Substituted pyrrolidines as G-protein coupled receptor 43 agonists

Abstract

The present invention is directed to a process for the preparation of aryl-pyrrolidine carboxylic acid derivative compounds which are useful in treating metabolic diseases, said process consisting of coupling an aryl-pyrrolidine compound with an aryl carboxylic acid compound followed by alkaline or acidic treatment, hydrogenolysis or treatment with a fluoride of the ester intermediate to afford novel aryl-pyrrolidine carboxylic acid derivative compounds.

Claims

1. A process for the preparation of a compound of formula Ib-1b: ##STR00086## wherein: D is C(?O); L.sup.2 is a single bond; R, R.sup.1, R.sup.2, R.sup.3, R.sup.3 and R.sup.4 are independently H; Ar.sup.1 is 2-chlorophenyl, 2-fluorophenyl or 2,3-difluorophenyl; and Ar.sup.2 is aryl, heteroaryl, cycloalkyl or monocyclic heterocyclyl, each of which being optionally substituted by one or more group(s) selected from halo, cyano, nitro, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, benzoxazol-2-yl, heteroarylalkyl, hydroxy, hydroxyalkyl, alkoxy, haloalkoxy, alkoxyalkoxy, cycloalkyloxy, cycloalkylalkyloxy, heterocyclyloxy, aryloxy, heteroaryloxy, alkoxyalkyl, haloalkoxyalkyl, arylalkyloxy, heteroarylalkyloxy, aryloxyalkyl, heteroaryloxyalkyl, amino, alkylamino, arylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, alkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, arylcarbamoyl, heteroarylcarbamoyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl, alkylsulfamoyl, arylsulfamoyl, heteroarylsulfamoyl, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino and oxo, wherein each of said substituent group(s) may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; or two Ar.sup.2 substituents form an alkylenedioxy group or a haloalkylenedioxy group, wherein each of said alkylenedioxy or haloalkylenedioxy groups may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; or fused to the Ar.sup.2 cycloalkyl group may be one or more aryl or heteroaryl moieties, wherein each of said aryl or heteroaryl moieties may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; wherein the process consists of: a) coupling a compound of formula A: ##STR00087## wherein: R is methyl, ethyl, tert-butyl, benzyl, allyl, phenacyl, methoxymethyl, methylthiomethyl, 2-methoxyethoxymethyl, 2-trimethylsilylethyl or tert-butyldiphenylsilyl; and (i) R.sup.8 is Cl or F; and R.sup.9 is H; or (ii) R.sup.8 is F; and R.sup.9 is F; with a compound of formula B: ##STR00088## wherein: R is Cl or -OL, wherein L is benzotriazol-1-yl, 7-azabenzotriazol-1-yl or imidazol-1-yl; and Ar.sup.2 is aryl, heteroaryl, cycloalkyl or monocyclic heterocyclyl, each of which being optionally substituted by one or more group(s) selected from halo, cyano, nitro, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, benzoxazol-2-yl, heteroarylalkyl, hydroxy, hydroxyalkyl, alkoxy, haloalkoxy, alkoxyalkoxy, cycloalkyloxy, cycloalkylalkyloxy, heterocyclyloxy, aryloxy, heteroaryloxy, alkoxyalkyl, haloalkoxyalkyl, arylalkyloxy, heteroarylalkyloxy, aryloxyalkyl, heteroaryloxyalkyl, amino, alkylamino, arylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, alkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, arylcarbamoyl, heteroarylcarbamoyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl, alkylsulfamoyl, arylsulfamoyl, heteroarylsulfamoyl, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino and oxo, wherein each of said substituent group(s) may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; or two Ar.sup.2 substituents form an alkylenedioxy group or a haloalkylenedioxy group, wherein each of said alkylenedioxy or haloalkylenedioxy groups may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; or fused to the Ar.sup.2 cycloalkyl group may be one or more aryl or heteroaryl moieties, wherein each of said aryl or heteroaryl moieties may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; in the presence of a base, to provide a compound of the formula: ##STR00089## wherein: R is methyl, ethyl, tert-butyl, benzyl, allyl, phenacyl, methoxymethyl, methylthiomethyl, 2-methoxyethoxymethyl, 2-trimethylsilylethyl or tert-butyldiphenylsilyl; (i) R.sup.8 is Cl or F; and R.sup.9 is H; or R.sup.8 is F; and R.sup.9 is F; and Ar.sup.2 is aryl, heteroaryl, cycloalkyl or monocyclic heterocyclyl, each of which being optionally substituted by one or more group(s) selected from halo, cyano, nitro, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, benzoxazol-2-yl, heteroarylalkyl, hydroxy, hydroxyalkyl, alkoxy, haloalkoxy, alkoxyalkoxy, cycloalkyloxy, cycloalkylalkyloxy, heterocyclyloxy, aryloxy, heteroaryloxy, alkoxyalkyl, haloalkoxyalkyl, arylalkyloxy, heteroarylalkyloxy, aryloxyalkyl, heteroaryloxyalkyl, amino, alkylamino, arylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, alkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, arylcarbamoyl, heteroarylcarbamoyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl, alkylsulfamoyl, arylsulfamoyl, heteroarylsulfamoyl, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino and oxo, wherein each of said substituent group(s) may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; or two Ar.sup.2 substituents form an alkylenedioxy group or a haloalkylenedioxy group, wherein each of said alkylenedioxy or haloalkylenedioxy groups may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; or fused to the Ar.sup.2 cycloalkyl group may be one or more aryl or heteroaryl moieties, wherein each of said aryl or heteroaryl moieties may be optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, C.sub.5-C.sub.12 aryl optionally substituted by a chloro or methyl group, heteroaryl, heteroalkyl, hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylamino, alkylcarbonylamino, haloalkylcarbonylamino, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, haloalkylsulfonylamino, oxo, and haloalkoxyalkyl; and b) (i) treating the compound provided in step a) above with a base, an acid or a fluoride; or (ii) hydrogenolysis of the compound provided in step a) above, to provide a compound of formula Ib-1b.

2. The process according to claim 1, wherein the compound of formula A is methyl (2S,5R)-5-(2-chlorophenyl)pyrrolidine-2-carboxylate.

3. The process according to claim 1, wherein the compound of formula B is 2 methoxybiphenyl-4-carbonyl chloride.

Description

BIOLOGY EXAMPLES

Brief Description of the Drawings

(1) FIG. 1 represents the effect of compounds 1; 2; 4; 5; 8; 10; 11 and 13 on isoprenaline-induced lipolysis in adipocytes isolated from normal rat. Compounds are tested at 30 ?M final concentration.

(2) FIGS. 2A and 2B represent the inhibition of blood glucose concentration in OGTT assay following bi-daily injection (at 50 mg/kg) of compound 1 during 28 days.

MEMBRANE BINDING ASSAY: GTP?S BINDING ASSAY

(3) The following assay can be used for determination of GPR43 activation. When a GPCR is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyses the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP. The non-hydrolysable GTP analog, [.sup.35S]GTP?S, was used to demonstrate enhance binding of [.sup.35S]GTP?S to membranes expressing receptors. The assay uses the ability of GPCR to stimulate [.sup.35S]GTP?S binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous or not endogenous GPCR.

(4) Preparation of Membrane Extracts:

(5) Membrane extracts were prepared from cells expressing the human GPR43 receptor (hGPR43) as follows: the medium was aspirated and the cells were scraped from the plates in Ca.sup.++ and Mg.sup.++-free Phosphate-buffered saline (PBS). The cells were then centrifuged for 3 min at 1500 g and the pellets were resuspended in buffer A (15 mM Tris-HCl pH 7.5, 2 mM MgCl.sub.2, 0.3 mM EDTA, 1 mM EGTA) and homogenized in a glass homogenizer. The crude membrane fraction was collected by two consecutive centrifugation steps at 40.000?g for 25 min separated by a washing step in buffer A. The final pellet was resuspended in 500 ?l of buffer B (75 mM Tris-HCl pH 7.5, 12.5 mM MgCl.sub.2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose) and flash frozen in liquid nitrogen. Protein content was assayed by the Folin method.

(6) GTP?S Assay (SPA Method):

(7) The assay was used to determine the activity of the compounds of the invention. The [.sup.35S]GTP?S assay was incubated in 20 mM HEPES pH7.4, 100 mM NaCl, 10 ?g/ml saponin, 30 mM of MgCl.sub.2, 10 ?M of GDP, 5 ?g membrane-expressing hGPR43, 250 ?g of wheatgerm agglutinin beads (Amersham, ref: RPNQ001), a range concentration of compounds of the invention (from 30 ?M to 1 nM) in a final volume of 100 ?l for 30 min at room temperature. The SCFA propionate was used at 1 mM final concentration as positive control. The plates were then centrifuged for 10 minutes at 2000 rpm, incubated for 2 hours at room temperature and counted for 1 min in a scintillation counter (TopCount, PerkinElmer). The results of the tested compounds are reported as the concentration of the compound required to reach 50% (EC.sub.50) of the maximum level of the activation induced by these compounds.

(8) When tested in the assay described above and by way of illustration the compounds in Table 3 activate GPR43 receptor. The EC.sub.50 value obtained is represented as follows: +++ means EC.sub.50<200 nM; ++ means 200 nM?EC.sub.50?1 ?M; + means EC.sub.50>1 ?M.

(9) TABLE-US-00008 TABLE 3 Compounds EC.sub.50 values in GTP?.sup.35S assay. Compound n.sup.o EC.sub.50 (nM) 1 +++ 2 +++ 3 +++ 4 +++ 5 +++ 6 +++ 8 ++ 9 ++ 10 ++ 11 ++ 12 ++ 13 ++ 14 ++ 15 ++ 16 ++ 17 ++ 18 ++ 19 ++ 20 ++ 21 ++ 23 + 24 + 26 + 27 + 30 + 31 + 32 + 33 + 34 + 35 + 36 + 38 + 39 + 40 + 41 + 42 + 43 + 44 + 45 + 47 + 48 + 49 + 52 + 53 + 58 + 59 + 77 +++ 78 ++ 83 + 88 + 89 ++ 91 ++ 96 ++ 99 ++ 102 + 105 + 107 + 108 + 109 + 113 +++ 114 + 116 ++ 117 ++ 120 + 121 ++ 122 +++ 123 +++ 125 ++ 126 +++ 127 +++ 128 +++ 129 +++ 130 +++ 131 + 132 +++ 133 ++ 134 ++ 135 +++ 136 ++ 137 ++ 138 +++ 140 +++ 141 ++ 143 + 149 ++ 150 ++ 151 ++ 153 + 155 + 156 ++ 157 +++ 160 ++ 161 +++ 162 + 169 + 174 + 176 + 177 + 178 ++ 179 + 183 + 184 ++ 189 ++ 191 ++ 192 ++ 193 +++ 194 ++ 195 ++ 196 +++ 197 +++ 198 +++ 199 +++ 200 +++ 201 +++ 202 +++ 203 ++ 204 +++ 206 + 207 + 224 +++ 225 +++ 226 ++ 227 +++ 228 +++ 229 +++ 230 + 231 + 232 +++ 233 ++ 234 + 235 +++ 236 +++ 237 ++ 239 ++ 240 ++ 242 ++ 246 +++ 247 +++ 261 ++ 268 +++ 269 +++ 272 ++ 273 ++ 274 ++ 275 +++ 276 ++ 278 ++ 279 + 280 + 281 ++ 283 +++ 284 ++ 285 + 286 + 287 ++ 288 ++ 289 +++ 290 +++ 291 ++ 292 +++ 293 ++ 294 ++ 295 + 296 + 297 +++ 298 +++ 299 +++ 300 ++ 301 ++ 302 +++ 303 ++ 304 + 305 + 306 + 307 +++ 308 +++ 309 ++ 310 ++ 311 +++ 312 +++ 313 ++ 314 +++ 315 +++ 316 +++ 318 + 319 ++ 320 +++ 321 ++ 322 ++ 323 ++ 324 ++ 325 +++ 326 + 327 ++ 328 +++ 329 ++ 330 ++ 331 ++ 332 + 333 + 334 ++ 335 + 336 + 337 +++ 338 +++ 339 ++ 340 +++ 341 + 342 +++ 343 +++ 344 ++ 345 +++ 346 +++ 347 +++ 348 +++ 349 ++ 350 +++ 351 +++ 352 + 353 ++ 354 +++ 355 +++ 356 +++ 357 +++ 358 ++ 359 ++ 360 +++ 361 +++ 362 +++ 363 ++ 364 + 365 ++ 366 +++ 367 +++ 368 +++ 369 ++ 370 + 371 +++ 372 +++ 373 ++ 374 ++ 375 ++ 386 ++ 387 +++ 388 +++ 389 +++ 390 +++ 391 + 392 + 393 + 395 ++ 396 ++ 397 ++ 398 +++ 399 +++ 400 ++ 401 +++ 402 + 403 + 404 ++ 405 +++ 406 +++ 407 ++ 408 +++ 409 +++ 410 ++ 411 ++ 412 ++ 413 +++ 414 + 415 +++ 416 +++ 417 +++ 418 ++ 419 +++ 420 +++ 421 +++
Radioligand Binding (RLB) Assay with Cell Membrane Extracts from Human GPR43 Recombinant Cell Line

(10) Human GPR43 radioligand binding assay is performed by adding successively in the wells of a 96 well plate (Master Block, Greiner, 786201), 50 ?l of compound of the invention at increasing concentrations (diluted in assay buffer: 50 mM Tris pH 7.4), 25 ?l radiolabeled antagonist (i.e. compound no277 described in EP10305100.9) diluted in assay buffer and 25 ?l cell membrane extracts (10 protein/well). The final concentration of radiolabeled antagonist in the assay is 10 nM. The plate is incubated 60 min at 25? C. in a water bath and then filtered over GF/B filters (Perkin Elmer, 6005177, presoaked in 0.05% Brij for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The filters are washed 3 times with 0.5 ml of ice-cold wash buffer (50 mM Tris pH 7.4). 50 ?l of Microscint 20 (Packard), is added and the plate is incubated 15 min on an orbital shaker and then counted with a TopCount? for 1 min/well.

(11) In Table 4 biological results obtained using the RLB assay as described above with compounds of the invention are set out in tabulated form. In this table, the constant of inhibition of radioligand binding carried out by the compound of the invention (Ki) is given. The Ki values (nM) obtained is represented as follows: +++ means Ki<1 ?M; ++ means 1 ?M?Ki?2 ?M; + means 2 ?M<Ki.

(12) TABLE-US-00009 TABLE 4 Compounds Ki values in RLB assay. Compound n.sup.o Ki (nM) 376 +++ 377 + 378 ++ 379 + 380 ++ 381 ++ 382 + 383 + 384 + 385 +++ 394 +
Cell Based Assay: Calcium Flux. The Aequorin-Based Assay.

(13) The following assay can be used for determination of GPR43 activation. The aequorin assay uses the responsiveness of mitochondrial apoaequorin to intracellular calcium release induced by the activation of GPCRs (Stables et al., 1997, Anal. Biochem. 252:115-126; Detheux et al., 2000, J. Exp. Med., 192 1501-1508). Briefly, GPCR-expressing clones are transfected to coexpress mitochondrial apoaequorin and G?16. Cells expressing GPR43 receptor are incubated with 5 ?M Coelenterazine H (Molecular Probes) for 4 hours at room temperature, washed in DMEM-F12 culture medium and resuspended at a concentration of 0.5?10.sup.6 cells/ml (the amount can be changed for optimization). Cells are then mixed with test compounds and light emission by the aequorin is recorded with a luminometer for 30 sec. Results are expressed as Relative Light Units (RLU). Controls include assays using cells not expressing GPR43 (mock transfected), in order to exclude possible non-specific effects of the candidate compound.

(14) Aequorin activity or intracellular calcium levels are changed if light intensity increases or decreases by 10% or more in a sample of cells, expressing a GPR43 and treated with a compound of the invention, relative to a sample of cells expressing the GPR43 but not treated with the compound of the invention or relative to a sample of cells not expressing the GPR43 (mock-transfected cells) but treated with the compound of the invention.

(15) Cell Based Assay: Intracellular Inositol-Phosphate Accumulation Assay. (Gq-Associated Receptor)

(16) The following assay can be used for determination of GPR43 activation. On day 1, GPR43-expressing cells in mid-log phase are detached with PBS-EDTA, centrifuged at 2000?g for 2 min and resuspended in medium without antibiotics. After counting, cells are resuspended at 4?10.sup.5 cells/ml (the amount can be changed for optimization) in medium without antibiotics, distributed in a 96 well plate (10 ?l/well) and the plate is incubated overnight at 37? C. with 5% CO.sub.2. On day 2, the medium is removed and the compounds of the invention, at increasing concentrations, are added (24 ?l/well) and the plate is incubated for 30 min. at 37? C. in a humidified atmosphere of 95% air with 5% CO.sub.2. The IPI concentrations are then estimated using the IPI-HTRF assay kit (Cisbio international, France) following the manufacturer recommendations.

(17) Cell Based Assay: cAMP Accumulation Assay (G.sub.i/o Associated Receptor)

(18) The following assay can be used for determination of GPR43 activation. Cells expressing GPR43 in mid-log phase and grown in media without antibiotics are detached with PBS-EDTA, centrifuged and resuspended in media without antibiotics. Cells are counted and resuspended in assay buffer at 4.2?10.sup.5 cells/ml. 96 well plates are filled with 12 ?l of cells (5?10.sup.3 cells/well), 6 ?l of compound of the invention at increasing concentrations and 6 ?l of Forskolin (final concentration of 10 ?M). The plate is then incubated for 30 min. at room temperature. After addition of the lysis buffer, cAMP concentrations are estimated, according to the manufacturer specification, with the HTRF kit from Cis-Bio International.

(19) In Vitro Assays to Assess Compound Activity in 3T3-L1 Cell Line.

(20) 3T3-L1 adipocytes cell line has been described as cellular model to assess compounds mimicking insulin-mediated effect such as inhibition of lipolysis and activation of glucose uptake.

(21) Lipolysis.

(22) 3T3-L1 cells (ATCC) are cultured in Dulbecco's modified eagle's medium (DMEM) containing 10% (v/v) bovine serum (fresh regular medium) in 24 well plate. On day 0 (2 days after 3T3-L1 preadipocytcs reached confluence), cells are induced to differentiate by insulin (10 ?g/ml), IBMX (0.5 mM) and dexamethasone (1 ?M). On day 3 and every other 3.sup.rd day thereafter, fresh regular medium is substituted until day 14.

(23) On day 14, the medium is removed and cells are washed twice with 1 ml of a wash buffer (Hank's balanced salt solution). The wash solution is removed and the SCFA or the tested compounds, or a combination of both, are added at the desired concentration in Hank's buffer supplemented with 2% BSA-FAF and incubated for 10 minutes ? 37? C. Then, isoproterenol (100 nM) is added to induce lipolysis and incubate for 30 minutes at 37? C. The supernatants are collected in a glycerol-free container. 25 ?l (the amount can be changed for optimization) of cell-free supernatants are dispensed in 96-well microtiter plate, 25 ?l of free glycerol assay reagent (Chemicon, the amount can be changed for optimization) is added in each well and the assay plate is incubated for 15 minutes at room temperature. The absorbance is recorded with a spectrophotometer at 540 or 560 nm. Using the supernatants, the free fatty acids amount can be assessed using the NEFA assay kit (Wako) according the manufacturer's recommendations.

(24) Glucose Uptake.

(25) 3T3-L1 cells are differentiated as described previously with or without of 30 ?M of tested compounds (the concentration can be changed for optimization) during the 14 days of differentiation. The day of the experiment, the cells are washed twice with a KREBS-Ringer bicarbonate (pH 7.3) supplemented with 2 mM sodium pyruvate and starved for 30 minutes in the same buffer at 37? C. in an atmosphere containing 5% CO2 and 95% O2. Various amount of SCFA, tested compounds or combination of both are then added with or without 10 nM of insulin (the amount can be changed for optimization) for 30 minutes at 37? C. in an atmosphere containing 5% CO2 and 95% O2. Then, D-(.sup.3H)-2 deoxyglucose (0.2 ?Ci/well) and D-2-deoxyglucose (0.1 mM) is added for 30 minutes. To stop the reaction, the cells are immersed in ice-cold saline buffer, washed for 30 min, and then dissolved in NaOH 1M at 55? C. for 60 minutes. NaOH is neutralized with HCl 1M. The 3H labeled radioactivity of an aliquot of the extract is counted in the presence of a scintillation buffer.

(26) In Vitro Assays to Assess Compound Activity in NCI-H716 Cell Line.

(27) Human intestinal cell line NCI-H716 has been described as cellular model to assess compounds mimicking nutrient-mediated effect such as glucagon-like peptide-1 (GLP-1) secretion.

(28) GLP-1 Release.

(29) NCI-H716 cells (ATCC, Manassas) are cultured in Dulbecco's modified eagle's medium (DMEM) containing 10% (v/v) bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 ?g/ml streptomycin in 75 ml flask. Cell adhesion and endocrine differentiation is initiated by growing cells in 96-well plate coated with matrigel in High Glucose DMEM containing 10% (v/v) bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 ?g/ml streptomycin for 2 days.

(30) On day 2, the medium is removed and cells are washed once with a pre-warmed wash buffer (Phosphate Buffered salt solution). The wash solution is removed and the SCFA or the tested compounds, or a combination of both, are added at the desired concentration in High Glucose DMEM containing 0.1% (v/v) bovine serum and incubated for 2 hours at 37? C. The supernatants are collected in a container. Using the cell-free supernatants, the GLP-1 amount is assessed using a GLP-1 specific ELISA assay kit according the manufacturer's recommendations (ALPCON).

(31) Ex Vivo Assays to Assess Compound Activity in Adipocytes from Normal Rat or Mice and High-Fat Diet Fed Mice.

(32) Mice C56Black6 male are housed in Makrolon type IV group housing cages (56?35?20 cm.sup.3) throughout the experimental phase. Animals' cages litters are changed once a week. They are housed in groups of 10 animals at 12 light dark (at 8 h 30 pm lights off), 22+/?2? C. and 50+/?5% relative humidity. Animals are acclimated one week. During the whole phase, standard diet or diet high in energy from fat (Research Diets, New Brunswick, N.J.) and tap water are provided ad libitum. The animals are 16 weeks old at the time of the study.

(33) For keeping only mice that have responded to the high fat diet, fasted glycemia are measured in these mice just before performing the ex-vivo study.

(34) Glucose Uptake Assay in Isolated Adipocytes.

(35) Animals are killed by cervical dislocation and epididymal fat pads are removed and digested in collagenase buffer at 370? C./120 rpm for approximately 50 minutes. The digest is filtered through gauze to recover the adipocytes, which are washed and resuspended in Krebs-Ringer Hepes (KRH) buffer containing 1% BSA, 200 nM adenosine and 2 mM glucose.

(36) Isolated adipocytes are washed in glucose-free KRH-buffer and resuspended to 30%. Adipocytes are then incubated at 370? C./80 rpm with either the tested compound (30 ?M, 10 ?M and 1 ?M) in the presence or absence of insulin (10 nM) for 30 min 2-deoxyglucose and 2-deoxy-D-[1-.sup.3H]-glucose (.sup.3H-2-DOG) are added and incubation continued for 10 min. The reactions are then stopped by addition of cytochalasin b followed by centrifugation through dinonylphthalate to recover the adipocytes. The uptake of .sup.3H-2-DOG- was measured by scintillation. Each data point is investigated in triplicates in two independent experiments.

(37) Lipolysis Assay in Isolated Adipocytes.

(38) Isolated adipocytes are diluted to 5% in KRH-buffer and are pre-treated with the tested compound (30 ?M, 100/1 and 10/1) for 30 min at 370? C./120 rpm. After the pre-treatment, Isoprenaline (1 ?M) is added to the adipocytes followed by 30 min incubation at 37? C./150 rpm. The reactions are put on ice and the buffer is assayed spectrophotometrically for the production of NADH.sup.+ from glycerol breakdown in reactions catalyzed by glycerol kinase and glycerol-3-phosphate dehydrogenase and/or Non Esterified Fatty Acid (NEFA). Each data point is investigated in triplicates in two independent experiments.

(39) According to the method described above and by way of illustration the compounds no1; 2; 4; 5; 8; 10; 11 and 13 inhibit isoprenaline-induced lipolysis in adipocytes from normal rat, at the concentration of 30 ?M (FIG. 1).

(40) In Vivo Assay to Assess Compound Activity in Rodent Diabetes Model.

(41) Genetic Rodent Models:

(42) Rodent models of T2D associated with obesity and insulin resistance have been developed. Genetic models such as db/db and ob/ob in mice and fa/fa in Zucker rats have been developed for understanding the pathophysiology of disease and testing candidate therapeutic compounds. The homozygous animals, C57 Black/6-db/db mice developed by Jackson Laboratory are obese, hyperglycemic, hyperinsulinemic and insulin resistant (J Clin Invest, 1990, 85:962-967), whereas heterozygotes are lean and normoglycemic. In the db/db model, mice progressively develop insulinopenia with age, a feature commonly observed in late stages of human T2D when sugar levels are insufficiently controlled. Since this model resembles that of human T2D, the compounds are tested for activities including, but not limited to, lowering of plasma glucose and triglycerides. Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant, and the fa/fa mutation may be the rat equivalent of the murine db mutation. Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed under standard laboratory conditions at 22? C. and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using one touch basic glucose monitor system (Lifescan). Mice that have plasma glucose levels between 250 to 500 mg/dl are used. Each treatment group consists of several mice that are distributed so that the mean of glucose levels are equivalent in each group at the start of the study. Db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide compounds of the invention, saline, or an irrelevant compound to the mice intravenously (i.v). Blood is sampled from the tail vein at intervals thereafter and analyzed for blood glucose concentrations. Significant differences between groups (comparing compounds of the invention to saline-treated) are evaluated using Student t-test.

(43) Ob/ob or obese mice are leptin-deficient mice that eat excessively and become profoundly obese, hyperinsulinemic and hyperglycemic. It is an animal model of type II diabetes. Such model can be used for oral glucose tolerance tests (OGTTs). A total of sixteen (16) male ob/ob mice (6 weeks of age) were obtained from Harlan. Upon arrival to the animal unit, mice were housed 4 per cage in rodent cages mounted with feeders containing regular chow. The mice were put in a 12/12 h light-dark cycle (light from 0600-1800 h) with controlled temperature conditions (22-24? C.). Fed blood glucose and body-weight was measured on day ?2 in the morning between 08:00 AM and 09:00 AM. Animals were randomized into 2 groups according to fed glucose levels (on day ?2). The 16 mice with blood glucose and body-weight closest to the mean were distributed into the following groups: Group 1: Vehicle p.o. bi-daily, (n=8) and Group 2: Compound of the invention, p.o., bi-daily, (n=8).

(44) Day 1 is the first day of dosing. Animals were dosed with compounds of the invention at 07:00 AM and 04:00 PM for 28 days. On the evening of day 27, food was removed and mice were transferred to clean cages. Mice were fasted for the subsequent 17 hours until the OGTT was performed. At ?15 min, blood glucose was measured (using a glucose analyzer) and animals were dosed with compounds of the invention or vehicle. At time point 0, blood glucose was measured again and glucose was administered by oral gavage (1 g/kg glucose). The blood glucose was then measured at time points 15, 30, 45, 60 and 120 minutes. The blood glucose area under the curve (AUC) from time ?15 to 120 min was then calculated (GraphPad software). The percentages of AUC inhibition induced by compounds of the invention were calculated as follows: % of AUC inhibition: [1?(AUC compound/AUC vehicule)]*100.

(45) When tested in the above-described assay, the compound 1 showed a % of AUC inhibition of 40%, indicating that compound 1 is able to significantly reduce the level of blood glucose in diabetic animal model (FIGS. 2A and 2B).

(46) The High-Fat Diet Fed Mouse:

(47) This model was originally introduced by Surwit et al. in 1988. The model has shown to be accompanied by insulin resistance, as determined by intravenous glucose tolerance tests, and of insufficient islet compensation to the insulin resistance. The model has, accordingly, been used in studies on pathophysiology of impaired glucose tolerance (IGT) and type 2 diabetes and for development of new treatments.

(48) C57BL/6J mice are maintained in a temperature-controlled room (22? C.) on a 12-h light-dark cycle. One week after arrival, mice are divided into two groups and are fed either a high-fat diet or received continuous feeding of a normal diet for up to 12 months. On caloric basis, the high-fat diet consist of 58% fat from lard, 25.6% carbohydrate, and 16.4% protein (total 23.4 kJ/g), whereas the normal diet contains 11.4% fat, 62.8% carbohydrate, and 25.8% protein (total 12.6 kJ/g). Food intake and body weight are measured once a week, and blood samples are taken at indicated time points from the intraorbital retrobulbar plexus from nonfasted anesthetized mice.

(49) For intravenous glucose tolerance tests (IVGTTs), 4-h fasted mice are anesthetized with 7.2 mg/kg fluanison/fenlanyl and 15.3 mg/kg midazolam. Thereafter a blood sample is taken from the retrobulbar, intraorbital, capillary plexus, after which D-glucose (1 g/kg) is injected intravenously in a tail vein (volume load 10 l/g). Additional blood samples are taken at 1, 5, 10, 20, 50, and 75 min after injection. Following immediate centrifugation at 4 C, plasma is separated and stored at ?20 C until analysis. For oral glucose tolerance tests (OGTTs), 16-h fasted anesthetized mice are given 150 mg glucose by gavage through a gastric tube (outer diameter 1.2 mm), which is inserted in the stomach. Blood samples are taken at 0, 15, 30, 60, 90, and 120 min after glucose administration and handled as above.

(50) Administration of the compounds: Five-week-old mice are fed a high-fat or a normal diet for 8 weeks. After 4 weeks, the mice are additionally given the compound of the invention in their drinking water (0.3 mg/ml, the amount can be changed for optimization. Control groups are given tap water without compound. After another 4 weeks, the mice are subjected to an OGTT as described above. Insulin and glucose measurements: Insulin is determined enzymatically using an ELISA assay kit (Linco Research, St. Charles, Mo.). Plasma glucose is determined by the glucose oxidase method.

(51) In Vivo Assay to Assess Compound Anti-Obesity Activity in Rodent Model.

(52) Mouse Acute Food Intake and Weight Change:

(53) Male C57BL/6N wild-type mice are weighed and vehicle or the tested compounds are administered by oral gavage to male mice approximately 30 min prior to the onset of the dark phase of the light cycle. Mice are fed ad libitum in the dark phase following dosing. A preweighed aliquot of a highly palatable medium high fat diet is provided in the food hopper of the cage 5 min prior to the onset of the dark phase of the light cycle and weighed 2 and 18 h after the onset of the dark phase of the light cycle.

(54) Acute Studies in Diet-Induced Obesity (DIO) Rats:

(55) For acute experiments, male Sprague-Dawley DIO rats from Charles River Laboratories are raised from 4 weeks of age on a diet moderately high fat (32% kcal) and high in sucrose (25% kcal). Animals are used at 12 weeks of age and are maintained on a 12/12 h light dark cycle. The rats are randomized into groups (n=6/group) for the tested compounds and vehicle dosing. Rats are weighed 17 h after dosing to determine effects on overnight body weight gain. The tested compounds are administered orally or s.c. at amount desired 1 h before the start of the dark cycle. Powdered food is provided in food cups which are weighed continuously at 5 min intervals over 18 h and the data are recorded using a computerized system.

(56) Chronic Studies in Diet-Induced Obesity Rats:

(57) For the 14-day chronic experiment, male Sprague-Dawley DIO rats are obtained as described above. Animals are used at 15 weeks of age and are maintained on a 12/12 hour light-dark cycle. Rats are conditioned to dosing for 4 days prior to baseline measurements, using an oral gavage or a s.c. route of vehicle. Thereafter, animals are dosed daily with vehicle or compound by oral gavage or s.c. The tested compound or vehicle is administered 1 h before the dark cycle for 14 days. Body composition is measured by dual energy X-ray densitometry (DEXAscan) 5 days prior to the study and at the end of the 14-day study. Daily endpoints included body weight and food intake.

(58) In Vivo Assay to Assess Compound Anti-Lipolytic Activity in Rodent Model.

(59) Male C57BL/6N wild-type are housed one per cage in a room maintained on a 12 h light/dark cycle under constant temperature (22-25? C.) with ad libitum access to food and water. The anti-lipolytic effects of the tested compounds are studied in awake mice. Animals are fasted overnight before experimental use. On the day of the experiment, animals are put in metabolic cages and left undisturbed to acclimate to the environment for 1-2 h. blood samples are taken at indicated time points from the intraorbital retrobulbar plexus. A 1% sodium citrate saline solution is used to flush the lines. A pre-treatment blood sample is obtained from each animal to determine baseline values for free fatty acids (FFA) and triglycerides (TG). The tested compounds are given via oral gavage, sc injection, iv injection or ip injection for each different series of experiments. Blood samples are collected into pre-cooled tubes pre-coated with heparin (200 ?l blood, Li-heparin, Sarstedt) for determination triglycerides and glycerol and in tri-potassium EDTA added sodium fluoride (200 ?l blood, K.sub.3-EDTA, 1.6 mg/mL+1% NaF, Sarstedt) for determination of plasma free fatty acids. The tubes are placed on wet ice pending processing. Blood samples will be centrifuged at 4000?g, at 4? C., 15 min the resulting plasma will be transferred into non-coated tubes and stored at ?80? C. until analyses. The plasma is thawed at 4? C. for determinations of FFA and TG using commercial kits (Wako Chemicals).

(60) While embodiments of the invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation ant it is understood that various changes may be made without departing from the spirit and scope of the invention.