TABLE I
__-~ Compd
I't-ItISl~: ~ I ~ C I ~ f i : ( l S l D I . ; S
O F S'-RlrnS~l'l'lr.'lI:lJ
sol,.en,sys,Pma
c
B
~
D
_.__ . ...
..
E 0,.31
I.'
0 . .i2 0.08 0.80 0 . x3 I), 84 S 1-Isoperitenylt ubercidin 0 . 8.5 0.84 0 . so 0 . 76 N6-Isopentenyltubercidin 0.81 0.8.5 0.4s 0.4i 0.04 0 67 0.66 0,6qi N l-Allytuberciditi 0 . s" 0,72 0 . .i.i 0.62 0 , 70 0 so N6-AIlyltriberciditi 0 . s.; 0.7.5 0.60 0.61 0.0:: 0 77 S 1-2-Phenosyet hyladenosiiie S6-2-Pheiiosyet hyladeircisiiie 0,so 0 S.5 ( 1 , s:i 0.6l) 0.47 0 , 7:; 0.07 0 , 7s 0.69 0.61 0.42 0.76 S l-Reiixyladenoriiie 0.73 ( 1 . s3 0 . S6 0.71 I)..;7 0 i!) N"-Reii~yIadeiiosiiie 0.0% 0 . s2 0 . S.5 0 .7 3 0 64 0.6:3 S I-n-Hesyladeiiosiiie 0 . s2 0 .S i 0 , SR 0.77 0 . 7., 0 . X!) N"-n-HesyIadetiosiiie 0 .S 2 0.6!1 0.62 0 . 5.7 N 1-n-Peiityladeiiosiiie 0 . 0'2 0.s1 0.00 0.73 I ) ,7.5 li S!I 0 .i 9 0 . si N6-n-Peiityladellosine 0 . so 0.S1 0,SR 0,66 0 . .io I).S2 N6-Phenyladenosine 0 .S i 0.72 0.40 0 71 0 , ,5:< ( ) . s.7 Ns-2-Et hoxyethyladeno*ine 0 . 7:; 0 , s2 0 , s.5 0 .6 i ( ) . .-I I [).is N6--2-Thienyladeiiosiii~ 0 . .-I7 I1. :3:< 0 , ?.-I I ) ,,i2 0.14 0 . !52 .idenosine 0.62 0.4s 0.:34 0 17 Tribercidiir (7-deaxaadeiiosirie) 0.23 0.60 0 .i 2 0 . 6:3 0 6h 0.66 S 1-Isopent eiiyladenosiiie 0.0.5 0 , 7s 0 . S7 0 , so 0.74: OLSS S6-Isopeiiteiiyladeliosiiie 0 .i s 0 . H6 0.72 0 . 7s 0 , i!l 0 61 0.46 0.7 3 Se-Fiirf iiryIadeiiorine* T h e solveiit systems rised for descending chromatography (Whatman No. 1 paper) (measrired by volrinie): -4,E;tOAc-n-PrOH-H.O (4:1 : 2 ) (ripper phase): B, i-PrOH-HGNH4OH ( 7 : 2 :1 ) ; C, i-PrOH-l% aqrieons (NH,)?SOd (2: 1 ) : I), I'-PrOH-coiiceiitrated HCH 2 0 1680: 170: 144): E, n-BriOH-HnO-ronceritrated ?\",OH (86: 14:5); F, n-BiiOH-AcOH-HrO i5:3:2). * Prircha.sed material. '1
TABLE I1 ~TV-.4BSOKPlION 8PEC'I.R.\ O F N6-SUBSTITVTED P U R I N E S U C L E O S I D E S p H 7.0 _-____ p H 1.0 ,-------pf{
______
Comprl
Tiiherridiii N 1-Isopeiitenylt uberridin" S6-Ihopeiitenyltrtbercidiii S '-AllyltIibeic.idin" S6-Allylt Irbercidiii X 1-2-Phenoxyet hyladeiiosiiie" N6-2-Pheliosyet hyladeiro-ine N l-Beiizyladeiioriiie,l NG-Beiixyladenosiiie
Xmxl;. m p
______ e
x
10-3
27 1 273 27.5, 230 273 274.5, 231 26 1 263 260 264
--___
hnr. mr
L
x
lo-:$
12.0---_-_ hm,,x.
mr
t
x
10-a
270 27:: 27.5 273 27,i, 210 26 1 26s 260 26h 2*;9 267.-5, 210 260 267.3, 210
270 272 ish 265,10-4 >10-4 i X lo-' io-^ x
10-6
1.7 X 6 x lo-' 3.6 x 10-5 :j
x
io-,?
t,ion of the adenosine analog tubercidin (7-deazaadenosine) decreased its act,ivity in the two leukemic cell lines but enhanced it, in Burkitt's lymphoma. It' is of iiit,erest that unlike IP,4, which inhibits line 6410 but not L K I D , K6-isopentenyltubercidin is inactive in line 6410 but is cytotoxic in LKID. The cultures of normal leukocytes (RPJII-5287) were insensitive to all of the K6-substBit'uted adenosines test,ed. Anot,her normal human leucocyt'e cell line ( R P I I I 7666, Gerner) was found to be moderately sensitive to S6-isopentenyladenosine.1YThe alkylated tubercidins were more inhibit.ory in the normal cell line than was tubercidin. The growth of Sarcoma 180 cells in culture (Table VI) was inhibited by some of these adensoine analogs. the most effective one being S6-furfuryladenosine. The adenine derivative, S6-furfurladenine, on t'he other hand was noninhibitory. Tubercidin was quit'e eff ective in this system, while it,s alkyl derivatives were less inhibitory. The act'ivity of S6-isopentenyltubercidin parallels that of I P A and because of the greater metabolic stability of 7-deazaadenosine as compared to adenosine renders it interesting for further study. (13) .I. T. G r a r e , .Jr., iinguhlished data.
A moderate, but qignificant increase in the wrvival time of mice bearing leukemia L1210 rewlted from treatmerit with S6-2-ethoxyethyl- and Sfi-furfuryladenorine (140 and 138%, reipectively, at 200 mg kg X 6), Sfi-benzyladenositie (111% at 125 m g k g X G ) , and Sfi-phenyladenosine (132% at 100 m g k g X 6). The remaining compouiidh produced increases in survival time ranging from 117 to 132%. Some of these compound, also exerted activity against Ehrlich ascites carcinoma and interfered with the replication of the hpleen focus forming virus, a member of the Friend leukemia viruh complex. l4 -\lost of the adenoiine analogs inhibited the growth of Escheizchaa colz at to 10-j -11, whereas the tubercidin derivatives showed little or no activity. In Streptococcus f'aecalis, however, the Sfi-substituted adenosines showed marked stimulation of growth. Since in this cell system adenosine itself is growth stimulatory it remain5 to be determined whether the effect of the analogs is exerted following cleavage of the side chain. Tubercidin i+ highly inhibitory in S. faecalis ( 2 X M), while the Sfi-allylanalog is noninihibitory and the S6-isopentenyltubercidin i. oriy moderately inhibitory (5 X -11). From the point of view of itructure-activity relationships it i i interesting to note that the Sfi-substituted adenine baqes which are good cytokinins yield nucleoside. which are inhibitory to mammalian celli.
Experimental Section IIelting points were determined on a IIel-Temp melting point apparatus and are not corrected. Uv spectra were obtained on a Cary 1Iodel 14 recording spectrophotometer. Opt,ical rotation was measured on a Jasco l l o d e l OIIl)-U17 5 optical rotatory dispersion recorder. The solvent systems nsed for descending chromatography are given in Table I below. Whatman paper No. 1 was used. Adenosine was piirchased from Nutritional Biochemicals Corp., 6-chloro-9-p-~-ribofiiransy1-9H-piirine and 6-fitrfurplamiiio-9-p-~-ribof~iraiios\-lpiirinefrom K & K Laborat,ories, Inc. Where analyse.: are indicated only b> elements, analytical resiilt,sobtained for those elements are within &0.4Cl, of the theoretical valiies. N6-2-Phenoxyethyladenosine (I).--Adenosine (2.67 g, 10 mmoles) was stirred and heated at 80" for a few miniites in 50 ml of DMF iint,il dissolved to a clear sollition. At 50", @-bromo(14) R . .\. Steeveu, personal comrniiniration.
*hciweti :itioiit sO-0Oc; priidric*t.. Heatiiig 2 hi. loiiger, showed i i o iir yield. T h e itqiieoiis soliitioii was filtered and the filtrate \vas evapoi.atetl to dryiiess. A 2..54-cm coliimii was prepared with 150 g of Celite 34521 a i d 70 ml of lower phase solvent A (EtOA4c-n-PrOH-Hp0, 4 : 1 : 2 ) and packed according to the met,hod of Hall.?* T h e evaporated prodrict Tva.; dissolved in the upper phaje of solvent X aiid :tbs:irbed on the coliimii. T h e coliimii was developed with the lipper phase scilveiit A at a flow rate of 125 ml hi.. The o p t i d deirsity of the eiiiiietit n-as moiiitored o i i a Bec*kniaii I)U-C\. -pecti,ciI)hotiiiiietei. aiid rec.oi,ded 011 a floiieywell recoidei,. The fiwt f i w , t i ( i i i h iotalliiig lL7 nil c~iiitaiiiedoiily the prodiict. Utirhairged tiihei.(idiii w a ~eliited . after 550 ml of hcilveiit. T h e solrit i o i i ( ~ i i i t : t i i r i i i g the pi,odrict was evaporated t o a syi'iip with a flash evapcii'atot', aiid i,eevaporated three times with 25-nil additions of abroliite EtOH t o dehydrate the giim. The grim ,tallized iisiiig N e C N as the solvelit. T h e crystalh washed with EtpO, and dried in t a c i t o a t 78" over P,O:: yield 0.393 g (63C;;), mp 160-162', [ C Y ] * ~ I I -71.8" i c 0.1044, 3 0 s EtOH-HpO). -4nal. (Ci,HisO,N,) C, H, S . N6-Allyltubercidin (X). ( a ) Allyl Bromide.-Commercial reagent grade material was fractioiiated and the fractioii boiling :it 60-70' was collected. ( b ) N1-AlIyltubercidin.-Tiiber~idiii ((),.TO() g , 1 .Ss mmoles), allyl t)i,omide (0.6S4 g, >.64 mmoles), aiid 7 nil o f IIJIF (frechly tlist,illed) were ireated a,. oiitlitied f o r I S . ( c ) W-AlIyltubercidin.-The syriip (~ititaiiiitigNL-allyltiihei,vidiii was ti,eated 13s oritliired for I S : yield 0.:310 g (54";) (based o i l total tither(-idiii),m p 130" der, CY]^% -65" (c 0.07675 g , :30(; EtOH-H?O). .4na(. (Ci4Hi&IS;I.O.lHnO) C, H, S. Assay of Cytokinin Activity.-The cytokiiiiii effect of the compoitiids was assayed with callris derived from the pit,h of PViscotisiii 38 tobacro plaiits iihiiig the method aiid media of llinashige aiid Skoog.2:' Resrtlts are given iii Table 111. Microbial Assay Procedures.-The microbial assays were cwt,ied o i i i according to techniqiies prtblished previoiisly.24 Eschrrichiu coli K-12 was grown iii the syiithetic mediiim of (;ray and Tatiim.25 S . ,faecalis 8043 was growii i n the mediiim of Flyiiii, d a(.,?6i i i the abseiice of piiriiies aiid pyrimidines, biit with 1 mpg of folic acid ml. T h e ccimpoiuids were added to ihe mediiim at 10-"-10-8 -11 for the tiibercidiiir; and 10-5-10-' .I/ for the K6-adeiiosirie aiialogs. Iiesiilts are given iii Table \-I. Effects on the Growth of Sarcoma 180 Cells in 1'ifro.-The u;mp iiiiids were added in aqiieoiis soliitioii to t,he ciiltiirea of SI80 cells growii as monolayers in T-15 flasks iri Eagle'sz7mediiim containing .ir; hor-e seriim. The cell5 were exposed to the comporuids for 6 days iiivolving three changes of the mediiim. Iliiriiig this time the eoiitrol ciiltiires increased The qiiairtity of cells was estimated by the
of Grad!, d rt1.,28 aiid by ploteiii d e t e r ~ ~ i i i i t t i o i i , ~The . . ? ~i.esiilt> :we giveir i i i Table \'I. Effect on Viability of Human Cells in 17i/ro.-Thc ~ ~ i t i i p i i~t l ih i t a.ere tested iii several hrimaii rrll lines gruwii iii hiispeiisioii nilt ~ i r e . These ~~ included cultures of myeloblastic leukemia (cell line RP?rII-6410), lymphoblastic leukemia (cell line R P N I LKID), Burkitt.'s tumor lymphoma (cell line R P J I I - H R I K ) , and ( i f iiormal human leiirncytes (free of viriis particles) ( R P l l I (cell line 3287). The c~c~nip~iiitrtis \ v ( ~ itlisholved ~ i l l I)irlhecwi phiisph;tte-biift'el.etl saliiie:" :tiid :itldrd t o l o i i viability (Table \.) :is mensiii~etlby dye exclii~ioti~ iisiiig a 1'; soliition of trypaii blite.:" The viability of the coiitroltem stitdied, being 85-94 ) r t h e LKII) system, 65-71 system, and 66-fQC; for the iiornial cells. Iii the .titdies dealiiig with the gt'ciwth-stimiilatiirg eW'ec.1:. of 1 he c ~ o m p ~ i i i i i d(Table s I \ ' ) , e 6410 re11 liite was growit f o r 5 days with vefeediitg at :: d;i t~esiiltitrgi i i six- to eightfold (*ell miiltiplic~atioiiwith ixthei. ,tatit viability. Thei,efoi,e, act iial cell c ~ i i i i i were t~ determilied. Effect of W-Substituted Adenosine on Leukemia L1210 in 1'wo.-Female I)BA/2 FIa-1)l) mice weighiiig 16-20 g were obtaiiied from the IlPJlI breeding c d o n y . Each animal was iiioriilated interperitoiieally with 106 cell> of lerikemia L1210 and treated oiic*edaily for 6 coiiseciitive days starting the day aft.ei, tiimoi' iiiocwlat ioti.
(21) T r a d e name of Jolins-.\Ianville Co. ( 2 2 ) R . H . Hall, Bzorhemirtri/. 4 , 661 (1965). (23) T. AIiirasiige a i d F . Skoog, I'hu.zol. Plnntorum. 16, 4 7 3 (1962). (24) A . I3locli a n d C. Coutsoreorgoporilos. Biochemiatr~.5, 3345 (1966); .\, I%locli, A I , H . Fleysiier, R . Tliedford, R . .\I. AIane. a n d R . H. Hall, .I. .Wed. Chem., 9 , 886 (1966). (25) C . H . G r a y and E. I,. Tatrim, Pro?. S i i t i . .Ar,nd. Si.?. 1.S.. 30, 404 (1911). (26) L. l f . FIynn. V, 13. \Yilliams. 1%. I.. O'Dell, a n d .\. G . Hogan. A n d . Chem., 23, 180 (19.51). ( 2 i ) H . Eagle, ,Si.ieiire, 130, 132 ( 1 0 5 9 ) .
( 2 8 ) .I. I;. G r a f l y , \ V . I..Liimmis. and C'. G . dtnitll. C r i i , w r K e . 5 . . 20, 1114 (1964). ( 2 9 ) 0. H . Loivry. N. ,I. Rosellroiinli. . \ , I,. 12arr, a n d R. .J. Randall. .I. Bioi. Chem.. 192, 265 (19.51). (30) G . E. AIoore a n d K. Ulricli. .I. Surgicrrl Re,\. 6 , 2 i O (1965): G . E . hloore. E. I t o , I
