HRP20040520A2 - Resolution (r,s)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorphenyl)-5-methoxycarbonyl-6-methyl-1,4- lipase catalysed - Google Patents
Resolution (r,s)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorphenyl)-5-methoxycarbonyl-6-methyl-1,4- lipase catalysed Download PDFInfo
- Publication number
- HRP20040520A2 HRP20040520A2 HR20040520A HRP20040520A HRP20040520A2 HR P20040520 A2 HRP20040520 A2 HR P20040520A2 HR 20040520 A HR20040520 A HR 20040520A HR P20040520 A HRP20040520 A HR P20040520A HR P20040520 A2 HRP20040520 A2 HR P20040520A2
- Authority
- HR
- Croatia
- Prior art keywords
- methyl
- dihydropyridine
- enantiomerically pure
- aminoethoxymethyl
- methoxycarbonyl
- Prior art date
Links
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- 230000007306 turnover Effects 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
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Description
Područje tehnike na koje se izum odnosi. Technical field to which the invention relates.
Int. klas.C 07 F Int. class C 07 F
Izum se odnosi na postupak priprave oba enantiomera 2-(2-aminoetoksimetil)-3-etoksimetil–4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1). Spoj 1 (amlodipin) najznačajniji je predstavnik skupine lijekova (1,4-dihidropiridini) koji se koriste u liječenju hipertenzije, angine pectoris i sl. koronarnih bolesti. The invention relates to the procedure for the preparation of both enantiomers of 2-(2-aminoethoxymethyl)-3-ethoxymethyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (1). Compound 1 (amlodipine) is the most significant representative of the group of drugs (1,4-dihydropyridines) used in the treatment of hypertension, angina pectoris and similar coronary diseases.
[image] [image]
Na molekulskoj razini amlodipin se veže na α1 podjedinicu proteina kalcijevog kanala posljedica čega je smanjenje ulaska kalcijevih iona u stanicu, a time opuštanja krvnih žila i srca. Budući da je receptor na kalcijevom kanalu protein sastavljen od kiralnih prirodnih aminokiselina, za očekivati je da će enantiomeri spoja 1 različito interagirati sa aktivnim mjestom. Arrowsmith i suradnici [J. Med. Chem., 29 (1986) 1696-1702] su dokazali navedenu pretpostavku te izmjerili aktivnost odgovarajućeg (-)-enantiomera koji je oko 1000x aktivniji od (+)-enantiomera. Isti autori prvi su pripravili oba enantiomera spoja 1 kromatografskom separacijom dijastereomernog intermedijera sa derivatom bademove kiseline. Navedena sinteza, zbog velikog broja sintetskih stupnjeva i kromatografske separacije, nije ekonomična. Goldmann i suradnici [J. Med. Chem., 35 (1992) 3341-3344] objavili su određivanje apsolutne konfiguracije enantiomera spoja 1 te su dokazali da samo S-(-)-enantiomer spoja 1 iz racemata ima djelovanje kao blokator kalcijevih kanala. Posljedica toga je mogućnost korištenja polovične doze aktivne supstancije s istom farmakološkom aktivnosti. Otkriće da R-(+)-enantiomer spoja 1 posjeduje djelovanje kao antihiperlipemik dalo je važnost istraživanju novih metoda priprave oba enantiomera spoja 1. At the molecular level, amlodipine binds to the α1 subunit of the calcium channel protein, the consequence of which is a reduction in the entry of calcium ions into the cell, thereby relaxing blood vessels and the heart. Since the calcium channel receptor is a protein composed of chiral natural amino acids, it is to be expected that the enantiomers of compound 1 will interact differently with the active site. Arrowsmith and colleagues [J. Honey. Chem., 29 (1986) 1696-1702] proved the mentioned assumption and measured the activity of the corresponding (-)-enantiomer, which is about 1000x more active than the (+)-enantiomer. The same authors were the first to prepare both enantiomers of compound 1 by chromatographic separation of the diastereomeric intermediate with a mandelic acid derivative. The aforementioned synthesis, due to the large number of synthetic stages and chromatographic separation, is not economical. Goldmann and colleagues [J. Honey. Chem., 35 (1992) 3341-3344] published the determination of the absolute enantiomer configuration of compound 1 and proved that only the S-(-)-enantiomer of compound 1 from the racemate has an effect as a calcium channel blocker. As a result, it is possible to use half the dose of the active substance with the same pharmacological activity. The discovery that the R-(+)-enantiomer of compound 1 has an antihyperlipemic effect gave importance to the research of new methods of preparation of both enantiomers of compound 1.
Pregledom patentne literature pronađena je totalna sinteza enantiomerno čistog spoja 1 [Cooper UK Pat. 2 233 974 A, 1991] u devet sintetskih stupnjeva sa stupnjem resolucije odgovarajuće azido-1,4-dihidropiridin-karboksilne kiseline preko dijastereomerne cinhonidinske soli. Nedostatak navedene metode je veliki broj sintetskih stupnjeva te nisko ukupno iskorištenje. Ostale metode baziraju se na resoluciji racemičnog spoja 1 preko dijastereomernih soli bilo sa prirodnom bilo neprirodnom vinskom kiselinom. Jedna od metoda [Spargo, WO 95/25722, 1995] bazira se na resoluciji u dimetilsulfoksidu kao otapalu prilikom čega dolazi do kristalizacije dijastereomerne soli u obliku mono-DMSO solvata. Za pripravu S-(-)-enantiomera spoja 1 korištena je neprirodna D-vinska kiselina. Prema drugim postupcima za istu resoluciju korišteni su deuterirani dimetilsulfoksid [Xitian, US Pat. 30,028,031, 2003] i dimetilacetamid [Senanayake i suradnici, US Pat. 30, 130,321, 2002] kao otapala. Osim preko tartaratnih soli opisana je i resolucija preko odgovarajućih hidrogentartaratnih soli [Joshi i suradnici, US Pat. 30,176,706, 2002]. Osnovni nedostatak objavljenih metoda resolucije su upotreba toksičnih otapala u postupku kao i mogućnost koprecipitacije neželjenog dijastereomera čime dolazi do pada optičke čistoće produkta te nužnost upotrebe višestruke prekristalizacije. A review of the patent literature found the total synthesis of enantiomerically pure compound 1 [Cooper UK Pat. 2 233 974 A, 1991] in nine synthetic steps with step resolution of the corresponding azido-1,4-dihydropyridine-carboxylic acid via the diastereomeric cinchonidine salt. The disadvantage of the mentioned method is the large number of synthetic stages and the low overall yield. Other methods are based on the resolution of racemic compound 1 via diastereomeric salts either with natural or unnatural tartaric acid. One of the methods [Spargo, WO 95/25722, 1995] is based on resolution in dimethylsulfoxide as a solvent, during which crystallization of the diastereomeric salt occurs in the form of a mono-DMSO solvate. Unnatural D-tartaric acid was used to prepare the S-(-)-enantiomer of compound 1. According to other procedures for the same resolution, deuterated dimethylsulfoxide was used [Xitian, US Pat. 30,028,031, 2003] and dimethylacetamide [Senanayake et al., US Pat. 30, 130,321, 2002] as solvents. In addition to using tartrate salts, resolution using corresponding hydrogen tartrate salts has also been described [Joshi et al., US Pat. 30,176,706, 2002]. The main disadvantage of the published resolution methods is the use of toxic solvents in the process, as well as the possibility of coprecipitation of the unwanted diastereomer, which leads to a decrease in the optical purity of the product, and the necessity of using multiple recrystallization.
Rezultat ovoga izuma je nova i vrlo efikasna metoda resolucije racemata amlodipina 1 putem enantioselektivne acilacije katalizirane lipazama uz upotrebu lako dostupnih i neotrovnih otapala. Metoda omogućuje pripravu, ovisno o upotrebljenom katalizatoru pripravu, i S- i R-enantiomera amlodipina 1 u visokom iskorištenju vrlo visoke optičke čistoće. The result of this invention is a new and very efficient method of resolution of the racemate of amlodipine 1 by means of enantioselective acylation catalyzed by lipases with the use of readily available and non-toxic solvents. The method enables the preparation, depending on the catalyst used, of both S- and R-enantiomers of amlodipine 1 in high yield with very high optical purity.
Lipaze su enzimi koji u živim organizmima prvenstveno služe u prvom stupnju metabolizma (hidroliza) triacilglicerola (masti i ulja) do odgovarajućih masnih kiselina i glicerola koji se dalje metaboliziraju djelovanjem drugih enzima. Zbog efikasnih metoda izolacije lipaza bilo iz eukariotskih ili prokariotskih organizama njihova upotreba kao katalizatora u izoliranom stanju je detaljno ispitana u različitim otapalima i temperaturama na različitim tipovima organskih spojeva. Osnovna karakteristika lipaza je visoka enantioselektivnost kemijske transformacije u točno određenim uvjetima. Razlog tome je prisustvo kiralnog aktivnog mjesta u enzimu gdje dolazi do nastanka dijastereoselektivnog prijelaznog kompleksa različite stabilnosti za pojedini enantiomer iz racemata. Navedena prednost lipaza vrlo uspješno je iskorištena u ovom izumu čiji rezultat omogućuje pripravu oba enantiomera amlodipina (S-(-)-1, R-(+)-1) u visokoj optičkoj čistoći prema Shemi 1. Prema našem izumu lipaza izolirana iz roda i vrste Pseudomonas cepacia enantioselektivno katalizira resoluciju racemičnog amlodipina (1) uz nastanak odgovarajućeg R-acilnog derivata A i neizreagiranog S-(-)-amlodipina (S-(-)-1) visoke optičke čistoće (~100%) što omogućava efikasnu metodu njegove priprave. Uz upotrebu lipaze B izolirane iz roda i vrste Candida antarctica racemat amlodipina (1) uspješno je transformiran do odgovarajućeg S-acilnog derivata B i neizreagiranog R-(+)-amlodipina (R-(+)-1) visoke optičke čistoće (~100%) pa je metoda pogodna i za pripravu R-(+)-amlodipina (R-(+)-1). Lipases are enzymes that in living organisms primarily serve in the first stage of metabolism (hydrolysis) of triacylglycerols (fats and oils) to corresponding fatty acids and glycerol, which are further metabolized by the action of other enzymes. Due to the efficient methods of isolating lipases from either eukaryotic or prokaryotic organisms, their use as catalysts in the isolated state has been thoroughly tested in different solvents and temperatures on different types of organic compounds. The basic characteristic of lipases is the high enantioselectivity of chemical transformation under precisely defined conditions. The reason for this is the presence of a chiral active site in the enzyme, where a diastereoselective transition complex of different stability is formed for each enantiomer from the racemate. The mentioned advantage of lipase was very successfully used in this invention, the result of which enables the preparation of both enantiomers of amlodipine (S-(-)-1, R-(+)-1) in high optical purity according to Scheme 1. According to our invention, lipase isolated from the genus and species Pseudomonas cepacia enantioselectively catalyzes the resolution of racemic amlodipine (1) with the formation of the corresponding R-acyl derivative A and unreacted S-(-)-amlodipine (S-(-)-1) of high optical purity (~100%), which enables an efficient method of its preparations. Using lipase B isolated from Candida antarctica racemate amlodipine (1) was successfully transformed to the corresponding S-acyl derivative B and unreacted R-(+)-amlodipine (R-(+)-1) of high optical purity (~100 %) so the method is also suitable for the preparation of R-(+)-amlodipine (R-(+)-1).
Shema 1 Scheme 1
[image] [image]
Navedeni izum (za oba enantiomera) moguće je provesti u različitim otapalima poput C5-C14 ravnolančanih alkana, izooktana, izopentana, izoheksana, ciklopentana, metilciklopentana, cikloheksana, metilcikloheksana, tetrahidronaftalena, cis-dekahidronaftalena, trans-dekahidronaftalena, smjese dekahidronaftalena bilo kojeg omjera, toluena, o-ksilena, m-ksilena, p-ksilena, smjese ksilena bilo kojeg omjera, etilbenzena, kumena, tetrahidrofurana, 2-metiltetrahidrofurana, dioksana, 1,2-dimetoksietana, dietilenglikoldimetil- ili dietil-etera, trietilenglikoldimetil- ili dietil-etera, anisola, orto, meta ili para-metilanisola, 1-metoksinaftalena, dietiletera, metil-t-butiletera, diizopropiletera, acetonitrila, propionitrila, butironitrila, pentanonitrila, metanola, etanola, n-propanola, i-propanola te ostalih ravnolančanih ili razgranatih C4-C10 alkohola. Temperatura na kojoj se reakcija može provesti kreće se između 0 i 70°C, pretežno na 40°C. Posebna vrijednost izuma je mogućnost upotrebe različitih acilnih donora bez znatnijeg pada enantioselektivnosti u reakciji koji se mogu opisati općom formulom R1OOCR2 gdje je R1= Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, n-pentil, neo-pentil, n-heksil, izoamil, 2-metoksietil, cikloheksil, CH2X, CHX2, ili CX3 gdje su X= F, Cl, Br, I istovrsni ili mješoviti; a R2 = Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, n ili razgranati-C5-C10, cikloheksil, fenil, o, m ili p-monosupstituirani fenil, polisupstituirani fenil ili heteroaril. Količina acilnog donora u reakciji moguća je od stehiometrijske do nekoliko tisuća ekvivalenata u suvišku. Reakcije resolucije se provode do konverzije polaznog spoja od 50% što je idealna vrijednost za pripravu enantiomerno čistih S-(-)-amlodipina (S-(-)-1) i R-(+)-amlodipina (R-(+)-1), a vrijeme (ovisno o otapalu i temperaturi kreće se između 5 minuta i nekoliko dana) se određuje HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Kromatogram separacije jednog primjera modelnih spojeva s enantiomerima 2, 3, S-(-)-1 i R-(+)-1 prikazan je na slici 1 s redosljedom izlaska odnosno retencijama za 3 (2,87 min), 2 (4,28 min), R-(+)-1 (6,52 min) te S-(-)-1 (8,78 min). The mentioned invention (for both enantiomers) can be carried out in different solvents such as C5-C14 straight-chain alkanes, isooctane, isopentane, isohexane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, tetrahydronaphthalene, cis-decahydronaphthalene, trans-decahydronaphthalene, a mixture of decahydronaphthalene in any ratio, toluene, o-xylene, m-xylene, p-xylene, mixtures of xylene in any ratio, ethylbenzene, cumene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyleneglycoldimethyl- or diethyl-ether, triethyleneglycoldimethyl- or diethyl- ether, anisole, ortho, meta or para-methylanisole, 1-methoxynaphthalene, diethylether, methyl-t-butylether, diisopropylether, acetonitrile, propionitrile, butyronitrile, pentanonitrile, methanol, ethanol, n-propanol, i-propanol and other straight-chain or branched C4-C10 alcohols. The temperature at which the reaction can be carried out is between 0 and 70°C, mostly at 40°C. The special value of the invention is the possibility of using different acyl donors without a significant drop in enantioselectivity in the reaction, which can be described by the general formula R1OOCR2 where R1= Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, n-pentyl, neo-pentyl, n-hexyl, isoamyl, 2-methoxyethyl, cyclohexyl, CH2X, CHX2, or CX3 where X= F, Cl, Br, I are the same or mixed; and R2 = Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, n or branched-C5-C10, cyclohexyl, phenyl, o, m or p-monosubstituted phenyl, polysubstituted phenyl or heteroaryl. The amount of acyl donor in the reaction can be from stoichiometric to several thousand equivalents in excess. Resolution reactions are carried out until the conversion of the starting compound is 50%, which is the ideal value for the preparation of enantiomerically pure S-(-)-amlodipine (S-(-)-1) and R-(+)-amlodipine (R-(+)- 1), and the time (depending on the solvent and temperature ranges between 5 minutes and several days) is determined by HPLC technique on an Ultron ES OVM column with 20 mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase. The separation chromatogram of one example of model compounds with enantiomers 2, 3, S-(-)-1 and R-(+)-1 is shown in Figure 1 with the order of exit and retention for 3 (2.87 min), 2 (4, 28 min), R-(+)-1 (6.52 min) and S-(-)-1 (8.78 min).
Slika 1 Picture 1
[image] [image]
Standardi spojeva 2 i 3 za ispitivanje kromatografske separacije su pripravljeni iz enantiomerno čistih S-(-)-1 i R-(+)-1 s acetanhidridom, a moguće ih je pripraviti i kromatografskom separacijom reakcijske smjese dobivene nakon enzimske separacije amlodipina (1) prema našem izumu. The standards of compounds 2 and 3 for testing chromatographic separation are prepared from enantiomerically pure S-(-)-1 and R-(+)-1 with acetic anhydride, and they can also be prepared by chromatographic separation of the reaction mixture obtained after the enzymatic separation of amlodipine (1) according to our invention.
[image] [image]
Ovisno o željenom parametru koji se želi postići reakciju je moguće prekinuti nešto prije ili kasnije od idealne konverzije od 50%. Dodatak bezvodnih kalcijevih soli opće formule CaX2 gdje je X = F, Cl, Br, I, CF3SO3 i dr., u reakcijsku smjesu pogodno utjeće na aktivnost lipaza što rezultira željenim produktom veće optičke čistoće. Uz upotrebu optimalnih uvjeta, resolucije je moguće efikasno provesti sa katalitičkom količinom lipaza pa sve do suviška lipaza prema supstratu, racematu amlodipina (1). Zbog velike razlike u polarnosti i bazičnosti između produkata opće formule A i S-(-)-amlodipina (S-(-)-1), moguća je njihova separacija kromatografijom na koloni silikagela ili kiselo-baznom ekstrakcijom iz sirove reakcijske smjese. Nakon provedene reakcije lipaze je moguće regenerirati iz reakcijske smjese odsisavanjem bez gubitka aktivnosti što povećava njihov obrtni broj (engl. "turnover number") i omogućava višestruku upotrebu u reakcijama. Depending on the desired parameter to be achieved, it is possible to stop the reaction a little earlier or later than the ideal conversion of 50%. The addition of anhydrous calcium salts of the general formula CaX2, where X = F, Cl, Br, I, CF3SO3, etc., to the reaction mixture has a favorable effect on the activity of lipases, which results in the desired product of higher optical purity. With the use of optimal conditions, resolutions can be efficiently carried out with a catalytic amount of lipase up to an excess of lipase towards the substrate, the racemate of amlodipine (1). Due to the large difference in polarity and basicity between the products of the general formula A and S-(-)-amlodipine (S-(-)-1), their separation is possible by chromatography on a silica gel column or by acid-base extraction from the crude reaction mixture. After the reaction, lipases can be regenerated from the reaction mixture by suction without losing activity, which increases their turnover number and enables multiple use in reactions.
Primjer 1 Example 1
Resolucija (R, S)-amlodipina, (R, S)-(+/-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1) izopropilacetatom u prisustvu Pseudomonas cepacia lipaze Resolution of (R, S)-Amlodipine, (R, S)-(+/-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1 ,4-dihydropyridine (1) with isopropylacetate in the presence of Pseudomonas cepacia lipase
U otopinu amlodipina (1, 2 g) u tetrahidrofuranu (200 ml) zagrijanu na 40°C dodana je odjednom liofilizirana Pseudomonas cepacia lipaza (2 g) te izopropilacetat (100 ml). Reakcijska smjesa je mješana na 40°C tijekom vremena potrebnog da se postigne konverzija od 50% što je praćeno HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Nakon toga reakcijska smjesa je ohlađena do sobne temperature, lipaza je odsisana i isprana s malo tetrahidrofurana. Matičnica je uparena do suha i kromatografirana na koloni silikagela uz diklormetan : 2-propanol : trietilamin (9.5 : 0.5 : 0.3) kao eluens (Rf = 0.27). Dobiven je S-(-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (S-(-)-1) u obliku žućkastog ulja (0,90 g), ev >99% prema HPLC. [image] (c = 1 u CH2Cl2) = -20,7°, r.t. (S-(-)-1)= 8,78 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Lyophilized Pseudomonas cepacia lipase (2 g) and isopropyl acetate (100 ml) were added to a solution of amlodipine (1.2 g) in tetrahydrofuran (200 ml) heated to 40°C. The reaction mixture was stirred at 40°C for the time required to achieve 50% conversion as monitored by HPLC technique on an Ultron ES OVM column with 20mmol Na2HPO4 buffer/acetonitrile (80:20) as mobile phase. After that, the reaction mixture was cooled to room temperature, the lipase was suctioned off and washed with a little tetrahydrofuran. The mother liquor was evaporated to dryness and chromatographed on a silica gel column with dichloromethane : 2-propanol : triethylamine (9.5 : 0.5 : 0.3) as eluent (Rf = 0.27). S-(-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (S-(-)-1) was obtained in the form of a yellowish oil (0.90 g), ev >99% according to HPLC. [image] (c = 1 in CH2Cl2) = -20.7°, r.t. (S-(-)-1)= 8.78 min with 20 mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
IR (KBr) υ: 3360, 3040, 2950, 2920, 2865, 2830, 1665, 1630, 1585, 1465, 1410, 1370, 1350, 1325, 1300, 1265, 1225, 1190, 1170, 1105, 1080, 1025, 1000, 930, 890, 850, 840, 820, 800, 770, 745, 720, 685, 670, 640, 600. IR (KBr) υ: 3360, 3040, 2950, 2920, 2865, 2830, 1665, 1630, 1585, 1465, 1410, 1370, 1350, 1325, 1300, 1265, 1225, 1190, 1170, 1025, 10105 1000, 930, 890, 850, 840, 820, 800, 770, 745, 720, 685, 670, 640, 600.
1H-NMR (DMSO-d6) δ: 1,10 (m, 3H, CH2CH3); 2,31 (s, 3H, CH3); 2,74 (m, 2H); 3,45 (m, 2H); 3,51 (s, 3H, OCH3); 3,96 (q, 2H, CH2CH3, J = 13,85); 4,57 (d, 1H, HCH, J = 14,63); 4,67 (d, 1H, HCH, J = 15,56); 5,31 (s, 1H, CH); 7,11 – 7,14 (m, 1H, arom.); 7.20 – 7.28 (m, 2H, arom.); 7.34 – 7.37 (m, 1H, arom.). 1H-NMR (DMSO-d 6 ) δ: 1.10 (m, 3H, CH 2 CH 3 ); 2.31 (s, 3H, CH3); 2.74 (m, 2H); 3.45 (m, 2H); 3.51 (s, 3H, OCH3); 3.96 (q, 2H, CH 2 CH 3 , J = 13.85); 4.57 (d, 1H, HCH, J = 14.63); 4.67 (d, 1H, HCH, J = 15.56); 5.31 (s, 1H, CH); 7.11 – 7.14 (m, 1H, arom.); 7.20 – 7.28 (m, 2H, arom.); 7.34 – 7.37 (m, 1H, arom.).
13C-NMR (DMSO-d6) δ: 14,10; 18,06; 36,80; 40,98; 50,55; 59;35; 66,80; 73,26; 101,59; 102,08; 127,61; 127,93; 129,16; 131,30; 145,89; 146,29; 146,63; 166,68; 167,53. 13C-NMR (DMSO-d6) δ: 14.10; 18.06; 36.80; 40.98; 50.55; 59;35; 66.80; 73.26; 101.59; 102.08; 127.61; 127.93; 129.16; 131.30; 145.89; 146.29; 146.63; 166.68; 167.53.
Primjer 2 Example 2
Resolucija (R, S)-amlodipina, (R, S)-(+/-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1) izopropilacetatom u prisustvu Candida antarctica B lipaze Resolution of (R, S)-Amlodipine, (R, S)-(+/-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1 ,4-dihydropyridine (1) with isopropylacetate in the presence of Candida antarctica B lipase
U otopinu amlodipina (1, 2 g) u toluenu (200 ml) zagrijanu na 40°C dodana je odjednom liofilizirana Candida antarctica B lipaza (2 g) te izopropilacetat (100 ml). Reakcijska smjesa je mješana na 40°C tijekom vremena potrebnog da se postigne konverzija od 50% što je praćeno HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Nakon toga reakcijska smjesa je ohlađena do sobne temperature, lipaza je odsisana i isprana s malo toluena. Matičnica je uparena do suha i kromatografirana na koloni silikagela uz diklormetan : 2-propanol : trietilamin (9.5 : 0.5 : 0.3) kao eluens (Rf = 0.27). Dobiven je R-(+)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (R-(+)-1) u obliku žućkastog ulja (0,92 g), ev >99% prema HPLC. [image] (c = 1 u CH2Cl2) = +21,3°, r.t. (R-(+)-1)= 6,52 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Lyophilized Candida antarctica B lipase (2 g) and isopropyl acetate (100 ml) were added to a solution of amlodipine (1.2 g) in toluene (200 ml) heated to 40°C. The reaction mixture was stirred at 40°C for the time required to achieve 50% conversion as monitored by HPLC technique on an Ultron ES OVM column with 20mmol Na2HPO4 buffer/acetonitrile (80:20) as mobile phase. After that, the reaction mixture was cooled to room temperature, the lipase was sucked off and washed with a little toluene. The mother liquor was evaporated to dryness and chromatographed on a silica gel column with dichloromethane : 2-propanol : triethylamine (9.5 : 0.5 : 0.3) as eluent (Rf = 0.27). R-(+)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (R-(+)-1) was obtained in the form of a yellowish oil (0.92 g), ev >99% according to HPLC. [image] (c = 1 in CH2Cl2) = +21.3°, r.t. (R-(+)-1)= 6.52 min with 20mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
IR (KBr) υ: 3360, 3040, 2950, 2920, 2865, 2830, 1665, 1630, 1585, 1465, 1410, 1370, 1350, 1325, 1300, 1265, 1225, 1190, 1170, 1105, 1080, 1025, 1000, 930, 890, 850, 840, 820, 800, 770, 745, 720, 685, 670, 640, 600. IR (KBr) υ: 3360, 3040, 2950, 2920, 2865, 2830, 1665, 1630, 1585, 1465, 1410, 1370, 1350, 1325, 1300, 1265, 1225, 1190, 1170, 1025, 10105 1000, 930, 890, 850, 840, 820, 800, 770, 745, 720, 685, 670, 640, 600.
1H-NMR (DMSO-d6) δ: 1,10 (m, 3H, CH2CH3); 2,31 (s, 3H, CH3); 2,74 (m, 2H); 3,45 (m, 2H); 3,51 (s, 3H, OCH3); 3,96 (q, 2H, CH2CH3, J = 13,85); 4,57 (d, 1H, HCH, J = 14,63); 4,67 (d, 1H, HCH, J = 15,56); 5,31 (s, 1H, CH); 7,11 – 7,14 (m, 1H, arom.); 7.20 – 7.28 (m, 2H, arom.); 7.34 – 7.37 (m, 1H, arom.). 1H-NMR (DMSO-d 6 ) δ: 1.10 (m, 3H, CH 2 CH 3 ); 2.31 (s, 3H, CH3); 2.74 (m, 2H); 3.45 (m, 2H); 3.51 (s, 3H, OCH3); 3.96 (q, 2H, CH 2 CH 3 , J = 13.85); 4.57 (d, 1H, HCH, J = 14.63); 4.67 (d, 1H, HCH, J = 15.56); 5.31 (s, 1H, CH); 7.11 – 7.14 (m, 1H, arom.); 7.20 – 7.28 (m, 2H, arom.); 7.34 – 7.37 (m, 1H, arom.).
13C-NMR (DMSO-d6) δ: 14.10, 18.06, 36.80, 40.98, 50.55, 59.35, 66.80, 73.26, 101.59, 102.08, 127.61, 127.93, 129.16, 131.30, 145.89, 146.29, 146.63, 166.68, 167.53. 13c-NMR (DMSO-D6) Δ: 14.10, 18.06, 36.80, 40.98, 50.55, 59.35, 66.80, 73.26, 101.59, 102.08, 127.61, 127.93, 129.16, 131.30, 145.89, 146.29, 146.63, 166.68, 167.53.
Primjer 3 Example 3
Resolucija (R, S)-amlodipina, (R, S)-(+/-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1) izopropilacetatom u prisustvu Pseudomonas cepacia lipaze Resolution of (R, S)-Amlodipine, (R, S)-(+/-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1 ,4-dihydropyridine (1) with isopropylacetate in the presence of Pseudomonas cepacia lipase
U otopinu amlodipina (1, 2 g) u tetrahidrofuranu (150 ml) zagrijanu na 50°C dodana je odjednom liofilizirana Pseudomonas cepacia lipaza (1 g) te izopropilacetat (100 ml). Reakcijska smjesa je mješana na 50°C tijekom vremena potrebnog da se postigne konverzija od 50% što je praćeno HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Nakon toga reakcijska smjesa je ohlađena do sobne temperature, lipaza je odsisana i isprana s toplim tetrahidrofuranom. Matičnica je uparena do suha i kromatografirana na koloni silikagela uz diklormetan : 2-propanol : trietilamin (9.5 : 0.5 : 0.3) kao eluens (Rf = 0.27). Dobiven je S-(-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (S-(-)-1) u obliku žućkastog ulja (0,84 g) čiji spektroskopski podaci odgovaraju produktu dobivenom u primjeru 1, ev >99% prema HPLC. [image] (c = 1 u CH2Cl2) = -21,0°, r.t. (S-(-)-1)= 8,78 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Lyophilized Pseudomonas cepacia lipase (1 g) and isopropyl acetate (100 ml) were added to a solution of amlodipine (1.2 g) in tetrahydrofuran (150 ml) heated to 50°C. The reaction mixture was stirred at 50°C for the time required to achieve 50% conversion which was monitored by HPLC technique on an Ultron ES OVM column with 20mmol Na2HPO4 buffer/acetonitrile (80:20) as mobile phase. After that, the reaction mixture was cooled to room temperature, the lipase was suctioned off and washed with warm tetrahydrofuran. The mother liquor was evaporated to dryness and chromatographed on a silica gel column with dichloromethane : 2-propanol : triethylamine (9.5 : 0.5 : 0.3) as eluent (Rf = 0.27). S-(-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (S-(-)-1) was obtained in the form of a yellowish oil (0.84 g) whose spectroscopic data correspond to the product obtained in example 1, ev >99% according to HPLC. [image] (c = 1 in CH2Cl2) = -21.0°, r.t. (S-(-)-1)= 8.78 min with 20 mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
Primjer 4 Example 4
Resolucija (R, S)-amlodipina, (R, S)-(+/-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1) izopropilacetatom u prisustvu Candida antarctica B lipaze Resolution of (R, S)-Amlodipine, (R, S)-(+/-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1 ,4-dihydropyridine (1) with isopropylacetate in the presence of Candida antarctica B lipase
U otopinu amlodipina (1, 2 g) u smjesi n-heptana i toluena (8 : 2) (200 ml) zagrijanu na 50°C dodana je odjednom liofilizirana Candida antarctica B lipaza (2 g) te izopropilacetat (100 ml). Reakcijska smjesa je mješana na 50°C tijekom vremena potrebnog da se postigne konverzija od 50% što je praćeno HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Nakon toga reakcijska smjesa je ohlađena do sobne temperature, lipaza je odsisana i isprana n-heptanom. Matičnica je uparena do suha u visokom vakuumu te kromatografirana na koloni silikagela uz diklormetan : 2-propanol : trietilamin (9.5 : 0.5 : 0.3) kao eluens (Rf = 0.27). Dobiven je R-(+)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (R-(+)-1) u obliku žućkastog ulja (0,93 g) čiji spektroskopski podaci odgovaraju produktu dobivenom u primjeru 2, ev >99% prema HPLC. [image] (c = 1 u CH2Cl2) = +21,7°, r.t. (R-(+)-1)= 6,52 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Lyophilized Candida antarctica B lipase (2 g) and isopropyl acetate (100 ml) were added to a solution of amlodipine (1.2 g) in a mixture of n-heptane and toluene (8:2) (200 ml) heated to 50°C. The reaction mixture was stirred at 50°C for the time required to achieve 50% conversion which was monitored by HPLC technique on an Ultron ES OVM column with 20mmol Na2HPO4 buffer/acetonitrile (80:20) as mobile phase. After that, the reaction mixture was cooled to room temperature, the lipase was sucked off and washed with n-heptane. The mother liquor was evaporated to dryness under high vacuum and chromatographed on a silica gel column with dichloromethane : 2-propanol : triethylamine (9.5 : 0.5 : 0.3) as eluent (Rf = 0.27). R-(+)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (R-(+)-1) was obtained in the form of a yellowish oil (0.93 g) whose spectroscopic data correspond to the product obtained in example 2, ev >99% according to HPLC. [image] (c = 1 in CH2Cl2) = +21.7°, r.t. (R-(+)-1)= 6.52 min with 20mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
Primjer 5 Example 5
Resolucija (R, S)-amlodipina, (R, S)-(+/-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1) izopropilacetatom u prisustvu Pseudomonas cepacia lipaze Resolution of (R, S)-Amlodipine, (R, S)-(+/-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1 ,4-dihydropyridine (1) with isopropylacetate in the presence of Pseudomonas cepacia lipase
U otopinu amlodipina (1, 2 g) u izopropanolu (200 ml) zagrijanu na 40°C dodana je odjednom liofilizirana Pseudomonas cepacia lipaza (2 g), izopropilacetat (100 ml) te mljeveni bezvodni kalcijev klorid (1 g). Reakcijska smjesa je mješana na 40°C tijekom vremena potrebnog da se postigne konverzija od 50% što je praćeno HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Nakon toga reakcijska smjesa je ohlađena do sobne temperature, lipaza je odsisana i isprana obilno izopropanolom. Matičnica je uparena do suha, suspendirana u diklormetanu (50 ml) te ekstrahirana vodom (3x25 ml). Organski ekstrakt je osušen nad bezvodnim natrijevim sulfatom, profiltriran i uparen. Ostatak je kromatografiran na koloni silikagela uz diklormetan : 2-propanol : trietilamin (9.5 : 0.5 : 0.3) kao eluens (Rf = 0.27). Dobiven je S-(-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (S-(-)-1) u obliku žućkastog ulja (0,94 g) čiji spektroskopski podaci odgovaraju produktu dobivenom u primjerima 1 i 3, ev >99% prema HPLC. [image] (c = 1 u CH2Cl2) = -21,5°, r.t. (S-(-)-1)= 8,78 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Lyophilized Pseudomonas cepacia lipase (2 g), isopropyl acetate (100 ml) and ground anhydrous calcium chloride (1 g) were added to a solution of amlodipine (1.2 g) in isopropanol (200 ml) heated to 40°C. The reaction mixture was stirred at 40°C for the time required to achieve 50% conversion as monitored by HPLC technique on an Ultron ES OVM column with 20mmol Na2HPO4 buffer/acetonitrile (80:20) as mobile phase. After that, the reaction mixture was cooled to room temperature, the lipase was sucked off and washed abundantly with isopropanol. The mother plant was evaporated to dryness, suspended in dichloromethane (50 ml) and extracted with water (3x25 ml). The organic extract was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was chromatographed on a silica gel column with dichloromethane : 2-propanol : triethylamine (9.5 : 0.5 : 0.3) as eluent (Rf = 0.27). S-(-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (S-(-)-1) was obtained in the form of a yellowish oil (0.94 g) whose spectroscopic data correspond to the product obtained in examples 1 and 3, ev >99% according to HPLC. [image] (c = 1 in CH2Cl2) = -21.5°, r.t. (S-(-)-1)= 8.78 min with 20 mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
Primjer 6 Example 6
Resolucija (R, S)-amlodipina, (R, S)-(+/-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1) etilacetatom u prisustvu Candida antarctica B lipaze Resolution of (R, S)-Amlodipine, (R, S)-(+/-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1 ,4-dihydropyridine (1) with ethyl acetate in the presence of Candida antarctica B lipase
U otopinu amlodipina (1, 2 g) u apsolutnom etanolu (200 ml) dodana je odjednom liofilizirana Candida antarctica B lipaza (2 g), etilacetat (100 ml) te praškasti bezvodni kalcijev klorid (1 g). Reakcijska smjesa je zatim mješana na 40°C tijekom vremena potrebnog da se postigne konverzija od 50% što je praćeno HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Nakon toga reakcijska smjesa je ohlađena do sobne temperature, lipaza je odsisana i isprana etanolom. Matičnica je uparena do suha, suspendirana u diklormetanu (50 ml) te ekstrahirana vodom (3x25 ml). Organski ekstrakt je osušen nad bezvodnim natrijevim sulfatom, profiltriran i uparen. Ostatak je kromatografiran na koloni silikagela uz diklormetan : 2-propanol : trietilamin (9.5 : 0.5 : 0.3) kao eluens (Rf = 0.27). Dobiven je R-(+)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (R-(+)-1) u obliku žućkastog ulja (0,9 g) čiji spektroskopski podaci odgovaraju produktu dobivenom u primjerima 2 i 4, ev >99% prema HPLC. [image] (c = 1 u CH2Cl2) = +22,9°, r.t. (R-(+)-1)= 6,52 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Lyophilized Candida antarctica B lipase (2 g), ethyl acetate (100 ml) and powdered anhydrous calcium chloride (1 g) were added to a solution of amlodipine (1.2 g) in absolute ethanol (200 ml). The reaction mixture was then stirred at 40°C for the time required to achieve 50% conversion as monitored by HPLC technique on an Ultron ES OVM column with 20mmol Na2HPO4 buffer/acetonitrile (80:20) as mobile phase. After that, the reaction mixture was cooled to room temperature, the lipase was sucked off and washed with ethanol. The mother plant was evaporated to dryness, suspended in dichloromethane (50 ml) and extracted with water (3x25 ml). The organic extract was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was chromatographed on a silica gel column with dichloromethane : 2-propanol : triethylamine (9.5 : 0.5 : 0.3) as eluent (Rf = 0.27). R-(+)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (R-(+)-1) was obtained in the form of a yellowish oil (0.9 g) whose spectroscopic data correspond to the product obtained in examples 2 and 4, ev >99% according to HPLC. [image] (c = 1 in CH2Cl2) = +22.9°, r.t. (R-(+)-1)= 6.52 min with 20mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
Primjer 7 Example 7
Resolucija (R, S)-amlodipina, (R, S)-(+/-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1) izoamilacetatom u prisustvu Pseudomonas cepacia lipaze Resolution of (R, S)-Amlodipine, (R, S)-(+/-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1 ,4-dihydropyridine (1) with isoamyl acetate in the presence of Pseudomonas cepacia lipase
U otopinu amlodipina (1, 2 g) u t-butilmetileteru (200 ml) na sobnoj temperaturi dodana je odjednom liofilizirana Pseudomonas cepacia lipaza (2 g) te izoamilacetat (100 ml). Reakcijska smjesa je mješana na sobnoj temperaturi tijekom vremena potrebnog da se postigne konverzija od 50% što je praćeno HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Nakon toga lipaza je odsisana i isprana s malo t-butilmetiletera. Matičnica je uparena u visokom vakuumu do suha i kromatografirana na koloni silikagela uz diklormetan : 2-propanol : trietilamin (9.5 : 0.5 : 0.3) kao eluens (Rf = 0.27). Dobiven je S-(-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (S-(-)-1) u obliku žućkastog ulja (0,81 g) čiji spektroskopski podaci odgovaraju produktu dobivenom u primjerima 1, 3 i 5, ev >99% prema HPLC. [image] (c = 1 u CH2Cl2) = -20,8°, r.t. (S-(-)-1)= 8,78 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Lyophilized Pseudomonas cepacia lipase (2 g) and isoamyl acetate (100 ml) were added to a solution of amlodipine (1.2 g) in t-butylmethylether (200 ml) at room temperature. The reaction mixture was stirred at room temperature for the time required to achieve 50% conversion which was monitored by HPLC technique on an Ultron ES OVM column with 20mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase. After that, the lipase was suctioned off and washed with a little t-butylmethylether. The mother liquor was evaporated to dryness under high vacuum and chromatographed on a silica gel column with dichloromethane : 2-propanol : triethylamine (9.5 : 0.5 : 0.3) as eluent (Rf = 0.27). S-(-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (S-(-)-1) was obtained in the form of a yellowish oil (0.81 g) whose spectroscopic data correspond to the product obtained in examples 1, 3 and 5, ev >99% according to HPLC. [image] (c = 1 in CH2Cl2) = -20.8°, r.t. (S-(-)-1)= 8.78 min with 20 mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
Primjer 8 Example 8
Resolucija (R, S)-amlodipina, (R, S)-(+/-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (1) izopropilacetatom u prisustvu Candida antarctica B lipaze Resolution of (R, S)-Amlodipine, (R, S)-(+/-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1 ,4-dihydropyridine (1) with isopropylacetate in the presence of Candida antarctica B lipase
U otopinu amlodipina (1, 2 g) u acetonitrilu (200 ml) na sobnoj temperaturi dodana je odjednom liofilizirana Candida antarctica B lipaza (1 g) te izopropilacetat (100 ml). Reakcijska smjesa je mješana na sobnoj temperaturi tijekom vremena potrebnog da se postigne konverzija od 50% što je praćeno HPLC tehnikom na koloni Ultron ES OVM uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Nakon toga lipaza je odsisana i isprana s malo acetonitrila. Matičnica je uparena do suha i kromatografirana na koloni silikagela uz diklormetan : 2-propanol : trietilamin (9.5 : 0.5 : 0.3) kao eluens (Rf = 0.27). Dobiven je R-(+)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (R-(+)-1) u obliku žućkastog ulja (0,89 g) čiji spektroskopski podaci odgovaraju produktu dobivenom u primjerima 2, 4 i 6, ev >99% prema HPLC. [image] (c = 1 u CH2Cl2) = +21,0°, r.t. (R-(+)-1)= 6,52 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. Lyophilized Candida antarctica B lipase (1 g) and isopropyl acetate (100 ml) were added to a solution of amlodipine (1.2 g) in acetonitrile (200 ml) at room temperature. The reaction mixture was stirred at room temperature for the time required to achieve 50% conversion which was monitored by HPLC technique on an Ultron ES OVM column with 20mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase. After that, the lipase was suctioned off and washed with a little acetonitrile. The mother liquor was evaporated to dryness and chromatographed on a silica gel column with dichloromethane : 2-propanol : triethylamine (9.5 : 0.5 : 0.3) as eluent (Rf = 0.27). R-(+)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (R-(+)-1) was obtained in the form of a yellowish oil (0.89 g) whose spectroscopic data correspond to the product obtained in examples 2, 4 and 6, ev >99% according to HPLC. [image] (c = 1 in CH2Cl2) = +21.0°, r.t. (R-(+)-1)= 6.52 min with 20mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
Primjer 9 Example 9
S-(-)-2-(2-(N-acetil)aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (2) S-(-)-2-(2-(N-acetyl)aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (2)
U otopinu S-(-)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (S-(-)-1; 1,02 g) u kloroformu (10 ml) dokapan je odjednom acetanhidrid (0,31) uz mješanje na sobnoj temperaturi. Reakcijska smjesa je mješana tijekom 24 sata, nakon čega je u reakcijsku smjesu ulijana voda (10 ml), te je zalužena s NaHCO3 do pH 7,00. Slojevi su odijeljeni, a vodeni je dodatno ekstrahiran s diklormetanom (5 ml). Spojeni organski slojevi osušeni su nad bezvodnim Na2SO4, profiltrirani i upareni. Ostatak je otopljen u etilacetatu (2,5 ml), te je dokapan diizopropileter (7,5 ml). Nastala suspenzija ostavljena je na -10 °C tijekom 36 sati. Nakon odsisavanja dobiveno je 0,75 g (57 %) spoja 2 u obliku žućkastih kristalića, Rf = 0,51; jedna mrlja na TLC uz diklormetan : metanol (9 : 1) kao eluens; t.t. 129,0-130,5 °C; [image] (c = 1 u CH2Cl2) = -25,7°, r.t. (2)= 4,28 min uz 20mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. In a solution of S-(-)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (S-(-)-1; 1.02 g) in chloroform (10 ml), acetic anhydride (0.31) was added dropwise all at once with stirring at room temperature. The reaction mixture was stirred for 24 hours, after which water (10 ml) was poured into the reaction mixture, and it was alkalized with NaHCO3 to pH 7.00. The layers were separated, and the aqueous one was additionally extracted with dichloromethane (5 ml). The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated. The residue was dissolved in ethyl acetate (2.5 ml), and diisopropyl ether (7.5 ml) was added dropwise. The resulting suspension was left at -10 °C for 36 hours. After suction, 0.75 g (57 %) of compound 2 was obtained in the form of yellowish crystals, Rf = 0.51; one spot on TLC with dichloromethane : methanol (9 : 1) as eluent; d.p. 129.0-130.5 °C; [image] (c = 1 in CH2Cl2) = -25.7°, r.t. (2)= 4.28 min with 20 mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
IR (KBr) υ: 3354, 3057, 2982, 2951, 2891, 1922, 1683, 1661, 1607, 1540, 1479, 1459, 1438, 1413, 1382, 1367, 1340, 1316, 1288, 1265, 1213, 1190, 1171, 1114, 1104, 1035, 1007, 958, 944, 880, 865, 834, 820, 790, 777, 760, 749, 735, 703, 678, 651, 619, 599, 570, 514, 459. IR (KBr) υ: 3354, 3057, 2982, 2951, 2891, 1922, 1683, 1661, 1607, 1540, 1479, 1459, 1438, 1413, 1382, 1367, 1340, 1316, 1288, 1190, 1215, 1265 1171, 1114, 1104, 1035, 1007, 958, 944, 880, 865, 834, 820, 790, 777, 760, 749, 735, 703, 678, 651, 619, 599, 570, 514, 459.
1H-NMR (CDCl3) δ: 1,11 (t, 3H, CH3CH2, J = 7,12); 1,94 (s, 3H, CH3); 2,29 (s, 3H, CH3); 3,38 - 3,59 (m, 7H, OCH3+CH2CH2); 3,94 - 4,00 (m, 2H, CH2CH3); 4,59 (d, 1H, HCH, J = 15,68); 4,68 (d, 1H, HCH, J = 15,67); 5,33 (s, 1H, CH); 6,01 (s, 1H, NH); 6,95 - 6,98 (m, 1H, arom.); 7,05 - 7,07 (m, 1H, arom.); 7,15 (d, 1H, arom., J = 7,94); 7,19 (s, 1H, NH); 7,30 (dd, 1H, arom., J1 = 1,46; J2 = 7,79). 1H-NMR (CDCl 3 ) δ: 1.11 (t, 3H, CH 3 CH 2 , J = 7.12); 1.94 (s, 3H, CH3); 2.29 (s, 3H, CH3); 3.38 - 3.59 (m, 7H, OCH 3 +CH 2 CH 2 ); 3.94 - 4.00 (m, 2H, CH2CH3); 4.59 (d, 1H, HCH, J = 15.68); 4.68 (d, 1H, HCH, J = 15.67); 5.33 (s, 1H, CH); 6.01 (s, 1H, NH); 6.95 - 6.98 (m, 1H, arom.); 7.05 - 7.07 (m, 1H, arom.); 7.15 (d, 1H, arom., J = 7.94); 7.19 (s, 1H, NH); 7.30 (dd, 1H, arom., J1 = 1.46; J2 = 7.79).
13C-NMR (CDCl3) δ: 13,73; 18,76; 22,81; 36,72; 38,78; 50,23; 59,30; 67,47; 70,07; 101,21; 103,33; 126,35; 126,86; 128,73; 130,96; 131,83; 143,73; 144,44; 145,24; 166,66; 167,54; 170,16. 13C-NMR (CDCl3) δ: 13.73; 18.76; 22.81; 36.72; 38.78; 50.23; 59.30; 67.47; 70.07; 101.21; 103.33; 126.35; 126.86; 128.73; 130.96; 131.83; 143.73; 144.44; 145.24; 166.66; 167.54; 170,16.
Primjer 10 Example 10
R-(+)-2-(2-(N-acetil)aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridin (3) R-(+)-2-(2-(N-acetyl)aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (3)
U otopinu R-(+)-2-(2-aminoetoksimetil)-3-etoksikarbonil-4-(2-klorfenil)-5-metoksikarbonil-6-metil-1,4-dihidropiridina (R-(+)-1; 1,02 g) u kloroformu (10 ml) dokapan je odjednom acetanhidrid (0,31) uz mješanje na sobnoj temperaturi. Reakcijska smjesa je mješana tijekom 24 sata, nakon čega je u reakcijsku smjesu ulijana voda (10 ml), te je zalužena s NaHCO3 do pH 7,00. Slojevi su odijeljeni, a vodeni je dodatno ekstrahiran s diklormetanom (5 ml). Spojeni organski slojevi osušeni su nad bezvodnim Na2SO4, profiltrirani i upareni. Ostatak je digeriran u diizopropileteru (20 ml). Nastala suspenzija ostavljena je na -10 °C tijekom 24 sata. Nakon odsisavanja izlučenih kristala dobiveno je 0,80 g (61 %) spoja 3 u obliku blijedožućkastih kristalića, Rf = 0,48; jedna mrlja na TLC uz diklormetan : metanol (9 : 1) kao eluens; t.t. 101,5-104,0 °C; [image] (c = 1 u CH2Cl2) = +20,3°, r.t. (3)= 2,87 min uz 20 mmol Na2HPO4 pufer/acetonitril (80 : 20) kao mobilnu fazu. In a solution of R-(+)-2-(2-aminoethoxymethyl)-3-ethoxycarbonyl-4-(2-chlorophenyl)-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine (R-(+)-1); 1.02 g) in chloroform (10 ml), acetic anhydride (0.31) was added dropwise all at once with stirring at room temperature. The reaction mixture was stirred for 24 hours, after which water (10 ml) was poured into the reaction mixture, and it was alkalized with NaHCO3 to pH 7.00. The layers were separated, and the aqueous one was additionally extracted with dichloromethane (5 ml). The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated. The residue was digested in diisopropyl ether (20 ml). The resulting suspension was left at -10 °C for 24 hours. After suctioning off the separated crystals, 0.80 g (61 %) of compound 3 was obtained in the form of pale yellow crystals, Rf = 0.48; one spot on TLC with dichloromethane : methanol (9 : 1) as eluent; d.p. 101.5-104.0 °C; [image] (c = 1 in CH2Cl2) = +20.3°, r.t. (3)= 2.87 min with 20 mmol Na2HPO4 buffer/acetonitrile (80 : 20) as mobile phase.
IR (KBr) υ: 3352, 3080, 2981, 2951, 1703, 1695, 1665, 1646, 1607, 1551, 1486, 1434, 1387, 1375, 1366, 1342, 1311, 1279, 1239, 1206, 1190, 1123, 1098, 1038, 1000, 968, 942, 916, 869, 850, 838, 784, 756, 738, 704, 686, 662, 637, 617, 580. IR (KBr) υ: 3352, 3080, 2981, 2951, 1703, 1695, 1665, 1646, 1607, 1551, 1486, 1434, 1387, 1375, 1366, 1342, 1311, 1279, 1239, 1193, 1106, 1106 1098, 1038, 1000, 968, 942, 916, 869, 850, 838, 784, 756, 738, 704, 686, 662, 637, 617, 580.
1H-NMR (CDCl3) δ: 1,18 (t, 3H, CH3CH2, J = 7,11); 2,01 (s, 3H, CH3); 2,37 (s, 3H, CH3); 3,37 - 3,68 (m, 7H, OCH3+CH2CH2); 4,01 - 4,12 (m, 2H, CH2CH3); 4,66 (d, 1H, HCH, J = 15,61); 4,76 (d, 1H, HCH, J = 15,64); 5,41 (s, 1H, CH); 6,27 (s, 1H, NH); 7,01 – 7,07 (m, 1H, arom.); 7,11 - 7,16 (m, 1H, arom.); 7,21 – 7,24 (m, 1H, arom.); 7,30 (s, 1H, NH); 7,38 (dd, 1H, arom., J1 = 1,53; J2 = 7,70). 1H-NMR (CDCl 3 ) δ: 1.18 (t, 3H, CH 3 CH 2 , J = 7.11); 2.01 (s, 3H, CH3); 2.37 (s, 3H, CH3); 3.37 - 3.68 (m, 7H, OCH3+CH2CH2); 4.01 - 4.12 (m, 2H, CH2CH3); 4.66 (d, 1H, HCH, J = 15.61); 4.76 (d, 1H, HCH, J = 15.64); 5.41 (s, 1H, CH); 6.27 (s, 1H, NH); 7.01 – 7.07 (m, 1H, arom.); 7.11 - 7.16 (m, 1H, arom.); 7.21 – 7.24 (m, 1H, arom.); 7.30 (s, 1H, NH); 7.38 (dd, 1H, arom., J1 = 1.53; J2 = 7.70).
13C-NMR (CDCl3) δ: 14,05; 19,06; 23,10; 36,99; 39,08; 50,56; 59,63; 67,75; 70,36; 101,51; 103,60; 126, 68; 127,19; 129,03; 131,26; 132,11; 144,12; 144,79; 145,56; 166,98; 167,89; 170, 56. 13C-NMR (CDCl3) δ: 14.05; 19.06; 23.10; 36.99; 39.08; 50.56; 59.63; 67.75; 70.36; 101.51; 103.60; 126, 68; 127.19; 129.03; 131.26; 132.11; 144.12; 144.79; 145.56; 166.98; 167.89; 170, 56.
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