May 1969
PHAHIVACOLOGICALLY
plain the greater potency of the analog compared to adenosine. In order to investigate the influence of similar 2-chloro substitution o n the pharmacological activity of AMP and ADP we have synthesized 2-chloroadenosine 5'-phosphate (2-chloro-AMP) and 2-chloroadenosine 5'-diphosphate (2-chloro-ADP) and compared the effects of these analogs with those of AMP and ADP on the arterial blood pressure of rats and on the aggregation of sheep platelets. In addition, a preliminary study has been made of the substrate specificity of these analog nucleotides for the enzyme adenylate kinase (E.C. 2.7.4.3) 3vhich catalyzes the reaction 2.kDP3-
e AMPZ-
f ilTP4-
Adenylate kinase is present in many tissues8 arid in plasma' and could conceivably be responsible for the interconversioii of 2-chloro-ARIP and 2-chloro-ADP in vivo. Chemistry.- 2 - Chloro-AMP was prepared from 2-chloroadenosiiie by the two routes outlined in Chart I. In the first, 2-chloroadenosine (I) was aceton-
495
ACTIVEXCCLEOTIDES
Khorana.13 Purificatioii of the crude reaction product by ion-exchange chromatography followed by preparative paper Chromatography resulted in a 3271 yield of pure diammonjum 2-chloro-ADP. Pharmacology.-Arterial blood pressure was measured in rats anesthetized with sodium pentobarbital. h cannula was irisertcd into the left common carotid artery and the blood pressure was recorded via a Statham pressure transducer on a Grass polygraph. Compounds dissolved in normal saline mere injected into the jugular vein. 2-Chloro-AMP caused a fall in arterial blood pressure which was greater than that caused by AMP, and which lasted considerably longer. 2-Chloro-ADP was found to be slightly more potent than ADP but its effect on rat arterial blood pressure was of much greater duration. These results and those obtained with 2-chloroadenosine are compared in Table I with the effects of adenosine. TABLE I EFFECTS OF SUCLEOSIDES A K D SUCLEOTIDES ON RATBLOODPRESSURE 4 v duration of blood presa. Compd
X o l a r potency ratioa
Adenosine 2-Chloroadenosine
fall, min
1.o
0.7 8.2 + 0.7 15 AMP 1.1 + 0.13 0.5 2-Chloro-hAIP 6.6 31 0.4 6 ADP 23.5 5 4.9 1 2-Chloro-BDP 30.7 zt 4.8 10 a bIean results (ASE) obtained by comparing the effectiveness of the compound with that of adenosine on four or more animals. Doses used were such as to cause a 30T0 fall in arterial blood pressure. :(;
'
xc
Y
atcd by the method of Hampton,lo arid the resultiiig cyclic ketal (11) was phosphorylated by the procedure of Tener" to give pure crystalline 2-chloroadenosine-5'monophosphoric acid (111) in 61% yield from I. 2-Chloro-AMP was also obtained by the direct phosphorylation of 2-chloroadenosine with POC1, in trimethyl phosphate, a procedure based on that reported recently by Yoshikawa, et a1.12 2-Chloroadenosine was only partially phosphorylated under the reaction conditions described by Yoshikawa, et al., for the synthesis of AMP from adenosine, and it was found necessary to use a large excess of POC13 and a prolonged reaction time in order to obtain complete phosphorylation of the analog. I n this way 2-chloro-AMP was isolated in 429; yield. 2-Chloro-ADP (V) was synthesized from 2-chloroAMP by the morpholidate procedure of Moffatt and ( 8 ) L. Noda, Enzymes, 6, 139 (1962). (9) R. J. Haslam and D. C. B. Mills, Biochem. J., 103, 773 (196i). (10) A. Hampton, J . d m e r . Chem. Soc., 83, 3640 (1961). (11) G. 1%.Tener, ibid., 83, 159 (1961). (12) M. Yoshikawa, T. K a t o , a n d T. Takenishi, Tetrahedron Lett., 5065 (1967).
The aggregation of platelets in citrated sheep plateletrich plasma was studied by a turbidimetric t e c h n i q ~ e ~ * > ~ 5 in which the changes in the optical density of the platelet suspension caused by added ADP were recorded on a potentiometric recorder. Inhibitors of the ADPmediated aggregation were incubated in the plateletrich plasma at 37" for varying periods of time prior to the addition of ADP. Initial rates of aggregation were measured and compared to the rate of the control response to 1.5 p M ADP. In Table I1 the potencies of the inhibitors AMP, TABLE I1 EFFECTS
OF SUCLEOSIDES A S D SUCLEOTIDES O S T€fE
AkGGREG.4TION O F SHEEP PLlTELETS Molar potency ratio
ADP aggregates 1.o 2-Chloro-ADP aggregates 9.0 Adenosine inhibits aggregation 1.0" 2-Chloroadenosine inhibits aggregation 4.0a AMP inhibits aggregation 0.1" 2-Chloro-AAiP inhibits aggregation 0. l a a Inhibitors preincubated for 2 min prior to the addition of ADP.
2-chloro-AMP, and 2-chloroadenosine are compared to that of adenosine. The preincubation period was 2 min. AMP and 2-chloro-AMP were of similar potency and were only one-tenth as active as adenosine (13) J. G. h l o f f a t t and H. G. Khorana, J . Amer. Chem. Soc., 83, 649 (1961). (14) G. 1'. R. Born, Nature, 194, 927 (1962). (15) &I. H. hlaguire and F. hlichal, ibzd., 217, 571 (1068).
O
5 1 IO
20
30
M i n u t e s pre-incubation
Figure I . -I3fec.t, of preincubation OIL the potenvy of inhibitors .WP-mediated platelet aggregation. Initial rates of aggregat i o i l iiiducetl hj- 1.5 p.ll .\DP were measured: adenosine (0 .. - o), 2-ckiloroadenosine ( 0 . - . . - . - 01, A l I P ( O- --- -. .. -CI, and 2-chloro-AklIP ( w ' ' ' . ). concentrstioris :ire those which caused 50(, inhibition. I J ~
I
periods of prcxiiicutmtioii o n the iiihibitory poteiicies of atleiiosine, 2-cliloro~deiiosiiic,X1\IP. a ~ i d2-chloro-AMP. 2-('hloro-ADP ha5 bceii found to hc a very poiverful :rggrcg:itoi, of sheep aiicl human blood platelets with i i i n ~times the aggregating poteiicy of ADP. Thc aggrrgat ioii caausc~l 1)s 2-chloro-;\DP is reversible. Thcsc findings have beeii reported in tlrtail cilse\vhcrt.ll Adenylate Kinase.- 2-('hloro--431P atid 2-~hloro11131' \ v ( w s u h t r a t c s for adrnylat(. liiii~isc. \ Y h c a t h c x i z y i n ~ froni rahhit niusclc was iiicubatcd wit 1i 2-chloro--iMP and ATP arid thc resulting mixture cx:iiiiiiictl't)>-paprr ctironiatof;raph?-,t h r w adenine iiuc1t.ot i c k c~ompoiicnts,thr riioiiopliosphat~~, diphosphate, aiitl triphosphatc, \verp fouiid to l w present. Similarly 2-1~hloro-AI)P\vhc311 iiiw1):itc.tl with :&nyl:itc kiriaw gavc. : \ t i tyuilil)i~ium xiiixturv of P-chloiwX?cIP. 2-c*hloro.\ 1II', : i l l I l 2-c.hlor0-ATP. (hi
Discussion
Experimental Section
' o q x i r d to :de~iosiiic>:it it? optiiiiuiii potmiq ( 2 min preiiicuhatioii) the ~iiolarpotency ratio of LKCt' :is ai1 inhibitor of AIDP-mediateclplatelet aggregatioli was 0 . 1 after 2 min prciiicuhatioii: this value decreased to 0.04 wftw 30 riiiri prei~icubation (cf. Figurc 1). 2-('hlo1~o-X11Pd S 0 h:~d 21 iiiolni, potc>iicy ratio of 0 . 1
l'aper i , l i ~ i ) i i i ~ i ~ i ) ~ uiiless r ~ i i ) l i01~ Iicrwise stated w:is c*urrietlr i u l l)y the ascending twhiiiqiie (111 \\'hatm:rn So. t paper in I 111, following solvent systclms: ( A ) n-BuOH-HJI (S6:14), ( I1 1 i-PrOH4.25 .I/ S H 4 H C 0 3( 2 : l :ind ( C ) isobutyric wid--I 11 S H 4 0 H (100:60). l'iiriiip derivatives were located on papw lly
Alay It)(jY
I'HAHMACOLOGICALLY
ohservation under uv hgnl,, and phosphates were detected by spraying with molybdate-HClOa.'g Relevant Rf values are shown in Table 111. Melting points were determined on a Reichert apparatus and are uncorrected. U v spectra were measured on a Perkin-Elmer 350 Fipectrophotometer. Where analyses are indicated only by symbols of the elements, analytical results obtained for those elements were within f 0 . 4 ( , of the theoretical values. Evaporations were carried out under reduced pressure at temperatures less than :35", and solids were dried in ~ C I ( over O Py05at room temperature, unless otherwise noted. Reagent purity pyridine was rendered anhydrous by storing it over CaHy. Commercial grade trimethj-1 phosphate was distilled and the fraction of hp 192-lY3' was stored over Linde 3Iolecular Sieve (Type 4h j. 2-Chloroadenosine was synthesized in this institute by a modification of the procedure of l I o n t gomery and Hewson,20in which 2,6-dichloropurine u-as fused with l-0-acetyl-2,3,5-tri-~-benzoyl-~-~-ribofuranose2l without a catalyst and the product was treated with methanolic XH3. Adenylate kinase ( 2 mg/ml of solution, Eoehringer Corp.) was used undiluted. 2-Cliloro-2',3'-0-isopropylideneadenosine (11) was prepared in 85L; yield from 2-chloroadenosine ( I ) by the method of of Hampton.'o The product (mp 238-241 " ) was chromatographically homogeneous in solvent systems A and B and was used for the synthesis of I11 without further purification. h small amount was recrystallized once from lIe?CO and three times from HyO to give the analytical sample, mp 2323-241 '. -1na2. (Ci,HieClSjO,) C, H , S . 2-Cl:loroadenosine 5'-I'hosphate (111). ( i ) A solution of I1 (2.74 g, 8 mmoles) in pyridine (50 ml) was treated with pyridiniuni @-cyanoethyl phosphate (32 ml of a 1 mmole!ml of pyridine stock solution)" and the resulting mixture was evaporated t o an oil. Traces of moisture were removed by three additions and evaporations of anhydrous pyridine (each 30 ml). The residue was then dissolved in anhydrous pyridine (50 ml), treated with dicyclohexylcarhodiimide (16 g), and allowed t o stand at room temperature for 18 hr. H?O (10 ml) was then added and after 1 hr the reaction mixture was concentrated to dryness. Following a further addition and evaporation of HZO (50 ml), the white solid residue was suspended in 0.4 -1.r LiOH (320 ml) and refluxed for 1 hr. The mixture was cooled and filtered. The filtrate TTas applied t o a column (3.2 X 25 em) of Bio-Rad AGSOJV-S4 (H+) ion-exchange resin and washed through with H20 until the effluent was neutral. The eluate was adjusted t o p H 2.8 with aqueous Ba(OH)?, heated under reflux for 1.5 hr, then concent,rated t o ca. 400 ml. -4queous Ba(OH)2was added t o p H 7.5 and the preripitated harium phosphate was removed by centrifugation. The supernatant was treated with twice its volume of EtOH and, after 1 hr at room temperature, the white precipitated barium salt of I11 was collected hy centrifugation and washed with I:tOH, ;\Ie&O, and Et20 t o give 4.69 g of the dry salt. Paper chromatography in solvent system B revealed the presence of a minor unidentified contaminant (Rf 0.07). T h e Ba salt (400 mg) was dissolved in H,O (ca. 10 ml) with the aid of a little Bio-Rad .1G50m'-S4 (H') and passed t,hrough a column (1 X 20 em) of the same resin. The combined e!i!uent and washings were neutralized with SH,OH, evaporated t o a glass, and dissolved in 3 ml of solvent B . This solution was applied t o a column (2.5 X 35 e m ) of Whatman cellulose powder packed in solvent B and the column was eluted with the same solvent. Fractions of 20 ml were collected; I11 emerged in fractions 12-16. These were pooled and concentrated t o dryness. Itepeat,ed evaporations of H,O removed residual S H 4 H C 0 3 and the glasslike S H , salt was dissolved in H20 ( 5 ml) and converted t o the free acid by passage through the 1 X 20 em cation-exchange cwlumn (H+ form). Evaporation of the eluate j-ielded a white crystalline residue of I11 (199 mg, from 11) which was (~hrc)matographicallypare and free fr norganic phosphate in solvent systems B and C. I t was recrystallized twice from H?O t o give the analytical sample as white needles, mp 159-160" dec, A,,, (0.1 .\ HCIj 265 mp (E 14,200). :lnai. (C,oH,,CIK,O,P. 1.5H20) C, H, N . (ii) I (755 mg, 2.5 mmoles) was dissolved with stirring in an ice-cold solution of POC13 (2.28 ml, 25 mmoles), H 2 0 (0.045 ml, 2.5 inmoles), and 30 ml of dry trimethyl phosphate. After 24 hr
ACTIVE S U C L E O T l U E S
a t 3 " , the clear solution was poured over ice (100 g ) with stirring and the p H was brought t o 9 with concentrated aqueous LiOH. The mixture was kept at room temperat,ure for 2 hr and the p H was maintained at 9 by further additions of I i O H . Iithium phosphate was removed by centrifugation and washed ( H 2 0 ) until no further uv-absorbing material could be extracted. The combined supernatants (ea. 180 ml) p-ere extracted twice with 100 ml of CHCI, to remove (lleO)aPO, diluted to 300 ml, treated with Ba(OAc)? (1.28 g, 5 mmoles), then allowed to stand overnight at room temperature. The resulting precipitate of Bar(PO4)? was removed by centrifugation and washed (HsO), and the supernatant and xashings were concentrated to ca. 50 ml. Two volumes of KtOH were added and the precipitated nucleotide E a salt was collected by centrifugation and washed once with I{:tOH-H20 (2:1, 30 mi), then with EtOH, lIe?CO, and litYO to give 1.57 g of product. Paper chromatography in solvent B shoned two u v - a h r b i n g spots, one corresponding in K fto I11 and the other at Ki 0.05. The two spots were eluted with HrO and shown to have identical A, values, 264 mp. The E a salt (300 mg) was subjected t o column chromatographjin a manner identical with that described in ( i ) above. Fractions 12-17 from the cellulose column were pooled and worked up to give 82 mg (42'.; yield) of a white crystalline solid, which was and had the same Rf value orpholidate ( I V ) was prepared on a 0.25-mmole scale from I11 by the general method for synthesis of nucleoside 5'-phosphoromorpholidates descrihed by lloffatt and I-toiosicity of these compoiinds, and also o f ;r;'-deoxy-2-fliioz.oadeuoaine and F)-8-o-x~lof~iranosyl-~-fl~ior(iadeiiirie, t o a riiiinbet of HEp-9 (.ell lines i n mli lire has been determined. The data permit certain conclii4oiis coiiceriiiiig the probable metabolism and niech:mimi of action of these nucleosides.
'2-B'luoro:tdeiiosirie2is readily analiolizec13-5 hut not c:it~Jiolizcd"-" in cells in culturr or in uico. I t is highly highly toxic to ant1 hiis hroailspcctrum mtibncterial activity.1i,12I t has a sl-nergistic vffect on the antimicrobial action of acti~iobolin'~ aiid is ;LII iiihibitor of blood-platelet aggregation. l4 The liroatl a l i d high-level tiiologic activity of 2-fluoroadcnosine has iiiatle tlu: study of other nucleosidcls of 2-fluoroadeiiino tlclsirahle. The preparation of 2-ami1io-2'-tlroxyade1iosi1ic (8-6) iii 1.7:; over-all yield and its a anoiiier in 1.3,'; over-all yieltl froni 2-amino-6-chloropurine by the conveiitioiliil c:hloromcrcuri procedure has been rcportetl.15 hi :ti1 (+fortto improve the yields of both anomers of 6 aiicl t o ol)t:Lin analytical samples of thtsc conipouiids, thtbir preparatioii from the chloronierruri derivative o f 2-1~eiix~~1iiido-N-~ieiizoyladeiiiii~~~ aiitl :3,~-di-C)-(p-ciilorol~eiizoyl)-2-tlcox\--n-erythro-pe1ito~ur~~1ios~l rhloriilcl; wis iiivcstigated m i d found t o givr 4 :is a i i npproxiinatcly 1: 1 mixture of a and moiiiers ill a total yitld of 36c/;, (Schenie I ) . Trcatineiit of 4 with S u O J l c i i i t tic usual niaiiiier resulted i i i ticcoriipoaitioii of tiiv I iuc*lcositle,whercns treatniei i t v i t li inct,haiiolic S H :It .' i rcmovcd only t.ht I,-clilorol)eiizo?-l groups. Tht. iijiportd by fiiiidi. f r ~ i ntlir S(iutlirin IZehealrli I i i h t i t i i t t . , I'oundatior~, arid t h e C'aiiwr Chemotherapj, National Serviw i'rntrr, National Canwr Institritr. Natioii:rl I n i t i n i t w of Ht.iilth, i P ) J . .4. Montgomery atid K. H r w w i i , .I. . I mer. ( ' h e m . Suc., 79, 4 K. Hariseii. J . B i o l . Cheni., 242, d I. I,. Briinrtt, Jr.. ibid., 242, 1 , Y. I). S:inipoii, and 31. 1,. llrlorli. .Ivc/I, ier, ibid., 102. 77 (IWA). .-L ntimicrobial .Iyen18 C h e m o i t ~ e r a p l i .1 W!,.
NH. I
p C I C , , H 4 C O O C a
p-ClC,H,COO 4.R=SHHi
+
H
O
C
W
+
HO 6
5 , R = I'(
",I
Inik. Bioc/iemisir!/, 4, l 2 X J (I!J(i:). ril)
S. 1;rFderikwii. .Ir.ch. B i o c h r m . R i o p h ! / s . , 113, :i83 (1966).
i 101
13. E. S k i i i ~ ~ J. x , -4. AIoiitgo~ner>.I. I
