6 - 0 ISOSTEHES ~ ~ 013. UHACIL . ~ K DT HMISE ~
Jaiiuary 1969
TAHLE I1 REL.~,LIVE TOXICITIES U F 6-0x1 .\.vu 6-Az.\ Isosmass O F UI~.ICIL . ~ S DTHYMINE
thymine.--A pievioris repot,t has described the synthesis of these compciiinds through the neqiierice: amiiioxyacetic acid -+ ethyl amiiioxyacetate -+ ethyl ureidooxyacetate 6-oxadihydropyriniiditie;6.6 however, iii the presetit study an alternate route was iitilized which is somewhat shorter. Ethyl ureidooxyacetate was synthesized by treating 20 g of hydroxyiirea'3 in 150 ml of EtOH with a solution of 5.83 g of N a in 100 ml of EtOH. The resulting solution was stirred for abolit 30 min a t room temperatiire, and then 42.3 g of ethyl bromoacetate was added dropwise over a period of about 2 hr; finally, the reaction mixtiire was stirred for an additional 48 hr at room temperatiire. The solvent u-as removed in z'acuo, the semisolid residiie wan repeatedly extracted (hot EtOA4c),and, ripon removal of the nolvent, there u-ab recovered 12.2 g of product which was rwrystallized (EtOAc), mp 122-124" (1it.j mp 125'). Cj-clization of ethyl iireidooxyacetate to 6-oxadihydrouracil was effected by dissolviiig 3 g of ester in EtOH f.50 ml) co~itaiiiiiig1 eyiiiv of NaOEt and allowiiig the reactioii mixture to staiid at rooni --f
r
Rlicroorganisiii
0 0 . 06 0.2 0 .6
lL
6 2
6 6 6
600 2000 >2000
>2000 2000 >2000
0.2
0.6
>2000
>2000
0.2
0.06
200
>2000
0.2
0.0B
60
0.6
111
~ - O ~ ; . I ~ ~ H Y ~ I ~GI(ON'TH O ~ I ~ . I\NCHI BLI T I O N
IN
f,. arabinoslas
lil- r K . \ C I L .\SU ~ E l U v . L ' r l v E s
Grojytli response, L/c transi1iission"------------------
ti-Oxadiliydrouracil, f i g / 1111
Concn for coiiiplete groivtli inllili, pg /tiil----ti-Oxa6-Oxadiliydro6-.lzadiliydro6-.lzauracil uracil tliyniine thymine
->
E. coli 9723 E. coli IT E . coli Texas L. dextranicum 8086 L. arabinosus 17-5 S . jaecalia 8043
T.\IjLE l h V E l L s . \ L OF
153
h-one
0.02
28 64 !I3
23 27 34 94
Uracil, pg/iiil--------0.06
___-_ 0.2
0.6
0.02
23
23
19
33 87 94
21 20 97
.)*I
-_
Uridine, pg/ml----0.06 0.2
24 24 44 9U
2 88 01 6 89 78 20 83 1)eterniiiied iisitig a Spectrotiic. 2u speclrophotonieter ai 600 nip.
temperatiire for 4 hr to proditce a gelatiiioiis mixture. The solveiit wah removed in L ' U C ~ aiid , the resrilting residue was dibsolved iii a miiiirnal qiiaiitity of H,O; the soliition was adjiisied to aboiit p H 7 with coiiceiitrated HC1 aiid the resulting precipitate wah recrystallized (HlO) t ( J yield 2.95 g of produc.t,-mp lX0-18lo (1it.j m r ~182"). 6-Oxadihydrot'hymine was prepared in a comparable two-step synthe*iu by coiidetisiiig 3 g of hydroxyiirea with 9 g of ethyl a-iodopropi:)iiate iii the presetice of EtOH (200 ml) containing 2.2 g of KOH, and heatiiig the reactioii mixtiire under refliix for aboiit 2 hi,. The d v e i i t was removed in vacuo, aiid the resdting oily 1,esidrie was takeii iip iii EtOH (100 ml) coritaiiiing 1 eqriiv of SaOEt aiid allowed to staiid a t room temperatiire for 2 hr. Aftel, removal of the wlveiit, the yeslilting solid residrie was dissolved i i i I1,O atid adiitsted t o u€I 6 with coiiceiitrated IlCl nt id t hc precipiiated inaterial wa.~rec tallixed from II,O to yield l.!)g ( J f prodrict, nip l.i2-l.iBo (lit.6 nip 133').
Results and Discussion 0-Oxadihydrouracil is significantly inhibitory to growth of :t number of microorganisms as indicated in Table 11. Two representative systems were chosen for more ext'ensive study, L . arabinosus and E . coli, and the reversal of toxicity by uracil and/or its conjugate derivat'ives is presented in Tables I11 arid IV, respectively. In I,. arabinosus, 6-oxadihydrouracil is reversed in :t c.ompetitivelike manner by uracil over a 30-fold range of concentrations with an inhibition index (rat,io of analog to metabolite required to inhibit growth cmnpletely) of about 10 at the upper levels (Table 111). Uridine also reverses the toxicity of 6-oxadihydrouracil in a comparable fashion with an inhibition index of about 10 over a 30-fold range of increasing substrate concentration. Uridylic acid even a t high concentrations is relatively inactive in reversing the toxicity of ~i-oxadihydrouracil,presumably due to the inability of such phosphate derivatives to penetrate cell walls. < I : < ) 11. L)eghenglii, 0i.u. S j j r , . , 40, 6 0 (l!ItiO)
0.6
23
22
31 64 97
29
Uridylic acid, 20 pg /!Ill
23 29
83 71 84
l'recursors of uracil biosynthesis and related conipoullds esserltially irleffective iI1 reversing t,he iiihibit,ory effect, of 6-oxadihydrouracil, l4 and compar-
lisv m s . OF ~ ~B-OXAUIHYDRO UIL.LCIL G a o MYH IN HIHI ,PION I N E . coli TEXIS BY URACIL 6-Oxadihydrouracil, rg/ml
0 0.6
7
ti 20
----Groivtli 7 -
None
10 19 94
response, yo transmissiona-Uracil, pg/nil 0.06 0.2 0.6
19
18
2.0
19
19
34 83
43
l!) 83
60 200 0 Determilied using a S p e c k " 600 mu.
24 90
-*>
1 3
20 ipectrophoiometer set at
able results have also been reported in the case of 6a ~ a u r a c i l . ~I n this respect it should be noted that t'he aza analog possesses potential pyrimidinelike nuclear structure, whereas the corresponding oxa derivative cannot' taut'omerixe to such an aromaticlike ring system because of the divalent, character of tmheoxygen atom. Thus, thc oxa ;~nalogis in reality ari isostere of dihydrouracil. Since both of these analogs appear tmo function as metabolite antagonists of uracil in these microbiological systems, and neither are reversed appreciably by dihydrouracil, orotic acid, or dihydroorotic acid, the enzymic sit,e of inhibition mould suggest a metabolic block after the formation of orotic acid in (14) The following metabolites did not produce any significant reversal of growth inhibition induced by 2 p&ml of 6-oxadihydrouracil a t their limit of soli1bilit)- in t h e assay medium: &alanine. P-iireidopropionic acid, X-
carI>ainuylaayariic w i d , orotic acid, dilisdrourotic acid. and diliydrouracil.
