Nucleosides and Nucleotides as Potential Therapeutic Agents

and, 3) the glutamine synthetase deadenylylating enzyme. Equation 4 shows the adenylylated and unadenylylated forms of glutamine synthetase, with their re- spective properties, as well as the enzymes which inter- convert the two forms, and their effectors.

of the products of glutamine metabolism. In this form glutamine synthetase activity is thus further diminished by the accumulated nitrogenous metabolites, which has as its result the lowering of the level of glutamine in the cell.

Activated by glutamine. Inhibited by a-ketoglutarate and UTP Adenylyltransferase

Unadenylylated glutamine synthetase \ Adenylylated glutamine synthetase (4) \ Deadenylylating enzyme

Activated by a-ketoglutarate and UTP. Inhibited by glutamine and glutamate

More active form Resistant to feedback inhibition

Mgz+ specific

It is clear from this scheme that conditions of nitrogen starvation, with a correspondingly low nitrogen saturation ratio, lead to activation of the deadenylylating enzyme as well as to an inhibition of the adenylyltransferase, owing to the alterations in the relative levels of glutamine and a-ketoglutarate. This results in the formation of relatively unadenylylated glutamine synthetase, the form of the enzyme which is intrinsically more active and less sensitive to feedback inhibition. In this condition, ammonia will be converted into nitrogenous metabolites with high efficiency.

On the other hand, with high levels of the nitrogen saturation ratio, as might be expected with a surfeit of nitrogen-containing compounds,thedeadenylylating enzyme would be inhibited and the adenylyltransferase activated. The result of this reciprocal enzyme control would be to convert the glutamine synthetase to the adenylylated form, which is less active and additionally more susceptible to inhibition by several

Less active form More sensitive to feedback inhibition

MnZ+ specific

The three-enzyme nitrogen regulatory system has as its most obvious role the preservation of homeostasis in cellular nitrogen metabolism; any shift away from the “normal” nitrogen saturation ratio is accompanied by adjustments in the three enzymes which tend to restore the ratio toward the preexisting level. These intricate controls have as an ancillary effect the preservation of cellular ATP, since both glutamine synthesis and adenylylation of glutamine synthetase consume ATP; in fact, the adenylylation-deadenylyla- tion system without reciprocal controJ would be a wasteful ATPase activity. The reason for the nucleotide controls in the adenylylation and deadenyl- ylation phenomena is as yet obscure; a possible link between glutamine synthesis and polynucleotide metabolism is suggested by the effects of ribonucleic acid species on the deadenylylating system.

Received: October 17, 1969 [A 783 1E1 German version: Angew. Chem. 82, 689 (1970)

Nucleosides and Nucleotides as Potential Therapeutic Agents

By T. Y . Shen[*l

The present article summarizes recent progress in the study of nucleoside derivatives as antiviral and immunosuppressive agents. A number of 5’-substituted 5‘-deoxy nucleosides have been found to be permeable and nonincorporable antimetabolites of 5’-nucleotides. N6-isopenfenyladenosine and analogs show certain promising immunosuppressive activities. Encouraged by the antibody-stimulating efJect of oligonucleotides, we have developed a convenient synthesis of oligonucleotides using fully pro fected phosphorylated intermediates. A group of tetradeoxyribonucleotides, dApdApdApdX, was prepared for biological and physical evaluations. Nucleotide derivatives may prove valuable in the treatment of’ several immunological disorders.

1. Introduction discussion is to highlight several biomedical targets of interest to medicinal chemists and to indicate the therapeutic potentials of nucleosides and nucleotides 111.

A brief review of some recent studies of nucleosides and nucleotides in the field of chemotherapy and immunology is presented below. The purpose of this

[*I Dr. T. Y . Shen

[I] Adapted from presentations at the Symposium on Recent Advances in Nucleoside Chemistry. 156th American Chemical Society Meeting, Atlantic City, New Jersey, Sept. 11, 1968, and at the Gordon Conference on Carbohydrate Chemistry, New Hampshire, June 1969.

Merck Sharp and Dohme Research Laboratories Rahway, New Jersey 07065 (USA)

678 Angew. Chem. internat. Edit. Vol. 9 (1970) 1 No. 9

During the past fifteen years, extensive chemical and biological studies of nucleosides and some nucleotide analogs have been stimulated mainly by their anti- tumor and antibiotic properties. The cytotoxic or bactericidal activities of these antimetabolites are often attributable to the inhibition of enzyme systems involved in the biosynthesis of nucleic acids, such as nucleotide reductases, kinases, and polymerases, or to the formation of fraudulent nucleic acids through incorporation [21. Several nucleoside antibiotics such as puromycin, blasticidins, and gugerotin inhibit protein synthesis, either by chain termination or by interference with the normal function of peptide synthetase or ribosomes 133. However, since these com- ponents are also essential in the biosynthesis of protein and nucleic acids in normal cells, the therapeutic value of nucleoside antimetabolites are frequently diminished by their potential toxicity. The need for a better therapeutic index is particularly important in the application of nucleoside analogs to viral chemotherapy 141 or metabolic diseases. Lack of incorporation into normal cells (Le. , lack of muta- genic effect) and a high degree of selectivity toxicity are almost mandatory for any useful agent.

2. Inhibitors of DNA Viruses

2.1. 2'-Deoxyribo- and Arabinonucleoside Analogs

The first clinical application of a nucleoside as antiviral agent with an adequate therapeutic index was demonstrated by a well-known thymidine analog, S-iodo-2'-deoxyuridine ( 1 ) (idoxuridine), in the treatment of an eye infection, herpes keratitis 151. The apparent safety in this case was accomplished by a unique physiological phenomenon, namely, ophthal- mic infections are topical and not unlike an in vitro system. Since then several more potent DNA-synthesis inhibitors or thymidine antimetabolites, such as trifluorothymidine (2) [61, cytosine arabinoside (cytarabine) (3) [TI, and its 5-fluoro analog (4) [*I, have also shown similar efficacy. A homolog of thymidine, 5-ethyl-2'-deoxyuridine (Sa), was also reported to have antiherpes activity [gal.

[2] J . A . Montgomery, Progr. Drug Res. 8, 431 (1965). [3] R . E . Monro and K . A . Murcker, J. Molecular Biol. 25, 347 (1967); M . Yukioka and S . Morisuwa, J . Biochemistry (Japan) 66, 241 (1969). [4] For summary see: Antiviral Substances. Ann. New York Acad. Sci. 130 (1965); W. H . Prusoff. Pharmacol. Rev. 19, 209 (1967). 151 H. E. Kaufman, E. L . Marfolu, and C. Dohlman, Arch. Oph- thalmol. 68, 235 (1962). [6] H. E. Kaufman and C. Heidelberger, Science (Washington) 145,585 (1964); R . A . Hyndiuk and H. E . Kaufman, Invest. Oph- thalmol. 5, 424 (1966). 171 D . A . Buthala, Proc. SOC. exp. Med. 69, 115 (1964). [8] R. Duschinsky, T . Gubriel, M . Hoffner, J . Berger, E . Tits- worth, E . Grunberg, J . H . Burchenal, and J . J . Fox, J. med. Chem. 9, 566 (1966); T . Y . Shen and W. V. Ruyle, US-Pat. 3 328 388 and unpublished results. [8aI K. K . Gauri, G. Mulorny, and W. Schiff, Chemotherapy 14, 129 (1969), and literature cited there.

In our laboratory another analog, 5-methylamino-Z'- deoxyuridine (MADU) ( 5 ) , was investigated [91. As found by Visser and his co-workers, MADU is only poorly active as a thymidine antagonist in the bacterial system f101. SurprisingIy, in tissue culture and rabbit-eye systems MADU is as effective as idoxuridine against herpes simplex [111. The high degree of selective toxicity against herpes virus was further dramatized by its lack of inhibition against other DNA and RNA viruses. It wassuggested that the phosphorylated derivatives of MADU block the utilization of thymidine 5'-triphosphate for DNA synthesis. The selectivity of MADU is thus the conse- quence of an enhanced phosphorylating capacity of infected cells 1123. Unfortunately the activity of MADU against systemic herpes infections proved to be a disappointment.

The in vivo activity of nucleosides is often limited by their unfavorable distribution or metabolic charac- teristicsrl31. It was observed recently that the in vivo immunosuppressive activity of 5'-adamantoyl cytarabine (6) is more potent and longer lasting than that of the parent nucleoside (3) [141. Selective localization, sustained hydrolysis, and resistance to enzymatic degradation were suggested as possible explanations. It would seem that similar derivatization of active nucleosides with an in vivo cleavable group, a tech- nique well studied in the production of other drugs, may well expand the potential application of other nucleoside analogs.

HOCH, d OH

A O N HOCK, 4

OH

R = I (Idoxuridine) ( I ) R = H (Cytarabine) (3)

CF3 (2) F (4) NHCH3 (5) CH2CH3 (SO)

More recently a purine nucleoside, adenine arabinoside (7) [151, was described as a broad-spectrum anti-DNA viral agent in vituo and in vivo"6J. I t is probably superior to idoxuridine in the treatment of herpes keratitis but its utility in systemic viral infections remains to be established. In view of the recent impli-

191 T . Y . Shen, J . F. McPherson, and B. 0. Linn, J. med. Chem. 9, 366 (1966). 1101 D . W. Visser, S . Kubat, and M . Lieb, Biochim. biophysica Acta 76, 463 (1963); S. Kubat and D. W. Visser, ibid. 80, 680 (1964). I l l ] M. M . Nemes and M. R. Hilleman, Proc. SOC. exp. Biol. Med. 119, 515 (1965). [12] R . W. Burg and M . M . Nemes, Federat. Meetings Abstract (1970). 1131 K . Gerzon and D . Kau, J. med. Chem. 10, 189 (1967). 1141 G. D. Gray, M . M . Mickelson, and J . A. Crim, Biochem. Pharmacol. 18, 2163 (1969). I151 W. W. Lee, A . Benitez, L . Goodman, and B. R . Baker, J. Amer. chem. SOC. 82, 2648 (1960). 1161 F. M . Schubel, Chemotherapy 13, 321 (1968).

Angew. Chem. internat. Edit. J Vol. 9 (19701 1 No. 9 679

cation of herpes virus in infectious mononucleosis, Burkitt's lymphoma [171, and cervical carcinoma a safe and more effective compound is obviously very desirable.

2.2. Search for Novel Structures

HOCH, d OH

( 7)

The potential role of soluble RNAs as regulators of nucleic acid metabolism has often been discussed [191.

The significance of various minor o r abnormal nucleotides present is particularly intriguing. In our study of pyrimidine nucleosides as novel antimetabolites it was noted that several thiopyrimidine nucleosides, such as 4-thiouridine (8) 1201, 2-thio- cytidine (9), and the 5-substituted derivatives (10) [211

and ( 1 1 ) [221, have been detected in t-RNA. Alteration of 3 5 s incorporation into t-RNA has also been observed after a phage infection 1231.

2-Thiocytosine arabinoside (12) was synthesized in our laboratory by the ring-opening of a cyclo-

HO OH

kf HO OH

HOCH,

HO d OH

( lo) , R = CH,NHCH, ( I ] ) , R = CH,CO,CH,

(171 G . Henle, W . Henle, and V. Diehl, Proc. nat. Acad. Sci. USA 59, 94 (1968); L . N . Chessim, P . R . Glade, J . A . Kasel, H . L. Moses, R . B . Heberman, and Y . Hirschaut, Ann. intern. Med. 69, 333 (1968). [18] W . E . Rawls, W . A . F. Tompkins, M . E . Fiqueroa, and J . L . Melnick, Science (Washington) 161, 1256 (1968). [19] M . J . Robins, R . H . Hall, and R. Thedford, Biochemistry 6 , 1837 (1967). [20] M. N . Lipsett, J. biol. Chemistry 240, 3975 (1965). [21] J . Carbon, H . David, and M . H . Strrdier, Science (Washing- ton) 161, 1146 (1968). [22] L. Baczynsky, K . Biemann, and R. H . Hall, Science (Washing- ton) 159, 1481 (1968); L. Baczynsky, K . Biemann, M. H . Fley- sher, and R . H . Hall, Canad. J . Biochem. 47, 1202 (1969). 1231 S. B. Weiss, Wen Tah Shu, J . W. Foft, and N . H . Scherberg Proc. nat. Acad. Sci. USA 61, 114 (1968).

TrOCH, H,S/CH,OH

100°C P

TrOCH, y&J OH

HOCH, d d H (12)

nucleoside [241, but to our disappointment it does not possess any significant antiviral properties.

So far, anti-DNA viral activity seems to be mostly associated with pyrimidine analogs with 2'-deoxy-~- ribose and D-arabinose moieties. A group of compounds with pyrimidine bases such as uracil, thymine, 5-bromouracil, and cytosine attached to modified pentofuranoses such as 3-amino-2-3-dideoxyribose (13), 2,3-anhydroribose (14), 2,3-dideoxyribose ( I S ) , or its 2,3-didehydro analog (16), and several hexo- pyranoses are all inactive 1251. Recently, the 2',3'-didehydro analog of 5-fluorouridine was found to be an anti-tumor agent [*61, but its antiviral activity has not been described.

A group of branched chain nucleosides, e.g., 2'-C- methyl and 3'-C-methyl homologs of adenosine and cytidine, was synthesized recently 127,281. Compared

[24] W . V. Ruyle and T . Y. Shen, J. med. Chem. IO, 331 (1967). (251 Unpublished results from our laboratories. 1261 T . A . Khwaja and C . Heidelberger, J . med. Chem. 10, 1066 (1967). [27] E. Walton, S. R. Jenkins, R . F. Nutf , M. Zimmermann, and F. W . Holly, J. Amer. chem. SOC. 88, 4524 (1966). [28] E . Walton, F. W . Holly, S . R . Jenkins, R . F. Nutf , and M . M . Nemes, J. med. Chem. 12, 306 (1969).

680 Angew. Chem. internat. Edit. / Vol. 9 (1970) 1 No. 9

with 3'-deoxyadenosine (cordycepin), both 3'-C- methyladenosine (18) and 3'-C-methyIcytidine (19) are much less toxic to cell cultures L291. However, both showed interesting activity against vaccinia infection in mice when administered at doses of 1-2 mg and are superior to the standard drug, Marboran. The corresponding 2'-C-methyl analogs are much less active [281.

NH2 NH2

HOCH,

k?J HO OH HO OH

3. Search for Inhibitors of RNA Viruses

3.1. Structure Information of RNA Polymerase

In the search for anti-RNA viral agents, our attention was directed to the unique synthesis of viral RNA, which involves a single-stranded RNA template, a double-stranded replicative intermediate, and a viral specific polymerase. The complexity of viral repli- cation has generated much speculation and many hypotheses 1301. I t would seem that novel reversible or irreversible inhibitors derived from nucleosides, in addition to their therapeutic value, might also be useful as molecular probes of the active site in polymerase and help to elucidate its mechanism. Current studies of RNA polymerases indicate that the incoming nucleoside triphosphate may interact first with the active site to form a reactive nucleoside-5'-phosphoryl- enzyme intermediate which then participates in coupling with the 3'-hydroxy group of the growing polymer. The sequence of reaction is similar to that in polynucleotide ligase [311 (an enzyme whose activity is changed significantly after viral infection and which may turn out to be a chemotherapeutic target also) 1321.

n - enzyme I1

(ligase) + or DPX

w HO OH ""dv (enzyme-pA)

[29] H . T. Shigeura, unpublished observations. [30] Nature (London) 219, 675 (1968). 1311 2. W. Hall and I . R . Lehman, J. biol. Chemistry 244, 43 (1969). [321 J . Sambrook and A. J . Shatkin, J. Virology 4 , 719 (1969).

The active site of RNA polymerase contains a histidyl residue 1331 which is essential for chain elongation. Analogous to the active site in nucleoside diphospho- kinases [341, the histidyl group may form a phosphoryl- imidazole intermediate (20), with the nucleoside triphosphate.

h N*NII

+ A T P +

0

Speculatively, a concerted activation (step 1) and transfer (step 2) sequence may be visualized as shown in Scheme 1.

Scheme 1 .

3.2. Models of the Enzyme Intermediate

As a model of this active intermediate we have synthesized the crystalline compound (21) [351. The site of phosphorylation is tentatively assigned as N-3, based on the observation that the N-3-phosphoryl derivatives

0 0 II 11

CH,-C-NH-CH-C-NH-CH, t AMP --+ I c H2 h

N+,NH

[33] A. Ishihama and J . Hurwitz, J. biol. Chemistry 244, 6680 (1 969). [34] P . L. Pedersen, J. biol. Chemistry 243, 4305 (1968). [3S] K . H . Boswell and T. Y. Shen, unpublished.

Angew. Chem. infernat. Edit. Vol. 9 (1970) / No. 9 68 1

of histidine are generally more stable than those phosphorylated at N-1 [361. Unlike many adenylic acid derivatives, which have ORD spectra similar to that of 5’-AMP with a negative Cotton effect at 277 nm, compound (21) gave an anomalous ORD spectrum with a positive Cotton effect near 270 nm. Whether the reversal of Cotton effect is indicative of a conformational change due to the intramolecular interaction of the imidazole moiety and adenine chromophore remains speculative, but any correlation of the physicochemical properties with those of the active intermediate of polymerase would certainly be worth investigating. A similar reversal of the Cotton effect is found for the 5’-adenylyl derivative of imidazole, pyrazole, and 2-hydroxy- pyridine 1351.

The possibility of using these analogs of an active intermediate to interfere with the enzyme action are still being investigated. I t is assumed that in the normal course of polymerization the formation of the active intermediate (imidazole-p-A) is followed by transfer of p-A to the growing chain of the polynucleotide. This would require either a conformational change of the active site, e.g. , prompted by the elimination of the charged pyrophosphate group, or a translocation of the active intermediate itself. One could imagine that these analogs may well interfere with the transfer step. Classical antimetabolites of the substrate ATP, with their charged triphosphate-like side chain, will probably interfere only with the activation step and the formation of the active intermediate.

3.3. 5‘-Substituted Nucleoside Analogs as Nucleotide Equivalents

The best known classical antimetabolites of nucleotides are their methylene phosphonate isosteres, e.g., compounds (22) [371 and (23) [381. In both cases an oxygen linkage is replaced by a methylene group that is non- cleavable under physiological conditions.

Depending on which oxygen atom is replaced by the methylene group, these analogs are either substrates or inhibitors 1371. However, their practical applications are obviously restricted by their poor permeability properties. To circumvent this barrier, we decided to investigate a group of nucleoside derivatives bearing a polar organic moiety a t the 5’-position which might mimic the triphosphate-Mg2+ side chain of the polym-

[36] D . E. Hultquist, Biochim. biophysica Acta 153, 329 (1968). [371 T. C. Meyers, K . Nakamura, and A . B. Danielsaheh, J. org. Chemistry 30, 1517 (1965). [38] G . H. Jonesand J . G. Moffat, J. Amer. chem. SOC. 90,5337 (1968).

erase substrate [391. These 5’-substituted nucleosides probably permeate easily into cells. In addition, they are attractive for several important reasons. Firstly, with their 5’-hydroxyl group replaced by a substituent they are totally nonincorporable, thus fulfilling our safety requirement mentioned above. Secondly, unlike most nucleoside antimetabolites, any inhibitory action of a 5‘-substituted nucleoside should not depend upon its prior enzymatic conversion into a nucleotide, and resistance development is therefore less likely 1403.

Furthermore, as recently shown by 5’-deoxy-5’- methylthioadenosine [411, 5’-substituted adenosine derivatives might be more resistant to catabolic enzymes such as adenosine deaminase and purine nucleoside phosphorylase. Although unknown at the beginning of our study, the feasibility of using a 5‘-substituted nucleoside as kinase or polymerase inhibitors was strongly indicated by the recent disclosure of 5‘-deoxy- 5‘-fluorothymidine (24) as a competitive inhibitor, not of thymidine to its kinase, but of thymidylic acid to thymidylate kinase [421. Another example of a 5’- substituted nucleoside analog is the naturally occurring antibiotic nucleocidin (2s) 1431 whose structure was elucidated recently.

0 H3cc~0 oH (24)

Our endeavor to construct an organic equivalent of the 5’-triphosphate-Mg2+ complex was much ham- pered by the uncertainty on the configuration of nucleotides such as the ATP-MgZ+ complex. In general, a cyclic chelating structure formed by the P and y phosphates and Mgz+ is favored, but the possible

WJ OH

1391 T . Y. Shen, Abstracts of Papers, 154th Amer. Chem. SOC. Meeting, Sept. 1967, Chicago, Ill. , p. 29. [40] B. R . Baker and P . M. Tanna, J. pharmac. Sci. 54, 1774 (1965). 1411 A . E. Pegg and H . G . Williams-Ashman, Biochem. J. 11-7, 241 (1969). 1421 P . Langen u. G. Kowollik, Europ. J. Biochem.6, 344 (1968). [431 G . 0. Morton, J . E. Lancaster, G . E . Van Lear, W. Fulmor, and W . E. Meyer, J. Amer. chem. SOC. 91, 1535 (1969).

682 Angew. Chem. internat. Edit. / Vol. 9 (1970) 1 No. 9

involvement of a ring nitrogen in the coordination remains to be established. Very recently structures (26) involving the participation of N7, instead of the 6-amino group, have been suggested on the basis of relevant X-ray data 1441. The three-dimensional structure of ATP in the hydrated disodium salt, with the phosphate chain folded back towards the purine base, was reported by another group (451.

HN=C=NCH,

RO d OR (31) yv RO R

R = H or Y=NH-CO-NHz, NH-C(NH)-NH2, R-R = isopropyIidene NH-CS-NH2, NH-C(NH)-NHC4Hg

3.4. Derivatives of S'-Amino-5'-deoxy Nucleosides The protected 5'-arnino-S'-deoxyadenosine (32) was phosphorylated with dibenzyl phosphite and N-chloro- succinimide in methylene chloride to the phosphor- amidate (33). The isopropylidene group was first removed by dilute formic acid to give (34), which, on

Our initial plan was to explore various nucleoside derivatives which have a cyclic or chelating structure at the 5'-position to mimic the triphosphate side chain, and the 5'-amino-5'-deoxy and S'azido-5'-deoxy derivatives of adenosine (27) and (28), respectively [461,

were chosen as key intermediates for their synthesis. These selections turned out to be particularly for- tuitous since both compounds exhibited antiviral activity per se; inhibition of parainfluenza I11 was demonstrated 1471 at 8-30 tJ-g/ml in tissue cultures with therapeutic indices ranging from 16 to 128, depending upon the severity of the infection. Encouraged by these findings, we synthesized a number of simple derivatives from the versatile 5'-tosylate (29) by similar nucleophilic displacement reactions. To minimize formation of the cyclonucleoside (30), the N6- formyl and 6-methylthio derivatives were employed in some cases.

OxO (32) (34), R = H H3C CH3

0

- HO OH

(35)

R hydrogenolysis in the presence of sodium hydroxide afforded a product, believed to be the desired 5'-aza AMP, that was too unstable for cyclization to the

TsOCH

NH2 +

HO OH

HOCH, iiJ H

NHC=O

J H

NHC=O

0 0 x H3C CH3

0 It

R X- = N3, NH2, CH3NH, (CH&N, CH3-C(O)S, -N=C=NH,

--SC=N

= CH3S, HCNH, (CI, NH2, SH) TsOCHz d (37)

OTs O$H

l o Several guanidine and urea derivatives were readily prepared from the cyanamide precursor (31).

I441 M . Sundaralingam, Biopolymers 7, 821 (1969). [45] 0. Kennard, N . W . Isaacs, J . C . Coppola, A. J . Kirby, S . Warren, W . D . S . Motherwell, D . G . Watson, D . L. Wampler, D . H. Chenery, A. C. Larson, K . A. Kerr, and L. R . DiSanse- verino, Nature (London) 225, 333 (1970). [46] W . Jahn, Chem. Ber. 98, 1705 (1965). I471 M . M . Nemes, unpublished observations. (39)

Angew. Chem. internat. Edit. J Vol. 9 (19701 No. 9 683

desired 5’-aza-3’,5‘-cyclic AMP. A similar observation regarding the stability of (35) was reported recently 1481.

In the 2‘-deoxyribonucleoside series, 2’-deoxyadenosine, a known antimetabolite, was sulfonated to give a mixture of mono- and di-tosylates. Treatment of their formyl derivatives (36) and (37), respectively, with sodium azide gave the corresponding azido derivatives (38) and (39). The former was reduced to 5‘-amino-2‘-5’-dideoxyadenosine (40) [491.

P A / C

o r ’ : H15 NHZ

3.5. 5‘-Substituted Arabinosyl Nucleosides - (411 4

PmdCH2 d PmdCHz

k$ The importance of arabinosyl nucleosides in the therapeutic field has been amply demonstrated by cytosine-, adenine- and 6-methylthiopurine arabinosides. Cytosine arabinoside was readily transformed to its 5‘-amino analog (41).

OR

itself [51J. The 4-keto group in (45) was converted to the 4-amino via the methylthio intermediate, and the protecting groups were removed to give (41). The arabinosyl halide (44) was condensed with adenine and then deblocked to give 5‘-amino-5‘-deoxyadenine arabinoside (46). The 6-mercaptopurine analog (47) was obtained in a small yield from the protected 6- chloropurine precursor by treatment with thiourea. In this case the removal of protecting groups was com- plicated by the formation of disulfide and other de- composition products.

P‘ R’

NHAc NHAc

OAc OAc

O N r3

R’= NH2,C1 b R R’ = NH,, C1

In order to develop a versatile route to study 5‘- substituted arabinosyl nucleosides, we first synthesized methyl 5‘-azidoarabinofuranoside (42) as a potential intermediate. Unexpectedly, attempted mild acid hydrolysis yielded a new crystalline compound which was found to be the piperidone (43) [501.

On the other hand, when a phthalimido group was placed at the 5’-position, the protected arabinosyl halide (44) could be prepared readily from the corresponding arabinosyl p-nitrobenzoate and used in the standard Hilbert-Johnson reaction. The $-nucleoside (45) was formed as the predominant product from the a-chloride. The stereospecificity is reminiscent of our earlier experience with 2,3,5-tri-O-benzylarabino- syl halide in the synthesis of cytosine arabinoside

[48] B. Jastorff and H. Hettler, Tetrahedron Letters 1969, 2543. [49] An independent synthesis of this compound was recently reported: M . G . Stout, M . J . Robins, R . K . Olsen, and R . K . Robins, J. med. Chem. 12, 658 (1969). [50] W. V. Ruyle, unpublished observations.

OH

(46/ , R2 = NH, (47). RZ = SH

The P-configuration of these purine arabinosides was corroborated by the similarity of their ORD spectra with that of adenosine. Previous workers have shown that the configuration of the 2‘-hydroxyl group has no significant effect on the ORD of these nucleosides.

3.6. Biological Properties of 5’-Substituted Nucleosides

The new analogs exhibited moderate to good cyto- toxicity vs. HeLa cells at concentrations of 1 milli- mole/] 1291. The 5’-azido-5‘-deoxy derivatives of both

[ S t ] T. Y. Shen, H . M. Lewis, and W . V . Ruyle, J. org. Chemistry 30, 835 (1965).

684 Angew. Chem. internat. Edit. / Vol. 9 (1970) J NO. 9

adenosine and 6-methylthiopurine riboside are highly toxic at this level. Several analogs, e.g., the 5’-amino, 5’-azido, 5‘-ureido, and 5‘-ethoxycarbamyl derivatives of adenosine, were shown to inhibit the conversion of adenosine to its phosphorylated derivatives in ascites cells to cn. 60 ”/, at concentrations of 0.9 mmole/l r291;

however, no significant antiviral activity was un- covered. The inhibition of adenosine utilization by these compounds is reminiscent of the inhibition of thymidine utilization by 5’-deoxy-5’-fluorothymidine (24) mentioned earlier. As part of our systematic structure modifications, the 5‘-azido derivative (49) of 6-isopentenyladenosine (48) (6-IPA) L521 was also synthesized. This derivative is again a nonincorporable antimetabolite. Yet, like 6-IPA, it inhibited HeLa cells to almost 100 ”/, at a concentration of 1 mmole/l, although its potency was somewhat less at lower concentrations.

p 3

CII,

(481, K = OH (6- IPA) (49), R = N,

KCH,

I T 0 OFT

4. Immunological Effects of Nucleosides and Nucleotides

4.1. Immunosuppressive Nucleosides

by their potential application as immunosuppressive

4.2. Properties of Cytokinin Derivatives

Kinetin riboside (52), 6-IPA, and other cytokinin derivatives represent a new group of immunosuppressants. The primary effect of 6-TPA was suggested as interference with RNA and protein synthesis [Sjl. It has recently been observed that, depending on the concentration and on the particular stage of the cell cycle, 6-IPA has a biphasic effect on the transfornia- tion and mitosis of human lymphocytes treated with phytohemagglutinin. This biphasic response is reminiscent of the antibody stimulating or inhibitory effect of another RNA synthesis inbibitor, 6-MP, in several in viva systems (561.

6-IPA and cytokinins also exert an indirect influence on certain humoral immune responses mediated by nucleic acids. The participation of two or more cell types, macrophages and lymphocytes, in the primary response has been demonstrated 157,581. In the case of certain soluble antigens, a 4 S RNA or a RNA-protein complex is released by macrophage after phagocytosis of the antigen. This RNA-containing material, the nature of which is still being elucidated, is possibly involved in the sensitization of lymphocytes 1591.

Coincidentally, nucleic acid digests or oligonucleotides have been shown to have an adjuvant effect in en- hancing the phagocytosis of antigen by macrophage and in stimulating the production of antibodies by sensitized lymphocytes (Fig. 1) 1601. For reasons not yet understood, the antibody stimulating effect is inhibited by 6-IPA, kinetin riboside (521, and other analogs [611.

stiniuIation by oIigonucIeotides kinetin analogs

1 I lymphocyte Our interest in 6-IPA analogs was further stimulated antigen - macrophage- - ox‘

phagocytosis HNA - protein sensit ization +

agents. Nucleoside antimetabolites such as 6-mercaptopurine, imuran, and cytarabine (3) have been used in clinical treatment of various immunological disorders. 6-Mercaptopurine arabinoside (50) and 6- methylthiopurine riboside (51) and its periodate oxidation product have also been studied in the laboratory 1531. Using the hemagglutinin response to sheep erythrocytes as a model for humoral antibody production and graft versus host reactions as re- presentative cell-mediated immune reactions, the mechanism of action of these immunosuppressants is gradually being elucidated 1531. It is instructive to realize that these DNA synthesis inhibitors are very effective in suppressing the proliferation of sensitized lymphocytes and cell-rnediated immunity, but the biochemical blockades imposed are easily overcome. Humoral antibody response is generally not inhibited by these compounds 1541.

[521 K . Biemann, S . Tsunakawa, K. Sonnenbichler, H. Feld- mann, D . Diifting, and H. G. Zarhair, Angew. Chem. 78, 600 (1966); Angew. Chem. internat. Edit. 5. 590 (1966). [531 J . P. Bell, M . L . Faures, G . A. LePnge, and A . P . Kimball, Cancer Res. 28, 782 (1968). [541 R . H. Gisler and J . P. Be“, Biochem. Pharmacol. 18, 2115, 2123 (1969).

complex

antibody production

Fig. 1. Effect of cytokinin o n antibody production.

Cytokinins are usually alkylated purine derivatives 1621, but a pyrimidine cytokinin, 6-methyluracil (pseudothymine) (53), was described recently 1631. Jt would be of interest to compare the immunosuppressive activity of pseudothymine nucleosides 1641.

[55] R. C. Gallo, J . Whang-Peng, and S. Perry, Science (Washing- ton) 165, 400 (1969). I561 E. Gabrielson and R . A. Good, Adv. Immunology 6, 148 (1967). I571 M. Fishman and F. L. Adler, J . exp. Med. 117, 595 (1963). [58] M . Fishman, Annu. Rev. Microbiol. 23, 199 (1969). [59] A. A. Gottlieb, V . R . Glisin, and P . Doty, Proc. nat. Acad. Sci. USA 57, 1849 (1967); A. A. Gottlieh and D . S. Straus, J. biol. Chemistry 244, 3324 (1969). [60] W . Braun and W . Firshein, Bacteriol. Rev. 31, 83 (1967). I611 O.J. Plescia and W . Braun: Nucleic Acids in Immunology. Springer-Verlag, Wien-New York 1968, p. 347. [621 J . P. Helgeson, Science (Washington) 161, 974 (1968). [631 B. I. Pozsar and Gy. Matolcsy, Life Sciences 7, 699 (1968). I641 M . W. Winkley and R . K. Robins, J. org. Chemistry 33, 2822 (1968).

Angew. Chem. infernat. Edit. j Vol. 9 (1970) J No. 9 68 5

Another alkylated purine, 1-methyladenine (54), is a potent meiosis inducer which stimulates ovulation in star fish at a concentration of 0.02 pg/ml[651. The corresponding 1-ethyl homolog is much less active. We have noted that I-isopentenyl adenine (55), an intermediate in the synthesis of N6-isopentenyladenine, inhibits thymidine incorporation in the HeLa cells at 1 mmole/l but increases thymidine and uridine incorporation in phytohemagglutinin-stimulated rat spleen cells at 10 pg/ml[661.

Both 6-IPA (48) and its 2-methylthio derivative have been found to occupy positions adjacent to certain anticodon regions in t-RNA 167,681. The special loca- tion of these bases and the effectiveness of hydrophobic N6 side chains have led to the suggestion that the cytokinin in t-RNA functions in the codon-anticodon interaction, possibly with an increasing ribosomal binding by the N6-substituents. Whether the regulatory effect of exogenous cytokinins is achieved by the incorporation into t-RNA per se or by an indirect influence on the “pool” of cytokinin-containing t-RNA species remains to be elucidated. The asymmetric N6- alkyl side chains in several cytokinins preferentially adopt the (S)-configuration

The effects of these nucleoside analogs on mammalian cells have received much attention recently. Differen- tial toxicity to certain leukemic cells and normal lymphocytes has been observed with a group of N6- alkylated adenosines [70-721. Obviously an extension of this kind of inhibitory action to immunological disorders would be of considerable interest.

SH P

HOCH, d OH

(50)

HO OH

1!4 (511,R = SCH, (52),R = NHCH, 0

L65] H . Kanatani, H . Sirai, K . Nakanishi, and T . Kurokowa, Nature (London) 221, 273 (1969). [66] H . T . Shigeura and T . L . Feldbush, unpublished observations. [67] W. J . Burrows, D. J. Armstrong, F. Skoog, J . M . Hecht, J.T. A . Boyle, N.J. Leonard, and J. Occolowitz, Science(Washing- ton) 161, 691 (1968). [681 D. J . Armstrong, W. J . Borrows, F. Skoog, K . L . Roy, and D. Soll, Proc. nat. Acad. Sci. USA 63, 834 (1969). [69] K . Koshimizu, A . Kobayashi, T. Fukita, and T. Mitsui, Phytochemistry I , 1989 (1968). [70] N. J. Leonard, S. M . Hecht, F. Skoog, and R . Y. Schmitz, Proc. nat. Acad. Sci. USA 59, 15 (1968).

5. Oligonucleotides

5.1. Adjuvant Effect of Oligonucleotides

In our laboratory we were also intrigued by the enhancement of host resistance to infection by oligonucleotides C601. In the original immunological experi- ments an antibody-stimulation effect was observed with a gross mixture of DNA digest and the optimal size of the oligomers from DNA digest was estimated to be tri- to hexanucleotides. Our aim then was to delineate the active structural features of oligomers, and, if possible, to shed further light on the mechanism of this stimulating effect. Base sequence, charge distribution, molecular conformation, nature of back- bone, and stability to nuclease cleavage were some of the parameters to be evaluated. The cellular uptake of oligonucleotides and the site of action, whether membrane or cytoplasmic, also required clarification (see Fig. 2).

Fig. 2. Immune stimulating effect of oligonucleotides.

1. Enhancement of phagocytic activity.

2. Reduction of induction period in antibody production.

3. Increase of deoxynucleotide kinase activity (dCMP, dGMP).

4. Protection against bacterial infection in vivo.

Active structures: Oligonucleotides (n = 3-6)

Poly C , Poly A (Poly C + Poly I).

5.2. Synthesis of Oligo(2’-deoxynucleotides)

As an initial step, a group of tetradeoxynucleotides of the general type dApdApdApdX (56) with dX = dA, dG, dI, T, and dC were synthesized by a modified procedure which incorporates the advantages of several well known techniques [73,74a,74bl.

N6 - Benzoyl-5’- 0-monomethoxytrityl-2‘- deoxyadenosine (57) was used as starting material. Treatment with trichloroethyl phosphate and mesitylenesulfonyl chloride in pyridine gives the 3’-phosphorylated derivative (58). Condensation with the 5‘-hydroxyl of N6-benzoyl-2‘-deoxyadenosine was also carried out in pyridine in the presence of triisopropylbenzenesulfonyl chloride to give the dinucleotide (59). The sequence of 3‘-phosphorylation and 5‘-condensation was re- peated to give the trinucleotide (60) and this in turn was condensed with appropriately protected deoxyadenosine, deoxyguanoside, and deoxycytidine. 2’- Deoxyinosine and thymidine were used directly. The use of fully protected intermediates enabled us to employ silica gel column chromatography for their isolation and to obtain 10-20 mg of products without

[71] M . H . Fleysher, M . T. Hakala, A . Bloch, and R . H. Hall, J. med. Chem. I I , 717 (1968). [I21 M. H. Fleysher, A . Bloch, M . T . Hakala, and C. A . Nichol, Abstracts of Papers, 156th Amer. Chem. SOC. Meeting, Sept. 1968, Atlantic City, N. J. Medi 25. [73] H . G . Khorana et al., J. Amer. chem. SOC. 89, 2148, 2185, 2195 (1967). [74] a) F. Eckstein and J. Rizk, Chem. Ber. 102, 2362 (1969); b) R . L. Lersinger and K . K . Ogilvie, J. Amer. chem. SOC. 89, 4801 (1967).

686 Angew. Chem. internat. Edit. 1 Yol. 9 (1970) No. 9

(56),X = A, T, G, C, I

t O = P - OCH, CC 1,

I 1. Zn-Cu/DMF ( 5 0 % ) 0-CH, ABZ *

- (CII,CC I J

2. NHiOH * -(C,H,CO)

’ii’ 9

P + HO-P-OCHzCCl, -

I OH

(57) OH

> (56)

(58) +

I OH

I 0 I

O= P - OCHzC C1, I

0-CH, ADZ

9 O=P-OCH,CC1,

b-CH, AHZ w P

O=P-OCH,CCl, + I

0-CH, ARz HOCH, X w P w - OH

0-P -OCH,CCl,

AH (60)

0 7

O=P-OCHzCC1, I

0-CH, X

’cr>l OH

0-YH, X

too much difficulty. The protecting groups in the tetranucleotide (61) were removed sequentially by treatment with Zn/Cu couple, dilute ammonia, and aqueous acetic acid. The selection of trichloroethyl as our phosphate-protecting group also conferred ver- satility to this approach for further structure modifications. The scope of this synthetic sequence was recently extended to the oligoribonucleotide series in which the 2’-tetrahydropyranyl was used as protected intermediate [751. Conversion of our oligodeoxyribo- nucleotides to cyclic (circular) analogs in order to increase their resistance to nucleases and to enhance cellular uptake is still under study; we are particularly interested in the physicochemical properties of the products.

5.3. Potential Applications of Oligonucleotides

Another potential application of nucleotide derivatives lies in the area of autoimmune diseases, such as systemic lupus erythmatosus (SLE) and other chronic degenerative disorders, which are characterized by the production of anti-nuclear antibodies directed against nucleic acids and nucleoproteins. A variety of antibodies to native DNA, single-stranded DNA, and even double-stranded RNA are found in patients with SLE [76,771. These antibodies react with nucleic acids to form antigen-antibody complexes which cause in- flammation along the basement membrane of renal glomerulus leading to the development of glomerular lesions. On considering the formation of pathogenic antigen-antibody complexes one is reminded of the fact that the binding of antigen with antibody is analogous to enzyme-substrate interactions. I t is well established that the interaction of polysaccharide and its antibody can be inhibited by chemical analogs of the determinant group, such as oligosaccharides 1781. Similarly, a partial inhibition of SLE sera by tetra- or pentanucleotides was demonstrated several years ago [791. With more precise information on the nature of binding involved it is conceivable that one may find oligonucleotide derivatives not only with higher affin- ity but also capable of cross-reacting with a large group of SLE sera. The formation of soluble antigen-antibody complexes between oligonucleotide analogs and anti-DNA antibodies would presumably minimize immunological lesions and possibly facilitate the induction of tolerance (see Fig. 3) [SO].

The concept of using derivatives of oligonucleotides or much simplified “structure equivalents” to mimic or to compete with biologically active polynucleotides should be applicable to other important problems of

[75] T . Neilson, Chem. Commun. 1969, 1139. [76] D . Koffler, R . I . Carr, V . Angello, T. Fiezi, and H. G . Kun- kel, Science (Washington) 166, 1649 (1969). 1771 P . H. Schur and M . Monroe, Proc. nat. Acad. Sci. USA 63, 1108 (1969). [78] E. A. Kabat: Structural Concepts in Immunology and Immunochemistry. Holt, Rinehart and Winston, New York 1968. [79] L. Levine and B. D. Stollar, Progr. Allergy 12, 161 (1968). 1801 A . R . Boyns and J . Hardwicke, Immunology 15,263 (1968).

Angew. Chern. internat. Edit. / Vof. 9 (1970) / No. 9 687

anti-DNA (Ab-Ag) - glomerular antibody + DNA (Ag - complex iit lesions (Ab) ‘ complement

+ Oligonucleotides (Ad’ Soluble

i- + Ab-Ag‘ (non-pathogenic?)

Fig. 3. Pathogenesis in systemic lupus erythmatosus.

medicinal interest such as interferon induction, viral RNA polymerase inhibition, specific t-RNA inhibition, and episome transfer. In each case one may assume that the interaction of nucleic acid with a protein molecule or a receptor involves only one region or several regions of oligonucleotides and that the specificity is determined by the base sequence and secondary structure of those regions. The feasibility of this kind of approach is clearly indicated by recent sequence determinations of QP and R17 phage RNA. The base sequence of QB phage was determined from both the 3’- [81,82J and 5’-terminals @31, and no less than 175 nucleotides from the 5’-terminus with a well- defined loop-like secondary structure have been elucidated @31. The identification of a specific binding region should enable us to design chemical structures either to compete for the protein binding site or to interact with the oligonucleotide region itself by means of intercalation, base pairing, or formation of other hydrogen bonds.

6. Novel Nucleoside Derivatives

6.1. “Double-Headed” Nucleosides

To expIore nucleoside analogs which might show a selectivity for certain base-sequences on interaction with nucleic acid, we have utilized the versatile 5’-0- tosyl and 5‘-amino-5‘-deoxy derivatives of nucleosides described above to synthesize novel ribonucleosides and 2’-deoxyribonucleosides which possess a second purine or pyrimidine moiety at the 5’-carbon C84351.

It is hoped that with the proper selection and spacing of the two bases, one may utilize one base for Watson- Crick type pairing and the other to intercalate between the next two adjacent nucleotides in a polynucleotide.

[81] U. Rensing and I . T. August, Nature (London) 224, 853 (1969). 1821 H. L. Weith and P. T. Gilham, Science (Washington) 166, 1004 (1969). [83] M . A. Billiter, J . E. Dahlberg, H. M. Goodman, J . Hindley, and C. Weissmann, Nature (London) 224, 1083, 1055 (1969). [84] R. Fecher, K . H. Boswell, J . Wittick, and T. Y. Shen, J. Amer. chern. SOC. 92, 1400 (1970). 1851 K. H . Boswell and T. Y. Shen, unpublished.

TsOCH, Th NH2

(62) OH

OH I I

OH (63)

The specificity of these interactions will be the basis for selective binding of three nucleotides by one double-headed unit. The nucleoside (63) gave an unusual CD spectrum 1841, presumably due to intramolecular interactions between the two proximate bases. These compounds are useful models for the study of base-base interactions. The pronounced Cotton effect may also serve as a convenient marker in following the intermolecular interactions between these compounds and polynucleotides.

7. Conclusion

The medical applications of nucleotide derivatives have not received much attention in the past partly because of the difficulties in chemical synthesis and partly because of their poor absorption and cellular permeability characteristics. However, recent developments in synthetic methods and isolation techniques have brought oligomers within the sphere of medicinal chemical explorations. New discoveries, such as the use of double-stranded polynucleotides as interferon- inducers in viral chemotherapy 186,871, have broadened the traditional concept of using only monomeric drugs. The growing knowledge of membrane receptor sites and membrane enzymes such as ATPase in the regulation of cellular activities also provides new ex- tracellular targets for drug action. With the structure elucidation of nucleic acids and the understanding of their interaction with protein receptors, further advances in the application of nucleoside and nucleotide derivatives as novel therapeutic agents are certainly to be expected.

Received: April 20, 1970 [A 779 IEI German version: Angew. Chem. 82, 729 (1970)

[86] G . P. Lampson, A. A . Tjfel l , A . K . Field, M . M . Nemes, and M . R. Hilleman Proc. nat. Acad. Sci. USA 58, 782 (1967). [87] J . H . Park and S . Baron, Science (Washington) 162, 811 (1968).

688 Angew. Chem. internat. Edit. VoI. 9 (1970) No. 9

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Nucleosides and Nucleotides as Potential Therapeutic Agents