Purine Nucleosides. XXV. The Synthesis of Certain Derivatives of 5’-Amino-5’-deoxy- and 5’-Amino-2’,5’-dideox~-~-1~-ribofuranos~lpurin~~ as Purine Nucleotide Analogs’
-O-i.;opi,opvlidene-3’-arniiio-3’-tieoxvnucleoside derivatives of adenosine 12a) and giianosiiic. 12b) were isolated and characterized for the first, time. The agnthehis o f .i’-amiric)-~‘,.i’-dideoxyadenosiiie ( 1 1 ) has IIOR been acc:omplished by a similar roiite. Treatment, of the .i’-:miino piirine nucleosides 2a, 2b, tirid 11 with RIeS02C1 provided the corresponding 3’-.\’-methanesulfonylamino purine nucleoside derivatives. *\ppropriate deblocking yielded 5’-?;-methanesulfonylamino-.i’-deoxyadeiiosine(sa),5’-T-niethanesulfonylamino.i’-deoxygaanosiiie (6b), and 3‘-.V-methanesulfonylamino-2‘,.‘,’-dideoxyadenosiiie(12), respectively. Similar reaction of t,he requisite 5’-amirio purine nucleosides 2a, 2b, and 11 with etjhyl chlorocarbonat’e followed by appropriat,e deblocking procedures gave .~‘-S-ethoxyrarboIiylamilio-,i‘-deoxy adenosine (5a), 3‘-S-ethoxycarbonylartiiiio-.i’-deox?-guanosine (5b), arid j’-.\’-ethoxycarbonylamino-:!’,3’-dideoxyadenosirie( l o ) , respectively. The rat ionnle for consideration of these nricleoside derivativrs HP 3’-1iucleotide analogs is presented and discussed.
.I major problem inherent iri the utilization of analogs of nucleic acid constituents in chemotherapy is the fact that the nucleotide is often the biologically active dcrivatiw :it the molecular level within the cell. The base or nucleoside must be converted to the nucleotide intracellularly for effective action. Resistance to drug rcyorise often develops when the required enzymes fail to perform this “lethal synthesih” of the nucleotide. ,ittempts t o employ the riucleotide derivatives directly as chemotheritpeutic agents have been generally unsuccevful due t o rnzynintic cleavage to thc hetrrocyclic base or nucleoside during transport or by failure of the highly polar nucleotides to cross cellular membranes. These problems have been reviewed ill detai1.2~i.in approach to this problem has been the simiilatioii of :I Iiucleotide ( ; . e . , 5’-adenylic acid) by :Itletiin-!3-yl311;:Inoic acid,.? in which a. more weakly ionized carboxylic Licit1 \vas employed for binding a t t h r phosphoric acid hit e. Such :tii :tpproacli has been partially successful in isolated enzyme systems since adenin-9-ylvaleric acid inhibits both lactic dehydrogenase and glutamic dehydrogenase? although uracil-l-valeric acid failed to mimic 2’-deouyuridylate :tnd did not inhibit thymidylate sy~ithetase.~The inhibitory activity of 3(ti-mercaptopurin-y-ylva1eraniide)salicylic acid was found to be about one-fourth that of F-thioinosirlic acid :ig:iinst succinoadenylate k i n o s y n t h e t : ~ . ~ Since O W of the major considerations in the design of :my drug is its transport characteristics, riucleotide :~nalogswhich would be polar but nonionic were chosen for the present investigation. Adenosine ;’-phosphate is presumed to possess a proton at S’ arid an anionic rixyg~ri011 the phosphate group (an assumption which h:th h c t i confirmed in the solid state by X-ray analysis6). -1pp:irently it is this ionic structure which prevents ( 1 ) TI& work \vas supported 1)y RePearcli Grant C.\-08109 from the Sational Cancer Institute of t h e Sational Institutes of Health and by Research Grant GI3-5446 from t h e XIoleciilar Iiiology Division of t h e Sational Science Foundarion. ( 2 ) 13. R . Baker and H. 6 . Sachdev. J . P h a r m . Sci., 62, 933 (1963). ( 3 ) 13. R . Baker, “Design of .\rtive-Site-Uirected Irreversible Enzyme 1nhil)irors. The Organic Chemistry of t h e Enzymic Active-Site,” .John \Viley and Sons, Ino., New 1-ork, K . Y,,l 9 6 i . Chapters 5 and 6. (4) 1%. R. Baker and G. E. Chtieda, J . Pharm. Sci., 64, 25 (196.5). [.j) 13. R. Baker a n d P. AI. Tanna. ibid., 64, 1609 (1966). (6) J . Kraiir, Acta C r y s t . , 16, 79 (19fi3!.
passage of the nucleotide through the cellular metilbratie.’ Thus nucleosides which possess un-ionized groups which could simulate 5’-phosphate binding but which mould still enter the cell as potent inhibitors pose excellent candidates for chemotherapy. Baker states8 that ‘‘ . .such inhibitors would not need actiwitiori for inhibition of enzymes operating on nucleotides and thus would not be subject to the type of drug resistance resulting by mutational loss of :I kinase or pyrophosphor3-lnHe.” Indeed, nucleocidin, n potent nucleoside antibiotic of recently proposed structure .j’-O-sulfamoyl-4’-C-fluoroade~iosine,~ could be envisaged as such an un-ionized nucleotide aiidog which lends support to this concept. 111 the prehent study derivatives of 5’-tlmino-5’deoxy-~-r~-ribofiiraiiosylpuriries were selected for investigation. I t was hoped that the S’-amino functiorl would give added stability against both chemical and enzymatic hydrolysis of the group selected as ti simuh t o r of the 3’-phosphate functioii. The syntheses of 5’-amino-.7’-dcoxyadenosine :tiid 3’-arnino4‘-deoxyguanosine have been reported by Jahn.’” I n the> present study, N6-formy1-Y’,:3’-O-isopropylidene-5’azido-5 ’-deoxysdenosinc (la) was prepared essentially by the method of Jahn‘” and isolated crystalline : t r d characterized for the first timc. The desired 2’,3’-Oisopropylidene-5’-amino-5’-deoxyadenosine(2a) intet mediate was obtained crystalline by deformylation of la in methanolic SH, followed by catalytic reductioii of the 5’-azido group (see Scheme I). Treatment of 2a with methanesulfonyl chloride in pyridine gave 2 ‘,,?’-O-isopropylidene-j‘-N- methanesulfonylamino - 5‘ deoxyadenosine (4a). Removal of the isopropylideiie group was achieved with 50% aqueous formic mid to yield 5‘-h’-methanesulfonylamino-.5‘-deoxyadenosine (64. The method of Jahn’” w t h utilized to introduce thc ;’-amino function into the guanosine molecule except that DAIF proved to be superior to DbfSO as a solvent for introduction of the azide group. The 2’,3’-O-isopropylidene-5’-azido-.i’-deoxyguanosinel b was sep:i( * . lleidelberger and K I, hlukherjee, Cancer Res., 22, 815 (1962). (8)U R. Raker and P. X.Tanna, J . Phnrm. Sez., 64, 1774 (1965). 1 0 ) G. 0 Morton, J 14’. Lancauter, G. E. Van Lear, W.Fulmor, a n d W 1 h1e)er J A m Chem Soc , 91, 1335 (1969) (10) LV J a h n , r h e m Ber , 98, 1705 (19h3)
(7)
PURISESUCLEOSIDES. XXV
July 1969 SCHEME I
1a.B = N6-formyladenine-9 b, B = guanine-9
2a, B = adenine-9 b, B = guanine-9
J
3a,B = adenine-9 b, B = guanine-9
.1 OH OH 5a.B = adenine-9 b. B guanine4
1
4a,B = adenine4 b, B = guanine-9
1 OH OH 6a.B = adenine4 b. B = guanine4
659
fully controlled conditions in pyridine-NEt3, methanesulfonyl chloride reacted selectively with the 5’-amino group of 11 to give an 85% yield of Z‘-N-methanesulfonylamino-2’, 5’-dideoxyadenosine (12). The proposal of Baker and Tannag that the 5’carbamoyloxy group can simulate phosphate binding and the synthesis by Mertes and Coats14 of several (3’+3’)-carbonate andogs of dinucleotides stimulated interest in the ethoxycarbonylamino group as a second simulator for the 5’-phosphate. Treatment of 2’,3’O-isopropylidene-5’-amino-5’-deoxyadenosine (2a) with ethyl chlorocarbonate gave an excellent yield of 2’,3’O-isopropylidene-5‘-N-ethoxycarbonylamino- 5’ - deoxyadenosine (3a). Removal of the isopropylidene group gave 5’-N-ethoxycarbonylami1~o-5’-deoxyaderiosine (5a). Similarly 2’, 3’-O-isopropylidene-5’-amino-5’deoxyguanosine (2b) and ethyl chlorocarbonate gave 2’,3’- 0 - isopropylidene - 5’- N - et hoxycarbonylamino -5‘deoxyguanosine (3b). Removal of the isopropylidene group gave 5’-N-ethoxycarbonylamino-5’-deoxyguanosine (5b). Treatment of 5’-amino-2’,5’-dideoxyadenosine (1 1) with ethyl chlorocarbonate in pyridine under carefully controlled conditions gave selective reaction a t the 5’amino group to yield 5’-N-ethoxycarbonylamino-2‘,5‘-dideoxyadenosine (10). Preliminary antiviral activity has been assayed in African green monkey kidney cells BSC1 in vitro used for the growth of Herpes simplex virus.15 6’-N-lIethanesulfonylamino-3’-deoxyadenosine (sa) has been shownlZ to possess superior antiviral activity to 5’amino-3’-deoxyadenosine16 in this system. j’-N-lIethanesulfonylamino-5’-deoxyadenosinewas not toxic to host cells at 2.5 pmoles/l. and at this concentration exhibited 73% inhibition of virus gromth.lZ Several of these nucleotide analogs exhibited significant antibacterial activity (see Table I).
rated from 2’,3’-O-isopropylideneguanosineN3+5’cyclonucleosidell by fractional crystallization of the mixture from water. By this procedure l b was obtained crystalline and mas adequately characterized for the first time. Reduction of l b provided crystalline 2’,3‘-0-isopropylidene-5’-amino-5‘-deoxyguanosine (2b) which served as the starting material for the 5’-amino5‘-deoxyguanosine nucleotide analogs. Treatment of 2b with methanesulfonyl chloride in chloroform-pyridine provided 2’,3’-O-isopropylidene-5’-N-methaneTABLE I sulfonylamino4‘-deoxyguanosine (4b). Removal of EFFECT OF NUCLEOTIDE AYALOGS O N THE GROWTH OF the isopropylidene blocking group with 50% aqueous Escherichia col+ formic acid readily gave 5’-N-methanesulfonylaminoCompound 507, growth inhih, Ai j‘-deoxyguanosine (6b). 5 ’-S-Ethoxycarbonylamino-5 ’Preliminary antiviral activity exhibited by 5’deoxyadenosine (5a) 4 . 5 x 10-6 N - methanesulfonylamino - 5‘ - deoxyadenosine12 (sa) 3 ‘-.Y-llethanesulfonylamino-3 prompted us to prepare 5’-N-methanesulfonylaminodeoxyadeiiosine (sa) 4 . 4 x 10-4 2‘,5‘-dideoxyadenosine (12) as a nucleotide analog of 5 ’-A~-Methanesulfonylamino-2’,5’2’-deoxyadenosine 5’-phosphate. For this purpose dideoxyadenosine (12) 4 . 2 x 10-4 5’-amino-2‘,5‘-dideoxyadenosine(11) was required. a The other nucleotide analogs reported in the present study 5‘-0-p-Toluenesulfonyl-2’-deoxyadenosine (7) was prewere without effect a t 10-3 11.1. These preliminary results pared by the method of Robins, et aZ.,l3and purified by were kindly supplied by Dr. ,4. Bloch, Roswell Park Memorial Institute, Buffalo, K.Y. crystallization from EtOAc and EtOH. j’-O-p-Toluenesulfonyl-2’-deoxyadenosine(7) was then treated with acetic-formic anhydride to yield 3‘-0,N6-diExperimental Section l7 formyl-5’-O-p-toluenesulfonyl-2’-deoxyndenosine (8) Melting points were determined on a Fischer-Johns block and which was obtained as a crystalline solid (see Scheme are uncorrected. Spectral data were obtained on Beckman DK-2 11). Treatment of 8 with NaN3 in D l I F gave a good (uv), Beckman IR-5.4 (ir), or T’arian A-60 (nmr) instruments. yield of 3‘-0,N6-diformyl-5’-aeido-2’,5’-dideoxyadeno-Evaporations were accomplished using a Buchler rotating evaporator under reduced (aspirator or oil vacuum pump) pressure sine (9). The formyl groups were then removed with methanolic NH3 and the 5’-azido group was reduced (14) M. P. Mertes a n d E. A. Coats, J . M e d . C h e m . , la, 154 (1969). with 5% Pd-C and Hz to give an excellent yield of (15) A. Diwan, C. N . Gowdy. R. K. Robins, and W. H. Prusoff, J. Gen. Vzrol., 8, 393 (1968). 5’-amino-2’,5’-dideoxyadenosine (11). Under careI-
(11) R. E. Holmes a n d R. K. Robins, J . Ore. Chem., 2 8 , 3483 (1963). (12) A . R. Diwan, R. K. Robins. a n d W.H. Prusoff, Experzentzn, as, 98 (1969). (13) 11. J. Robins, J. R. RlcCarthl, Jr., and R. K. Robins, Bzochemistry, 5, 224 (1966).
(16) T. Neilson, W. V. Ruyle. R. L. Bugianesi, K. H. Boswell, and T. Y. Shen. Abstracts. 154th National Meeting of t h e American Chemical Society, Chicago, I!1., Sept 1967, P 29. (17) Spectral d a t a (uv) are given for compounds 2a and lb. Spectra not given are directly comparable t o these for t h e respective nucleoside series.
I I(’( 1st I
NH
I
7
iwI 9
8
0H 10 iitiles. othetwise stated. Hydrogeiiatims were effected usiiig :I Pttrr apparat 11s at de*ignated 11, pressure and cat.alyt. Where analyses are indicated only by ~~ynibols of the elements, atial>-tical resiilts obt,ained for thow elenlent.: were r i t h i n t 0 . 4 f ; of the I heoret ical valiies.
OH
01: 11
12
5 ‘-.l~-Methanesulfonylamino-5’-deoxyadenosine (6a).---A soliilion of 0.92 g (0.0024 mol) of 4a in 20 nil of 50c X L ~ allowed t o staiid for G day- ni i’ooni t
solvents were evaporated and Et011 was added t o the resriltiiig d e a i qyriip. Precipit:itiuii of a white solid occiirred upon stiriiiig .~6-Formyl-2‘,3’-O-isopropylidene-5’-azido-5‘-deoxyadenosineiiiid the mixtiire \vas evaporated t,o dryriesi. The resulting so (la).---To a solution of S6-formyl-.i’-O-tosyl-%’,:~’-O-i~opropyli-war recry..tallized f i v m F:tOH-E:tOAc (2: 1 ) to give 0.8 g (97( of rieedle,. of 6a, 4ntrrs :it 150--170°. .1ntrl. (CllH16TBOiS) tieneadeiioiine10 f32.0 g, 0.065 mol) i n 250 ml of D X 3 0 wah I i >s. ~rdded13.8 g (0.243 mol) of ?;aszi and the resrilting mixture wa-. 2’,3’-O-Isopropylidene-5’-~~’-ethoxycarbonylamino-5’-deoxy.;tired at 65-70” for 30 min. The reaction niixt,iire way theii adenosine ( 3 a b A solritioii of 1.1 nil (0.014 mol) of rt#hyl poiired into 800 In1 of CHC~.I-H.O f l :1). The IaJ-ers n-ere c,hlorocalboiiate i i i 30 in1 i i f TIIF w i h added drnpwise over :t ieparatrd and the aqueoiis phase was extracted with three i5-ml period of 15 iiiiii 10 :I rnsyrietic:ally s t i r w l solritioii of 4.0 g port inns of CEICI:,. The conibined organic phase was washed (0.013 mol) of 2a i i i 150 nil of d1.y TITI? :tiid 2.2 nil of Et:,S :it with three 75-1111 portioiis of 1110 and dried (\Ig804). The r i i o m 1erriI)ei~atiirc. I:r:jSfl+ CI- wa‘: renioved hy filtration :tiid mixtiire wah filtered and the filtrate wa. evaporated t o dryiieh.. the filtrate was evaporated t o dryiiess. The re.*:ulliiig solid foam The i,es:ultiiig white foam was di\solved i l l CIICl3-hIeO1I : i t i d \\-:I, dissolved i i i :5 :igairi evaporated to give a white uolid. 1 dropwi,-e to I I. of Is, inp 101-~J04”. pl~oductf r o m Et011 ‘The resriliiiig precipit .\ small sample for a N I I ~ :I sniall sttniple t o give needles, inp c~nlllnirl uf Ilerr1r:~l :t 2’,.7’-O-Isopropylidene-5’-amino-5’-deoxyadenosine (2aL (Ci,,H:2N6031 ‘2, 11, Y. ‘To 200 nil of NeOH prePatr1rated with 317, at -lOo was added 5’-~\--Ethoxycarbonylamino-5’-deoxyadenosine (Eia).-~-.Ad r i 11 g (0.039 mol) of l a aiid the resiiltitig wliition was allowed t o tioil nf 0.y g (0.002 mol) of 3a in 16 nil of 50:; aqrieuii5 Ir(:OaIi itaiid for 113 hr a i room teniperature i i i i~ sealed vessel. This was allowed t o - t a i i t l for 24 hi, :it rooin ternperatrire. l‘hr wliiiioii was evaporated a d the rcwilting syrrip was di~,olveti soliilinii ~ : i -w:ipnnited t o dryiiess nud the reaidite was UIi i i 4.; nil of MeOII, filtered, arid cooled for h--10 d a y at 0’. ,orated twice ii-iiig I11OFI. ’I’he iwiilting solid was IYThe crystalline prodilrt (10.7 g, 8:3ci) wa- filtered, di.olved iii iallized fi.oni EtOI-I--l.. The 2-~1ni11o-d-h>-drc hiopteriri, x:~iitIrq)hitoir of the t r y ~ ~ n i i i ~ ~ i ~ r t i a l i'oxarithopterin were f o i i n d to tie effective iieithei, :i> < l i t
Iiiitially dihydrofolate redurtase from 7'. cquiperduni was prep:ii,cd exactly as oiitliried in ref 1; in later studies acetone powtler::, prepared from :1-6 x 1U'" trypanosomes, were extracted
with i nil of p l l 7.0 pho.~phatebuffer (0. I J l ) arid the exti':i(,ts mr'e centrifuged for 10" a t 100,OOOg and 4' in ii 13eckrii:in .\Iode1 L 1,efrigerated wntrifLtge; the resulting supernatant so111tiona served as the soiirce of enzyme. This modifiedprocedirre for obtaining tr>-pnnosomal w e d becaure of the high loss uf enzymr itc during t,he dialyhis itep employed in (nir origii roceditre. It yielded an enzynie preparation m-hich did 1 tly, with rerpevt to sensitivity t,o varioiis inhibitors, from the enzyme preparatioik made by the previous procedure. In thi.: connect,ion, it i h m r t h pointing oiit that Schrecker arid Hitennekens5 found 1 ti:i1 dihpdrofolate rediic,iii.;e in crlide extracts of chicken liver aiitl this enzyme after partid purificntiori showed similar nensitivit). to the diamiiiopteridirie inhibitor, aminopterin. Acetone powd e r , ~were ~ prepared from swtioii; of chiclieii liver, the powder> were extracted as fnr the trypniiohunial preparation, the estracts were dialyzed overnight against 100 vol of p H 5 . 5 biiffer 1 0.U1 .lf), and the dialyzed rn:tmial was used for erizymat.ic :issays. Acetone powders were prepared also from i;ection.: of rat, liver and were estracted a n d dialyzed as described for t h r h chicken liver preparations. .-I similar preparation' of (dihydro i folat,e reductase, obtained hy high-speed ceritrifugation of a rat liver homogenate, W:LS iisrti b)- Hampshire and her colleagiio~~ for evaliiating the inhibitor>-poteric'y of a series of 2,4-diamino-5arylazopyrimidiiies . Iteduced riicotirinmide-ndeniii~, dinucleotide p1ioaph:cte (SADPH) TVXX purchased from P. I,. Biochemicals, Iiic., l l i l waiikee, Wis. : folic acid war piirchased from Calbiochem, Lo. .hgeles, Calif. Dihydrofolate x a s prepared by the method of Futtermang and alw by t,he procedure of Friedkin and hii cdleagues.'o S o differences were observed between the rates of enzymatic rednctiori of d drofolate prepared by the different methods. Reference> t o ithehes of pteridines carried out i i i this laboratory are given in the tables.
( 1 ) A preliminary account of a portion of this work mas presented a t thr. I'all Meeting of t h e American Society for Pharmacology a n d Experimental Therapeutics, Washington. D. C., .\up 1967: J. .I. lIeCormack and J. , J . Jaffe. I'hnrmacologist, 9 , 193 (1468). ( 2 ) J. .J. Jaffe and J. J. McCormack, M o L . f'hurmacol., 3, 359 (1967). ( 3 ) .J. J. Rurchall and G . H. Hitchings, i 6 i d . , 1, 126 (1965). 14) See. for example, (a) 13. R. Baker and U-T. Ho, J . I'hurm. Sci., 63, 1 1 3 i (1964); (b) 13. R. Baker and B-T. Ho, ? b i d l . . 66, 470 (1966); (c) I?. I?. I3akP.r and G. J. Lonrrnr. .I. .?fed. C h e m . , 10, 1113 (1967): Id) t i . R. Baker. t t , u / , , 11, 483 (1968).
t.5) .j. \V, Si,liiecker n n i i 1.. \ I . Ihirnnekens, Biochem. Pliarmacul., 13, i : i I (l'J64). 16) 8. F . Zakrzewski, ./. Hie/. C l i r m . . 256, 1 7 i 6 (1960). ( 7 ) 9. E'. Zakrsenski a n d i'. i. Nirhol, .I. Phnrmueol. B s p l l . Therap.. 157, 162 (1962). (81 J. Haminhire. 1'. IIeljlwin, \ . rll. Triggle, D. J. Triggle, and S. Vi cstablishecl t h:it seemingly inial1 ch:inge\ i t i chemlcal structure can produce niarbed alterations in the abllity of :in agent to inhibit :t particular reductabe. 1T7e have iiiitiated a similar comparative htudy of the relatioiithip between the chemical ttructure of variouq 2,4-di:Imiiiopyrimidines and related heterocyclic byitems, and their ability to inhibit dihydrofolatc reductases from 2'. cyiiipcrrlunz. chickeri liver, and rat liver. It ->IL\ hoped that huch :I htudy would provide information \r-hich might prove useful iii the design of new agelit. for i i w i i i the chemotherapy of trypanosomia.;es > t i i d other protozoal disea
Experimental Section
