A new series of pyridoxine and pyridoxal chloro and fluorophosphonates have been synthesized. Partially protected pyridoxine 1, 2 or pyridoxal 4 reacted with methyl phosphonic dichloride in the presence of triethylamine at -10 °C. Thus, the following chlorophosphonate derivatives were obtained: 3,4'-0-isopropylidenepyridoxine-5'-0- methylchlorophosphonate (5), 4',5'-0-isobutylidenepyridoxine-3-0- methylchlorophosphonate (6), and monoethylacetalpyridoxal-3-0- methylchlorophosphonate (7). Similarly, from adequate starting compounds 1,3 and unprotected pyridoxal (PL) in the reaction with methyl phosphonic difluoride and triethylamine, near O"C, the corresponding 3-0 or 5'-0-methylfluorophosphonateswere obtained; 3,4'-0 isopropylidenepyridoxine-5'-0-methylfluorophosphonate(8), 4-metoxymethylpyridoxine- 5'-0-methylfluorophosphonate (9) and pyridoxal- 5'-0-methylfluorophosphonate (10)

Butula, Ljubica

Elshani, Sadik

Matijević-Sosa, Julija

University of Zagreb Repository

Synthesis of some pyridoxine and pyridoxal halophosphonates

A new series of pyridoxine and pyridoxal chloro and fluorophosphonates have been synthesized. Partially protected pyridoxine 1, 2 or pyridoxal 4 reacted with methyl phosphonic dichloride in the presence of triethylamine at -10 °C. Thus, the following chlorophosphonate derivatives were obtained: 3,4\u27-0-isopropylidenepyridoxine-5\u27-0- methylchlorophosphonate (5), 4\u27,5\u27-0-isobutylidenepyridoxine-3-0- methylchlorophosphonate (6), and monoethylacetalpyridoxal-3-0- methylchlorophosphonate (7). Similarly, from adequate starting compounds 1,3 and unprotected pyridoxal (PL) in the reaction with methyl phosphonic difluoride and triethylamine, near O"C, the corresponding 3-0 or 5\u27-0-methylfluorophosphonateswere obtained; 3,4\u27-0 isopropylidenepyridoxine-5\u27-0-methylfluorophosphonate(8), 4-metoxymethylpyridoxine- 5\u27-0-methylfluorophosphonate (9) and pyridoxal- 5\u27-0-methylfluorophosphonate (10)

Sadik Elshani

Ljubica Butula

Julija Matijević-Sosa

Hrčak - Portal of scientific journals of Croatia

CROATICA CHEMICA ACTA CCACAA 69 (3) 1239-1249 (1996)ISSN-00l1-1643CCA-2372 Original Scientific paperSynthesis of Some Pyridoxine and PyridoxalHalophosphonates.Sadik Elshani, a Ljubica Butula, b* and Julija Matijeuić-Sosasa Department of Chemistry, The College of Arts and Sciences,University of Idaho, Moscow, Idaho, USAb Faculty of Pharmacy and Biochemistry, University of Zagreb,10000 Zagreb, CroatiaReceived May 9, 1995; revised January 3, 1996; accepted March 6, 1996A new series of pyridoxine and pyridoxal chloro and fluorophosphon-ates have been synthesized. Partially protected pyridoxine 1, 2 orpyridoxal 4 reacted with methyl phosphonic dichloride in the presenceof triethylamine at -10 °C. Thus, the following chlorophosphonatederivatives were obtained: 3,4'-0-isopropylidenepyridoxine-5'-0-methylchlorophosphonate (5), 4',5'-0-isobutylidenepyridoxine-3-0-methylchlorophosphonate (6), and monoethylacetalpyridoxal-3-0-methylchlorophosphonate (7). Similarly, from adequate startingcompounds 1,3 and unprotected pyridoxal (PL) in the reaction withmethyl phosphonic difluoride and triethylamine, near O"C, the corre-sponding 3-0 or 5'-0-methylfluorophosphonateswere obtained; 3,4'-0-isopropylidenepyridoxine-5'-0-methylfluorophosphonate(8), 4-metoxy-methylpyridoxine-5'-0-methylfluorophosphonate (9) and pyridoxal-5'-0-methylfluorophosphonate (10).INTRODUCTIONPyridoxal-5'-phosphate (PLP) is a molecule which appears as a coenzymein a group of enzymes such as aminotransferase.l=' glycogen phosphorylase,"decarboxylase'' and many others. Extensive research has been carried outin order to clarify the catalytic activity of the enzymes, as well as to define* Author to whom correspondence should be addressed.1240 S. ELSHANI ET AL.the structural interrelationship and the significance of individual functionalgroups in the molecule, at position-46-9 and position-č.l?In our recent work,11,12a synthetic pathway for the fluorination of somepyridoxine derivatives (PN) or pyridoxal-5'-phosphate (PLP) is described.A preliminary biological study!' on the activity of the fluorinated coen-zyme of aminotransferase, pyridoxal-5'-fluorophosphate (PL-FP), when re-constituted with an apoenzyme of aminotransferase, has proved that it func-tions as a coenzyme and is fully catalytically competent. This indicates thatsubstitution of the hydroxyl group in the phosphate group for fluorine doesnot specifically impair the binding of the coenzyme to the protein. Further-more, the fluorine presence in the phosphate moiety does not alt er the co-enzyme alignment in the protein cavity. It is believed that it has no delete-rious effect on the coenzyme molecule's role in catalysis.To investigate further the relationship of coenzyme structure in thephosphate moiety towards activity, we describe in this paper the synthesesof similar compounds to those of fluorophosphates+-V with the P~Fbond, thefluorophosphonates. These are compounds with an -O-P(O)XCH3 group,where X- is a chloro or fluoro atom. The group is introduced at different po-sitions of PN or PL molecules. The synthesis was carried out by the methodsdescribed by Tammelin et al.13-15and Cheymol et al.16 where an aminoal-cohol reacts with methyl phosphonic dichloride or methyl phosphonic di-fluoride, yielding the corresponding methylchloro or methylfluorophosphon-ates (Scheme 1).R-CH2-0H + CH3P(O)X2---------.,---> R-CH2-O- f(O)X + TEA x HXEt3N,-lO CCH3X = Cl or FScheme 1.The main reason for the synthesis of chlorophosphonate derivatives wastheir capability to serve as starting products for the synthesis of fluorophos-phonates, in the reaction with NaF. In our previous work,l2 in the reactionof dichlorophosphate and NaF in the presence of crown-ether, a number ofdifluorophosphates were obtained. Nevertheless, no similar method could beapplied to the synthesis of fluorophosphonates due to the instability of start-ing chlorophosphonates 5-7.Monard and Quinshon.!" Zeffert, IS and Benshop et aP9 have describedanother reaction procedure for the synthesis of methylfluorophosphonates,where, instead of methyl phosphonic difluoride, a mixture of both, methylphosphonic dichloride and methyl phosphonic difluoride, in a molar ratio1 : 1, was used. The reaction mechanism was proposed by Zeffert.l'' DuringSYNTHESIS OF SOME PYRIDOXINE AND PYRIDOXAL HALOPHOSPHONATES 1241the reaction, in its first step, a mixed reagent of an methyphosphonicfluoro-chloride (CH3P(0)CIF) is formed, which reacts further with the alcoholicgroup. The reaction proceeds in such amanner that, finally, a compoundwith a stronger P-F bond is formed.RESULTS AND DISCUSSIONAn experimental procedure for the synthesis of some methylhalophos-phonates, starting with some protected pyridoxine (PN) and pyridoxal (PL)as well as with unprotected pyridoxal, is described. The following protectedPN and PL derivatives were prepared as starting materials: 3,4'-0-isopropy-lidenepyridoxine (1),12,20 4,5' -O-isobutylidenepyridoxine (2),21,22 4' -G-metliyl-pyridoxine (3),23 and monoethylacetalpyridoxal (4). 242 3 4The synthesis of methylchlorophosphonates of PN and PL derivati vesDerivatives of pyridoxine 1, 2 and pyridoxal 4 (1 mmole) react with aslight excess of methyl phosphonic dichloride (1,1 or up to 1.4 mmole) andEt3N (4-10 mmole) at -10 °C, yielding the eorresponding methylchlorophos-phonates, 5, 6, and 7, in good yields (Scheme 2). This reaction shows thatboth the hydroxymethyl group at position 5' and the hydroxyl group at po-sition 3 reaet with methyl phosphonic dichloride. This is why unproteetedPN or PL, in the reaction with methyl phosphonic diehloride, gave a mixtureof several components detected by TLC ( solvent system a or b). No separa-tion of these produets could be achieved using the standard procedure forseparation and purifieation.The following methylchlorophosphonates were isolated: 3,4' -O-isopropyl-idenepyridoxine-5'-0-methylchlorophosphonate (5), 4',5'-0-isobutylidenepyri-doxine-3-0-methylchlorophosphonate (6) and monoethylacetalpyridoxal- 3-0-methylchlorophosphonate (7).1242 S. ELSHANI ET AL.5H3C0~CH3O ijOIIHC-P-O3 IClH C N31,2,467Scheme2.The reaction progress as well as the purity of the isolated methylchlo-rophosphonates 5, 6, and 7 were monitored by TLe. The isolated methyl-chlorophosphonates were difficult to purify. Therefore, analyses were carriedout on freshly prepared samples (yellow oils) with out purification. The re-·action conditions and analytical data are listed in Table 1.The synthesis of methylfluorophosphonates of PN deriuatiues and PLSimilarly, in the reaction of 1, 3, or PL , where hydroxymethyl groupsat position 5'- of the pyridine ring are available for the reaction with methylphosphonic difluoride, 5'-O-methylfluorophosphonates 8, 9 and 10 were ob-tained. (Scheme 3, Table II - method A).TABLEI~ :i t"l rn ul o ,." tn ~ t"l -o ;a a ~ z t"l ~ o ~ :00 s ~ ~ > S "d 6 rn :;l o z ;:; t,J enPyridoxine-andpyridoxal-O-methylchlorophosphonates5-7NoRatio"Prod.YieldMolecularAnalysisCalc.lFoundIR(Film)(%)Formula%CRu/cm-111.14585C12R17NCI04P47.155.602960-2740(-CR2,-CR3),1610(Py),1320-1200(P-(305.699)47.355.88CR3,P=O),1070(C-O-C),1030(P-O)21.16680C13R19NCI04P48.855.922980-2760(-CR,-CR2,-CR3),1600(Py),1310(P-(319.726)48.726.09CR3),1270-1220(P=O),1080(C-0-C),1060(P-O)31.410768CllR15NCI04P45.305.182980,2880(-CR,-CR2,-CR3),1570(Py),(291.673)44.925.581315-1200(P-CR3,P=O),1060(C-0-C),1040(P-O)'"Ratioof'reagentsICH3P(OlClzandTEA!inmmoletoonemmoleof'thestartingcompound.TABLEIIPyridoxine-andpyridoxal-O-methylfluorophosphonates8-10YieldMolecularAnalysisIR(Film)NoRatio*Meth.Prod.(%)FormulaCalc./Foundu/cm-1%CRN1-2.54A860C12R17NF04P49.825.924.843000,2930(-CR2,-CR3),1600(Py),1315(P-CR3),114B893(289.247)49.696.014.571290-1220(P=0),1080(C-O-C),1030(P-O)ClOR15NF04P45.635.742940-2500(-CR2,-CR3;/N+-H'O-/assoc.),3-26A983~.~~1600(Py),1320(P-CR3),1280-1240(P=O),1070(263.210)45.385.92.(C-O-C),1030(P-O)PL-26A1086CgRllNF04P43.734.485662980(~CR2,-CR3),2740-2500(N+-W'0-,(247.166)43.284.315'41assoc.),1570(Py),1310(P-CR3),1260-1180.(P=O),1080(C-0-C),1060(P-O)'"Ratioofreagents:CH3P(O)FZandTEA(rnethodAl;CH3P(OlFz,CI-13P(OlClzandTEA(metodElinmmoletoonemmoleofthestaringcompound.,.... tv *""" v.:>1244 S. ELSJlANI ET AL.1,3,PLCH20CH3 ~H0)(iCH O-P-CH2 I 3FH C N3910Scheme 3.The followingmethylfluorophosphonates were prepared: 3,4' -O-isopropy-lidenepyridoxine-5'-O-methylfluorophosphonate (8); 4'-O-methylpyridoxine-5' -O-methylfluorophosphonate(9) and pyridoxal-5' -O-methylfluorophospho-na te (10).In the reaction with a mixed reagent (methyl phosphonic dichloride andmethyl phosphonic difluoride; method B) and compound 3 or PL, the corre-sponding 9 and 10 were obtained in simmilar yields (between 60 to 86 per-cent). Only compound 1 and mixed reagents gave 3, 4'-O-isopropylidene-5'-O-methylfluorophosphonate (8) in a better yield (93%) than with methylphosphonic difluoride alone (Scheme 4).The reaction conditions and analytical data are listed in Table II.An attempt to prepare 3-0-methylfluorophosphonates starting from 2, 3or 4 derivatives, with the free hydroxyl group at position 3, by using bothsingle and mixed fluorinated agents, failed.SYNTHESIS OF SOME PYRIDOXINE AND PYRIDOXAL I-IALOPHOSP]-IONATES 1245H3CH3ctX)C~O-~-CH3HC N:;:::;-'38CH3P(O)CI2 + CH3P(O)F2•Scheme 4.NMR SPECTRA13CNMR spectra were recorded in deuterated solvents (CDC13).Chemi-cal shifts are reported as 8/ppm uersus TMS as internal standard. The reso-nance of C-5'- is in the range from 63 to 68 ppm, and the aromatic carbonsfrom 98 to 151 ppm. The 13CNMRchemical shifts for compounds 8, 9 and10 are listed in Table III.TABLE III13CNMR chemical shifts of PN and PL derivatives 8-10 (CDCl3/TMS)Com-pound Chemical schifts, o/ppm8148.89 (C-6); 145.85 (C-3); 139.31 (C-2); 125.88 (C-4); 123.86 (C-5) 3JCp ==5.86 Hz (d) 100.31(0-C-0); 63.00 (C-5'); 2Jcp == 5.86 Hz (d); 57.98 (C-4');17.81 (C-2'); 10.33 (CH3-P); lJpC == 146.53 Hz (d) 2Jcp == 26.37 Hz (d)9151.16 (C-3); 148.83 (C-6); 138.26(C-2); 133.18 (C-5); 3Jcp == 14.65 Hz «n,127.60 (C-4) 4Jcp == 7.32 Hz (d); 67.03 (C-4'); 63.65 (C-5') 2Jcp == 5.86 Hz(d); 45.78 (OCH3); 18.98 (C-2'); 12.61 (CH3-P) lJcp == 145.30 Hz (d) 2Jcp== 36.57 Hz (d)10174.22 (C-4'); 146.38 (C-6); 144.57 (C-3); 135.25 (C-5) 3JcP == 5.86 Hz (d);130.38 (C-2); 98.05 (C-4); 68.55 (C-5'); 18.05 (C-2'); 12.22 (CH3-P) 1Js«: ==143.60 Hz (d) 2Jcp == 38.09 Hz (d)Chemical shifts in the 19FNMR of C-5' -O-substituted compounds in therange from (-)54 to (-)59 ppm and doublet splitting of signals, due to I = 1/2of 31pnucleus, indicate the P-F bond. Coupling constant s are in the rangeof 940-1043.5 Hz. These valu es are in good agreement with the data pub-lished for same phosphofluorides.š'' The 19FNMR data of compounds 8-10are listed in Table N.1246 S. ELSHANI ET AL.TABLE IV19FNMR of methylfluorophosphonates 8-10(CDC13/CFC13)Compound Chemical Shifts, J(P-F)lHz8/ppm-54.7 (d) 9608 -58.0 (d) 1040.5-59.3 (d) 1043.5-54.3 (d) 9599 -57.7 (d) 1043-59.0 (d) 103410 -54.5 (d) 940EXPERIMENTALReagents and materials: Pyridoxine hydrochloride was kindly donated by »Pli-va«, Pharmaceutical, Chemical, Food and Cosmetic Industry, Zagreb, Croatia. Pyri-doxal hydrochloride, anhydrous, was purchased from »Sigma- Chemical Co. Thinlayer chromatography (TLC) was performed on Macherey Nagel, Sil-NMR!UV 254polygram plates, and Whatman, PE Sil GfUVpolyester plates, Darmstadt, Germany.General remarks; Melting points (m.p.), not corrected, were determined on a Tot-toli apparatus. TLC solvent systems were used, (a) dichloromethane-methanol (9 : 1) or(b) isopropanol-ammonia-water (7 : 1 : 2). Spots were detected with a UV lamp (254and 366 nm) or in iodine vapour. IR spectra were recorded as films on a Perkin EI-mer 457 grating spectrometer. 13CNMR spectra were recorded on a JEOL FX-100spectrometer in CDC13and (CD3)CO. 19FNMR spectra were recorded on a Varian80 A FT NMR spectrometer operating at 74.84 MHz. Spectra widths of 8000-2000 Hzand 16 K addresses were used, giving digital resolution of ±0.02 ppm for chemicalshifts and ±l Hz coupling constants. The samples were dissolved in DMSO-d6 orCDC13using deuterium lock. 19FNMRchemical shifts, expressed in 8/ppm, are ref-erenced to CFC13as internal standard.Abbreuiations: r.t. room temperature; Et3N, triethylamine; PL, pyridoxal; PN,pyridoxine; TLC, thin layer chromatography.Methylchlorophosphonates of pyridoxine and pyridoxal derivatives.General procedure. Chosen PN derivatives 1, 2 and 4 (1 mmole), together withEt3N (4-10 mmole) in 5 ml of chloroform, were slowly added under vigorous stirringto a cooled solution (-10 °C) of methyl phosphonic dichloride (1.1-1.4 mmole), dis-solved in chloroform (2-3 ml), for 30 minutes. Stirring was continued an additional1-1.5 hrs at room temperature. The course of reaction was monitored by TLC (a or b),and the disappearance of the starting compound indicated the end point of the re-action.SYNTHESIS OF SOME PYRIDOXINE AND PYRIDOXAL HALOPHOSPHONATES 1247The resulting solution was concentrated in vacuum; dry ether was added to theresidue and the precipitated Et3N hydrochloride was filtered off. The ether was flashevaporated, yielding a pale yellow oily residue. The raw products thus obtained werekept in tightly closed bottles and refrigerated. The purity of isolated compounds wasdetermined by TLC, elemental analyses and IR (film) spectra. Table I.3-0,4'-O-isopropylidenepyridoxine-5'-O-methylchlorophosphonate (5) was obtainedstarting from compound 1 (209 mg, 1 mmole) dissolved together with Et3N (0.6 ml,4 mmole) in 5 ml of chloroformin reaction with methyl phosphonic dichloride (145 mg,1.1 mmole, in 2 ml of chloroform). Yield: 260 mg (85%).4'-O,5'-O-isobutylidenepyridoxine-3-0-methylchlorophosphonate (6).Methyl phosphonicdichloride (145 mg, 1.1 mmole) in 2 ml of chloroform and PN derivative 2, (223 mg,1 mmole), together with Et3N (0.9 ml, 6 mmole), in chloroform (5 ml), at -10 °C gavecompound 6. Yield: 255 mg (80%)Monoethylacetal pyridoxal-3-0-methylchlorophosphonate (7) was obtained fromPL derivative as hydrochloride 4, (231 mg, 1 mmole), Et3N (1.5 ml, 10 mmole) andmethyl phosphonic dichloride (190 mg, 1.4 mmole) according to the general proce-dure. Yield: 199 mg (68%).MethylfZuorophosphonates of pyridoxine derivati ves and pyridoxalMETHODATo the solution of methyl phosphonic difluoride (2-2.5 mmole, 0.14-0.17 ml) indry chlorofom, a mixture of protected PN 1 or 3 derivative or unprotected PL(1mmole) and Et3N (4-6 mmole), in chloroform, was added dropwise, at -10 °C, dur-ing 20-50 minutes. When addition was completed, the reaction was continued understirring for another 30 min. at rt. The end point of the reaction was determined byTLC. The excess of solvent was evaporated and Et3N hydrogenfluoride was precipi-tated with ether. The products were isolated from the reaction mixture in a similarmanner as chloroderivatives. Thus, compounds 8, 9 and 10 were isolated as purpleoily products, which were more stable than chlorophosphonate derivatives. Elemen-tal analysis and IR (film) data of TLC pure products are listed in Table II.3,4' -O-isopropylidenepyridoxine-5' -O-methylfluorophosphonate (8) was synthe-sized in the reaction of methyl phosphonic difluoride (0.17 ml, 2.5 mmole) in chlo-roform (3-5 ml) and the mixture of compound 1 (209 mg, 1 mmole) and Et3N (0.6 ml,4 mmole) dissolved in 3 ml of chloroform, under stirring at -10 °C. Yield: 174 mg(60%).4'-O-methylpyridoxine-5'-O-methylfluorophosphonate (9). Into the solution of me-thyl phosphonic difluoride in chloroform (0.14 ml, 2 mmole, 3 ml), a mixture of com-pound 3, as hydrochloride (220 mg, 1 mmole) and Et3N (0.9 ml, 6 mmole) in 5 ml ofchlorofom, was added under vigorous stirring, at -10 °C, during 20 minutes. The pu-rity of the dark oily product was determined by TLC (b). Yield: 220 mg (83.5%).Pyridoxal-5'-O-methylfluorophosphonate (10). The solution of pyridoxal hydro-chloride (204 mg, 1 mmole) and Et3N (0.9 ml, 6 mmole) in chloroform (6 ml) wasadded to methyl phosphonic difluoride (0.14 ml, 2 mmole) in chloroform under stir-ring at -10 °C, during 30 minutes. Evaporation of solvents gave an oily TLC(b) pureresidue. Yield: 212 mg (86%).1248 S. ELSHANI ET AL.METHOD B3,4'-O-isopropilidenepyridaxine-5'-O-methyl{luorophosphonate (8). A mixture ofequal quantity of methyl phosphonic dichloride (128 mg, 1 mmole) and methyl phos-phonic difluoride (100 mg, 0.072 ml, 1 mmole) was dissolved in 2 ml of dry chloro-form and cooled to - 10 °C. Into the mixture, while stirring vigorously, a solutian of 1(1 mmole) and Et3N (0.6 ml, 4 mmole) in dry chloroform (3 ml) was succesivly added,during 30 minutes. The stirring was continued for another 30 minute s at r.t. Thesolvent was evapara ted and then 5 ml of dry ether was added. The precipitated Et3N-hydrofluoridewas filtered offand the ether was flash-evaporated. The purity of the oilyresidue was determined by TLC (b). Yield: 270 mg (93%).REFERENCE S1. E. E. SneH, Vitam. Harm. 28 (1970) 265.2. A. E. Braunstein and M. M. Schemzakin, Biokhimija 18 (1953) 393.3. D. E. Metzler, M. Ikawa and E. E. SneH,J. Am. Chem. Soc. 76 (1954) 648.4. G. T. Cori and F. Cori, J. Biol. Chem. 135 (1940) 733.5. L. Schirch and W.T. Jenkins, J. Biol. Chem. 239 (1964) 3797 and 3801.6. W. Korytnik, S. C. Srivastava, N. Angelino, P. G. G. Potti and B. Paul, J. Med.Chem. 16 (1971) 1096.7. W.Korytnik and B. Paul, J. Med. Chem. 13 (1970) 187.8. W.Korytnik, H. Ahrens and N. Angelino, Tetrahedron 26 (1970) 5415.9. W. Korytnik, A. C. Ghosh, P. G. G. Potti and S. Srivastava, J. Med. Chem. 19(1976) 999.10. W.Korytnik and B. Lachmann, J. Med. Chem. 14 (1971) 641.11. Lj. Butula, S. Elshani and M. Flogel, Acta Pharm Yug. 41 (1991) 1.12. S. Elshani, Lj. Butula, 1.Butula, D. Vikić-Topić,A. Berton and A. Irriarte, Croat.Chem. Acta 64 (2) (1991) 161.13. L. E. Tammelin, Acta Chem. Scand. 11 (1957) 859.14. L. E. Tammelin, Arkiv Kemi. 12 (1958) 287.15. L. E. Tammelin, Svensk. Kem. Tidskr. 70 (1958) 157.16. J. Cheymol, P. Chabrier, Th. Nguen Thanh and G. Cheymol, Compt. Rend. 261(1965) 3237.17. Ch. Monard and J. Quinshon, BuZZ.Chim. Soc. Fr. (1961) 1084.18. B. Zeffert, P. Coulter and H. Tannenbaum, J. Am. Chem. Soc. 82 (1960) 3843.19. H. Benshop, G. R. van der Berg and G. W.Krapy, Recueil 89 (1970) 1025.20. W.Korytnik and W.Wiedemann, J. Chem. Soc. (1962) 2531.21. D. Szlampek-Nesteruk, A. Rudnicki and T. Sikorska, Przem. Chem. 54 (1975) 238.22. S. D. L'vova, Z. J. Har and V.J. Gunar, Khim. Farm. Zh. 12 (12) 1978) 106.23. S. Harris, D. Heyl and K. Folkers, J. Am. Chem. Soc. 66 (1944) 2088.24. D. Heyl, S. Harris and K. Folkers, J. Am. Chem. Soc. 73 (1951) 3436.25. G. S. Reddy and R. Schmutzler, Z. Naturforsch 256 (1970) 1199.

Synthesis of Some Pyridoxine and Pyridoxal Halophosphonates

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CROATICA CHEMICA ACTA CCACAA 69 (3) 1239-1249 (1996)ISSN-00l1-1643CCA-2372 Original Scientific paperSynthesis of Some Pyridoxine and PyridoxalHalophosphonates.Sadik Elshani, a Ljubica Butula, b* and Julija Matijeuić-Sosasa Department of Chemistry, The College of Arts and Sciences,University of Idaho, Moscow, Idaho, USAb Faculty of Pharmacy and Biochemistry, University of Zagreb,10000 Zagreb, CroatiaReceived May 9, 1995; revised January 3, 1996; accepted March 6, 1996A new series of pyridoxine and pyridoxal chloro and fluorophosphon-ates have been synthesized. Partially protected pyridoxine 1, 2 orpyridoxal 4 reacted with methyl phosphonic dichloride in the presenceof triethylamine at -10 °C. Thus, the following chlorophosphonatederivatives were obtained: 3,4'-0-isopropylidenepyridoxine-5'-0-methylchlorophosphonate (5), 4',5'-0-isobutylidenepyridoxine-3-0-methylchlorophosphonate (6), and monoethylacetalpyridoxal-3-0-methylchlorophosphonate (7). Similarly, from adequate startingcompounds 1,3 and unprotected pyridoxal (PL) in the reaction withmethyl phosphonic difluoride and triethylamine, near O"C, the corre-sponding 3-0 or 5'-0-methylfluorophosphonateswere obtained; 3,4'-0-isopropylidenepyridoxine-5'-0-methylfluorophosphonate(8), 4-metoxy-methylpyridoxine-5'-0-methylfluorophosphonate (9) and pyridoxal-5'-0-methylfluorophosphonate (10).INTRODUCTIONPyridoxal-5'-phosphate (PLP) is a molecule which appears as a coenzymein a group of enzymes such as aminotransferase.l=' glycogen phosphorylase,"decarboxylase'' and many others. Extensive research has been carried outin order to clarify the catalytic activity of the enzymes, as well as to define* Author to whom correspondence should be addressed.1240 S. ELSHANI ET AL.the structural interrelationship and the significance of individual functionalgroups in the molecule, at position-46-9 and position-č.l?In our recent work,11,12a synthetic pathway for the fluorination of somepyridoxine derivatives (PN) or pyridoxal-5'-phosphate (PLP) is described.A preliminary biological study!' on the activity of the fluorinated coen-zyme of aminotransferase, pyridoxal-5'-fluorophosphate (PL-FP), when re-constituted with an apoenzyme of aminotransferase, has proved that it func-tions as a coenzyme and is fully catalytically competent. This indicates thatsubstitution of the hydroxyl group in the phosphate group for fluorine doesnot specifically impair the binding of the coenzyme to the protein. Further-more, the fluorine presence in the phosphate moiety does not alt er the co-enzyme alignment in the protein cavity. It is believed that it has no delete-rious effect on the coenzyme molecule's role in catalysis.To investigate further the relationship of coenzyme structure in thephosphate moiety towards activity, we describe in this paper the synthesesof similar compounds to those of fluorophosphates+-V with the P~Fbond, thefluorophosphonates. These are compounds with an -O-P(O)XCH3 group,where X- is a chloro or fluoro atom. The group is introduced at different po-sitions of PN or PL molecules. The synthesis was carried out by the methodsdescribed by Tammelin et al.13-15and Cheymol et al.16 where an aminoal-cohol reacts with methyl phosphonic dichloride or methyl phosphonic di-fluoride, yielding the corresponding methylchloro or methylfluorophosphon-ates (Scheme 1).R-CH2-0H + CH3P(O)X2---------.,---> R-CH2-O- f(O)X + TEA x HXEt3N,-lO CCH3X = Cl or FScheme 1.The main reason for the synthesis of chlorophosphonate derivatives wastheir capability to serve as starting products for the synthesis of fluorophos-phonates, in the reaction with NaF. In our previous work,l2 in the reactionof dichlorophosphate and NaF in the presence of crown-ether, a number ofdifluorophosphates were obtained. Nevertheless, no similar method could beapplied to the synthesis of fluorophosphonates due to the instability of start-ing chlorophosphonates 5-7.Monard and Quinshon.!" Zeffert, IS and Benshop et aP9 have describedanother reaction procedure for the synthesis of methylfluorophosphonates,where, instead of methyl phosphonic difluoride, a mixture of both, methylphosphonic dichloride and methyl phosphonic difluoride, in a molar ratio1 : 1, was used. The reaction mechanism was proposed by Zeffert.l'' DuringSYNTHESIS OF SOME PYRIDOXINE AND PYRIDOXAL HALOPHOSPHONATES 1241the reaction, in its first step, a mixed reagent of an methyphosphonicfluoro-chloride (CH3P(0)CIF) is formed, which reacts further with the alcoholicgroup. The reaction proceeds in such amanner that, finally, a compoundwith a stronger P-F bond is formed.RESULTS AND DISCUSSIONAn experimental procedure for the synthesis of some methylhalophos-phonates, starting with some protected pyridoxine (PN) and pyridoxal (PL)as well as with unprotected pyridoxal, is described. The following protectedPN and PL derivatives were prepared as starting materials: 3,4'-0-isopropy-lidenepyridoxine (1),12,20 4,5' -O-isobutylidenepyridoxine (2),21,22 4' -G-metliyl-pyridoxine (3),23 and monoethylacetalpyridoxal (4). 242 3 4The synthesis of methylchlorophosphonates of PN and PL derivati vesDerivatives of pyridoxine 1, 2 and pyridoxal 4 (1 mmole) react with aslight excess of methyl phosphonic dichloride (1,1 or up to 1.4 mmole) andEt3N (4-10 mmole) at -10 °C, yielding the eorresponding methylchlorophos-phonates, 5, 6, and 7, in good yields (Scheme 2). This reaction shows thatboth the hydroxymethyl group at position 5' and the hydroxyl group at po-sition 3 reaet with methyl phosphonic dichloride. This is why unproteetedPN or PL, in the reaction with methyl phosphonic diehloride, gave a mixtureof several components detected by TLC ( solvent system a or b). No separa-tion of these produets could be achieved using the standard procedure forseparation and purifieation.The following methylchlorophosphonates were isolated: 3,4' -O-isopropyl-idenepyridoxine-5'-0-methylchlorophosphonate (5), 4',5'-0-isobutylidenepyri-doxine-3-0-methylchlorophosphonate (6) and monoethylacetalpyridoxal- 3-0-methylchlorophosphonate (7).1242 S. ELSHANI ET AL.5H3C0~CH3O ijOIIHC-P-O3 IClH C N31,2,467Scheme2.The reaction progress as well as the purity of the isolated methylchlo-rophosphonates 5, 6, and 7 were monitored by TLe. The isolated methyl-chlorophosphonates were difficult to purify. Therefore, analyses were carriedout on freshly prepared samples (yellow oils) with out purification. The re-·action conditions and analytical data are listed in Table 1.The synthesis of methylfluorophosphonates of PN deriuatiues and PLSimilarly, in the reaction of 1, 3, or PL , where hydroxymethyl groupsat position 5'- of the pyridine ring are available for the reaction with methylphosphonic difluoride, 5'-O-methylfluorophosphonates 8, 9 and 10 were ob-tained. (Scheme 3, Table II - method A).TABLEI~ :i t"l rn ul o ,." tn ~ t"l -o ;a a ~ z t"l ~ o ~ :00 s ~ ~ > S "d 6 rn :;l o z ;:; t,J enPyridoxine-andpyridoxal-O-methylchlorophosphonates5-7NoRatio"Prod.YieldMolecularAnalysisCalc.lFoundIR(Film)(%)Formula%CRu/cm-111.14585C12R17NCI04P47.155.602960-2740(-CR2,-CR3),1610(Py),1320-1200(P-(305.699)47.355.88CR3,P=O),1070(C-O-C),1030(P-O)21.16680C13R19NCI04P48.855.922980-2760(-CR,-CR2,-CR3),1600(Py),1310(P-(319.726)48.726.09CR3),1270-1220(P=O),1080(C-0-C),1060(P-O)31.410768CllR15NCI04P45.305.182980,2880(-CR,-CR2,-CR3),1570(Py),(291.673)44.925.581315-1200(P-CR3,P=O),1060(C-0-C),1040(P-O)'"Ratioof'reagentsICH3P(OlClzandTEA!inmmoletoonemmoleof'thestartingcompound.TABLEIIPyridoxine-andpyridoxal-O-methylfluorophosphonates8-10YieldMolecularAnalysisIR(Film)NoRatio*Meth.Prod.(%)FormulaCalc./Foundu/cm-1%CRN1-2.54A860C12R17NF04P49.825.924.843000,2930(-CR2,-CR3),1600(Py),1315(P-CR3),114B893(289.247)49.696.014.571290-1220(P=0),1080(C-O-C),1030(P-O)ClOR15NF04P45.635.742940-2500(-CR2,-CR3;/N+-H'O-/assoc.),3-26A983~.~~1600(Py),1320(P-CR3),1280-1240(P=O),1070(263.210)45.385.92.(C-O-C),1030(P-O)PL-26A1086CgRllNF04P43.734.485662980(~CR2,-CR3),2740-2500(N+-W'0-,(247.166)43.284.315'41assoc.),1570(Py),1310(P-CR3),1260-1180.(P=O),1080(C-0-C),1060(P-O)'"Ratioofreagents:CH3P(O)FZandTEA(rnethodAl;CH3P(OlFz,CI-13P(OlClzandTEA(metodElinmmoletoonemmoleofthestaringcompound.,.... tv *""" v.:>1244 S. ELSJlANI ET AL.1,3,PLCH20CH3 ~H0)(iCH O-P-CH2 I 3FH C N3910Scheme 3.The followingmethylfluorophosphonates were prepared: 3,4' -O-isopropy-lidenepyridoxine-5'-O-methylfluorophosphonate (8); 4'-O-methylpyridoxine-5' -O-methylfluorophosphonate(9) and pyridoxal-5' -O-methylfluorophospho-na te (10).In the reaction with a mixed reagent (methyl phosphonic dichloride andmethyl phosphonic difluoride; method B) and compound 3 or PL, the corre-sponding 9 and 10 were obtained in simmilar yields (between 60 to 86 per-cent). Only compound 1 and mixed reagents gave 3, 4'-O-isopropylidene-5'-O-methylfluorophosphonate (8) in a better yield (93%) than with methylphosphonic difluoride alone (Scheme 4).The reaction conditions and analytical data are listed in Table II.An attempt to prepare 3-0-methylfluorophosphonates starting from 2, 3or 4 derivatives, with the free hydroxyl group at position 3, by using bothsingle and mixed fluorinated agents, failed.SYNTHESIS OF SOME PYRIDOXINE AND PYRIDOXAL I-IALOPHOSP]-IONATES 1245H3CH3ctX)C~O-~-CH3HC N:;:::;-'38CH3P(O)CI2 + CH3P(O)F2•Scheme 4.NMR SPECTRA13CNMR spectra were recorded in deuterated solvents (CDC13).Chemi-cal shifts are reported as 8/ppm uersus TMS as internal standard. The reso-nance of C-5'- is in the range from 63 to 68 ppm, and the aromatic carbonsfrom 98 to 151 ppm. The 13CNMRchemical shifts for compounds 8, 9 and10 are listed in Table III.TABLE III13CNMR chemical shifts of PN and PL derivatives 8-10 (CDCl3/TMS)Com-pound Chemical schifts, o/ppm8148.89 (C-6); 145.85 (C-3); 139.31 (C-2); 125.88 (C-4); 123.86 (C-5) 3JCp ==5.86 Hz (d) 100.31(0-C-0); 63.00 (C-5'); 2Jcp == 5.86 Hz (d); 57.98 (C-4');17.81 (C-2'); 10.33 (CH3-P); lJpC == 146.53 Hz (d) 2Jcp == 26.37 Hz (d)9151.16 (C-3); 148.83 (C-6); 138.26(C-2); 133.18 (C-5); 3Jcp == 14.65 Hz «n,127.60 (C-4) 4Jcp == 7.32 Hz (d); 67.03 (C-4'); 63.65 (C-5') 2Jcp == 5.86 Hz(d); 45.78 (OCH3); 18.98 (C-2'); 12.61 (CH3-P) lJcp == 145.30 Hz (d) 2Jcp== 36.57 Hz (d)10174.22 (C-4'); 146.38 (C-6); 144.57 (C-3); 135.25 (C-5) 3JcP == 5.86 Hz (d);130.38 (C-2); 98.05 (C-4); 68.55 (C-5'); 18.05 (C-2'); 12.22 (CH3-P) 1Js«: ==143.60 Hz (d) 2Jcp == 38.09 Hz (d)Chemical shifts in the 19FNMR of C-5' -O-substituted compounds in therange from (-)54 to (-)59 ppm and doublet splitting of signals, due to I = 1/2of 31pnucleus, indicate the P-F bond. Coupling constant s are in the rangeof 940-1043.5 Hz. These valu es are in good agreement with the data pub-lished for same phosphofluorides.š'' The 19FNMR data of compounds 8-10are listed in Table N.1246 S. ELSHANI ET AL.TABLE IV19FNMR of methylfluorophosphonates 8-10(CDC13/CFC13)Compound Chemical Shifts, J(P-F)lHz8/ppm-54.7 (d) 9608 -58.0 (d) 1040.5-59.3 (d) 1043.5-54.3 (d) 9599 -57.7 (d) 1043-59.0 (d) 103410 -54.5 (d) 940EXPERIMENTALReagents and materials: Pyridoxine hydrochloride was kindly donated by »Pli-va«, Pharmaceutical, Chemical, Food and Cosmetic Industry, Zagreb, Croatia. Pyri-doxal hydrochloride, anhydrous, was purchased from »Sigma- Chemical Co. Thinlayer chromatography (TLC) was performed on Macherey Nagel, Sil-NMR!UV 254polygram plates, and Whatman, PE Sil GfUVpolyester plates, Darmstadt, Germany.General remarks; Melting points (m.p.), not corrected, were determined on a Tot-toli apparatus. TLC solvent systems were used, (a) dichloromethane-methanol (9 : 1) or(b) isopropanol-ammonia-water (7 : 1 : 2). Spots were detected with a UV lamp (254and 366 nm) or in iodine vapour. IR spectra were recorded as films on a Perkin EI-mer 457 grating spectrometer. 13CNMR spectra were recorded on a JEOL FX-100spectrometer in CDC13and (CD3)CO. 19FNMR spectra were recorded on a Varian80 A FT NMR spectrometer operating at 74.84 MHz. Spectra widths of 8000-2000 Hzand 16 K addresses were used, giving digital resolution of ±0.02 ppm for chemicalshifts and ±l Hz coupling constants. The samples were dissolved in DMSO-d6 orCDC13using deuterium lock. 19FNMRchemical shifts, expressed in 8/ppm, are ref-erenced to CFC13as internal standard.Abbreuiations: r.t. room temperature; Et3N, triethylamine; PL, pyridoxal; PN,pyridoxine; TLC, thin layer chromatography.Methylchlorophosphonates of pyridoxine and pyridoxal derivatives.General procedure. Chosen PN derivatives 1, 2 and 4 (1 mmole), together withEt3N (4-10 mmole) in 5 ml of chloroform, were slowly added under vigorous stirringto a cooled solution (-10 °C) of methyl phosphonic dichloride (1.1-1.4 mmole), dis-solved in chloroform (2-3 ml), for 30 minutes. Stirring was continued an additional1-1.5 hrs at room temperature. The course of reaction was monitored by TLC (a or b),and the disappearance of the starting compound indicated the end point of the re-action.SYNTHESIS OF SOME PYRIDOXINE AND PYRIDOXAL HALOPHOSPHONATES 1247The resulting solution was concentrated in vacuum; dry ether was added to theresidue and the precipitated Et3N hydrochloride was filtered off. The ether was flashevaporated, yielding a pale yellow oily residue. The raw products thus obtained werekept in tightly closed bottles and refrigerated. The purity of isolated compounds wasdetermined by TLC, elemental analyses and IR (film) spectra. Table I.3-0,4'-O-isopropylidenepyridoxine-5'-O-methylchlorophosphonate (5) was obtainedstarting from compound 1 (209 mg, 1 mmole) dissolved together with Et3N (0.6 ml,4 mmole) in 5 ml of chloroformin reaction with methyl phosphonic dichloride (145 mg,1.1 mmole, in 2 ml of chloroform). Yield: 260 mg (85%).4'-O,5'-O-isobutylidenepyridoxine-3-0-methylchlorophosphonate (6).Methyl phosphonicdichloride (145 mg, 1.1 mmole) in 2 ml of chloroform and PN derivative 2, (223 mg,1 mmole), together with Et3N (0.9 ml, 6 mmole), in chloroform (5 ml), at -10 °C gavecompound 6. Yield: 255 mg (80%)Monoethylacetal pyridoxal-3-0-methylchlorophosphonate (7) was obtained fromPL derivative as hydrochloride 4, (231 mg, 1 mmole), Et3N (1.5 ml, 10 mmole) andmethyl phosphonic dichloride (190 mg, 1.4 mmole) according to the general proce-dure. Yield: 199 mg (68%).MethylfZuorophosphonates of pyridoxine derivati ves and pyridoxalMETHODATo the solution of methyl phosphonic difluoride (2-2.5 mmole, 0.14-0.17 ml) indry chlorofom, a mixture of protected PN 1 or 3 derivative or unprotected PL(1mmole) and Et3N (4-6 mmole), in chloroform, was added dropwise, at -10 °C, dur-ing 20-50 minutes. When addition was completed, the reaction was continued understirring for another 30 min. at rt. The end point of the reaction was determined byTLC. The excess of solvent was evaporated and Et3N hydrogenfluoride was precipi-tated with ether. The products were isolated from the reaction mixture in a similarmanner as chloroderivatives. Thus, compounds 8, 9 and 10 were isolated as purpleoily products, which were more stable than chlorophosphonate derivatives. Elemen-tal analysis and IR (film) data of TLC pure products are listed in Table II.3,4' -O-isopropylidenepyridoxine-5' -O-methylfluorophosphonate (8) was synthe-sized in the reaction of methyl phosphonic difluoride (0.17 ml, 2.5 mmole) in chlo-roform (3-5 ml) and the mixture of compound 1 (209 mg, 1 mmole) and Et3N (0.6 ml,4 mmole) dissolved in 3 ml of chloroform, under stirring at -10 °C. Yield: 174 mg(60%).4'-O-methylpyridoxine-5'-O-methylfluorophosphonate (9). Into the solution of me-thyl phosphonic difluoride in chloroform (0.14 ml, 2 mmole, 3 ml), a mixture of com-pound 3, as hydrochloride (220 mg, 1 mmole) and Et3N (0.9 ml, 6 mmole) in 5 ml ofchlorofom, was added under vigorous stirring, at -10 °C, during 20 minutes. The pu-rity of the dark oily product was determined by TLC (b). Yield: 220 mg (83.5%).Pyridoxal-5'-O-methylfluorophosphonate (10). The solution of pyridoxal hydro-chloride (204 mg, 1 mmole) and Et3N (0.9 ml, 6 mmole) in chloroform (6 ml) wasadded to methyl phosphonic difluoride (0.14 ml, 2 mmole) in chloroform under stir-ring at -10 °C, during 30 minutes. Evaporation of solvents gave an oily TLC(b) pureresidue. Yield: 212 mg (86%).1248 S. ELSHANI ET AL.METHOD B3,4'-O-isopropilidenepyridaxine-5'-O-methyl{luorophosphonate (8). A mixture ofequal quantity of methyl phosphonic dichloride (128 mg, 1 mmole) and methyl phos-phonic difluoride (100 mg, 0.072 ml, 1 mmole) was dissolved in 2 ml of dry chloro-form and cooled to - 10 °C. Into the mixture, while stirring vigorously, a solutian of 1(1 mmole) and Et3N (0.6 ml, 4 mmole) in dry chloroform (3 ml) was succesivly added,during 30 minutes. The stirring was continued for another 30 minute s at r.t. Thesolvent was evapara ted and then 5 ml of dry ether was added. The precipitated Et3N-hydrofluoridewas filtered offand the ether was flash-evaporated. 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Synthesis of some pyridoxine and pyridoxal halophosphonates

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