US005750729A United States Patent (19) 11) Patent Number: 5,750,729 Alexander et al. 45) Date of Patent: May 12, 1998

54 COMPOUNDS AND METHODS FOR Darby, G. "In search of the perfect antiviral." Antiviral MAKING AND USNG SAME Chem & Chemo 6:54-63 (1995). Engel, R., "Phosphonates as Analogues of Natural Phos 75 Inventors: Petr Alexander. San Mateo; Ernest J. phates." Chem Rev 77(3):349-367 (1977). Prisbe, Los Altos, both of Calif. Engstler et al. "N-(4-nitrophenyl)oxamic acid and related N-acylanilines are non competitive inhibitors of Vibrio 73) Assignee: Gilead Sciences,ne. Foster City, Calif. cholerae sialidase but do not inhibit trypanozoma cruzi or tryponosoma bruceii trans-sialidases." Helvetica Chimica (21) Appl. No.: 806,575 Acta 77:1166-1173 (1994). Hansen et al., "Synthesis of 22 Filed: Feb. 25, 1997 3'-Azido-2,3'-dideoxy-beta-D-arabino-hexopyranosyl Nucleosides." Lebigs Ann. Chem 1079-1082 (1990). Related U.S. Application Data Jasko et al. "5'-Phosphonates of Ribonucleosides and 62) Division of Ser. No. 615,669, Mar. 13, 1996, which is a 2'-Deoxyribonucleosides: Synthesis and Antiviral Activity.” division of Ser. No. 384,504, Feb. 1, 1995, Pat No. 5,659, Nucl5 & Nuclt 12(8):879-893 (1993). 023. Jois et al., "Synthesis and Antiviral Evaluation of Some Novel 1.2.4Triazolo 4,3-beta 1.2.4 triazole Nucleoside (51) Int. Cl...... C07F 9/28; CO7F 327/00; Analogs." J Het Chem 30:1289-1292 (1993). CO7F 333/32; CO7G 3/00 Kambler et al., "gamma-phosphono-gamma-lactones. The 52 U.S. Cl...... 549/216; 549/5; 549/13; use of allyl esters as easily removabe phosphonate protect 549/21; 549/62:536/4.1; 536/7.1; 536/6 ing groups." CanJ Chem 63:823-827 (1985). 58) Field of Search ...... 536/4.1, 7.1, 6. Kim et al. "Synthesis and HIV Activity of Phosphonate 536/18.5, 18.6; 549/5, 13, 21, 22. 62,216 Isosteres of D4T Monophosphate." Bioorg Med Chem Lett 5(2):367-370 (1992). 56) References Cited Kira et al., "Anti-Herpes Activities of Isonucleoside Ana U.S. PATENT DOCUMENTS logues with Variable Bases at the 2"Position." Nucls & Nuclt 14(3-5):571-574 (1995). 5,059,690 10/1991 Zahler et al...... 544/276 Miller et al., "Gene transfer and antisense nucleic acid 5,276,143 1/1994 Sabesan et al. . techniques." Parasitology Today 10(3):92-97 (1994). 5,314,893 5/1994 Tino et al...... 514/274 5,414,000 5/1995 Tino et al...... 514/261 Mitchell et al. "Boron trifluoride-methanol complex as a 5,659,023 8/1997 Alexander et al...... 536/22.1 non-depurinating detritylating agent in DNA synthesis." Nuc Acids Res 18(17):5321 (1990). FOREIGN PATENT DOCUMENTS Neidlein et al., "172. Synthesen und Untersuchengen von O 398 231 A2 11/1990 European Pat. Off. . Oxazolo2.3-alphalisoindlol-9b(2H)-yllphosphonaten WO 92/13869 8/1992 WIPO und-phosphinaten: eine neue Klasse von Heterocyclen." WO95/O7919 3/1995 WIPO Helvetica Chimica Acta 76:2407-2417 (1993). WO9507920 3/1995 WIPO Otmar et al., "15-(adenine-9-yl)-5-deoxypentofuranosyl phosphonates-a novel type of nucleotide analogs related to OTHER PUBLICATIONS HPMPA," Collect Czech Czech Chem Commun Aerschot et al., "Synthesis and Anti-HIV Activity of 58:2159-2179 (1993). Dideoxyctiline Analogues Containing a Pyranose Carbohy Pattenden et al. "Maleic anhydrides in synthesis. Preparation drate Moiety.” NUCLS & NUCLT 10(1-3):589-590 (1991). of furan-2(5h)-one phosphonate derivatives and a new Alexander et al., "Synthesis and Antiviral Activity of Pyra synthesis of pulvinic acids and pulvinone analogues." J nosylphosphonic Acid Nucleotide Analogues." J Med Chem Chem Soc Perkin Trans I 1:2357-2361 (1991). 39:1321-1330 (1996). Perez-Perez et al., "Phosphonates Derivatives of 2'. Augustyns et al., "Sugar Modified Oligonucleotides." 3'-Dideoxy-2',3'Didehydro-Pentopyranosyl Nucleosides." NUCLS & NUCLT 10(1-3):587-588 (1991). Nucls & Nuclt 14(3-5):707-710 (1995). Bergstromet al., "Organoiron-Mediated Alkylation of Phos (List continued on next page.) phite Esters: Synthesis of (Dicarbonyl) (eta5-cyclopentadienyl)iron-Derived Nucleoside Phospho Primary Examiner-W. Gary Jones nate Esters." J Org. Chem 57:873-876 (1992). Assistant Examiner-Jeffrey Fredman Bessodes et al., “Synthesis of Unsaturated 4'-Azido Pyra Attorney, Agent, or Firm-Max D. Hensley nosyl Thymines as Potential Antiviral and anti-HIV 57 ABSTRACT Agents." J Chem Soc Perkin Trans 13035-3039 (1990). Caruthers, M.H., "Chapter 1: Synthesis of Oligonucleotides In accordance with this invention novel compounds are and Oligonucleotide Analogues." Oligodeoxynucleotides: provided that are selected from saturated and unsaturated Antisense Inhibitors of Gene Expression (J.S. Cohen, Ed.) pyrans and furans substituted with at least a phosphonate pp. 7-24 (1989). group and a heterocyclic base. These compounds are useful Cinatlet al. "Failure of Antiretroviral Therapy: Role of Viral as antiinfectives, flame retardants, diagnostic oligonucle and Cellular Factors." Intervirology 37:307-314 (1994). otides and immunogens. Crane et al., "Isonucleosides from Glucosamine." J. Carbo hydrates Nucl Nuclt 7(5):281-296 (1980). 1 Claim, No Drawings 5,750,729 Page 2

- OTHER PUBLICATIONS Wu-Pong, Susanna, "Oligonucleotides: Opportunities for Perez-Perez et al., “Synthesis and Antiviral Activity of Drug Therapy and Research." Pharmaceutical Technology Phosphonate Derivatives of Enantioneric pp. 102-114 (1994). Dihydro-2H-Pyranyl Nucleosides." Bioorg Med Chem Lett Xiong et al., "Kinetic Analysis of the Interaction of Cido 5(11):1115-1118 (1995). fovir Diphosphate with Human Cytomegalovirus DNA Renault et al. "Synthesis and antiviral evaluation of furo pyrimidine diones cyclic and acyclic, nucleoside ana Polymerase." Biochem Pharm 51:1563-1567 (1996). logues." Heterocycles 41(5):937–945 (1995). Yokota et al. "Inhibitory effects of acyclic nucleoside phos Rosowsky et al., "Synthesis of 3'-O-Propargylthymidine phonate analogues on hepatitis B virus DNA synthesis in as a Candidate Antiretroviral Agent." Nucl & Nuclt HB611 cells." Antiviral Chem & Chemo 5(2):57-63 (1994). 8(4):491-497 (1989). Schmitz et al., "Nonradioactive Labeling of Oligonucleotide Booma et al. "Stereoselective C-3 Substitution of 1.5 anhy in Vitro with the Hapten Digoxigenin by Tailing with dro 2-deoxy-2-formyl-3,4,6-tri-o-methylhex-1-enitols: Terminal Transferase.” Anal Biochem 192:222-231 (1991). entry to ribo and xylo series". J. Chem. Soc. Perkin Trans. Stull et al., "Antigene, Ribozyme and Aptamer Nucleic Acid Drugs: Progress and Prospects.” Pharm Res 12(4):465-483 1. pp. 393-394, 1993. (1995). Booma et al. "A Novel tandem ferrier rearrangement cycli Tino et al., "Synthesis and Antiviral Activity of Novel sation. Synthesis of Chiral pyrano (2,3-b)(1) benzopyran Isonucleoside Analogs." JMed Chem36:1221-1229 (1993). Verheggen et al. "Synthesis and Antiherpes Virus Activity system". Tetrahedron Lett. 34(42):6757-6760, 1993. of 1.5-Anhydrohexitol Nucleosides." J Med Chem Liem et al. "Synthesis of 23-anhydroglycosyl phospho 36:2033-2040 (1993). nates'. Australian J. Chem. 49:371-377, 1996. Wagner et al., "Gene inhibition using antisense oligodeoxy nucleotides,” Nature 372:333-335 (1994). Barnes et al., "Synthesis of 2-deoxy-alpha and b-d-arabino Weil, Edward D., "Phosphorus-Based Flame Retardants." hexopyranosyl phosphonic acids and related compounds: Handbook of Organophosphorus Chemicals 683 and analogues of early intermediates in the shikimate pathway". 709–711 (1992). J. Chem. Soc. Perkins Trans 1. pp. 431-438. 1996. 5,750,729 1 2 COMPOUNDS AND METHODS FOR It is an object of this invention to provide antiviral MAKING AND USNG SAME compounds having an improved selectivity index, i.e., that are less toxic yet more efficacious than nucleotide analogues This is a divisional of application Ser. No. 08/615,669 known heretofore. fied on Mar. 13, 1996 which is a divisional of Ser. No, It is another object to prepare compounds that are suitable 08/384504 fied on Feb. 1, 1995 now U.S. Pat. No. 5.659, for facilitating the labelling of oligonucleotide probes and O23. polypeptides. This application is cross referenced to related copending It is an additional object to provide compounds useful in application Ser. No. 08/615,670 filed on Mar. 13, 1996. This application relates to nucleotide analogues and to the preparation of fire retardant resins. their use in diagnostic and therapeutic methods. It relates to O It is a further object to obtain nucleotide analogues that immunogens and oligonucleotides containing such nucle are useful as anti-infective agents. otide analogues. Nucleotide analogues containing phosphonate groups are SUMMARY OF THE INVENTION disclosed for example in U.S. Pat. Nos. 4,659,825, 4,808, The objects of this invention are accomplished by novel 716, 4,724,233, 5,142,051, 5,302.585,5208,221.5,352.786, 15 compounds having structure (1)

5,356,886 and in EPpublication numbers 269,947, 481.214. 630,381, 369,409, 454,427 and 618,214. EP 398,231 dis (1) closes a structure

25 wherein where B is a purine or pyrimidine base, X and X’ are H or the dashes indicate the positions of optional double bonds; C-C alkyl, the broken line designates an optional double # designates chiral centers, which are numbered; bond, Y and X are unsubstituted or substituted C-C alkyl n is 0 or 1; or together they constitute an oxygen atom or methylene Yindependently is OH, -OR. --OCH(R)OC(O)R. an group in which event the broken line is a single bond. oligonucleotide. --OPRT, a monophosphate, a Verheggen et al. “J. Med. Chem." 36:2033-2040 (1993) diphosphate, an amino acid amidate, a polypeptide disclose various antiviral pyrans as shown below amidate, -NHR, or -N(R); PRT is a protecting group; HO (a) Z is CHOR. halo substituted C-C alkyl, CH=CH2. 35 CECH, -CHN, CH, a detectable label or H; B is a heterocyclic base; R" and R' independently are H. CN. N. halo, OR, C-C alkyl or C-C alkyl substituted by N. OH, HO (b) 40 halo, CN or OR, or R." and R' may be joined together to form a 1,2-diol protecting group, a bond, or -CH-; R" is Hor, when R' and R' are joined together to form a bond, R is H or F; 45 R" and R" are H or may be joined together to form a HO (c) bond; R’ independently is H, -C(O)R. PRT, an oligonucleotide, a monophosphate or a diphosphate; R independently is unsubstituted alkyl, aryl, alkenyl, 50 alkynyl, alkaryl, alkynylaryl or alkenylaryl; said groups where H is substituted by halo, carboxy, hydroxyl, (d) cyano, nitro, N-morpholino, or amino; and/or said groups where -CH2- has been substituted by NH. S. or O; 55 Z and one Y group may be joined together to form a ring OH if Z is CH-OH and Y is OH: one Y group and R' or R' may be joined together to wherein B are defined pyrimidine and purine heterocycles form a ring if Y is OH and said R' or R' group and X is a defined heterogroup. Verheggen et al. disclose the 60 comprises OH. provided that ring positions 1-2 of the 4-hydroxymethyl and 4-phosphonylmethoxy analogues of furan or pyran ring are saturated and, when R" is OH compound (b). which is cyclized with Y. then R'' and the Patom are U.S. Pat. No. 5,276.143 discloses incorporating certain located on the same side of the ring; dideoxyfructonucleosides and deoxyfructonucleotides into when Y and Z or Y and R' or R' form a ring, the Patom oligonucleotides. 65 becomes chiral center #6; U.S. Pat. No. 5314,893 discloses various antiviral tet when n=1 the pyran ring is saturated or is unsaturated at rahydropyrams. the 1-2 or 2-3 positions; 5,750,729 3 4 when n=0, R" and R'' are H; and A further embodiment provides a method for treatment of the salts thereof. viral infections comprising administering to a subject a In another embodiment of the invention a method is therapeutically effective amount of a compound having provided for the detection of a target nucleic acid sequence structure (1) comprising: 5 (a) providing a labelled oligonucleotide probe having (1) structure (1) Z. (1) o o 10 I B X P(Y) A. R- wherein #, B, Y. Z. R. R. R. R. R., n and the dashed Rid lines are defined above. R. Is R" 15 The invention herein also includes novel synthetic meth ods. One such method comprises wherein (a) providing a compound having structure (I) the dashes indicate the positions of optional double bonds; # designates chiral centers, which are numbered; Z. (T) n is 0 or 1; 2O O Yindependently is OH, -OR. --OCH(R)OC(O)R’, an PRTO oligonucleotide. --OPRT, a monophosphate, a A. diphosphate, an amino acid amidate, a polypeptide amidate, -NHR'. or -N(R); OPRT PRT is a protecting group; 25 wherein PRT is defined above, and Z is CHOPRT halo Z is CHOR. halo substituted C-C alkyl, CH=CH substituted C-C alkyl, CH=CH2. C=CH, -CH2N. CECH,- -CHN. CH, a detectable label or H; c.reacting theth compoundd of structure (I)() withwi P(OPRT) 3. B is a heterocyclic base; in the presence of a Lewis Acid; and R" and R' independently are H, CN, N halo. OR 30 (c) recovering from the reaction mixture of step (b) a C-C, alkyl or C-C, alkyl substituted by N, OH, compound having structure (II) halo, CN or OR, or R' and R' may be joined together to form a 1.2-diol protecting group, a bond, or Z. (II) -CH2-; O O R" is Hor, when R" and R' are joined together to form 35 a bond, R is H or F: PRTO P(OPRT R" and R' are H or may be joined together to form a bond; This method is unexpectedly superior to the same method R’ independently is H. -C(O)R, PRT, an 40 using dialkyl phosphonic acid in that far less by-products are oligonucleotide, a monophosphate or a diphosphate; produced and yields accordingly increase from about 20% to R’ independently is unsubstituted alkyl, aryl, alkenyl, greater than about 90%. alkynyl, alkaryl, alkynylaryl or alkenylaryl; said groups Another method of this invention also relates to double where H is substituted by halo, carboxy, hydroxyl, cyano, bond migration in pyranose derivatives. It comprises nitro, N-morpholino, or amino; and/or said groups where (a) providing a compound having structure (III) -CH- has been substituted by NH. S. or O; Z and one Y group may be joined together to form a ring Z. (III) if Z is CH-OH and Y is OH; O one Y group and R' or R' may be joined together to B P(OPRT) form a ring if Y is OH and said R' or R' group 50 comprises OH, provided that ring positions 1-2 of the furan or pyran ring are saturated and, when R' is OH (b) treating the compound with a base; and which is cyclized with Y. then R' and the Patom are (c) recovering from the reaction mixture of step (b) a located on the same side of the ring; compound having structure (IV) when Y and Z or Y and R' or R' form a ring, the Patom 55 becomes chiral center f0; Z. (IV) when n=1 the pyran ring is saturated or is unsaturated at O the 1-2 or 2-3-positions; when n=0, R" and R' are H; 60 B A P(OPR(OPRT provided that one Y is an oligonucleotide having a sequence that is complementary to that of the target wherein B is defined above, and Zand PRT are defined in the sequence; and preceding method. the salts thereof; DETALED DESCRIPTION OF THE (b) hybridizing the labelled oligonucleotide probe to the 65 INVENTION target nucleic acid; and As used herein, and unless modified by the immediate (c) detecting the bound labelled oligonucleotide probe. context: 1. Alkyl means C-C branched, normal or cyclic 5,750,729 5 6 saturated hydrocarbons and includes methyl ethyl, propyl, typically will be selected from H. OR, OH, lower alkyl, cyclopropyl, isopropyl, n-, sec-, iso- and tertbutyl cyclobu azido or fluoro. An atomic R' substituent is not bound to an tyl and the like. 2. Alkenyl means C-C branched, normal unsaturated C atom. Either the (R) or (S) configuration of or cyclic hydrocarbons containing at least 1 (generally 1-3) R' or R' are acceptable, and R and R' may possess the cis or trans oriented conjugated or unconjugated double same or different stereochemistry. R' or R' like Y or Z. is bond, including ethenyl, propenyl, isopropenyl, 1-, 2- and a convenient site for substitution of an oligonucleotide or a 3-butenyl, 1- and 2-isobutenyl and the like. 3. Alkynyl detectable label for use in diagnostics. means C-C branched, normal, or cyclic hydrocarbons In one embodiment of the invention at least one R' or bearing at least 1 (generally 1-3) triple bond, e.g., R" is OH and is internally cyclized by dehydration with a 2-propynyl. 4. Aryl means an unsaturated resonant cyclic or hydroxyl group of the phosphonate, whereby compounds fused polycyclic ring structure containing at least one 3-6 having the partial structures (3) or (4) are obtained. membered ring containing ring atoms solely of carbon or of carbon and at least one N-, S- or O- heteroatom, including (3) phenyl, 2- and 3-pyrrolyl, 2- and 3-thienyl, 2- and Y 4-imidazolyl, 2-, 4- and 5-oxazolyl. 3- and 4-isoxazolyl 2 15 4-and 5-thiazolyl, 3-, 4- and 5-isothiazolyl, 3- and v.)(CH) 4-pyrazolyl. 2-, 3- and 4-pyridinyl or 2-, 4- and R (4) 5-pyrimidinyl. 5. Alkenylaryl means alkenyl substituted with at least 1 (generally 1-3) aryl group and bonded SN- 4' through the alkenyl or aryl moiety to the remainder of the compound through saturated or unsaturated carbon. 6. Alky N/s Y nylaryl means alkynyl substituted with at least 1 (generally O 1-3) aryl group and bonded through the alkenyl or aryl wherein Y. n, R" and R" are defined above. Cyclization moiety to the remainder of the compound through saturated introduces chirality into the P atom, designated #6. Y in or unsaturated carbon. 25 structures (2)–(4) is (R) or (S), or may be racemic. The Stereochemistry is depicted according to carbohydrate cyclic oxygen atom in structure (3) will be located on the convention with the furan or pyran oxygen at the rear of the same side of the plane of the furan or pyran ring as the molecule, and the stereoconfiguration of the substituents phosphorous atom. indicated by their position above and below the plane of the R" typically is H, whether RandR' are joined to form ring. The bonds that project from the side of the ring 30 a bond or not, but may be fluoro when R'' and R' form a (substituents of unsaturated ring carbon atoms) are coplanar bond. R' and R typically are H, but may be joined with the ring (unless indicated to the contrary by the bond together to form a bond. When n=1, only one of R" and R' designation ot, which designates the genus of diastereomers or RandR'? may be taken together to form a bond; when and racemates). Hydrogen atoms bound to ring carbons may n=0, R'' and R' are H. not show but shall be understood to occupy any undesig 35 n. In general the pyranose ring is preferred, i.e. n=1. nated valence of the ring carbon atoms. Such hydrogen Moreover, preferably the 2-3 carbon bond of this ring is a atoms generally are in the opposite configuration of any double bond. group or atom that also may be bound to the same saturated Group R typically is Horan ester-forming group -C(O) carbon atom. R bound to any one or more of the pyran or furan hydroxyl Group Z typically is CHOH, CHOR. H. CH or vinyl. substituents R', R' or Z. Synthetic reactions may require Generally, it is in the D configuration, but may be in the L that R’=PRT. R. is not critical and can vary widely. configuration or racemic. When Z contains a reactive func Principally, R groups serve as protecting groups during tionality like OH the reactive group generally is protected synthetic reactions or groups that are capable of cleavage in (see infra) throughout the synthesis, and is deprotected as vivo (generally, esters) to yield the free hydroxyl. Note that one of the last steps in the synthesis, Thus in CHOR, R 45 some -CO)R are capable of acting as PRT (see infra). commonly is PRT. Z is a convenient site for the attachment Group R also is not a critical functionality and may vary of oligonucleotide or of detectable moieties such as biotin, widely. R for example includes C-C aryl (including fluorescent groups and the like using methods known perse. phenyl, 2- and 3-pyrrolyl. 2- and 3-thienyl, 2- and In other embodiments where Z is CHOH, Z is internally 4-imidazolyl, 2-, 4- and 5-oxazolyl. 3- and 4-isoxazolyl 2-, cyclized by dehydration with a hydroxyl group of the 4-and 5-thiazolyl, 3-, 4- and 5-isothiazolyl. 3- and phosphonate, whereby compounds having the partial struc 4-pyrazolyl, 1-, 2-, 3- and 4-pyridinyl, and 1-, 2-, 4- and ture (2) are obtained (the omitted structural subunit is found 5-pyrimidinyl), C-C aryl substituted with halo, alkyl in structure (1); in this and the following structures where C-C alkoxy, CN, NO, OH. carboxy, carboxyester, thiol, R", R'' and R" are not depicted they are H or a bond as thiolester, C-C2 haloalkyl (1-6 halogen atoms), C-C2 dictated by their context): 55 alkenyl or C-C alkynyl including 2-, 3- and 4-alkoxyphenyl (C-C alkyl). 2-, 3- and (2) 4-methoxyphenyl, 2-, 3- and 4-ethoxyphenyl. 2.3-, 2,4- 2.5-, 2,6-, 3.4- and 3.5-diethoxyphenyl, 2- and 3-carboethoxy-4-hydroxyphenyl, 2- and 3-ethoxy-4- hydroxyphenyl, 2- and 3-ethoxy-5-hydroxyphenyl, 2- and 3-ethoxy-6-hydroxyphenyl, 2-, 3- and 4-O-acetylphenyl, 2-, 3- and 4-dimethylaminophenyl, 2-, 3- and 4-methylmercaptophenyl, 2-, 3- and 4-halophenyl (including 2-, 3- and 4-fluorophenyl and 2-, 3- and wherein Y and R' are defined above. 65 4-chlorophenyl, 2.3-, 2,4-, 2,5-, 2,6-, 34- and 3,5- Groups R' and R' are saturated C atom substituents or dimethylphenyl, 2.3-, 2.4-, 2.5-, 2,6-, 3.4- and 3.5- are taken together to form a bond. R' or R' groups biscarboxyethylphenyl, 2.3-, 2,4-, 2.5-, 2,6-, 34- and 3.5- 5,750,729 7 8 dimethoxyphenyl, 2.3-, 24-, 2.5-. 2,6-, 3,4- and 3.5- PRT is a protecting group used to prevent side reactions dihalophenyl (including 2,4-difluorophenyl and 3.5- with the protected group during synthetic procedures. For difluorophenyl), 2-, 3-and 4-haloalkylphenyl (1 to 5 halogen the most part the decision as to which groups to protect, atoms, C-C alkyl including 4-trifluoromethylphenyl), 2-, when to do so, and the nature of the PRT will be dependent 3- and 4-cyanophenyl, 2-, 3- and 4-nitrophenyl, 2-, 3- and upon the chemistry of the reaction to be protected against 4-haloalkylbenzyl (1 to 5 halogen atoms, C-C alkyl (e.g., acidic, basic, oxidative, reductive or other conditions) including 4-trifluoromethylbenzyl and 2-, 3- and and the intended direction of the synthesis. The PRT groups 4-trichloromethylphenyl and 2-, 3- and 4 do not need to be, and generally are not, the same if the trichloromethylphenyl), 4-N-methylpiperidinyl, 3-N- compound is substituted with multiple PRT. In general, PRT methylpiperidinyl. 1-ethylpiperazinyl, benzyl, alkylsali will be used to protect hydroxyl or amino groups. The latter cylphenyl (C-C alkyl, including 2-, 3- and O are found on some heterocyclic bases, while hydroxyl PRT 4-ethylsalicylphenyl). 2-3- and 4-acetylphenyl, 1.8- (-OPRT) are used to protect Y. R' and Zhydroxyl groups, dihydroxynaphthyl (-O-CoH-OH) and aryloxy ethyl The order of deprotection to yield free hydroxyl also is C-C aryl (including phenoxy ethyl), 2,2'- dependent upon the intended direction of the synthesis and dihydroxybiphenyl, alkoxy ethyl (C-C alkyl including the reaction conditions to be encountered. Typically the Z. -CH-CH-O-CH (methoxyethyl), alkyl substituted 15 B, Y and R' groups all will be protected and thereafter will be deprotected in any order as required. For example, by OH or by 1 to 3 halo atoms (including -CH, -CH heterosubstitution at the R' groups generally will require (CH3)2, -C(CH), -CH2CH, -(CH2)CH3. -(CH2) that one ROH be unprotected or differently protected than CH, -(CH2)CH, -(CH2)6CH, -CH2CHF. the other R' (see scheme 1). In other embodiments, the Y -CHCHCl, -CHCF and -CHCCl3), 2-, 3- and hydroxyl groups and/or B amino groups are not protected 4-N-N-dialkylaminophenol, -CHCH-N(CH), but Z and R' are -CHOPRT or -OPRT, respectively. A very large number of hydroxy protecting groups and corresponding chemical cleavage reactions are described in N1NN O; "Protective Groups in Organic Chemistry". Theodora W. 25 Greene (John Wiley & Sons. Inc., New York, 1991, ISBN 0-471-62301-6) ("Greene"). As will be seen from the dis -N-2-propylmorpholino, 2.3-dihydro-6-hydroxyindene. cussion below, some R groups described above also are sesamol, catechol monoester, -CH2-C(O)-N(R'), capable of acting as PRT, wherein each R" is the same or different H or C-C alkyl, Typical hydroxy protecting groups are described in -CH2-S(O)(R'), -CH2-S(O)2(R'), -CH2-CH(OC 30 Greene at pages 14-118 and include Ethers (Methyl); Sub (O)CHR)-CH(OC(O)CHR"), cholesteryl, a 5 or 6 stituted Methyl Ethers (Methoxymethyl, Methylthiomethyl, carbon monosaccharide, disaccharide or oligosaccharide (3 t-Butylthiomethyl. (Phenyldimethylsilyl)methoxymethyl, to 9 monosaccharide residues), enolpyruvate (HOOC-C Benzyloxymethyl, p-Methoxybenzyloxymethyl. (=CH)O), glycerol, ot-D-B-diglycerides (wherein the fatty (4-Methoxyphenoxy) methyl, Guaiacol methyl, acids composing glyceride lipids generally are naturally 35 t-Butoxymethyl, 4-Pentenyloxymethyl, Siloxymethyl. occurring Saturated or unsaturated C-2, C-1s or C-o 2-Methoxyethoxymethyl. 2.2.2-Trichloroethoxymethyl, Bis fatty acids such as linoleic, lauric, myristic, palmitic, stearic, (2-chloroethoxy)methyl, 2-(Trimethylsilyl)ethoxymethyl. oleic, palmitoleic, linolenic and the like fatty acids). Tetrahydropy ranyl, 3-Bromotetrahydropyranyl. trimethoxybenzyl, triethoxybenzyl, 2-alkyl pyridinyl (C- Tetrahydropthiopyranyl, 1-Methoxycyclohexyl, 4 alkyl), M e th ox y t e t r a h y dro p y r a n y , 4 - Me th oxy t e tra hydroth i opy r a nyl, 4-Methoxytetrahydropthiopyranyl SS-Dioxido. 1-(2- Chloro-4-methyl)phenyl)-4-methoxypiperidin-4-yl. 35. 14 Dioxan-2-yl. Tetrahydrofuranyl. Tetrahydrothiofuranyl, 2.3, 45 3a. 4,5,6,7,7a-Octahydro-7,8,8-trimethyl-4,7- methanobenzofuran-2-yl)); Substituted Ethyl Ethers (1-Ethoxyethyl, 1-(2-Chloroethoxy)ethyl, 1-Methyl-1-

methoxyethyl, 1-Methyl-1-benzyloxyethyl, 1-Methyl-1- benzyloxy-2-fluoroethyl, 2.2.2-Trichloroethyl, 50 2-Trimethylsilylethyl 2-(Phenylselenyl)ethyl, t-Butyl, Allyl p-Chlorophenyl, p-Methoxyphenyl. 2.4- Dinitrophenyl, Benzyl); Substituted Benzyl Ethers (p-Methoxybenzyl, 3,4-Dimethoxybenzyl, o-Nitrobenzyl, p-Nitrobenzyl, p-Halobenzyl, 2,6-Dichlorobenzyl, C-C alkylene-C-C aryl (including benzyl, -CH2 55 p-Cyanobenzyl, p-Phenylbenzyl. 2- and 4-Picolyl, pyrrolyl, -CH2-thienyl, -CH-imidazolyl, -CH 3-Methyl-2-picolyl N-Oxido, Diphenylmethyl, pip'- oxazolyl, -CH2-isoxazolyl, -CH2-thiazolyl, -CH2 Dinitrobenzhydryl, 5-Dibenzosuberyl, Triphenylmethyl, isothiazolyl, -CH-pyrazolyl, -CH-pyridinyl and or - N a ph thy 1 dip he n y 1 m e thy 1 . -CH-pyrimidinyl) substituted in the aryl moiety by 3 to 5 p-methoxyphenyldiphenylmethyl, Di(p-methoxyphenyl) halogen atoms or 1 to 2 atoms or groups selected from phenylmethyl, Tri(p-methoxyphenyl)methyl, 4-(4'- halogen. C-C alkoxy (including methoxy and ethoxy), Bromophenacyloxy)phenyldiphenylmethyl, 44'4"-Tris(4, cyano, nitro, OH, C-C haloalkyl (1 to 6 halogen atoms; 5-dichlorophthalimidophenyl)methyl, 4,4'4"-Tris including-CH2-CC), C-C alkyl (including methyl and (levulinoyloxyphenyl)methyl, 4.4'.4"-Tris ethyl), C-C alkenyl or C-C alkynyl, and other com (benxoyloxyphenyl)methyl 3-(Imidazol-1-ylmethyl)bis(4. pounds set forth in Table 7 below. The hydroxyl groups of 65 4"-dimethoxyphenyl)methyl, 1,1-Bis(4-methoxyphenyl)-1'- Z. R' and/or R' optionally are substituted with one of pyrenylmethyl, 9-Anthryl, 9-(9-Phenyl)xanthenyl, 9-(9- groups III, IV or V disclosed in WO94/21604. Phenyl-10-oxo)anthryl, 1.3-Benzodithiolan-2-yl. 5,750,729 9 10 Benzisothiazolyl S.S.-Dioxido); Silyl Ethers (Trimethylsilyl, Triethylsilyl. Triisopropylsilyl, Dimethylisopropylsilyl. TABLE a Diethyl isopropylsily, Dimethylth exylsilyl, t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Tribenzylsilyl, r 2- 7 r 7- - Tri-p-xylylsilyl, Triphenylsilyl, Diphenylmethylsilyl, O O O "S-" t-Butylmethoxyphenylsilyl); Esters (Formate, D3 O Benzoylformate, Acetate. Choroacetate. Dichloroacetate, Trichloroacetate. Trifluoroacetate. Methoxyacetate, Triphenylmethoxyacetate, Phenoxyacetate. 7 v. 7 v. 10 N1 N1 p-Chlorophenoxyacetate. p-poly-Phenylacetate. I oano 3-Phenylpropionate, 4-Oxopentanoate (Levulinate). 4.4- O (Ethylenedithio)pentanoate, Pivaloate, Adamantoate. 7- rv Crotonate, 4-Methoxy crotonate, Benzoate, O O p-Phenylbenzoate, 2.4.6-Trimethylbenzoate (Mesitoate)); 15 Carbonates (Methyl, 9-Fluorenylmethyl, Ethyl, 2.2.2- 7 v. Trichloroethyl, 2-(Trimethylsilyl)ethyl 2-(Phenylsulfonyl) N1 ethyl, 2-(Triphenylphosphonio)ethyl, Isobutyl, Vinyl, Allyl, Rio 1 So p-Nitrophenyl, Benzyl, p-Methoxybenzyl, 3.4- Dimethoxybenzyl, o-Nitrobenzyl, p-Nitrobenzyl, S-Benzyl Thiocarbonate, 4-Ethoxy-1-naphthyl Methyl 4. -? r, ro- 'o Dithiocarbonate); Groups With Assisted Cleavage R14O N o2so (2-Iodobenzoate, 4-Azidobutyrate, 4-Niotro-4- methylpentanoate, o-(Dibromomethyl)benzoate. 25 7- r 2-Formylbenzenesulfonate, 2-(Methylthiomethoxy)ethyl O O Carbonate, 4- (Methylthiomethoxy)buty rate. 2-(Methylthiomethoxymethyl)benzoate); Miscellaneous Esters (2,6-Dichloro-4-methylphenoxyacetate, 2,6- ro- v. Dichloro-4-(1.1.3.3-tetramethylbutyl)phenoxyacetate, 24 p1 B is (1, 1 - dimethylpropyl) phen oxyacetate. 30 R1401 So Chorodiphenylacetate, Isobutyrate. Monosuccinoate, (E)-2- Methyl-2-butenoate (Tigloate), o-(Methoxycarbonyl) wherein R' is C-C alkyl. benzoate, p-poly-Benzoate, oc-Naphthoate, Nitrate, Alkyl Group Y typically will be OH or convertible to OH by N.N,N',N'-Tetramethylphosphoro diamidate. 35 chemical or biological means. For in vivo hydrolysis Y N-Phenylcarbamate. Borate, Dimethylphosphinothioyl. 2,4- usually is OR in which R is phenyl or substituted phenyl Dinitrophenylsulfenate); and Sulfonates (Sulfate, Methane as described below or Yis-OCH(R)OC(O)R. Y is OPRT sulfonate (Mesylate). Benzylsulfonate, Tosylate). in intermediates for the most part. Certain end uses for More typically, hydroxy protecting groups include sub intermediate compounds of the invention contemplate Y=an oligonucleotide or protein. stituted methyl ethers, substituted benzyl ethers, silyl ethers. Particularly useful Y groups are alkylacyloxymethyl and esters including sulfonic acid esters, still more typically, groups and their derivatives, including -CH trialkylsilyl ethers, tosylates and acetates. (CHCHOCH)OC(O)C(CH). Typical 1.2-diol protecting groups (thus, generally where two R' OH groups are taken together with a protecting 45 functionality) are described in Greene at pages 118-142 and include Cyclic Acetals and Ketals (Methylene, Ethylidene, 1 -t-Butyl ethyl idene, 1-Phenylethylide ne, N-0 (4-Methoxyphenyl)ethylidene, 2.2.2-Trichloroethylidene. O Acetonide (Isopropylidene), Cyclopentylidene, 50 Cyclohexylidene, Cycloheptylidene, Benzylidene. -CHOC(O)CH, -CH2OC(O)COCH), -CH p-Methoxybenzylidene, 2.4-Dimethoxybenzylidene. 3.4- (CHOCH)OC(O)C(CH), -CH(CH(CH))OC(O)C Dimethoxybenzylidene, 2-Nitrobenzylidene); Cyclic Ortho (CH), -CHOC(O)CHCHCCH), -CH2OC(O)CH, Esters (Methoxymethylene, Ethoxymethylene, -CHOC(O)CH, -CHOC(O)CoHs. -CHOC(O) Dimethoxymethylene, 1-Methoxyethyl idene, 55 CHCH, -CHOC(O)CH(CH), -CHOC(O)C(CH) 1-Ethoxyethylidine, 1.2-Dimethoxyethylidene. and -CHOC(O)CHCH (O-Methoxybenzylidene, 1-(N.N-Dimethylamino) Y also may be an amino acid residue. In general, the ethylidene Derivative. O-(N.N-Dimethylamino)benzylidene amino acid residue has the structure R'OC(O)CH(R)NH-. where R' is R. one or more additional amino acid residues Derivative, 2-Oxacyclopentylidene); Sillyl Derivatives (Di linked via peptide bonds, or H and R is alkyl substituted t-butylsilylene Group, 1,3-(1,1,3,3- with amino, carboxyl, amide, carboxyl ester, hydroxyl, Tetraisopropyl disiloxany idene), and Tetra-t- C-C, aryl, guanidinyl, imidazolyl, indolyl, sulfhydryl butoxydisiloxane-1,3-diylidene), Cyclic Carbonates, Cyclic sulfoxide, and/or alkylphosphate. Ordinarily R' is RandR Boronates, Ethyl Boronate and Phenyl Boronate. is a side chain of a naturally occurring amino acid. With More typically, 1,2-diol protecting groups include those 65 respect to the carboxyl-containing side chains it will be shown in Table 1a, still more typically, epoxides, acetonides, understood that if the C atom of the subject carboxyl is cyclic ketals and aryl acetals. linked by 5 or less atoms to the phosphoamide N then the 5,750,729 11 12 carboxyl optionally will be blocked, e.g. by esterification C.-methylaspartic acid, O-methylglutamic acid, with R or amidation wherein the ester or amide bonds are 1-aminocyclopropane-1-carboxylic acid; isoleucine. hydrolyzable in vivo. R also is taken together with the alloisoleucine, tert-leucine, B-methyltryptophan and amino acido N to form a proline residue (R-CH2)3-). C.-amino-3-ethyl-3-phenylpropionic acid residues; However, R is generally a side group such as H. -CH3. 5 3-phenylserinyl; -CH(CH), CH-CH(CH), -CHCH-CH2CH, Aliphatic O-amino-B-hydroxy acids such as serine, -CH-CH, -CHCH-S-CH, -CH2OH, -CH B-hydroxy leucine, B-hydroxy nor leucine, (OH)-CH, -CH2-SH, -CH-CHOH, -CH2 B-hydroxynorvaline, and O-amino-f-hydroxystearic CO-NH. -CH-CH-CO-NH, -CH-COOH. acid residues; -CH-CH-COOH, -(CH2)-NH and -(CH2)- 10 o-Amino, o-, y-, 8- or e-hydroxy acids such as NH-CONH2)-NH2. R also includes 1-guanidinoprop-3- homoserine, Y-hydroxynorvaline, 8-hydroxynorvaline yl, benzyl, 4-hydroxybenzyl, imidazol-4-yl, indol-3-yl, and epsilon-hydroxynorleucine residues; canavinyl and methoxyphenyl and ethoxyphenyl. canalinyl; Y-hydroxyornithinyl; When the amino acid residue contains one or more chiral 2-hexosaminic acids such as D-glucosaminic acid or centers, any of the D. L., meso, threo or erythro (as 15 appropriate) racemates, scalemates or mixtures thereof may D-galactosaminic acid residues; be used as group Y. In general, if the intermediates are to be o-Amino-3-thiols such as penicillamine, B-thiolnorvaline hydrolyzed non-enzymatically (as would be the case where or B-thiolbutyrine residues; the compounds are used as chemical intermediates for the Other sulfur containing amino acid residues including free acids), D isomers are useful. On the other hand, L cysteine; homocystine; B-phenylmethionine; methion isomers are more versatile since they can be susceptible to ine; S-allyl-L-cysteine sulfoxide; 2-thiolhistidine; cys both non-enzymatic and enzymatic hydrolysis, and are more tathionine; and thiol ethers of cysteine or homocys efficiently transported by amino acid or dipeptidyl transport teine; systems in the gastrointestinal tract. Phenylalanine, tryptophan and ring-substituted o. amino Examples of suitable amino acid residues include the 25 acids such as the phenyl- or cyclohexylamino acids following: O-aminophenylacetic acid, O-aminocyclohexylacetic acid and O-amino-B-cyclohexylpropionic acid; pheny Glycyl; lalanine analogues and derivatives comprising aryl, Aminopolycarboxylic acids, e.g., aspartic acid. lower alkyl hydroxy, guanidino, oxyalkylether, nitro. B-hydroxy as partic acid glutamic acid, 30 sulfur or halo-substituted phenyl (e.g. tyrosine, meth B-hydroxyglutamic acid, B-methylaspartic acid, yltyrosine and o-chloro-, p-chloro-3,4-dicloro, o-, m B-methylglutamic acid, B.B-dimethylaspartic acid, or p-methyl-, 2,4,6-trimethyl-, 2-ethoxy-5-nitro, Y-hydroxyglutamic acid, B.Y-dihydroxyglutamic acid, 2-hydroxy-5-nitro and p-nitrophenylalanine); furyl-, 3-phenylglutamic acid, y-methyleneglutamic acid. thienyl-, pyridyl-, pyrimidinyl-, purine or naphthylala 3-amino adipic acid, 2-aminopimelic acid. 35 nines; and tryptophan analogues and derivatives 2-aminosuberic acid and 2-aminosebacic acid residues; including kynure nine, 3-hydroxykynure nine. Amino acid amides such as glutaminyl and asparaginyl; 2-hydroxytryptophan and 4-carboxytryptophan resi Polyamino- or polybasic-monocarboxylic acids such as dues; arginine, lysine, B-aminoalanine, Y-aminobutyrine, o-Amino substituted amino acid residues including sar ornithine, citruline. homoarginine, homocitrulline, cosine (N-methylglycine). N-benzylglycine, 5-hydroxy-2,6-diaminohexanoic acid (commonly. N-methylalanine, N-benzylala nine, hydroxylysine, including allohydroxylysine), diami N-methylphenylalanine, N-benzylphenylalanine, nobutyric and histidine residues; N-methylvaline and N-benzylvaline; and Diaminodicarboxylic acids such as O.O'-diaminosuccinic o-Hydroxy and substituted ot-hydroxy amino acid resi acid, o.o'-diaminoglutaric acid, O.O'-diaminoadipic 45 dues including serine, threonine, allothreonine, phos acid, o.o'-diaminopimelic acid, C.O'-diamino-3- phoserine and phosphothreonine residues. hydroxypimelic acid, O.O'-diaminosuberic acid, O.O.'- Any one of the foregoing or other known amino acids are diaminoazelaic acid, and O,Oc-diaminosebacic acid suitably employed in this invention particularly if they are residues; capable of autocatalytically hydrolyzing the P-amidate Imino acids such as proline, 4- or 3-hydroxy-2- 50 bond. Thus, they should contain a free carboxyl group, or pyrrollidine carboxylic acid (commonly. should do so upon hydrolysis in vivo. hydroxyproline, including allohydroxyproline). Of particular interest are hydrophobic residues such as Y-methylproline, pipecolic acid, 5-hydroxypipecolic mono-ordi-alkyl or aryl amino acids, cycloalkylamino acids acid, -N(CHCOOR), wherein n and R are as and the like. These residues contribute to cell permeability defined above, and azetidine-2-carboxylic acid resi 55 by increasing the partition coefficient of the nucleotide dues; analogue amidate. Typically, the residue does not contain a A mono- or di-alkyl (typically C-C branched or normal) sulfhydryl or guanidino substituent. amino acid such as alanine, valine, leucine, allylglycine, Y optionally is a polypeptide radical. Polypeptides com butyrine, norvaline, norleucine, heptyline. 0-methylserine. prise dipeptides (2 residues), or polypeptides of 3, 5, 10 and o-amino-o-methyl-y-hydroxyvaleric acid, O-amino-O- up to 100 or more residues. They include enzymes (e.g., methyl-8-hydroxyvaleric acid, O-amino-O-methyl-e- hydrogen peroxidase) as well as antibodies or proteins of hydroxycaproic acid, isovaline, O-methylglutamic acid, any type against which one wishes to raise an immune O-aminoisobutyric acid, O-aminodiethylacetic acid, response. The nature and identity of the polypeptide may O-aminodiisopropylacetic acid, O-aminodi-n-propylacetic vary widely. The polypeptide optionally contains a pepti acid. ot-aminodiisobutylacetic acid, O-aminodi-n- 65 dolytic enzyme cleavage site at the peptide bond between butylacetic acid, O-aminoethylisopropylacetic acid. the first and second residues adjacent to the phosphorus o-amino-n-propylacetic acid, O-aminodiisoamyacetic acid, atom. Such cleavage sites are flanked by enzymatic recog 5,750,729 13 14 nition structures, e.g. a particular sequence of residues to hydrolysis by proteases common to the brush border such recognized by a peptidolytic enzyme. as aminopeptidase N (EC 3.4.11.2). In addition, di- or Peptidolytic enzymes for cleaving the polypeptide con tripeptides with amino acid residues can be selected on the jugates of this invention are well known, and in particular basis of their relative resistance to hydrolysis by proteases include carboxypeptidases. Carboxypeptidases digest 5 found in the lumen of the intestine. For example, tripeptides polypeptides by removing C-terminal residues, and are or oligopeptides lacking asp and/or glu are poor substrates specific in many instances for particular C-terminal for aminopeptidase A (EC 3.4.11.7), di- or tripeptides lack ing amino acid residues on the N-terminal side of hydro sequences. Such enzymes and their substrate requirements phobicamino acids (leu, tyr, phe, val, trp) are poor substrates in general are well known. For example, a dipeptide (having for endopeptidase 24.11 (EC 3.4.24.11), and peptides lack a given pair of residues and a free carboxyl terminus) is ing a pro residue at the penultimate position at a free covalently bonded through its O-amino group to the phos carboxyl terminus are poor substrates for carboxypeptidase phorus atom of the nucleotide analogue herein. It is expected P (EC 3.4.17). Similar considerations can also be applied to that this peptide will be cleaved by the appropriate pepti the selection of peptides that are either relatively resistant or dolytic enzyme, leaving the carboxyl of the proximal amino relatively susceptible to hydrolysis by cytosolic, renal, acid residue to autocatalytically cleave the amidate bond. 5 hepatic, serum or other peptidases. Such poorly cleaved Suitable dipeptidyl groups (designated by their single polypeptide amidates are immunogens or are useful for letter code) are AA, AR, AN. AD, AC, AE, AQ, AG, AH., AI, bonding to proteins in order to prepare immunogens. AL, AK AM, AF, AP, ASAT, AWAY, AV, RA, RR, RN, Group B. B is a heterocyclic base. It typically is capable RD, RC, RE, RO, RG, RH, RI, RL, RK, RM, RF, RP, RS. of participating in Watson-Crick base pairing, or is a pro RT, RW, RY, RV.NA, NRNN, ND, NC, NQ, NG, tected analogue thereof. It includes any naturally-occurring NS D heterocycle found in nucleic acids, nucleotides or nucleo D M. DF sides of living organisms. The heterocyclic bases generally are the purine, pyrimidine or related heterocycles shown in E. 25 formulas (6)–(9). K. s (6) GD, GC, G H E. k i. 30 LN, D, IC, IK, f R. KN, KD, KC, KLS. (7) S. i. 35 FA, FR, FN, FD, FC, FE FQ FG MYE. FT, E. tS D, (8) s ST, S W. SY, SV, TA, TR, TN, r D TC TE, TQ, T G, TH

YE. YQ, YG, YH. YI, YL, YK, YM, YF, YP Ys s s 45 YY.YV, VA, VR, VN, VD, VC, VE, VQ, VG, VH, VI, VL, WK, VM, VF, VP VS, VT. V.W. VY and VV. O (9) Tripeptides are also useful as Y. The sequence -X4-pro X5- (where X4 is any amino acid residue and X5 is an amino acid residue, a carboxyl ester of proline, or hydrogen) will 50 be cleaved by luminal carboxypeptidase to yield X4 with a free carboxyl, which in turn autocatalytically cleaves the phosphono amidate bond. X5 usually will be a benzyl ester of the carboxy group of X5. Wherein Dipeptide or tripeptide species can be selected on the 55 R is H, OH, F, Cl, Br, I, OR, SH, SR, NH, or NHR: basis of known transport properties and/or susceptibility to R is N, CF, CC1, CBI, CI, CR, CSR, or COR; peptidases that can affect transport to intestinal mucosal or R is H. C-C alkyl, C-C alkenyl, C-C alkynyl or other cell types. Dipeptides and tripeptides lacking an C-C aryl-alkyl unsubstituted, or H substituted by O-amino group are transport substrates for the peptide OH.F, Cl, Br or I, or CH, substituted by O. NH or NR; transporter found in brush border membrane of intestinal R includes CH, CHCH, CHCH, CHCHBr, mucosal cells (Bai. J. P. F. "Pharm Res.”9:969-978 (1992). CHCHCl, CHCHF, CHCCH, CHCHCH, CH, Transport competent peptides can thus be used to enhance CHOH, CHOCH. CHOCH, CHOCCH, bioavailability of Y amidate compounds. Di- or tripeptides CHOCHCHCH, CHCH, CHCHOH. having one or more amino acids in the D configuration are CHCHOCH, CHCHOCH, CHCHOCCH, also compatible with peptide transport and can be utilized in 65 CHCHOCHCHCH and CHCHOCH,; Yamidate compounds. Amino acids in the D configuration R is N or CH: can be used to reduce the susceptibility of a di- or tripeptide R" is N, CH, CCN, CCF CC=CH or CC(O)NH; 5,750,729 15 16 R' is H. OH, NH, SH, SR (such as SCH SCHCH 1,1-dimethylpropy nyl, di(2-pyridyl)methyl, SCHCCH. SCHCHCH, or SCH), NH(R) (such as 2-furanylmethyl, 2-Iodoethyl, Isobornyl, Isobutyl, NH(CH), NH (CHCH) NH(CHCCH). Isonicotinyl, p-(p'-Methoxyphenylazo)benzyl, NH(CHCHCH) or NH(CH); N(R), such as 1-methylcyclobutyl, 1-methylcyclohexyl, 1-methyl-1- N(CHCH); or halogen (F, Cl, Br or I); cyclopropylmethyl, 1-methyl-1-(3,5-dimethoxyphenyl) R' is H, OH. F. Cl, Br, I, SR (such as SCH. SCHCH ethyl, 1-methyl-1-(p-phenylazophenyl)ethyl, 1-methy-1- SCHCCH. SCHCHCH or SCH), OR, NH, phenylethyl, 1-methyl-1-(4-pyridyl)ethyl, phenyl, N(R) or NHR'; and p-(phenylazo)benzyl, 2,4,6-tri-t-butylphenyl, R" is O, S or Se. 4-(trimethylammonium)benzyl, 2,4,6-trimethylbenzyl); Specific heterocyclic bases include the purines O Amides (N-formyl N-acetyl. N-choroacetyl. hypoxanthine, inosine, xanthine, 2-aminopurine. 2.6- N-trichoroacetyl. N-trifluoroacetyl, N-phenylacetyl, N-3- diaminopurine, 2-amino-6-chloropurine, adenine, guanine. phenylpropionyl, N-picolinoyl, N-3-pyridylcarboxamide. 6-thio-2-aminopurine and the 8-aza, 7-deaza-8-aza, 1-deaza, N-benzoylphenylalanyl, N-benzoyl, N-p-phenylbenzoyl); 7-deaza or 3-deaza derivatives of each of the foregoing Amides With Assisted Cleavage (N-o-nitrophenylacetyl, purines; and the pyrimidines cytosine, thymine, uracil, 15 N-o-nitrophenoxyacetyl. N-acetoacetyl, (N'- 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; dithiobenzyloxycarbonylamino)acetyl, N-3-(p- 5-iodocytosine; 5-bromocytosine; 5-methylcytosine; hydroxyphenyl)propionyl, N-3-(o-nitrophenyl)propionyl, 5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; N-2-methyl-2-(o-nitrophenoxy)propionyl. N-2-methyl-2-(o- 5-iodouracil; 5-bronouracil; 5-trifluoromethyluracil; phenylazophenoxy)propionyl. N-4-chlorobutyryl, N-3- 5-methoxymethyluracil; 5-ethynyluracil; and methyl-3-nitrobutyryl, N-o-nitro cinnamoyl 5-propynyluracil. Cytosine or 5-halo- and 5-C-C-alkyl N-acetylmethionine, N-o-nitrobenzoyl N-o- cytosine are preferred where Z-CHOR. For the other Z (benzoyloxymethyl)benzoyl. 4.5-diphenyl-3-oxazolin-2- groups, B ordinarily is a purine or its monoaza or mon one); Cyclic Imide Derivatives (N-phthalimide. Odeaza analogue. N-dithiasuccinoyl N-2,3-diphenylmaleoyl. N-2.5- B includes both protected and unprotected forms of the 25 dimethylpyrrolyl, N- 1, 14.4- heterocyclic bases. Protecting groups for exocyclic amines tetramethyldisilylazacyclopentane adduct,5-substituted 13 of heterocyclic bases such as adenine, cytosine, 2.6- dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 13 diaminopurine and the like are known. The selection of a dibenzyl-13-5-triazacyclohexan-2-one. 1-substituted 3.5- protecting group will be apparent to the ordinary artisan and dinitro-4-pyridonyl); N-Alkyl and N-Aryl Amines will depend on the nature of the labile group and the 30 (N-methyl N-allyl. N-(2-(trimethylsilyl)ethoxymethyl. chemistry which the protecting group is expected to encoun N-3-acetoxypropyl. N-(1-isopropyl-4-nitro-2-oxo-3- te. pyrrolin-3-yl), Quaternary Ammonium Salts, N-benzyl, Typical amino protecting groups are described in Greene N-di(4-methoxyphenyl)methyl, N-5-dibenzosuberyl. at pages 315-385 and include Carbamates (methyl and N-triphenylmethyl, N-(4-methoxyphenyl)diphenylmethyl. ethyl, 9-fluorenylmethyl, 9(2-sulfo)fluoroenylmethyl. 9-(2. 35 N-9-phenylfluorenyl, N-2,7-dichloro-9-fluorenylmethylene. 7-dibromo)fluorenylmethyl, 2,7-di-t-buthyl-9-(10.10 N-ferrocenylmethyl N-2-picolylamine N-oxide). Imine dioxo-10.10.10, 10-tetrahydrothioxanthyl)methyl, Derivatives (N-1,1-dimethylthiomethylene, N-benzylidene, 4-methoxyphenacyl); Substituted Ethyl (2.2.2-trichoroethyl. N-p-methoxybenylidene. N-diphenylmethylene, N-(2- 2-trimethylsilylethyl 2-phenylethyl. 1-(1-adamantyl)-1- pyridyl)me sity 1 methylene, N(N',N'- methylethyl, 1,1-dimethyl-2-haloethyl, 1,1-dimethyl-2.2- dimethylaminomethylene, N.N'-isopropylidene, N-p- dibromoethyl, 1.1-dimethyl-2.2.2-trichloroethyl, 1-methyl nitrobenzylidene. N-salicylidene, N-5-chlorosalicylidene. 1-(4-biphenylyl)ethyl, 1-(3,5-di-t-butylphenyl)-1- N-(5-chloro-2-hydroxyphenyl)phenylmethylene. methylethyl, 2-(2'- and 4'-pyridyl)ethyl. 2-(N.N- N-cyclohexylidene); Enamine Derivatives (N-(5.5- dicyclohexylcarboxamido)ethyl, t-butyl, 1-adamantyl, dimethyl-3-oxo-1-cyclohexenyl)); N-Metal Derivatives vinyl, allyl, 1-isopropylallyl, cinnamyl, 4-nitrocinnamyl, 45 (N-borane derivatives. N-diphenylborinic acid derivatives, 8-quinolyl. N-hydroxypiperidinyl, alkyldithio. benzyl, N-phenyl(pentacarbonylchromium- or -tungsten)carbenyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl. N-copper or N-zinc chelate); N-N Derivatives (N-nitro. p-chorobenzyl, 2,4-dichlorobenzyl, 4-methylsulfinylbenzyl, N-nitro so, N-oxide); N-P Derivatives 9-anthrylmethyl, diphenylmethyl); Groups With Assisted (N-diphenylphosphinyl, N-dimethylthiophosphinyl. Cleavage (2-methylthioethyl 2-methylsulfonylethyl 2-(p- 50 N-diphenylthiophosphinyl, N-dialkyl phosphoryl. toluene sulfonyl)ethyl, 2-(1,3-dithianyl)methyl. N-dibenzyl phosphoryl, N-diphenyl phosphoryl); N-Si 4-methylthiophenyl, 2,4-dimethylthiophenyl. Derivatives; N-S Derivatives; N-Sulfenyl Derivatives 2-phosphonioethyl, 2-triphenylphosphonioisopropyl, 1.1- (N-benzenesulfenyl, N-o-nitrobenzenesulfenyl, N-2,4- dimethyl-2-cyanoethyl, m-chloro-p-acyloxybenzyl. dinitrobenzenesulfenyl. N-pentachlorobenzenesulfenyl. p-(dihydroxyboryl)benzyl, 5-benzisoxazolylmethyl, 55 N - 2 - nitro - 4 - methoxy be n ze n e sulfe nyl. 2-(trifluoromethyl)-6-chromonylmethyl); Groups Capable N-triphenylmethylsulfenyl, N-3-nitropyridinesulfenyl); and of Photolytic Cleavage (m-nitrophenyl. 3.5- N-sulfonyl Derivatives (N-p-toluene sulfonyl, dimethoxybenzyl, o-nitrobenzyl, 3,4-dimethoxy-6- N-benzene sulfonyl, N-2, 3, 6-trim ethyl-4- nitrobenzyl, phenyl(o-nitrophenyl)methyl); Urea-Type methoxybenzene sulfonyl. N-2,4,6- Derivatives (phenothiazinyl-(10)-carbonyl, N'-p- trimethoxybenzene sulfonyl. N-2,6-dimethyl-4- to i u e n e s u f on y a m in oc a rib on y 1, methoxybenzenesulfonyl. N- pentamethylbenzenesulfonyl, N'-phenylaminothiocarbonyl); Miscellaneous Carbamates N-2,3,5,6,-tetramethyl-4-methoxybenzenesulfonyl, N-4- (t-amyl. S-benzyl thiocarbamate, p-cyanobenzyl, methoxybenzene sulfonyl, N-2,4,6- cyclobutyl, cyclohexyl, cyclopentyl, cyclopropylmethyl, trimethylbenzene sulfonyl, N-2,6-dimethoxy-4- p-decyl oxybenzyl. diisopropylmethyl. 2,2- 65 methylbenzenesulfonyl, N-2.2.5.7.8-pentamethylchroman dimethoxycarbonylvinyl. o-(N,N-dimethylcarboxamido) 6-sulfonyl, N-methane sulfonyl, N-B- benzyl, 1.1-dimethyl-3-(N.N-dimethylcarboxamido)propyl. trimethylsilyethanesulfonyl, N-9-anthracenesulfonyl, N-4- 5,750.729 17 18 (4', 8' -dimethoxynaphthylmethyl)benzene sulfonyl. N-benzylsulfony 1, N-trifluoromethylsulfonyl, TABLE 1-continued N-phenacylsulfonyl). Nucleus Structures More typically, protected amino groups include carbam 5 Z. O O ates and amides, still more typically, -NHC(O)R’ or o o -N=CRN(R). P(OH)2 P(OH)2 B Z.

BV - BV - O Exemplary Compounds of the Invention 10 1. 12 Z. O B O B FoH, By way of example and not limitation, embodiment 15 compounds are named below in tabular format (Table 5). Generally, each compound is depicted as a substituted rea Poh). ea B nucleus in which the nucleus is designated by capital letter O F (s) and each substituent is designated in order by lower case 13 14 15 letter or number. Table 1 is a schedule of nuclei which differ 20 Z.

principally by the position and presence or absence of ring unsaturation, the stereochemistry of the ring substituents, the O O size of the ring and substitution at the 2 and 3 positions. Each Z. nucleus is given a numerical designation from Table 1. and this designation appears first in each compound name. ROH), BV - ROH). B OH: Similarly, Tables 2, 3 and 4 list the selected Z, B. R." and O O R1b Rile O R" substituents, again by letter or number designation. R", 6 17 18 R" and R', if not shown herein, are H or bonds as dictated 2. by valence. Accordingly, each named compound will be depicted by a number followed by upper and lower case 30 B. Z. O B O O letters designating the Z and B substitutents and, as P(OH) P(OH)2 Kit, P(OH) appropriate, one or two more numbers designating the R' A A N" / and R' substituents. R' is H in structures 19–26. Thus. structure (28), scheme 2, is represented by 20.A.c.12 or R9 Rib 26.A.c.12 and compound (30), scheme 2, is represented by 35 19 2O 2 9.A.c.12, where B=cytosine. O O O O O O Z 2. B Z TABLE 1. P(OH)2 RibA P(OH) Rib P(OH 4o B B A Nucleus Structures Rlk Z. O Z. O Z. O 22 23 24 B o P(OH) B o P(OH) B o P(OH)2 Z. Rib R1a 45 BA O 9 O BA O Z. P(OH)2 W. A. P(OH2 R. R.7. R. R1b Ria Rile R1b. ROH). Ol O2 O3 R1b. O 50 25 26 27 2. O Z. O O o o o Z. Z. Z. B P(OH) B P(OH), B P(OH O Z. (- O B. |- O Rib Ria 55 Rib Rh ROH). ROH). ROH). R15 Rla O Ric O Rla O 04 05 O6 28 29 3O

Z. O O Z. O 6C O O O o o o o o o B P(OH), B s P(OH) B P(OH)2 B P(OH) B P(OH), B P(OH)2 Z. Z. R1b. R1b Rila

O7 O8 09 65 Ria Ria Rib 31 32 33

5,750,729 35

TABLE 5-continued O9.H.h; 09.H.i; 09.H.j; 09.H.k; 09.H.l; 09.I.a; 09.Ib; 09.I.c; 09.Id; 09.I.e; 09.I.f; O9.Ig; 09.I.h; 09.I.ii; 09.Ij; 09:Ik; 09.Ll; 10.A.a; 10.A.b; 10.A.c; 10. Ad; 10.A.e; 10.Af; 10.B.a; 10.B.b; 10.B.c; 10.B.d; 10.B.e; 10.B.f, 10.C.a; 10.C.b; 10.C.c; 10.C.d; 10.C.e; 10.C.f. 10.D.a; 10.D.b; 10.D.c.; 10.D.d; 10.D.e; 10.D.f. 11.A.a; 11.A.b; 11.A.c; 11. Ad; 11.A.e., 11.A.f; 11.B.a; 11.B.b; 11.B.c; 11.B.d; 11.B.e; 11.B.f., 11.C.a; 11.O.b; 1.C.c; 11.C.d; 11 Ce; 11.C.f; 11.D.a; 11.D.b; 11.D.c; 11.D.d; 11.De; 11.D.f. 12.A.a; 12.A.b.; 12.A.c; 12.A.d; 12.A.e; 12.A.f; 12.A.g; 12.A.h; 12.A.i; 12.A.j; 12. Ak; 12.A.l; 12.B.a, 12.B.b; 12.B.c; 12.B.d; 12.Be; 12.B.f; 12.Bg; 12.B.h; 12.B.i; 12.B.j; 12.B.k; 12.B.1; 12.C.a; 12.O.b; 12.C.c; 12.O.d; 12.O.e; 12.C.f, 12.O.g; 12.C.h; 12.C.i; 12.C.j; 12.O.k; 12.C.l; 12.D.a; 12.D.b; 12.D.c; 12.D.d; 12.De; 12.D.f; 12.D.g; 12.D.h; 12.D.i; 12.D.j; 12.D.k; 12.D.l; 12.E.a; 12.E.b; 12E.c; 12E.d; 12E.e; 12E.f; 12E.g.; 12E.h; 12.E.i; 12 Fi; 12 Fj; 12 Fk; 12 Fl; 12.G.a; 12.G.b; 12.G.c; 12.G.d; 12.G.e; 12.G.f; 12.G.g; 12.Gh; 12.G.i; 12.G.j; 12.G.k; 12.G.l; 12 Ha; 12.H.b; 12.H.c; 12.H.d; 12.He; 12.H.f. 12.H.g; 12.H.h; 12.Hi; 12.H.j; 12.H.k; 12.H.l; 12.I.a; 12.Ib; 12.I.c; 12.Id; 12.Ile; 12.I.f; 12.Lig, 12...h; 12.I.i; 12.I.j; 12 Ik; 12.I.l., 13.A.a; 13.A.b; 13.A.c; 13. Ad; 13.A.e., 13A.f. 13.B.a; 13.B.b; 13.B.c; 13.B.d; 13.B.e; 13.B.f. 13.C.a; 13.C.b; 13.C.c; 13.C.d; 13.C.e; 13.C.f. 13.D.a; 13.D.b; 3.D.c; 13.D.d; 13.D.e; 13.D.f. 14A.a; 14.A.b. 14.A.c; 14Ad; 14.A.e; 14.A.f, 14.A.g; 14.A.h; 14.A.i; 14 Aj; 14 Ak; 14.A.l; 14.B.a; 14.B.b; 14.B.c; 14.B.d; 14.B.e; 14.B.f. 14.Bg; 14.B.h; 14.B.i; 14.B.j; 14.B.k; 14.B.l; 14.C..a; 14.C.,b; 14.C.c; 14.C.d; 14.C.e; 14.C.f; 14.C.g; 14.C.h; 14.C.i; 14.C.j; 14.C.k; 14.C.l; 14.D.a; 14.D.b; 14.D.c; 14.D.d; 14.D.e; 4.D.f. 14.D.g; 14D h; 14.D.i; 14.D.j; 14.D.k; 14.D.l; 14E.a; 14.E.b; 14E.c; 14.E.d; 14E.e; 14E.f; 14E.g.; 14E.h; 14.E.i; 14E.j; 14Fj; 14Fk; 14 Fl; 14.C.a; 14.C.b; 14.G.c; 14.G.d; 14.G.e; 14.G.f, 14.G.g; 14.C.h; 14.G.i; 14.G.j; 14.G.k; 14.g. l; 14.Ha; 14.H.b; 14.H.c; 14 Hid; 14.He; 14.H.f. 14.H.g; 14.H.h; 14.H.i; 14.H.j; 14.H.k; 14.H.l; 14.I.a; 14.I.b; 14.I.c; 14.I.d; 14.I.e; 14.f; 14.1.g; 14.I.h; 14.Li; 14.I.j; 14.I.k; 14.I.l; 15.A.a; 15.A.b; 15.A.c; 15.A.d; 15.A.e; 15.Af; 15.B.a; 15.B.b; 15.B.c; 15.B.d; 15Be; 15.B.f. 15.C..a; 15.O.b; 15.C.c; 15.O.d: 15.C.e; 15.C.f. 15.D.a; 15.D.b; 5D.c; 15.D.d; 15.De; 15.D.f. 16.A.a; 16.A.b.; 16.A.c; 16.A.d; 16.A.e: 16.A.f. 16.B.a. 16.B.b; 16.B.c; 16.Bd; 16.B.e; 16.B.f. 16.O.a; 16.C.b; 16.O.c; 16.C.d; 16.C.e; 16.C.f. 16.D.a; 16.D.b; 16.D.c.; 16.D.d; 16.D.e; 16.D.f. 17.A.a; 7.A.b.; 17Ac; 17.A.d.; 17.A.e; 17.Af; 17.8.a; 17.B.b.; 17B.c; 17.B.d.; 17.B.e; 17.B.f; 17.Ca; 17.C.b. 17.O.c; 17.C.d.; 17.C.e; 17.C.f; 7.D.a; 17.D.b; 7.D.c.; 17.D.d; 17.D.e; 17.D.f.

Utilities tain a free hydroxyl group (ordinarily one or both of the R' groups or Z) linked to the pyran or (preferably) the furan The compounds of this invention are useful perse or as 35 ring, they optionally are incorporated internally into the intermediates in the preparation of polymers having a wide sequence of the oligonucleotide. Terminally incorporated variety of diagnostic, therapeutic and industrial utilities. diphosphoryl compounds of this invention which contain no The compounds are useful in the preparation of polyphos free hydroxyl capable of participating in chain elongation phonate flame retardants. The compounds of this invention also are useful in DNA sequencing in essentially the same that contain nonresonant sites of unsaturation, e.g. enolpy manner as deoxyNTPs have been used in the past (see ran and R or Z groups, are incorporated into polyvinyl example 8 of U.S. Pat. No. 5.276.143). The nucleotide polymers by methods or analogous methods heretofore analogues of the invention (when diphosphorylated) are employed with known vinylphosphonates. The compounds useful as chain terminators for dideoxynucleotide-type DNA of this invention that do not already contain vinyl groups or sequencing protocols, provided that the nucleotide analogue the like are useful nonetheless as intermediates preparing 45 lacks a free hydroxyl group suitable for polymerase medi vinylphosphonate monomers. These monomers are copoly ated chain elongation. These compounds will not have a free merized with vinyl resins by free radical catalysis methods hydroxyl group at R". R' or Zand do not possess a cyclic already known per se, e.g. by use of persulfate or electron structure incorporting the phosphorus atom (although such beam. Other methods of incorporation of the compounds of excluded structures can be intermediates). The nucleotide this invention into polymeric resins will be readily apparent 50 analogue is included in a kit with other reagents (such as to the skilled artisan, so it is not necessary to use vinyl klenow polymerase or T4 polymerase. dNTPs, etc) needed intermediates, for DNA sequencing (Otvos. et al. "Nucl. Acids Res." The compounds of this invention are useful as interme 15:1763-1777 (1987). diates in preparing labelled oligonucleotide probes or, when If the oligonucleotide-incorporated compound of this Y is an oligonucleotide, are directly useful in assays for 55 invention is binding-competent for its complementary target nucleic acid sequences. Typically, the phosphonate sequence, i.e., if it is capable of base-pairing, then this group of the compounds of this invention is covalently nucleotide monomer will participate in hybridization. It is bonded to the terminus of an oligonucleotide having a not necessary, however, that the incorporated nucleotide predetermined sequence, although any hydroxyl group is analogue of this invention base pair or otherwise participate useful for this purpose. The structure or sequence of the in hybridization. If it is located at the terminus of the oligonucleotide is not important except insofar as it is oligonucleotide it will be useful as an immunological rec binding-competent for its complementary sequence. Many ognition site to detect the presence of the oligonucleotide oligonucleotides having this property are well known. e.g. when used as a probe or as a linker for a detectable group. conventional phosphodiester or phosphorothioate oligo The compounds of this invention are useful as linkers or nucleotides. 65 spacers in preparing affinity absorption matrices, immobi The compounds of this invention generally will be ter lized enzymes for process control, or immunoassay reagents. minally incorporated into the oligonucleotide. If they con The compounds herein contain a multiplicity of functional 5,750,729 37 38 groups that are suitable as sites for cross-linking desired (including African swine fever virus and Japanese encepha substances. For example, it is conventional to link affinity litis virus), togaviruses (including Venezuelan equine reagents such as hormones, peptides, antibodies, drugs, and encephalomyelitis virus), influenza viruses (types A-C), the like to insoluble substrates. These insolublized reagents retroviruses (HIV 1, HTV 2, HTLV I, HTLV II, SIV, HBV. are employed in known fashion to absorb binding partners 5 FeLV. FIV. MoMSV), adenoviruses (types 1-8), poxviruses for the affinity reagents from manufactured preparations, (vaccinia virus), enteroviruses (polio virus type 1-3, hepa diagnostic samples and other impure mixtures. Similarly, titis A virus), gastroenteritis viruses (Norwalk viruses, immobilized enzymes are used to perform catalytic conver rotaviruses), hantaviruses (Hantaan virus), papovaviruses, sions with facile recovery of enzyme. Bifunctional com rhinoviruses, parainfluinza virus types 1-4, rabies virus, pounds are commonly used to link analytes to detectable RSV, hepatitis viruses A, B, C and E, and the like. The groups in preparing diagnostic reagents. 10 structure (1) compounds in which Z is CHOR are believed Many functional groups in the compounds of this inven to be most effective against DNA viruses, while those in tion are suitable for use in cross-linking. For example, the which Z is CH, CH=CH, CHN. C=CH, or haloalkyl phosphonic acid is used to form esters with alcohols or are believed to be most effective against RNA and retrovi amides with amines. The Z, R' or R' groups substituted 5 ruses. Compounds in which Z is H are believed to be with OH. azido (which is reduced to amino if desired before effective to at least some degree against DNA, RNA and cross-linking), CN or halo are suitable sites. Similarly, the retroviruses. Hepatitis B viruses and HIV are believed to be amino, halo, acyl and other reactive sites (e.g., the 8-purine most effectively treated by the B-L compounds of this position) found on group B are suitable. Suitable protection invention. of reactive groups will be used where necessary while The antiviral activity of individual nucleotide analogues is assembling the cross-linked reagent. In general, the com determined by routine assay of antiviral (or other pounds here are used by linking them through phosphonic antimicrobial) activity using enzyme inhibition assays, tis acid to the hydroxyl or amino groups of the linking partner sue culture assays, animal model assays and the like as will in the same fashion as shown herein and covalently bonded be understood by those skilled in the art. to the other binding partner through a Z, B.R' or R' group. 25 Protozoan parasite infections are treated using the com For example a first binding partner such as a steroid hor pounds of the invention. The term protozoa includes those mone is esterified to the phosphonic acid of this invention members of the subphyla Sarcomastigophora and Sporozoa and then this conjugate is cross-linked to cyanogen bromide of the phylum Protozoa, More particularly, the term proto activated Sepaharose, whereby immobilized steroid is zoa as used herein includes genera of parasitic protozoa obtained. Other chemistries for conjugation are well known. 30 which are important to man because they either cause See for example Maggio, "Enzyme-Immunoassay" (CRC. disease in man or in his domestic animals. These genera for 1988, pp 71-135) and references cited therein. the most part are classified in the superclass Mastighphora of The oligonucleotides of this invention are labelled with the subphylum Sarcomastigophora and the class Telosporea any conventional detectable label, e.g. a fluorescent moiety of the subphylum Sporozoa in the classification according to such as fluorescein, radioisotopes such as C or H. stable 35 Baker (1969). Illustrative genera of these parasitic protozoa free radicals, avidin, biotin and the like all of which previ include Histomonas, Pneumocystis, Trypanosoma. Giardia, ously have been used as labels for immunoassays or diag Trichomonas, Eimeria. Isopora, Leishmania, Entamoeba. nostic probes. The label will be present on the oligonucle Toxoplasma and Plasmodium. Parasitic protozoans include otide or on the residue of the nucleotide analogue of this Plasmodium falciparum, Plasmodium berghei, Plasmodium invention. Suitable labelling methods are well known and malariae, Plasmodium vivax, Leishmania braziliensis, are readily used with reactive groups such as hydroxyl, allyl Leishmania donovani, Trypanosoma cruzi, Trypanosoma and the like. A simple method is to label the compound of brucei, Trypanosoma rhodesiense, Pneumocystis carini, this invention with H by proton exchange. The compounds Entamoeba histolytica, Trichomonas vaginalis and the like also are biotinylated using conventional methods. See for (de Vries, E., et al. "Mol. Biochem. Parasitol" 47:43-50 instance U.S. Pat. No. 5.276,143 for analogous structures. 45 (1991)). The compounds in which Z is CHOR and B is However, the oligonucleotides of this invention also are 3-deazaadenine are particularly interesting for malarial para useful directly in diagnostic probe assays without an exog sites. enous detectable label. In one embodiment of this Nucleoside analogues of the invention are used to treat alternative, antibodies are raised against the nucleotide ana yeast or fungal infections caused by Candida glabrata, logues of this invention. Such antibodies (which in turn are 50 Candida tropicalis, Candida albicans, and other Candida labelled or used in a double antibody configuration) bind to species Cryptococcus species including Cryptococcus the analogue of this invention and thereby are useful in neoformans, Blastomyces species including Blastonyces detecting the presence of the bound probe that incorporates dermatidis, Torulopsis species including Torulopsis the nucleotide analogue of this invention. glabrata, Coccidioides species including Coccidioides The compounds of the invention, particularly those in 55 immitis, Aspergillus species and the like. which the phosphonate and base have the same absolute The therapeutically useful compounds of this invention in stereochemistry, are useful for treatment of microbial which hydroxyl or amino groups are protected are useful as infections, for treatment of tumors or for other indications oral or sustained release forms. In these uses the protecting described below. Microbial infections treatable by the com group is removed in vivo, e.g., hydrolyzed or oxidized, so as pounds of this invention include viruses, parasites, yeasts to yield the free amino or hydroxyl. This is necessary in the and fungi, but it is believed that the compounds are most case of the phosphonate hydroxyl groups. Suitable esters or effective against viruses, which constitutes the preferred amidates for this utility are selected based on the substrate utility, Exemplary viral infections include infections caused specificity of esterases and/or carboxypeptidases expected to by DNA or RNA viruses including herpesviruses (CMV, be found within cells where precursor hydrolysis is desired. HSV 1, HSV 2, EBV, varicella zoster virus, bovid herpes 65 To the extent that the specificity of these enzymes is virus type 1, equid herpesvirus type 1), papillomaviruses unknown, one will screen a plurality of nucleotide analogues (HPV types 1-55; carcinogenic HPV), flaviviruses of this invention until the desired substrate specificity is 5,750.729 39 40 found. This will be apparent from the appearance of free water-miscible ointment base. Alternatively, the active phosphonate or of antimicrobial activity. One generally ingredients may be formulated in a cream with an oil-in selects compounds that are (i) not hydrolyzed or hydrolyzed Water cream base. comparatively slowly in the upper gut, (ii) gut and cell If desired, the aqueous phase of the cream base may permeable and (iii) hydrolyzed in the cell cytoplasm and/or include, for example, at least 30% wiv of a polyhydric systemic circulation. Screens with cells from particular alcohol, i.e. an alcohol having two or more hydroxyl groups tissues are used to identify precursors that are released in such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, organs susceptible to a target viral or microbial infection, glycerol and polyethylene glycol (including PEG 400) and e.g. in the case of liver, precursor drugs capable of hydroly mixtures thereof. The topical formulations may desirably sis in the liver. Other infections, e.g. CMV or HIV, option 10 include a compound which enhances absorption or penetra ally are treated with a precursor that is hydrolyzed at tion of the active ingredient through the skin or other substantially the same rate and to substantially the same affected areas. Examples of such dermal penetration enhanc degree in all tissues. Assays known in the art are suitable for ers include dimethyl sulphoxide and related analogues. these purposes, including intestinal lumen stability, cell The oily phase of the emulsions of this invention may be permeation, liver homogenate stability and plasma stability 5 constituted from known ingredients in a known manner. This assays. These assays are used to determine the bioavailabil phase may comprise an emulsifier alone, or a mixture of at ity characteristics of the precursors. However, even if the least one emulsifier with a fat or an oil or with both a fat and derivatives are not converted in vivo they remain useful as an oil. Preferably, a hydrophilic emulsifier is included chemical intermediates. together with a lipophilic emulsifier which acts as a stabi The nucleotide analogues of the invention also can be (1) 20 lizer. It is also preferred to include both an oil and a fat. applied to tissue culture systems to eliminate or reduce viral Emulsion stabilizers suitable for use in the formulation of spread or growth during the production of biopharmaceuti the present invention include Tweene 60, Span(s) 80, ceto cals or other products (such as proteins or vaccines), (2) used stearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl to eliminate or reduce viral spread or growth in clinical mono-stearate and sodium lauryl sulfate. Suitable oils or fats samples (such as blood), and (3) used to stop growth of 25 include straight or branched chain, mono- or dibasic alkyl tissue culture or bacterial cells (using toxic amounts of esters such as di-isoadipate, isocetyl stearate, propylene compound) while leaving the cells to carry on with protein glycol diester of coconut fatty acids, isopropyl myristate, production. decyl oleate, isopropyl palmitate, butyl stearate or Pharmaceutical formulations. Compounds of the inven 2-ethylhexyl palmitate. These may be used alone or in tion and their physiologically acceptable salts (hereafter 30 combination depending on the properties required. collectively referred to as the active ingredients) are formu Alternatively, high melting point lipids such as white soft lated for administration by any route appropriate to the paraffin and/or liquid paraffin or other mineral oils can be condition to be treated. The compounds and formulations used. preferably will be sterile. Formulations suitable for topical administration to the eye The active ingredients are placed into pharmaceutical 35 also include eye drops wherein the active ingredient is formulations. The formulations, both for veterinary and for dissolved or suspended in a suitable carrier, especially an human use, comprise at least one active ingredient, as above aqueous solvent for the active ingredient. The active ingre defined, together with one or more acceptable carriers there dient is typically is present in such formulations in a for and optionally other therapeutic ingredients. The carrier concentration of 0.01 to 20% by weight. (s) must be "acceptable" in the sense of being compatible Formulations suitable for nasal administration wherein with the other ingredients of the formulation and not del the carrier is a solid include a coarse powder having a eterious to the recipient. particle size for example in the range 20 to 500 microns The formulations conveniently are presented in unit dos (including particle sizes in a range between 20 and 500 age form and may be prepared by any of the methods well 45 microns in increments of 5 microns such as 30 microns, 35 known in the art of pharmacy. In general the formulations microns, etc), which is administered by rapid inhalation are prepared by uniformly and intimately bringing into through the nasal passage from a container of the powder. association the active ingredient with liquid carriers or finely Suitable formulations wherein the carrier is a liquid, for divided solid carriers or both, and then, if necessary, shaping administration as for example a nasal spray or as nasal the product. 50 drops, include aqueous or oily solutions of the active ingre Formulations of the present invention suitable for oral dient. Formulations suitable for aerosol administration may administration may be presented as discrete units such as be prepared according to conventional methods and may be capsules, cachets or tablets each containing a predetermined delivered with other therapeutic agents such as pentamidine amount of the active ingredient; as a powder or granules; as for treatment of pneumocystis pneumonia. solution or a suspension in an aqueous liquid or a non 55 Formulations suitable for vaginal administration may be aqueous liquid; or as an oil-in-water liquid emulsion or a presented as pessaries, tampons, creams, gels, pastes, foams water-in-oil liquid emulsion. The active ingredient may also or spray formulations containing in addition to the active be presented as a bolus, electuary or paste. ingredient such carriers as are known in the art to be For external infections of the eye or other external tissues appropriate. e.g. mouth and skin, the formulations are preferably applied Formulations suitable for parenteral administration as a topical ointment or cream containing the active include aqueous and non-aqueous sterile injection solutions ingredient(s) in an amount of, for example, 0.075 to 20% which may contain anti-oxidants, buffers, bacteriostats and wfw (including active ingredient(s) in a range between 0.1% solutes which render the formulation isotonic with the blood and 20% in increments of 0.1% w/w such as 0.6% w/w, of the intended recipient; and aqueous and non-aqueous 0.7% w/w, etc), typically 0.2 to 15% w/w and most typically 65 sterile suspensions which may include suspending agents 0.5 to 10% w/w. When formulated in an ointment, the active and thickening agents. The formulations may be presented in ingredients may be employed with either a paraffinic or a unit-dose or multi-dose containers, for example sealed 5,750,729 41 42 ampoules and vials, and may be stored in a freeze-dried Compounds such as those of structures (2), (3), and (4) (lyophilized) condition requiring only the addition of the generally are expected to have a higher oral bioavailability sterile liquid carrier, for example water for injections, imme than the corresponding uncyclized nucleotide analogue and diately prior to use. Extemporaneous injection solutions and or exhibit reduced toxicity when compared with the same suspensions may be prepared from sterile powders, granules dose of the corresponding uncyclized nucleotide analogue. and tablets of the kind previously described. Preferred unit Doses will be adjusted accordingly. dosage formulations are those containing a daily dose or unit The compounds of the invention optionally are employed daily sub-dose, as herein above recited, or an appropriate in combination with other therapeutic agents for the treat fraction thereof, of an active ingredient. ment or prophylaxis of the infections or conditions indicated The present invention further provides veterinary compo 10 above. Examples of such further therapeutic agents include sitions comprising at least one active ingredient as above agents that are effective for the treatment or prophylaxis of defined together with a veterinary carrier therefor. Veteri viral, parasitic or bacterial infections or associated condi nary carriers are materials for administering the composition tions or for treatment of tumors or related conditions. These and may be solid, liquid or gaseous materials which are include 3'-azido-3'-deoxythymidine (zidovudine, AZT), 15 2'-deoxy-3'-thiacytidine (3TC). 2',3'-dideoxy-2',3'- otherwise inert or acceptable in the veterinary art and are didehydroadenosine (D4A). 2',3'-dideoxy-2',3'- compatible with the active ingredient. These veterinary didehydrothymidine (D4T). carbovir (carbocyclic 2',3'- compositions may be administered orally, parenterally or by dideoxy-2',3'-didehydroguanosine). 3'-azido-2',3'- any other desired route. dideoxyuridine. 5-fluorothymidine. (E)-5-(2-bromovinyl)- Compounds of the invention can be used to provide 2'-deoxyuridine (BVDU), 2-chlorodeoxyadenosine, controlled release pharmaceutical formulations containing 2-deoxycoformycin, 5-fluorouracil. 5-fluorouridine, as active ingredient one or more compounds of the invention 5-fluoro-2'-deoxyuridine, 5-trifluoromethyl-2'- in which the release of the active ingredient is controlled and regulated to allow less frequent dosing or to improve the deoxyuridine, 6-azauridine, 5-fluoroorotic acid, pharmacokinetic or toxicity profile of a given compound. In methotrexate, triacetyluridine, 1-(2'-deoxy-2'-fluoro-1-3- 25 arabinosyl)-5-iodocytidine (FIAC), tetrahydro-imidazo(4.5. general, the compounds are administered from controlled 1-jk)-(1,4)-benzodiazepin-2(1H)-thione (TIBO). 2'-nor release systems such as the intravitreal implant of WO cyclicGMP, 6-methoxypurine arabinoside (ara-M), 92/14450 or U.S. Pat. No. 5,098.443, or the matrices of U.S. 6-methoxypurine arabinoside 2'-O-valerate. cytosine arabi Pat. No. 4,740.365 or U.S. Pat. No. 5,141,752, although noside (ara-C). 2',3'-dideoxynucleosides such as 2',3'- many others are known and are suitable for use herein. 30 dideoxycytidine (ddC). 2',3'-dideoxyadenosine (ddA) and Therapeutic Administration. Suitable routes for adminis 2',3'-dideoxyinosine (dd), acyclic nucleosides such as tration include oral, rectal, nasal, topical (including ocular, acyclovir, penciclovir, famciclovir, ganciclovir. HPMPC. buccal and sublingual), vaginal and parenteral (including PMEA, PMEG, PMPA, PMPDAP, FPMPA, HPMPA and subcutaneous, intramuscular, intravitreal, intravenous, HPMPDAP, (2R, 5R)-9-tetrahydro-5-(phosphonometh intradermal, intrathecal and epidural). The preferred route of 35 oxy)-2-furanyladenine, (2R, 5R)-1-tetrahydro-5- administration will depend upon the condition of the patient, (phosphonomethoxy)-2-furanylthymine, other antivirals the toxicity of the compound and the site of infection, among including ribavirin (adenine arabinoside), 2-thio-6- other considerations known to the clinician. azauridine, tubercidin, aurintricarboxylic acid, For each of the above-indicated theapeutic indications the 3-deazaneoplanocin, neoplanocin, rimantidine, adamantine, amount required of an active ingredient (as above defined) and foscarnet (trisodium phosphonoformate), antibacterial will depend upon a number of factors including the severity agents including bactericidal fluoroquinolones of the condition to be treated, the infectious agent, whether (ciprofloxacin, pefloxacin and the like), aminoglycoside the use is prophylactic or to treat an acute infection, the site bactericidal antibiotics (streptomycin, gentamicin, amicacin of infection or pathology (e.g. CMV retinitis is treated and the like), f-lactamase inhibitors (cephalosporins, peni systemically or by intravitreal injection) and other factors 45 cillins and the like), other antibacterials including ultimately at the discretion of the attending physician or tetracycline, isoniazid, rifampin, cefoperazone. claithromy veterinarian. In general, however, a suitable dose for con cin and azithromycin, antiparasite or antifungal agents sideration by the clinician will be in the range of analogous including pentamidine (1.5-bis(4-aminophenoxy)pentane), methoxyphosphonates (see supra), taking into account dif 9-deazainosine, sulfamethoxazole. Sulfadiazine, ferences in potency, generally 0.1 to 250 mg per kilogram 50 quinapyramine, quinine, fluconazole, ketoconazole, bodyweight of recipient per dose (including active itraconazole, Amphotericin B, 5-fluorocytosine, ingredient(s) in a range between 0.1 mg and 250 mg/Kg/ clotrimazole, hexadecylphosphocholine and nystatin, renal dose in increments of 0.5 mg/Kg/dose such as 2.5 mg/Kg/ excretion inhibitors such as probenicid, nucleoside transport dose, 3.0 mg/Kg/dose, 3.5 mg/Kgfdose, etc), typically in the inhibitors such as dipyridamole, dila Zep and range 0.5 to 50 mg per kilogram body weight per dose and 55 nitrobenzylthioinosine, immunomodulators such as FK506, most usually in the range 1 to 15 mg per kilogram body cyclosporin A, thymosino-1, cytokines including TNF and weight per dose. Unless otherwise indicated all weights of TGF-B, interferons including IFN-O, IFN-B and IFN-Y, active ingredient are calculated as a compound of structure interleukins including interleukin 1,2,3,4, 5, 6, 7, 8, 10, 12. (1) wherein Y is not a polymer. 13, macrophage/granulocyte colony stimulating factors The desired dose is administered at appropriate intervals including GM-CSF, G-CSF, M-CSF, cytokine antagonists in unit dosage forms, usually with a relatively higher induc including anti-TNF antibodies, anti-interleukin antibodies, tion dose and lower, less frequent maintenance doses. The soluble interleukin receptors, protein kinase C inhibitors compounds also are used prophylactically, for example, by and, particularly in treatment of HTV.cotherapy with IFN-O, administration on about from 1 to 7 days before viral IL-2 or IL-12. infection. HPV tumors or growths and herpes lesions often 65 Immunogens and Antibodies. The compounds of this are treated topically, either by local injection or by topical invention are used as immunogens to prepare antibodies gels, ointments or the like. capable of binding specifically to the compounds or their 5,750,729 43 44 metabolic products. The immunogenic compositions are or through a furan or pyran ring hydroxyl group (usually at useful as intermediates in the preparation of antibodies for the R', R' or Z positions). Alternatively, the precursor use in diagnostic or quality control assays for the com compound is cross-linked through the phosphonate, typi pounds or their metabolic products. The antibodies are cally by amidation or esterification of the phosphonate by useful for measuring the presence, absence or amounts of the the polypeptide itself or by a cross-linking functionality compounds by any convenient homogenous or heterogenous covalently bonded to the polypeptide, whereby Y is an procedure such as fluorescence polarization immunoassay, immunogenic protein having more than 50 amino acid fluorescence immunoassay (using fluorescent labels such as residues, usually less than 1000. The conjugates are pre fluorescein and the like), radioimmunoassay, enzyme immu pared in conventional fashion. For example. noassay (using enzyme indicators such as alkaline N-hydroxy succinimide, succinic anhydride or phosphatase, horseradish peroxidase, glucose oxidase, ure alkN=C=Nalk are useful in preparing the conjugates of ase and the like) and nephelometric inhibition assay by this invention. Animals typically are immunized against the described methods (WO 92/22639). Competitive-type immunogenic conjugates and monoclonal antibodies pre assays usually require the antibody, and a tracer (such as a pared in conventional fashion. fluorescent or radio label) conjugated to the compound to be 15 assayed. The antibodies directed against the compounds of Synthetic Methods this invention desirably will not cross-react with naturally The Table 1 saturated compounds are made by the fol occurring nucleotides or nucleosides. lowing scheme 1. Table 1 unsaturated compounds are made The immunogens of this invention contain the precursor by scheme 2 or straight-forward variations thereof. Scheme or hydrolytic products in association with an immunogenic 2 also is readily adapted to make saturated Table 1 com substance such as a protein or peptide. Immunogenic sub pounds by adding the R' groups across the double bond, for stances include adjuvants such as Freund's adjuvant, immu example, via expoxidation, hydroxylation, reduction or nogenic proteins such as viral, bacterial, yeast, plant and halogenation. Table 1 compounds having R' and R'joined animal polypeptides, in particular keyhole limpet with the heterocyclic ring to form cyclopropyl are produced hemocyanin, serum albumin, bovine thyroglobulin or soy 25 by carbene addition across the double bond of the corre bean trypsin inhibitor, and immunogenic polysaccharides. sponding unsaturated ring. Table 1 compound 14 is made by Methods for the manufacture hapten immunogens are the method of scheme 3a. Compounds of this invention in conventional per se, and are useful here, taking into account which Y is CH. H. CH=CH, alkylhalo, CHN or CECH the functional groups that are available for cross-linking. are made by the procedures of scheme 3 or variations thereof The polypeptide immunogen (or a polypeptide that is 30 that will be apparent to the ordinary artisan in this field. desired to be made immunogenic by cross-linking to a These schemes are used with any base B. provided that the compound of this invention) may be conjugated to a site on base is suitably protected during synthesis. Other com the heterocyclic base rather than to the phosphonate moiety. pounds of this invention are made by variations of these In general, the site will be an amino group located on the schemes that will be readily apparent to those skilled in the purine or pyrimidine moiety of the nucleotide phosphonate. 35 art. at the 5 position of pyrimidines (such as cytosine or uracil), These schemes employ conventional abbreviations for at the 1 position of purines (such as adenosine or guanine) known reagents.

Scheme 1

CHOAc CHOAc O O AcO OAccie. Aco Br G>

AcOSt. OAC AcO5". OAC

(10) (11) n = 1 or O, alternatively, H replaces acetate at the 2-, 3 and/or 6-positions (and/or 5-acetate if n = 0)

CHOAc CHOH O O O O 1) TrCl AcO ( P(OiPr), T G HO P(OiPr). 2)acetone, TsoHor G 2) if n = 0 or if 2- or (CH) (CH), 3-Ac replaced with f OAc f OH H then: dihydropyran, AcO HO Tso (12) (13) 5,750,729 45 -continued Scheme 1

CHOTr CHOTr O O heterocyclic O O HO P(OiPr). Priaps B P(OiPr). THF (CH) (CH) f O O O O DC DX (14) (15)

O O HO OH

(15) (16) 1) AcOH 1) TBDMSC) 2) TMSBr imidazole, DMF lutidine, CH3CN 2TrfC)

CHOH CHOTr O O O O B - kor, B - Poir, to oH -lasers Trfo, OTBDMS (17) (18) Na or TBAX DMF X=N, halogen, or OR

CHOH CHOTr O O. O O. B POH).

Scheme 2

OAc OAc O P(oiPra O BF, EtO NH3 oy or G P(OiPr). Se AcO AcOV (21) (22) commercially available tri-O-acetyl-D-glucal 5,750,729 47 -continued Scheme 2

OTr OT r

O O hetero- B P(OiPr). -- cyclic base P(OiPr) 3P, HO DEAD, HF (24) (25) separate C. and anomers, carry forward individually CHBr, (3 is shown to right) 80% zinc-copper o-anomer heterocyclic base AcOH only Ph3P, DEAD, THF

CT OH OH O O O o o B B P(OiPr)2 c B P(OiPr). MOHGH fP(OiPr). O (26) (29) (31) TMSBr TMSBr base lutidine lutidine CHCN CHCN

OT OH OH R B O B/ FoHo. B O P(OH) A. P(OiPr).

(27) (30) (32) 1) 80% AcOH 2) TMSBr, lutidine, CHCN

OH O OTr O o o B P(OiPr). B P(OiPr) OH < 80% AcOH |- O A P(OH)2 CH (32b) OH CH, (32a) o (28) TSN B POH). lutidine CHCN (32c) CH

5,750,729 S1 52 -continued The step in scheme 2 where (21) is converted to (22) by Scheme 3a double bond migration is inventive. A protected, Z-substituted pyran (I) is treated in accord with this step, and OAc any Lewis Acid (e.g. HCl, ZnCl2 BFOEt, SnCl and the like) is suitable. The reaction solvent is not critical; any high NH/MeOH boiling (>100° C.) organic solvent is satisfactory. The phos OAC P(OiPr) phite PRT group is not critical and usually falls within R, AcO (40c) typically lower alkyl (C-C) and preferably iPr or ethyl. OH 10 Also inventive is the step in scheme 2 where the double O bond in (26) migrates to the 1-2 position as shown for O compound (27). A protected, Z-substituted pyran (III) is OH P(OiPr) -Ele treated with any base, whether organic or inorganic, where HO (40d) upon the double bond spontaneously migrates to a position 15 o, to the phosphate atom. The Banomers require a stronger O base than do the O anomers. This method has the additional O O advantage of removing benzyl protecting groups from any n RuO exocyclic amino groups of B. PRT in this method generally O OH P(OiPr). Ge. is selected from R as noted in the previous paragraph. (40e) The various diastereomers falling within the scope of this O invention are readily produced from the corresponding sug ars using the foregoing schemes. For example. Ph O O DAST 25 (41) O P(OiPr). Ele (40f) O

O 30 R1b Rila O O n - OK is produced from D-rhamnal. O F P(OiPr) E; SO (40g) (42) F 35 O

-( O O OP(OiPr) AcOH G R1b Ria (4Oh) is produced from D-glactal, F (43) HO

O O P(OiPr) Eile (40i) F OT (43a) O O P(OiPr). heterocyclic G base (40) PhP, DEAD, R1b Ria THF

OTr is produced from D-xylal. OH In general, other suitable sugars or unsaturated analogues O O O O thereof will be readily apparent starting materials. L-isomers B I of the claimed compounds are derived from the correspond P(OiPr). Taro Y P(OH), ing L-sugars or analogues thereof, taking into account the (4ok 2) TMSBrlutidine (401) stereochemical shift upon substitution by heterocylic base CHCN F. 65 (see scheme 2). Aside from the foregoing, the following Table 6 lists each of the sugars from which the Table 1 structures are derived. 5,750,729 S3 S4 removed under acid, base or hydrogenolysis conditions or TABLE 6 are removed with an esterase according to conventional methods. 1. D-allose 18. D-gulose 35. D-ribose 2. D-glucose 19. L-galactose 36. L-lyxose Synthesis of compounds of structure (1) having amino 3. D-altrose 20. D-allose 37. D-xylose acids such as tyr, cys, ser and thr is accomplished by 4. D-Imanose 21. D-galactose 38. L-xylose optionally protecting hydroxyl or thiol groups using protect 5. D-glucal 22. L-glucose 39. D-lyxose ing groups know in the art. For example, the hydroxyl group 6. L-galactal 23. L-allose 40. D-arabinose 7. D-glucal 24. L-gulose 41. D-xylose of ser, thr or tyr can be protected using benzyl, ethyl and the 8. L-galactal 25. D-glucose 42. D-lyxose like and the thiol group of cys can be protected using trityl, 9. D-glucal 26. L-mannose 43. D-arabinose 10 p-methylbenzyl and the like. The choice of a protecting 10. D-galactal 27. L-gulose 44. D-ribose 11. L-glucal 28. D-alose 45. L-arabinose group will depend on the stability of the bis-amidate toward 12. L-galactal 29. D-glucose 46. L-ribose conditions used to remove a particular protecting group. 13. D-glucal 30. D-altrose 47. L-xylose Appropriate protecting groups can be selected or determined 4. 2-deoxy-D-glucose 31. Lidose 48. L-arabinose by the skilled artisan using routine methods. 15. D-galactal 32. L-gulose 49. L-ribose 15 Synthesis of compounds in which Y is N-alkylamine is 16. L-galactal 33. L-talose 50. L-xylose 17. L-glucal 34. L-galactose accomplished essentially as described by Saito. "Chem. Pharm. Bull." 39:3207 (1991). Amidate-ester synthesis. Synthesis of mixed amidate If racemization occurs during any step in the synthesis the ester nucleotide analogues where one Y is an amino acid various diastereomers at chiral atoms 1-6 are separated using standard methods such as HPLC, RPLC or crystalli ester and the other is a group of the formula OR is zation. accomplished by conversion of the nucleotide analogue di It is also within the scope of this invention to use or bis-ester to the corresponding mono ester by treatment compounds falling within the claims as intermediates for with a base such as ammonia to remove one ester group. The other claimed compounds, for example as shown in scheme resulting mono ester is then converted to a mixed amidate 3a or as will be readily apparent to those skilled in the art, 25 ester as described for synthesis of bis amidate compounds. e.g. interconversion of heterocyclic bases. Oligonucleotides. Compounds of this invention where Y Bis amidate synthesis. Synthesis of bis-phosphoroamidate is an oligonucleotide are prepared from parental monomers nucleotide analogues of structure (1) where Ygroups are the in which Y is OH. The monomers are converted to the same and are an amino acid, dipeptide, tripeptide or reactive intermediate using conventional chemistry, for polypeptide is accomplished by conversion of the phospho 30 example the method of Uhlmann et al., "Chemical Reviews” nic acid nucleotide analogue to the corresponding bis 90(4):543 at 553, part c and FIG. 23 (1990) or Mazur et al., phosphoroamidate compound. The diacid is suspended in “Tet. Let." 40(20):3949 at scheme (1) and page 3955 (1984). approximately 2 equivalents of the Y reactant in a solvent For example, an oligonucleotide chain is synthesized on a such as dry pyridine or DMF (dimethylformamide) option matrix such as controlled pore glass in the 3'-5" or 5' to 3' ally containing a non-nucleophilic organic base such as 35 direction, whereby the 3' or 5' ends, respectively of the triethylamine (about 3 to 10 equivalents). The dehydration oligonucleotide are bonded to the matrix and the oligonucle step is accomplished by modification of a described reaction otide is protected except for the terminal 5" or 3' hydroxyl. (Mukaiyama, T. et al., "J. Am. Chem. Soc.”94:8528-8532 respectively, of the last nucleotide. The protected (1972)) by adding a 1:1 mixture of triphenylphosphine (reg. o-chlorophenyl derivative of the structure 1 compound is no. 603-35-0; Aldrich) and 2,2'-dipyridyl disulfide (2 to 4 prepared, analogous to the starting material shown in FIG. equivalents; reg. no. 2127-03-9; Aldrich) in pyridine to the 23 of Uhlmann et al. This is covalently bonded to a terminal nucleotide analogue?amino acid mixture and (a) stirring at OH of the oligonucleotide using the Uhlmann et al. method. room temperature for about 4 to 16 hours or (b) heating to Alternatively, the compound of this invention is converted 60° C. to 100° C. (including any temperature in one degree to the intermediate that is analogous to compound 12 of C increments between 60° and 100° C. such as 70, 80 or 45 Mazur et al. This analogue is added to the oligonucleotide 90° C.) for about 4 to 16 hours. The resulting reaction using essentially the dinucleotide preparative chemistry mixture is then concentrated and the final bis-amidate prod shown on page 3955 of Mazur et al. The pyridinium salt of uct is recovered and purified by conventional methods. the compound of this invention (without free hydroxyl An alternative reaction suitable for synthesizing most groups) is condensed with the free 5' or 3' end of the amidate compounds is to convert the phosphonate to the 50 otherwise protected oligonucleotide in the same way Mazur corresponding chloridate by reaction with thionyl chloride in et al. condense phosphonate 12 with a second nucleoside solvent (DMF) as described in EP 481 214. An amino acid, unit using DCC in dry pyridine in the presence of DoweX 50. dipeptide or other molecule bearing a free amine is then After reaction by either method, the oligonucleotide is reacted with the chloridate to yield the corresponding mono separated from the matrix (if present during the addition of amidate (where the phosphonate is cyclized) or bis-amidate. 55 the compound of this invention) and deprotected. Synthesis of compounds of structure (1) having amino Alternatively, the compounds of this invention are chemi acids that contain amino, guanidino or carboxyl groups cally converted to nucleotide triphosphate analogues. This is (such as lys, arg, his, asn, gln, lys-lys, arg-arg, lys-arg and accomplished using known reactions, for example reaction the like) is accomplished by the same method, but using of the phosphonate with tris(tri-n-butylammonium) pyro protected amine or carboxyl groups. After synthesis of the phosphate in DMF. The resulting deoxyNTP or NTP ana protected amidate compound, the protecting groups are logue (where R' or R' are unprotected hydroxy) is incor removed by conventional methods. Suitable protecting porated enzymatically into the oligonucleotide using ligase groups are well known (see supra) and include acid labile or polymerase. groups such as p-tosyl, BOC (t-butoxycarbonyl) and FMOC Esters. Hydroxy esters, especially at the phosphonate, are (fluorene methoxycarbonyl) for protecting amine groups. 65 generally synthesized as known in the art or as shown below. Groups such as t-butyl, methyl, ethyl, benzyl and the like For instance, see the methods described in EP 481 214 or can be used to protect carboxyl groups. These groups are Mukaiyama, T. et al. “J. Am. Chem. Soc." 94:8528–8532 5,750,729 SS 56 (1972) . Dialkyl phosphonate esters are synthesized via particularly useful for preparing acyloxyalkyl esters by conversion of a dichlorophosphonate chloridate (Quast. H. treatment of the cyclic compound with the corresponding et al. "Synthesis" 7:489-490 (1974); Quast. H. et al. "Syn acyloxyalkylhalide. In an exemplary method for the prepa thesis" 7:490 (1974); Moedritzer, K. et al., “Synth. Reac. ration of acyloxyalkyl esters of the cyclic compounds, Inorg. Met-Org. Chem." 5:417-27 (1974); Stowell, M. H. triethylamine and RC(O)OCH2Cl are reacted with the B. et al. "Tet. Lett." 31:3261–3262 (1990)) to a correspond cyclic compound. The stoichiometric proportion of triethy ing dialkylester (or dialkylamide) by reaction with alcohols lamine: RC(O)OCH.Cl: cyclic compound may be selected (or amines). Monoalkylesters (or mono alkylamides) are to be 1-2:1-2:1. Use of such low proportions of reactants obtained by hydrolysis of the disubstituted phosphonate in may lessen side reactions with any exocyclic amino group of base (NaOH, KOH and the like). Disubstituted diacyloxy B and thereby greatly improve yields. alkyl phosphonates are obtained by reaction of the unsub Each of the following schemes exemplify compound (30) stituted phosphonate with a substituted chloromethyl ester as the nucleotide analogue. However, any B is employed in (RC(O)OCH(R)Cl). A corresponding monosubstituted place of cytosine, provided that any exocyclic oxo or amino acyloxyalkyl phosphonate is obtained by hydrolysis in acid groups are protected as required. Also, step 3 of scheme 4 or base. 15 will be omitted when B contains no exocyclic amine. Bis esters having R groups such as aryl, substituted aryl, alkaryl or substituted alkaryl (such as phenyl, alkoxyphenyl. Scheme 4 benzyl, alkoxybenzyl) are also synthesized by reaction of the phosphonic acid with thionyl chloride and a catalytic NH amount of DMF in a solvent such as acetonitrile. The resulting dichloridate is then reacted with about 4, 5 or 6 N 1 equivalents of the sodium or potassium alkoxide or a sodium or potassium aryloxide obtained from reaction with sodium oes N hydride or potassium hydride and the alcohol (such as phenol, benzyl alcohol and the like) in a solvent such as THF 25 9Ye° NoH or acetonitrile at a reduced temperature (below about -70 C., preferably about -76° C. to -78° C.). The internally cyclized analogues having structures (2), (3) and (4) are prepared by a number of suitable methods from the free hydroxy phosphonic acid. These methods 30 (45) include treatment with DCC in DMF, reaction with Wils 1) DCC or meier's reagent (CICH=N(CH),Cl), or methods of phos 2) (a) Vilsmeier's Reagent phate activation known per se. In one embodiment of this (b) hydrolysis invention for the preparation of the corresponding cyclized H compound from the parental phosphonate nucleotide, the 35 phosphonate is (a) treated with CICH=N(CH)Cl to yield NH a NCY the phosphonylchloridate and (b) optionally the phospho nylchoridate is reacted with a nucleophile (preferably at low temperature, e.g. lower than about -20° C.) such as an r r alcohol or amine to produce one of the intermediates 4N N oH 9 es N o described above. In a further step the product of steps (a) or f O (b) is subject to hydrolysis or protonolysis (typically acid o P-OH\ 1) Wilsmeier's OncPa protonolysis) respectively to yield the cyclic compound. OH Reagent G Vilsmeier's reagent is advantageously produced in situ by combining SOCl PCls, POCl, COCl or the like with 45 DMF. Advantageously, the product of step (a) is not purified or separated from the reaction mixture before being reacted (44) (46) with the nucleophile, a distinct economic advantage for this 2) Nucleophile synthetic route. (Nul) Internally cyclized dihydroxy compounds of this inven tion where the remaining Y group is an ester or amide H typically are made by reacting the appropriate cyclic com a 1. pound with SOCl/DMF to yield the activated phosphonyl NH NC chloride (see scheme 4), followed by treatment with the corresponding nucleophile (e.g. alkoxide, phenolate, amine, 55 etc.) to yield the protected intermediate formamidine which r r is subsequently hydrolyzed to the target compound (48). es N es N Alternatively, esters can also be prepared as depicted in ye9 re' scheme 5. The N-O- protected intermediate phosphonate OnN (3) Protonolysis O YNu diester is obtained. The N- and O- protecting groups are subsequently removed followed by treatment of the phos phonate diester with NaH leading to cyclization yielding target compound. A third method for the synthesis of esters of the cyclic compounds entails alkylation of the cyclic (48) (47) compounds using common alkylating agents RLv (where 65 Lv is a leaving group) such as alkyl halides, tosylates, diazoalkanes and the like (see scheme 6). This method is 5,750,729 57 58 Scheme 5 Mixed bis amidate synthesis. Synthesis of compounds of structure (1) where both Y are amino acids or where one Y NHz NH is an amino acid and the other is an amine (NH2, NHR'. N(R)) is accomplished by direct conversion as described N e N a above for bis amidates followed by separation of the final products. Another method to synthesize mixed bis amidates is amidation of an appropriate phosphonate monoester. es -OTf f es - OTr f followed by removal of the ester group under conditions that do not remove the first amide. Synthesis of phosphonate P-ORW P-ORV monoester compounds has been described (EP 481 214). oR3 NH4OHEtOH OR3 O Such compounds are then converted to the mixed bis amide by condensation with a second amino acid or amine as described (i.e., using a 1:1 mixture of triphenylphosphine (49) (50) and 2,2'-dipyridyl disulfide) to yield the final product. 5 Mono amidate synthesis. Synthesis of compounds of 8O, AcOH structure (1) where one Y is an amino acid and the other is OH is accomplished essentially as described for bis amidate NH NH synthesis. Cyclic phosphonates are prepared by direct dehy dration of the corresponding dihydroxy nucleotide analogue using DCC (dicyclohexylcarbo-diimide) or 4-morpholino 20 N,N'-dicyclohexylcarboxamide as described (Ho et al. O r alsr “Mol. Pharmacol." 41:197-202 (1992)). The cyclic phos N O N OH af phonate is condensed with an amino acid ester in the O pé NaHADMF o P-OR presence of a 1:1 mixture of triphenylphosphine and 2,2'- ors (NaHDMF 'or' 25 dipyridyl disulfide in a suitable solvent such as pyridine or DMF, Protected heterocyclic base compounds. The present invention includes nucleotide analogues that comprise a (52) (51) protected heterocyclic base. These compounds are useful as synthetic intermediates and/or, as antiinfective or antiviral 30 agents per se. Exocyclic amines of the compounds of this invention are Scheme 5 protected by reacting the nucleotide analogue with RC(O) NH NH C1 or (CHO),CHR. The exocyclic amine groups such as the N-amine of cytosine, the N-amine of adenine and the 1 1 35 N-amine of guanine are protected. Suitable methods are essentially as described (Gilliam. "Anal. Biochem." 157:199 (1986); Gallo-Rodriguez. "J. Med. Chem." 37:636 (1994); Maillard, "J. Pharm. Sci.” 83:46 (1994)). The exemplary reaction schemes used to synthesize pro tected and cyclized heterocyclic base compounds shown below utilize compound (30) where B=cytosine. NHC(O)R3 45 - 1) SOC/DMF S (52)-ESR3COC N 2 Methods for linking cholesteryl, saccharide and other (44) 2) RoNa! (52)- it 4. moieties to reactive groups have been described (Hadfield, O N O "Adv. Pharmacol. Chemother.” 20:21 (1984); Gouyette, O “Tet. Lett." 30:6019 (1989); Ksander, “J. Med. Chem." 50 Y 37:1823 (1994), and are adapted here to substitute such Y NoR3 moieties into the phosphonate). Bis esters are converted to monoesters by chemical hydrolysis in base or acid according to the bis ester used. For (55) VE example, treatment with NaOH (0.5 to 2N) or NHOH in a NHC(OR3 solvent such as THF (tetrahydrofuran), dioxane or an alco 55 hol for 1 to 24 hours at 22° to 90° is suitable for most esters. (52). TMSCDMF 1 The choice of solvent will depend on the characteristics of 2) RCOCLPyridine the bis ester used. The hydrolytic stability of the phospho 3)H3Ot 4N O N O nate bis esters is unequal and provides a means for obtaining O the monoester. Selection of hydrolysis conditions is deter M mined by routine testing. Alkaline hydrolysis yields the Y phosphonate monoester and a corresponding alcohol or YoH phenol. Other Y groups are then linked to the monoester using reagents and conditions (i.e., a 1:1 mixture of triph (56) enylphosphine (PPh) and 2,2'-dipyridyl disulfide in a suit 65 able solvent such as pyridine or DMF) essentially as wherein R is as defined above. Either procedure is readily described for synthesis of bis amidates. adapted to compounds in which the heterocyclic base is, for 5,750,729 59 60 instance, adenine, guanine, 2,6-diaminopurine or -continued 2-aminopurine where an exocyclic amine is linked to the NHC(O)R3 base. The cyclic esters may comprise a single isomer or a scalemic mixture at the phosphorus atom. Low temperature N reaction conditions (lower than about -20°. e.g., about -20° to about -40°C. or about -40° to about -80 C.) may tend es N O to favor single isomer products, while reaction at higher O A temperatures (above about -20°, e.g. -20° to 40°C.) may Y result in a scalemic mix. The pivaloyloxymethyl ester syn N thesis may yield a scalemic mixture at the phosphorus atom. O OCHOC(O)R When and if a scalemic mixture is obtained, the isomers are (57) V. conveniently separated by HPLC, although the mixture can be used, for example, as a synthetic intermediate or as an active antimicrobial agent, without resolution at the 6 site. wherein R is defined above. A method to obtain heterocyclic bases comprising the 15 The amine protecting group =CRN(R) is incorporated C(O)R protecting group is accomplished as follows using into an exocyclic amine to yield protected heterocyclic base the acyl chloride (RC(O)Cl) using compound (30) as an compounds as follows example N=CRN(R3) (R-NCR'(OMe); NHC(O)R (52) A rose 1 (50) Es N als N O O O N OTr O 25 y/ A. N O P-OR3 OR3 OR3 (58) V (57) V. Exemplary R alkyl groups include methyl, ethyl, propyl. 1) Detritylation isopropyl, cyclopropyl, butyl, isobutyl and cyclobutyl. In 2) TMSBr general, both Ralkyl groups will be the same. The reaction can be carried out in dry DMF at room temperature (about NHC(O)R3 35 20°-30° C.) as previously described (Kerr et al. "J. Pharm. Sci.” 83:582 (1994); Kerr et al. "J. Med. Chem.” 35:1996 N (1992)). or DMF can be substituted with CHCN and 4 A molecular sieves. Exemplary compounds include species where R is hydrogen, alkyl (including ethyl, propyl. O N OH O M isopropyl), aryl (including phenyl) or acyloxymethyl. Pro O P-OH tected heterocyclic bases where R is hydrogen are stable under neutral anhydrous conditions and are generally labile OH under acidic aqueous conditions. When R is methyl, the (58) V 45 protecting group is more stable to aqueous acidic or basic 1) Detritylation conditions. 2)NaH/THF DCC/Morpholine Pyridine Compounds containing a protected heterocyclic base and 1 or 2 amino acids, dipeptides or oligopeptides attached to the phosphorus atom via an amidate linkage are obtained as (55) TMSBre (56) 50 described for synthesis of bis-amidate or amidate-ester com pounds. wherein Tr is the hydroxyl protecting group trityl. The Table 7 lists Rester and Y amidate moieties that can be detritylation step is accomplished by acid treatment, such as bonded via oxygen or directly, respectively, to the phospho 80% acetic acid at about 20° to 80° C. for 4-24 hours. The 55 rus atom. Esters of structures 1-5, 8-10 and 16, 17, 19-22 R. moiety is removed using a Lewis acid such as TMSBr to are synthesized by reacting a nucleotide analogue having a yield the free phosphonate. free hydroxyl with the corresponding halide (chloride or Phosphonate compounds comprising a C-Co 1-acyloxy acyl chloride and the like) and N N-dicylohexyl-N- 1-alkyl or a C-C 1-acyloxy-1-alkyl-1-aryl ester group are morpholine carboxamidine (or another base such as DBU. prepared as follows triethylamine. CsCO, N,N-dimethylaniline and the like) in DMF (or other solvent such as acetonitrile or 6 R3-C(O)-O-CHCI N-methylpyrrollidone.) Esters of structures 5-7, 11, 12, 21, (56) TBA or N,N-Dicyclohexyl G and 23–26 are synthesized by reaction of the alcohol or -N-morpholine alkoxide salt (or the corresponding amines in the case of carboxamidine 65 compounds such as 13, 14 and 15) with the monochloro DMF phosphonate or dichlorophosphonate or another activated phosphonate. 5,750,729 61 62 To the extent any compound of this invention cannot be TABLE T produced by one of the foregoing schemes other methods -CH-CO)-N(R')* will be apparent to the artisan referring to conventional . -CH-S(O)(R') methods (see for instance Liotta et al. "Compendium of . -CH-S(O)(R) Organic Synthesis Methods” (John Wiley & Sons. New . -CH-O-C(O)-CH-CH 3-cholesteryl York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; 3-pyridyl Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, N-ethylmorpholino Louis S. Hegedus and Leroy Wade. 1977; Vol. 4, Leroy G. -CH-O-C(O)-CH -CH-O-C(O)-CHCH 10 Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 1. -CH-O-C(O)-C(CH) 6, Michael B. Smith; March, J., “Advanced Organic . -CH-CC Chemistry. Third Edition", (John Wiley & Sons, New York, ... -CH -NH-CH-CO)O-CHCH 1985); as well as “Comprehensive Organic Synthesis. -NCE)-CHCOO-CHCH, Selectivity, Strategy & Efficiency in Modern Organic Chem 15 istry. In 9 Volumes", Barry M. Trost, Editor-in-Chief . -CH-O-C(O)-CHis . -CH-O-C(O)-CH(CH) (Pergamon Press, New York, 1993 printing). , -CH-C#H(OC(O)CHR). CH-(OC(O)CHR)* All citations are hereby expressly incorporated by refer 19. ence. The following examples are illustrative and do not -CHC(O)N O limit the scope of this invention.

2O. EXAMPLE 1. 25 N Synthesis of Diisopropyl (4,6-di-O-acetyl-2,3- O H dideoxy-o-and -3-D-erythro-hex-2-enopyranosyl) 21. H phosphonate (22) 30 O

OHHO (21)-(22) HO 35 22. N To solution of (2.72 g.10 mmol) of 3,4,6-tri-O-acetyl1.2- dideoxy-D-arabino-hex-1-enopyranose (3,4,6-tri-O-acetyl -CH-O-C(O) -() D-glucal) and (3.12 g. 15 mmol) of triisopropylphosphite in 23. N dry toluene (20 ml) was added boron trifluoride etherate (0.18 ml, 1.5 mmol). The mixture was heated to 100° C. for 4 h. The solvent was removed, residue was codistilled three times with toluene (50 ml), and purified by column chro matography on silica gel, eluting with hexane-ethyl acetate 45 4:1 to afford (22) as a colorless oil (3.145g, 83%). (Modified procedure from H. Paulsen. "J. Thiem: Chem. Ber." -) 106:3850-3876 (1973).) 50 25. CHCHC(O)O EXAMPLE 2

55 Synthesis of Diisopropyl (2,3-dideoxy-o-and-f-D- 26 OCH erythro-hex-2-enopyranonate (23)

-CH -- OCH (22)-(23) OCH -Each R' is the same or different (includes methyl, ethyl, propyl, isopropyl Compound (23), (3.79 g, 10 mmol) was dissolved in and t-butyl). saturated solution of NH in MeOH and stirred at ambient i-chiral center is (R), (S) or racemate. 65 temperature for 6 h. The solvent was removed and the Other esters that are suitable for use herein are described residue codistilled with toluene. Drying afforded (23), 2.94 in EP 632,048. g as a mixture of o. and B anomers 1:1 (100%). 5,750.729 63 EXAMPLE 3 EXAMPLE 5 Synthesis of Diisopropyl-(2,3-dideoxy-6- triphenylmethyl-O-D-erythro-hex-2-enopyranosyl) phosphonate (24a) and Diisopropyl (2,3-dideoxy-6- Synthesis of Diisopropyl 4-(cytosin-1-yl)-2,3- O-triphenylmethyl-B-D-erythro-hex-2- dideoxy-6-O-triphenyl-methyl-B-D-threo-hex-2- enopyranosyl) phosphonate (24b) enopyranosyl phosphonate (25")

OTr OTr 10 NH (25')-Ge N . (23) - G f -- HO W/ Po-C) HO W es N OTr O 15 (24a) (24b) '. Fo-C).

To a solution of compound (23), (2.94 g. 10 mmol) in - toluene (50 ml) was added triphenylmethylchloride (2.79g. 20 10 mmol) and pyridine (3 ml). Mixture was stirred at ambient temperature for 24 h. Toluene was added (150 ml) and the mixture was extracted with 1M hydrochloric acid, a saturated solution of NaHCO and water. The organic phase Compound (25') (0.557 g. 0.76 mmol) was treated with was dried (MgSO), filtered, and evaporated to leave an oil 25 saturated solution of NH in MeOH (20 ml) for 16 h at which was purified by column chromatography on silica gel, ambient temperature. The solvent was removed and product eluting with hexane-ethyl acetate (5:1. 3:1) to give pale was purified by silica gel chromatography to give (25") (0.38 yellow oils of (24a) ((or anomer, 2.09 g. 39%) and (24b) (B g. 80%): 'H NMR (CDC) & 1.40 (m. 12H), 3.32 (m. 1H). anomer, 2.46g, .46%); (24a): 'HNMR (CDC1) & 6 1.28 (m. 30 3.48 (m. 1H). 4.04 (m. 1H), 4.72 (d. J=17.5 Hz1H), 5.12 (m. 12H), 3.40 (m.2H), 3.53 (m.1H). 4.22 (m. 1H), 4.53 (d. 2H). 5.65 (m. 1H), 5.82 (d.J-7.1 1H), 6.20 (m. 1H), 6.30 (m, J=17.5 Hz, 1H), 4.76 (m. 1H), 5.94 (.2H), 7.28 (m. 15H). (24b): "H NMR (CDC) 81.32 (m. 12H), 3.27 (m. 1H).3.35 1H), 7.1-7.6 (m. 15H), 7.83 (d, J-7.1 1H). (m. 1H). 4.07 (brs, 1H), 4.21 (m. 1H), 4.42 (m. 1H), 4.77 (m, 1H), 5.88 (m. 1H). 6.02 (m, 1H), 7.28 (m. 15H). 35 EXAMPLE 6 EXAMPLE 4 Synthesis of Diisopropyl (4-(N-benzoylcytosin-1- yl)-2,3-dideoxy-6-O-triphenylmethyl-B-D-threo-hex 40 Synthesis of Diisopropyl 4-(cytosin-1-yl)-2,3- 2-enopyranosyl) phosphonate (25') dideoxy-3-D-threo-hex-2-enopyranosyl) O phosphonate (29) 45 NH (24b) - G) (25")-Go N Gr / \ OTr o, on y- N/ P(O-C), 50 o O P(O-C), (25)

- (29) To a 0° C. stirred solution of triphenylphosphine (0.786 g. 55 3 mmol) in anhydrous THF (20 ml) under dry nitrogen atmosphere, was added diethylazodicarboxylate (0.522 g. 3 mmol). After the mixture was stirred for 30 min, compound (24b) was added followed by N-benzoylcytosine. After the Compound (25") (0.38 g. 0.6 mmol) was dissolved in mixture was stirred at 0° C. for 24 h. the solvent was 80% acetic acid and stirred at ambient temperature for 16 h. removed and the residue was purified by column chroma The solvent was removed and product isolated by silica gel tography on silica gel (CHCI-MeOH) to give (25') (0.557 chromatography to give (29) (0.195 g, 83%): "H NMR g, 38%): "H NMR (CDCl) 8 139 (m.12H), 3.14 (m. 2H). (CDC1,) 8 135(m. 12 H). 3.28 (m. 1H), 3.56 (m. 1H), 3.88 3.92 (m, 1H), 4.62 (m, 1H), 4.85 (m.2H), 5.53 (m. 1H), 6.05 65 (m. 1H), 4.58 (d. J=18 Hz, 1H), 4.77 (m, 2H), 5.40 (m. 1H), (m. 1H), 6.42 (d, J=7.2 1H), 7.10-7.60 (m. 20H), 7.92 (d. 5.79 (d, J-7.2 Hz, 1H), 6.04 (m. 1H), 6.34 (m. 1H), 7.98 (d, J=7.2 1H). J=7.2 Hz, 1H). 5,750.729 6S 66 EXAMPLE 7 EXAMPLE 9

Synthesis of 4-(cytosin-1-yl)-2,3-dideoxy-B-D- 5 Synthesis of 4-(cytosin-1-yl)-B-D-threo threo-hex-2-enopyranosylphosphonic acid (30") hexopyranosylphosphonic acid (32)

NH NH (29) - Ge N t O (31') - Ge N 1. N OH o oss on P(OH), FOH), 15

(30) (32)

20 To a solution of (29) (0.195 g, 0.5 mmol) in acetonitrile (5 ml) was added 2.4-lutidine (0.6 ml. 5 mmol) and trim To a solution of (32) (0.195 g, 0.49 mmol) in acetonitrile ethylbromosilane (0.66 ml, 5 mmol) and the mixture was (5 ml) was added 2.4-lutidine (0.6 ml, 5 mmol) and trim stirred for 24 h. Solvent was evaporated and the dry residue ethylbromosilane (0.66 ml, 5 mmol) and the mixture was was coevaporated with methanol (10 ml). The residue was 25 stirred for 24 h. Solvent was evaporated and dry residue was dissolved in water (5 ml) and applied on DoweX 1 AcO coevaporated with methanol (10 ml). The residue was dis form) (20 ml), eluting with water (300 ml) and 0.1M acetic solved in water (5 ml) and applied on Dowex 1 (AcO-form) acid. Product-containing fractions were collected, evapo (20 ml), eluting with water (300 ml) and 0.1 M acetic acid. rated and the residue was precipitated from H2O-acetonitrile Product containing fractions were collected, evaporated and to give white precipitate of (30) (0.136 g, 90%): mp residue was precipitated from H2O-acetonitrile to give a 250°-300° C. decomp.; H NMR (CDC) 83.50-3.74 (m. white precipitate of (32) (0.136 g.90%): mp 217° C.; "H 2H), 4.01 (m. 1H), 4.58 (dd J=18.0, 1.83 Hz, 1H), 5.29 (m, NMR (CDC1) & 1.71 (m. 2H), 2.00 (m. 2H), 3.49 (m. 2H), 1H), 5.96 (m. 1H), 6.18 (d. J-7.9 Hz, 1H), 6.53 (m. 1H), 3.73 (m. 1H), 3.91 (m. 1H), 4.59 (m. 1H), 6.05 (d. J=8.0 Hz, 8.26 (d.J-7.9 Hz, 1H); HRMS,304,0692 (M+H -glycerol), 35 1H), 8.51 (d.J-8.0 Hz, 1H). HRMS. 306.0859 (M-H calcd for CHNOP304,0698. -glycerol), calcd for CHNOP306.0855.

EXAMPLE 8 EXAMPLE O

Synthesis of Diisopropyl 4-(cytosin-1-yl)-2,3- dideoxy-B-D-threo-hexopyranosyl phosphonate Synthesis of Diisopropyl (4-(N-benzoylcytosin-1- (31') yl)-2,3-dideoxy-6-O-triphenylmethyl-O-D-threo 45 hex-2-enopyranosyl phosphonate (26) NH O

(29) - G NH or -oh , 50 (24a)-se N o P(O-C), Or es N O

55 (31') Po-C),

(26) Compound (29) (0.195 g, 0.5 mmol) was dissolved in 60 methanol (10 ml), catalytic amount of 10%Pd/C was added and mixture was hydrogenated by H. under 200 Psi for 4 hours. Then mixture was filtered through celite and evapo Starting from compound (24a) and repeating the process of ration afforded 0.195 g of (31') as a white solid: "H NMR example 4, compound (26) was prepared: "H NMR (CDCl3) (CDC1) 8 1.27 (m. 12 H), 1.75 (m. 2H), 2.00 (m. 2H), 3.49 65 8 1.38 (m, 12H), 3.15 (m. 2H), 3.95 ((m, 1H), 4.62 (m. 1H), (m. 1H), 3.67 (m. 1H), 3.88 (m, 2H), 4.76 (m. 2H), 4.97 (m. 4.85 (m. 2H), 5.52 (m. 1H), 6.08 (m.2H), 6.40 (d. J-7.8Hz 1H), 5.71 (m, 1H), 8.14 (m. 1H). 1H), 7.1-7.4 (m. 20H), 7.92 (d. J=7.8 1H).

5,750,729 69 70 Starting from compound (59) and repeating the proce (m. 1H), 4.21 (m. 1H), 5.04 (d. J-7.8 Hz 1H), 5.76 (m. 1H). dures from examples 1 to 3, and from 10 to 13, compound 6.06 (d. J=7.8 Hz 1H), 7.54 (d. J=7.8 Hz 1H); HRMS. (61) was prepared: mp 290° C.; H NMR (CDC) 82.14 (m. 288.0754 (M-H -glycerol), calcd for CHNOP 1H), 2.91 (m. 1H), 3.53 (m. 1H).3.72 (m, 1H), 4.08 (m. 1H), 288.0749. 5.12 (d.J-8.1 Hz 1H), 5.76 (m. 1H), 6.05 (d. J=7.5 Hz 1H), 5 7.56 (d. J=7.5 Hz 1H).

EXAMPLE 6 EXAMPLE 8 Synthesis of Diisopropyl 23,6-trideoxy-o-and-B-L- erythro-hex-2-enopyranosylphosphonate (63) 10

O O Synthesis of 4-(adenine-9-yl)-2,3-dideoxy-D-threo AcO O HO I hex-1-enopyranosyl phosphonic acid (28") P(O-C) A NH OAC 1 N W (62) (63) (24a) - G Cy OH N O O Starting from 34-di-O-acetyl-1,2,6-trideoxy-L-arabino hex-1-enopyranose (3.4-di-O-acetyl-6-deoxy-L-glucal) (62) A. P(OH) and repeating the procedures from examples 1 and 2 com pound (63) was made as a mixture of O. and B anomers: "H (28") NMR (CDC1) 81.25 (m. 15H). 3.32 (m. 1H), 3.82 (m. 1H). 25 4.38 (m. 1H), 4.72 (m. 2H), 5.98 (m. 2H). EXAMPLE 17 Synthesis of 4-(cytosin-1-yl)-2,3,6-trideoxy-3-L- Starting from compound (24a) and using the procedures threo-hex-2-enopyranosylphosphonic acid (64) and 30 from examples 4, 5, 6 and 7 (except instead of 4-(cytosin-1-yl)-23.6-trideoxy-L-threo-hex-1- N-benzoylcytosine. N-benzoyladenine was used) com enopyranosylphosphonic acid (65) pound (28") was prepared: "H NMR (CDC) 82.43 (m, 1H), 3.08 (m. 1H), 3.33 (m, 1H), 3.61 (m. 1H), 4.34 (m. 1H). 35 5.34 (m. 1H). 5.35 (d. J=6.84 Hz, 1H). 5.87 (n, 1H), 8.27 (s, 1H), 8.45 (s. 1H).

EXAMPLE 19

Synthesis of 4-(adenine-9-yl)-2-3,6-trideoxy-L- 45 threo-hex-1-enopyranosylphosphonic acid (65') O O.

50 N (-oil (63)-> ?? A. N Sa N (65')

55 NH

Starting from mixture of anomers of compound (63) and repeating procedures from examples 4.5, and 7, compounds (64) and (65) were prepared. The ol, and B anomers were 60 Starting from compound (63) and using the procedures separated after deprotection step (example 5) by silica gel from examples 4, 5, and 7 (except instead of chromatography: (64); mp 288°C.; H NMR (CDCl)81.03 N-benzoylcytosine, N-benzoyladenine was used) com (d. J=6.54 Hz, 3H). 4.03 (m. 1H), 4.50 (d. J-1758 Hz 1H). pound (65') was prepared: mp 151° C.: "H NMR (CDC) 8 5.12 (m. 1H). 5.94 (m, 1H), 6.04 (d. J=7.3 Hz 1H), 6.43 (m. 1.00 (d.J-6.35 Hz 3H), 2.37 (m. 1H), 2.99 (m. 1H), 4.37 (m, 1H), 8.05 (d. J-7.3). HRMS, 288,0756 (M+H -glycerol), 65 1H), 4.99 (d. J=6.84 Hz 1H), 5.78 (m. 1H), 8.10 (s, 1H), 8.24 calcd for CHNOP 288.0749. (65): mp 285° C.; "H (s, 1H) HRMS. 312.0862 (M+H -glycerol), calcd for NMR (CDC1) 8 1.16 (d, J-6.3 Hz 3H), 2.20 (m, 1H), 2.88 CHNOP312,0861. 5,750.729 71 72 EXAMPLE 20 EXAMPLE 21 Synthesis of 4-(thymin-1-yl)-23.6-trideoxy-O-L- threo-hex-2-enopyranosyl phosphonic acid (66) 4 Compounds made in the foregoing examples were (thymin-1-yl)-2,3,6-trideoxy-B-L-threo-hex-2- assayed for inhibitory effect against human CMV using the enopyranosylphosphonic acid (64") and 4-(thymin standard plaque inhibition assay (Lurain. "J. Vir.” 1-yl)-2,3,6-trideoxy-L-threo-hex-1- 66:7146 7152 (1992)). The cells used for the toxicity assay enopyranosylphosphonic acid (65") were normal human dermal fibroblasts. The results are shown in Table 8 below.

O O P(OH) TABLE 8 Compound Tested ICs (IM) CCso (M) (30) 10 >1000 O N (28) 40 >1000 15 (32) 8O >1000 (63)-> 51 (64) 200 >1000 HN (65) 300 >1000 O (66) O A related known compound, HPMPC, was more potent but also more toxic than the best compound of the invention O tested here, exhibiting an ICso of 0.5 micromolar and a CCo -W P(OH)2 of 200 micromolar. O N The claims shall be construed to exclude any subject s 25 matter that, at the date of the invention, would not have been HN patentable under applicable statutory and judicial authority. We claim: O (64") 1. A method comprising O O Poh, (a) providing a compound having structure (I) A Z (I) O N O s PRTO HN 35 A O (65") OPRT (b) reacting the compound of structure (I) with P(OPRT) Starting from compound (63) and using procedures from in the presence of a Lewis Acid; and examples 4, 5, and 7 (except instead of N-benzoylcytosine, N-benzoylthymine was used). The mixture was separated (c) recovering from the reaction mixture of step (b) a after deprotection of base (example 5) and each isolated compound having structure (II) compound was deprotected by trimethylbromosilane (example 7): (66): "H NMR (CDC) 8 1.00 (d. J=6.59 Hz Z. (II) 3H), 1.86 (s.3H), 3.92 (m. 1H), 444 (m. 1H), 4.97 (m, 1H), 45 O O 5.82 (m. 1H), 6.53 (mm. 1H), 7.41 (s, 1H). (64'): 'H NMR (CDCl) 8 1.06 (d. J=5.86 Hz3H), 1.87 s.3H).3.99 (m. 1H). PRTO P(OPRT) 4.41 (m. 1H), 4.99 (m. 1H). 5.86 (m. 1H). 648 (m. 1H), 7.38 (s. 1H); HRMS 325.0578 (M-H -glycerol), calcd for CHNONaP325.0565. (65"): "H NMR (CDC) 81.17 50 wherein PRT is a protecting group and Z is CHOPRT, halo (d. J=1.17 Hz 3H), 1.88 (d, J-0.99 Hz 3H), 2.16 (m. 1H), substituted C-C alkyl, CH=CH CECH, -CHN 2.83 (m, 1H), 4.16 (m. 1H), 4.91 (m. 1H), 5.61 (m. 1H), 7.42 CH, or H. (d, J=0.98 Hz 1H); HRMS325.0573 (M+H -glycerol), calcd for CHNONaP325.0578.