Patent Application: US-201214118379-A

Abstract:
the invention relates to hypophosphorous acid derivatives of formula wherein — x is h or oh , — r represents one or several radicals r 1 - r 5 , identical or different , two of r 1 - r 5 optionally occupying the same position on the phenyl group , one to four of r 1 - r 5 being h and the others being selected in the group comprising - 0 - n — cooh ; — s — n — cooh ; — nh — n — cooh ; - 0 -— cooh ; — o — n — oh ; or ′, — r ′ being a c 1 - c 3 alkyl radical ; — oh ; — cooh ; halogen , particularly — f , — ci , — br , — i , — cf 3 ; — ocf 3 ; — n0 2 ; — ch ═ ch — cooh ; — n — cooh ; o — n — p0 3 h 2 ; o — n — p0 3 h 2 ; o — n — s0 3 h ; o — n — conhoh ; o — n - tetrazol ; o — n - hydroxyisoxazol — n = 1 to 5 , preferably 1 - 3 ; said hypophosrous acid derivatives being diastereoisomers or enantiomers .

Description:
as illustrated hereinafter in the examples , the pharmacological peculiarity of the above compounds lies in their strong agonist activity on mglu4r . depending on the substitutions , they may also be selective with respect to other metabotrobic receptors , particularly mglu7 or mglu8 , resulting in specific properties . they may thus have a higher agonist effect on mglu7r compared to all known agonists , both subtypes mglu4 and mglu7 acting then in synergy for the treatment of pain . it will be measured that such a simultaneous activation has a high therapeutic interest . in another embodiment , they may have mglu8 / 4 ec 50 ratio of interest , particularly above 100 . derivatives of a first preferred group have a ec 50 with respect to mglu4 ≦ 0 . 2 1 . 1m . particularly preferred derivatives of said group also have a ec 50 with respect to mglu7 ≦ 15 μm . they include derivatives of formula ( i ) wherein x =— oh and r represents one substituent in para , or two substituents in meta and para , or three substituents , particularly two in meta and one in para , respectively . in particularly preferred derivatives of said first group , the phenyl group is substituted by one substituent . a particularly preferred substituent is — o —( ch 2 ) n — cooh with n = 1 or — s —( ch 2 ) n — cooh with n = 1 . in other preferred derivatives of said first group , two substituents are present on the phenyl group , one being — o —( ch 2 ), — cooh with preferably n = 1 , and the other is or ′, with r ′ preferably being — ch 3 or cf 3 . in still other preferred derivatives , three substituents are present , one being — o —( ch 2 ) n — cooh with preferably n = 1 , the second one is or ′, with r ′ preferably being — ch 3 and the third one is — f , — cl , — i or — no 2 . derivatives of a second preferred group have a ec 50 with respect to mglu4 & lt ; about 1 μm . they include derivatives of formula ( i ) wherein x =— oh or h and r represents one substituent in para , or two substituents in meta and para , or three substituents , particularly two in meta and one in para , respectively . in particularly preferred derivatives of said second group , the phenyl group is substituted by one substituent . a particularly preferred substituent is — o — ch 2 ) n — cooh with n = 1 and x = h ; or — o —( ch 2 ) n — po 3 h 2 with n = 1 and x =— oh ; or —( ch 2 ) n — cooh with n = 2 and x =— oh ; or — ch ═ ch — cooh and x =— oh . in other preferred derivatives of said second group , two substituents are present on the phenyl group , one being — o —( ch 2 ) n — cooh with preferably n = 1 , and the other is or ′, with r ′ preferably being — ch 3 , or is oh , or is no 2 ; or one is s —( ch 2 ) n1 — cooh with preferably n1 = 1 and the other is no 2 . in other preferred derivatives , three substituents are present , one being — o —( ch 2 ) n — cooh with preferably n = 1 and the two others , identical or different , being selected from r as above defined or or ′, with r ′ preferably being — ch 3 , or — no 2 , — f , — cl , or — i . more particularly , the invention thus relates to the simultaneous activation of mglur subtypes 4 and 7 for the treatment of neuropathic and inflammatory pain comprising using an effective therapeutic amount of said derivatives . other derivatives of interest have a ec 50 with respect to mglur7 receptors above 15 μm , even above 100 μm . they correspond to derivatives having a high selectivity for mglur4 . other derivatives of high value have a high selectivity with respect to mglu8 with ec 50 mglu8 & gt ; 10 / ec 50 mglu4 & lt ; 0 . 5 . the invention thus also relates to pharmaceutical compositions comprising an effective amount of at least one of the hypophosphorous acid derivatives such as above defined in combination with a pharmaceutically acceptable carrier . advantageously , the pharmaceutical compositions of the invention are under a form suitable for an administration by the oral route , such as tablets , pills or capsules . they preferably comprise 1 to 100 mg of active ingredient per dose unit . alternatively , the pharmaceutical compositions of the invention are under a form suitable for an administration by injection , such as injectable solutions for the intravenous , subcutaneous or intramuscular route . such compositions advantageously comprise 1 to 30 mg of active ingredient per dose unit . the invention also relates to a method for treating neuropathic and inflammatory pain comprising simultaneously activating glutamate metabotropic receptors sub - groups 4 and 7 by administering to a patient in need thereof an efficient amount of at least one hypophosphorous acid derivative of formula ( i ) as above defined . the derivates of the invention are advantageously obtained by using the methods disclosed in above mentioned wo 2007 / 052169 . other characteristics and advantages of the invention are given in the following examples with reference to fig1 - 5 , which represent , respectively , fig1 : the lack of effect of intrathecal administration of derivative 1 in healthy rats when pain is evoked by a mechanical stimulus , fig2 : the antihyperalgesic effect of intrathecal administration of derivative 1 on the vocalization threshold to paw pressure in the carrageenan inflammatory pain model , fig3 : the antihyperalgesic effect of intrathecal administration of derivative 1 on the vocalization threshold to paw pressure in the cci neuropathic pain model , fig4 : the antihyperalgesic effect of intrathecal administration of derivate a on the vocalization threshold to paw pressure in the cci neuropathic model ( decrease when odn against mglu4r are injected ), fig5 : the antihyperalgesic effect of derivative 1 ( i . p .) in a rat model of inflammatory pain (“ carrageenan ”) treated . fig6 : the mechanical antihyperalgesic effect of derivative 2 is profoundly impaired in inflamed genetically modified mice lacking the gene encoding for the mglu4 receptor fig7 : the knockdown of spinal mglu 4 expression by antisense oligonucleotides reduces mechanical antihyperalgesia induced by derivative 2 or acpt - i all chemicals and solvents were purchased from commercial suppliers ( acros , aldrich ) and used as received . z - l - vinyl glycine methyl ester was purchased from ascent scientific ltd ( north somerset , uk ). solvents for reactions were dried on 4 å molecular sieves ( carlo erba sds ). 1 h ( 250 . 13 or 500 . 16 mhz ), 13 c ( 62 . 9 or 125 . 78 mhz ) and 31 p ( 101 . 25 or 202 . 47 mhz ) nmr spectra were recorded on an arx 250 or an avance ii 500 bruker spectrometer . chemical shifts ( 6 , ppm ) are given with reference to residual 1 h or 13 c of deuterated solvents ( cdcl 3 7 . 24 , 77 . 00 ; cd 3 od 3 . 31 , 49 . 0 ; d 2 o 4 . 80 ) or external reference ( h 3 po 4 95 %). product visualization was achieved with 2 % ( w / v ) ninhydrin in ethanol . thin layer chromatography ( tlc ) system for routine monitoring the course of certain reactions and confirming the purity of analytical samples employed aluminium - backed silica gel plates ( merck dc kieselgel 60 f 254 ): ch 2 cl 2 / meoh or cyclohexane / ethyl acetate were used as developing solvents and detection of spots was made by uv light and / or by iodine vapours . merck silica gel ( 230 - 400 mesh ) was used for flash chromatography . optical rotations were measured at the sodium d line ( 589 nm ) at room temperature with a perkin - elmer 341 polarimeter using a 1 dm path length cell . mass spectra ( ms ) were recorded with a lcq - advantage ( thermofinnigan ) mass spectrometer with positive ( esi +) or negative ( esi −) electrospray ionization ( ionization tension 4 . 5 kv , injection temperature 240 ° c .). hplc analyses were carried out on a gilson analytical instrument with a 321 pump , column temperature of crownpak columns was controlled with an igloo - cil peltier effect thermostat , eluted peaks were detected by a uv - vis 156 detector and retention times are reported in minutes . two columns were used on analytic scale : a daicel crownpak cr (+) column ( 150 mm × 4 mm ), further designed as “ crownpak ”, eluted with ph 2 . 0 perchloric acid at a 0 . 4 ml · min − 1 flow rate , and a phenomenex rp polar column ( 250 mm × 4 . 6 mm , 4 μm ), further designed as “ rp polar ”, eluted at a 0 . 5 ml · min − 1 flow rate with the following method : solvent a : water / formic acid 1000 : 1 , solvent b : water / acetonitrile / formic acid 900 : 100 : 1 , 100 % a for 10 min , linear gradient to 100 % b from 10 to 30 min , 100 % b from 30 to 40 min . preparative scale hplc was performed with a daicel crownpak cr (+) 150 × 10 mm column , further designed as “ preparative crownpak ”, eluted with a ph 2 . 0 hydrochloric acid aqueous solution . hplc - ms analyses were performed on a thermo finnigan lcq advantage instrument as described above , equipped for hplc with a phenomenex rp polar column ( 250 mm × 4 . 6 mm , 4 μm ). products were eluted with the following gradient using solvent a ( water / acetonitrile / formic acid 950 : 50 : 1 ) and solvent b ( water / acetonitrile / formic acid 900 : 100 : 1 ), 100 % a for 10 min , linear increase from 0 to 100 % b between 10 and 20 min , 100 % b from 20 to 30 min . purity of the tested compounds was established by analytical hplc - ms or by hplc and was at least 95 %. 1 was synthesized according to previously published procedure . ( wo2007 / 052169 , selvam et al jmedchem 2010 ). to a solution of h - phosphinic acid 1 ( 1 . 0 mmol , 1 eq ) and aldehyde ( x = oh ) or halide ( x = h ) ( 2 . 2 eq ) in 2 . 0 ml of dichloromethane at 0 ° c . under an argon atmosphere was added dropwise n , o - bis ( trimethysilyl ) acetamide ( bsa ) ( 4 . 4 eq ). the mixture was allowed to warm to room temperature and stirred overnight , then cooled to 0 ° c . and 25 ml of 1n hcl were added , then extracted with ethyl acetate . the organic layer was concentrated in vacuo . this residue was dissolved in 10 ml of water , the ph was adjusted to 7 using saturated sodium hydrogenocarbonate solution , then extracted with ethylacetate ( 2 × 50 ml ). the organic layer was separated , and the aqueous phase was treated with aqueous hcl to adjust the ph to 1 . the aqueous phase was extracted with ethyl acetate ( 2 × 50 ml ). the combined acidic organic extracts were dried over magnesium sulfate , filtered and concentrated in vacuo . to a solution of 1 . 0 mmol of h - phosphinic acid 1 and 2 . 2 mmol of aldehyde ( x = oh ) or bromide ( x = h ) in 2 . 0 ml of dry dichloromethane at 0 ° c . under argon was added dropwise n , o - bis ( trimethylsilyl )- acetamide ( bsa , 1 . 08 ml , 4 . 4 mmol ). the mixture was allowed to warm to room temperature and stirred overnight under argon , then cooled to 0 ° c . 1n hydrochloric acid ( 20 ml ) was added , then extracted with ethyl acetate ( 2 × 100 ml ). the combined organic extracts were dried over magnesium sulfate , filtered and concentrated under vacuum . the crude product , still containing aldehyde or bromide in excess , was directly deprotected . the crude product synthesised by general procedure a or b was dissolved in 6n hydrochloric acid ( 10 . 0 ml ). the mixture was stirred at 100 ° c . for 5 h , then cooled to room temperature . the solution was diluted with ethyl acetate ( 50 ml ) and water ( 10 ml ). the separated organic layer was extracted with 1n hydrochloric acid ( 3 × 10 ml ). the combined aqueous phases were concentrated under vacuum , then the residue was purified using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water or 1n nh 4 oh elution ). if necessary , another purification was made on an anion exchange chromatography ( dowex ag 1 - x4 , aco − , 200 - 400 mesh ). the loading solution was prepared in 500 ml , of freshly boiled and cooled pure water ( the ph was adjusted to 5 ). the resin was first eluted with freshly boiled and cooled water , then with formic acid . the crude product synthesised by general procedure a or b was dissolved in 6n hydrochloric acid ( 5 . 0 ml ). the mixture was stirred at 100 ° c . for 5 h , then cooled to room temperature . the solution was diluted with ethyl acetate ( 50 ml ) and water ( 10 ml ). the separated organic layer was extracted with of 1n hydrochloric acid ( 3 × 10 ml ). the combined aqueous phases were concentrated under vacuum . the residue was purified using an anion exchange chromatography ( dowex ag 1 - x4 , aco − , 200 - 400 mesh ). the loading solution was prepared in 500 ml of freshly boiled and cooled pure water ( the ph was adjusted to 5 ). the resin was first eluted with freshly boiled and cooled water , then with dilute hcl . to a solution of phenol ( 1 eq ) in dry acetone , anhydrous potassium carbonate ( 1 . 3 eq ) was added at room temperature . after 10 minutes , ethylbromoacetate ( 1 . 5 eq ) was added and the mixture was refluxed and monitored by tlc until completion . after cooling , the solvent was removed under reduced pressure . the residue was added with water , then extracted with dichloromethane and washed with brine . the combined organic extracts were dried over magnesium sulfate , filtered and concentrated under vacuum . ethanol or isopropanol was added and evaporated by azeotropic distillation in order to eliminate the ethylbromoacetate in excess . the solvent was evaporated to dryness , affording a residue which was chromatogaphed on a silica gel column ( dichloromethane / ethyl acetate as eluent ) to afford aryloxyacetic acid ethyl ester . nabh 4 ( 1 . 1 eq ) was added to a stirred and ice - bath cooled solution of aldehyde ( 1 eq ) in thf and water ( 50 / 50 ). the reaction mixture was stirred for 45 minutes at 0 ° c . and , after distilling off the organic solvent , the aqueous residue was added with water ( 50 ml ) and then extracted with dichloromethane ( 50 ml ). the organic layer was washed with 10 % na 2 co 3 ( 20 ml ) and brine ( 20 ml ), then dried over na 2 so 4 , filtered , and evaporated in vacuo to dryness , affording a residue which was chromatographed on a silica gel column ( dichloromethane / ethyl acetate as eluent ). pbr 3 ( 1 eq ) was carefully added to a solution of alcohol ( 1 eq ) in distilled dichloromethane at 0 ° c . under argon . the reaction mixture was stirred at 0 ° c . for 3 h and extracted with dichloromethane . the organic layer was washed with brine , dried over na 2 so 4 , filtered , and evaporated in vacuo to dryness without further purification . the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 252 mg , 0 . 8 mmol ) and 4 - formylphenoxyacetic acid ( 317 mg , 1 . 8 mmol ). 629 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 od ): δ 49 . 3 . the previous compound was deprotected according to general procedure c . 150 mg of pure derivative 2 were obtained ( 54 % yield , 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 71 - 194 ( m , 2h ), 1 . 98 - 2 . 00 ( m , 2h ), 3 . 95 ( bs , 1h ), 4 . 58 ( s , 2h ), 4 . 81 ( bs , 1h ), 6 . 85 ( d , j = 8 . 3 hz , 2h ), 7 . 25 ( d , j = 7 . 8 hz , 2h ). 31 p nmr ( 101 mhz , d 2 o ): δ 55 . 4 . 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 8 and 22 . 9 ( 2d , j = 89 hz ), 24 . 1 , 54 . 5 ( d , j = 15 hz ), 66 . 2 , 73 . 0 ( d , j = 111 hz ), 110 . 0 , 130 . 0 , 131 . 3 , 158 . 5 , 172 . 8 , 174 . 7 . hplc - ms : t r = 6 . 05 min . hplc ( crownpak , t = 5 . 0 ° c ., detection λ = 210 / 254 nm ): t r = 18 . 4 min . the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 292 mg , 0 . 9 mmol ) and methyl 4 - formyl - 2 - methoxyphenoxyacetate ( 457 mg , 2 . 0 mmol ). 863 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 od ): δ 47 . 4 . the previous compound was deprotected according to general procedure d . the pure compound was obtained with a purification on anion exchange resin on 95 mg of crude product on a 14 × 1 cm column . the desired product was eluted with a 1 . 0 × 10 − 2 n hydrochloric acid aqueous solution . 61 mg of pure derivative 3 were obtained ( 18 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 73 - 1 . 90 ( m , 2h ), 2 . 04 - 2 . 11 ( m , 2h ), 3 . 86 ( s , 3h ), 4 . 03 ( q , j = 6 . 0 hz , 1h ), 4 . 76 ( s , 2h ), 4 . 87 ( d , j = 8 . 5 hz , 1h ), 6 . 93 ( d , j = 8 . 5 hz , 1h ), 6 . 97 ( d , j = 8 . 5 hz , 1h ), 7 . 11 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 56 . 2 . 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 7 ( 2d , j = 89 hz ), 24 . 0 , 54 . 4 ( d , j = 15 hz ), 57 . 2 , 66 . 8 , 73 . 0 ( d , j = 111 hz ), 112 . 6 , 114 . 6 , 121 . 2 , 131 . 9 , 147 . 7 , 149 . 8 , 172 . 6 , 174 . 3 . ms ( esi ): m / z 376 . 0 ( m − 1 ). hplc ( crownpak , t = 10 . 0 ° c ., detection λ = 210 / 254 nm ): t r = 15 . 4 min . hplc ( rp polar , detection λ = 230 / 270 nm ): t r = 10 . 0 min . the compound was prepared according to general procedure b with h - phosphinic acid ( 292 mg , 0 . 9 mmol ) and 4 - formyl - 2 , 6 - dimethoxyphenoxyacetic acid ( 250 mg , 1 . 0 mmol ). 31 p nmr ( 101 mhz , cd 3 od ): δ 47 . 8 . the previous compound was deprotected according to general procedure d . the pure compound was obtained with a purification on anion exchange resin on 300 mg of crude product on a 13 × 2 cm column . the desired product was eluted with a 1 . 5 × 10 − 2 n hydrochloric acid aqueous solution . 155 mg of pure derivative 4 were obtained ( 42 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 85 - 1 . 96 ( m , 2h ), 2 . 06 - 2 . 11 ( m , 2h ), 3 . 81 ( 2s , 6h ), 4 . 03 ( m , 1h ), 4 . 59 ( m , 2h ), 4 . 95 ( d , j = 8 . 5 hz , 1h ), 6 . 77 ( s , 2h ). 31 p nmr ( 101 mhz , d 2 o ): δ 57 . 6 . 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 8 ( d , j = 89 hz ), 24 . 0 , 54 . 4 ( d , j = 15 hz ), 57 . 6 , 70 . 8 , 73 . 2 ( d , j = 110 hz ), 105 . 9 , 134 . 5 , 136 . 4 , 153 . 2 , 172 . 5 , 174 . 8 . ms ( esi ): m / z 405 . 9 ( m − 1 ). hplc ( crownpak , t = 10 . 0 ° c ., detection λ = 210 / 254 nm ): t r = 22 . 7 and 25 . 0 min . hplc ( rp polar , detection λ = 230 / 270 nm ): t r = 14 . 7 min . to a solution of 5 - nitrovanilline ( 591 mg , 3 . 0 mmol ) in dmf ( 9 . 0 ml ) was added cesium carbonate ( 2 . 00 g , 6 . 16 mmol ), followed by ethyl bromoacetate ( 3 . 33 ml , 30 mmol ). the red mixture was left under stirring at 110 ° c . until it became yellow , and then for further 3 minutes . after evaporation of dmf under reduced pressure , 22 ml of water were added and the mixture was extracted with dichloromethane ( 60 ml ). the organic phase was washed with brine ( 15 ml ), dried over magnesium sulfate and concentrated under vacuum . the residue was purified by column chromatography over silica gel ( eluent : dichloromethane ) to give 625 mg of a solid ( 74 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 30 ( t , j = 7 . 2 hz , 3h ), 4 . 01 ( s , 3h ), 4 . 27 ( q , j = 7 . 2 hz , 2h ), 4 . 91 ( s , 2h ), 7 . 65 ( s , 1h ), 7 . 90 ( s , 1h ), 9 . 95 ( s , 1h ). 13 c nmr ( 63 mhz , cdcl 3 ): δ 14 . 6 , 57 . 2 , 62 . 0 , 70 . 0 , 113 . 9 , 120 . 4 , 132 . 0 , 145 . 2 , 145 . 9 , 154 . 2 , 168 . 5 , 189 . 3 . the compound was prepared according to general procedure a with h - phosphinic acid 1 ( 243 mg , 0 . 77 mmol ) and ethyl 4 - formyl - 2 - methoxy - 6 - nitrophenoxyacetate ( 481 mg , 1 . 7 mmol ). 169 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 cocd 3 ): δ 44 . 8 . [(( 3s )- 3 - amino - 3 - carboxy ) propyl ][( 3 - methoxy - 5 - nitro - 4 -( carboxymethoxy ) phenyl ) hydroxymethyl ] phosphinic acid ( derivative 5 ). the previous compound was deprotected according to general procedure c . 42 . 4 mg of pure product derivative 5 were obtained ( 13 % yield , 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 63 - 1 . 90 ( m , 2h ), 1 . 97 - 2 . 17 ( m , 2h ), 3 . 84 ( s , 3h ), 4 . 03 ( m , 1h ), 4 . 72 ( s , 2h ), 4 . 90 ( d , j = 9 . 5 hz , 1h ), 7 . 31 ( s , 1h ), 7 . 43 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 52 . 1 . 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 1 and 23 . 2 ( 2d , j = 89 hz ), 24 . 2 , 54 . 6 ( d , j = 14 hz ), 71 . 2 , 72 . 9 ( d , j = 108 hz ), 115 . 8 , 117 . 5 , 136 . 3 , 140 . 9 , 144 . 9 , 154 . 3 , 172 . 8 , 174 . 3 . ms ( esi ): m / z 423 . 1 ( m + 1 ), 421 . 0 ( m − 1 ). hplc - ms ( method a ): t r = 9 . 51 min . to a solution of 3 - fluoro - 4 - hydroxy - 5 - methoxybenzaldehyde ( 302 mg , 1 . 78 mmol ) in acetone ( 20 ml ) was added cesium carbonate ( 765 mg , 2 . 35 mmol ), followed by ethyl bromoacetate ( 0 . 30 ml , 2 . 7 mmol ). the mixture was refluxed in acetone under stirring for 1 hour . after evaporation of acetone under reduced pressure , 100 ml of water were added and the mixture was extracted with dichloromethane ( 200 ml ). the organic phase was washed with brine ( 100 ml ), dried over magnesium sulfate and concentrated under vacuum . the residue was purified by column chromatography over silica gel ( eluent : dichloromethane ) to give 361 mg of an oil ( 79 % yield ). 1 h nmr ( 500 mhz , cd 3 cocd 3 ): δ 1 . 25 ( t , j = 7 . 2 hz , 3h ), 3 . 98 ( s , 3h ), 4 . 20 ( q , j = 7 . 2 hz , 2h ), 4 . 89 ( t , j = 1 . 6 hz 2h ), 7 . 34 ( dd , j = 10 . 3 hz , 1 . 6 hz , 1h ), 7 . 41 ( s , 1h ), 9 . 88 ( d , j = 1 . 5 hz , 1h ). 13 c nmr ( 126 mhz , cd 3 cocd 3 ): δ 15 . 0 , 57 . 6 , 62 . 1 , 70 . 3 ( d , j = 1 . 9 hz ), 110 . 0 , 111 . 8 ( d , j = 20 . 6 hz ), 133 . 1 ( d , j = 7 . 2 hz ), 141 . 8 , ( d , j = 12 . 7 hz ), 154 . 8 ( d , j = 4 . 5 hz ), 155 . 0 d , j = 46 . 1 hz ) 169 . 7 , 191 . 3 . the compound was prepared according to general procedure a with h - phosphinic acid ( 465 mg , 1 . 48 mmol ) and ethyl 2 -( 2 - fluoro - 4 - formyl - 6 - methoxyphenoxy ) acetate ( 361 mg , 1 . 41 mmol ). 1 . 3 g of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 od ): δ 47 . 6 . ms ( esi ): m / z 570 . 1 ( m − 1 ) the previous compound was deprotected according to general procedure c . 115 . 0 mg of pure derivative 6 were obtained ( 21 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 80 - 1 . 80 ( m , 2h ), 1 . 99 - 2 . 08 ( m , 2h ), 3 . 77 ( s , 3h ), 3 . 95 ( m , 1h ), 4 . 60 ( s , 2h ), 4 . 74 ( d , j = 9 . 7 hz , 1h ), 6 . 77 ( d , j = 11 . 3 hz , 1h ), 6 . 83 ( s , 1h ). 31 p nmr ( 202 mhz , d 2 o ): δ 40 . 3 . 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 0 and 23 . 8 ( dd , j = 89 . 4 ; 7 . 7 hz ), 24 . 4 , 54 . 8 ( d , j = 14 . 1 hz ), 57 . 7 , 71 . 0 , 73 . 5 ( d , j = 107 . 7 hz ), 108 . 5 , 108 . 8 ( d , j = 20 . 9 hz ), 135 . 0 ( d , j = 12 . 8 hz ), 135 . 7 ( d , j = 8 . 5 hz ), 153 . 7 , 156 . 0 ( d , j = 244 hz ), 173 . 1 , 174 . 6 . ms ( esi ): m / z 396 . 0 ( m + 1 ). hplc - ms ( method a ): t r = 7 . 91 min . to a solution of 5 - chlorovanilline ( 505 mg , 2 . 7 mmol ) in acetone ( 25 ml ) was added cesium carbonate ( 1 . 16 g , 3 . 56 mmol ), followed by ethyl bromoacetate ( 0 . 45 ml , 4 . 05 mmol ). the mixture was refluxed in acetone under stirring for 1 hour . after evaporation of acetone under reduced pressure , 100 ml of water were added and the mixture was extracted with dichloromethane ( 200 ml ). the organic phase was washed with brine ( 100 ml ), dried over magnesium sulfate and concentrated under vacuum . the residue was purified by column chromatography over silica gel ( eluent : dichloromethane ) to give 330 mg of an oil ( 45 % yield ). 1 h nmr ( 250 mhz , cd 3 cocd 3 ): δ 1 . 27 ( t , j = 7 . 2 hz , 3h ), 3 . 98 ( s , 3h ), 4 . 22 ( q , j = 7 . 2 hz , 2h ), 4 . 87 ( s , 2h ), 7 . 50 ( s , 1h ), 7 . 58 ( s , 1h ), 9 . 90 ( s , 1h ). 13 c nmr ( 126 mhz , cd 3 cocd 3 ): δ 15 . 1 , 57 . 5 , 62 . 1 , 70 . 5 , 112 . 3 , 125 . 8 , 128 . 9 , 134 . 0 , 149 . 7 , 154 . 5 , 169 . 5 , 191 . 2 . the compound was prepared according to general procedure a with h - phosphinic acid ( 398 mg , 1 . 3 mmol ) and ethyl 2 -( 2 - chloro - 4 - formyl - 6 - methoxyphenoxy ) acetate ( 330 mg , 1 . 2 mmol ). 819 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 cocd 3 ): δ 48 . 2 . ms ( esi ): m / z 587 . 8 ( m + 1 ), 585 . 9 ( m − 1 ) the previous compound was deprotected according to general procedure c . 120 . 0 mg of pure derivative 7 were obtained ( 26 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 78 - 1 . 87 ( m , 2h ), 2 . 05 - 2 . 11 ( m , 2h ), 3 . 78 ( s , 3h ), 4 . 03 ( m , 1h ), 4 . 57 ( s , 2h ), 4 . 85 ( d , j = 9 . 2 hz , 1h ), 6 . 98 ( s , 1h ), 7 . 02 ( s , 1h ). 31 p nmr ( 202 mhz , d 2 o ): δ 43 . 6 . 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 5 and 23 . 3 ( dd , j = 88 . 3 ; 6 . 6 hz ), 24 . 1 , 54 . 5 ( d , j = 14 . 6 hz ), 57 . 6 , 70 . 6 , 72 . 9 ( d , j = 108 . 9 hz ), 111 . 8 , 121 . 5 , 128 . 4 , 136 . 0 , 143 . 5 , 154 . 0 , 172 . 7 , 174 . 4 . ms ( esi ): m / z 410 . 0 ( m − 1 ). hplc - ms ( method a ): t r = 9 . 00 min . to a solution of 5 - iodovanilline ( 502 mg , 1 . 8 mmol ) in acetone ( 25 ml ) was added cesium carbonate ( 766 mg , 2 . 35 mmol ), followed by ethyl bromoacetate ( 0 . 30 ml , 2 . 7 mmol ). the mixture was refluxed in acetone under stirring for 1 hour . after evaporation of acetone under reduced pressure , 100 ml of water were added and the mixture was extracted with dichloromethane ( 200 ml ). the organic phase was washed with brine ( 100 ml ), dried over magnesium sulfate and concentrated under vacuum . the residue was purified by column chromatography over silica gel ( eluent : dichloromethane ) to give 320 mg of a yellow solid ( 49 % yield ). 1 h nmr ( 250 mhz , cd 3 cocd 3 ): δ 1 . 28 ( t , j = 7 . 2 hz , 3h ), 3 . 97 ( s , 3h ), 4 . 23 ( q , j = 7 . 2 hz , 2h ), 4 . 85 ( s , 2h ), 7 . 55 ( s , 1h ), 7 . 95 ( s , 1h ), 9 . 89 ( s , 1h ). 13 c nmr ( 126 mhz , cd 3 cocd 3 ): δ 15 . 2 , 57 . 4 , 62 . 1 , 70 . 5 , 92 . 4 , 113 . 9 , 135 . 1 , 135 . 5 , 153 . 1 ( 2c ), 169 . 4 , 169 . 5 , 191 . 0 . the compound was prepared according to general procedure a with h - phosphinic acid ( 289 mg , 0 . 92 mmol ) and ethyl 2 -( 2 - iodo - 4 - formyl - 6 - methoxyphenoxy ) acetate ( 320 mg , 0 . 88 mmol ). 516 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 cocd 3 ): δ 48 . 1 . ms ( esi ): m / z 677 . 9 ( m − 1 ) the previous compound was deprotected according to general procedure c . 90 . 0 mg of pure derivative 8 were obtained ( 20 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 73 - 1 . 77 ( m , 2h ), 2 . 05 - 2 . 08 ( m , 2h ), 3 . 81 ( s , 3h ), 4 . 00 ( m , 1h ), 4 . 56 ( s , 2h ), 4 . 78 ( d , j = 9 . 4 hz , 1h ), 7 . 07 ( s , 1h ), 7 . 40 ( s , 1h ). 31 p nmr ( 202 mhz , d 2 o ): δ 40 . 4 . 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 1 and 23 . 9 ( dd , j = 89 . 2 ; 10 . 4 hz ), 24 . 5 , 54 . 9 ( d , j = 13 . 9 hz ), 57 . 5 , 70 . 6 , 73 . 1 ( d , j = 108 . 0 hz ), 92 . 2 , 113 . 6 , 130 . 1 , 138 . 2 , 147 . 1 , 152 . 9 , 173 . 2 , 174 . 5 . ms ( esi ): m / z 501 . 9 ( m − 1 ). hplc - ms ( method a ): t r = 12 . 15 min . to a solution of 3 , 5 - dichloro - 4 - hydroxybenzaldehyde ( 302 mg , 1 . 6 mmol ) in acetone ( 15 ml ) was added cesium carbonate ( 700 mg , 2 . 15 mmol ), followed by ethyl bromoacetate ( 0 . 27 ml , 2 . 43 mmol ). the mixture was refluxed in acetone under stirring for 1 hour . after evaporation of acetone under reduced pressure , 100 ml , of water were added and the mixture was extracted with dichloromethane ( 200 ml ). the organic phase was washed with brine ( 100 ml ), dried over magnesium sulfate and concentrated under vacuum . the residue was purified by column chromatography over silica gel ( eluent : dichloromethane ) to give 264 mg of an oil ( 59 % yield ). 1 h nmr ( 500 mhz , cdcl 3 ): δ 1 . 29 ( t , j = 7 . 2 hz , 3h ), 4 . 26 ( q , j = 7 . 2 hz , 2h ), 4 . 85 ( s , 2h ), 7 . 96 ( s , 2h ), 9 . 98 ( s , 1h ). 13 c nmr ( 126 mhz , cd 3 cocd 3 ): δ 15 . 0 , 62 . 3 , 70 . 8 , 131 . 2 , 131 . 4 , 135 . 4 , 156 . 4 , 168 . 4 , 190 . 5 . the compound was prepared according to general procedure a with h - phosphinic acid ( 315 mg , 1 . 0 mmol ) and ethyl 2 -( 2 , 6 - dichloro - 4 - formylphenoxy ) acetate ( 264 mg , 0 . 956 mmol ). 553 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 cocd 3 ): δ 47 . 3 ms ( esi ): m / z 591 . 0 ( m − 1 ) the previous compound was deprotected according to general procedure c . 102 . 0 mg of pure derivative 9 were obtained ( 26 % yield , 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 69 - 1 . 81 ( m , 2h ), 2 . 02 - 2 . 12 ( m , 2h ), 4 . 03 ( m , 1h ), 4 . 69 ( s , 2h ), 4 . 78 ( d , j = 9 . 6 hz , 1h ), 7 . 41 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 39 . 3 . 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 6 and 24 . 4 ( dd , j = 78 . 7 ; 13 . 8 hz ), 24 . 8 , 55 . 5 , 70 . 8 , 73 . 2 ( d , j = 117 . 4 hz ), 128 . 7 , 129 . 8 , 138 . 6 , 150 . 1 , 174 . 3 , 174 . 4 ms ( esi ): m / z 413 . 9 ( m − 1 ). hplc ms ( method a ): t r = 11 . 34 min . to a solution of 4 - hydroxy - 3 , 5 - dimethylbenzaldehyde ( 500 mg , 3 . 33 mmol ) in acetone ( 20 ml ) was added cesium carbonate ( 1 . 41 g , 4 . 33 mmol ), followed by ethyl bromoacetate ( 0 . 555 ml , 5 mmol ). the mixture was refluxed in acetone under stirring for 1 hour . after evaporation of acetone under reduced pressure , 100 ml of water were added and the mixture was extracted with dichloromethane ( 200 ml ). the organic phase was washed with brine ( 100 ml ), dried over magnesium sulfate and concentrated under vacuum . the residue was purified by column chromatography over silica gel ( eluent : dichloromethane ) to give 580 mg of an oil ( 74 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 25 ( t , j = 6 . 9 hz , 3h ), 4 . 27 ( s , 6h ), 4 . 21 ( q , j = 6 . 9 hz , 2h ), 4 . 38 ( s , 2h ), 7 . 44 ( s , 2h ), 9 . 77 ( s , 1h ). 13 c nmr ( 126 mhz , cdcl 3 ): δ 15 . 2 , 17 . 4 , 62 . 2 , 70 . 1 , 131 . 6 , 132 . 8 , 133 . 8 , 161 . 7 , 169 . 5 , 192 . 1 . the compound was prepared according to general procedure a with h - phosphinic acid ( 809 mg , 2 . 57 mmol ) and ethyl 2 -( 4 - formyl - 2 , 6 - dimethylphenoxy ) acetate ( 580 mg , 2 . 46 mmol ). 1 . 4 g of crude product were obtained . 31 p nmr ( 101 mhz , cdcl 33 ): δ 50 . 1 ms ( esi ): m / z 550 . 1 ( m − 1 ) the previous compound was deprotected according to general procedure c . 238 . 0 mg of pure derivative 10 were obtained ( 26 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 70 - 1 . 86 ( m , 2h ), 1 . 98 - 2 . 06 ( m , 2h ) 2 . 16 ( s , 6h ), 3 . 97 ( m , 1h ), 4 . 41 ( s , 2h ), 4 . 77 ( d , j = 8 . 7 hz , 1h ), 7 . 03 ( s , 2h ). 31 p nmr ( 202 mhz , d 2 o ): δ 43 . 3 . 13 c nmr ( 126 mhz , d 2 o ): δ 16 . 8 , 22 . 6 and 23 . 3 ( dd , j = 88 . 5 ; 8 . 7 hz ), 24 . 2 , 54 . 6 ( d , j = 14 . 5 hz ), 69 . 9 , 73 . 2 ( d , j = 109 . 3 hz ), 128 . 9 , 132 . 6 , 134 . 5 , 155 . 4 , 172 . 9 , 174 . 6 ms ( esi ): m / z 374 . 1 ( m − 1 ), 376 . 0 ( m + 1 ). hplc - ms ( method a ): t r = 8 . 33 min . the compound was prepared according to general procedure b with h - phosphinic acid ( 315 mg , 1 . 0 mmol ) and ( e )- 4 - formyl - phenylacrylic acid ( 388 mg , 2 . 2 mmol ). the reaction was stirred for 48 h to achieve complete conversion . 31 p nmr ( 101 mhz , cd 3 od ): δ 48 . 7 . the previous compound was deprotected according to general procedure c . after the cation exchange resin , 355 mg of compound were obtained . a second purification on an anion exchange resin ( ag 1 - x4 , 200 - 400 mesh , 18 × 2 . 5 cm , aco − ) was necessary to obtain a pure compound . the crude compound was diluted in 500 ml of pure and freshly boiled and cooled water , and the ph was brought to 9 ( with a freshly prepared 1n sodium hydroxide aqueous solution ). the resin was eluted with formic acid ( 1n to 5n ) and the desired compound was eluted with 3n formic acid . 158 mg of pure derivative 11 were obtained ( 46 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 76 - 1 . 87 ( m , 2h ), 2 . 05 - 2 . 16 ( m , 2h ), 4 . 05 ( t , j = 6 . 0 hz , 1h ), 4 . 94 ( d , j = 10 . 0 hz , 1h ), 6 . 47 and 6 . 48 ( 2d , j = 16 . 0 hz , 1h ), 7 . 45 ( d , j = 7 . 5 hz , 2h ), 7 . 61 ( d , j = 5 . 5 hz , 2h ), 7 . 67 and 7 . 68 ( 2d , j = 16 . 0 hz , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 53 . 0 . 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 2 ( d , j = 86 hz ), 24 . 4 , 54 . 8 , 74 . 0 ( d , j = 106 hz ), 118 . 9 , 128 . 9 , 129 . 9 , 135 . 1 , 141 . 3 , 147 . 5 , 172 . 4 , 173 . 0 . hplc - ms : t r = 7 . 49 min . the compound was prepared according to a previously described procedure ( a ) battistuzzi , g . ; cacchi , s . ; fabrizi , g . ; bernini , r . 3 - arylpropanoate esters through the palladium - catalyzed reaction of aryl halides with acrolein diethyl acetal . synlett 2003 , 8 , 1133 - 1136 . b ) giannini , g . ; marzi , m . ; pezzi , r . ; brunetti , t . ; battistuzzi , g . ; di marzo , m . ; cabri , w . ; vesci , l . ; pisano , c . n - hydroxy -( 4 - oxime )- cinnamide : a versatile scaffold for the synthesis of novel histone deacetilase ( hdac ) inhibitors . bioorg . med . chem . lett . 2009 , 19 , 2346 - 2349 ). to a solution of 4 - bromobenzaldehyde ( 740 mg , 4 . 0 mmol ), acrolein diethylacetal ( 1 . 83 ml , 12 mmol ), tetra - n - butylammonium chloride ( 1 . 11 g , 4 . 0 mmol ), tributylamine ( 1 . 91 ml , 8 . 0 mmol ) in 16 ml of dmf , palladium ( ii ) acetate ( 27 mg , 0 . 12 mmol ) was added . the mixture was warmed at 90 ° c . and stirred overnight . after cooling , the reaction mixture was diluted with 2n hcl ( 60 ml ) and extracted with diethyl ether ( 60 ml × 3 ). the organic layer was dried over magnesium sulfate and concentrated under reduced pressure . the residue was purified by column chromatography over silica gel ( eluent : 5 - 15 % etoac / cyclohexane ) to give 538 mg of a yellow oil ( 65 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 25 ( t , j = 7 . 0 hz , 3h ), 2 . 68 ( d , j = 7 . 5 hz , 2h ) 3 . 06 ( d , j = 7 . 5 hz , 2h ), 4 . 14 ( q , j = 7 . 0 hz , 2h ), 7 . 40 ( d , j = 8 . 2 hz , 2h ), 7 . 83 ( d , j = 8 . 2 hz , 2h ), 10 . 00 ( s , 1h ). 13 c nmr ( 63 mhz , cdcl 3 ): δ 14 . 6 , 31 . 5 , 35 . 7 , 61 . 1 , 129 . 4 , 130 . 4 , 135 . 2 , 148 . 3 , 172 . 8 , 192 . 4 . the compound was prepared according to general procedure a with h - phosphinic acid 1 ( 375 mg , 1 . 19 mmol ) and ethyl 3 -( 4 - formyl )- phenylpropanoate ( 540 mg , 2 . 62 mmol ). 336 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 cocd 3 ): δ 45 . 6 . the previous compound was deprotected according to general procedure c . 152 . 6 mg of pure product derivative 12 were obtained ( 37 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 74 - 1 . 99 ( m , 2h ), 2 . 01 - 2 . 15 ( m , 2h ), 2 . 68 ( t , j = 7 . 3 hz , 2h ), 2 . 91 ( t , j = 7 . 3 hz , 2h ), 4 . 05 ( m , 1h ), 4 . 94 ( d , j = 9 . 0 hz , 1h ), 7 . 28 ( d , j = 8 . 0 hz , 2h ), 7 . 35 ( d , j = 8 . 0 hz , 2h ). 31 p nmr ( 101 mhz , d 2 o ): δ 55 . 7 . 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 7 and 22 . 8 ( 2d , j = 89 hz ), 24 . 1 , 31 . 4 , 36 . 7 , 54 . 5 ( d , j = 14 hz ), 73 . 5 ( d , j = 109 hz ), 128 . 7 , 129 . 9 , 135 . 8 , 142 . 3 , 172 . 7 , 179 . 3 . ms ( esi ): m / z 346 . 0 ( m + 1 ), 344 . 0 ( m − 1 ). hplc - ms ( method a ): t r = 9 . 27 min . the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 304 mg , 1 . 0 mmol ) and 4 -( 2 - hydroxyethoxy ) benzaldehyde ( 353 mg , 2 . 2 mmol ). 773 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 od ): δ 49 . 2 . the previous compound was deprotected according to general procedure d . the purification on anion exchange column was performed with 73 mg of crude product on a 21 × 1 cm column . the desired product was eluted with a 2 . 5 × 10 − 2 n hydrochloric acid aqueous solution . 49 . 0 mg of pure derivative 13 were obtained ( 15 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 78 - 1 . 87 ( m , 2h ), 2 . 06 - 2 . 09 ( m , 2h ), 3 . 87 ( m , 2h ), 4 . 03 ( q , j = 5 . 0 hz , 1h ), 4 . 11 ( m , 2h ), 4 . 88 ( d , j = 7 . 5 hz , 1h ), 7 . 00 ( dd , j = 2 . 5 / 8 . 0 hz , 2h ), 7 . 35 ( dd , j = 3 . 0 / 8 . 0 hz , 2h ). 31 p nmr ( 101 mhz , d 2 o ): δ 55 . 1 . 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 9 and 23 . 0 ( 2d , j = 89 hz ), 24 . 2 , 54 . 6 ( d , j = 15 hz ), 61 . 6 , 70 . 8 , 73 . 2 ( d , j = 111 hz ), 116 . 3 , 130 . 0 , 130 . 7 , 159 . 6 , 172 . 9 . ms ( esi ): m / z 332 . 0 ( m − 1 ). hplc ( crownpak , t = 10 . 0 ° c ., detection λ = 210 / 254 nm ): t r = 9 . 9 min . hplc ( rp polar , detection λ = 230 / 270 nm ): t r = 8 . 7 min . to a solution of 4 - hydroxybenzaldehyde ( 152 mg , 1 . 25 mmol ) in dmf ( 20 ml ) was added potassium carbonate ( 223 mg , 1 . 62 mmol ), followed by diisopropyl bromomethylphosphonate ( 484 mg , 1 . 87 mmol ). the mixture was heating in dmf at 90 ° c . under stirring for 17 hours . after evaporation of dmf under reduced pressure , 100 ml of water were added and the mixture was extracted with ethyl acetate ( 3 * 200 ml ). the organic phase was washed with brine ( 100 ml ), dried over magnesium sulfate and concentrated under vacuum . the residue was purified by column chromatography over silica gel ( eluent : cyclohexane / ethyl acetate ( 5 : 5 )) to give 98 . 2 mg of an oil ( 26 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 33 ( t , j = 6 . 0 hz , 12h ), 4 . 25 ( d , j = 10 . 2 hz , 2h ), 4 . 82 ( q , j = 6 . 0 hz , 2h ), 7 . 05 ( d , j = 8 . 7 hz , 2h ), 7 . 81 ( d , j = 8 . 7 hz , 2h ), 9 . 87 ( s , 1h ). 13 c nmr ( 126 mhz , cdcl 3 ): δ 25 . 4 ( dd , j = 12 . 7 ; 3 . 4 hz ), 64 . 0 ( d , j = 171 . 0 hz ) 73 . 3 ( d , j = 6 . 2 hz ), 116 . 3 , 132 . 2 , 133 . 3 , 164 . 8 ( d , j = 13 . 4 hz ), 192 . 0 . 31 p nmr ( 202 mhz , cdcl 3 ): δ 16 . 0 the compound was prepared according to general procedure a with h - phosphinic acid ( 68 . 8 mg , 0 . 22 mmol ) and diisopropyl ( 4 - formylphenoxy ) methylphosphonate ( 98 . 2 mg , 0 . 327 mmol ). 115 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 od ): δ 47 . 7 ; 18 . 2 ms ( esi ): m / z 616 . 0 ( m + 1 ) the previous compound was deprotected according to general procedure c . after the cation exchange resin , 52 . 1 mg of compound were obtained . a second purification on an anion exchange resin ( ag 1 - x4 , 200 - 400 mesh , 10 . 5 × 1 . 5 cm , aco − ) was necessary to obtain a pure compound . the crude compound was diluted in 150 ml of pure and freshly boiled and cooled water , and the ph was brought to 9 ( with a freshly prepared 1n sodium hydroxide aqueous solution ). the resin was eluted with formic acid ( 1 . 8n to 3 . 5n ) and the desired compound was eluted with 3n formic acid . 14 . 6 mg of pure derivative 14 were obtained ( 17 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 62 - 1 . 77 ( m , 2h ), 2 . 01 - 2 . 09 ( m , 2h ), 3 . 9 ( m , 1h ), 4 . 12 ( d , j = 9 . 2 hz , 2h ), 4 . 76 ( d , j = 7 . 6 hz , 1h ), 7 . 02 ( d , j = 7 . 0 hz , 2h ), 7 . 34 ( d , j = 7 . 0 hz , 2h ). 31 p nmr ( 202 mhz , d 2 o ): δ 39 . 4 ; 14 . 7 . 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 4 ( dd , j = 89 . 6 ; 16 . 4 hz ), 24 . 9 , 55 . 6 , 65 . 6 ( d , j = 158 . 3 hz ), 73 . 9 ( d , j = 109 . 3 hz ), 116 . 1 , 129 . 8 , 132 . 0 , 160 . 0 ( d , j = 12 . 2 hz ), 174 . 1 . hplc - ms ( method a ): t r = 5 . 14 , ( esi ): m / z 383 . 9 ( m + 1 ) m / z 382 . 0 ( m − 1 ) the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 315 mg , 1 . 0 mmol ) and ethyl 3 - formylphenoxyacetate ( 458 mg , 2 . 2 mmol ). the reaction had to be stirred for 48 h to achieve complete conversion . 31 p nmr ( 101 mhz , cd 3 od ): δ 57 . 8 . the previous compound was deprotected according to general procedure c . 229 mg of pure product derivative 15 were obtained ( 66 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 79 - 1 . 93 ( m , 2h ), 2 . 05 - 2 . 12 ( m , 2h ), 4 . 05 ( q , j = 6 . 0 hz , 1h ), 4 . 94 ( d , j = 9 . 5 hz , 1h ), 6 . 92 ( d , j = 8 . 0 hz , 1h ), 6 . 99 ( d , j = 1 . 5 hz , 1h ), 7 . 05 ( d , j = 8 . 0 hz , 1h ), 7 . 33 ( t , j = 8 . 0 hz , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 56 . 6 . 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 6 ( 2d , j = 89 hz ), 23 . 9 , 54 . 4 ( d , j = 15 hz ), 66 . 2 , 73 . 2 ( d , j = 109 hz ), 114 . 5 , 115 . 8 , 122 . 0 , 131 . 4 , 139 . 6 , 158 . 6 , 172 . 5 , 174 . 6 . ms ( esi ): m / z 346 . 1 ( m − 1 ). hplc ( crownpak , t = 10 . 0 ° c ., detection λ = 210 / 254 nm ): t r = 20 . 6 and 22 . 9 min . the diastereoisomers of derivative 15 were separated by hplc using the preparative crownpak column at 4 . 0 ° c . with a 1 . 0 ml · min − 1 flow , a 2 ml injection loop , and a dual uv detection at 210 and 254 nm . 21 injections were performed in order to obtain enough product for pharmacological tests . each injection was prepared with 2 . 5 mg of derivative 15 in 1 . 5 ml of ph 2 . 0 hydrochloric acid . the diasteroisomer with the shortest retention time was named - i and the other one - ii . 18 mg of pure dia - i derivative 16 and 22 mg of pure dia - ii derivative 17 were obtained . derivative 16 - dial : 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 78 ( m , 2h ), 2 . 08 ( m , 2h ), 4 . 03 ( bs , 1h ), 4 . 76 ( s , 2h ), 4 . 88 ( d , j = 9 . 5 hz , 1h ), 6 . 93 ( d , j = 7 . 5 hz , 1h ), 7 . 02 ( bs , 1h ), 7 . 08 ( d , j = 7 . 0 hz , 1h ), 7 . 35 ( dd , j = 7 . 5 / 8 . 0 hz , 1h ). 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 9 ( d , j = 88 hz ), 24 . 1 , 54 . 6 ( d , j = 14 hz ), 66 . 4 , 73 . 6 ( d , j = 108 hz ), 114 . 5 , 115 . 7 , 122 . 1 , 131 . 4 , 140 . 2 , 158 . 6 , 172 . 8 , 174 . 8 . ms ( esi ): m / z 346 . 1 ( m − 1 ). hplc ( crownpak , t = 4 . 0 ° c ., detection λ = 210 / 254 nm ): t r = 21 . 3 min . [ α ] d 20 : + 3 . 3 ( h 2 o , c 0 . 9 ). derivative 17 - diaii : 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 73 - 1 . 88 ( m , 2h ), 2 . 08 - 2 . 13 ( m , 2h ), 4 . 03 ( bs , 1h ), 4 . 76 ( s , 2h ), 4 . 91 ( d , j = 7 . 5 hz , 1h ), 6 . 93 ( d , j = 6 . 0 hz , 1h ), 7 . 01 ( bs , 1h ), 7 . 07 ( d , j = 4 . 0 hz , 1h ), 7 . 35 ( bs , 1h ). 13 c nmr ( 126 mhz , d 2 o ): δ 22 . 8 ( d , j = 89 hz ), 24 . 0 , 54 . 6 , 66 . 3 , 73 . 9 , 114 . 5 , 115 . 8 , 122 . 1 , 131 . 4 , 139 . 9 , 158 . 6 , 172 . 7 , 174 . 7 . ms ( esi ): m / z 346 . 1 ( m − 1 ). hplc ( crownpak , t = 4 . 0 ° c ., detection λ = 210 / 254 nm ): t r = 23 . 9 min . [ α ] d 20 : + 17 . 5 ( h 2 o , c 1 . 1 ). to a suspension of 3 , 4 - dihydroxybenzaldehyde ( 691 mg , 5 . 0 mmol ) in dimethylformamide ( 10 . 0 ml ) was added potassium carbonate ( 2 . 76 g , 4 eq ). this mixture was stirred for 30 min at room temperature then cooled at 0 ° c . ethylbromoacetate ( 1 . 66 ml , 15 . 0 mmol ) was added , and the mixture was allowed to warm at room temperature and stirred for 19 h . the mixture was then diluted in water ( 50 ml ) and extracted with ethyl acetate ( 2 × 150 ml ). the combined organic phases were washed with brine ( 20 ml ), dried with magnesium sulfate , filtered and concentrated under vacuum . a brown oil was obtained and filtered on a silica gel layer , using cyclohexane / ethyl acetate ( 1 : 1 , 250 ml ). the filtrates containing the desired product were concentrated under vacuum . this orange oil , still containing traces of dimethylformamide , was directly engaged in the following step . a sample of crude 3 , 4 - bis ( ethoxycarbonylmethoxy ) benzaldehyde was purified by silica gel chromatography ( eluant cyclohexane / ethyl acetate in gradient from 3 : 1 to 6 : 4 ) to afford pure compound that was used for the nmr characterization . 1 h nmr ( 500 mhz , cdcl 3 ): δ 1 . 23 ( 2t , j = 7 . 5 hz , 6h ), 4 . 20 ( q , j = 7 . 5 hz , 4h ), 4 . 72 and 4 . 75 ( 2s , 4h ), 6 . 89 ( d , j = 8 . 0 hz , 1h ), 7 . 33 ( d , j = 1 . 5 hz , 1h ), 7 . 41 ( dd , j = 1 . 5 / 8 . 0 hz , 1h ), 9 . 77 ( s , 1h ). 13 c nmr ( 126 mhz , cdcl 3 ): δ 15 . 5 , 62 . 8 and 62 . 9 , 67 . 5 and 67 . 6 , 114 . 5 and 115 . 0 , 128 . 1 , 132 . 4 , 149 . 6 , 154 . 4 , 169 . 4 and 169 . 7 , 191 . 8 . tlc : r f = 0 . 5 ( cyclohexane / ethyl acetate 1 : 1 ). this compound was synthesized according to general procedure b with 1 ( 282 mg , 0 . 9 mmol ) and the previously synthesized 3 , 4 - bis ( ethoxycarbonylmethoxy ) benzaldehyde . 31 p nmr ( 101 mhz , cd 3 od ): δ 48 . 9 . the deprotection was achieved according to general procedure c . two cation exchange columns were necessary to obtain the pure compound . 51 mg of a pure fraction of derivative 18 were obtained ( 13 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 73 - 1 . 86 ( m , 2h ), 2 . 03 - 2 . 09 ( m , 2h ), 4 . 03 ( q , j = 6 . 0 hz , 1h ), 4 . 79 and 4 . 80 ( 2s , 4h ), 4 . 85 ( d , j = 9 . 0 hz , 1h ), 6 . 98 ( d , j = 8 . 5 hz , 1h ), 7 . 0 - 7 . 04 ( m , 2h ). 31 p nmr ( 101 mhz , d 2 o ): δ 53 . 0 . 13 c nmr ( 126 mhz , d 3 o ): δ 23 . 0 and 23 . 1 ( 2d , j = 89 hz ), 24 . 3 , 54 . 7 ( d , j = 15 hz ), 67 . 1 , 73 . 4 ( d , j = 109 hz ), 114 . 2 , 115 . 5 , 122 . 6 , 132 . 7 , 148 . 0 and 148 . 1 , 173 . 0 , 174 . 6 . hplc - ms ( method a ): t r = 6 . 25 ( esi ): m / z 422 . 0 ( m + 1 ) m / z 420 . 0 ( m − 1 ). the compound was prepared according to general procedure e with 5 - chlorovanillin ( 2 . 7 mmol ) in dry acetone ( 25 ml ), anhydrous k 2 co 3 ( 3 . 5 mmol ), and ethylbromoacetate ( 4 . 05 mmol ) at reflux for 1 h30 . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . 957 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane ). 330 mg of purified compound were obtained ( 45 % yield ). 1 h nmr ( 250 mhz , cd 3 cocd 3 ): δ 1 . 27 ( t , j = 7 . 2 hz , 3h ), 3 . 98 ( s , 3h ), 4 . 22 ( q , j = 7 . 2 hz , 2h ), 4 . 87 ( s , 2h ), 7 . 50 ( s , 1h ), 7 . 58 ( s , 1h ), 9 . 90 ( s , 1h ) 13 c nmr ( 126 mhz , cd 3 cocd 3 ): δ 15 . 1 , 57 . 5 , 62 . 1 , 70 . 5 , 112 . 3 , 125 . 8 , 128 . 9 , 134 . 0 , 149 . 7 , 154 . 5 , 169 . 5 , 191 . 2 . ms ( esi ): m / z 272 . 9 and 274 . 9 [ m + h ] + . the compound was prepared according to general procedure e with 5 - iodovanillin ( 1 . 8 mmol ) in dry acetone ( 25 ml ), anhydrous k 2 co 3 ( 2 . 4 mmol ), and ethylbromoacetate ( 2 . 7 mmol ) at reflux for 1 h15 . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . 673 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane ). 320 mg of purified compound were obtained ( 49 % yield ). 1 h nmr ( 250 mhz , cd 3 cocd 3 ): δ 1 . 28 ( t , j = 7 . 2 hz , 3h ), 3 . 97 ( s , 3h ), 4 . 23 ( q , j = 7 . 2 hz , 2h ), 4 . 85 ( s , 2h ), 7 . 55 ( s , 1h ), 7 . 95 ( s , 1h ), 9 . 90 ( s , 1h ) 13 c nmr ( 126 mhz , cd 3 cocd 3 ): δ 15 . 2 , 57 . 4 , 62 . 1 , 70 . 5 , 92 . 4 , 113 . 9 , 135 . 1 , 135 . 5 , 153 . 1 , 169 . 4 , 191 . 1 . ms ( esi ): m / z 364 . 9 [ m + h ] + . the compound was prepared according to general procedure e with 3 - fluoro - 4 - hydroxy - 5 - methoxybenzaldehyde ( 1 . 8 mmol ) in dry acetone ( 20 ml ), anhydrous k 2 co 3 ( 2 . 4 mmol ), and ethylbromoacetate ( 2 . 7 mmol ) at reflux for 1 h . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . 395 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane ). 361 mg of purified compound were obtained ( 79 % yield ). 1 h nmr ( 500 mhz , cd 3 cocd 3 ): δ 1 . 25 ( t , j =, 3h ), 3 . 98 ( s , 3h ), 4 . 21 ( q , j =, 2h ), 4 . 89 ( s , 2h ), 7 . 34 ( dd , j = 10 . 3 and 1 . 6 hz , 1h ), 7 . 40 ( t , j = 1 . 6 hz , 1h ), 9 . 88 ( d , j = 1 . 6 hz , 1h ) 13 c nmr ( 126 mhz , cd 3 cocd 3 ): δ 15 . 0 , 57 . 6 , 62 . 1 , 70 . 3 , 110 . 0 , 111 . 8 ( d , j = 20 . 6 hz ), 133 . 1 ( d , j = 7 . 2 hz ), 141 . 8 ( d , j = 12 . 7 hz ), 154 . 5 ( d , j = 4 . 5 hz ), 156 . 0 ( d , j = 246 hz ), 169 . 7 , 191 . 3 . ms ( esi ): m / z 256 . 9 [ m + h ] + . the compound was prepared according to general procedure e with 3 , 5 - dichloro - 4 - hydroxybenzaldehyde ( 1 . 6 mmol ) in dry acetone ( 15 ml ), anhydrous k 2 co 3 ( 2 . 1 mmol ), and ethylbromoacetate ( 2 . 4 mmol ) at reflux for 1 h . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . 354 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane ). 264 mg of purified compound were obtained ( 60 % yield ). 1 h nmr ( 500 mhz , cd 3 cocd 3 ): δ 1 . 32 ( t , j = 7 . 2 hz , 3h ), 4 . 30 ( q , j = 7 . 2 hz , 2h ), 4 . 74 ( s , 2h ), 7 . 82 ( s , 2h ), 9 . 67 ( s , 1h ) 13 c nmr ( 126 mhz , cd 3 cocd 3 ): δ 15 . 0 , 62 . 3 , 70 . 8 , 131 . 2 , 131 . 4 , 135 . 4 , 156 . 4 , 168 . 4 , 190 . 5 the compound was prepared according to general procedure e with 3 , 5 - dimethyl - 4 - hydroxybenzaldehyde ( 3 . 3 mmol ) in dry acetone ( 20 ml ), anhydrous k 2 co 3 ( 4 . 3 mmol ), and ethylbromoacetate ( 5 mmol ) at reflux for 1 h . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . 774 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane ). 580 mg of purified compound were obtained ( 74 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 26 ( t , j = 7 . 2 hz , 3h ), 2 . 33 ( s , 6h ), 4 . 23 ( q , j = 7 . 2 hz , 2h ), 4 . 42 ( s , 2h ), 7 . 50 ( s , 2h ), 9 . 82 ( s , 1h ) 13 c nmr ( 126 mhz , cdcl 3 ): δ 15 . 2 , 17 . 4 , 62 . 2 , 70 . 1 , 131 . 6 , 132 . 8 , 133 . 8 , 161 . 7 , 169 . 5 , 192 . 1 the compound was prepared according to general procedure e with 4 - hydroxybenzaldehyde ( 1 . 2 mmol ) in dry dmf ( 20 ml ), anhydrous k 2 co 3 ( 1 . 6 mmol ), and diisopropyl bromomethyl phosphonate ( 1 . 9 mmol ) heating at 110 ° c . overnight . isopropanol was added and evaporated by azeotropic distillation with diisopropyl bromomethyl phosphonate . 418 mg of crude product were obtained and purified by silica gel chromatography ( eluent : cyclohexane / ethyl acetate 5 / 5 ). 98 mg of purified compound were obtained ( 26 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 32 ( d , j = 6 . 0 hz , 6h ), 1 . 34 ( d , j = 6 . 0 hz , 6h ), 4 . 27 ( d , j = 10 . 2 hz , 4 . 78 ( q , j = 6 . 0 hz , 1h ), 4 . 83 ( q , 6 . 0 hz , 1h ), 7 . 05 ( d , j = 8 . 7 hz , 2h ), 7 . 82 ( d , j = 8 . 7 hz , 2h ), 9 . 87 ( s , 1h ) 13 c nmr ( 126 mhz , cdcl 3 ): δ 25 . 3 , 25 . 3 , 25 . 4 , 25 . 5 ( 4 ch 3 isopropyl ), 63 . 7 ( d , j = 171 hz ), 73 . 2 , 73 . 3 ( 2ch isopropyl ), 116 . 3 , 132 . 2 , 133 . 3 , 164 . 8 , 192 . 0 . 31 p nmr ( 202 mhz , cdcl 3 ): δ 16 . 0 . ms ( esi ): m / z 300 . 9 [ m + h ] + . the compound was prepared according to general procedure e with 3 - ethoxy - 4 - hydroxybenzaldehyde ( 2 . 4 mmol ) in dry acetone ( 15 ml ), anhydrous k 2 co 3 ( 3 . 1 mmol ), and ethylbromoacetate ( 3 . 6 mmol ) at reflux for 1 h30 . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . 565 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane ). 385 mg of purified compound were obtained ( 63 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 31 ( t , j = 7 . 0 hz , 3h ), 1 . 49 ( t , j = 7 . 0 hz , 3h ), 4 . 18 ( q , j = 7 . 0 hz , 2h ), 4 . 31 ( q , j = 7 . 0 hz , 2h ), 4 . 79 ( s , 2h ), 6 . 91 ( d , j = 8 . 0 hz , 1h ), 7 . 42 ( d , j = 8 . 0 hz , 1h ), 7 . 45 ( s , 1h ) 9 . 86 ( s , 1h ) 13 c nmr ( 63 mhz , cdcl 3 ): δ 14 . 0 , 14 . 5 , 61 . 2 , 64 . 4 , 65 . 9 , 111 . 2 , 113 . 0 , 125 . 5 , 131 . 0 , 149 . 1 , 152 . 7 , 168 . 1 , 191 . 5 . ms ( esi ): m / z 253 . 0 [ m + h ] + . the compound was prepared according to general procedure e with 4 - hydroxy - 3 -( trifluoromethoxy ) benzaldehyde ( 1 . 5 mmol ) in dry acetone ( 15 ml ), anhydrous k 2 co 3 ( 1 . 9 mmol ), and ethylbromoacetate ( 2 . 2 mmol ) at reflux for 1 h . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . 311 mg of pure compound were obtained ( 73 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 22 ( t , j = 7 . 0 hz , 3h ), 4 . 19 ( q , j = 7 . 0 hz , 2h ), 4 . 76 ( s , 2h ), 6 . 99 ( d , j = 9 . 0 hz , 1h ), 7 . 72 ( d , j = 9 . 0 hz , 1h ), 7 . 74 ( s , 1h ) 9 . 82 ( s , 1h ) 13 c nmr ( 63 mhz , cdcl 3 ): δ 13 . 8 , 61 . 6 , 65 . 6 , 113 . 6 , 120 . 5 ( d , j = 259 hz ), 123 . 3 , 130 . 4 , 130 . 5 , 138 . 5 , 155 . 1 , 167 . 3 , 189 . 4 the compound was prepared according to general procedure e with 4 - hydroxy - 3 -( trifluoromethyl ) benzaldehyde ( 1 . 3 mmol ) in dry acetone ( 20 ml ), anhydrous k 2 co 3 ( 1 . 7 mmol ), and ethylbromoacetate ( 2 . 2 mmol ) at reflux for 1 h . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . the crude product was purified by silica gel chromatography ( eluent dichloromethane ). 215 mg of purified compound were obtained ( 59 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 30 ( t , j = 7 . 0 hz , 3h ), 4 . 28 ( q , j = 7 . 2 hz , 2h ), 4 . 85 ( s , 2h ), 7 . 02 ( d , j = 8 . 7 hz , 1h ), 8 . 04 ( dd , j = 8 . 9 and 2 . 0 hz , 1h ), 8 . 16 ( d , j = 2 . 0 hz , 1h ) 9 . 95 ( s , 1h ) 13 c nmr ( 63 mhz , cdcl 3 ): δ 13 . 8 , 61 . 6 , 65 . 5 , 112 . 9 , 120 . 5 ( d , j = 32 . 0 hz ), 122 . 8 ( d , j = 273 hz ), 128 . 9 , 129 . 6 , 135 . 0 , 160 . 1 , 167 . 2 , 189 . 6 ms ( esi ): m / z 573 . 1 [ 2m + na ] + . the compound was prepared according to general procedure e with vanillin ( 5 . 3 mmol ) in dry acetone ( 20 ml ), anhydrous k 2 co 3 ( 6 . 8 mmol ), and ethylbromoacetate ( 6 . 3 mmol ) at reflux for 1 h . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . 980 mg of pure compound were obtained ( 78 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 23 ( m , 3h ), 3 . 88 ( s , 3h ), 4 . 21 ( m , 2h ), 4 . 78 ( s , 2h ), 6 . 90 ( m , 1h ), 7 . 38 ( m 2h ), 9 . 80 ( s , 1h ) 13 c nmr ( 63 mhz , cdcl 3 ): δ 13 . 9 , 55 . 7 , 61 . 2 , 65 . 6 , 109 . 8 , 112 . 3 , 125 . 7 , 130 . 8 , 149 . 7 , 152 . 4 , 167 . 9 , 190 . 6 ms ( esi ): m / z 253 . 0 [ m + h ] + . the compound was prepared according to general procedure e with 4 - hydroxybenzaldehyde ( 3 . 3 mmol ) in dry acetone ( 15 ml ), anhydrous k 2 co 3 ( 4 . 3 mmol ), and ethyl - 2 bromopropionate ( 3 . 6 mmol ) at reflux for 2 h45 . isopropanol was added and evaporated by azeotropic distillation with ethyl - 2 - bromopropionate . 609 mg of pure compound were obtained ( 83 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 10 ( t , j = 7 . 1 hz , 3h ), 1 . 52 ( d , j = 6 . 8 hz , 3h ), 4 . 10 ( q , j = 7 . 1 hz , 2h ), 4 . 76 ( q , j = 6 . 8 hz , 1h ) 6 . 85 ( d , j = 8 . 7 hz 2h ), 7 . 70 ( d , j = 8 . 9 hz , 2h ), 9 . 74 ( s , 1h ) 13 c nmr ( 63 mhz , cdcl 3 ): δ 14 . 0 , 18 . 2 , 61 . 4 , 72 . 4 , 115 . 1 , 130 . 4 , 131 . 8 , 162 . 4 , 171 . 1 , 190 . 5 the compound was prepared according to general procedure e with 3 , 4 dihydroxybenzaldehyde ( 3 . 7 mmol ) in dry acetone ( 20 ml ), anhydrous k 2 co 3 ( 2 . 3 mmol ), and ethylbromoacetate ( 3 . 4 mmol ) at 0 °. the mixture was stirred at room temperature for 20 h . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate the crude product was purified by silica gel chromatography ( eluent dichloromethane ). 112 mg of purified compound were obtained ( 15 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 31 ( t , j = 7 . 1 hz , 3h ), 4 . 28 ( q , j = 7 . 1 hz , 2h ), 4 . 78 ( q , j = 6 . 8 hz , 1h ) 6 . 95 ( d , j = 8 . 8 hz , 1h ), 7 . 46 ( d , j = 8 . 9 hz , 1h ), 7 . 55 ( s , 1h ), 9 . 86 ( s , 1h ) 13 c nmr ( 63 mhz , cdcl 3 ): δ 15 . 2 , 63 . 5 , 67 . 1 , 113 . 9 , 117 . 1 , 125 . 3 , 132 . 8 , 148 . 6 , 152 . 4 , 170 . 8 , 193 . 3 the compound was prepared according to general procedure e with syringaldehyde ( 2 . 1 mmol ) in dry acetone ( 10 ml ), anhydrous k 2 co 3 ( 2 . 7 mmol ), and ethylbromoacetate ( 2 . 5 mmol ) at reflux for 4 h . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . the crude product was purified by silica gel chromatography ( eluent dichloromethane ). 221 mg of purified compound were obtained ( 31 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 30 ( t , j = 7 . 2 hz , 3h ), 4 . 24 ( q , j = 7 . 4 hz , 2h ), 4 . 81 ( s , 2h ), 4 . 84 ( s , 2h ), 6 . 98 ( d , j = 8 . 7 hz , 1h ), 8 . 0 ( dd , j = 8 . 9 and 1 . 9 hz , 1h ), 8 . 44 ( d , j = 1 . 9 hz , 1h ), 9 . 91 ( s , 1h ) 13 c nmr ( 126 mhz , cdcl 3 ): δ 15 . 3 , 62 . 7 , 67 . 2 , 115 . 0 , 121 . 3 , 131 . 2 , 135 . 9 , 135 . 9 ( 2c ), 163 . 3 , 165 . 0 , 168 . 9 , 191 . 2 to a solution of ethyl - 2 - mercaptoacetate ( 0 . 7 mmol ) in dry dmf ( 2 . 5 ml ), sodium hydride ( 0 . 85 mmol ) was added 0 ° c . after 15 minutes , 4 - fluoro - 3 - nitrobenzaldehyde ( 0 . 7 mmol ) was added and the mixture was heated at 60 ° c . overnight and monitored by tlc until completion . after cooling , the solvent was removed under reduced pressure . the residue was chromatogaphed on a silica gel column ( cyclogexane / ethyl acetate 4 / 1 as eluent ) to give 46 mg of ethyl 2 -( 4 - formyl - 2 - nitrophenylthio ) acetate as yellow compound ( yield 24 %). 1 h nmr , ( 250 mhz , cdcl 3 ): δ 1 . 25 ( t , j = 7 . 5 hz , 3h ), 3 . 80 ( s , 2h ), 4 . 21 ( q , j = 7 . 75 , 2h ), 7 . 64 ( d , j = 9 . 75 , 1h ), 8 . 03 ( d , j = 9 . 75 , 1h ), 8 . 68 ( s , 1h ), 10 . 00 ( s , 1h ) 13 c nmr ( 126 mhz , cdcl 3 ): δ 15 . 4 , 36 . 5 , 63 . 8 , 128 . 5 , 129 . 3 , 133 . 9 , 145 . 6 , 147 . 1 , 169 . 5 , 190 . 1 . the compound was prepared according to general procedure e with 4 - hydroxy - 3 - nitrobenzaldehyde ( 1 . 8 mmol ) in dry dmf ( 15 ml ), anhydrous k 2 co 3 ( 2 . 2 mmol ), and ethylbromoacetate ( 2 . 2 mmol ) at 80 ° c . overnight . ethanol was added and evaporated by azeotropic distillation with ethylbromoacetate . the crude product was purified by silica gel chromatography ( eluent cyclohexane / ethylacetate 9 / 1 ). 166 mg of purified compound were obtained ( 37 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 28 ( t , j = 7 . 25 , 3h ), 4 . 26 ( q , 2h ), 4 . 87 ( s , 2h ), 7 . 07 ( d , j = 9 . 75 , 1h ), 8 . 04 ( d , j = 9 . 75 , 1h ), 8 . 06 ( s , 1h ), 9 . 93 ( s , 1h ) the compound was prepared according to general procedure e . to a solution of ethyl - 2 - mercaptoacetate ( 1 . 7 mmol ) in dry dmf ( 5 ml ), sodium hydride ( 2 . 0 mmol ) was added at 0 ° c . after 10 minutes , 4 - bromobenzaldehyde ( 1 . 4 mmol ) was added and the mixture was heated for 3 hours . after cooling , the solvent was removed under reduced pressure . the residue was chromatogaphed on a silica gel column ( cyclohexane / ethyl acetate 9 / 1 as eluent ) to give 188 mg of ethyl 2 -( 4 - formylphenythio ) acetate ( yield 59 %). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 23 ( t , j = 7 . 5 hz , 3h ), 3 . 73 ( s , 2h ), 4 . 18 ( q , 2h ), 7 . 42 ( d , j = 8 . 75 hz , 2h ), 7 . 76 ( d , j = 8 . 75 hz , 2h ), 9 . 93 ( s , 1h ). the compound was prepared according to general procedure e . to a solution of p - toluidine ( 4 . 7 mmol ) in dry acetone ( 20 ml ), ethylbromoacetate ( 4 . 7 mmol ) was added . the mixture was refluxed for 3 hours . after cooling , the solvent was removed under reduced pressure . the residue was chromatogaphed on a silica gel column ( dichloromethane / ethyl acetate 9 / 1 as eluent ) to give 326 mg of ethyl 2 -( p - tolylamino ) acetate ( yield 36 %). 1 h nmr ( 500 mhz , cdcl 3 ): δ 1 . 29 ( t , j = 7 . 4 hz , 3h ), 2 . 28 ( s , 3h ), 3 . 90 ( s , 2h ), 4 . 26 ( q , j = 7 . 4 hz , 2h ), 6 . 58 ( d , j = 8 . 2 hz , 2h ), 7 . 05 ( d , j = 8 . 2 , 2h ) 13 c nmr ( 126 mhz , cdcl 3 ): δ 15 . 6 , 21 . 8 , 47 . 7 , 62 . 5 , 114 . 8 , 131 . 2 , 146 . 3 , 172 . 8 . ms ( esi ): m / z 193 . 9 [ m + h ] + to a solution of ethyl 2 -( p - tolylamino ) acetate ( 1 . 69 mmol ) in methanol / water ( 20 ml / 20 ml ) was added the 2 , 3 dichloro - 5 , 6 - dicyanobenzoquinone ( 2 . 87 mmol ) and the mixture was stirred for 4 hours at room temperature . the residue was chromatographed on a silica gel column ( dichloromethane / ethyl acetate 8 / 2 as eluent ) to give 88 mg of ethyl 2 -( 4 - formylphenylamino ) acetate . ( yield 25 %). ms ( esi ): m / z 208 . 0 [ m + h ] + a stirring solution of 2 , 6 - difluorophenol ( 2 g , 15 . 4 mmol ) and hexamethylenetetramine ( 2 . 16 g , 15 . 4 mmol ) in tfa ( 16 ml ) was heated at reflux under argon overnight . on cooling to room temperature the solvent was evaporated in vacuum and the crude residue was taken up in dcm ( 35 ml ). the mixture was washed with an aqueous solution of nahco 3 ( sat .) and the separated aqueous layer acidified to ph 1 with concentrated hcl . the aqueous layer was extracted with dcm ( 2 × 25 ml ), the combined organic fractions dried ( mgso 4 ) and evaporated in vacuum to afford desired product as a cream solid ( 1 . 76 g , 73 %). rf = 0 . 39 ( sio 2 ; cyclohexane / etoac ( 6 / 4 ); uv ) 1 h nmr ( 300 mhz ; cdcl 3 ) δ : 6 . 38 ( br , 1h ); 7 . 50 ( d , 2h , j = 6 . 6 hz ); 9 . 82 ( s , 1h ). 13 c nmr ( 75 mhz ; cdcl 3 ) δ : 113 . 2 ( dd , j cf = 13 . 8 hz and 7 . 8 hz ); 128 . 2 ( t , j cf = 6 . 0 hz ); 139 . 0 ( t , j cf = 16 . 1 hz ); 151 . 9 ( dd , j cf = 246 . 9 hz and 5 . 3 hz ); 189 . 1 . 19 f nmr ( 282 mhz ; cdcl 3 ) δ : − 52 . 2 656 mg ( 4 . 74 mmol ) of k 2 co 3 and 0 . 44 ml ( 3 . 95 mmol ) of ethyl 2 - bromoacetate were added to a solution of 3 , 5 - difluoro - 4 - hydroxybenzaldehyde ( 0 . 5 g , 3 . 16 mmol ) in 40 ml of acetone . the reaction mixture was refluxed under argon during 1 hour . after cooling , acetone was evaporated under vacuum . the mixture was diluted in 40 ml of dcm and washed with brine ( 3 ×). the combined organic phase was dried under mgso 4 . evaporation of the solvent and chromatography of the oily residue [ sio 2 , cyclochexane / dcm ( 8 / 2 )] afforded a yellowish oil ( 580 mg , 75 %). rf = 0 . 23 ( sio 2 ; cyclohexane / dcm : 1 / 1 ; uv ). 1 h nmr ( 250 mhz ; cdcl 3 ) δ ( ppm ): 1 . 31 ( t , 3h , j = 7 . 5 hz ); 4 . 28 ( q , 2h , j = 7 . 5 hz ); 4 . 92 ( s , 2h ); 7 . 48 ( d , 2h , j = 8 . 75 hz ); 9 . 86 ( s , 1h ). 13 c nmr ( 125 mhz ; cdcl 3 ) δ ( ppm ): 15 . 5 ; 63 . 1 ; 70 . 6 ; 114 . 9 ( dd , j cf = 17 . 6 hz and 6 . 0 hz ); 132 . 1 ( t , j cf = 7 . 0 hz ); 141 . 3 ( t , j cf = 13 . 6 hz ); 156 . 3 ( dd , j cf = 251 . 1 hz and 5 . 6 hz ); 169 . 4 ; 190 . 1 . 19 f nmr ( 282 mhz ; cdcl 3 ) δ ( ppm ): − 50 . 9 . the compound was prepared according to general procedure f with ethyl 2 -( 2 - chloro - 4 - formyl - 6 - methoxyphenoxy ) acetate ( 1 . 5 mmol ) in thf ( 20 ml ) and water ( 20 ml ) and nabh 4 ( 1 . 5 mmol ). 355 mg of compound were obtained . this compound was used without further purification . 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 31 ( t , j = 7 . 1 hz , 3h ), 3 . 86 ( s , 3h ), 4 . 25 ( q , j = 7 . 1 hz , 2h ), 4 . 55 ( s , 2h ), 4 . 60 ( s , 2h ) 6 . 95 ( s , 1h ), 6 . 98 ( s , 1h ). ms ( esi ): m / z 274 . 9 and 276 . 9 [ m + h ] + , 570 . 7 and 572 . 7 [ 2m + na ] + the compound was prepared according to general procedure f with ethyl 2 -( 2 - fluoro - 4 - formyl - 6 - methoxyphenoxy ) acetate ( 1 . 4 mmol ) in thf ( 20 ml ) and water ( 20 ml ) and nabh 4 ( 1 . 4 mmol ). 220 mg of compound were obtained . this compound was used without further purification . 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 33 ( t , j = 7 . 1 hz , 3h ), 3 . 90 ( s , 3h ), 4 . 28 ( q , j = 7 . 1 hz , 2h ), 4 . 65 ( s , 2h ), 4 . 71 ( s , 2h ) 6 . 95 ( s , 1h ), 6 . 76 ( m , 2h ). ms ( esi ): m / z 538 . 8 [ 2m + na ] + the compound was prepared according to general procedure f with ethyl 2 -( 4 - formyl - 2 -( trifluoromethoxyphenoxy )) acetate ( 2 . 5 mmol ) in thf ( 40 ml ) and water ( 40 ml ) and nabh 4 ( 2 . 7 mmol ). 526 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane ). 140 mg of purified compound were obtained ( 19 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 26 ( t , j = 7 . 1 hz , 3h ), 2 . 97 ( s , 1h , oh ), 4 . 22 ( q , j = 7 . 2 hz , 2h ), 4 . 54 ( s , 2h ), 4 . 67 ( s , 2h ) 6 . 85 ( d , j = 8 . 6 hz , 1h ), 7 . 15 ( d , j = 8 . 3 hz , 1h ), 7 . 24 ( s , 1h ).) 13 c nmr ( 63 mhz , cdcl 3 ): δ 13 . 9 , 61 . 5 , 63 . 6 , 66 . 2 , 114 . 4 , 120 . 6 ( d , j = 257 hz ), 121 . 7 , 126 . 1 , 135 . 4 , 138 . 2 , 149 . 4 , 168 . 6 . ms ( esi ): m / z 610 . 7 [ 2m + na ] + the compound was prepared according to general procedure f with ethyl 2 -( 4 - formyl - 2 -( trifluoromethylphenoxy )) acetate ( 2 . 7 mmol ) in thf ( 40 ml ) and water ( 40 ml ) and nabh 4 ( 2 . 9 mmol ). 611 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane / ethyl acetate 9 : 1 ). 408 mg of purified compound were obtained ( 55 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 27 ( t , j = 7 . 0 hz , 3h ), 3 . 29 ( s , 1h , oh ), 4 . 24 ( q , j = 7 . 1 hz , 2h ), 4 . 53 ( s , 2h ), 4 . 67 ( s , 2h ) 6 . 81 ( d , j = 8 . 4 hz , 1h ), 7 . 37 ( d , j = 8 . 2 hz , 1h ), 7 . 53 ( s , 1h ).) 13 c nmr ( 63 mhz , cdcl 3 ): δ 13 . 9 , 61 . 6 , 63 . 7 , 65 . 8 , 113 . 0 , 119 . 6 ( q , j = 30 . 4 hz ) 123 . 4 ( d , j = 273 hz ), 126 . 1 ( q , j = 5 . 1 hz ), 131 . 7 , 134 . 1 , 155 . 0 , 168 . 4 . ms ( esi ): m / z 300 . 9 [ m + na ] + the compound was prepared according to general procedure f with ethyl 2 -( 4 - formyl - 2 - methoxyphenoxy ) acetate ( 5 . 9 mmol ) in thf ( 40 ml ) and water ( 40 ml ) and nabh 4 ( 6 . 5 mmol ). 437 mg of crude product were obtained and purified by silica gel chromatography ( eluent dichloromethane ). 249 mg of purified compound were obtained ( 18 % yield ). 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 19 ( t , j = 7 . 0 hz , 3h ), 3 . 15 ( s , 1h , oh ), 3 . 74 ( s , 3h ) 4 . 15 ( q , j = 7 . 1 hz , 2h ), 4 . 46 ( s , 2h ), 4 . 55 ( s , 2h ) 6 . 70 ( s , 1h ), 6 . 71 ( s , 1h ), 6 . 83 ( s , 1h ).) 13 c nmr ( 63 mhz , cdcl 3 ): δ 14 . 1 , 55 . 7 , 61 . 2 , 64 . 5 , 66 . 5 , 111 . 0 , 114 . 3 , 118 . 9 , 135 . 7 , 146 . 4 , 149 . 6 , 169 . 1 . ms ( esi ): m / z 502 . 7 [ 2m + na ] + the compound was prepared according to general procedure g with ethyl 2 -( 2 - chloro - 4 -( hydroxymethyl )- 6 - methoxyphenoxy ) acetate ( 1 . 3 mmol ) in dichloromethane ( 12 ml ) and pbr 3 ( 1 . 3 mmol ). 338 mg of compound were obtained ( 78 % yield ). this compound was used without further purification . 1 h nmr ( 250 mhz , cd 3 od ): δ 1 . 29 ( m , 3h ), 3 . 83 ( s , 3h ), 4 . 15 ( m , 2h ), 4 . 55 ( s , 2h ), 4 . 94 ( s , 2h ) 6 . 80 ( d , j = 2 . 0 hz , 1h ), 6 . 85 ( d , j = 2 . 0 hz , 1h ). ms ( esi ): m / z 336 . 9 [ m − h ] − , 360 . 8 [ m + na ] + the compound was prepared according to general procedure g with ethyl 2 -( 2 - fluoro - 4 -( hydroxymethyl )- 6 - methoxyphenoxy ) acetate ( 0 . 4 mmol ) in dichloromethane ( 5 ml ) and pbr 3 ( 0 . 4 mmol ). 67 mg of compound were obtained ( 54 % yield ). this compound was used without further purification . 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 29 ( t , j = 7 . 0 hz , 3h ), 3 . 89 ( s , 3h ), 4 . 25 ( q , j = 7 . 0 hz , 2h ), 4 . 41 ( s , 2h ), 4 . 71 ( s , 2h ) 6 . 74 ( m , 1h ), 6 . 81 ( m , 1h ). ms ( esi ): m / z 319 . 9 [ m + h ] + the compound was prepared according to general procedure g with ethyl 2 -( 4 - hydroxymethyl - 2 -( trifluoromethoxyphenoxy )) acetate ( 0 . 5 mmol ) in dichloromethane ( 5 ml ) and pbr 3 ( 0 . 5 mmol ). 139 mg of compound were obtained ( 82 % yield ). this compound was used without further purification . 1 h nmr ( 250 mhz , cdcl 3 ): δ 1 . 28 ( t , j = 7 . 2 hz , 3h ), 4 . 26 ( q , j = 7 . 0 hz , 2h ), 4 . 44 ( s , 2h ), 4 . 69 ( s , 2h ), 7 . 25 ( d , j = 8 . 6 hz , 1h ), 7 . 27 ( d , j = 8 . 7 hz , 1h ), 7 . 32 ( s , 1h ). 13 c nmr ( 126 mhz , cdcl 3 ): δ 13 . 6 , 31 . 7 , 61 . 2 , 65 . 8 , 114 . 1 , 122 . 0 ( d , j = 258 hz ), 123 . 6 , 128 . 1 , 131 . 5 , 137 . 9 , 150 . 0 , 167 . 7 . ms ( esi ): m / z 378 . 9 ( 100 %), 379 . 8 ( 13 %), 380 . 9 ( 100 %), 381 . 9 ( 13 %) [ m + na ] + the compound was prepared according to general procedure g with ethyl 2 -( 4 - hydroxymethyl - 2 -( trifluoromethylphenoxy )) acetate ( 1 . 5 mmol ) in dichloromethane ( 10 ml ) and pbr 3 ( 1 . 5 mmol ). 421 mg of compound were obtained ( 82 % yield ). this compound was used without further purification . 1 h nmr ( 500 mhz , cdcl 3 ): δ 1 . 26 ( t , j = 7 . 1 hz , 3h ), 4 . 10 ( q , j = 7 . 1 hz , 2h ), 4 . 46 ( s , 2h ), 4 . 70 ( s , 2h ), 6 . 86 ( d , j = 8 . 6 hz , 1h ), 7 . 49 ( dd , j = 8 . 7 and 2 . 1 hz , 1h ), 7 . 62 ( d , j = 2 . 1 hz , 1h ). 13 c nmr ( 126 mhz , cdcl 3 ): δ 15 . 4 , 33 . 5 , 63 . 0 , 67 . 2 , 114 . 6 , 121 . 0 ( q , j = 32 . 0 hz ), 124 . 0 ( d , j = 272 . 0 hz ), 128 . 6 , 132 . 3 , 135 . 3 , 157 . 1 , 169 . 3 . the compound was prepared according to general procedure g with ethyl 2 -( 4 - hydroxymethyl - 2 - methoxyphenoxy )) acetate ( 1 . 0 mmol ) in dichloromethane ( 5 ml ) and pbr 3 ( 1 . 0 mmol ). 261 mg of compound were obtained ( 83 % yield ). this compound was used without further purification . 1 h nmr ( 500 mhz , cdcl 3 ): δ 1 . 23 ( t , j = 7 . 2 hz , 3h ), 3 . 81 ( s , 3h ) 4 . 22 ( q , j = 7 . 2 hz , 2h ), 4 . 42 ( s , 2h ), 4 . 62 ( s , 2h ), 6 . 71 ( d , j = 8 . 3 hz , 1h ), 6 . 84 ( dd , j = 8 . 3 and 1 . 9 hz , 1h ), 6 . 89 ( d , j = 1 . 9 hz , 1h ). 13 c nmr ( 126 mhz , cdcl 3 ): δ 14 . 2 , 34 . 0 , 55 . 9 , 61 . 3 , 66 . 4 , 112 . 9 , 114 . 0 , 121 . 4 131 . 9 , 147 . 4 , 149 . 6 , 168 . 7 ms ( esi ): m / z 324 . 9 ( 100 %), 325 . 9 ( 13 %), 326 . 8 ( 100 %), 327 . 9 ( 13 %) [ m + na ] + the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 1 . 5 mmol ), ethyl 2 -( 2 - ethoxy - 4 - formylphenoxy ) acetate ( 1 . 5 mmol ) and bsa ( 6 . 1 mmol ). 1 . 5 g of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cd 3 oh ): δ 47 . 9 . ms ( esi ): m / z 566 . 1 [ m − h ] − the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 1 . 0 mmol ), ethyl 2 -( 4 - formyl - 2 -( trifluoromethoxyphenoxy )) acetate ( 1 . 0 mmol ) and bsa ( 4 . 2 mmol ). 684 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cd 3 oh ): δ 47 . 9 . ms ( esi ): m / z 607 . 9 [ m + h ] + and 1214 . 9 [ 2m + h ] + the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 8 mmol ), ethyl 2 -( 4 - formyl - 2 -( trifluoromethylphenoxy )) acetate ( 0 . 8 mmol ) and bsa ( 3 . 12 mmol ). 219 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cdcl 3 ): δ 50 . 8 the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 6 mmol ), ethyl 2 -( 4 - formyl - 2 - hydroxyphenoxy ) acetate ( 0 . 6 mmol ) and bsa ( 2 . 4 mmol ). 308 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cdcl 3 ): δ 47 . 7 the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 7 mmol ), ethyl 3 -( 2 -( 2 - etoxy - 2 - oxo ethoxy )- 5 - formylphenyl )- 3 - oxopropanoate ( 0 . 7 mmol ) and bsa ( 2 . 8 mmol ). 467 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cdcl 3 ): δ 50 . 3 . ms ( esi ): m / z 652 . 1 [ m − h ] − the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 66 mmol ), 2 -( 4 - formyl - 2 - nitrophenoxy ) acetate ( 0 . 66 mmol ) and bsa ( 2 . 64 mmol ). 492 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cdcl 3 ): δ = 39 . 7 the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 17 mmol ), ethyl 2 -( 4 - formyl - 2 - nitrophenylthio ) acetate ( 0 . 17 mmol ) and bsa ( 0 . 68 mmol ). 140 mg of crude product were obtained and used without further purification . the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 84 mmol ), ethyl 2 -( 4 - formylphenythio ) acetate ( 0 . 84 mmol ) and bsa ( 3 . 36 mmol ). 294 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cdcl 3 ): δ = 41 . 9 the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 43 mmol ), ethyl 2 -( 4 - formylphenylamino ) acetate ( 0 . 43 mmol ) and bsa ( 1 . 7 mmol ). 367 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cdcl 3 ): δ = 48 . 0 the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 99 mmol ), ethyl 2 -( 4 - formylphenylamino ) acetate ( 0 . 99 mmol ) and bsa ( 3 . 96 mmol ). 552 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cdcl 3 ): δ = 50 . 0 the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 2 . 7 mmol ), ethyl 2 -( 4 - formylphenoxy ) propanoate ( 2 . 7 mmol ) and bsa ( 11 . 0 mmol ). 750 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , cdcl 3 ): δ 47 . 7 . ms ( esi ): m / z 536 . 1 [ m − h ] − the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 315 mg , 1 . 0 mmol ) and 4 - bromomethyl - phenoxyacetic acid ( 539 mg , 2 . 2 mmol ). 967 mg of crude product were obtained . 31 p nmr ( 101 mhz , cd 3 od ): δ 50 . 7 . the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 9 mmol ), ethyl 2 -( 4 -( bromomethyl )- 2 - methoxyphenoxy ) acetate ( 0 . 9 mmol ) and bsa ( 3 . 5 mmol ). 555 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , meod ): δ 53 . 0 . ms ( esi ): m / z 536 . 0 [ m − h ] − and 1072 . 9 [ 2m − h ] − the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 4 mmol ), ethyl 2 -( 4 -( bromomethyl )- 2 -( trifluoromethoxy ) phenoxy ) acetate ( 0 . 4 mmol ) and bsa ( 1 . 6 mmol ). 293 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , meod ): δ 51 . 2 . ms ( esi ): m / z 590 . 0 [ m − h ] − and 1180 . 9 [ 2m − h ] − the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 8 mmol ), ethyl 2 -( 4 -( bromomethyl )- 2 -( trifluoromethyl ) phenoxy ) acetate ( 0 . 8 mmol ) and bsa ( 3 . 2 mmol ). 555 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , meod ): δ 48 . 6 . ms ( esi ): m / z 574 . 0 [ m − h ] − and 1148 . 8 [ 2m − h ] − the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 0 . 6 mmol ), ethyl 2 -( 4 -( bromomethyl )- 2 - fluoro - 6 - methoxyphenoxy ) acetate ( 0 . 6 mmol ) and bsa ( 2 . 5 mmol ). 510 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , meod ): δ 48 . 5 the compound was prepared according to general procedure b with h - phosphinic acid 1 ( 1 mmol ), ethyl 2 -( 4 -( bromomethyl )- 2 - chloro - 6 - methoxyphenoxy ) acetate ( 1 mmol ) and bsa ( 4 mmol ). 307 mg of crude product were obtained and used without further purification . 31 p nmr ( 101 mhz , meod ): δ 41 . 9 . ms ( esi ): m / z 569 . 9 [ m − h ] − the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). another purification was made on an anion exchange chromatography ( dowex ag 1 - x4 , aco − , 200 - 400 mesh ). the resin was first eluted with freshly boiled and cooled water , then with formic acid 2 . 5m . 37 mg of pure derivative 19 were obtained ( 6 % yield in 2 steps )). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 34 ( t , j = 7 hz , 3h ), 1 . 77 ( m , 2h ), 2 . 08 ( m , 2h ), 4 . 03 ( m , 1h ), 4 . 08 ( q , j = 7 hz , 2h ), 4 . 70 ( s , 2h ), 4 . 88 ( d , j = 8 hz ), 6 . 92 ( m , 2h ), 7 . 07 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 44 . 4 . 13 c nmr ( 63 mhz , d 2 o ): δ 13 . 8 , 21 . 7 ( d , j = 123 hz ), 22 . 6 , 52 . 9 , 65 . 1 , 65 . 6 , 71 . 8 ( d , j = 110 hz ), 112 . 5 , 113 . 6 , 119 . 8 , 130 . 8 , 146 . 5 , 147 . 5 , 171 . 3 , 173 . 1 . ms ( esi ): m / z 383 . 9 [ m + h ] + , 391 . 9 [ m − h ] − . hplc ms t r = 8 . 27 min . the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution . 127 mg of pure derivative 20 were obtained ( 28 % yield in 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 67 ( m , 2h ), 2 . 06 ( m , 2h ), 3 . 90 ( m , 1h ), 4 . 80 ( m , 3h ), 7 . 06 ( d , j = 8 hz , 1h ), 7 . 33 ( d , j = 8 hz , 1h ), 7 . 40 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 38 . 4 . 13 c nmr ( 63 mhz , d 2 o ): δ 22 . 2 ( d , j = 85 hz ), 23 . 2 , 54 . 1 , 65 . 8 , 71 . 8 ( d , j = 109 hz ), 114 . 5 , 120 . 4 ( d , j = 258 hz ), 121 . 5 , 126 . 6 , 132 . 1 , 137 . 5 , 149 . 0 , 172 . 1 173 . 8 . ms ( esi ): m / z 431 . 9 [ m + h ] + , 430 . 0 [ m − h ] − . hplc ms t r = 12 . 52 min . the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). another purification was made on an anion exchange chromatography ( dowex ag 1 - x4 , aco − , 200 - 400 mesh ). the resin was first eluted with freshly boiled and cooled water , then with formic acid . 16 mg of pure derivative 21 were obtained ( 4 % yield in 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 73 ( m , 2h ), 2 . 08 ( m , 2h ), 3 . 99 ( q , j = 6 hz , 1h ), 4 . 82 ( s , 2h ), 4 . 84 ( d , j = 8 hz , 1h ), 7 . 07 ( d , j = 9 hz , 1h ), 7 . 57 ( d , j = 8 hz , 1h ), 7 . 68 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 39 . 1 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 1 ( d , j = 87 hz ), 24 . 6 , 55 . 3 , 67 . 3 , 73 . 3 ( d , j = 107 hz ), 115 . 1 , 119 . 5 ( d , j = 31 hz ), 125 . 0 ( d , j = 273 hz ), 127 . 3 , 132 . 4 , 133 . 7 , 156 . 3 , 173 . 5 , 174 . 5 . ms ( esi ): m / z 415 . 9 [ m + h ] + . hplc ms t r = 12 . 74 min . the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). another purification was made on an anion exchange chromatography ( dowex ag 1 - x4 , aco − , 200 - 400 mesh ). the resin was first eluted with freshly boiled and cooled water , then with formic acid 2 . 3m . 17 . 9 mg of pure derivative 22 were obtained ( 9 % yield in 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 72 ( m , 2h ), 2 . 04 ( m , 2h ), 3 . 95 ( t , j = 6 . 8 hz , 1h ), 4 . 72 ( s , 2h ), 4 . 73 ( d , j = 9 hz , 1h ), 6 . 89 ( m , 2h ), 6 . 96 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 40 . 3 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 5 ( d , j = 89 hz ), 24 . 6 ( d , j = 2 hz ), 55 . 2 ( d , j = 15 hz ), 67 . 4 , 73 . 8 ( d , j = 110 hz ) 115 . 1 , 116 . 2 , 120 . 8 , 127 . 5 , 133 . 4 , 146 . 4 , 146 . 7 , 173 . 6 , 175 . 2 . ms ( esi ): m / z 363 . 9 [ m + h ] + . hplc ms t r = 6 . 49 min . the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). another purification was made on an anion exchange chromatography ( dowex ag 1 - x4 , aco − , 200 - 400 mesh ). the resin was first eluted with freshly boiled and cooled water , then with formic acid 4m . 40 mg of pure derivative 23 were obtained ( 16 % yield in 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 71 ( m , 2h ), 2 . 06 ( m , 2h ), 3 . 96 ( q , j = 5 . 7 hz , 1h ), 4 . 80 ( s , 2h ), 4 . 82 ( d , j = 8 . 8 hz , 1h ), 7 . 04 ( d , j = 8 . 8 hz , 1h ), 7 . 56 ( d , j = 8 . 8 hz , 1h ), 7 . 82 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 39 . 4 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 6 ( d , j = 90 hz ), 24 . 6 , 55 . 1 , 67 . 6 , 73 . 3 ( d , j = 108 hz ), 115 . 5 , 120 . 8 , 131 . 6 , 132 . 9 , 134 . 6 , 157 . 7 , 171 . 1 , 173 . 5 , 174 . 7 ms ( esi ): m / z 392 . 0 [ m + h ] + . hplc ms t r = 6 . 93 min . the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). 75 mg of pure derivative 24 were obtained ( 29 % yield in 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 74 ( m , 2h ), 2 . 09 ( m , 2h ), 4 . 00 ( m , 1h ), 4 . 86 ( s , 2h ), 4 . 85 ( d , j = 8 . 5 hz , 1h ), 7 . 16 ( d , j = 9 . 25 , 1h ), 7 . 63 ( d , j = 9 . 25 , 1h ), 7 . 97 ( s , 1h ). 13 c nmr ( 63 mhz , d 2 o ): δ 22 . 2 ( d , j = 87 hz ), 23 . 3 , 53 . 5 , 66 . 3 , 71 . 5 ( d , j = 107 hz ), 115 . 5 , 124 . 4 , 132 . 0 , 133 . 7 , 138 . 9 , 150 . 5 , 171 . 8 , 172 . 6 . 31 p nmr ( 101 mhz , d 2 o ) δ 39 . 7 . ms ( esi ): m / z 393 . 0 [ m + h ] + . hplc ms : t r = 8 . 02 min the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). 17 mg of pure derivative 25 were obtained ( 24 % yield in 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 92 ( m , 2h ), 2 . 11 ( m , 2h ), 3 . 97 ( m , 3h ), 4 . 87 ( d , j = 9 . 75 , 1h ), 7 . 45 ( d , j = 7 . 5 , 1h ), 7 . 65 ( d , j = 7 . 75 , 1h ), 8 . 24 ( s , 1h ) 13 c nmr ( 500 mhz , d 2 o ): δ 23 . 8 ( d , j = 87 hz ), 250 , 36 . 3 , 55 . 4 , 73 . 2 ( d , j = 107 hz ), 125 . 7 , 128 . 7 , 134 . 3 , 135 . 8 , 138 . 7 , 147 . 5 , 173 . 7 , 174 . 9 . 31 p nmr ( 101 mhz , d 2 o ) δ 38 . 5 . ms ( esi ): m / z 408 . 9 . 0 [ m + h ] + . the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). 88 mg of pure derivative 26 were obtained ( 28 % yield in 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 70 ( m , 2h ), 2 . 06 ( m , 2h ), 3 . 78 ( s , 2h ,) 3 . 93 ( m , 1h ), 4 . 81 ( d , j = 9 . 5 hz , 1h ), 7 . 40 ( m , 4h ) 13 c nmr ( 250 mhz , d 2 o ): δ 21 . 5 ( d , j = 87 hz ), 21 . 6 , 34 . 6 , 52 . 0 , 71 . 2 ( d , j = 111 hz ), 125 . 1 , 126 . 2 , 128 . 2 , 132 . 6 , 135 . 6 , 139 . 9 , 170 . 8 , 172 . 8 . 31 p nmr ( 101 mhz , d 2 o ): δ = 41 . 92 . ms ( esi ): m / z 363 . 9 [ m + h ] + . hplc - ms : t r = 9 . 38 min a solution of (( s )- 3 -((( benzyloxy ) carbonyl ) amino )- 4 - methoxy - 4 - oxobutyl )(( 4 -( 2 - ethoxy - 2 - oxoethoxy )- 3 , 5 - difluorophenyl )( hydroxy ) methyl ) phosphinic acid ( 552 mg , 0 . 99 mmol ) in 6 n hcl ( 6m ) was refluxed during 3 h . then , the mixture was cooled to room temperature and the solvent was evaporated under vacuum . the residue was diluted in etoac ( 50 ml ) and extracted with 1 n hcl ( 2 × 80 ml ). the combined aqueous layers were washed twice with etoac and the solvent was evaporated to afford 238 mg ( 0 . 62 mmol ) of crude product which was purified by cation ( and anion ) exchange resin chromatography . cation exchange resin chromatography : 238 mg of crude compound were deposited on a dowex ag 50w - x4 , h + , 50 - 100 mesh . the compound was eluted with water and 85 mg of desired product were collected . anion exchange resin chromatography : 85 mg of compound were deposited on a dowex ag 50w - x4 , aco − , 200 - 400 mesh . the compound was eluted with boiled water and formic acid . 27 mg ( yield 7 % on 2 steps ) of pure product derivative 28 were collected ( 2 m hcooh ). 1 h nmr ( 250 mhz ; d 2 o ) δ ( ppm ): 1 . 87 ( m , 2h ); 2 . 17 ( m , 2h ); 4 . 13 ( m , 1h ); 4 . 87 ( s , 2h ); 4 . 94 ( d , j hp = 9 . 4 hz , 1h ); 7 . 13 ( d , j = 9 . 1 hz , 2h ). 13 c nmr ( 63 mhz ; d 2 o ) δ ( ppm ): 21 . 7 ( d , jcp = 91 . 0 hz ); 22 . 8 ; 53 . 1 ( d , jcp = 14 . 8 hz ); 69 . 8 ; 71 . 3 ( d , jcp = 109 . 5 hz ); 110 . 8 ( dd , j cf = 23 . 3 hz and 4 . 0 hz ); 133 . 2 ( t , jcf = 14 . 0 hz ); 134 . 0 ( t , jcf = 8 . 1 hz ); 154 . 7 ( dd , j cf = 246 . 6 hz and 5 . 4 hz ); 171 . 4 ; 172 . 9 . 31 p nmr ( 200 mhz ; d 2 o ) δ ( ppm ): 41 . 5 ( s ). 19 f nmr ( 376 mhz : d 2 o ) δ ( ppm ): − 129 . 0 ( s ) ms ( esi ) m / z : 384 [ m + h + ]. hplc : t r = 10 . 1 min the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( fr , 50 - 100 mesh , water elution ). 259 mg of pure derivative 29 were obtained ( 26 % yield in 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 52 ( d , j = 7 hz , 3h ), 1 . 69 ( m , 2h ), 2 . 04 ( m , 2h ), 3 . 93 ( m , 1h ), 4 . 80 ( d , j = 8 hz , 1h ), 4 . 85 ( q , j = 7 hz , 1h ), 6 . 89 ( d , j = 9 hz , 2h ), 7 . 30 ( d , j = 9 hz , 2h ). 31 p nmr ( 101 mhz , d 2 o ): δ 40 . 8 13 c nmr ( 126 mhz , d 2 o ): δ 19 . 0 , 23 . 3 ( d , j = 85 hz ), 24 . 3 , 54 . 9 ( d , j = 13 hz ), 73 . 2 ( d , j = 108 hz ), 73 . 9 , 116 . 3 , 129 . 9 , 131 . 9 , 157 . 8 , 173 . 3 , 178 . 0 . ms ( esi ): m / z 361 . 9 [ m + h ] + . hplc - ms t r = 8 . 43 min . the protected derivative was deprotected according to general procedure c . 54 . 0 mg of pure product derivative 30 were obtained ( 16 % yield , 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 70 - 190 ( m , 2h ), 2 . 03 - 2 . 10 ( m , 2h ), 3 . 13 ( d , j = 16 . 5 hz , 2h ), 4 . 04 ( t , j = 6 . 0 hz , 1h ), 4 . 67 ( s , 2h ), 6 . 91 ( d , j = 8 . 5 hz , 2h ), 7 . 19 ( 2d , j = 8 . 5 hz , 2h ). 31 p nmr ( 101 mhz , d 2 o ): δ 60 . 8 . 13 c nmr ( 126 mhz , d 2 o ): δ 24 . 0 , 24 . 6 ( d , j = 92 hz ), 36 . 7 ( d , j = 88 hz ), 54 . 4 ( d , j = 15 hz ), 66 . 3 , 116 . 4 , 126 . 4 , 132 . 4 , 132 . 5 , 157 . 7 , 172 . 6 , 174 . 7 . ms ( esi ): m / z 330 . 2 ( m − 1 ). hplc - ms : t r = 8 . 02 min . hplc ( rp polar , elution water / acetonitrile / formic acid 900 : 100 : 1 , detection λ = 240 / 270 nm ): t r = 8 . 3 min . [ α ] d 20 : + 7 . 8 ( h 2 o , c 0 . 2 ). the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). 55 mg of pure derivative 31 were obtained ( 18 % yield in 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 64 ( m , 2h ), 2 . 01 ( m , 2h ), 2 . 96 ( d , j = 17 hz , 2h ), 3 . 78 ( s , 3h ), 3 . 88 ( t , j = 6 hz , 1h ), 4 . 59 ( s , 2h ), 6 . 72 ( d , j = 8 hz , 1h ), 6 . 77 ( d , j = 8 hz , 1h ), 6 . 88 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 44 . 1 13 c nmr ( 126 mhz , d 2 o ): δ 24 . 6 , 25 . 4 ( d , j = 92 hz ), 37 . 9 ( d , j = 87 hz ), 55 . 0 ( d , j = 14 hz ), 57 . 2 , 67 . 0 , 114 . 9 , 115 . 3 , 123 . 5 , 128 . 7 , 146 . 6 , 149 . 7 , 173 . 5 , 174 . 8 . ms ( esi ): m / z 362 . 0 [ m + h ] + 360 . 1 [ m − h ] − . hplc ms t r = 10 . 27 min . [ α ] d 20 + 10 . 8 ( h 2 o , c 1 . 0 ). the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). another purification was made on an anion exchange chromatography ( dowex ag 1 - x4 , aco − , 200 - 400 mesh ). the resin was first eluted with freshly boiled and cooled water , then with formic acid 3m . 27 mg of pure derivative 32 were obtained ( 17 % yield in 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 78 ( m , 2h ), 2 . 03 ( m , 2h ), 3 . 12 ( d , j = 17 hz , 2h ), 4 . 01 ( t , j = 6 hz , 1h ), 4 . 80 ( s , 2h ), 6 . 98 ( d , j = 9 hz , 1h ), 7 . 1 ( d , j = 8 hz , 1h ), 7 . 2 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 48 . 2 13 c nmr ( 126 mhz , d 2 o ): δ 23 . 9 , 24 . 8 ( d , j = 93 hz ), 36 . 6 ( d , j = 88 hz ), 54 . 3 ( d , j = 15 hz ), 67 . 1 , 116 . 4 , 121 . 8 ( d , j = 256 hz ), 125 . 5 ( d , j = 4 hz ), 127 . 5 ( d , j = 8 hz ), 130 . 8 ( d , j = 9 hz ), 139 . 0 , 149 . 9 , 172 . 5 , 174 . 0 ms ( esi ): m / z 416 . 0 [ m + h ] + . hplc ms t r = 23 . 35 min . [ α ] d 20 + 9 . 2 ( h 2 o , c 1 . 0 ). the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). another purification was made on an anion exchange chromatography ( dowex ag 1 - x4 , aco − , 200 - 400 mesh ). the resin was first eluted with freshly boiled and cooled water , then with formic acid 2 . 5m . 38 mg of pure derivative 33 were obtained ( 12 % yield in 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 64 ( m , 2h ), 2 . 01 ( m , 2h ), 3 . 00 ( d , j = 16 hz , 2h ), 3 . 93 ( t , j = 6 hz , 1h ), 4 . 74 ( s , 2h ), 6 . 97 ( d , j = 9 hz , 1h ), 7 . 37 ( d , j = 8 hz , 1h ), 7 . 49 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 43 . 2 13 c nmr ( 126 mhz , d 2 o ): δ 24 . 5 , 25 . 5 ( d , j = 92 hz ), 37 . 3 ( d , j = 86 hz ), 55 . 0 ( d , j = 15 hz ), 67 . 2 , 115 . 4 , 119 . 7 ( d , j = 29 hz ), 124 . 9 ( d , j = 272 hz ), 127 . 8 , 129 . 6 , 136 . 2 , 167 . 2 , 173 . 3 , 174 . 4 ms ( esi ): m / z 399 . 9 [ m + h ] + . hplc ms t r = 21 . 72 min . [ α ] d 20 + 8 . 0 ( h 2 o , c 1 . 0 ). the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). 70 mg of pure derivative 34 were obtained ( 31 % yield in 2 steps ). 1 h nmr ( 250 mhz , d 2 o ): δ 1 . 64 ( m , 2h ), 2 . 03 ( m , 2h ), 2 . 98 ( d , j = 17 hz , 2h ), 3 . 85 ( s , 3h ), 3 . 90 ( t , j = 6 hz , 1h ), 4 . 68 ( s , 2h ), 6 . 73 ( d , j = 11 hz , 1h ), 6 . 78 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 41 . 0 13 c nmr ( 126 mhz , d 2 o ): δ 24 . 6 , 25 . 6 ( d , j = 92 hz ), 38 . 3 ( d , j = 86 hz ), 55 . 0 ( d , j = 15 hz ), 57 . 7 , 71 . 1 , 111 . 3 , 111 . 5 , 131 . 6 , 134 . 3 , 153 . 8 , 156 . 1 ( d , j = 245 ), 173 . 4 , 174 . 8 . ms ( esi ): m / z 380 . 0 [ m + h ] + . hplc ms : t r = 11 . 91 min . [ α ] d 20 + 8 . 4 ( h 2 o , c 1 . 0 ). the compound was prepared according to general procedure c applied to the protected derivative . the purification was performed using a dowex ag 50w - x4 cation exchange resin column ( h + , 50 - 100 mesh , water elution ). 6 mg of pure derivative 35 were obtained ( 1 % yield in 2 steps ). 1 h nmr ( 500 mhz , d 2 o ): δ 1 . 63 ( m , 2h ), 2 . 03 ( m , 2h ), 2 . 98 ( d , j = 17 hz , 2h ), 3 . 83 ( s , 3h ), 3 . 90 ( t , j = 6 hz , 1h ), 4 . 61 ( s , 2h ), 6 . 90 ( s , 1h ), 6 . 93 ( s , 1h ). 31 p nmr ( 101 mhz , d 2 o ): δ 40 . 4 13 c nmr ( 126 mhz , d 2 o ): δ 24 . 8 , 25 . 8 ( d , j = 91 hz ), 38 . 5 ( d , j = 88 hz ), 55 . 3 ( d , j = 15 hz ), 57 . 6 , 70 . 9 , 114 . 6 , 124 . 0 , 128 . 3 , 133 . 1 , 142 . 6 , 154 . 0 , 173 . 8 , 174 . 8 . ms ( esi ): m / z 395 . 9 ( 100 %), 397 . 9 ( 32 %), 398 . 9 ( 6 %) [ m + h ] + . hplc ms t r = 17 . 49 min . [ α ] d 20 + 1 . 1 ( h 2 o , c 0 . 2 ). agonist activities ( ec50 ) of derivatives 1 to 35 have been determined for group iii metabotropic glutamate receptors ( mglu4 , mglu6 , mglu7 and mglu8 ) expressed in transfected cells by measuring the ligand - induced intracellular calcium release via the fluorescent probe fluo4 - am . the ec50 &# 39 ; s are compared to those of l - ap4 ( l - 2 - amino - 4 - phosphonobutyric acid ) used as a reference . it results from the examination of the above data that derivatives 2 to 4 , 6 to 8 and 20 have a high activity with respect to mglu4r . at low concentrations , these compounds are mglu4r selective . advantageously , derivates 2 , 3 , 6 to 8 and 20 display a higher affinity on mglu7 than ever known , then producing an advantagenous synergistic effect at higher concentrations through the activation of both mglu4 and mglu7 for pain treatment compared to reference derivative l - ap4 . derivates 4 , 5 and 30 are particularly selective for mglu4 , with a decreased activity on mglu7r compared to the reference derivative . antihyperalgesic properties of derivative 2 have been tested in vivo on healthy animals and animal models for inflammatory ( inflammation caused by injection of carrageenan ) or neuropathic ( chronic constriction of the sciatic nerve ) pain . after the induction period of the neuropathy or inflammation , various amounts of derivative 2 dissolved in serum have been injected in rats , ( from 0 . 5 to 15 μg / rat , i . t . from 1 to 30 mg / kg , i . p .) on time - course of vocalization threshold to paw pressure . the results obtained on healthy animals are given on fig1 . they are expressed by the time course curves of means +/− sem of vocalization threshold in grams ( n = 6 - 8 rats per group ) ( a ) or by the area under the curve ( b ). morphine ( 10 μg / rat , i . t .) is used as a positive control . significantly different from the vehicle group , *** p & lt ; 0 . 001 . as previously shown for other agonists of mglurs of group iii , derivative 2 does not modify the perception of the mechanical acute pain in healthy animal ( fig1 ). on the contrary , derivative 2 abolishes the excess of mechanical pain observed on animals with neuropathic or inflammatory pains in a dose and time - dependent manner ( fig2 and 3 ). fig2 illustrates the effect of intrathecal administration of derivative 2 on the vocalization threshold to paw pressure in the carrageenan inflammatory pain model ( effect of derivative 2 ( 0 . 5 , 1 , 5 , 10 μg / rat , i . t .) on time - course of vocalization threshold to paw pressure ). morphine ( 10 μg / rat , i . t .) is used as a positive control . results are expressed by the time course curves of means +/− sem of vocalization threshold in grams ( n = 6 - 8 rats per group ) ( a ) or by the area under the curve ( b ), significantly different from the vehicle group , * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 . moreover , while derivative 2 also dose - dependently reduced the mechanical hypersensitivity measured using von frey filaments eliciting innocuous to noxious mechanical stimuli in a model of inflammation induced by carrageenan in c57bl6 mice , intrathecal administration of derivative 2 ( 10 μg / mice , i . t .) is significantly decreased in mice lacking mglur4 receptor as compared to their wild - type littermates in this inflammatory pain model ( carrageenan ) fig3 . fig4 illustrates the effect of intrathecal administration of derivative 2 on the vocalization threshold to paw pressure in the cci neuropathic pain model ( effect of derivative 2 ( 1 , 5 , 10 μg / rat , i . t .) on time - course of vocalization threshold to paw pressure ). gabapentin ( 100 mg / kg , p . o .) is used as a positive control . results are expressed by the time course curves of means +/− sem of vocalization threshold in grams ( n = 7 - 8 rats per group ) ( a ) or by the area under the curve ( b ), significantly different from the vehicle group , ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 . the antihyperalgesic effect of derivative 2 is significantly higher than the one of gabapentine ( 100 mg / kg , per os ) used as control in the neuropathy model and close to the one of morphine used as control on the inflammatory pain . the effect of intrathecal administration of derivative 2 ( 10 μg / rat , i . t .) on the vocalization threshold to paw pressure in the cci neuropathic pain model after mglur4 or mismatch odn treatment is illustrated by fig4 . results are expressed by the time course curves of means +/− sem of vocalization threshold in grams ( n = 5 - 8 rats per group ) ( a ) or by the area under the curve ( b ), significantly different from the considered group , * p & lt ; 0 . 05 , *** p & lt ; 0 . 001 . on neuropathic rats treated with antisens oligonucleotides against mglu4 , the antihyperalgesic effect of derivative 2 is significantly reduced with respect to the control conditions ( saline or “ mismatch ” antisens targeting no target ), illustrating that a great part of derivative 2 effect is induced via mglu4 r . fig5 illustrates the effect of systemic injection of derivative 2 on the vocalization threshold to paw pressure in a rat model of inflammatory pain ( carageenan ) ( effect of derivative 2 ( 1 , 10 , 30 mg / kg , i . p .)) on time - course of vocalization threshold to paw pressure ). results are expressed by the time course curves of means +/− sem of vocalization threshold in grams ( n = 7 - 8 rats per group ) ( a ) or by the area under the curve ( b ), significantly different from the vehicle group , * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 . the above results demonstrate the strong antihyperalgesic potential of derivative 2 in chronic pains of inflammatory or neuropathic origin . adult male sprague - dawley rats weighting 175 - 200 g were purchased from charles river . c57 / bl6 mice were from elevage janvier , and homozygous mglu4 (−/−) and wild - type (+/+) littermates were generated from crosses between heterozygous animals . animals were housed under controlled environmental conditions ( 22 ° c . ; 55 % humidity ) and kept under a 12 / 12 h light / dark cycle , with food and water ad libitum for a week prior to start the experiments in order to acclimatize . animal care and experiments were carried out in accordance with the committee for research and ethical issues of the iasp ( zimmermann m ., 1983 ; goudet c ., 2008 ). the animals were submitted to the paw pressure test previously described by randall and selitto ( 1957 ). nociceptive thresholds , expressed in grams ( g ), were measured with a ugo basile analgesimeter ( apelex , tip diameter of the probe : 1 mm , weight : 30 g ) by applying an increasing pressure to the right hind paw of rats until a squeak ( vocalization threshold ) was obtained ( cut - off was 750 g except carrageenan treated animals for which the cut - off was 500 g ). the treatments were done after the measurement of two consecutive stable vocalization threshold values and their effects were assessed 15 , 30 , 45 , 60 , 90 , and 120 min after . mechanical allodynia and hyperalgesia were assessed using the von frey hair filaments of 3 different bending forces ( 0 . 07 g , 0 . 6 g , 1 . 4 g ). for each filament , 5 stimuli were applied with an interval of 3 - 5 s . thresholds to mechanically induced vocalization were assessed with rats presenting an hyperalgesia elicited by a subcutaneous injection of 2 - carrageenan ( 200 μg ) into the right hind paw ( 200 μl ). four hours later , vocalization thresholds were significantly decreased from 336 ± 6 g to 137 ± 2 g . unilateral peripheral mononeuropathy was induced according to the method described by bennett and xie ( 1988 ). briefly , after determining vocalization thresholds , rats were anesthetized with sodium pentobarbital ( 50 mg / kg i . p .) and four chromic gut ( 5 - 0 ) ligatures were tied loosely ( with about 1 - mm spacing ) around the right common sciatic nerve . the nerve was constricted to a barely discernible degree , so that circulation through the epineurial vasculature was not interrupted . only animals presenting a decrease ≧ 15 % of the presurgery value of vocalization threshold were selected , i . e ., 90 % of ligated animals ( presurgery value : 379 ± 7 g , pre - drug value : 188 ± 7 g ). for all experiments , treatments were randomized and administered according to the method of blocks in order to assess the effect of the different treatments at the same time interval to avoid uncontrolled influences ( one block includes a number of animals corresponding to the number of the different treatments administered ; all animals in a same block are tested in the same short laps of time ; the number of blocks corresponds to the number of animals per treatment ). different animals were used for each experiment ( n = 6 - 10 per treatment , according to the experiments ) performed in a blinded manner in a quiet room and animal behaviors were observed by a single experimenter . intrathecal injections were performed , under isoflurane anesthesia ( 4 % induction , 2 % maintenance ), as previously described ( mestre et al ., 1994 ). briefly , the anesthetized rat was held in one hand by the pelvic girdle and a 25 - gauge x1 - inch needle connected to a 25 μl hamilton syringe was inserted into the subarachnoidal space between lumbar vertebrae l5 and l6 , until a tail flick was elicited . the syringe was held in position for few seconds after the injection of a volume of 10 μl / rat . gabapentin , derivative 2 and morphine were dissolved in saline ( 0 . 9 % nacl , b . braun , melsungen , germany ). all solutions were prepared immediately prior to injection . as odn was designed based on rat mglur4 sequence in regions lacking known splice variants . they were synthesized by eurogentec and sequences were as follows : as - mglur4 , 5 ′- cggatagagttcatgtgg - 3 ′. one odn with scramble arrangement in the base composition compared to the 18 - mer specific as was used as control for sequence - independent effects of odn treatments . a blast search revealed that this mismatch odns were not complementary to any registered nucleotide sequences . i . t . administrations of odns ( 12 . 5 μg / rat ) or saline were performed in a volume of 10 μl via direct transcutaneous injection ( with a 25 - gauge needle connected to a 25 μl hamilton syringe ) between the l5 and l6 dorsal spinous processes ( mestre et al ., 1994 ) under animal anaesthesia with isofluran ( 3 . 5 %). this treatment was repeated twice daily for 4 days ( days 1 - 4 ). pain scores were determined using standard methods in strict conformity with ethical standards ( zimmermann m ., 1983 ) before odn treatments and then on day 4 in the afternoon . treatments were randomized and all experiments were performed blind by the same experimenter using the method of equal blocks to avoid any uncontrollable environmental influence that might induce a modification in behavioural response . for the mechanical pain test , the results are expressed as vocalization thresholds , in grams ( g ). to investigate global effects , areas under the time - course curves ( aucs , g · min ) of the antihyperalgesic effects were calculated from individual scores at each time , using the trapezoidal method . data were analyzed by a two - way anova followed , when the f value was significant , by a dunnett &# 39 ; s test , when the time - course of the effects was studied . one - way anova followed by a student - newman - keuls &# 39 ; test was used to analyze the effect of the different treatments determined by the aucs . the level of statistical significance was set at p & lt ; 0 . 05 . metabotropic glutamate receptors were transiently transfected in hek293 cells by electroporation as described elsewhere ( brabet i . et al ., 1998 ) and plated in 96 - well microplates . the high affinity glutamate transporter eaac1 was co - transfected with the receptor in order to avoid any influence of glutamate released by the cells in the assay medium . in the experiments carried out by the inventors , group - iii mglurs were co - transfected with a chimeric g - protein which couples the activation of the receptor to the phospholipase - c ( plc ) pathway . thus receptor activation induces production of inositol phosphate ( ip ) which in turn induces intracellular ca 2 + release . receptor activity was then determined by measurement of the ip production or ca release as already described ( goudet c . et al ., pnas 2004 ). for intracellular calcium measurements , cells expressing mglurs were loaded with ca2 +- sensitive fluorescent dye fluo - 4 am ( invitrogen , cergy - pontoise , france ) dissolved in hanks &# 39 ; balanced salt solution ( hbss , invitrogen , cergy - pontoise , france ) containing 2 . 5 mm probenicid ( sigma - aldrich chemie , saint - quentin fallavier , france ) for 1 h at 37 ° c ., then washed and incubated with hbss containing probenecid . a drug plate was prepared with the various concentrations of agonist to be tested and drug solution was added in each well after 20 s of recording . fluorescence signals ( excitation 485 nm , emission 525 nm ) were measured by using the fluorescence microplate reader flexstation iii ( molecular devices , saint - grégoire , france ) at sampling intervals of 1 . 5 s for 60 s . all points are realized in triplicate . the dose - response curves were fitted using the graphpad prism program and the following equation : y =[( ymax − ymin )/( 1 +( x / ec 50 ) n )]+ ymin where ec 50 is the concentration of the compound necessary to obtain the half maximal effect and n is the hill coefficient . bennett g . j . and xie y . k ., pain 33 ( 1988 ), pp . 87 - 107 . mestre c ., pelissier t ., fialip j ., wilcox g . and eschalier a ., j . pharmacol . toxicol . methods 32 ( 1994 ), pp . 197 - 200 . randall l . o . and selitto j . j ., arch . int . pharmacodyn . ther . 111 ( 1957 ), pp . 409 - 419 . gomeza , j ., mary , s ., brabet , i ., parmentier , m . l ., restituito , s ., bockaert , j ., and pin , j . p . 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