341
Vitamin B6Analogs. Synthesis and Biological Activity of Homologs of Pyridoxal 5'-Phosphate1
The synthesis from acyclic precursors of norpyridoxal 5'-phosphate (S'IIa) and a-methylpyridosal j'-phuuphate (VIIb), compounds in which the methyl group at position2of pyridoxal 5'-phosphate ( P L P ) has been replaced by H or C2H5,is described. Both compounds replace P L P as a coenzyme for purified glutamate-oxaloacetate apotransaminase (GOT) of pig heart and for crystalline apotryptophanase (TPase) from Escherichia coli, hut with varying effectiveness. S'IIa is a more efficient coenzyme than P L P for GOT, as judged either by its affinity for the apoenzyme, or the maximum velocity of the react.ion catalyzed by t,he reconstituted enzyme; l ? I b is less effective than P L P for both criteria. Both T I I a and VIIb are less effective than P L P as coenzymes for TPase. The results show that, the methyl group of P L P is not a prerequisite for the coenzymatic activity of this compound.
l'or a closer assessment of the relation of structure to function of vitamin B6, a comparative study of the (+hemica1arid enzymological properties of analogs of pyridoxal phosphate in which the methyl group a t position 2 is replaced by hydrogen or by an ethyl group is important. The latter compound has been synthesized3 but is riot currently available; the former compound has not been made. Both of the corresponding analogs of pyridoxine, 3-hydroxy-4,5-bis(hydroxymet hyl)pyridine4 and 2-et hyl-3-hydroxy-4,.i-bis( hydroxymethyl)pyridine,6 have been previously synthesized but only from difficultly available starting materials or in poor over-all yield. To obtain these compounds in sufficient amounts for subsequent synthesis of the corresponding analogs of pyridoxal phosphate, their synthesis via the more recently developed oxazole pathway' was undertaken. The appropriate K-formyl-a-amino acid esters I a itlid I b (Scheme I ) were prepared by formylation of the corresponding aniirio acid esters (Sheehan and Ya11g9). 1"nylatioii of the ester hydrochlorides resulted in lower yields. Cyclization of I b with P205in chloroform by the proredure of Karrer, et aI.,'O gave the oxazole I I b in 47y0 yield, while I I a could be prepared from I a in only 3-3y0 yield under the same conditions." Altered reaction conditions (see Experimental Section) gave no better results. The 5-ethoxyoxazoles I I a and I I b underwent Diels-Alder condensation with diethyl nialeate to give the bicyclic compounds 111,which were not isolated but were converted to the diesters IVa or IVb, respectively, with ethanolic HCI. IVa was isolated as its hydrochloride in 53y0 yield arid IVb as the (1) Supported in part by a grant (AL1-15i5) from t h e U. S. Public Heltltli Service. (2) To xliutn inquiries should be addressed. (8) SI. Ikawa and E. E. h e l l , J . Am. Chem. Soc., 76, 6 3 i ( 1 Y 3 1 ) . (4) 13. Van der \\ 81, Til. J. de Uuer, and 1%.0 . IIuisman, R e c . Trac. C h i n , 80, 2 0 3 (1961). ( 5 ) P. A I . Gadekar. J . L. Frederick, and E. C. de Henau, J . ,\fed. I'harTfl. Chem., 6, 531 (1962). (6) H. .i. Harris and .i. S . \\ilson, .I. Am. Chem. Soc., 63, 2526 (1941). ( i )G. T a . Kundratyeve and C. Huang, Dokl. Akad. N a u k . S S S R , 141, 628, 861 (1961). (8) E. E. Harris. R . A Firestune, K. Pfister, 3rd. R. R. Boettcher, F. ,J. Crosb, K . B. C'urrie, h l . hlonltco, E. R. Peterson, and IV. Reuter, J . O r y . Chem., 27, 2705 (1982). (9) T. C . Sheehan and D. H . Y m g . J . Am. Chem. Soc., 80, 1154 (1958). ( I O ) P. Karrer, E. .\Igamiclii, H. C . Storm, and R. \\idmer. Hell,. Chim. B c l a , 8 , 20.5 (1925). (11) T h i s is another example of the lower rate of formation and lower stability of less siihstituted ring compounds. as compared with alkyl s u b stituted V I I E S . For furtlier examples, see L. Eliel, "Stereochemistry of Carbon Coinpuunds," 1IcGraw Hill llook Co., Inc.. New York N. Y.. 1962 . p 196.
SCHEME I
! I R Io
H
HNI CI - R C-OEt HC 1 I I1 0 0
PzO5, chloroform
HCI, ethanol
LiAIH4
I
II VV ab:: R R == H CH2CH3
EtO$uCOzEt
C02Et
R=H I l l b: R'CHzCH3
1110:
CH20H
Ho@H20H
1
110. R = H Ilb: R=CHzCH3
1 1 I 15'
la: R = H I b: R = C H z C H j
OEt
HC=O
MnOa
R
H o ~ c H 2 0 H
R Va: R = H Vb: R = C H z C H j
1
Vla: R= H VI b: R = C H 2 C H j p-Toluidine H3P04 t p205
0 0 cHC-0 H Z 0 p 0 3 H i
R
VI1 a : R = H VI1 b: R 'CH2CH3
free ester in 51% yield. Reduction of the free diesters with LiA1H4I2gave t'he pyridoxine analogs Va and T'b which were isolated as their hydrochlorides in 30 and SS7& yield, respectively. Oxidation of Va arid T'b with manganese dioxide gave the corresponding analogs of pyridoxal VIa arid VIb which were converted by phosphorylation of a11 appropriate Schiff baseL3t(J the cwrresponding aiialogs of pyridoxal phosphate VIIa arid VIIb. Previous studies reviewed e l s e ~ h e r e 'have ~ shoivn that compound Vb (a-methylpyridoxine) does riot replace pyridoxine as a vitamin for Cemfosfomdla ulmi xiid A%"wriyces cat~lsbet~gensis or for ruts depleted of vitamin Be prior t o testing the analog, but acts instead as a competitive aiitngonist of pyridosine. However, (12) R. G. Jones and E. C. Iiornfeld, J . Am. Chem. Soc., 73, 107 (1951). (1:i) M. Llurakami. A I . Iwanami, and T. Numata, Japanebe Patent 18,749 (1985); Chen. Abstr., 56, 2068 (1966). (14) E. E. Snell, I'itamius Hurmones, 16, i i (1Y58).
TABLE I1 . ~ n s o i : i ~ ~ 31 i . ~\xInt.\ o~
ANI)
;\IoL.\R ABSO~~B.\KCE OF Ax.\r,oc:s OF l'wxuos.\L _..__
Compound
0.1 S HCI
w-~~eth!.lpyridi,unl(1'1h)
'LS!)(SOOO)
I'yl~idl~x:\l
28s (91 00)
. A N D PultIuos.\L
l'rtosl~1t.\wcL
Amax, m p (e)--------
.- ._
p H 7.0h
3 1 s(~400'1
0.1 A' N a O H
239 (S300) 300 (.iiOO) 302 ( I SOO) 240 (s:zoo)
I n acid phenylhydrazineC
41 n (21 ,
.m)
410 (23,000)
300 (6100) 3!10 (1700)
S(Irpyridoxa1 (T-Ia)
283 (6400)
313 (4200)
241 (S900) 299 (;,000) 383 (.i00) 390 (6000)
41.i (18,.i00)
295 (8200) 389 (.i800) 410 124,000) 33.5 (1,500) Pyi.itlox:tl 5'-phoq~h:lte" 2x3 (72(10) 3% (.-),iOO) 3sn (ti600) 410 w,,;oo) 3x4 (l:;oo) Noi,pyritlcix:il .i'-pho.?phate (YIIa)rL 200 (47(10) 351 (2900) 3s2 (43(10) 413 (21 ,000) 32s (900) '6 Qitaiititative determinations of organic phosphate showed OOC,, of the theoretical for pyridosal phosphate, 9Ss for a-methylpyridosal phosphate, and 83% for nnrpyridoxal phosphate. The triie molar absorbancies of the latter compolind, I herefore, may be Procetiilre of R'ada slid Sne112Lfor determilintion I n 0.1 -21 potassiiim phosph:tte briffer. somewhat higher than those tabulated. of pyridoxal and pyridoxal phosphate.
a-lIethylpyridoxa1 j'-phosphate (TIIb)cL
B. 4-Ethyl-5-ethoxyoxazole (IIb).-P?Os (100 g ) was suspeiided in 15(30 ml of freshly distilled, dry CHCI,. T o the refliisiirg siihpeiisioii, 102.7 g of I b was added during a period of 1 hi,. Oiie hoiu later a j0-g portion of PyOswas introdiiced, and :rfter 2 til. ariother 50 g of PpOj was added. The siispensioii was i,cHiised for a total of 5 hr. After standing overnight at room tcamper:it tire) the chloroform was siphoned off from the iiisolitble lltrnps of P20r,concentrated to 150 ml, and extracted with a little concentrated ayiieous KaOH. The insoliible P205 was dissolved by slowly adding S a O H pellets arid ice so that the temperatiire wa3 kept a t 0-5' arid the pH a t 3-5. After solution \vas complete, the NaOH extract of the CHCL layer was added and the pH broiight to 9. T h e alkaline soliition was extracted once with ,500 ml and three times with 300 ml of ether. The ether extracts Jvere combined with the chloroform layer and concentrated at atmospheric pressiire imtil the boiling point began to rise above $0'. The residiie was distilled under vaciium; the oxazole IIt) di3tilled at 71-73' 120 m m ) ; yield 46.0 g (477;); t P 4 1.002; A:], I "228 mp ( E 4160); nmr spectrum (no solvent), 6 pi'otoii:, as two siiperimposed triplets a t 1.19 and 1.3%ppm, 2 protons as a qiiadruplet a t 2.50 ppm, 2 protons as a qiiadriiplet n t 4.24 ppm, 1 proton as a singlet a t 7.69 ppm. A n d . Calcd for CiHliSO?: C, 50.56; H, i . 8 5 . Found: C, 59.63; H, 7.38. Pyridinedicarboxylic Acid Esters IVa and IVb. A. Diethyl 3-Hydroxypyridine-4,5-dicarboxylate(IVa) Hydrochloride.-IIa (13.3 mmoles) was mixed with 2 equiv of diethyl maleate and heated in a roiind-bottom flask with an air-cooled condenser for .i hr at 1l.i'. After cooling, 13 ml of dry HC1 (1.14 * \ ) in absoliit,e E t O H was added and the mixture was heated oii a steam hath for 20 min. By cooling to -20" and adding ether, 1.03 g of I\.a. TIC1 precipitated. From the mother liqnor two further cwps of 0.69 and 0.24 g coiild be collected; tot8al yield, 1.95,g (.j;
