Naj. 1967
1-DEAZAURIDINE B S D
321
2’-DEOXl~-~-DEAZAURIDIXE
CHARTI
There have been no reports of synthetic deaza analogs of nucleosides where the nitrogen bonded to the sugar has been replaced by carbon. Although several C-glycosyl compounds have been found in plantsjgthe sole compounds reported in bacteria and mammals are pseudouridine (2) and the formycin antibiotics.’O 0
o8
HOH-C
‘ RO
(9) L. J. Haynes, d d r n n . Carbohgdrnte Chem., 18, 227 (1963). (10) (a) F. F. Davis and F.K. hllen, J . B i d . Chem., 227, 907 (1957); (b) C . Su and F . IY. Allen, Biochim. B i o p h y s . A c t a , 32, 393 (1959); ( e ) IT. E. Colin, J . Biol. Chem., 235, 1488 (1960); (d) R. K. Robins, L. E. Townsend, I‘. Cassidy, J. F . Gerster, -1.F. Lewis, and R. L. Miller, J . Heterocyclic Chem., 3, 110 (1966). (11) (a) R.11.. Langley. J . A m . C h e m . SOC.,78, 2156 (1956); (b) T. L . V . Ulbricht, Tetrahedron, 6, 225 (1959); (c) R. Shapiro and R. W. Chambers, J . A m . C h e m . SOC.,83, 3920 (1961); (d) T. L. V. Ulbrioht, Angeu. C h e n . I n t e r n . E d . E n d . , 1, 4 i 6 (1962); (e) JV. h s b u n a n d S. B. Binkley. J . Org. C h e m . , 31, 2215 i1966). (12) 11. Bobek, J . F a r k a s , and F. Sorm, Tetrahedron Letters. 27, 3115 (1966). (13) (a) H. Gilman, Org. Reactions, 8, 258 (1954); (b) S. T’. S u n t h a n k a r a n d H. Gilman, J . Org. Chem., 16, 8 (1951). (14) H. J. den Hertog, J. P. Wibant. F. R . Schepman, a n d h.v a n der \Tal, K e c . T r a v . Chim.,69, i o 0 (1950).
OR
/
RO
I
HO 2 OH
The synthesis of 1-deazauridine 5’-nionophosphate (la) and 2’-deoxy-l-deazauridine 5’-monophosphate (lb) requires the formation of a C-C glycosyl linkage between the pyridine portion and C1of the respective sugar. Appropriate protection of the oxygen functions of the sugar and the pyridine would permit the use of a route involving alkylation of the protected halosugar by the pyridine derivative. Successful application of this approach has been reported using organometallic (lithium) derivatives of pyrimidines” and triazines.12 Th~iq,initial attempts in the synthesis of the title Compounds centered on the procedure used in the synthe$is of pseudouridine.llc >Letallations with n-butyllithium involving the replacement of a hydrogen atom of an aromatic system adjacent to a hetero atom are ~ o m m o n . ’ ~Model reactions (Chart I) in the pyridine series showed that 2,6dimethoxypyridine (3a) was converted to the 3 4 t h ium derivative (4a) using n-butyllithium. The position and extent of metallation mas established by treatmerit of 4a with solid CO, to give a 25% yield of 2,6dimethoxynicotiiiic acid (5a). Alternatively, 4a v a s prepared cia the halopyridine derivative. Dibrominatiori of 3a according to the method of den Hertog, et u1.,l4 gave 3,5-dibromo-2,6-dimethoxypyridine(6a). l\lonodebromination of 6a by treatment with 1 equiv of n-butyllithium followed by hydrolysis gave 3-bromo2,6-dimethoxypyridine (7a). The latter also mas obtained by low-temperature bromination of 3a. Using the monobromo compound 7a for metallation substantially improved the yield of 4a since treatment with COS gave 55% carboxylation (5a). The relative ease of hydrogenolysis of a benzyl ether under mild conditions prompted the use of the benzyl protective group in the pyridine derivative. 2,6-Diberizyloxypyridine (3b) was prepared by treatment of
/
0 OJ-NAO H
H
8
-
10
n
O b A O H 9 a, R = CH,; b, R
=
CH,C,,H,
2,6-dichloropyridine with sodium benzyloxide. Studies on the ease of hydrogenolysis revealed that controlled reduction to 2,6-dihydroxypyridine (8) ( 2 moles of H,) or glutarimide (9) (3 moles of H2) was feasible. Following the sequence in Chart I, 2,6-dibenzyloxypyridine (3b) was treated with bromine and a base to give 3-bromo-2,6-diberizyloxypyridine (7b) which was converted to the lithium compound (4b). The position of metallation was shown by carboxylation to 5b. Hurd and demonstrated in the reaction of an organolithium compound with halosugars that the reactivity of the lithium compound prohibited selective reaction a t the halogen-bearing carbon. Poor yields in the pseudouridine synthesis1lc .cries for the riboRe (14) and the 2'-deosgribosc. (181 rompounds. Ubiiig 3-(2--deosyribofuraiio~yl~-~,(~-clibemyloxypyridine (18) the furanosyl nature of t I i v ring was established b y lacali of periodate roiisumptioii a t p H 7 (Table I ) . A4t~1pH of 1 ..jslow conmniptioii ol' :\yerage 0.95 mole of period:tte,'inole of 18 TWS ohscrvcvl. iiidicaat iiig equilibration t o t lie ribopglaiiosc. 4t ruvt ure (19). .Idditioiial .. 1hu-. hwse ocluilibrat,ioii results i i i :til apprositn:ir(~ (i0:40 ratio nitli the ribofurano,;e structure (18j ])IY>dominating. 1he nnir spectrum of ;~-("deoxy-D-ribofuraiiohyl)-".ciclibeiizyloxypyridirie (18) supports the assigned ,strlli>ture. S o attempt has been made t o assign the coiifiguration a t the ariomeric position due t o the complex pattern observed which suggest's a mixture of the 01 niitl 3 :momers. Hydrogenolysis of the diberizyloxy compouncls 14 ;ind 18 was hampered by instability of the produt ('
r .
r.
tioii at, 340 nip, t,he wuveleIigt,h usccl in thc spcvt photometric aisay for both eiizynics. Thuq in w ~ i trast to preliminary reports,29 none of the c*onipouiid.: displayed significant inhibition. The results are summarized in Table 11. ,lbsorpti(i11 uf light at 340 nip TABLE I1 1:ESULl'S
O F E K Z Y M E ISHIBITIOS
T1~yrriidvlate ----s>.ntlietase"-~-;\lax concn Inliili, tested. .If CC
Ctiinyd
the attendnnt problems in purification. Catalytic reduction of 14 or 18 in ethanol-hydrochloric acid follo\\-ed by lyophilization, extraction, and precipitatiori from alcohol-ether mixtures gave t'he respective products 3-n-ribosyl-2,6-dihydroxypyridine (21) and 3-( 2-deoxy-n-ribosyl) -2, 6-dihydroxypyridine (20). tempts to employ chromatography or crystallization for purification resulted in rapid decomposition to insoluble red materials. The instability of 20 and 21 prohibited studies to determine the cyclic structure of the sugar and the anomeric configuration a t Clt. Several authors*a have noted the ease of chemical oxidation of 2,6-, 2,3-, and 3,B-dihydroxypyridines to the corresponding azaquinones. -4lt'hough Ames and coworliersZ3bwere able to isolat'e 4-methyl- and 4,s-dimethyl-2,3,6-trihydroxypyridine the unsubstit'uted 2,3,(5-triol has not been reported. Reductive acetylat'ioii (zinc-acet'ic anhydride) of 2,3,6-hydroxyazaquinone is reported t o yield 2,3,6-triaceto~ypyridirle.~~~ Solutions of 2,B-dihydroxypyridine a t pH values varying froni 2 to 12.5 were found to air oxidize a t varying rates with alkaline and neutral oxidations proceeding i I i several hours. The ult'raviolet absorption pattern observed for the oxidation product,? was dependent on the pH of the oxidation media. Ensign and Rit,teribergZ4 also studied this oxidation and reported that, 2,6-dihydroxypyridirie undergoes spontaneous oxidation to give a pignient that has t,he charact'erist,ics of azaquinones which arise from the chemical oxidation of trihydroxypyridines. Thus, the instability of the reducation products 20 and 21 is attributed to spontaneous air oxidation to the corresponding 'L,S,B-trihydroxp coinpounds follon-ed by rapid oxidation in neutral or basic media t'o 0- or p-azaquinones.25 Biological Studies.-The in vitro studies were carried out on purified preparations of t'hymidylate synthetase and dihydrofolate reductase. Det'ails of the assay method in this laboratory have been d e s c r i b d Z 6 Thymidylate synthetase vas isolated according to the method of Wnhba and Friedkinz7fromE. coZiB. Dihydrofolat'e reductnw was purified from chicken liver according to the procedure of 1\Iathews and Huenrielieiis.28 Difficulties wei'e encaountered in the assay of the hydroxypyridiriw 8, 20, and 21 due t o iiistability in the assay media and the attendant changes in absorp(23) (a) J. H. I3oyer a n d S. Kruger, J . A m . Chem. Soc., 79, 3552 (1957); (b) I). E. .\nnes, R. E. Bowman, a n d T. I;. Gre Chem. Soc., 3008 (1953); ( c ) .I. A . RIoore a n d I:. .I. Marascia, .I. Ani. Chem. Soc., 81, 6049 (1959). (24) J. C. Ensixn m i l S. C'. R i t t P n l i r r p , .I. R i d . C ' h ~ n i . , 239, 2283 ( I 9 f i A ) . 123) Professor .James McCliesnry a n d Professor Ralph .\dams, UniverFity of Kansas, are acknoa lediied for useful disctiasions on t h i s point. (26) SI. 1'. Slertes and h-. R. Patel, J . X e d . Cirem., 9 , 868 (1966). ( 2 7 ) .I,J. . \\'ahha a n d SI, Friedkin, J . Biol. Chem., 237, 3794 (1982). (288) ('. I\. S l a t l i e a s and I:. SI. Huennekena, ibid., 238, 3536 (1963).
8
4.2x
10-5
