the reaction mixture was heated refluxed at 135° C. for 2 hours. LCMS indicated that; 50% of starting material remained unreacted. Additional 1.68 g, 12.42 mmol of additional 4,N,N-trimethylaniline and 0.95 g, 6.21 mmol POCl ₃ were added to the reaction mixture at room temperature and refluxed at 135°C for 1 h LCMS indicated that 10% of starting material remained unreacted. Additional 0.84 g, 6.21 mmol 4,N,N-trimethylaniline (total 6.0 eq.) and 0.48 g, 3.11 mmol POCl ₃ (total 3 eq.) were additionally added to the reaction mixture at room temperature temperature and heated under reflux at 135°C for 1 hour.

In the second step, 4.67 g, 44.75 mmol methoxyacetic acid hydrazide (total 7.2 eq.) was added to the reaction mixture, followed by the addition of 7.71 g, 59.66 mmol N,N-diisopropylethylamine at room temperature and heated with reflux at 100°C for 1 h. The reaction mixture was cooled to room temperature and neutralized with aq. NaHCO ₃ solution (25.0 ml). The organic layer was extracted with ethyl acetate (3x75 ml) followed by the addition of DCM (3x50.0 ml) and washed with brine. The EtOAc-organic layer was separated by filtration of the insoluble precipitate (pure product 0.805 g) and the combined organic layers were dried over anhydrous MgSO ₄ , concentrated under reduced pressure. The crude product was purified by Combiflash chromatography (mobile phase: 0-10% MeOH:EtOAc) to give an additional 0.8 g of a yellow solid. Total yield for the last two steps of ^{Intermediate A} ,3,5,9,11,13,15-heptaene-5-carboxylate) amounted to 72.58%.

m/z calculated for C ₁₇ H ₁₆ ClN ₅ O ₃ [M+H]+: 374; found: 374.1.

Synthesis of Intermediate B (15-chloro-9-(methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^2.6 .0 ^8.12 ]heptadeca-1(17),3,5,9 ,11,13,15-heptaene-5-carboxylic acid).

Intermediate A (0.4 g, 1.07 mmol) was dissolved in THF/H2O/MeOH (3.2/4.8/8.0 v/v/mL). 0.05 g, 2.14 mmol LiOH was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was acidified with aq. 2 N HCl solution, the precipitate was collected and washed with deionized water. After drying 0.36 g of Intermediate B (15-chloro-9-(methoxymethyl)-2,4,8,10,11pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1(17),3, 5,9,11,13,15-heptaene-5-carboxylic acid) was obtained as a white solid.

m/z was calculated for C ₁₅ H ₁₂ ClN ₅ O ₃ [M+H]+: 346; found: 345.9.

Intermediate C (ethyl-15-chloro-9-(phenoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^2.6 .0 ^8.12 ]heptadeca-1(17),3.5, 9,11,13,15-heptaene-5-carboxylate) and D (ethyl 15-chloro-9-(benzyloxymethyl)-2,4,8,10,11pentaazatetracyclo[11.4.0.0 ^2.6 .0 ^8.12 ]heptadeca-1(17),3,5,9,11,13,15-heptaene-5-carboxylate) was synthesized analogously to Intermediate A using 2-phenoxyacetohydrazide and 2-(benzyloxy)acetohydrazide instead of 2-methoxyacetohydrazide, respectively.

Scheme 29 illustrates some selected examples using Intermediate A to generate new analogues.

- 131 040488

Scheme 29

Synthesis of Compound 233:

.N O'N XG Compound 233

Acetoxime (0.22 g, 0.31 mmol) was dissolved in anhydrous THF (0.5 ml). 0.38 ml, 0.62 mmol 1.6 M n-BuLi was added dropwise and the reaction mixture was stirred at 0-5° C. for 1 h in a separate flask. A solution of Intermediate A (0.05 g, 0.13 mmol) in 1.0 ml THF was added via cannula at 0-5° C. and the reaction mixture was stirred for 16 h with gradual warming at room temperature. LCMS indicated starting material and intermediate m/z: 374.1 (45/14%, confluence of two peaks), m/z 401 (10%), m/z 402 (18%).

The reaction mixture was quenched with 0.03 ml conc. H2SO4 followed by the addition of 0.03 ml deionized water and refluxed for 2 h. LCMS indicated starting material, product and intermediate m/z: 374.1 (43%), m/z 383 (40%), m /z 402 (17%).

The reaction mixture was concentrated under reduced pressure and neutralized with aq. NaHCO ₃ solution, the precipitate was collected and washed with deionized water. After drying, 22.0 mg of a crude precipitate was obtained. The compound was purified with prep. TLC plates using 1:99 MeOH:CHCl3.

Synthesis of Compound 238:

AL ^Cl compound 238

Step 1 Intermediate A (0.045 g, 0.12 mmol) was dissolved in anhydrous toluene (3.0 mL). 0.05 ml, 0.25 mmol aminoethanol (35.0 eq.) was added and the reaction mixture was heated at reflux for 16 h. Toluene was evaporated and the reaction mixture was dissolved in DCM (25.0 ml). The DCM layer was washed with brine followed by deionized water, separated and dried over anhydrous MgSO ₄ . Evaporation of the organic layer provided 38.3 mg of the corresponding amide. LCMS showed product formation m/z: 389.

Step 2 The above amide (0.038 g, 0.09 mmol) was dissolved in dry DCM (2.0 mL). To the reaction mixture was added 0.026 ml, 0.2 mmol DAST (2.0 eq.) at 0°C and stirred for 1.5 h at 0°C. 0.065 g of solid K ₂ CO ₃ (4.8 eq.) was added at 0° C. and the reaction mixture was stirred for 30 min. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x15.0 ml). The organic layer was washed with saline, separated and dried over anhydrous MgSO ₄ . Evaporation of the solvent provided 36 mg of a white solid, m/z calculated

- 132 040488 for C ₁₇ H ₁₅ ClN ₆ O2 [M+H]+: 371; found: 371.

Synthesis of Compound 239:

connection 239

Step 1i Intermediate A (0.05 g, 0.13 mmol) was dissolved in anhydrous toluene (3.0 mL). 0.28 g, 2.67 mmol aminoethanol (20.0 eq.) was added and the reaction mixture was heated at reflux for 16 hours. Toluene was evaporated and the reaction mixture was dissolved in DCM (25.0 ml). The DCM layer was washed with brine followed by deionized water, separated and dried over anhydrous MgSO ₄ . Evaporation of the organic layer provided the amide. LCMS showed product formation m/z: 431.

Step 2 The above amide (0.057 g, 0.13 mmol) was dissolved in dry DCM (2.0 ml). To the reaction mixture was added 0.035 ml, 0.3 mmol DAST (2.0 eq.) at 0°C and stirred for 1.5 h at 0°C. LCMS indicated the formation of product m/z 413. 0.088 g of solid K ₂ CO ₃ (4.8 eq.) was added at 0° C. and the reaction mixture was stirred for 30 min. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x15.0 ml). The organic layer was washed with saline, separated and dried over anhydrous MgSO ₄ . Concentration of the organic layer provided a product which was triturated with 20/80 hexane/EtOAc to give a solid which was collected by filtration and dried: 49.4 mg (89%).

Synthesis of Compound 243:

YY ci-^^Yp-N ^N o - compound 243

Step 1 Intermediate A (0.05 g, 0.13 mmol) was dissolved in anhydrous toluene (3.0 mL). 0.02 ml, 2.67 mmol amino alcohol (20.0 eq.) was added and the reaction mixture was heated at reflux for 16 h. LCMS indicated starting material on the left. Xylene (3.0 ml) was placed, 10.0 eq. 3-aminobutan-1-ol and the reaction mixture was heated under reflux for 16 hours. Finally, a total of 40.0 eq. aminoethanol was required to convert all of the starting material into the product by refluxing with xylene. The reaction mixture was cooled to 0°C and the precipitate was filtered off. The filtrate was extracted with DCM (4x15.0 ml). The DCM layer was washed with brine followed by deionized water, separated and dried over anhydrous MgSO4. Evaporation of the organic layer provided the corresponding amide. LCMS showed product formation m/z: 403.

Step 2 The above amide (0.054 g, 0.13 mmol) was dissolved in dry DCM (2.0 mL). To the reaction mixture was added 0.05 ml, 0.33 mmol DAST at 0°C and stirred for 1.5 h at 0°C. LCMS showed product formation. Was added 0.09 g of solid K ₂ CO ₃ at 0°C and the reaction mixture was gradually heated to room temperature. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x15.0 ml). The organic layer was washed with saline, separated and dried over anhydrous MgSO ₄ . Evaporation of the solvent provided the crude product. Purification was carried out using prep. TLC, mobile phase: 95:05, DCM:MeOH.

m/z calculated for C ₁₈ H ₁₇ ClN ₆ O ₂ [M+H] ⁺ : 385; found: 385.

Synthesis of Compound 244:

connection 244

The above Compound 243 (0.011 g, 0.03 mmol) was dissolved in toluene (2.0 ml). 0.010 g, 0.04 mmol DDQ was added and the reaction mixture was stirred at 50°C for 1 h. LCMS showed starting material m/z 385 and a small amount of product m/z 383. The reaction mixture was stirred at 60°C for 3 h LCMS showed starting material m/z 385, product m/z 383. The reaction mixture was stirred at 70°C for 2 h. The reaction mixture was stirred at 40°C for 16 h. The toluene was evaporated and the crude product was purified with prep. TLC plates. Mobile phase DCM:McOH, 95:05 v/v yielding 4.4 mg of product, m/z calculated for

- 133 040488

C18H15ClN6O2 [M+H]+ : 383; found: 383.

Synthesis of Compound 249:

The above Compound 238 (0.016 g, 0.05 mmol) was dissolved in toluene (2.0 ml). 0.015 g, 0.07 mmol DDQ was added and the reaction mixture was stirred at 50°C for 1 h. LCMS indicated starting material m/z 371. The reaction mixture was stirred at 60°C for 5 h. LCMS indicated starting material m/z 371, product m/z 369 and by-product m/z 407. The reaction mixture was stirred at 30° C. for 16 h. LCMS indicated starting material m/z 371, product m/z 369 and by-product m/z 407. the mixture was stirred at 65° C. for 4 hours. LCMS indicated product m/z 369, side product m/z 407 and a small amount of starting material. The toluene was evaporated and the crude product was purified with prep. TLC plates. Mobile phase DCM:MeOH, 95:05 v/v to give 2.3 mg of product.

m/z calculated for C ₁₇ H ₁₃ ClN ₆ O ₂ [M+H]+: 369; found: 369. Synthesis of Compound 256:

compound 256

Step 1 Intermediate A (0.1 g, 0.27 mmol) was dissolved in anhydrous THF (3.0 mL). 0.67 ml, 0.67 mmol of a 1.0 M solution of DIBAL in THF was added dropwise and the reaction mixture was stirred at 05°C for 2 h. LCMS showed the formation of an alcohol reduction product m/z 332. 0 ml) followed by the addition of water (0.5 ml). Added a saturated solution of NaHCO ₃ and the precipitate was filtered through a pad of celite. The product was extracted using DCM (3x25.0 ml). The combined DCM layers were washed with brine, separated and dried over anhydrous Na ₂ SO ₄ . Solvent evaporation provided 46.1 mg [15-chloro-9-(methoxymethyl)-2,4,8,10,11pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1(17),3.5 ,9,11,13,15-heptaen-5-yl]methanol as a solid, 51.9% yield.

m/z calculated for C ₁₅ H ₁₄ ClN ₅ O ₂ [M+H]+: 332; found: 332.

Step 2 The above alcohol (0.05 g, 0.14 mmol) was dissolved in anhydrous DCM (3.0 ml). 0.09 g of Dess-Martin periodinane was added and the reaction mixture was stirred at room temperature for 2 hours. LCMS indicated the formation of product m/z 330. The reaction was quenched with 1N NaOH solution (2 ml). A saturated solution of NaHCO ₃ was added and the product was extracted using DCM (3x20.0 ml). The combined DCM layers were washed with brine, separated and dried over anhydrous Na ₂ SO ₄ . Evaporation of the solvent provided the desired aldehyde (15-chloro-9-(methoxymethyl)2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1(17),3.5, 9,11,13,15-heptaene-5-carbaldehyde) as a solid product, quantitative yield.

m/z calculated for C ₁₅ H ₁₂ ClN ₅ O ₂ [M+H]+: 330; found: 330.

Step 3. A 1.6 M solution of n-BuLi in hexane (0.68 ml, 1.08 mmol) was added dropwise to a 1.4 ml, 0.86 mmol solution of trimethylsilyldiazomethane in hexane dissolved in 3.0 ml of THF, at -78°C. The reaction mixture was stirred at -78°C for 30.0 min. The aldehyde obtained in Step 2 (0.142 g, 0.43 mmol) in a solution in 3.0 ml THF was added dropwise to the reaction mixture at -78° C. and gradually warmed to room temperature. LCMS showed product m/z 326 and starting material m/z 330. The reaction mixture was quenched with saturated NH ₄ Cl solution. The product was extracted using DCM (3x15.0 ml). The combined DCM layers were washed with brine, separated and dried over anhydrous Na ₂ SO ₄ . The crude product was purified using an ISCO Combiflash purification system, mobile phase: ethyl acetate/hexane. 19.0 mg of Compound 256 was obtained and 71.6 mg of starting material was isolated.

m/z calculated for C ₁₆ H ₁₂ ClN ₅ O [M+H]+: 326; found: 326.

Synthesis of Compound 285:

Compound 285

Compound 256 (0.025 g, 0.08 mmol) was dissolved in degassed DMF (2.0 mL). To the reaction mixture was added 0.03 ml, 0.23 mmol of iodobenzene, followed by the addition of 0.06 ml, 0.41 mmol of TEA.

- 134 040488

The reaction mixture was stirred at room temperature. A mixture of 0.04 g, 0.04 mmol Pd(PPh ₃ ) ₄ and 0.003 g, 0.015 mmol CuI was added to the reaction mixture and stirred for 16 h. LCMS indicated the formation of product m/z 402. The reaction mixture was diluted with deionized water. The product was extracted using DCM (3x10.0 ml). The combined DCM layers were washed with brine, separated and dried over anhydrous Na ₂ SO ₄ . The crude reaction mixture was purified with prep. TLC plates. Mobile phase: EtOAc/MeOH.

m/z calculated for C ₂₂ H ₁₆ ClN ₅ O [M+H]+: 402; found: 402.

Synthesis of Compound 314:

ci

Aldehyde

^{Compound 314}

1(17),3,5,9,11,13,15-heptaene-5-carbaldehyde) from the synthesis of Compound 256; Step 2 (0.015 g, 0.04 mmol) and 0.011 g (0.055 mmol) TosMIC were dissolved in MeOH (2.5 ml). 0.013 g (0.09 mmol) K ₂ CO ₃ was added and the reaction mixture was stirred at 60° C. for 2 hours. LCMS indicated the formation of product m/z 369.1. MeOH was evaporated, the precipitate was dissolved in water and acidified with aq. a solution of 2n. HCl. The particles were filtered off and washed with deionized water to give 10.4 mg (62%) of Compound 314.

m/z calculated for C ₁₇ H ₁₃ ClN ₆ O ₂ [M+H]+: 369; found: 369.1.

Synthesis of Compound 339:

1,3-bis-(1-Adamantyl)imidazole chloride (0.07g, 0.01mmol), [(allyl)PdCl] ₂ (0.003g, 0.009mmol), CuI (0.004g, 0.02mmol) and Cs ₂ CO ₃ (0.04 g, 0.13 mmol) were added to the tube under nitrogen atmosphere. A mixture of diethyl ether and DMF (2:1, 2.0 ml) was added, followed by the addition of 0.03 g (0.09 mmol) of Compound 256 and 0.014 g (0.1 mmol) of MeI. The reaction mixture was stirred at 40.0° C. for 16 hours. LCMS showed the formation of product m/z 340.2. The reaction mixture was quenched with water (20.0 ml) and diluted with ethyl acetate (40.0 ml). The combined layers were filtered. The organic layer was separated and dried over anhydrous Na ₂ SO ₄ . Evaporation of the organic layer provided the crude product. Purification of the crude product was carried out using prep. TLC plates: mobile phase: EtOAc:MeOH, 97:03 v/v. ml to obtain 8.5 mg of Compound 339 (27%).

m/z calculated for C ₁₇ H ₁₄ ClN ₅ O [M+H]+: 340, found 340.2.

Synthesis of Compound 345:

Compound 345 was prepared analogously to compound 339 (Scheme 29) using ethyl iodide instead of methyl iodide to give the compound.

m/z calculated for C ₁₈ H ₁₆ ClN ₅ O [M+H]+: 354, found 354.2.

Synthesis of Compound 346:

Compound 346 was prepared analogously to compound 339 (Scheme 29) using benzyl bromide instead of methyl iodide to give the compound.

m/z calculated for C ₂₃ H ₁₈ ClN ₅ O [M+H]+: 416, found 416.3.

- 135 040488

Synthesis of Compound 329:

Connection 329

Alcohol [15-chloro-9-(methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^2.6 .0 ^8.12 ]heptadeca1(17),3,5,9,11,13 ,15-heptaen-5-yl]methanol.

Synthesis of Compound 254:

Compound 254

Isobutyronitrile (10.0 g, 144.70 mmol) was dissolved in EtOH:water (150:50 ml, v/v) followed by the addition of 10.0 g,

144.70 mmol hydroxylamine hydrochloride and 20.0 g, 144.70 mmol K ₂ CO ₃ . The reaction mixture was heated under reflux at 80° C. for 6 hours. The solvent was evaporated under reduced pressure and the resulting solid was treated with 150 ml of ethanol, sonicated, filtered and washed with 100 ml of ethanol. The combined filtrate was evaporated under reduced pressure and azeotropically distilled with toluene (3x25.0 ml) to give 8.1 g of N'-hydroxy-2-methylpropimidamide as a colorless liquid suspension (54.8% yield). The above amidoxime (1.37 g, 13.38 mmol) was azeotropically distilled with toluene (5x10 ml) before use and dissolved in 20.0 ml anhydrous THF. 0.27 g, 6.69 mmol NaH was added in three portions to the reaction mixture at 0° C. and stirred at ambient temperature for 30.0 minutes. 0.5 g, 1.34 mmol of Intermediate A was added and the reaction mixture was stirred for 45.0 minutes at ambient temperature and heated under reflux at 67° C. for 90.0 minutes. The solvent was evaporated under reduced pressure and the resulting yellow paste was treated with 25.0 ml of aq. saturated NaHCO ₃ solution. The precipitate was filtered through a funnel and washed with 10.0 ml of water and 10.0 ml of hexane to give 0.380 g of a solid (69.1% yield).

m/z calculated for C ₁₉ H ₁₈ ClN ₇ O ₂ [M+H]+: 412.0; found: 412.1.

Synthesis of Compound 215:

Compound 215

Alcohol [15-chloro-9-(methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^2.6 .0 ^8.12 ]heptadeca1(17),3,5,9,11,13 ,15-Heptaen-5-yl]methanol (prepared in Compound 256, Step 1) (34 mg, 0.1025 mmol) was suspended in dry THF (2 ml). HMPTA (36.7 mg, 0.205 mmol) was added followed by ethyl iodide (0.33 ml) and NaH (41 mg 60% oil suspension). The reaction mixture was stirred at RT for 5 min, then heated to 70°C overnight. The mixture was cooled and partitioned between EtOAc and brine. The organic phase was dried and concentrated to give an oil which was purified by column chromatography (0 to 10% MeOH in DCM) to give 3.7 mg of compound 215 as an oil.

Synthesis of Compound 274:

Compound 274

[15-Chloro-9-(methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^2.6 .0 ^8.12 ]heptadeca1(17),3,5,9,11,13, 15-heptaen-5-yl]methanol (0.02 g, 0.06 mmol) was dissolved in anhydrous THF (3.0 ml). 0.003 g NaH was added and the reaction mixture was stirred at room temperature for 30.0 minutes. 0.012 ml, 0.12 mmol of 2-bromopyridine was added dropwise and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was heated at reflux for an additional 2 h. LCMS indicated m/z 409. The reaction mixture was concentrated under reduced pressure and diluted with saturated NaHCO ₃ solution. The product was extracted using DCM (4x10.0 ml). The combined DCM layers were washed with brine, separated and dried over anhydrous Na2SO4. Purification was carried out using prep. TLC, mobile phase: 95:05,

DXM:MeOH. ~1.0 mg of product was obtained.

m/z calculated for C ₂₀ H ₁₇ ClN ₆ O2 [M+H]+: 409; found: 409.

Scheme 30 illustrates some selected examples using Intermediate B to generate new analogues.

Scheme 30 connection 264; R = H compound 332; R=4-OCH ₃ compound 334; R=4-Cl

Synthesis of Compound 234:

connection 234

Intermediate B (0.043 g, 0.12 mmol), 0.3 mmol EDC.HCl and 0.048 g, 0.31 mmol HOBt hydrate were dissolved in THF/DCM (1:1, v/v 1.5 ml) with followed by the addition of 0.09 ml, 0.62 mmol of trimethylaniline and 0.016 ml, 0.25 mmol of propargylamine. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with aq. ammonium chloride and extracted with ethyl acetate. The combined layers were washed with saline, separated and dried over anhydrous MgSO ₄ . Evaporation of the organic layer provided a crude product of 13.0 mg. The crude product was purified by preparative TLC plate, mobile phase: 5:95, MeOH, ethyl acetate.

m/z calculated for C ₁₈ H ₁₅ ClN ₆ O ₃ [M+H] ⁺ : 383; received: 383.1.

Synthesis of Compound 348:

Step 1. Similar to the synthesis of compound 234, Intermediate B was converted into 15-chloro-Ni-(2hydroxyphenyl)-9-(methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ² ' ⁶ .0 ⁸ ' ¹² ]heptadeca1(17),3,5,9,11,13,15-heptaene-5-carboxamide.

Step 2 The above amide (0.017 g, 0.038 mmol) was dissolved in anhydrous toluene (2.5 mL). p-Toluenesulfonic acid monohydrate (0.043 g, 0.23 mmol) was added and the reaction mixture was heated at reflux for 16 hours. LCMS showed a ratio of product to starting material; 50:50. Toluene was replaced with xylene (2.5 ml) and the reaction mixture was heated at 130° C. for 6 hours. LCMS indicated product m/z 419.2. The reaction mixture was concentrated and diluted with ethyl acetate (25.0 ml). The organic layer was washed with saturated NaHCO3 followed by brine. The organic layer was separated and dried over anhydrous Na ₂ SO ₄ . Evaporation of the solvent provided the crude product. Purification of the crude product was carried out using prep. TLC plates. Mobile phase: EtOAc:MeOH, 95:05 v/v to give compound 348.

Synthesis of Compound 240:

compound 240

Step 1 Intermediate B (0.05 g, 0.15 mmol) was dissolved in dry DCM (2.0 ml). 0.05 ml, 0.36 mmol trimethylamine (2.5 eq.) was added followed by 0.024 ml, 0.29 mmol oxalyl chloride (2.0 eq.) and the reaction mixture was stirred for 60 minutes at room temperature. To the reaction mixture was added 0.076 ml, 0.7 mmol amino alcohol (5.0 eq.) at 0° C. and stirred for 2.5 hours. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x15.0 ml). The combined organic layers were washed with brine, separated and dried over anhydrous MgSO ₄ . Evaporation of the organic layer provided 54.1 mg of amide. LCMS showed product formation m/z: 431.

Step 2. (2S)-2-amino-3-methylbutyl-15-chloro-9-(methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca -1(17),3,5,9,11,13,15-heptaene-5-carboxylate (0.027 g, 0.06 mmol) was dissolved in dry DCM (2.0 ml). To the reaction mixture was added 0.016 ml, 0.13 mmol DAST at 0°C and stirred for 3 hours at 0-5°C. LCMS showed product formation. 0.04 g of solid K2CO3 was added at 0°C and the reaction mixture was gradually warmed to room temperature. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x15.0 ml). The organic layer was washed with saline, separated and dried over anhydrous MgSO ₄ . Evaporation of the solvent provided the crude product. Purification was carried out using prep. TLC, mobile phase: 95:05, DCM:MeOH. Received 23.7 mg of a solid product.

Mass spectrum m/z calculated for C ₂₀ H ₂₁ ClN ₆ O ₂ [M+H]+: 413; found: 413.

Synthesis of Compound 246:

connection 246

Compound 240 was converted to Compound 246 using DDQ, toluene at 50° C. analogously to Compound 245 to give 5.5 mg (37%) Compound 246. LCMS showed product m/z: 411.

Synthesis of Compound 242:

compound 242

Step 1 Intermediate B (0.025 g, 0.07 mmol) was dissolved in dry DCM (2.0 ml). 0.03 ml, 0.21 mmol trimethylamine (3.0 eq.) was added followed by 0.015 ml, 0.18 mmol oxalyl chloride (2.5 eq.) and the reaction mixture was stirred for 60 minutes at room temperature. 0.05 g, 0.36 mmol (R,S)-2-amino-2-phenylethan-1-ol (5.0 eq.) was added to the reaction mixture at 0°C and stirred for 2.5 h at room temperature. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x15.0 ml). The combined organic layers were washed with brine, separated and dried over anhydrous MgSO ₄ . Evaporation of the organic layer provided the desired amide. LCMS showed product formation m/z: 465.

Step 2 The above amide (0.034 g, 0.07 mmol) was dissolved in dry DCM (2.0 ml). To the reaction mixture was added 0.03 ml, 0.22 mmol DAST at 0° C. and stirred at 0° C. for 1.5 h. LCMS indicated product formation. 0.05 g of solid K2CO3 was added at 0°C and the reaction mixture was gradually warmed to room temperature. The reaction mixture was diluted with aq. 138 040488 NaHCO3 solution and extracted with DCM (3x15.0 ml). The organic layer was washed with saline, separated and dried over anhydrous MgSO ₄ . Evaporation of the solvent provided the crude product. Purification was carried out using prep. TLC, mobile phase: 95:05, DCM:MeOH.

m/z calculated for C ₂₃ H ₁₉ ClN ₆ O2 [M+H]+: 447; found: 447.

Synthesis of Compound 245:

compound 245

Compound 242 (0.015 g, 0.03 mmol) was dissolved in toluene (1.5 ml). Added 0.009 g, 0.04 mmol DDQ and the reaction mixture was stirred at 50°C for 1.5 h. An additional 0.005 g, 0.022 mmol DDQ was added and the reaction mixture was stirred at 50° C. for 1.5 hours. Starting material m/z 447 and product m/z 445 in a ratio of 1:6. The reaction mixture was stirred at room temperature for 16 hours. Purification was carried out using prep. TLC, mobile phase: 95:05, DCM:MeOH. The m/z: 445 band was isolated to give 9.3 mg of a solid compound (62.4% yield).

m/z calculated for C ₂₃ H ₁₇ ClN ₆ O ₂ [M+H] ⁺ : 445; found: 445.

Synthesis of Compound 237:

connection 237

Step 1. Intermediate B (0.025 g, 0.07 mmol) was dissolved in dry DCM. 0.009 ml, 0.02 ml, 0.14 mmol trimethylamine was added, followed by 0.11 mmol oxalyl chloride, and the reaction mixture was stirred for 30 minutes at room temperature. To the reaction mixture was added 0.028 ml, 0.36 mmol of 3-amino-1-propanol at 0°C, stirred for 2.5 h and then concentrated. LCMS showed the formation of product m/z: 403, a small amount of starting material remained.

Step 2 The crude amide from Step 1 (0.018 g, 0.045 mmol) was dissolved in dry DCM (2.0 mL). To the reaction mixture was added 0.012 ml, 0.09 mmol DAST at -78°C and gradually heated to 0°C. 0.03 g of solid K2CO3 was added at -78°C and the reaction mixture was gradually warmed to room temperature. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x15.0 ml). The organic layer was washed with saline, separated and dried over anhydrous MgSO ₄ . Evaporation of the solvent provided 14.7 mg of compound 237 as a white solid.

m/z calculated for C ₁₈ H ₁₇ ClN ₆ O ₂ [M+H] ⁺ : 385; found: 385.1.

Synthesis of Compound 263:

Compound 263

Stage 1. Intermediate B (0.03 g, 0.09 mmol), 0.034 g, 0.17 mmol EDC.HCl and 0.027 g, 0.17 mmol HOBt.xH ₂ O were dissolved in anhydrous DCM (2.5 ml) . 0.024 g, 0.17 mmol of R-(-)-2-phenylglycinol was added and the reaction mixture was stirred for 6 hours at room temperature. LCMS showed product formation m/z 464.9. The reaction mixture was diluted with deionized water and extracted with DCM (3x10.0 ml). The combined DCM layers were washed with brine, separated and dried over anhydrous Na2SO4. Evaporation of the organic layer provided the crude product. Received liquid syrup.

m/z calculated for C ₂₃ H ₂₁ ClN ₆ O ₃ [M+H] ⁺ : 465; found: 464.9.

Step 2 The above amide (0.04 g, 0.086 mmol) was dissolved in dry DCM (2.0 ml). 0.03 ml, 0.21 mmol DAST was added and the reaction mixture was stirred at 0° C. for 2 hours. LCMS indicated product m/z 446.9. Was added 0.06 g of solid K ₂ CO ₃ at 0°C and the reaction mixture was gradually heated to room temperature. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x15.0 ml). The organic layer was washed with saline, separated and dried over anhydrous Na ₂ SO ₄ . Evaporation of the solvent provided crude

- 139 040488 product. Purification was carried out using prep. TLC, mobile phase: 95:05, DCM:MeOH. received

25.0 mg solid.

m/z calculated for C ₂₃ H ₁₉ ClN6O ₂ [M+H]+: 447; found: 446.9.

Synthesis of Compound 264:

Compound 264

Step 1 Intermediate B (0.03 g, 0.09 mmol), 0.034 g, 0.17 mmol EDC.HCl and 0.027 g, 0.17 mmol HOBt-H ₂ O were dissolved in anhydrous DCM (2.5 ml) . 0.024 g, 0.17 mmol S-(+)-2-phenylglycinol was added and the reaction mixture was stirred for 6 hours at room temperature. LCMS showed product formation m/z 464.9. The reaction mixture was diluted with deionized water and extracted with DCM (3x10.0 ml). The combined DCM layers were washed with brine, separated and dried over anhydrous Na ₂ SO ₄ . Evaporation of the organic layer provided the crude product. Received liquid syrup.

m/z calculated for C ₂₃ H ₂₁ ClN ₆ O ₃ [M+H] ⁺ : 465; found: 464.9.

Step 2 The above amide (0.04 g, 0.086 mmol) was dissolved in dry DCM (2.0 ml). 0.03 ml, 0.21 mmol DAST was added and the reaction mixture was stirred at 0° C. for 2 hours. LCMS indicated product m/z 446.9.

0.06 g of solid K2CO3 was added at 0°C and the reaction mixture was gradually warmed to room temperature. The reaction mixture was diluted with aq. NaHCO3 solution and extracted with DCM (3x15.0 ml). The organic layer was washed with saline, separated and dried over anhydrous Na ₂ SO ₄ . Evaporation of the solvent provided the crude product. Purification was carried out using prep. TLC, mobile phase: 95:05, DCM:MeOH. Received 26.4 mg of a solid product.

m/z calculated for C ₂₃ H ₁₉ ClN ₆ O ₂ [M+H] ⁺ : 447; found: 446.9.

Compounds 332, 334-338 were prepared using a synthetic method similar to that used for the synthesis of Compound 264 as shown in Scheme 30.

Compounds 180-182 were prepared using a synthetic method similar to that used for the synthesis of Compound 168 as shown in Scheme 27.

Compounds 183-193 were prepared using a synthetic method similar to that used for the synthesis of Compounds 169-179 as shown in Scheme 26.

Compounds 194 and 195 were prepared using a synthetic method similar to that depicted in Schemes 21 and 22.

Compounds 196-198 and 206 were obtained using a synthetic method that is similar to that shown in Scheme 18a.

Compound 202 was obtained using a synthesis method that is similar to the method used for the synthesis of Compound 129, as shown in Scheme 18a.

Compounds 199, 200, 204 and 205 were obtained using a synthetic method that is similar to that shown in Scheme 18b.

Compounds 201 and 203 were prepared using a synthetic method similar to that shown in Scheme 24.

Compounds 207-210 were prepared using a synthetic method that is similar to that depicted in Scheme 17.

Nitrile substituents in Compounds 207-210 were obtained similarly to those transformations shown in Scheme 22.

Compounds 211-214 were prepared using a synthetic method that is similar to that depicted in Scheme 20.

Compound 255 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 254.

Compound 259 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 243.

Compound 260 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 242.

Compound 261 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 256.

Compound 265 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 264.

Compound 266 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 264.

Compound 267 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 264.

Compound 268 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 263.

Compound 270 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 264.

Compound 271 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 264.

Compound 275 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 264.

Compound 276 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 245.

Compound 278 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 233.

Compound 281 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 233.

Compounds 282, 283, 286, 287 were prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 243.

Compound 288 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 256.

Compound 293 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 285.

Compounds 294, 295 and 296 were prepared from the respective starting materials using the synthetic routes described in Schemes 28 and 29; similar to compounds 243 and 244.

Compound 303 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 233.

Compound 304 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 264.

Compound 297 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 243.

Compound 307 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 285.

Compound 308 was prepared from the appropriate starting materials using the synthetic routes described in Scheme 28; similar to Intermediate A.

Compound 309 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 238.

Compound 310 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 285.

Compound 311 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 285.

Compound 312 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 244.

Compound 313 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 244.

Compound 315 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 314.

Compound 316 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 238.

Compound 317 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 29; similar to compound 238.

Synthesis of Compound 319

Compound 319

Step 1 Intermediate B (0.04 g, 0.12 mmol) was dissolved in anhydrous DMF (1.0 ml). HATU (0.088 g, 0.23 mmol) and triethylamine (0.048 ml, 0.35 mmol) were added to the reaction mixture, followed by (S)-2-amino-2-(2-fluorophenyl)ethαn-1-ol (0.044 g, 0.23 mmol). The reaction mixture was stirred at room temperature for 16 hours. LCMS showed product m/z 483.0 and a small amount of starting material. An additional 0.044 g (0.23 mmol) of (S)-2-amino-2-(2-fluorophenyl)ethan-1-ol was added at room temperature and the reaction mixture was stirred for another

- 141 040488

including LCMS showed the formation of the product m/z 483.0. The reaction mixture was diluted with deionized water and extracted with DCM (3x10.0 ml). The combined DCM layers were washed with brine, separated and dried over anhydrous Na ₂ SO ₄ . Evaporation of the organic layer provided the crude product.

m/z calculated for C ₂₃ H ₂₀ ClFN6O ₃ [M+H]+: 483; found: 483.0.

Step 2 The above amide (0.06 g, 0.12 mmol) was dissolved in dry DCM (2.5 ml). 0.03 ml (0.23 mmol) of DAST was added and the reaction mixture was stirred at 0° C. for 2 hours. LCMS indicated product m/z 465.2. Solid K2CO3 (0.06 g, 0.46 mmol) was added at 0° C. and the reaction mixture was gradually warmed to room temperature. The reaction mixture was diluted with aq. NaHCO ₃ solution and extracted with DCM (3x10.0 ml). The organic layer was washed with brine, separated and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product. Purification was carried out using prep. TLC, mobile phase: 95:05, DCM:MeOH. Pure product Compound 319 (44.2 mg) was obtained as a solid (82.2% yield).

m/z calculated for C ₂₃ H ₁₈ ClFN ₆ O ₃ [M+H] ⁺ : 465; found: 465.2.

Compound 320 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 319.

Compound 321 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 319.

Compound 322 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 319.

Compound 325 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to connection 320.

Compound 326 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to connection 320.

Compound 330 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 319.

Compound 331 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 319.

Compound 333 was prepared from the appropriate starting materials using the synthetic routes described in Schemes 28 and 30; similar to compound 319.

Compound 340 was prepared from the appropriate starting materials (chiral center derived from (2R)-2-amino-2-phenylethan-1-ol) using the synthetic routes described in Schemes 28 and 30; similar to compound 319.

Compound 343 was prepared from the appropriate starting materials (chiral center derived from (2S)-2-amino-2-phenylethan-1-ol) using the synthetic routes described in Schemes 28 and 30; similar to compound 319.

Synthesis of Compound 323:

Connection 323

Compound 319 (0.018 g, 0.04 mmol) was dissolved in toluene (2.0 ml). Added DDQ (0.011 g, 0.05 mmol) and the reaction mixture was stirred at 50°C for 3 h. . The reaction mixture was stirred at 65° C. for 2 h, LCMS indicated starting material m/z 465.2 and product m/z 463.2 in a ratio of 1:1.6. Additional DDQ (0.003 g, 0.012 mmol) was added and the reaction mixture was stirred at 75° C. for 5 hours. LCMS indicated completion of the reaction. The reaction mixture was concentrated under vacuum. Purification was carried out using prep. TLC, mobile phase: 80:20, EtOAc:hexane. Received; 5.8 mg of Compound 323.

m/z calculated for C ₂₃ H ₁₆ ClFN ₆ O ₂ [M+H] ⁺ : 463.2; found: 463.2.

Compound 324 was prepared from Compound 320 using the same conditions as those shown for the synthesis of Compound 323.

Synthesis of Compounds 305 and 306.

Scheme 31

Compound 288 Compound 305 Compound 306

- 142 040488

Compound 288 (0.015 g, 0.042 mmol) was dissolved in anhydrous THF (3.0 ml). 0.003 ml, 0.05 mmol methyl iodide was added at -78°C followed by 0.05 ml, 0.05 mmol 1.0 M LDA solution. The reaction mixture was stirred at -78°C and gradually warmed to room temperature. LCMS showed the formation of the main product m/z 368, unreacted starting material m/z 354 and dimethylated unknown product m/z 382.1. The reaction mixture was quenched with saturated NH ₄ Cl solution and extracted with EtOAC. The organic layer was dried and concentrated. Purification of the crude reaction mixture was carried out using prep. TLC plates, mobile phase: EtOAc:hexane 75:25 v/v ml to isolate three bands. By MS it was found that the first lane confirmed the m/z 354 of starting material, the second lane confirmed the m/z 368 of the monomethyl substituted product of Compound 305, and the third lane confirmed the m/z 382.1 of the dimethyl substituted product of Compound 306. 1H NMR data (CDCl ₃ ) confirmed the monomethyl substitution on the imidazole ring. Note: 1H NMR data confirmed the formation of products and the isolation of pure products.

Compound 216 was prepared analogously to compound 129 in Scheme 18a. MS: [M+1] = 395.

Compound 217 was prepared analogously to compound 129 in Scheme 18a. MS: [M+1] = 381.

Scheme 32

MeO' No.O

Meo

Meo

347 connection

Synthesis of Compound 218:

5-(ethoxycarbonyl)-16-methoxy-2,3,4,10,12-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca NRR' = NHMa: compound 24/

NRR' = NfCH^; connection 248

CljP^PPh^ .jun

Intermediate from

Schemes 27 (see Scheme 32). MeNHOMe, EDC

ArMgBr /

R= E: Compound 218

R= Cl: compound 219

1(17),3,5,8,10,13,15-heptaene-9-carboxylic acid from Scheme 27 (0.609 g, 1.65 mmol) while stirring in DMF (10 ml) at 0°C was added NaHCO ₃ (0.749 g, 8.9 mmol) and NBS (0.793 g, 4.45 mmol). Reactions by

- 143 040488 was allowed to flow to ambient temperature overnight. Then the reaction mixture was diluted with EtOAc, cooled to 0°C and sat. sodium thiosulfate. After foaming ceased, the organic layer was separated, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . Filtration and removal of solvent provided crude bromide which was purified by flash column chromatography using a gradient elution of 0 to 80% EtOAc in hexane. 424.2 mg (64%) was obtained as a yellowish solid.

MS: [M+1] = 405.

To the above bromide (286.7 mg, 0.709 mmol) in a thick-walled round bottom flask were added CuI (121.5 mg, 0.638 mmol), trimethylsilylacetylene (1.04 g, 10.7 mmol), triethylamine (0.717 g, 7.09 mmol ), dicyclohexyl(2',6'-dimethoxybiphenyl-2-yl)phosphine (0.349 g, 0.851 mmol) and 1,4-dioxane (2.5 ml; degassed). The reaction vessel was purged with nitrogen gas and bis(triphenylphosphine)palladium (II) dichloride (298.2 mg, 0.425 mmol) was added. The reaction mixture was stirred at rt. for 30 min, then heated at 100°C in a sealed tube for 16 h, diluted with EtOAc and washed with sat. NaHCO ₃ , brine and dried (MgSO ₄ ). Silica gel column chromatography of the filtered and concentrated reaction mixture using a gradient of 0 to 100% EtOAc in hexane provided 157.9 mg (53%) of the desired trimethylsilylacetylene product as a brownish solid.

MS: [M+1] = 422.

The trimethylsilylalkyne prepared above (128.7 mg, 0.305 mmol) was treated with lithium hydroxide (36.6 mg, 1.53 mmol) in a solvent mixture of THF (0.9 ml), water (0.75 ml) and MeOH (0.15 ml) at r.t. for 2 hours Then the mixture was acidified to pH 3-4 with dilute hydrochloric acid and was extracted with EtOAc (3x). It was found that the remaining sediment in aq. layer is the product and was collected by filtration and combined with the product isolated from the organic layer to give 95.6 mg of the acid as a yellowish solid.

To acid (95.6 mg, 0.298 mmol) in THF (1.3 mL) and dichloromethane (1.3 mL) was added N,O-dimethylhydroxylamine hydrochloride (232.4 mg, 2.38 mmol), EDC hydrochloride (456 .7 mg, 2.38 mmol), HOBt hydrate (91.2 mg) and triethylamine (0.833 ml, 5.93 mmol). After stirring for 16 h, the reaction mixture was diluted with EtOAc and washed with sat. _NH4Cl . The aqueous layer was separated and extracted with EtOAc (3x), the combined organic layer was washed with sat. NaHCO ₃ , brine and dried (MgSO4). Filtration followed by removal of the solvent provided 104.8 mg of the amide as a yellowish solid.

To the above Weinreb amide (20.1 mg, 0.0552 mmol) with stirring in anhydrous. THF (0.8 ml) cooled in an ice-salt bath was slowly added with a solution of 4-fluorophenylmagnesium bromide (1 M THF; 0.828 ml). The reaction mixture was stirred at ambient temperature over 4 h, then quenched with sat. NH ₄ Cl, extracted with EtOAc (3x), washed with sat. NaHCO ₃ , brine and dried (MgSO ₄ ). Rev. TLC of the filtered stock using 5% MeOH in DCM provided 2.0 mg of Compound 218 as a white to yellowish or grayish solid.

MS: [M+1] = 400.

Compound 219 was prepared analogously to compound 218 as shown in Scheme 32. MS: [M+1] = 416.

Synthesis of Compound 220:

Compound 220

5-Benzoyl-9-ethynyl-16-methoxy-2,3,4,10,12-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca1(17),3.5,8,10.13 ,15-heptaene (90.3 mg, 0.237 mmol; prepared analogously to 218) was stirred in THF (1.5 mL) at rt. NaBH ₄ (26.8 mg, 0.71 mmol) was added. After 1 h, the reaction was quenched with NH ₄ Cl for 5 min and extracted with EtOAc. The organic layer was separated and washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under vacuum provided a clear viscous oil which was treated with triethylsilane (241.9 mg, 2.08 mmol) and trifluoroacetic acid (0.32 ml) in DCM (1.5 ml) for 3 h. in a rotary evaporator to remove the solvent, diluted with EtOAc and washed with sat. _NaHC03 . The aqueous layer was separated and extracted with EtOAc, the combined organic layer was washed with brine and dried over MgSO ₄ . Rev. TLC of the filtered concentrate using 2% MeOH in DCM/EtOAc (1:1) provided 2.5 mg of Compound 220 as a clear filmy solid.

- 144 040488

MS: [M+1] = 370.

Compound 221 was prepared analogously to compound 220 as shown in Scheme 32. MS: [M+1] = 384.

Synthesis of Compound 344:

Bromide intermediate in Scheme 32 (26.5 mg, 0.0656 mmol), 2-(tri-n-butylstannyl)oxazole (58.7 mg, 0.164 mmol) and dichloro-1,1'-bis-(diphenylphosphino) adduct ferrocenepalladium(II) dichloromethane (7.2 mg) in dioxane (0.5 ml) was heated under nitrogen at 150° C. for 5 h. After cooling, the reaction mixture was diluted with ethyl acetate, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . Separation of prep. TLC using 5% MeOH in ethyl acetate as eluent provided 6.4 mg (25%) of the oxazole ethyl ester product as a yellowish solid.

The above ethyl ester (6.4 mg) was treated with lithium hydroxide (6.0 mg) in a mixture of solvents THF/H ₂ O/MeOH (6:5:1; 0.24 ml) for 16 h. Acetic acid (3 ml) and the reaction mixture was heated at 120°C for 4 hours After cooling, the reaction mixture was diluted with EtOAc, washed with sat. _NaHC03 . The aqueous layer was separated and extracted with EtOAc (3x). The combined organic solution was washed with brine and dried over MgSO ₄ . Rev. TLC using 8% MeOH in DCM as the eluent solvent system provided 1.6 mg (31% over two steps) of the decarboxylated compound 344 product as a yellowish solid.

MS: [M+1] = 321.

Synthesis of Compound 347:

The Stille oxazole coupling product of Scheme 32 (21.2 mg, 0.054 mmol) was treated with sodium borohydride (0.44 ml, 0.5 M in 2-methoxyethyl ether) at 0° C. for three days, then quenched with acetone (2 ml ) within 60 min. The reaction mixture was diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . The crude alcohol was obtained after filtration and removal of the solvent and was treated with phosphorus oxybromide (55 mg) in acetonitrile (1 ml) at 85°C for 7 h. After cooling, the reaction mixture was diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . Filtration and removal of the solvent under reduced pressure provided the crude bromide product as a greenish solid.

The above bromide was hydrogenated with a catalytic amount of 10% Pd on charcoal in EtOAc/MeOH (5 ml, 1:1 v/v) for 2 h. provided the desired methyl product, which was purified with prep. TLC (8% MeOH in DCM as eluent) gave 3.6 mg (20% over three steps) of compound 347 as a yellowish solid.

MS: [M+1] = 335.

- 145 040488

Scheme 33

Synthesis of Compound 222:

Compound 222

Cyan ether (407.1 mg, 1.16 mmol) was treated with lithium hydroxide (83.5 mg, 3.49 mmol) in a solvent mixture of THF (6 ml), water (5 ml) and MeOH (1 ml) at r. T. for 16 h, then concentrated under vacuum, acidified to pH 3-4 with dilute HCl and cooled to 0°C. The precipitate was collected by filtration, washed with a small amount of water and dried to give 271.9 mg (73%) of the acid as a greyish solid. This acid (271.9 mg) was suspended and stirred in THF (2 ml) at 0° C., to which a solution of borane dimethyl sulfide (2 M THF; 8.4 ml) was added dropwise. The reaction mixture was allowed to warm to ambient temperature overnight, cooled in an ice bath, quenched with MeOH (10 mL) over 2 h and concentrated in vacuo. The resulting solid residue was partitioned between DCM and sat. NaHCO ₃ and stirred for 20 min. The aqueous layer was separated and extracted with DCM (3x). The combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent provided 137.8 mg of the crude alcohol product as a yellowish waxy solid.

The above alcohol (137.8 mg) was treated with phosphorus oxybromide (256.3 mg, 0.894 mmol) in 1,4-dioxane (5 mL) at 100° C. for 3 hours. After cooling in an ice bath, the reaction mixture was treated with sat. NaHCO ₃ (15 ml) and EtOAc (15 ml) under stirring conditions for approximately 20 minutes. Main water the layer was separated and extracted with EtOAc (2x). The combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under vacuum provided the crude primary bromide as a solid paste, which was stored in the cold and used without further purification if necessary.

The above crude bromide (27.0 mg, 0.0727 mmol) was treated with 4-fluorophenol (65.2 mg, 0.585 mmol) and cesium carbonate (47.4 mg, 0.145 mmol) at rt. within 16 hours the Reaction mixture was diluted with EtOAc, washed with saline and dried over MgSO ₄ . Rev. TLC of the filtered concentrate using 5% MeOH in DCM/EtOAc (1:1) provided 1.2 mg of Compound 222 as a yellowish solid.

MS: [M+1] = 403.

Compound 223 was prepared analogously to compound 222 as shown in Scheme 33. MS: [M+1]=403 Compound 224 was prepared analogously to compound 222 as shown in Scheme 33. MS: [M+1]=385 Compound 225 was prepared analogously compound 222 as shown in Scheme 33. MS: [M+1] = 464.

- 146 040488

Scheme 34

Ethyl 1-(5-chloro-2-nitrophenyl)-5-(2-ethoxy-2-oxoethyl)-1H-1,2,3-triazole-4-carboxylate (21.2 g; prepared analogously to 14 in Scheme 11) was treated with tin(II) chloride hydrate (60 g) in a mixture of solvents EtOAc/EtOH (1:2, 300 ml) at 70°C for 3 h. HCl (40 ml; 37%) was added and heating was continued for 3 days. More tin(II) chloride hydrate (25 g) and HCl (15 ml) were added and heating was continued for 2 days. The reaction mixture was cooled, concentrated under reduced pressure to a brownish oil, diluted with EtOAc (250 ml) and carefully basified to pH 8-9 with sodium carbonate solution. The aqueous layer was separated and extracted several times with EtOAc. The combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent followed by recrystallization in MeOH provided 3.3 g (51%) of the cyclized monoester as a yellowish solid.

MS: [M+1] = 307.

Preparation of tert-butyl isocyanoacetate.

To a suspension of tert-butylglycinate hydrochloride (10.0 g, 60 mmol) in DCM (200 ml) was added EDC-HCl (14.9 g, 78 mmol) and triethylamine (12.5 ml, 89.8 mmol). The reaction mixture was cooled to -50°C, formic acid (3.4 ml, 89.8 mmol) in DCM (10 ml) was added slowly. The reaction mixture was stirred at -50° C. for 1 h, then at 4° C. for 3 h. Water (150 ml) was added. After 30 min stirring aq. the layer was separated and extracted with DCM (3x). The combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under reduced pressure provided 10 g (100%) of formylamide as a clear viscous oil.

¹ H-NMR (CDCl ₃ ) δ: 8.23 (1H, s), 6.17 (1H, br.s), 3.98 (2H, d, J=5.5 Hz) and 1.48 ( 9H, c).

To a solution of formylamide (10.5 g, 66 mmol) in DCM (180 ml) was added triethylamine (36.8 ml, 264 mmol). The solution was cooled in an ice-cold salt bath and POCl ₃ (7.4 ml, 79.2 mmol) was added slowly. The reaction mixture was stirred in a cold bath for 1 hour. Then sodium carbonate (7.7 g, 72.6 mmol) in water (90 ml) was added to the cold reaction mixture. After 15 minutes, the cold bath was removed and stirring was continued at ambient temperature for 1 hour. The aqueous layer was separated and extracted with DCM (3x). The combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under reduced pressure provided 7.9 g (84%) of t-butyl isocyanoacetate as a dark brown liquid;

¹ H-NMR (CDCl ₃ ) δ: 4.12 (2H, s) and 1.51 (9H, s).

- 147 040488

A solution of tert-butyl isocyanoacetate (1.51 g, 10.7 mmol) in DMF (43 ml) was cooled to -50° C. under nitrogen. Potassium tert-butoxide (1.05 g, 9.4 mmol; finely pressed) was added. After stirring for 1 h at -50°C, the 1,2,4-triazole intermediate (2.32 g, 6.48 mmol; prepared analogously to compound 20 in Scheme 11) was added to the resulting reddish clear solution and the reaction mixture was stirred to a temperature environment during the night. Saturation was added. _NaHCO3

(15 ml) and the reaction mixture was extracted with diethyl ether (5x), washed with brine and dried (MgSO ₄ ). Silica gel chromatography of the filtered concentrate using a gradient of 0 to 100% EtOAc in hexanes provided 2.5 g (89%) of the tert-butyl ether product imidazole as a yellowish solid.

MS: [M+1-tBu] = 374.

The above imidazole tert-butyl ester (1.1 g, 2.56 mmol) was treated with trifluoroacetic acid (13 ml) in DCM (13 ml) for 3 hours or until all starting tert-butyl ester was completely hydrolysed. The reaction mixture was then concentrated under reduced pressure. Residual TFA was removed by repeated addition and evaporation of toluene. The acid product was obtained as a dark brown viscous oil and was used without further purification.

MS: [M+1] = 374.

Ethyl-16-chloro-9-cyano-2,3,4,10,12-pentaazatetracyclo [ 11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1(17),3.5,8,10.13 ,15heptaene-5-carboxylate (477 mg, 1.34 mmol); (obtained analogously as ethyl-9-cyano-16-methoxy2,3,4,10,12-pentaazatetracyclo[ ^{11.4.0.0 2'6.08'12} ]heptadeca-1( ¹⁷ ),3,5,8 ^, 10,13,15-heptaene-5-carboxylate in Scheme 27) was treated with lithium hydroxide (80.5 mg, 3.36 mmol) in a solvent mixture of THF (6 ml), water (5 ml) and MeOH (1 ml) at k.t. within 16 hours the Reaction mixture was concentrated under reduced pressure, acidified to pH 3-4 dilute HCl and cooled to 0°C. The precipitate was collected by filtration, washed with a small amount of water and further dried to give 396.2 mg of crude triazolocarboxylic acid product.

MS: [M+1] = 327.

To a suspension of the above crude acid (396.2 mg) in anhydrous THF (7 ml) at 0° C. was added borane-dimethylsulfide complex (10.9 ml; 2 M THF) dropwise. The reaction mixture was left to warm to ambient temperature overnight and cooled to 0°C, then slowly quenched with MeOH. After 30 minutes of stirring, the reaction mixture was concentrated under vacuum. The resulting suspension was treated with MeOH, which was subsequently removed under vacuum. This process was repeated several times. The resulting residue was then treated with 5% MeOH in DCM and washed with sat. _NaHC03 . The aqueous layer was extracted with DCM (3x), the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent provided a mixture of the crude alcohol product ([M+1]=313) and the corresponding primary amide due to hydrolysis of the cyano group ([M+1]=331). 388.8 mg of this crude mixture was obtained and used without further purification.

The above mixture of alcohol (388.8 mg) was treated with phosphorus oxybromide (2.02 g) in 1,4-dioxane (10 ml) at 100° C. for 8 hours. The reaction mixture was cooled to 0° C. and sat. was carefully quenched. NaHCO ₃ (15 ml). After 20 minutes of stirring, the reaction mixture was extracted with EtOAc (3x), washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under reduced pressure provided the crude bromide as a viscous paste which was used in the next step without further purification.

Compound 226

Compound 226 was prepared analogously to Compound 222 in Scheme 33 using the bromide prepared above. MS: [M+1] = 389.

Compound 227 was prepared in the same manner as Compound 226 depicted in Scheme 34. MS: [M+1] = 407.

- 148 040488

Compound 228 was prepared in the same manner as Compound 226 depicted in Scheme 34.

MS: [M+1] = 407.

Scheme 35

Synthesis of Compound 229:

The benzyl analog 229 shown in Scheme 35 was prepared similarly to the benzyl compound 220 in Scheme 32. MS: [M+1] = 411.

Synthesis of Compound 230:

The ketone analog 230 shown in Scheme 35 was prepared similarly to ketone 218 in Scheme 32. MS: [M+1] = 474.

Synthesis of Compound 231:

The benzyl analog 231 shown in Scheme 35 was prepared similarly to the benzyl compound 220 in Scheme 32. MS: [M+1] = 460.

Scheme 36

Compound 63 (0.805 g, 1.78 mmol; from Scheme 18a) was treated with lithium hydroxide (0.128 g, 5.34 mmol) in a solvent mixture of THF (6 mL), water (5 mL) and MeOH (1 mL) at rt. T. for 16 hours Then the reaction mixture was concentrated under vacuum, acidified to pH 3-4 with dilute HCl. The resulting precipitate was collected by filtration, washed with water and dried to give 0.638 g of the acid as a yellow solid.

MS: [M+1] = 424.

The above acid (0.638 g, 1.5 mmol) was treated with NBS (1.61 g, 9 mmol) and NaHCO ₃ (1.51 g, 18 mmol) at rt. for 16 hours. The reaction mixture was cooled to 0°C, sat. sodium thiosulfate (aq.). This mixture was extracted with EtOAc (2x), washed with sat.

NaHCO3, brine and dried over MgSO ₄ . Silica gel chromatography of the filtered concentrate with a gradient of 0 to 100% EtOAc in hexane provided 0.580 g (72%) of the dibromo product as a yellowish solid.

MS: [M+1] = 538.

Compound 232 was prepared analogously to Compound 55 in Scheme 18a using the bromide obtained above. MS: [M+1] = 439.

Compound 235 was prepared analogously to Compound 55 in Scheme 18a using the bromide obtained above. MS: [M+1] = 440.

Compound 236 An alkyne moiety was prepared analogously to Compound 161 in Scheme 21. MS: [M+1] = 384.

Compound 241. The alkyne moiety was prepared analogously to Compound 161 in Scheme 21. MS: [M+1] = 385.

compound 247

Synthesis of Compound 247.

The bromide ester (13.9 mg, 0.0344 mmol) was treated with lithium hydroxide (10 mg) in a solvent mixture of THF (0.3 ml), water (0.25 ml) and MeOH (0.05 ml) at r. T. within 16 hours Then the reaction mixture was concentrated under vacuum, acidified to pH 3-4 diluted HCl and cooled to 0°C. The resulting precipitate was collected by filtration, washed with water and dried to give 9.5 mg (74%) of the acid as a light brown solid.

MS: [M+1] = 377.

To the above acid (5.1 mg, 0.0136 mmol) with stirring in DCM (0.15 ml) were added oxalyl chloride (8.6 mg, 0.0678 mmol) and DMF (5 μl). After stirring for 2 h, the solvent and excess reagent were removed under vacuum. The resulting residue was resuspended in DCM (0.15 ml), cooled in an ice-cold salt bath, and ethanolic methylamine (100 μl; 33%) was added dropwise. After 20 minutes of stirring, the reaction mixture was applied to prep. TLC plate and product were isolated using 5% MeOH in DCM as eluent. 4.3 mg (81%) Compound 247 was obtained as a white solid.

MS: [M+1] = 390.

Compound 248 was prepared analogously to Compound 247 as shown in Scheme 32. MS: [M+1] = 430.

To acid (108.0 mg, 0.335 mmol) suspended in DCM (2 mL) was added oxalyl chloride (170.1 mg, 1.34 mmol) slowly at 0°C, followed by DMF (20 μL). After the formation of bubbles ceased, the ice bath was removed and the reaction mixture was allowed to warm at rt. within 2 hours the Solvent and excess reagent were removed under vacuum. The resulting light brown solid was cooled to 0°C. A solution of NaBH ₄ (2.2 ml; 1.5 M in methoxyethoxyethane) was added. After 30 min, the reaction was quenched with 1N. HCl (0.2 ml) and stirring was continued until the formation of bubbles ceased. Added EtOAc (10 ml) and sat. NaHCO3 (10 ml) and stirred overnight. The aqueous layer was separated and extracted with EtOAc (3x); the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent provided 97.0 mg (94%) of the alcohol as a yellowish solid.

MS: [M+1] = 309.

The above alcohol (97.0 mg, 0.315 mmol) was treated with Dess-Martin periodinane (266.9 mg, 0.629 mmol) in DCM (2 mL) for 1 h. The reaction mixture was diluted with DCM, washed with sat. _NaHC03 . The aqueous layer was separated and extracted with DCM (3x), the combined organic layer was washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent under reduced pressure provided a quantitative yield of crude aldehyde as a brownish solid which was used without further purification.

Compound 250 was prepared analogously to compound 48 in Scheme 16 using the above aldehyde as shown in Scheme 33. MS: [M+1] = 362.

Compound 251 was prepared analogously to compound 250 as shown in Scheme 33. MS: [M+1] = 376.

- 150 040488

Compound 252 was prepared analogously to compound 250 as shown in Scheme 33. MS: [M+1] = 364.

Compound 253 was prepared analogously to compound 250 as shown in Scheme 33. MS: [M+1] = 452.

Scheme 37

The acid (16 in Scheme 15, X=OMe; 258.1 mg, 0.941 mmol) was treated with acetic acid (2 ml) at 120° C. for 5 hours. The solvent was then removed under vacuum. The solid was sonicated in water (7 ml), filtered, washed with water and dried to give 158.4 mg (73%) of the decarboxylated product as a brownish solid.

MS: [M+1] = 231.

connection 257

Compound 257 was prepared analogously to compound 167 in Scheme 11. MS: [M+1] = 364.

Compound 258 was prepared analogously to compound 167 in Scheme 11. MS: [M+1] = 336.

Synthesis of Compound 262:

Benzyltriphenylphosphonium bromide (29.0 mg, 0.0669 mmol) was stirred in THF (0.5 mL) chilled in an ice-cold salt bath. Sodium hydride (4.12 mg, 0.103 mmol; 60% oily suspension) was added. After 20 minutes stirring, aldehyde (15.8 mg, 0.0515 mmol) was added. The reaction mixture was allowed to slowly warm up to rt. for 4 h, then quenched sat. NH ₄ Cl , extracted with EtOAc (3x), washed with brine and dried over MgSO ₄ . Compound 262 was isolated by re-prep. TLC using 2% MeOH in DCM. 1.1 mg was isolated as a white solid.

MS: [M+1] = 381.

- 151 040488

The starting ester (76.4 mg, 0.235 mmol) was treated with N-bromosuccinamide (83.6 mg, 0.470 mmol) in acetonitrile (2.3 mL) at rt. in three days. To the reaction mixture was added sat. sodium thiosulfate. After 15 minutes of stirring, aq. the layer was separated and extracted with EtOAc (2x). The combined organic layer was washed with brine and dried over MgSO ₄ . The bromide product was isolated using prep. TLC using hexane:EtOAc = 1:3 as eluting solvent. 50.2 mg (52%) was obtained as a light brown foamy solid.

MS: [M+1] = 405.

To the above bromide (24.1 mg, 0.0596 mmol) under nitrogen were added phenylboronic acid (10.3 mg, 0.083 mmol), tetrakis-(triphenylphosphine)palladium(0) (6.9 mg, 0.006 mmol), dimethoxyethane (0.69 ml; degassed) and aq. Na ₂ CO ₃ solution (77 µl; 2 M). The reaction mixture was heated at 100°C for 5 h, cooled to rt, diluted with EtOAc, washed with sat. NaHCO ₃ , brine and dried over MgSO ₄ . Rev. TLC with hexane:EtOAc=1:3 provided 17.2 mg (72%) of the Suzuki coupling product as a yellowish amorphous material.

MS: [M+1] = 402.

Syntheses of Compounds 272, 273 and 277:

Compound 272 was prepared analogously to compound 167 in Scheme 11 starting from the above imidazole ester. MS: [M+1] = 440.

Compound 273 was prepared analogously to compound 167 in Scheme 11 starting from the above imidazole ester. MS: [M+1] = 412.

Compound 277 was prepared analogously to compound 167 in Scheme 11. MS: [M+1] = 378.

Compound 279 was prepared by a Suzuki coupling in a manner analogous to the synthetic route described above (see Scheme 37). MS: [M+1] = 436.

Synthesis of Compound 318.

Stage ^1. Bromide starting material 5-bromo-16-methoxy-2,3,4,10,12-pentaazatetracyclo[ ^{11.4.0.0 2'6.08'12} ]heptadeca-1( ¹⁷ ),3,5,8 ,10,13,15-heptaene-9-carboxylate (35.4 mg, 0.088 mmol) obtained in Scheme 37 was treated with CuI (13.3 mg, 0.07 mmol), X-Phos (35.9 mg, 0.088 mmol) in DME (0.55 ml) and TFA (0.122 ml). The flask was purged with N ₂ and Cl ₂ Pd(PPh ₃ ) ₂ (30.7 mg, 0.0438 mmol) was added to this mixture. The reaction mixture was heated to 100°C for 20 h, cooled and partitioned between EtOAc and water. The organic phase was dried and concentrated to give the crude product, as well as desilylated material, ethyl 5-ethynyl-16-methoxy-2,3,4,10,12-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ] heptadec-1(17),3,5,8,10,13,15-heptaene-9-carboxylate. The two compounds were separated using prep. TLC (hexane/EtOAc 1:2).

Stage 2. Ethyl-5-ethynyl-16-methoxy-2,3,4,10,12-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca1(17),3.5.8.10, 13,15-heptaene-9-carboxylate (6.2 mg, 0.0177 mmol) from Step 1 was dissolved in DMF (0.2 ml). To the solution were added iodobenzene (10.9 mg, 0.0532 mmol), TEA (12.5 μl), CuI (0.7 mg, 0.0035 mmol) and Pd(PPh ₃ ) ₄ (10.2 mg, 0.009 mmol). The mixture was stirred at rt. during the night. The mixture was diluted with water and extracted 3 times with EtOAc. The organic phase was washed with brine and dried. The conversion provided 9.7 mg of pure Compound 318.

connection 267 connection 280

Synthesis of Compound 280.

Dicyclohexyl[2-(2,4,6triisopropylphenyl)phenyl]phosphane (7.8 mg, 0.0164 mmol), cesium carbonate (22.3 mg, 0.0685 mmol) and acetonitrile (0.30 ml). The reaction flask was purged with nitrogen gas and dichloro-bis(acetonitrile)palladium(II) (1.42 mg, 0.0055 mol) was added. After stirring at rt. trimethylsilylacetylene (80.7 mg, 0.822 mmol) was added over 30 min and the reaction mixture was heated at 90° C. for 5 h, cooled to rt, diluted with EtOAc and washed with sat. _NaHC03 . The aqueous layer was separated and extracted with EtOAc (2x), the combined organic layer was washed with brine and dried over MgSO ₄ . Preparative TLC of the filtered concentrate using 5% MeOH in DCM/EtOAc (1:1) provided 4.1 mg of the trimethylsilylacetylene derivative as a yellowish solid.

MS: [M+1] = 489.

The above trimethylsilylacetylene (4.1 mg, 0.0084 mmol) was treated with potassium carbonate

- 152 040488 (1.2 mg, 0.0084 mmol) in methanol (0.2 ml) at RT within 3 h. Preparative TLC using

7% MeOH in DCM/EtOAc (1:1) as eluting solvent provided 1.6 mg Compound

280 as a yellowish solid.

MS: [M+1] = 417.

Syntheses of Compounds 284, 301 and 302.

Compound 284 was obtained similarly to MS: [M+1] = 403.

Compound 301 was obtained similarly to MS: [M+1] = 437.

Compound 302 was obtained similarly to MS: [M+1] = 435.

compound 280, based on compound compound 280, based on compound compound 280, based on compound

240.

264.

245.

Syntheses of Compounds 289-292.

Compound 289 was prepared analogously to compound 263 as shown in Scheme 30. MS: [M+1] = 399.

Compound 290 was prepared analogously to compound 263 as shown in Scheme 30. MS: [M+1] = 399. 291 was prepared analogously to compound 243 as shown in Scheme 29. MS: [M+1] = 337.

Compound 292 was prepared analogously to compound 243 as shown in Scheme 29. MS: [M+1] = 337.

(See Diagram 32)

Г ] / 1. UBH ₄ JLJx y

2. POBr ₃ ^MeO /° ^Me

Υ Y-CO ₂ Et 3. MeOH, NaH /

Ν ν connection 298

Synthesis of Compound 298.

The ester (107.9 mg, 0.264 mmol) in THF (2.4 ml) was treated with a solution of lithium borohydride (0.264 ml; 2 M THF) at 0°C. The reaction mixture was allowed to warm to ambient temperature over 4 h, then slowly quenched with sat. NaHCO ₃ , extracted with EtOAc (4x), washed with brine and dried over MgSO ₄ . Filtration and removal of the solvent provided 77.3 mg (86%) of the alcohol as a yellowish solid.

The above alcohol (16.4 mg, 0.0448 mmol) was treated with phosphorus oxybromide (25.7 mg, 0.0895 mmol) in 1,4-dioxane (0.5 ml) at 95° C. for 3 h. then cooled to

0°C, sat. NaHCO ₃ (5 ml) for 20 min and extracted with EtOAc (3x), washed with brine and dried over MgSO ₄ . Filtration and drying provided 16.6 mg of a yellowish solid which was dissolved in anhydrous MeOH (18 μl) and THF (0.35 ml). This mixture was cooled to 0° C. and NaH (9.2 mg; 60% suspension) was added. After stirring for 2 h at 0° C., the reaction was quenched with sat. NaHCO ₃ , extracted with EtOAc (3x), washed with brine and dried over MgSO ₄ . Rev. TLC using 10% MeOH in DCM provided 0.8 mg of Compound 298 as a yellowish solid.

MS: [M+1] = 381.

The starting alcohol (616 mg) was converted to the corresponding bromide as described previously (see Scheme 21). The resulting crude bromide was dissolved in anhydrous methanol (23 ml) and cooled to 0°C. NaH (932 mg; 60% suspension) was added in portions. After the formation of bubbles ceased, the reaction mixture was heated at reflux for 30 min, then cooled to rt. and treated with 2n. HCl (11 ml). The resulting precipitate was collected by filtration and the desired methyl ester was isolated by silica gel chromatography using a gradient elution of 0 to 10% MeOH in DCM. 217 mg was collected as a yellowish solid.

MS: [M+1] = 279.

R = Vp: connection 299

R = Me: 300

- 153 040488

Syntheses of Compounds 299 and 300.

Compound 299 was prepared analogously to Compound 289 from the methyl ester mediate. MS: [M+1] = 461.

Compound 300 was prepared analogously to Compound 289 from the methyl ester mediate. MS: [M+1] = 385.

Scheme 38 Using the above inter-Using the above inter-

Burgess reagent Dry THF, 70 °C, 5 h

Synthesis of Compound 327:

compound 327

Step 1. Isopropoxy analogue (15-chloro-9-[(propan-2-yloxy)methyl]-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1( 17),3,5,9,11,13,15-heptaene-5-carboxylic acid) Intermediate B was prepared similarly using the same reaction sequence as shown in Scheme 28. This compound (0.05 g, 0. 13 mmol) was dissolved in anhydrous DMF (2.5 ml). HATU (0.061 g, 0.16 mmol) and aminoacetaldehyde dimethyl acetal (0.029 ml, 0.27 mmol) were added to the reaction mixture, followed by 0.047 ml (0.26 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 4 hours. LCMS showed product m/z 461.3 and a small amount of starting material. An additional 0.030 g (0.08 mmol) of HATU and 0.029 ml (0.27 mmol) of aminoacetaldehyde dimethyl acetal were added to the reaction mixture and the reaction mixture was stirred for an additional 2 hours. LCMS indicated completion of the reaction. The reaction mixture was diluted with deionized water and extracted with ethyl acetate (3x15.0 ml). The combined ethyl acetate layers were washed with brine, separated and dried over anhydrous Na ₂ SO ₄ . Evaporation of the organic layer provided the crude product 15-chloro-N-(2,2-dimethoxyethyl)-9-[(propan-2yloxy)methyl]-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^2.6 . 0 ^8,12 ]heptadeca-1(17),3,5,9,11,13,15-heptaene-5carboxamide (62 mg, 100%).

m/z calculated for C ₂₁ H ₂₅ ClN ₆ O ₄ [M+H]+: 461; found: 461.3.

Step 2. The acetal fragment was removed by stirring the above compound (0.062 g, 0.13 mmol) in THF (2.0 ml) with 1.3 ml (1.3 mmol) of 1.0 M HCl solution at a temperature of 35-40° C for 16 hours. LCMS showed deprotected aldehyde m/z 415.3. The reaction mixture was diluted with ethyl acetate 30.0 ml and washed with saturated NaHCO ₃ followed by brine. The organic layer was separated and dried over anhydrous Na ₂ SO ₄ . Solvent evaporation provided the crude product (15-chloro-N-(2-oxoethyl)-9-[(propan-2-yloxy)methyl]2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8,12} ]heptadeca-1(17),3,5,9,11,13,15-heptaene-5-carboxamide (55 mg, 100%).

m/z calculated for C ₁₉ H ₁₉ ClN ₆ O ₃ [M+H]+: 415, found 415.3.

Step 3 The above aldehyde (0.055 g, 0.13 mmol) was dissolved in anhydrous THF (5.0 ml). Burgess reagent (0.064 g, 0.26 mmol) was added and the reaction mixture was heated at 70° C. for 2 hours. LCMS indicated a mixture of starting material m/z 415 and product; m/z 397.2. Additional Burgess reagent (0.032 g, 0.13 mmol) was added and the reaction mixture was heated at 70° C. for 3 hours. LCMS indicated product m/z 397.2. The reaction mixture was diluted with ethyl acetate (30.0 ml) and washed with saturated NaHCO ₃ followed by brine. The organic layer was separated and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product. Purification of the crude product was carried out using prep. TLC plates: mobile phase: EtOAc:MeOH, 96:04 v/v. ml. Received 13.5 mg of solid

Compounds 327 (25.4% yield).

m/z calculated for C ₁₉ H ₁₇ ClN ₆ O ₃ [M+H]+: 397, found 397.2.

Compound 341 was prepared analogously to Compound 327 as shown in Scheme 38.

Synthesis of Compound 349:

connection 349

^Intermediate C ⁽ prepared in Scheme 28) was ^converted to ^the corresponding carboxylic acid 1(17),3,5,9,11,13,15heptaene-5-carboxylic acid in a manner similar to the conversion of Intermediate A to Intermediate B. The compound was then converted to Compound 349 in a three-step sequence similar to compound 327 shown in Scheme 38 using the appropriate reagents.

Synthesis of Compound 350:

connection 350

Compound 350 was synthesized in the same manner as shown for compound 349 using the appropriate starting materials and the same reactions depicted in Scheme 38.

Synthesis of Compound 355:

compound 355

Compound 355 was synthesized in the same manner as shown for compound 349 using the appropriate starting materials and the same reactions depicted in Scheme 38.

Scheme 39

R

Synthesis of Compound 342:

Compound 342 R = CH ₂ CF ₃

Compound 352 R = CHEP

Compound 351 R = Ch^Ph

Compound 354 R = 2-F-Ph

Compound 353 R= 2-F-Ph (Pathway used for Comp. 353)

_CF3

Compound 342

- 155 040488

The above aldehyde, R = CH2CF3, (15-chloro-9-[(2,2,2-trifluoroethoxy)methyl]-2,4,8,10,11pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca -1(17),3,5,9,11,13,15-heptaene-5-carbaldehyde) was obtained similarly to the aldehyde shown in Scheme 29, (15-chloro-9-(methoxymethyl)-2,4,8, 10,11pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1(17),3,5,9,11,13,15-heptaene-5-carbaldehyde). This compound (0.04 g, 0.1 mmol) was dissolved in 3.0 ml dry MeOH and K2CO3 (0.028 g, 0.2 mmol) was added at room temperature. Ohira-Bestmann's reagent (0.02 ml, 0.14 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 16 hours. LCMS indicated the formation of product m/z 394.2. The reaction mixture was concentrated in vacuo and diluted with 20.0 ml aq. sodium bicarbonate solution. The precipitate was filtered off and washed with deionized water to give 34.0 mg of a solid after drying (85.9% yield).

m/z calculated for C ₁₇ H ₁₁ ClF ₃ N ₅ O [M+H]+: 394; found: 394.

Synthesis of Compound 351:

ci

Compound 351

The above aldehyde, R = CH ₂ Ph, 9-[(benzyloxy)methyl]-15-chloro-2,4,8,10,11pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1(17) ,3,5,9,11,13,15-heptaene-5-carbaldehyde was obtained similarly to the aldehyde shown in Scheme 29, (15-chloro-9-(methoxymethyl)-2,4,8,10,11pentaazatetracyclo[11.4. 0.0 ^2.6 .0 ^8.12 ]heptadeca-1(17),3,5,9,11,13,15-heptaene-5-carbaldehyde). This was converted to compound 351 in a similar manner to compound 342 as shown in Scheme 39.

Synthesis of Compound 353.

The above acetylene derivative compound 353 (15-chloro-5-ethynyl-9-(2-fluorophenoxymethyl)-2,4,8,10,11 -pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1 ( 17),3,5,9,11,13,15-heptaene) was obtained from ethyl 12-chloro-9-oxo-2,4,8-triazatricyclo[8.4.0.0 ^2,6 ]tetradeca-1(10) ,3,5,11,13-pentaene-5-carboxylate and 2-(2-fluorophenoxy)acetohydrazide with sequences similar to those described in Scheme 28.

Synthesis of Compound 328.

Aldehyde (15-methoxy-9-(methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^2.6 .0 ^8.12 ]heptadeca1(17),3,5,9,11,13 ,15-heptaene-5-carbaldehyde) was obtained similarly as described in Scheme 29 for 15chloro-9-(methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^2.6 .0 ^8.12 ]heptadeca-1(17),3,5,9,11,13,15heptaene-5-carbaldehyde. This compound was converted to Compound 328 similarly as shown in Scheme 39 for Compound 342 to give 5-ethynyl-15-methoxy-9-(methoxymethyl)-2,4,8,10,11pentaazatetracyclo[11.4.0.0 ^2.6 . 0 ^8,12 ]heptadeca-1(17),3,5,9,11,13,15-heptaene (Compound 328).

Syntheses of Compounds 352 and 354.

Compounds 352 and 354 were prepared respectively from compounds 351 and 353 in a manner similar to that described in Scheme 29 for compound 339.

Scheme 40

Synthesis of Compound 329.

Step 1 Compound 103 (described in Scheme 18a) (107 mg, 0.29 mmol) was stirred in THF (1.5 ml) at 0°C and DIBAL (0.73 ml, 1 M sol in hexane, 0. 73 mmol). The mixture was stirred for 2 hours. An additional 0.5 eq. DYBAL to bring the reaction to completion. After 30 min the mixture was quenched with saturated NaHCO ₃ and extracted with EtOAc (3x). The organic phase was washed with brine and dried (MgSO ₄ ). Concentration provided a crude product which was purified with prep. TLC (10% MeOH/DCM) yielding 57 mg (61%) [15-methoxy-9-156 040488 (methoxymethyl)-2,4,8,10,11-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8 ,12} ]heptadeca-1(17),3,5,9,11,13,15-heptaen-5yl]methanol.

Step 2 The above alcohol (18.9 mg, 0.0577 mmol) was stirred in AcCN (1 ml) and POBr ₃ (50 mg, 0.173 mmol) was added. The mixture was stirred at 90°C for 3 h, cooled, diluted with EtOAc and treated with sat. _NaHC03 . The mixture was extracted with EtOAc. The organic phases were combined, washed with brine, dried and concentrated to give the crude product, which was used directly in the next reaction.

Step 3 The above crude bromide was stirred with 10% Pd/C (catalyst quant.) in 8 ml 1:1 MeOH/EtOAc under an H ₂ filled balloon for 48 hours. The mixture was filtered through celite. Concentration provided a crude product which was purified with prep. TLC (15% MeOH/DCM) gave 11.3 mg of compound 329 as a white solid.

Synthesis of Compound 356:

Compound 356

Scheme 41

9-tert-Butyl-5-ethyl-16-chloro-2,3,4,10,12-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca1(17),3.5,8.10 ,13,15-heptaene-5,9-dicarboxylate (prepared in Scheme 34) (100.7 mg, 0.234 mmol) was treated with lithium hydroxide (28.1 mg, 1.17 mmol) in a solvent mixture of THF (0.6 ml ), water (0.5 ml) and MeOH (0.1 ml) for 12 h. The reaction mixture was then concentrated by removing more organic solvents under reduced pressure, resuspended in acetic acid (3 ml) and heated at 120° C for 20 hours. The resulting brownish clear solution was then added dropwise to 30 ml of cold water with stirring. The solution was then cooled in an ice bath for over 30 min. ^The resulting precipitate was collected by ^filtration , washed with ^water and further dried to give 45.4 mg ( ⁶⁴ % over two steps) -1(17),3,5,8,10,13,15-heptaene-9-carboxylic acid as a brownish solid.

MS: [M+1] = 302.

To the above monoacid (45.3 mg, 0.150 mmol) in DMF (0.5 mL) were added N,O-dimethylhydroxylamine hydrochloride (22.0 mg, 0.225 mmol), HATU (62.7 mg, 0.165 mmol) and N, N-diisopropylethylamine (58.2 mg, 0.450 mmol). After 2 hours stirring, the reaction mixture was diluted with EtOAc, washed with 0.5N. HCl, sat. NaHCO ₃ , brine and dried over MgSO ₄ . Filtration followed by removal of the solvent under vacuum provided 40.8 mg (79%) of Weinreb's amide as a yellowish solid.

MS: [M+1] = 345.

To the above amide (40.8 mg, 0.118 mmol) in anhydrous THF (0.5 ml) at -78°C was added a solution of diisobutylaluminum hydride (0.5 ml; 1 M in hexane). After stirring for 1 h, the reaction mixture was left to slowly warm to -10°C and cooled again to -78°C. Saturated aqueous potassium sodium tartrate solution (5 ml) was added and stirred for 60 minutes. The solution was extracted with EtOAc (4x), washed with brine, dried over MgSO ₄ . Filtration and removal of solvent provided 18.7 mg (55%) 16-chloro-2,3,4,10,12-pentaazatetracyclo[11.4.0.0 ^{2.6.0 8.12} ]heptadeca-1(17),3, 5,8,10,13,15-heptaene-9-carbaldehyde as a yellowish solid.

MS: [M+1] = 286.

To the above aldehyde (18.7 mg, 0.0655 mmol) with stirring in MeOH (1 mL) at rt. potassium carbonate (18.1 mg, 0.131 mmol) and dimethyl 1-diazo-2-oxopropylphosphonate (21.4 mg, 0.111 mmol) were added. After stirring for 16 h, the reaction mixture was diluted with EtOAc, washed with sat. NaHCO3, aq. the layer was separated and extracted twice with EtOAc; the combined organic solution was washed with saline and dried over MgSO ₄ . Filtration and removal of the solvent provided a crude mixture from which the desired alkyne compound 356 was isolated with prep. TLC using 5% MeOH in EtOAc/dichloromethane (1:1) as eluent. 8.7 mg of Compound 356 was obtained as a light yellow solid.

MS: [M+1] = 282.

Compounds 357-375, 382, 383, 385-398, 399-412, 414, 416 and 425-428 were prepared in a manner similar to that described in Schemes 29, 30 and 34 using the appropriate starting materials. For

Compounds 398 used trifluoroethyl iodide in the Sonogashira reaction.

Compounds 376-381 were prepared as shown in Scheme 21 prior to the introduction of the oxazoline moiety.

Compound 384 was obtained similarly to Compound 280 after the introduction of the oxazoline fragment.

Compound 413 was prepared as shown in Scheme 39 using benzyl bromide instead of methyl iodide.

Compound 415 was prepared analogously to Compound 280 starting from compound 452 and 3-methyl-1butyne. In this reaction mixture, two additional compounds were formed - compound 453 (bis-alkyne product) and compound 454 (the Cl atom of the A-ring substituted with an alkyne fragment).

Compounds 417-423 were prepared as shown in Scheme 39 using the appropriate substituted benzyl bromide instead of methyl iodide.

Compound 424 was prepared analogously to Compound 280 using bromide as starting material.

Compound 429 was prepared using the Mitsunobu reaction between phenol and imidazoyl methyl alcohol (see Scheme 15 and following examples of reaction conditions).

Compound 430 was prepared as shown in Scheme 29 by alkylation of imidazoyl methyl alcohol with BnBr using NaH in THF as the base medium.

Compounds 431-432 were prepared as shown in Scheme 29 by alkylation of imidazoylmethyl alcohol with an appropriately substituted benzyl bromide using NaH in THF as the basic medium.

Compounds 433-435 were prepared analogously to Compound 429 using an appropriately substituted phenol.

Compound 436 was prepared analogously to Compound 430 by alkylation of the corresponding starting alcohol with 2-F-benzyl bromide under the same basic conditions.

Compound 437 was prepared analogously to Compound 430 by alkylation of the corresponding starting alcohol with 3-F-benzyl bromide under the same basic conditions.

Compound 438 was prepared analogously to Compound 430 by alkylation of the corresponding starting alcohol with 3-N-benzyl bromide under the same basic conditions.

Compound 439 was prepared analogously to Compound 430 by alkylation of the corresponding starting alcohol with 2-N-benzyl bromide under the same basic conditions.

Compounds 440-443 were prepared analogously to Compound 429 using the Mitsunobu reaction between phenol and the corresponding primary alcohol.

Compounds 444-445 were prepared analogously to Compounds 274 and 215 as shown in Scheme 29 by alkylation of imidazoylmethyl alcohol with a suitably substituted benzyl bromide using NaH in THF as the basic medium.

Compounds 446-447: Starting from a similar aldehyde as shown in Scheme 39, these compounds were prepared similarly using olefination conditions as shown in Scheme 33.

Compound 448 was prepared starting from compound 356 and similarly to the condition in Scheme 29, the Sonogashira reaction was carried out using benzoyl chloride instead of Ph-I under PdCl ₂ (PPh ₃ )2 catalysis.

Compound 449 was prepared starting from compound 403, benzylation was carried out using BnBr as the alkylating agent and NaH in THF as the basic condition, heating if necessary.

Compound 450 was prepared starting from compound 403, benzylation was carried out using 3-F-BnBr as the alkylating agent and NaH in THF as the basic condition, heating if necessary.

Compound 451 was prepared starting from compound 403, benzylation was carried out using 2-F-BnBr as the alkylating agent and NaH in THF as the basic condition, heating if necessary.

Compound 455 was prepared starting from compound 256 and similarly to the condition in Scheme 29, the Sonogashira reaction was carried out using benzoyl chloride instead of Ph-I catalyzed by PdCl ₂ (PPh ₃ ) ₂ .

Compounds 452-454 were synthesized under the same conditions as compound 280 starting from compound 452 and 3-methyl-1-butyne.

Compounds 456-471 were prepared in a manner analogous to that described in Schemes 28 and 29; using appropriate source materials. NaH replaced KO'Bu to form an imidazole ring.

Compounds 180-471 were characterized by MS and 1H NMR. Characteristics of MS are given in table. 1.

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Table 1

Characterization of MS Compounds 180-471

Comm. No. Structure Observed MS (M+1) 180 x L? otTD MeO ^'^; X OD\ _R 460 181 L G ⁰ r x< l ί J ) MeO ^ pZ \ t \\___/ X\ // -XX 460 182 F o z z \ .·. 442 183 Ll3, _z , _; ..N3 N 'γ' LD MeO -'^ O—m u^A ⁷ Q- ^F 502 184 Z. Z o l^d Vz Jz □ o f 502 185 //L XD V LL 459

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186 ^fA< Y-N_OMe ^N 'N 396 187 YN'O F^YyN oEt 410 188 ъ Ύ 0 r° “Π 476 189 /Go .γ ΈΧ 476 190 /Γ° Υ 486 191 r Y ^CN γ ^Ν ί ΜβοΥΥ-Ν CL /¾. ύ Ο, 403 192 ΥΓψ Μθο^^γγ rc/% ^Ν 'Ν^ U 441 193 C/YCN ΥΥ ^Ν Λ ί Ύ ) CF ₃ MeO Ο^-;. ” Y and 453

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194 O Y L Hell ΧΧ o X 440 195 ..o' ¹ Ό D· 458 196 s F O L γ-ν About Z T 403 197 RLA XX and 389 198 N-l/ Ν-Ά 384 199 I C χχ o 426 200 ..1. •... O 414

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201 .,-. . MeoAyN o L ^Ν ν g 450 202 AA Cl _A ^N n U 443 203 >? A ) Ta zz _z a \a Q 3 485 204 A ⁰ ci^aa o.,-. ^nAV 436 205 p A T O 388 206 X ^N ^ ° ^N 1 j ''CI'^A _r N o II \___/ ^N V 412 207 Av G A ^CN ίΎ ) fA 369 208 2 φ O XZ Xp. ^z I .., 0 A ^z oAA 403

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209 Ji Meo .N g M ^CN 403 ^N v C> ^ci 210 MeSG M —CN Q 370 ^N V 211 na _co ^x ra r V 347 1— W 212 H ₃ SSG 423 1 Y ^N ^/ r> ---Br 213 II H _3ccr 441 I v- Έ ^ ^Ν ---Br [| \ / ^N -k 214 II H ₃ GCT i 437 r^X Λ 0-/ 215 J 360 cr o _x

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216 dCJ 395 217 381 218 2 δ d d 400 219 jCQ 6 MeO - N-% d ⁷ N _o 416 220 xx / o 370 221 /. /Nd__/ xx / o 384 222 nX ^CN MeoXX-^O-^^ ^l/ 403

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223 /AF Xa ) x ^meo n 403 224 p^cn p T /=4 MeO' - 'N ^N P 385 225 ^X- ^cn XX 7 /P ^{Br N/} n 464 226 / ^N Г 5-CN pP ^z Р/ ⁿ n 389 227 G Acn ^N V 407 228 GP ^N ^N 407 229 G HHg ^F x/x/NP uQ O pAp ^N v 411

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230 Cyd' ^{0 N} Y ΪΎ ) ¹ C./' ⁰ O F 474 231 uQ ' ^N v\ o F 460 232 MM UG MeO^^yN^o^ ^N d ^v U 439 233 γ o 383 234 ώ. O 383 235 z ^N " ^M e°- ^> zZx. _{rN 0} ⁿ -n U 440

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236 Αχ - WITH 384 237 AO Cl Ο _χ ^N V 385 238 Al /) / l > ci 4__ 371 239 DN cr 413 240 Az about 413 241 rV-s n ^N f ⁿ n U 385 242 O AV qAA^n o._ 447

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243 Cl \ _z °\ >4/ 385 244 x4V O 383 245 o and '' ^{C1 N} 0, ⁿ n 445 246 °C' O 411 247 s z \ Dx co f o ” 390 248 ₂ Ay^-A ₂ Lz 7-2 Q 430 249 §4 o 369

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250 δ 0 0 about ζΛz 362 251 .00™ jOO o MeO Np, N—/ N- / ' ⁿ N 376 252 p- “ MeO^^Y _N __y N' / ' ⁿ 'N 364 253 PF 0 χ-CN o ^N I <5 ⁿ 'n ^z 452 254 r ^z r ° 0p ^? 426 255 ?0 ) X'0 O 398 256 04 about 326

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257 /ch A MeO' ^: L 364 258 Μ /°~ ^Ν cll Ν- 2 'Ν 336 259 Λ Λ 399 260 gA°1 XX > SG - _Οχ/ ^Ν 'Ά 461 261 <Γ CI ν Ο^χ- ^Ν ν^ ^/ 340 262 Cj^CN MeoX^N'^^Q' ^N 'N ^Z 381 263 r ^N /°D jjTA 447

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264 Οη 1 /' α ^/χζ γ Ν o 447 265 r-yZ X) CI-^^y-N Ο... 461 266 φΧΟ CI-^^y-N Ο 467 267 s7 ^{l Ν} Α 427 268 ΥΥΥ ^α ίΑΎ 475 270 Ο A > ό d 475 271 < 461

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272 ^A 'VV 440 273 ίΗΓ 412 274 rUO rO “ΛζΡ ⁴ 409 275 Λ c ϋ 461 276 ..oo ^N ' lft I 473 277 h Hh 378 278 pD-O ^Cl '^ ^X fr ^rJ \_y ^o ^ 397

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279 w <m 1 o % A o us 1 J O ζΑΑ _ζ tZ ZZ 0 s f 2 436 280 .0 ·· / О ζ^ο ' y 417 281 X Z ₄ Z A. 2 G A o γο — 411 282 Oud γ o 385 283 A 7 l U ζγο 385 284 Ο-η Αγ ^Ν ^Ν ^Ζ> 403 285 A 402

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286 - Cl 0 ^N 'NT 413 287 /N 0-1 .£% ci -/V ⁿ ~n 1 413 9 QQ /N _ η Г pc:/ ^^^ιΓ ¹⁴ °Z τ J04 9 QQ // γύ ^ν ) ^Ν Ό 9 QQ Z O 27 MeoAAJ ^M A About 27 27 290 -NO-. icd A MeO ' ' ' n —ϊχ NV 399 -,νΟΑΑ 291 DH ) MeO Ν-Λ N-/N 337 292 r-l sur ....... 337 MeO-^^NA N- / ⁿ -n

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293 Y\ Cl Nb' 416 294 X......J _f γ o 385 295 Y γ O 385 296 γ, Y' And 383 297 ,^N P-y bmx Cl \ _y O\ ^N 'N 399 298 2 F OU F^ ΧΎΥ / vJ° 381 299 gas D) CI'^^^N'X_O ^Ν Χ 461

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300 ί Cl^ _z i pPp 1 385 301 I Tp ¹ O z 'OA 437 302 A / / r ^z ° AA A 435 303 jf Cl ,.N θ'-N 451 304 I Pi IJa o i Ί AAA 515 305 Cl syaΆ 368 306 Cl s~ Apr 382

- 176040488

307 Υ With 434 308 Γ YCO _z Et ΥΟ CI'^^yN^O^CH3 ^Ν Υ 442 309 .Ν ,Υ Υ γΝo^CFg Ύ 439 310 L-L ίΎ >ΟΙ'^^γ-Ν Υ ⁷ τ 444 311 A A 430 312 ιΓ X "> TL A O 451 313 X 9 Ο 513

- 177040488

314 About 369 315 At z L Z^x _zV about 397 316 C < XY / Cl __ _/ O _X ^- ^N 'rT 1 399 317 Χχ V and 385 318 G Lso ₂ E1 MeO ^ · ' / \u003d l -W) 426 319 'Xyi X O 465 320 1 £ 7A ζ^-Λ^ζ '= .. 1 τι 465

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321 О NА ^F Pf > I 493 322 XLp O 479 323 X about 463 324 No pl /^vΆ 463 325 pxMp Qω 479 326 /. z. P o _x Pa 4 o 493 327 Μ o 397

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328 322 329 xg ΜβίΛ^Ν 312 330 JCJ ^CI ^Y ^N 461 331 Qd X-λd And 477 332 Q ⁷ ?=\ Ζχ zdz 1 I o ° 1 Q o / 477 333 ABOUT Cl %λχ· O 477 334 ϋΧ O O Z 482

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335 ^F A1 , ^NA 483 336 & ζ^, ο O. h'j O 515 337 SU UA j ⁷ a ⁿ 5 ° - . ^G - ⁿ n 515 338 Ay Αχ Q CJ 497 339 ^0Ι ^^ϊΓ ^Ν \ A 340 340 o xy/:G O MeO' ¹ ^ Q. A 443 341 A ^N -N ^ 383

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342 yr _Z °x/ ^CF 3 ^N 'N 394 343 Al / z^. P about VAc o o 2 443 344 G 0 ABOUT X R-R 321 345 /N R 354 346 XX O cg ^ p ^ν ν 416 347 2 δ X Ζ /ЧX ^ζ 335 348 ^N O-//x C ^J_ ' ” -: . . 419

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349 _ A D GA < C-Q 431 350 ,T R xd o 445 351 l _C ,A1 ^c Τι ^N 'N \e^ o 402 352 _C1 ar ^{C| n} 'nA^ _v O 416 353 dh x o 406 354 ^C| -'XX%--'N /=l °Ύ F 420

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355 _Cl jcd ^c 4 /=\ ⁿ -n ohQ> F 449 356 Ί ¹ VZ zz 0 o 282 357 o 0 ^Z \AA ^Z z^o /=\ S w 403 358 4 °z <4ύ> 0 O 403 359 Ω X aAb VJ° 0 “Π 421 360 o 0 ςΑο O ^- ^ 421 361 Pa jA/^Ά α ν4 ^N V 438

- 184 040488

362 χ Cl P Lz ^IN 'N 0 O J ¹ 471 363 Cl χχ4 ^N V o-^ Y _T 369 364 ζΥΥ z Vz zY 0 about °, Ύτ 355 365 X Cl^ M YO £ G 471 366 MeO .3 ^z ^AA/ ^z / ⁰ % ^F '0 417 367 MeO ^x M xj 0 P 417 368 MeO ^x cif and ^N V cj 0 ABOUT 433

- 185 040488

369 O ABOUT / ao 0 P 435 370 xpav? ^NV 449 371 φ ъ ^ζ 4 0 ο J ¹ 467 372 S δ 0 ϊ \ ΐ\ 0 Οω 467 373 2 φ Ο 0 0 413 374 Ξ CD Ο ,0 ^ζ Ρ^Αγ ^ζ ζ ^ζ ο Ο \___/ 0 ο 468 375 <G ^z Κζ ζ-ζ 0 2 485

- 186 040488

376 O b vJ° o 417 377 O 0 1 417 378 . N Ρ-η X00O N'-2— 'Ν 435 379 0 0 τι 435 380 ο 0 F0 0% ^Z xXF>Xz ^Z Χ0 ^Ο 355 381 Ο /0 <χτ> ^J° 355 382 Ξ α > Ο .0 vJ° 365

- 187 040488

383 -Ν P^ ΝΝ 323 384 ryO / ί Ύ > ¹ y π q \ ^Ν OMe 403 385 ζαΤ^ ^Ν 'Ν 401 386 ^Ν'Ν 469 387 _n yv ^c| -^H ^ν ν 467 388 ίΜγ V 397 389 Ρ-η ^i^NCpzOFs ΜθΟ^^^νΙ Ν'Ν 465

- 188 040488

390 h NV 465 391 _M "AAJ ^ci ^^ ^{ci N} V 466 392 ^{c F3} mlH ^N V 483 393 _C |AA^7 N- X 'N 325 394 y _r ^N V 363 395 vy ^{5 N'N} 358 396 ^N Y 372

- 189 040488

397 o.h O\ 388 398 ^NV 364 399 X about 376 400 O h O 426 401 o A ^Ζ ΑΑγ ^Ζ 1 and 359 402 A _ ll ^N V 359 403 2 δ h ^Z A'^A ^Zill 278

- 190 040488

404 < δth 354 405 2 a> O U and 368 406 X. _ AL G Y — X # ^CF3 /cd ΜβΟ-^^^Νγ Ύ 422 407 A ° MeO^^m-y 355 408 1 l Y ^l ABOUT 355 409 _ /=\ χ γ / ^x ° ^Me MeO'^^^^N'X 384 410 γί Ύ 292

- 191 040488

411 ^ν'ν 372 412 φ ъ “Ο ο 438 413 ό b MeO - NO Ν' 2 ^ΙΝ 'Ν 368 414 Μθ0' ^Χχ Ο\ _Ν Ο Ν' 2 384 415 / 02 324 416 Q 296 417 οχ 5 MeOO/y Ο ^ν 'ν ^ζ 402

- 192 040488

418 those 5 MeO'^^NA 452 419 S φ O X AX 9 m 386 420 xO' MeO '^ ^/X NX N , / 'N 452 421 λ . ΤΊ 434 422 •b o 451 423 A 9 o o J ¹ 500 424 s φ o X ¹ 320

- 193 040488

425 Cl a Ζ-Λ T I 340 426 Ji cr A 368 427 cr Ai A 384 428 cr co O M i Xr. 438 429 jcr Mm ο-X ft __/ ^{OMe N} -A 408 f \____ _l 430 J XT ' ^H ^Dr\ / ^{0Me N} 'N 422 cA ^F 431 J Ύ ) U ^)Γ ^Ν \/ ^0Μ θ ^N 'N ^Z 440

- 194 040488

432 rr ^N V°Xr ^F ^pme ^N 'N 440 433 o A} ^C| ^^?gL_ N-yY ^N OMe 426 434 ciAxy _N N'X^ N OMe 426 435 (ΎΆύπ^ ^N OMe 426 436 xxlxs ^N W 396 437 XJ (> V 396 438 ^Yn'n 413

- 195 040488

439 O. x) oh X-Z Z-Z X about 413 440 about X b 364 441 O X GL a X 382 442 O X < a £ su l 382 443 LL. f oh oh 382 444 χ ^Ν l(ъ Ν £ \ ^N OMe 457 445 oz\ X) g ^f XX £ o 457

- 196 040488

446 C// ci \ /°a ^N 'hi 1 432 447 LL ό y wow Oh oh 450 448 „A ^{8 N} V 386 449 "1 X) _z AAb Ή z-z c φ 368 450 Well ζ,γΧΑζ U-z ζ-ζ Q 2 494 451 !ΐι 7Э ζ ^/ γ^^Α ^ζ: bz ζ-ζ 0 ο φ 2 386 452 □ th from 337

- 197 040488

453 / G 356 454 X 369 455 γτ ^ν ) About Cl __rme 430 456 d? O^^L-N o— fAf ^N 'N^ 434 457 PN ^a VV ^N A DD ^N '£ 384 458 N Ο-, A-O ^N'hA 369 459 θ-Ν ^{F Ν} 'Ν^ 418

- 198 040488

460 ΑΧ MgChrfr ⁿ V 421 461 .Ν ,Ά .οΑ 428 462 ΑΡ'Ν J Ύ > Μ Ν-Α 426 463 α Αα?Ι A ^γΝνΡ-' A 384 464 A A- 371 465 AT .mA ^N V 375 466 ? ΑΧ άο ^{N Ν} 'Ν^ 369

- 199040488

467 XX X LL 419 468 o %z%' X ² 427 469 2=4 / HD Oh Oh Z=/ 427 470 X Z. 7 >L Z t A / lA 405 471 AX') NV γίΧγ _ο - ^FN 'hJ 403

Conducting reactions similar and similar to those shown in Schemes 1-37, the following compounds are also specifically considered in this document.

Ar Λ _χ Ν-Χ N-° -CN MeSG VN II χ, ^Ν Α ,-Ν MeSG g/X o- CF _3ys

- 200 040488

pi _n J MeO'^Py-N_ phx,p - NJ ΓΎ ) T MeO^M/^ n -γ o __N- ₀ ,^νΡ Ν ^Λ Μ .nj ' pr ^ρ '''θ> γ_ _Ά ^Ν ΑΆ O Ν=/ M'? od MeO-^ΑΑ^Ν Q w / - ⁷ V .PI Ρ ^Ο MeO'^^PyN_у ⁿ -n Tj /X XX 'X LL AJ4 nP\P pAp ^F '^)/ ^N \/ ^O ^ _v - ^N ' ^N FcP GzS PP) JX-i. LL ^N PN XX 4 ji pp ^N A ^N VA X7

- 201 040488

O ft V K γ P F o ft About SG Z f a Υ J1 NO jctT^ ⁷ MeO'^ryj-N θ' .N Oo γ .N Ό N-Y N'Pv Jf D ) ^{v F} '^r ^N v/°Y^ II \_/ Y\ ^N 'NL? MM P-N ΜβΟ^ΌγΝ (Ύ Μ,Ο-Γ^νΡ,Ο-ΡΛ _F bA ^N *hl^ _F A or MeO'^^OV^N _o _\_ Q F MeO^^OyN ox, ^N ' Cr ^F F 3)0 O^'^'^TG'H O' “ΟΓχ ^b o ^.^^b

- 202 040488

dX ^Qdk ° ¹ ' Chl d jCO ^t СГVN p^ rVJ sda,? _{cf 3} II "-/>r \ ⁿ % o -d 0 -N .¾ « ^ν Ά O -N __.N-0 X^ ^N Λ XX / N'm'X ^N A ⁿ V ^x ) -N JJo XX / Cl ^N VX ^ ^N ₄ y^O Χ<Χ About N=/ N'O xcd ⁷ OddXX/C! Ν-γ ^N vd_ kJ ABOUT

- 203 040488

about 4 rWX ^ν ^Υ7 ^{Cl 1 Ν} '^Ο ^ ^Ν ν JZ° α— ^4' —ι^Ν_ ^ν -ν^ Χ ^ν χΑ ^Aa-^-Q ^N > χ 0|ΖχΖχ^ν F ^44Cl _r u ,> lX ^Ν 'Ν^ ' __Ν-ο d/G^ CI-^^^yN θ ^Ν -ν^ A W α-Π sSG> Api ^Ν Ρ'Ν ,ΆΓ^ ί D ) ^cl '^rV/°z ^ν -ν X W ΓΎ ) Ά' YM ,Ν~Ο χίΡ Νη/' ⁷ Ο

- 204 040488

XX) “ΎΎ Υν ν< γ ΓΥ ) N^ Ρ'Ν b Υ Υ γ ΥΥ -CN ΛΑ / Ρ"\ Μ II J > θΥν-Λ _/ ν-Ν Ο ' ^Ν Χ Ο ry Λ Υ —CN G ^~ο νΎ^ Γγ \ ί Ί ) II 1 ) γΥ0\-Ν ο^τ Η <5 ^Νν Υν to ^χ ) l γύ4 b Υ Ν~0 Λγ γ3 ο Lee 1 Υ ο Λ WITH. _ Ν=/ Υ /Γ° γγ γ UJ/ ΓΥ\ Ρ-Ν 4ι _ζ Ν^ΎΥ YQ γ Γ λ Λ °Ό ^Νν Ο Λ Y'? Λ # \|Λ— ίΎ ) ^Ν ^\7 ΓΎ ) Ν γ-y γ" 'Ν γ" Ν γ~P F ⁷

- 205 040488

Example 105 Evaluation of the activity of a positive allosteric modulator of the α5-containing GABAA receptor (GABAA-R).

Stage 1. Creation of clones of GABAA-P subunits (α5, β3, γ2, α1, α2 and α3) and obtaining the corresponding cRNAs.

Clones of the human GABA-P α5, β3, γ2, α1, α2 and α3 subunits are obtained from commercial sources (eg, OriGene, http://www.origene.com and Genescript, http://www.genescript.com). These clones are constructed into pRC, pCDM, pcDNA and pBluescript KSM vector (for oocyte expression) or other equivalent expression vectors. Standard transfection agents (eg FuGene, Lipofectamine 2000 or others) are used to transiently transfect host cells.

Stage 2. Functional analysis of GABAA-R for α5β3γ2, α1β3γ2, α2β3γ2 and α3β3γ2 subtypes in the Xenopus oocyte expression system.

cRNAs encoding α5, β3, γ2, α1, α2 and α3 subunits were transcribed in vitro using the T3 mMESSAGE mMACHINE Kit (Ambion) and administered (at a ratio of α:β:γ = 2:2:1 or other optimized conditions) into oocytes freshly prepared from Xenopus laevis. After two days of culture, GABA-dependent Cl channels from oocytes were displayed using a TEVC device (Warner Instruments, Inc., Foster City, CA). GABA, benzodiazepine and diazepam are used as reference compounds to test the system.

Step 3. Evaluate test compounds for positive allosteric modulator activity of the α5β3γ2 subtype and test off-target activity of α1 to α3 associated β3γ2 subtypes when _EC50 =5 μM threshold selectivity is reached: GABA-dependent Cl channels from oocytes are measured in a TEVC setup in the presence of test connections. The positive allosteric modulator activity of each of the test compounds is tested in a 5-point dose-response analysis. Test compounds include some control compounds (literature EC50 values for the α5β3γ2 subtype are in the range of 3-10 μM). _EC50 in the α5β3γ2 subtype is obtained for each compound. If the EC ₅₀ in α5β3γ2 is <5 μM, then the EC50 of the other three subtypes (α1β2γ2, α2β3γ2 and α3β3γ2) is further determined individually to investigate the selectivity of compounds in the α5β3γ2 subtype over other subtypes.

Step 4: Evaluate additional test compounds for the α5β3γ2 subtype and test off-target activity when an EC ₅₀ =0.5 μM threshold selectivity is reached. A second batch of test compounds is tested using the same strategy, but with a lower cut-off EC ₅₀ (0.5 μM). Again determine the EC ₅₀ α5β3γ2 subtype for each of the compounds. α1-α3 associated β3γ2 subtypes are investigated only if the EC ₅₀ for the α5-containing receptor is <0.5 μm.

Example 106 Evaluation of Compounds for Positive Allosteric Modulator Binding and Activity at _GABA.alpha.5 Receptors.

(A) Binding activity of test compounds on GABAA-R.

Preparation of Tissue Culture and Membrane: Binding was performed on Ltk cells stably expressing GABAA receptors: α1β1γ2, α2β3γ2, α3β3γ2 and α5β3γ2 (provided by Merck Co., NJ, USA). Cells were seeded in 100 mm culture plates in DMEM/F12 medium containing 10% serum and antibiotics in 5% CO ₂ and allowed to grow for 1-2 days. Then, GABA expression was induced with dexamethasone as follows: 0.5 μM for 1 day for α5-containing and 2 μM for 3 days for α1, α2 and α3-containing GABA-R. After induction, cells were harvested by scraping into Dulbecco's phosphate-buffered saline (DPBS, pH 7.4, Invitrogen, Carlsbad, CA, USA) and centrifuged at 150xg for 10 min. The pellet was washed twice by resuspension and centrifugation. Cell pellets from at least five different preparations were pooled, suspended in binding assay buffer (50 mM KH ₂ PO ₄ ; 1 mM EDTA; 0.2 M KCl, pH 7.4) and membranes prepared by sonication (3-5 times, 30 s) using Branson Sonifier 150 (G. Heinmann, Germany). Protein content was determined using the BCC method (Bio-Rad Labs, Reinach, Switzerland) with bovine serum albumin (Sigma Aldrich, St. Louis, MO, USA) as standard. Aliquots were prepared and stored at -20°C for further use in binding assays.

Ligand Binding: Saturation binding curves were generated by incubating membranes with increasing concentrations (0.01-8 nM) of [ ³ H]Ro 15-1788 (Flumazenil, 75-85 Ci/mmol, PerkinElmer, MA, USA) with non-specific binding measured in the presence of 10 μM diazepam. Inhibition of [ ³ H]Ro 15-1788 binding of the test compounds was performed at radioligand concentrations equal to or lower than the Kd values for α1, α2, α3 and a5-containing GABAA-P, determined from saturation curves.

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

Data Analysis: Saturation and inhibition curves were generated using GraphPad Prism software (GraphPad Software, Inc., CA, USA). The equilibrium dissociation constants (Ki values) of the unlabeled ligand were determined using the Cheng-Prusoff equation Ki=IC5o/(1+S/Kd), in which IC ₅₀ is the concentration of unlabeled ligand that inhibits 50% of [ ³ H] ligand binding; S is the radioligand concentration; and K _d is the equilibrium dissociation constant of the radioactive ligand. The logarithmic range of the compounds (1 nM - 10 μM) was used to determine the Kj values, which are presented as mean ± SD from triplicate analyses.

(B) Positive allosteric modulator activity of test compounds on the α5β2γ2 subtype GABAA-R.

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

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

Compounds of the present invention were investigated using the following methods.

GABA dose response.

1) 8 oocytes were placed in 8 OpusXpress chambers and hydrated with modified saline

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bart (MBS) at 3 ml/min. Glass electrodes filled with 3 M KCl (0.5-3 MΩ) were used.

The oocyte membrane potential had voltage clamping at -60 mV.

2) The average value of EC ₂₀ GABA, obtained from previous studies, was used five to six times to stabilize the oocytes. Oocytes were washed with MBS for 5-10 min between each application

GABA.

3) Conducted GABA dose-response to obtain the EC ₂₀ value of GABA.

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

1) New oocytes were used for a new study.

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

3) EC ₂₀ GABA was used to generate current (1 _gamk ). Oocytes were washed with MBS for 5-10 min.

4) 1 μM diazepam or methyl 3,5-diphenylpyridazine-4-carboxylate was pre-applied for 40 s followed by co-application of 1 μM diazepam or methyl 3,5-diphenylpyridazine-4-carboxylate and EC ₂₀ GABA to obtain 1 _study . 1 _research . divided by 1 _gamma to obtain potentiation (%).

Test compounds in multiple doses.

1) Repeat steps 1), 2) and 3) above in a control study.

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

3) Remove all tested oocytes, new oocytes were used and the above steps 1) and 2) were repeated for the second concentration of the same compound tested. Each oocyte was used for only one concentration study for one test compound. The steps were repeated for other test compounds.

In some embodiments, the compounds of this invention have a binding affinity (as represented by Ki) for a5-containing GABAA-Ps of less than 200 nM, less than 180 nM, less than 150 nM, or less than 100 nM. In some embodiments, the compounds of this invention have a binding affinity (as represented by Ki) for a5-containing GABA-Ps of less than 50 nM. In some embodiments, the compounds of this invention have a binding affinity (as represented by Ki) for a5-containing GABA-Ps of less than 10 nM.

In some embodiments, the compounds of this invention are selective for α5-containing GABAA-P over α1-containing GABAA-P. In some embodiments, the compounds are more than 50-fold, more than 100-fold, more than 500-fold, or more than 1000-fold selective for α5-containing GABAA-P relative to α1-containing GABA-P.

In some embodiments, the compounds of this invention have an EC ₅₀ for a5-containing GABAA-P less than 500 nM, less than 100 nM, or less than 50 nM. In some embodiments, the compounds of this invention have an EC ₅₀ for a5-containing GABAA-P less than 25 nM.

In some embodiments, the compounds of this invention potentiate α5-containing GABAA-Rs by greater than 10%, greater than 25%, greater than 50%, or greater than 75% at 100 nM. In some embodiments, the compounds potentiate α5-containing GABAA-P by more than 10%, more than 25%, more than 50%, or more than 75% at 1000 nM.

The results of screening studies of binding and functional activity of PAM are summarized in table. 2and 3.

Tab. 6 illustrates the Ki ranges of α5 GABA binding associated with the compounds of this invention.

________________________________________________Table 2

Ki values of GABA binding "5 (nM) < 100 nM 100-1000 nM > 1000 nM Connections 1, 2, 3, Connections 50, 110, Connections 116,

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4, 6, 7, θ, 9, 10, 113 115 119 124, 117, 121 123 131, eleven, 12, 44 .55 101, 125, 134, 136 138, 135, 140, 142, 143 103 105, 107, 108 139, 141, 143 144, 152, 154, 192, 193, 114 128 153, 158, 146 170, 191, 200, 204, 221, 229, 231, 162 163 164, 166, 201, 219, 220, 237, 234, 239 250- -253, 169 171, 172, 173, 240, 246, 247, 248, 262, 272, 279, 306, 174 175, 177, 179, 265- -267, 273, 274, 315, 329, 386, 389, 5, 47, 48 .49 51, 281, 283, 284, 286, 390, 429- -435, 446, 52, 53, 54 .56 102, 287, 292 297 -300, 447, 449, 450, 457, 104 106 111 112 303, 305, 309, 312, 458, 463, 466, 468 118 120 126 127 314, 316, 318, 321, 130 133 137, 145, 336, 347, 348, 352, 147 148, 149, 155, 363, 370, 371, 374, 156 157, 160, 165, 376, 378, 379, 382, 168 178, 45, 46, 384 392, 453, 460, 109 122, 129 132, 469, 470 150 151, 159, 161, 167 176, 180- -190, 194- -199, 202, 203, 205- -210, 216, 217, 218 222, 223- -227, 230 232, 233, 235, 236 238, 241- -245, 249 254- 261, 263, 264 268- 271, 275- 278 280, 282, 285, 288- -291, 293- -296, 301 302, 304, 307, 308 310, 311, 313, 317 319, 320, 322, 323 324, 325, 326, 327 328, 330, 331, 332 333, 334, 335, 337 338, 339, 340, 341 342, 343, 344, 345 346, 34 9- -351, 353- -62, 364, 366, 367- -369, 372, 373, 375 377, 383, 385, 387 388, 393, 394- 399 401- 427, 448, 451 452, 454- -456, 459 461, 462, 465, 467 471

Tab. 3 illustrates the ranges of functional potentiation of α5 GABA associated with the compounds of this invention:

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Table 3

Functional data of GABA "5 5-20% @ 100nM 20-50% @ 100nM > 50% @ 100 nM 10, 50, 51, 104, 112, 118, 121, 122, 133, 164, 166, 168, 190, 200, 206, 211, 215, 217, 223, 227, 229, 232, 233, 236, 241, 242, 244, 245, 254-257, 268, 285, 301, 302, 304, 308, 310, 311, 328, 331- 333, 335, 341, 349, 359, 367, 368, 385, 395, 396, 401, 404, 405, 407-409, 413, 417, 420, 423, 454 Connections 1, 2, 9, 11, 48, 45, 55, 109, 110, 111, 118, 120, 126, 127, 128, 130, 132, 137, 147, 148, 153, 155, 158, 162, 163, 175, 180-184, 187-189, 191, 195, 196, 198, 199, 202, 203, 205, 207, 210, 212, 213, 222, 224, 225, 226, 238, 243, 249, 254-257, 264, 290, 293, 313, 319, 320, 323-326, 330, 334, 337, 339, 340, 343, 345, 346, 351, 354, 355, 357, 358, 360, 361, 362, 397, 398, 399, 400, 387, Compounds 113, 114, 145, 149, 160, 171, 172, 173, 174, 176, 177, 178, 179, 185, 186, 194, 271, 350, 353, 356 402, 406, 414, 415, 418, 419, 421, 422

Selected compounds of this invention exhibit >10-fold binding selectivity for GABA α5 over GABA α1, GABA α2, or GABA α3. Some compounds of this application show more than 20-fold, 50-fold or 100-fold binding selectivity for GABA α5 compared to GABA α1, GABA α2 or GABA α3.

Tab. 4 illustrates the binding selectivity ranges of compounds for GABA α5 versus GABA α1, GABA α2, or GABA α3.

Table 4

Binding selectivity GABA "2 or GABA "3 for GABA "5 on comparison with GABA "1, from 20 - before 50 times from 50- before 100- > 100 -multiple multiple 323, 324, 344 . 127 147, . 158 164 . 165 128 163 166, 168, 130, 148, 162 , 169, 171, , 173, 176 , 183, 172, 174, 175, 177- 184, 206, 209 , 222, 198, , 205 242 , 244, 182, 194, 195, 202, 223, 254, 255 , 261 245, , 256- -258, 293, 203, 207, 210, 212, 350, 353, 354 , 403- 319, .320 323 , 326, 217, 218, 232, 233, 405, 408, 409 .411 330, , 339 340 , 343, 236, 243, 249, 260, 459 351, , 354, 355 , 366 264, 268, 270, 271, 367, , 373, 387 , 388, 275, 276, 285, 289, 419, 426 427 .448 290, 301, 302, 304, 452, .455 456 313, 320 , 324- -326, 332, 334, 335, 337, 340, 344 -34 6, 357— 362, 368, 372, 385, 393, 395 -399, 401, 402, 414, 415, 417, 418, 420-425, 454

Example 107 Effect of methyl 3,5-diphenylpyridazine-4-carboxylate on aging-disordered (AD) rats.

Methyl 3,5-diphenylpyridazine-4-carboxylate corresponding to compound number 6 in van Niel et

- 210 040488 al. J. Med. Chem. 48:6004-6011 (2005), is a selective agonist of a5-containing

GABA _a -R. It has an in vitro α5 potency of +27 (EC ₂₀ ). The effect of methyl 3,5-diphenylpyridazine-4carboxylate on rats with age-related disorders was studied using the RVL task. In addition, the occupation of receptors by methyl 3,5-diphenylpyridazine-4-carboxylate in a5-containing

GABA receptor.

(A) Effect of methyl 3,5-diphenylpyridazine-4-carboxylate on aging-impaired rats using the Radial Eight-arm Maze (REL) behavioral task.

The in vivo effect of methyl 3,5-diphenylpyridazine-4-carboxylate on retention of spatial memory in aging-impaired (AD) rats was assessed in the radial eight-armed maze (REL) behavioral task using vehicle control and four different dosage levels of methyl-3.5 -diphenylpyridazine-4-carboxylate (0.1, 0.3, 1 and 3 mg/kg, i.p.). Behavioral tasks of RVL were performed on eight rats with LN. All five treatment conditions (vehicle and four dosage levels) were tested in all eight rats.

The used device RVL consisted of eight sleeves located at the same distance. The raised arm of the labyrinth (7 cm wide x 75 cm long) was projected from each facet of the octagonal central platform (30 cm diameter, 51.5 cm high). The transparent sidewalls on the sleeves were 10 cm high and were angled at 65° to form an indentation. Food wells (4 cm diameter, 2 cm depth) were placed at the distal end of each sleeve. Froot Loops™ (Kellogg Company) was used as a reward. Blocks made from Plexiglas™ (30 cm high x 12 cm wide) can be fitted to prevent entry into either sleeve. Numerous additional maze stimuli surrounding the device were also provided.

VL rats were initially subjected to a pretreatment test (Chappell et al. Neuropharmacology, 37:481-487, 1998). The pretraining test consisted of a habituation phase (4 days), a training phase on standard win-biased tasks (18 days), and another training phase (14 days) in which a slight delay was introduced between the presentation of a subset of arms determined by the experimenter (e.g., five sleeves available and three sleeves blocked), and the completion of an eight-arm biased-to-win challenge (i.e. with all eight sleeves available).

During the habituation phase, rats were introduced to the maze in an 8-minute session over four consecutive days. In each of these sessions, food rewards were dispersed throughout the RVL, initially on the central platform and arms, and then gradually limited to the arms. After this habituation step, a standard test protocol was used in which a food pellet was placed at the end of each sleeve. Rats received one trial each day for 18 days. Each daily trial was terminated when all eight food pellets had been received or when 16 choices had been made or 15 minutes had elapsed. After completion of this training phase, a second training phase was conducted in which the memory requirements were increased by applying a small delay during the test. At the beginning of each test, three arms of the eight-arm maze were blocked. The rats were allowed to eat from the five sleeves, access to which was allowed during this initial data-gathering phase of the trial. The rats were then removed from the maze for 60 seconds, during which time the barriers on the maze were removed, thus allowing access to all eight arms. The rats were then placed back on the central platform and allowed to receive the rest of the food rewards during this phase of the test retention test. The identity and configuration of the blocked sleeves varied between trials.

The number of errors made by the LN rats during the retention test phase was monitored. An error occurred during the trial if rats entered a sleeve from which food had already been received in the pre-deferred component of the trial, or if a rat revisited a sleeve in a post-deferred session that it had already visited.

After completion of the pre-test test, the rats were tested with longer delay intervals, ie. a two-hour delay between the information stage (presentation of some blocked sleeves) and the retention test (presentation of all sleeves). During the delay interval, the rats remained away from the maze in the test room, on carts in their individual cages. VL rats were pretreated 30-40 min prior to daily trials with a single injection under the following five conditions: 1) vehicle control - 5% dimethyl sulfoxide, 25% polyethylene glycol 300, and 70% distilled water; 2) methyl 3,5-diphenylpyridazine-4-carboxylate at 0.1 mg/kg; 3) methyl 3,5-diphenylpyridazine-4carboxylate at 0.3 mg/kg; 4) methyl 3,5-diphenylpyridazine-4-carboxylate at 1 mg/kg; and 5) methyl 3,5-diphenylpyridazine-4-carboxylate at 3 mg/kg by intraperitoneal (ip) injection. Injections were made every other day with washing days in between. Each VL rat was treated under all five conditions during the testing period. Against any bias voltage, the effect of the drug was evaluated using an ascending-descending series of doses, i.e. a series of doses were given

- 211 040488 first in ascending order and then repeated in descending order. Therefore, each dose had two determinations.

Parametric statistics (paired t-test) were used to compare the performance of the retention test in LN rats in the 2-hour delay version of the RVL task in the context of different doses of methyl 3,5-diphenylpyridazine-4-carboxylate and vehicle control (see FIG. 1). The mean number of errors encountered in the trials was significantly less when treated with 3 mg/kg methyl 3,5-diphenylpyridazine-4-carboxylate (mean error ± standard error of the mean (SEM) = 1.31 ± 0.40), than when using the control filler (mean number of SEM errors=3.13±0.62). Relative to vehicle control treatment, methyl 3,5-diphenylpyridazine-4-carboxylate significantly improved memory performance at 3 mg/kg (t(7)=4.233, p=0.004).

The therapeutic dose of 3 mg/kg became ineffective when LN rats were simultaneously treated with 0.3 mg/kg TB21007, an α5-GABA _a -P inverse agonist. The mean number of errors made by rats with combined treatment with TB21007/methyl 3,5-diphenylpyridazine-4-carboxylate (0.3 mg/kg TB21007 with 3 mg/kg methyl-3,5-diphenylpyridazine-4-carboxylate) was 2.88 ±1.32 and was no different from vehicle control rats (3.13±1.17 mean errors). Thus, the effect of methyl 3,5-diphenylpyridazine-4-carboxylate on spatial memory is a GABAA α5 receptor-dependent effect (see Fig. 1).

(B) Effect of methyl 3,5-diphenylpyridazine-4-carboxylate on the occupation of α5-containing GABA receptors.

Animals.

Adult male Long Evans rats (265-295 g, Charles River, Portage, MI, n=4/group) were used to study the occupation of GABAA α5 receptors. Rats were housed individually in ventilated stainless steel racks with a 12:12 light/dark cycle. Food and water were available without restrictions. In additional studies to evaluate compound exposure at behaviorally active doses, young or old Long Evans rats (n=2-4/group) were used for these studies.

Connections.

Ro 15-4513 was used as a receptor occupancy indicator (Ro) for GABAA α5 receptor sites in the hippocampus and cerebellum. Ro 15-4513 was chosen as an indicator based on its selectivity for GABAA α5 receptors over other alpha-subunit containing GABA receptors, and because it has been successfully used in animal and human studies of GABAA α5 Ro (see for example, Lingford-Hughes et al., J. Cereb. Blood Flow Metab. 22:878-89 (2002); Pym et al., Br. J. Pharmacol. 146:817-825 (2005); and Maeda et al. , Synapse, 47:200-208 (2003)). Ro 15-4513 (1 μg/kg) was dissolved in 25% hydroxyl-propyl beta-cyclodextrin and administered iv 20' before RO estimates. Methyl 3,5-diphenylpyridazine-4-carboxylate (0.1-10 mg/kg) was synthesized by Nox Pharmaceuticals (India), dissolved in 25% hydroxyl-propyl beta-cyclodextrin and administered intravenously 15' before the introduction of the indicator . Compounds were administered in a volume of 0.5 ml/kg except for the highest dose of methyl 3,5-diphenylpyridazine-4-carboxylate (10 mg/kg) which was administered in a volume of 1 ml/kg due to solubility limitations.

Obtaining and analysis of tissues.

Rats were sacrificed by cervical dislocation 20' after the indicator injection. The entire brain was quickly removed and washed lightly with sterile water. Blood drained from the torso was collected in EDTA-coated Eppendorf tubes and stored on wet ice until completion of the study. The hippocampus and cerebellum were dissected and stored in 1.5 ml Eppendorf tubes and placed on wet ice until tissue was removed. From previously drug-naive rats, six brain cortical tissue samples were collected for use in creating blank and standard curve samples.

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

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

LC/MS/MS for methyl 3,5-diphenylpyridazine-4-carboxylate.

Receptor occupancy was determined using a ratio method that compared occupation in the hippocampus (an area of high density of GABA a5 receptors) with occupation in the cerebellum (an area of low density of GABA _{a a} 5 receptors) and additionally using a high dose of GABA _a α5 negative allosteric modulator L -655708 (10 mg/kg, i.v.) to determine complete occupation.

Vehicle administration followed by 1 μg/kg iv Ro 15-4513 indicator resulted in a >5-fold increase in Ro 15-4513 levels in the hippocampus (1.93 ± 0.05 ng/g) compared to the cerebellum (0. 36±0.02 ng/g). Methyl 3,5-diphenylpyridazine-4-carboxylate (0.01-10 mg/kg, iv) decreased Ro 15-4513 binding in the hippocampus in a dose dependent manner without affecting Ro 15-4513 levels in the cerebellum (Fig. 2) at 10 mg/kg IV, showing >90% occupancy (FIG. 3). Both methods of calculating RO giving very similar results with ED ₅₀ values for methyl 3,5-diphenylpyridazine-4-carboxylate as 1.8 mg/kg or 1.1 mg/kg were based on the ratio method or using L-755608 to determine occupation.

Exposure to methyl 3,5-diphenylpyridazine-4-carboxylate was below the limit of quantification (BQL) at 0.01 mg/kg IV in both plasma and hippocampus, but was detectable at low levels in the hippocampus at 0 .1 mg/kg IV (see Table 9). The effect on the hippocampus was linear, with a 10-fold increase in dose from 0.1 to 1 mg/kg IV resulting in a 12-fold increase in exposure. Increasing the dose from 1 to 10 mg/kg IV only increased the exposure 5-fold. Plasma exposure increased 12-fold as the dose was increased from 1 to 10 mg/kg IV.

In table. 5 shows the % occupation of GABAA a5 receptors by methyl 3,5-diphenylpyridazine-4carboxylate (0.01-10 mg/kg, i.v.).

Table 5

The effect of methyl-3,5-diphenylpyridazine-4-carboxylate on the hippocampus and plasma in an experimental group of young Long Evans rats

Dose (mg/kg, v.v.) %RO (method L- 655708) (SEM) %RO (ratio method) (SEM) Plasma ng/ml (SEM) hippocampus ng/g (SEM) 0.01 19, 2 (11.1) 15.7 (9.1) BQL BQL 0, 1 16.4 (4.9) 13.4 (4.0) BQL 14.6 (3.5) 1 38.5 (11.2) 31.5 (9.1) 62.8 (6.1) 180.0 (10.3) 10 110.0 (6.6) 90.2 (5.4) 763.5 (85.7) 947.2 (51.3)

Additional studies were performed in aged Long Evans rats to determine the effects at behaviorally appropriate doses in cognitive studies. Exposure to young Long Evans rats was also determined for association with receptor occupancy studies that were performed on young Long Evans rats. The effects on young and old Long Evans rats were relatively similar (Table 6, Fig. 4). A 3-fold increase in dose from 1 to 3 mg/kg ip resulted in greater than dose-proportional increases in both hippocampal and plasma exposure in young and old rats, with increases ranging from 4.5 to 6.6-fold .

Table 6

The effect of methyl-3,5-diphenylpyridazine-4-carboxylate on the hippocampus and plasma in an experimental group of young Long Evans rats

Young Young old old Dose (mg/kg, ip) hippocampus ng/g (SEM) Plasma ng/ml (SEM) hippocampus ng/g (SEM) Plasma ng/ml (SEM) 1 25.9 (1.7) 20.0 (1.4) 38.8 (21.7) 45.2 (29.6) 3 129, 1 (22.4) 132.9 (19.5) 177.5 (19.5) 196 (18.2)

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

These studies showed that methyl 3,5-diphenylpyridazine-4-carboxylate produced a dose-dependent increase in occupation of GABAA α5 receptors. Methyl 3,5-diphenylpyridazine-4-carboxylate has also shown positive effects on the brain with a brain/plasma ratio >1. Studies have further shown that methyl 3,5-diphenylpyridazine-4-carboxylate produced cognitive enhancing effects via positive allosteric modulation of the GABAA α5 receptor subtype.

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

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

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

Parametric statistics (paired t-test) were used to compare the performance of the retention test in rats with LN in the 4-hour delayed version of the RVL task in the context of various doses of ethyl-3-methoxy-7-methyl-9H-benzo[f]imidazo[1 ,5-a][1,2,4]triazolo[4,3-d][1,4]diazepine-10-carboxylate and vehicle control (see FIG. 5). Relative to treatment with vehicle control ethyl 3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4]diazepin- 10-carboxylate significantly improved memory performance at 3mg/kg (t(7)=4.13, p=0.004 or t(7)=3.08, p=0.018) and at 10mg/kg (t(7) =2.82, p=0.026).

Effect of ethyl 3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3-d][1,4]diazepine-10carboxylate on occupation The a5-containing GABAA receptor was also studied with a subsequent procedure which is essentially similar to that described in Example 107(B) (see above). This study showed that ethyl-3-methoxy-7-methyl-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[4,3d][1,4]diazepine-10 α-carboxylate (0.01-10 mg/kg, iv) reduced Ro 15-4513 binding in the hippocampus without affecting Ro 15-4513 levels in the cerebellum (Fig. 6) with a dose of 10 mg/kg, iv, showing >90% occupancy (FIG. 7).

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

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

Influence of 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4(5H)one in vivo on retention of spatial memory in rats with age-related disorders (AD) were assessed using the behavioral task of the Morris water maze. The water labyrinth is a pool surrounded by a new system of images in relation to the labyrinth. The water maze test protocol can be based on a modified water maze task that has been shown to be hippocampus dependent (de Hoz et al., Eur. J. Neurosci., 22:745-54, 2005; Steele and Morris, Hippocampus, 9:118-36, 1999).

Rats with age-related cognitive impairment were implanted with a unilateral cannula into the lateral ventricle. Stereotaxic coordinates were 1.0 mm posterior to the bregma, 1.5 mm lateral to the midline, and 3.5 mm ventral to the skull surface. After about a week of recovery, the rats were pre-trained in the water maze for 2 days (6 trials per day) to locate the submerged rescue platform below the surface of the pool, in which the location of the rescue platform changes every day. None

- 214 040488 intracerebroventricular (ICV) infusion was not performed during pre-treatment.

After pretreatment, rats received an ICV infusion or 100 µg of 6,6-dimethyl-3-(3hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one ( n=6) in 5 µl of DMSO or vehicle DMSO (n=5) 40 min before water maze training and study. The training consisted of 8 trials per day for 2 days, in which the hidden rescue platform remained in the same place. The rats were given 60 seconds to locate the platform with 60 second intervals between attempts. The rats were given a probe test (120 s) 24 hours after the end of training, in which the rescue platform was removed. There were four blocks during the training, in which each block had four training series of tests.

Rats treated with vehicle and 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7dihydro-2-benzothiophen-4(5H)-one showed a hidden platform approximately the same time at the beginning of training (block 1). In this training block, rats were treated with vehicle and 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one, both took approximately 24 seconds to find the rescue platform. However, rats treated with 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one could find platform more skillfully (i.e., faster) at the end of training (block 4) than vehicle-treated rats alone. In block 4, rats treated with 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one expended approximately 9 6 s to find the rescue platform, while vehicle-treated rats took approximately 19.69 s. These results show that 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7dihydro-2-benzothiophene-4(5H)-one improved learning ability in rats in the water maze (see Fig. 8A).

During the verification test, 24 hours after training, the rescue platform was removed. A search/swim model in rats was used to measure whether rats remember where the rescue platform was located during pre-training to test long-term memory in rats. In this test, the target annulus is a designated area 1.5 times the size of the rescue platform around the area where the platform was located during pre-training. The opposite annulus is a control region the same size as the target annulus that is located opposite the target annulus in the basin. If the rats had good long-term memory, they tended to search in the area adjacent to where the platform was located during pre-training (ie, the target annulus; not the opposite annulus). Annular time is the amount of time in seconds that the rat spent in the target or opposite annulus. The annulus crossing number (#) is the number of times the rat swam across the target or opposite annulus.

The vehicle-treated rats spent the same amount of time in the target annulus and the opposite annulus, indicating that these rats did not appear to remember where the platform was located during pretraining. In contrast, rats treated with 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2benzothiophen-4(5H)-one spent significantly more time in the target annulus and crossed the target annulus more frequently than the time they spent or the number of times they crossed the opposite annulus. These results show that 6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2benzothiophene4(5H)-one improved long-term memory in rats in the water task. maze (see Fig. 8B and 8C.

The compounds of the present invention showed a positive allosteric modulating effect on the GABAA a5 receptor (see, for example, Example 106). These compounds will enhance the effect of GABA on the GABA a5 receptor. Therefore, the compounds of the present invention should produce cognitive enhancement effects in age-impaired animals (such as rats) similar to those induced by other selective GABAA α5 receptor agonists such as methyl-3,5-diphenylpyridazine-4-carboxylate, ethyl-3- methoxy-7-methyl-9Hbenzo[1]imidazo[1,5-a] [1,2,4]triazolo[4,3-d] [1,4]diazepine-10-carboxylate and 6,6-dimethyl- 3-(3hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (see, for example, Examples 107109).

Claims (29)

CLAIM 1. Connection selected from: Compound Structure 344 CD O 0 ^Ζ \ΥΥχ ^Ζ °\Υ 356 K ¹ o 357 O b /=\ 2 VY 358 0 ^z \YYy ^Z 359 O 0 ί~\ t 0 -Π 360 About 0 z/ O - 216 040488 - 217 040488 368 CD O 0 ^z yxA ^z XL? 0 O 369 CD O 0 ^Z \X? ⁸ 370 MeO^^NA 371 CD O 0 ^Z A ^ζ γΜγ ^ζ XL? 0Ω 372 2 CD O 0 ^Z A ^z Yx/V z^o 0 q 373 2 CD O 0 ^z aaaa ^z z ^z O 0 374 2 CD O 0 z ^z o o \___/ 0 o - 218 040488 375 N ^F N - n 382 Ρ-η ΧΗΡγ N- 'N 383 MeSU^^AYA N- ^N N 385 ^C| ^C ^N 'N 386 DYA 387 npp ^N V 388 ddAA MeO^^Np NV - 219 040488 389 N Ο—, w ^CFs MeO^yX A 390 ΜθΟ^^νΛ ³ A 391 MeO ^Χ Ν% A 392 \γ MeO'^-^yY ^N Y 393 αν ci'^y Ά 394 yv MeoVyx^A ^Ν Υ 395 A _ /=\ jQ / ^С| ^^L ^N v - 220 040488 - 221 040488 403 CD ABOUT X ίι 404 CD ABOUT X in 405 Q II <YY> Az Z-z X about CD 2 406 2 CD ABOUT X q 407 2 CD O A 408 CD OK 409 CD ABOUT Y Z A<x Z GP qAZ CD - 222 040488 - 223 040488 - 224 040488 - 225 040488 or a pharmaceutically acceptable salt, enantiomer, diastereoisomer, Z-isomer, E-isomer or combination thereof. 2. Connection selected from: - 226 040488 - 227 040488 326 -O 327 N yr । 328 (4 U o f 330 χΑΑ 331 ^rA ,ss> CI'^^y-N_P\ 332 £ϊ 'ί jOj ^N 'N 333 fX ys-/~O .N _/Ζ ^Ν Υ^ \ x4 ΟΙ^^γ-Ν ο _χ - 228 040488 - 229 040488 341 cr A A ^N A A A 342 J A O^CF ₃ 343 A About zA MeO^ Ar ^N t a 345 cr Λ ,, ,N4 AT il Λ ^N v A 346 J Af Aχ A A- A A°ZO 348 J o-~ 349 £ cr A NX AZ \ AAA Η /Y- ^N 'N °3 N ^A °^ - 230 040488 - 231 040488 355 F/F cW ^Cl /=\ ⁿ ~ ⁿ mr F 384 F-b / ζχ,Ν-// N^'^F JO > ¹ F Π \_ ^N ^M \ ^N OMe 421 F-^-x ^VF ry _F II \ / \^-V Cl __/OMe ^N O 422 ΓΎ) CF CI^FyN_/OMe ⁿ O 423 FFZ F (/ y-ocF ₃ C! FxF\^n_OMe ⁿ O 429 __/OMe ^N O 430 f ⁿ Cl __/OMe ⁿ O - 232 040488 431 Cl 432 __/OMe 433 "YAG S|L\L^,s_ ^N OMe 434 rLDo-D CI^A\_ _N nI/A ^N OMe 435 XX /=\ ΓΎ ^ν '}' ^C AY\ _N Ύ, _Μ θ 444 N<r\ ^NOMe 445 < ^N χΓΓ ΝΧιλ ^N OMe - 233 040488 or a pharmaceutically acceptable salt, enantiomer, diastereomer, Z-isomer, E-isomer, or a combination thereof. 3. A pharmaceutical composition containing a compound according to any one of claims 1, 2 or a pharmaceutically acceptable salt, enantiomer, diastereomer, Z-isomer, E-isomer, or a combination thereof in a therapeutically effective amount and an acceptable carrier, adjuvant or excipient. 4. The pharmaceutical composition of claim 3, wherein said composition further comprises a second therapeutic agent. 5. The pharmaceutical composition of claim 4, wherein the second therapeutic agent is selected from an antipsychotic, memantine, and an acetylcholinesterase (AChE-I) inhibitor. - 234 040488 6. The pharmaceutical composition of claim 4, wherein the second therapeutic agent is an antipsychotic selected from aripiprazole, olanzapine, and ziprasidone, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof. 7. The pharmaceutical composition of claim 4, wherein the second therapeutic agent is memantine, a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof. 8. The pharmaceutical composition of claim 4 wherein the second therapeutic agent is an AChE-I selected from donepezil, galantamine and rivastigmine and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof. 9. A method of treating a cognitive impairment associated with a disorder of the central nervous system (CNS) in an object in need of such treatment, comprising the step of administering a compound according to any one of claims 1, 2 or a pharmaceutical composition according to any one of claims 3-8 . 10. The method of claim 9, wherein the CNS disorder is an age-related cognitive impairment. 11. The method of claim 9, wherein the CNS disorder is mild cognitive impairment (MCI). 12. The method of claim 9 wherein the CNS disorder is amnesic mild cognitive impairment (aMCI). 13. The method of claim 9 wherein the CNS disorder is dementia. 14. The method of claim 9 wherein the CNS disorder is Alzheimer's disease. 15. The method of claim 9 wherein the CNS disorder is schizophrenia or bipolar disorder. 16. The method of claim 9 wherein the CNS disorder is amyotrophic lateral sclerosis (ALS). 17. The method of claim 9 wherein the CNS disorder is post-traumatic stress disorder (PTSD). 18. The method of claim 9, wherein the CNS disorder is associated with cancer therapy. 19. The method of claim 9 wherein the CNS disorder is mental retardation. 20. The method of claim 9 wherein the CNS disorder is Parkinson's disease (PD). 21. The method of claim 9 wherein the CNS disorder is autism. 22. The method of claim 9, wherein the CNS disorder is compulsive behavior. 23. The method of claim 9 wherein the CNS disorder is drug addiction. 24. A method of treating brain cancer in a subject in need of such treatment, comprising the step of administering a compound according to any one of claims 1, 2, or a pharmaceutically acceptable salt, enantiomer, diastereomer, Z-isomer, E-isomer, or a combination thereof, or a pharmaceutical compositions according to any one of claims 3-8. 25. A method of treating a cognitive impairment associated with brain cancer in a subject in need of such treatment, comprising the step of administering a compound according to any one of claims 1, 2 or a pharmaceutically acceptable salt, enantiomer, diastereomer, Z-isomer, E- an isomer or a combination thereof or a pharmaceutical composition according to any one of claims 3-8. 26. A method of treating brain cancer or a cognitive impairment associated with brain cancer in a subject in need of such treatment, comprising the step of administering a compound or a pharmaceutical composition containing a compound, wherein the compound is selected from: - 235 040488 Compound Structure 1 H ₃ Sola < _m X N 2 L γγ ί £ ch 3 CQ Ν^ ^Η 3 4 YCO ₂ Et .Ν Λ Xa / O H ₃ CO N y ' 5 A -L 1 about γ4, o 6 /b ^ζ γΥΑ/ ^ζ 1 - o ΥΥ ° (1 J GP 9 sh Ax°n <Tx>^ η £ 10 t .b -p f A V\ ^Ζ ΥΛ/ ^Ζ ie Ϊ ° S' and 11 g y-co ₂ Et sh n ₃ ssg ύύ Ύ° 0 OCH ₃ 12 G ACO ₂ Et LE NV \ N o χΧ ^οΗ 3 H ₃ C 44 sh g i o Az Z-z Q LL - 236 040488 45 w 1 c 8 Υ Am 0 LL. 46 b ^ζ γΥΥγ ^ζ YA -° (/ / m 47 ^ζ γΑΑγ ^ζ γχ$ 48 Μ ΥΛ ζΆ o ι > /V) X_/ K 49 S o 0 τΥν k O fY ° M ^and 50 su ^s ° ₂ e, ΜθοΧΧνΧ Ν'- 2 ' Ν 51 M. _z y-CO ₂ Et TA p MeO '''^ N—/ 52 G >~CO ₂ Et <Άτ /-X Cu CT' MeO N'T N'Ά - 237 040488 53 g CO ₂ Et XY x yy~cN MeO ^N 'N 54 x £Jo= tM 107 . 0 o o ^Z v TV , ^z Fz Zz Q IL 108 g y-CO ₂ Et /X/ ^N \ ίΎ ) lL-Az 109 Xz ^yF ^Ay ^z & x 110 X F ² ; GL ^z yF_Ay ^z £ ° X ^z 111 g,>-CO ₂ Et _, N-X /Qj £A N=/ - 238 040488 - 239 040488 - 240 040488 - 241 040488 - 242 040488 170 N_yN lU \ ^N y} 180 y, y°,.-, ^N -/ W MeO^-^NX, 0-4 s A 181 ,n /'° Me° n4 0-4 \ Y Y Y N 182 AyA ^N V 194 z ^N A° ΜθοΥα^^,^/ iy] ^195n'n 207 r y-CN ΓΎ ) ΛΑ MeO^A/yy Y=/ 208 dy ^cN ίΎ ) (l MeO'^'A ^x _N y /==/ lUA - 243 040488 209 .N r A ^CN ΌΟ A Ν' /— ^{8 N} 'N 210 XX J A MeO / 211 0xxb A ^N Ή Ar 212 GA XX) H3s/LAA Qa 213 XX— ^Br XX / H ₃ CCT X 1 bK ^N ^/ \___k 214 ^Br / ^N - XX / H.ccAAA 1 >---k yV 218 r ^N y-= XX / x5 ΉιΧ N ' ₀ - 244 040488 219 C ^N p= Hell 220 R /DM yes / O MeO''^'-'^ ν-D 221 yar _z ίΎ ) A MeO'^P^'N'X^ 222 Yars Pj „ PVP ^N 'N 223 r pcN A l Meo— ⁿ 'n 224 ,.N G ;. -cn xb Meo'—U»R ^N 'N 225 )-CN Oj P MeQ '' ^ oD \ xA ^w 226 Г z)-CN r:' . ^α Ppiv ⁿ 'N 227 Г pCN ^α ΑΛρ ^N V - 245 040488 228 229 - Q her % Vz Z-z X 230 O-^ AV'^ CHAE zX^>d _z Cz zz P 231 __Ν·Ό J Λ ABOUT F 247 A ^Br MeoAA^ Χθ d|\ ^N NH / 248 A ^{Br me} ° ¹ ' x° ^N 0 250 oTo MeCH^^-D N—' fil - 2 ⁷ 'N - 246 040488 251 MeSG m 252 MeSU z xX. ABOUT ] zAypAz Lz Z-z Η 253 X IVeO' A™ o χΑΧ P 257 X MeO ,N O'N ,. _ΎN'N ^_ 258 J Mesu -Ν P-N ΟΛν Pa P 262 Meo' ' h* 0 272 Places P0>- 273 MeO ^- AFC ___A-f νΆ ADU R va 277 MeSg i .adl - 247 040488 279 Ac° _2Et MeO^^^NX /=\ SG 289 nJ A MeO''N^ nJ 290 oAA nJ 291 2 (D About Ay 292 2 s Q Z Vx-Ax ^z 298 oX ^z θχ AA X Φ 299 AA D) f T ) ΟΙ'^- ^/Χ Ν-γ O—N'n 300 318 r^CO,Et 4j>C / MeO N—l r^. ^7*0 376 Cl Q zP about b 377 Cl Ργ χγ-ΑΛθ ^Αα- ^Ν ν 378 Cl Ργ χχΑ ^{0 N} ν 379 cA Q ^ζ γΑΑγ ^ζ 0 380 Ργ ιί^Χ ^with Aa ^ν 'Ν 381 O X γ-Α or its pharmaceutically acceptable salt, enantiomer, diastereoisomer, Z-isomer, E-isomer or combinations thereof. - 248 040488 27. A method of treating brain cancer or a cognitive impairment associated with brain cancer in a subject in need of such treatment, comprising the step of administering a compound or a pharmaceutical composition containing a compound, wherein the compound is selected from: Compound Structure VX—CO ₂ Et 55 II l / 'Ό 56 II J / MeO^^YyN_ Λ- -CO ₂ Et 103 ΓΎΛ II\/v- ABOUT- 104 CD Λ f Ύ £ Q-CO ₂ E _t 105 eleven ) MeO '^y-'N Ν 'Ν \ Λ 0—OMe 106 ζM CO ₂ Et II λ / MeO^^YN II Λ-Rr Ν A ^Br - 249 040488 118 MCh /° ^Ν xYC MeO^^TyN_y—/ \ 119 ,Ν-Ο Ο ΓΎ ) _ ΜθΟ^^ΓΧ^,ν ο-/ \ 120 Λ-Η ,νΖ ο XY / /=χ MeO^^y-N_y_/ \ 122 "Ο MeO ο-Ζ\ 123 ^Χ Ο JO > MeO^^YyN ο-Ζ \ ^Ν Ό 124 Y ^F Υ ' MeO^^^yN ο-Ζ \ ^ν Ό ^w 128 Υ Ρ'Ν ^/Ο l nA ν^\ jQf ) MeO^^y-NOMe - 250 040488 129 P P MeO^ OMe 130 P PN ΟΑ-ΐγ _mind 131 P P MeO^MP^-N OMe 137 P Г X—CO ₂ Et PO F^^pp_/OPh P^ 138 P/°'N X X JX PC' _ph 141 X ^N AAp ^F ^[rPPPA NXA /7 'p 142 ΛA Op^jlX > MeO^^Pp O-/p Γρ- ⁷ 'p - 251 040488 147 ΛΑ A A 148 AaX Ji Λ / ^F /)_/° ΑΑ Ν A U ν 156 .':. ' ' 157 V F __oPh NV ⁷ 158 ix V 159 b :';. ' 162 N~O A ^Α MeO'^-'^vN QPh - 252 040488 163 YN'O MeO^YV-N^ oYY V-f 164 Y /Go dG MeO'Y'yN _/©Me 165 Ύ N~ ₀ Μ θοΧγΥγ-N_ 166 Χγ MeoYYyN^ 169 Y /° ΜβΟ^ΥγΝ_/OMe 171 Y Ν'Ο Υγ MeO^YyN QPh ^Ν Ν 172 γϊΎ (D - 253 040488 173 N~O MeO^XZx^N oEt 174 Φ A vx 175 // ^N '° ΟΑχ MeO^^Z4. _N O~XX G A ⁷ y ^N _C| l^ 176 7-0 XX 4 about CD 177 0-Oe hu A o CD 178 φ Ayp. όΤ - 254 040488 179 xd A o CD 2 183 ,N No XX/γ Mo' Ap _o A O' ¹¹ 184 ,NN~O _Ν χΗΧγ 185ANO. ³ :/. ^N ' NU 186 ,N NO __/OMe 187 N'O ίΑΧγ F N__^OEt NV ⁷ 188 N'O pX x F AMy,N_y ,, F ^N^ CT - 255 040488 Ax , ^N ~O 189 0 3 G ΟΣ 0- -P 190 F o ^N d VN Г v ^N 4 N~° °\zd. ^N.4 -CN 191 MeO^ C £ ^N V 0 0 “П z-CN 192 MeO' £ At - Y h-CN 193 MeO ^x £ £ S'y 0 -CN 196 Me(Y C £ ^N 4 0 0 ^N £-£ -CN 197 J mu. £ ^Nd - 256 040488 198 α^^γ-Ν 199 Α ^ρ /NY ίΎ ) Μ 200 Ah Χν ^Ν \ ^{F С|} LxL\_ Ν ^ν ~ l χζ 201 Ν'Ο dT A 1 202 Y ci ^^γ-Ν Oyx U 203 Λ ^Ν ~ ^Ο uddl ΜθΟ^ΑΧ,ν o /=\ 204 4-A ^χ,νΑ ^C| LchLx.^ O^^x and - 257 040488 205 J V V- P ill °C O-N 206 t cr V\_ _N ⁿ A oV 215 ΓΎ \ cY H \ / II /X ^N V o _x Λ -Br 216 J x ^N V y >— Br 217 cr ^N Y Br 232 MeO ^x Ai -“Ό 233_/ ⁰ '| ^N Cl ^x Ary ^N 'N o _x - 258 040488 234 _{Cl_A} 235 xxT MeO^^VN o k_/ XN ^ν Ά and 236 yr H ₃ CO^^£N 237 χΧ O 238 Wu..'/oh 239 Λ , XD y O 240 __ N /Nth X jTY ) _ Ν-χΛ Ν ο- - 259 040488 - 260 040488 249 Az/kJ Ν ίΎ ) ^C| Τ 254 Ρ'Ν γχγ ci^^yN_ _/Ο ^ 255 ^; :, Ο 256 : .. Τί 259 ΜΡ Μ ο 260 CI 261 μ5^ - 261 040488 263 264 ρτχ ^Ν Ό 265 ψχο ^C| __ η ^Ν -Χ 266 χ-νο CI __ Ρχ 267 ΙΑ ) Cl^^^y-N 0\ 268 ιχΧο I 270 ΑΧό CI_ρ. v 271 ΧΑΧ CI - 262 040488 ^274N -F 275 α _ρ^ ^ν Ό 276 κ ο 278 ( 0 280 1 /F0n\A £Ύ > Ν'Ν^ 281 10th 282 0άΟ 283 Ρίο - 263 040488 284 A YES* 285 Ρ - /=\ CI^PyN ^Ν 'Ν 286 CI^PyN ^Ν ν I 287 . X ρ CI^A ^Ν ν τ 288 / ^ΝΥΓ Ν'Ν^ 1 293 γΡ ci^^Pn ο^ 294 Ρ<°ϊ' ΓΎ > ^Cl ^^ιΓ ^Ν \-/ ⁰ ^ - 264 040488 295 ' ' ABOUT 296 Ν O-L 297 yN_/°^| /\/ ^N dba ci^AAn 301 About S4 H 302 XX'L ^ο \ 303 ,Ν Ρ'Ν AAx ΓΎ ) ^C| _y^CF ₃ 304 f ^N y_/°X ΑΑ Ό Cl __y^CFs - 265 040488 305 cr £4 1 306 cr £ A ^N C Q^z 307 Ji cr rs GU VN F / ^Οχ ^ 308 J C £ ^N y -CO ₂ Et °^CF ₃ 309 J C £ ^N y Nz Y °\Z ^CF 3 310 J £ vi 311 Jl cr <n Λz ⁿ 4 \-N Г .y__ ^N y 312 J C £ ^N d N-U 4 O^CF ₃ - 266 040488 313 ί N NiY~V° Ο J Cr ^|Γ ^Ν \7°' ^N 'N xYF ₃ a . Kl-A 329 ABOUT ,°\ 427 J .N 7 ^N O N'° N^\ OMe 428 ίΓ Cl .. .NY Y \ -Oa II ^N O Ν-Ο ⁴ n^cf ₃ OMe 456 o' 4 P'N Ch 1 ABOUT- 457 ck a ^N Y about 1 459 5 a ^N Y θ'Ν ⁴ Y 461 BY a ^N Y θ'Ν Υ 462 ABOUT A oi /Iλ- Ρ'Ν -Λ L Ο— 463 ( O - ^Ν Ό 465 aft hr A .NY jY ) Il .Υ ^N V Ο'Ν Λ ι Ν^\ Ο— 468 Z=\ w o ^Z V ^Z ^Y ^Z Υ / °Ύ ^Ζ X 469 z ⁼ 2 ^l - 267 040488 or its pharmaceutically acceptable salt, enantiomer, diastereoisomer, Z-isomer, E-isomer or combinations thereof. 28. The method of claim 24 or 25, wherein said brain cancer is medulloblastoma. 29. The method of claim 26 or 27, wherein said brain cancer is medulloblastoma.