Journal $Medicinal Chemistry 0 CopyTklht 1967 by the American Chemical Society
VOLUIIE10, NUMBER3
APRIL26, 1967
Irreversible Enzyme Inhibitors. LXXT.’.~B~ Inhibitors of Thymidine Phosphorylase. I. Mode of Ribofuranose Binding B. R.
BAKER
Department of Chemistry, Universzty of California, Sanfu Barbara, California 93106, and Department of .Zledicinal Chemistry, State University of .Yew Yorb ut Buflulo, Buffalo, S e w York Received October 18, 1966 5’-Deoxythymidine ( V ) was about as good a substrate for thymidine phosphorylase as was thymidine ( I ) ; both I n contrast, 3’-deoxythymidine (IV) was not a substrate and a 20-fold loss in had about the same apparent K,. binding, compared to thymidine, was noted; thus the 5‘-hydroxyl of thymidine is not necessary for binding, but the 3’-hydroxyl is necessary. Replacement of the furanose oxygen of thymidine (I) with methylene (TI)gave a greater than tenfold loss in binding, but this result could not necessarily be attributed to binding by the furanose oxygen. When the 5’-hydroxyl of thymidine (I) was acylated with the highly polar carbamoyl group (VIII), a 40fold 1 0 s in binding occurred; with less polar but bulkier acyl groups such as dimethylcarbamoyl (IX) or carbophenoxy, only a six- and twofold loss in binding occurred, respectively. The possible biological significance of repulbioii of polar groups a t 5’ position of thymidine is discussed; also discussed are further modifications of thymidine that could lead t,o more specific inhibitorb of thymidine phosphorylase from different species and tissues.
Thymidine phosphorylase is an enzyme that catalyzes pho.phorolysis of the nucleosidic linkage of pyrimidine 2’-cleoxynucleosides, such as thymidine (I), with forniatioii of 2-deoxy-a-D-ribofuranose l-phosphate (111) and the pyrimidine (11). This enzyme has been isolated from a variety of plant, animal, and bacterial
0
HOCH,
I
+
I1 HP04’-
+
HOCHz OH ’
popo(o-~ 2
1
OH
’
I11
-4lt’hough its main function appears to be c:ttabolic, some bacteria and tumors can utilize t’he reverse reaction anabolically under stress of certain dietary or genet’ic deficiencies. A particularly good source for the enzyme is Esch.eiichia coli B since thymicline phosphorylase is readily separable from uridine phosphorylase.3 I n contrast, from some mammalian sources.
(1) This work was generously supported b y grants Ch-05845, CA-05867, and C.1-08695 from the National Cancer Institute, U. S. Public Health Geri-ice. ( 2 ) For the previous paper of this series, see B.R . Baker and D. V. Santi, .I. 3 f e d . Chem., 10, 62 (196i). (3) X’. E. Razzell and H. G. Khorana. Biochim. Biophys. Acta, 28, 562 ( 1%X).
295
sources t’hese two enzymes are not’ separable and may be one and the same enzyme;*>j however, separation of these two enzymes from some mammalian sources has been achieved.6 There are two main chemotherapeutic interests in thymidine phosphorylase, namely, (a) its ability to convert ;-fluorouracil (FU)7to its 2’-deoxy-p-D-ribofuranoside (FUDR),’ the first step in the intracellular activation of FC to FUDRP7 via thymidine phosphorylase*,g and thymidine kinase,B~lO~ll and (b) the detoxification of prefornied FUDR7 by cleavage to FC,9then further catabolisni to a-fluoro-@-alanineby ot’her enzymes.” Since some tumors fail to detoxify FU or FUDR due to the genetic deletion13of t’hese e n z y m e ~ , ~it’ has ’ ~ been proposedI4 that the selective action of F U on certain tumor lines is due to the lack of one of the detoxification enzymes in a susceptible cell line, and its presence in less susceptible normal tissues. If an inhibitor of thymidine phosphorylase could be designed such that it would inhibit phosphorolysis of FUDR in a t’uinor cell line with little inhibition of the phosphorylase in normal tissues, then such an inhibitor would be a useful (4) T. A. Krenitsky, J. IT. Mellors, and R. I -ribofwanme)
(
I
H ( 1) -ribop>ranose)
SXIT
'0
'nould be compared with I-methylurnc~il(SIII) K L L ~ complesed 200-fold lesi efEertively than I'UDR. The four hydrouyal1iylur:ic~il. (STY-XVII) were complexed 2-%fold better thnii 1inethylui~acd, but 60-SO-fold less effertivcly t h m l~L711154 ss n-c4Fr9 7 ..i 31 22 Thymidine phouphor e from E . coli €3 was prepared n i i d a+a~-eclwith 0.4 m31 FUDRi as described in the Experimental Section. See ref 22b for synthesis of these compoi1nd.s. Ratio 1 1 f conceiitratiori of inhibitor to 0.4 m X FUDR e d m a t e d to give .?Or; inhibition. d Since 20% inhibition is readily detectable, t,he (aciiiceiit,ratioii for 50% inhibition i.3 a t lea?t four times greater t,haii the concentration measured.
:I-
,
H
0' 11
XXI\
n1ay 1967
IRREVERSIBLE ESZYNE ISHIBITORS. LXXV
301
Inhibition of Thymidine Phosphorylase.-An assay mis of 3.00 ml of buffer, 0.15 ml of enzyme solution, and 1.65 ml of water was prepared; if the substrate or inhibitor was dissolved in water then the assay mix contained 0.60 ml of DMSO and 1.05 ml of water. Five pairs of tubes were placed in a rack so that the back tubes could serve as zero-time tubes. I n each tube of the pairs 1 and 5 were placed 50 fil of DRISO and in pairs 2-4 were placed 50 pl of DMSO containing vaTing concentrations of inhibitors. The biggest error in the assay could occur if the Experimental Sectio~P DMSO solution was not placed near the bottom of the tube: if any droplets were above the half-way point of the tube, the Thymidine Phosphorylase.-E. coli B cells were broken in a tithe was replaced. French pre,wire cell a t 1400 kg/cm*. The 4 5 9 0 % ( N H ~ ) Z S O ~ In each of the ten tubes was placed 400 pl of assay mix and the fraction used for thymidylate synthetasezza~~a also contained contents were then mixed with a F’ibro Jr. Mixer3$after the addition thymidine pho~phorylase.~The final volume from 40 g of frozen to each tube. I n each of the back zero-time tubes 1-5 was placed cells w a s 4 i ml. 300 p1 of 1 Jf KOH, then the contents were mixed. T o the five Solutions. The buffer employed23 was 0.2 Jf in succinate and front tubes were added 50 p1 of 4 m J l FUDR at 30-aec intervals, in arsenate adjusted to p H 5.9. A 4 mM solution of F13DR7,34 the starting time being noted; care must be taken to get no dropwater was st.able indefinitely and was used a s the substrate.9 lets on the upper half of the tube and the contents of each tube Dimethyl sulfoxide (DMSO) wa* a reagent grade purchased were niised after each addition. Then 50 pl of 4 mM FUDR %-as from J. T. Baker Chemical Co. added to each back (zero-time) tube: when the additions Enzyme Standardization.--Ail assa>- mis wm prepared with were complete, the tube contents were mixed. 1.50 ml of buffer, 75 p1 of enzyme solution, 0.30 ml of DLISO, Assay.-Five 1-ml cuvettes were placed in front of each pair of arid 0.83 nil of water: total volume = 2.70 ml. If more or less tubes. The optical density of the back tubes was then read iii. enzyme solrition was employed, the water was adjusted accordeach of the cuvettes, the cuvettes being rinsed, dried, and reingly to give the same total volume. placed at the respective position in front of each pair of tubes. I n each of five t,est t,ubes was placed 0.45 ml of assay mix. To At 15 min from zero time, 300 pl of 3 A l KOH was added to the tribe 5 was added 300 pl of 1 JI aqueous KOH; this tube served front tribes 1-5 a t 30-see intervals, the contents being mised after a> the zero-time tube. To each of the tubes was added 50 fi1 each addition. The optical density of the front tubes was then of 4 m J I FUDR7 a t 30-see intervals, noting the starting time. read in the respective cuvettes. The optical density change The optical density of tube 5 was then read in a 1-ml cuvette on a (front minus back reading) was noted for each pair of Berkman DG spectrophotometer. tubes. At the end of 5 min, 300 p1 of 1 .If KOH was added to tube 1, Tubes 1 and 5 were enzyme controls, that is, the velocity withthen the optical density was read in the same cuvette used for oiit inhibitor, T’o; these VOvalues usually agreed within 375 and, tube 5. -4t 10, 20, and 30 miii from zero time, tubes 2-4 were if the difference was greater than 67
