CARBOCYCLIC C-NUCLEOSIDES DERIVED FROM FORMYCIN Except where reference is made to the work of others, the work described in this dissertation is my own or was done in collaboration with my advisory committee. This dissertation does not include proprietary or classified information. _______________________ Mingzhu He Certificate of Approval: Holly Ellis Associate Professor Chemistry and Biochemistry Stewart Schneller, Chair Professor Chemistry and Biochemistry Peter Livant Associate Professor Chemistry and Biochemistry Edward Parish Professor Chemistry and Biochemistry George T. Flowers Dean Graduate School CARBOCYCLIC C-NUCLEOSIDES DERIVED FROM FORMYCIN Mingzhu He A Dissertation Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Auburn, Alabama December, 19, 2008 iii CARBOCYCLIC C-NUCLEOSIDES DERIVED FROM FORMYCIN Mingzhu He Permission is granted to Auburn University to make copies of this dissertation at its discretion, upon requests of individuals or institutions and at their expense. The author reserves all publication rights. __________________________________ Signature of Author __________________________________ Date of Graduation iv For my parents Hong He and Linqing Cao v DISSERTATION ABSTRACT CARBOCYCLIC C-NUCLEOSIDES DERIVED FROM FORMYCIN Mingzhu He Doctor of Philosophy December, 19, 2008 (M. S., XiaMen University, China, 2003) 140 Typed Pages Directed by Stewart W. Schneller Analogs of naturally occurring nucleosides have served as structural models for the design of antitumor, antiviral, and antibacterial agents. Some modified nucleosides have become major therapeutic agents for the treatment of viral infectious diseases such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and the herpes viruses. Carbocyclic nucleosides (carbanucleosides) are nucleoside analogs in which the oxygen in the furanoses of traditional nucleosides is replaced by a methylene moiety. One relevant feature of these derivatives is their metabolic stability against phosphorylases as a consequence of the absence of the natural N-glycosidic bond. Much like carbanucleosides, C-nucleosides represent another class of nucleosides resistant to chemical and the enzymatic hydrolytic cleavage of the glycosidic bond. This results from the structural arrangement in which the ribofuranosyl moiety is liked to a heterocyclic base through a C-C bond rather than the traditional C-N bond. Recently, nucleoside analog discovery has been focused on the hybrid nucleosides, carbocyclic C-nucleosides. This has been due to the challenging enantiomeric syntheses posed by carbocyclic C- nucleosides. Consequently, no significant biological activities for this class of compounds have been reported. Thus, it was of interest to undertake a study of carbocyclic C- nucleosides. The main body of this research work deals with the progress towards carbocyclic formycin analogs as representative carbocyclic C-nucleosides by combining the structural components of formycin and 5'-noraristeromycin, a broadly active antiviral candidate that is devoid of the toxicity of aristeromycin. Synthesis of related N-methylated carbocyclic formycin analogs and their structural assignments are also described. vi vii ACKNOWLEDGMENTS The project represented in this dissertation would not have been possible without the help and guidance of many people to whom I owe much gratitude. First and foremost, I would like to thank my research advisor Dr. Stewart W. Schneller for his guidance and support with valuable knowledge during this work. I also want to thank him for his progressive mentorship and his words of encouragement. My gratitude is extended to distinguished members of my committee including Drs. Holly Ellis, Peter Livant, and Edward Parish who each contributed to the dissertation process as well. I sincerely acknowledge the Schneller group members who provided advice, friendship and encouragement. Gratitude is also extended to Department of Chemistry and Biochemistry, and National Institutes of Health for their financial support. And finally, I am deeply indebted to my family in China for their encouragement, support and endless love. viii Style manual or journal used: The Journal of Organic Chemistry Computer software used: Microsoft Word 2003, CS ChemBioDraw Ultra 11.0 ? 2008 Mingzhu He All Rights Reserved ix TABLE OF CONTENTS LIST OF SCHEMES.......................................................................................................... xi LIST OF FIGURES..........................................................................................................xiv LIST OF TABLES..........................................................................................................xvi INTRODUCTION..........................................................................................................1 Antiviral Activity via Inhibition of S-Adenosylhomocysteine Hydrolase...............6 Carbocyclic Nucleosides........................................................................................10 C-nucleosides as antiviral agents...........................................................................15 Hybrid nucleosides--Carbocyclic C-nucleosides...................................................21 A MODEL STUDY TO N-METHYL CARBOCYCLIC FORMYCIN ANALOGS .......27 Synthesis of pyrazole derivative ............................................................................28 Synthesis of the key nitro nitrile intermediate .......................................................29 Synthesis of the final N-methyl carbocyclic formycin analogs .............................30 Structural assignments for N 1 -methyl- and N 2 -methylated products .....................33 ENANTIOSELECTIVE SYNTHESIS OF CARBOCYCLIC 5'-NORFORMYCIN........37 Exploring the key step toward target compound 22 ...............................................37 Attempts to synthesize the key epoxide..................................................................37 Attempts to synthesize the cyclopentyl alkyne from allylic monoacetate 39.........41 x Synthesis of the cyclopentyl alkyne from cyclopentenone....................................44 Key step for the synthesis of target compound 22.................................................50 Enantioselective synthesis of carbocyclic 5?-norformycin (22).............................56 Enatioselective Synthesis of N-1 and N-2 Methylated Carbocyclic 5'-Norformycin Analogs ..................................................................................................................60 Attempted Synthesis of N-2 methylated 5?-norcarbofomycin 72 ..........................66 FUTURE DIRECTION: PROPOSED SYNTHESIS OF ANOTHER CARBOCYLIC C- NUCLEOSIDE-PYRAZOMYCIN ANALOGS................................................................67 BIOLOGICAL RESULTS.................................................................................................69 CONCLUSION..................................................................................................................75 EXPERIMENTAL SECTION...........................................................................................77 REFERENCES ................................................................................................................110 xi LIST OF SCHEMES Scheme 1............................................................................................................................12 Scheme 2............................................................................................................................13 Scheme 3 General strategy for the synthesis of Carbocyclic C-nucleosides.....................22 Scheme 4 Synthesis of pyrazole ring.................................................................................28 Scheme 5 Synthesis of key nitro nitrile compound ...........................................................29 Scheme 6 mechanism of cine substitution.........................................................................30 Scheme 7 Introducing methyl group to pyrazole ring .......................................................30 Scheme 8 Synthesis of (?)-2..............................................................................................31 Scheme 9 Synthesis of (?)-1..............................................................................................32 Scheme 10 Methylated pyrazole ring?s stability................................................................32 Scheme 11 Synthesis of Compounds (?)-12 and (?)-13....................................................36 Scheme 12 Retrosynthesis of epoxide 24 ..........................................................................38 Scheme 13 Synthesis of diol 27.........................................................................................39 Scheme 14 Attempted synthesis of diene 26 .....................................................................40 Scheme 15 Synthesis of 9-deaza-5?-noraristeromycin.......................................................41 Scheme 16 Retrosynthesis from monoacetate ...................................................................42 Scheme 17 Synthesis of (+)-monoacetate 39.....................................................................43 Scheme 18 Attempted synthesis of compound 45 .............................................................43 xii Scheme 19 Examples using conjugative addition to 2-cyclopentenone 46 .......................44 Scheme 20 Retrosynthesis from L-like-2-cyclopentenone 47...........................................45 Scheme 21 Synthesis of L-like-2-Cyclopentenone 47 from monoacetate 39....................45 Scheme 22 Route from Chu?s paper ..................................................................................46 Scheme 23 Synthesis of cyclopentyl protected L-like-2-cyclopentenone 53 ....................47 Scheme 24 General mechanism of Ring Closure Metathesis ............................................49 Scheme 25 Conjugate addition of acetylenic alane ...........................................................51 Scheme 26 Conjugate addition of alkynylboron reagents .................................................51 Scheme 27 Conjugate addition of trioctynylalumin reagent..............................................52 Scheme 28 Conjugate addition of mixed cuprate reagent .................................................52 Scheme 29 Nickel-catalyzed 1,4-addition of organoaluminum acetylide to S-trans enones ............................................................................................................................................53 Scheme 30 Key step for the synthesis of target compound ...............................................54 Scheme 31 Possible unwanted adol side reaction..............................................................55 Scheme 32 Synthesis of compound 65 ..............................................................................57 Scheme 33 Synthesis of nitronitrile compound 67 ............................................................58 Scheme 34 Displacement on the N-nitro group .................................................................59 Scheme 35 Synthesis of target compound 22 ....................................................................59 Scheme 36 Synthesis of methylated formycin derivatives ................................................61 Scheme 37 Methylation of 67............................................................................................62 Scheme 38 Possible reason for side product 79.................................................................62 Scheme 39 Synthesis of N-1 methylated 5?-norcarbofomycin 71......................................65 Scheme 40 Alternate route to synthesize 72......................................................................65 xiii Scheme 41 Synthesis of compound 72 ..............................................................................66 Scheme 42 Proposed synthesis of 85 and 87 .....................................................................68 xiv LIST OF FIGURES Figure 1 Naturally occurring nucleosides............................................................................1 Figure 2 Examples of coenzymes ........................................................................................2 Figure 3 Examples of Modified Nucleosides.......................................................................4 Figure 4 Metabolism of the anti-HIV agent AZT................................................................5 Figure 5 5?-capped structures...............................................................................................7 Figure 6 AdoMet metabolic cycle........................................................................................8 Figure 7 AdoMet/AdoHcy metabolism ...............................................................................9 Figure 8 Phosphorolysis of nucleosides.............................................................................10 Figure 9 Structures of Aristeromycin and Neplanocin A ..................................................11 Figure 10 Aristeromycin and neplanocin A analogs with base modified..........................13 Figure 11 Aristeromycin analogs with side chain modified ..............................................14 Figure 12 Naturally occurring C-nucleosides....................................................................15 Figure 13 Reduction of FTP by ribonucleotide reductase .................................................17 Figure 14 Metabolic conversions of formycin...................................................................18 Figure 15 N-methyl derivatives of formycin .....................................................................19 Figure 16 Structure of carbocyclic C-nucleoside...............................................................21 Figure 17 Examples of synthesized carbocyclic C-nucleosides ........................................22 Figure 18 Racemic Carbocyclic C-nucleosides .................................................................23 Figure 19 Exploring for the synthesis of Carbocyclic Formycin.......................................24 xv Figure 20 Synthesis of carbocyclic formycin analogs .......................................................25 Figure 21 Carbocyclic 5'-norformycin and its methylated versions ..................................26 Figure 22 (?)-1 and (?)-2 ...............................................................................................27 Figure 23 X-ray structure for compound (?)-8..................................................................31 Figure 24 Compounds (?)-12 and (?)-13...........................................................................35 Figure 25 ............................................................................................................................37 Figure 26 Grubbs Catalyst for RCM..................................................................................48 Figure 27 1 H NMR spectral data for compound 59 ...........................................................54 Figure 28 Chelation intermediate to 59 .............................................................................56 Figure 29 N-methyl carbocyclic 5?-norformycin analogs ..................................................60 Figure 30 Possible methylation sites of 22 ........................................................................61 xvi LIST OF TABLES Table 1 Selected 1 H NMR and 13 C NMR Spectral Data....................................................34 Table 2 comparative TLC Data for the N-methylated isomers..........................................34 Table 3 comparative TLC Data for the N-methylated isomers..........................................63 Table 4 Selected 1 H NMR and 13 C NMR Spectral Data....................................................64 Table 5 The spectrum of viruses to be assayed..................................................................69 Table 6 Antiviral Activity of Compounds against HSV-1, HSV-2, HCMV and VZV Based on Cytopathogenic Effect (CPE) Inhibition Assay .................................................70 Table 7 Antiviral Activity of Compounds against RSV A, Parainfluenza and SARS CoV Based on Neutral Red Visual Inhibition Assay .................................................................71 Table 8 Antiviral Activity of Compound 1 against Vaccinia Virus and Cowpox Virus Based on Cytopathogenic Effect (CPE) Inhibition Assay ................................................71 Table 9 Antiviral Activity of Compound 1 in Vero Cell Cultures ....................................72 Table 10 Antiviral Activity of Compound 1 in HEL Cell Cultures...................................72 Table 11 Antiviral Activity of Compound 1 against Cytomegalovirus in Human Embryonic Lung (HEL) Cells............................................................................................73 Table 12 Antiviral Activity of Compound 1 against Varicella-zoster in Human Embryonic Lung (HEL) Cells............................................................................................74 Table 13 Antiviral Activity of Compound 1 against HBV................................................74 INTRODUCTION Nucleosides are that involve binding a (a nitrogen heterocycle often referred to simply as a base) to a or ring. Examples include , , , , and (Figure 1). glycosylamines nucleobase ribofuranose deoxyribofuranose adenosine guanosine uridine cytidine thymidine It is known that nucleic acids, DNA and RNA, are the genetic material that cells and viruses use to produce faithful copies of themselves. 1 As the building blocks of DNA and RNA, the naturally occurring molecules of Figure 1 are vital to the functioning and multiplication of cells. 2 They are also important structural moieties in several coenzymes 1 Figure 1. Naturally occurring nucleosides. such as nicotinamide adenosine dinucleotide (NAD + ), nicotinamide adenine dinucleotide phosphate (NADP + ), flavin adenine dinucleotide (FAD) and coenzyme A (Figure 2). Figure 2. Examples of coenzymes. Nucleosides also play important roles in fundamental metabolic pathways. For example, adenosine is a major component of adenosine triphosphate (ATP) and is implicated in sleep regulation. 3 Thus, modified nucleosides affect these enzymes and display a range of biological activities potentially. 4-7 Because of the combined concern over emerging infections diseases and the increased possibility of bioterrorist attack, scientists are in an extensive search for new 2 3 drugs against viral infections. 8 In the latter case, particular attention has been paid to the orthopox family of viruses, especially to variola, the causative agent of smallpox or a similar virus, monkeypox virus. 9-13 For emerging threats, antiviral drugs are needed to be developed since vaccines for some widely occurring viruses such as hepatitis C virus (HCV), human immunodeficiency virus (HIV), Epstein-Barr virus (EBV) are not available 14 or are accompanied with side effects, such as the Hepatitis B virus (HBV) vaccine. 15 Since the advent of antiviral chemotherapy in 1959 with the discovery of the anti-herpes activity of 5-iodo-2?-deoxyuridine, 16 analogs of the natural nucleosides have served as structure models in the design of antitumor, antiviral, antibacterial agents. Some modified nucleosides have been synthesized and some of them have been clinically approved for treating different viruses. 17 Figure 3 shows the examples, ribavirin (?-D- ribofuranosyl-1,2,4-triazole-3-carboxamide) represents a base-modified nucleoside analog and it has a broad spectrum activity against both RNA and DNA viruses. Currently, it is used in the treatment of hepatitis C virus and respiratory syncytial infection. 18,19 The 5?-triphosphate of ribavirin inhibits viral RNA polymerase 20 and the viral specific mRNA capping enzyme guanylyltransferase. 21 Recently, ribavirin was shown to act as a RNA-virus mutagen, forcing RNA viruses into a lethal accumulation of errors, dubbed ?error catastrophe?. 22,23 And, obviously, this lethal mutagenesis might be enhanced by the inhibitory effect of ribavirin (in its 5?-monophosphate form) on inosine monophosphate (IMP) dehydrogenase and the consequent decrease in cellular guanosine triphosphate (GTP) pools. 22,23 Figure 3. Examples of Modified Nucleosides. Acyclovir ((9-hydroxyethoxy) methyl guanine), an acyclic nucleoside analog, inhibits the DNA polymerase of herpes simplex virus type 1, HSV-1. 24,25 Before it can interact with viral DNA synthesis, it needs to be phosphorylated intracellularly, in three steps, into the triphosphate form. The first phosphorylation step is ensured by the HSV- encoded thymidine kinase (TK), and is therefore confined to virus-infected cells. Subsequent phosphorylations are achieved by host cellular kinases. 24,26-28 4 Other nucleoside variations include the 2?,3?-dideoxynucleoside analogs: 3?-azido- 3?-deoxythymidine (AZT), 29,30 2?,3?-dideoxycytidine (ddC), 31 2?,3?-dideoxyinosine (ddI), 31 2?,3?-didehydro-3?-deoxythymidine (d4T), 32,33 (-)-dideoxy-3?-thiacytidine ((-)-3TC), 34,35 and Abacavir. These six nucleoside analogs have been licensed as anti-HIV drugs, and act by inhibiting the reverse transcriptase of human immunodeficiency virus. 36,37 All of these 2?,3?-dideoxynucleoside analogs act according to a similar ?recipe? as exemplified for AZT (Figure 4). They also must be phosphorylated consecutively inside the host cell by three cellular kinases to form the corresponding 5?-triphosphate derivative; this active metabolite can interact with the reverse transcription (RNA?DNA) reaction resulting in DNA chain termination since they lack the 3?-hydroxyl group necessary for the continued nucleic acid construction via a 3?, 5?-phosphodiester linkage. 38,39 N NH O O H 3 C O N 3 OH dThd Kinase N NH O O H 3 C O N 3 OP dTMP Kinase N NH O O H 3 C O N 3 O NDP Kinase PP N NH O O H 3 C O N 3 OPPP Inhibition of HIV reverse transcriptase/Chain termination AZT AZT-MP AZT-DP AZT-TP *dThd, (2'-deoxy)-thymidine; MP, monophosphate; NDP, nucleoside 5'-diphosphate; Figure 4. Metabolism of the anti-HIV agent AZT. 5 6 Antiviral Activity via Inhibition of S-Adenosylhomocysteine Hydrolase What about an approach to antiviral agents that does not require phosphorylation for antiviral effects? Viruses, like any other replicating species, require the methylation of the 5?-terminal residue of viral mRNA for forming the cap structure necessary for viral protein translation and replication. This 5?-capped structure (Figure 5) consists of a N 7 - methylguanosine residue linked at its 5?-hydroxy group to the 5?-end of the mRNA strand by an unusual 5?-5? triphosphate bridge. Additionally, a methyl group on the 2?-hydroxy group of the penultimate adenine nucleosides is also required. This methylated ?cap? is important in mRNA transport from the nucleus and for the following reasons: (1) efficient translation of the mRNA into proteins; (2) protection against ribonucleases and phosphatases; (3) mRNA splicing to ribosomes; and (4) the initiation of transcription of the viral mRNA. 40 Therefore interference of this 5?-capping process will definitely lead to the inhibition of viral replication. The capping process involves three enzymatic reactions (following steps) in which the initial mRNA 5?-triphosphate terminus is first cleaved to a diphosphate RNA (by RNA triphosphatase) followed by guanosine monophosphate (GMP) capping (by RNA guanylytransterase), and, finally, methylation at N7 position of guanine by RNA (guanine-7) methyltransferase. 41 (1) pppN(pN) n ? ppN(pN) n + P i (2) ppN(pN) n + pppG ? G(5?)pppN(pN) n + PP i (3) G(5?)pppN(pN) n + AdoMet ? m 7 G(5?)pppN(pN) n + AdoHcy Figure 5. 5?-capped structures. Both the sugar and base methylations at the 5'-terminus of mRNA are catalyzed by specific methyltransferases, which require S-adenosylmethionine (AdoMet) as the methyl donor. 42-44 As a universal methyl donor for numerous biological methylation processes, 45 AdoMet has been widely known as the second most widely used enzyme cofactor after ATP. 46 The methyl group is transferred when a bionucleophile attacks AdoMet; the nucleophile can be small molecules like tryptamine, medium size molecules such as phospholipids and macromolecules like mRNA. 47 During the methyl transfer, AdoMet is converted to S-adenosyl-L-homocysteine (AdoHcy), which can subsequently be hydrolyzed to form adenosine (Ado) and homocysteine (Hcy) through a reaction catalyzed by AdoHcy hydrolase (Figure 6). 48,49 This is a reversible reaction that normally favors the synthesis reaction (formation of AdoHcy), but the reaction is forced in the hydrolytic direction by further metabolism of adenosine and homocysteine: deamination (to inosine and AMP, respectively) or 7 phosphorylation of adenosine and remethylation of homocysteine to methionine through a reaction catalyzed by the methylcobalamin-dependent enzyme methionine synthase or combination of homocysteine with serine to form cystathionine. The cycle is completed by the conversion of methionine to AdoMet by AdoMet synthetase, which transfers the adenosyl part of ATP to methionine. This cycle is part of the general metabolism of sulfur-containing amino acid derivatives, itself regulated at the genetic level by AdoMet. 50 8 Figure 6. AdoMet metabolic cycle. Inhibition of AdoHcy hydrolase results in accumulation of AdoHcy, which is both the product and a feedback inhibitor of essential (AdoMet)-dependent methylation reactions (Figure 7). Such methylation reactions are required for final processing of the aforementioned 5?-capped structure of mRNA (as m 7 G(5?)pppN(pN) n ). 42,51,52 The increased concentration of AdoHcy lowers the ratio of AdoMet/AdoHcy and subsequently inhibits AdoMet transferases. This could lead to the inhibition of the transmethylation and 5?-capping, which is essential for viral protein formation and viral replication. 46 Figure 7. AdoMet/AdoHcy metabolism. Thus, AdoHcy hydrolase is an attractive pharmacological target as it leads to the inhibition of methyltransferases, and, in turn, it might be expected that the design of AdoHcy hydrolase inhibitors would lead to general suppression of protein synthesis. 9 Carbocyclic Nucleosides Nucleoside analogs form the basis of several medicinally important therapies, including antiviral and anticancer treatments. As described in the previous section, for example, acyclovir and AZT are currently in widespread clinical use as anti-herpetic and anti-AIDS medications. However, clinical applications of these nucleosides have been limited by some inherent side effects, such as toxicity and drug resistance, because of their close structural similarity to the natural nucleosides. 53 Another limitation is the instability of the glycosidic bond between the sugar moiety and the heterocyclic base of the nucleoside denied by medicinal agents. This bond undergoes phosphorolysis, which also occurs in normal nucleosides (Figure 8). 54 Figure 8 Phosphorolysis of nucleosides In an effort to overcome this, interest has shifted to the synthesis of carbocyclic nucleosides in which the furanose oxygen of traditional nucleosides has been replaced by a methylene group. 55-59 This change in structure imparted greater stability to the C-N bond against phosphorylases and availed increased lipophilicity, which is an added benefit for oral uptake and cellular penetration. 60-62 10 Two of the most important carbocyclic nucleosides are aristeromycin (Ari) and neplanocin A (NpcA), which are naturally occurring. As shown in Figure 9, they are different from each other only by the presence of a double bond between C-4? and C-6? of the carbocyclic ring of neplanocin A. Neplanocin A was isolated from the culture broth of Ampullariella regularis in 1979 63,64 and later synthesized, 65-70 while aristeromycin was synthesized 65-70 before its isolation from Streptomyces citricol in 1969. 71 Both compounds show broad antiviral activities and sparked the explosion of research for additional carbocyclic nucleosides with biological activity. The major mode of action for aristeromycin and neplanocin A is inhibition of AdoHcy hydrolase 47,51 and they represent first generation hydrolase inhibitors. AdoHcy hydrolase is widely recognized as a target for antiviral therapy (vide infra) arising from tight inhibitor enzyme binding as well as by the depletion of the essential hydrolase cofactor NAD + by consuming it and giving the inactive NADH enzyme form. Figure 9. Structures of Aristeromycin and Neplanocin A. Both aristeromycin and neplanocin A, like adenosine, are phosphorylated by aristeromycinous kinases 72 to their 5?-mono-, 5?-di- and 5?-tri-phosphate forms, which are a possible source of their undesirable toxicities. Adenosine kinase yields the monophosphate form of aristeromycin, resulting carbocyclic adenosine monophosphate 11 (AMP) serves as a substrate for AMP deaminase that leads to the inosine monophosphate (IMP) analog of aristeromycin. 73 It is then converted to the carbocyclic analog of guanosine monophosphate (GMP), a metabolite that inhibits the crucial cellular enzyme hypoxanthine (guanine)-phosphoribosyltransferase (HGPRTase), 74 which is involved in the purine salvage parthway. On the other hand, carbocyclic AMP yields carbocyclic ATP through series of phosphorylations. 72,75 The structural resemblance to ATP leads it to interfere with the fundamental cellular processes involving ATP and resulting undesirable side effects (Scheme 1). Scheme 1 12 Neplanocin A cytoxicity is also thought to form its triphosphate, which is known to be incorporated into RNA 76 and to be converted to neplanocyl methionine, under catalysis by adenosylmethionine synthase, a metabolite that may inhibit cellular RNA methylation. 76,77 Neplanocin A is also a substrate for adenosine deaminase and, consequently, is converted to an inactive form?neplanocin D. (Scheme 2) Scheme 2 With this discussion in mind, the efforts of several research groups have been channeled into the design of neplanocin A and aristeromycin analogs that would be devoid of phosphorylation while retaining the potent antiviral properties that reside in the inhibition of AdoHcy hydrolase (vide infra). For example, analogs of aristeromycin, 3- deazaaristeromycin 78 and 3-deaza-neplanocin A 79 were synthesized. These compounds (Figure 10) proved to be potent with less cytotoxicity, vis a vis the parent nucleoside. 80 13 Figure 10. Aristeromycin and neplanocin A analogs with base modified. The next development in this area came from the Schneller group. They synthesized 5?-noraristeromycin 81 and 5?-deoxyaristeromycin (Figure 11). 82 The latter one showed moderate activity but with no toxicity probably because it was incapable of 5?- phosphorylation, while the former one showed a very potent antiviral activity against vaccinia virus, hepatitis B virus, human cytomegalovirus, measles and influenza, with much less toxicity than aristeromycin and neplanocin A. 83,84 It?s believed that 5?- noraristeromycin does not undergo phosphorylation, for one or both of two reasons: (i) the shortened C-4? chain length does not allow the 4?-hydroxy group to reach the proper position in the binding site of the phosporylating kinase and/or (ii) the 4?-hydroxyl group of 5?-noraristeromycin is secondary and, as a consequence, may be less reactive than the primary 5?-hydroxyl of aristeromycin. H 3 C N N N N NH 2 HO OH HO N N N N NH 2 HO OH 5'-deoxyAri 5'-norAri 4' Figure 11. Aristeromycin analogs with side chain modified. In the aforementioned modified carbocyclic nucleosides, the goal was to retain antiviral activity via inhibition of AdoHcy hydrolase while eliminating the cytoxicity of resulting metabolites. The role of AdoHcy hydrolase in the biological activity of the compounds requires further study. 14 15 C-Nucleosides as antiviral agents C-Nucleosides are a unique class of nucleosides in which the ribofuranosyl moiety is linked to a heterocyclic base through a carbon-to-carbon bond instead of the traditional carbon-to-nitrogen bond. As a result, they are resistant to the chemical and the enzymatic hydrolytic cleavage of the glycosidic bond, much like carbocyclic nucleosides. C-Nucleosides have received considerable attention due not only to the potent metabolic stability but also to the interesting biological activities of the naturally occurring ones. 85 The first example of a natural C-nucleoside was Pseudouridine (?-uridine), which was isolated in 1957 86 as the fifth nucleoside obtained from ?soluble RNA,? and its structure was established in 1962 as 5-(?- D -ribofuranosyl) uracil (Figure 12). 87-89 It is now known that ?-uridine is present ubiquitously in active transfer RNA (tRNA), 90 and that certain tRNAs deficient in ?-uridine are incapable of participating in protein synthesis. 91 This C-nucleoside is formed enzymatically from uridine after assembly of the tRNA chain. 92-95 Later, a few C-nucleosides were isolated from nature, such as oxazinomycin, pyrazomycin, showdomycin, formycin A and formycin B (see Figure 12). They are antibiotics and exhibit anticancer and/or antiviral activity. 96-99 Oxazinomycin, which inhibits both gram-positive and gram-negative bacteria and Ehrlich ascites and sarcoma 180 (both solid and ascites) in mice, was discovered independently in two Japanese laboratories. 100,101 Pyrazomycin, isolated from the culture filtrates of Streptomyces candidus, 102 has been shown to be an inhibitor of a variety of viruses and tumors. 103 Showdomycin, elaborated by Streptomyces showdoensis, 104 has been found to inhibit gram-positive and gram-negative bacteria and Ehrlich ascites in vitro and HeLa in vitro. Figure 12. Naturally occurring C-nucleosides. Formycin A was isolated as an antibiotic from the rice mold Nocardia interforma by Hori et al. 105 and was identified as a C-nucleoside isomeric with the natural nucleic acid constituent adenosine by Koyama et al. 106 Formycin has growth inhibitory effects against Ehrlich carcinoma, mouse leukemia L1210, Yoshida rat sarcoma cells and HeLa cells, and Xanthomonas oryzae as well as exhibits some immunosuppressive activity. 100,107 Interest in formycin A stems from the fact that it can replace adenosine in a variety of biochemical reactions. 108,109 Formycin and its derivatives act as substrates for many adenosine specific enzymes including enzymes of nucleotide metabolism, RNA polymerase, polynucleotide phosphorylase, the pyrophosphorylase of tRNA and 16 adenosine kinase. 109,110 Formycin A is incorporated into RNA and DNA, 109 and its derivative, formycin 5'-triphosphate (FTP), acts as a source of biological energy and ribopolynucleotides with formycin replacing adenosine, at the binding site of t-RNA to ribosomes have shown no mistranslation of the messenger. 111 FTP can also substitute for ATP in aminoacyl-tRNA synthetase, and it is the first nucleotide analog containing an abnormal base that is capable of functioning in this reaction. Additionally, formycin 5'- triphosphate is a competitive in vitro inhibitor of the nucleoside triphosphate reductase of Lactobacillus leichmanni in the reduction of ATP, GTP and UTP; however, in the presence of positive effector 2?-deoxyguanosine triphosphate (dGTP), FTP can be reduced at about the same rate as ATP to 2?-deoxyformycin 5'-triphosphate (dFTP) (Figure 13), which is able to mimic dATP as activators of CTP reduction by ribonucleotide reductase. 112 Figure 13. Reduction of FTP by ribonucleotide reductase. Unfortunately, formycin is also a good substrate for the adenosine catabolic enzyme, adenosine deaminase (ADA) 113 that deaminates formycin, producing the oxygenated form, formycin B, which, in turn, may be converted to the xanthosine analog, 17 oxoformycin, by hepatic aldehyde oxidase (Figure 14). 114,115 Both formycin B and oxoformycin have shown little or no biological activity and low toxicity in experimental animals. 116 The rapid inactivation of formycin by animal tissues that contain adenosine deaminase has limited investigations of therapeutic activities of this potentially useful antimetabolite. O N N H N N NH 2 HO OH HO Formycin A O N NH H N N O HO OH HO Formycin B adenosine deaminase Aldehyde Oxidase O N H NH H N N O HO OH HO O O N N N N NH 2 HO OH HO Adenosine Oxoformycin Figure 14. Metabolic conversions of formycin. In order to increase the effectiveness of formycin as a chemotherapeutic agent, chemically modified formycins were synthesized 117,118 in attempt to decrease or abolish the activity of the formycin molecule with adenosine deaminase, while retaining the activity with adenosine kinase and other key enzymes of adenine nucleotide metabolism. A study 119 suggested that adenosine derivatives which exist in the anti conformation would be excellent substrates for adenosine deaminase, whereas those in the syn 18 rotameric conformation would have little or no substrate activity. This report prompted the synthesis of nucleosides designed to restrict rotation around the glycosyl bond of formycin and increase the proportion of nucleoside in the syn conformation. This hypothesis predicted that 1-methylformycin, which exists predominantly in the anti conformation (since there is no steric hindrance to rotation about the glycosyl bond), would be a good substrate for ADA. On the other hand, marked steric hindrance to rotation occurs with 2-methylformycin. This compound has been found to exist predominantly in the syn conformation in the crystalline state (Figure 15). 120 Figure 15. N-methyl derivatives of formycin. The above hypothesis consequently predicted that 2-methylformycin would be a poor substrate for ADA. Crabtree et al 121 described the effects which methylation of the individual nitrogen atoms of formycin have on the activity as a substrate for ADA. The data invalidate the hypothesis above: no substrate activity for ADA was detected with 1- methylformcin, whereas, 2-methylformycin displays substrate activity with erythrocyte ADA. However, another report from Makabe et al 122 on the enzymatic deamination of certain 1- and 2-alkylformycin showed that both 1-methylformycin and 2- methylformycin were deaminated by Takadiastase ADA and calf intestinal mucosa ADA. 19 20 The observed rate of deamination of 1-methylformycin was found to be approximately equal to the rate of deamination of formycin. By contrast, the rate of deamination of 2- methylformycin was very much slower than that of formycin and 1-methylformycin. In fact, no detectable deamination of 2-methylformycin was observed at enzyme concentrations which resulted in complete deamination of formycin and 1- methylformycin. These results suggest that the conformation (either syn or anti) of an adenosine analog is not the only consideration in determining ADA substrate activity. The lack of substrate activity for certain adenosine analogs maybe also due to specific chemical modifications of the compound which result in a change of the electronic, steric and/or structural parameters. It was also found that 2-methylformycin has relatively strong activity against vaccinia virus. Giziewice et al reported the two methylated derivatives had no toxicity on the primary rabbit kidney (PRK) cells used for evaluation of antiviral activity. 123 Hybrid nucleosides--Carbocyclic C-Nucleosides On the basis of the interesting chemical and biological properties of C- nucleosides and carbocyclic nucleosides, it was of interest to investigate hybrid nucleosides: carbocyclic C-nucleosides (Figure 16). Figure 16. Structure of carbocyclic C-nucleoside. The history of carbocyclic C-nucleosides dates back to the 1960s. 124 However, despite the long history of both carbocyclic and C-nucleosides, only a few carbocyclic C- nucleosides have been prepared, probably due to the synthetic difficulties of these compounds. To date, the preferred strategy for the synthesis of carbocyclic C-nucleoside analogs proceeds via the construction of functionalized carbafuranoses by Diels-Alder reactions (Scheme 3). During exploration of this pathway, a few versatile carbocyclic C- nucleoside precursors also arose. 125-129 In this research, Katagiri, Kaneko, and coworkers developed a method for the synthesis of carbocyclic pyrimidine C-nucleosides 125-127 and carbocyclic oxazinomycins, 130 while Cookson and coworkers 131 described a synthesis of imidazo-[1,5-a]pyridine carbocyclic C-nucleosides and their corresponding 2'-deoxy 21 derivatives. Koizumi and coworkers 132 reported the first enantioselective synthesis of the carbocyclic analog of showdomycin. Stoodley and coworkers 133 reported the synthesis of carbocyclic tiazofurin and its antipode, and Leumann et al. synthesized a series of carbocyclic pyrimidine C-nucleosides following the similar routes (Examples shown in Figure 17). 134 Scheme 3 General strategy for the synthesis of carbocyclic C-nucleosides Recently, research aimed at the development of the synthesis of carbocyclic 9- deazapurine nucleosides appeared. Chu et al. reported the synthesis of carbocyclic 9- deazaaristeromycin, 135 carbocyclic 9-deazainosine and other corresponding analogs. 136 Schneller et al. reported the synthesis of 9-deaza-5?-noraristeromycin. 137 Hong and coworkers described the first synthesis of 4'-branched carbocyclic C-nucleoside (Figure 17). 138 Furthermore, many of the carbocyclic C-nucleoside have been synthesized as racemic mixtures (Figure 18), probably due to the synthetic difficulties when seeking 22 enantiopure substances. Two such analogs of the bioactive C-nucleosides have been described: (?)-carbapyrazofurin, 139 (?)-carbashowdomycin. 140 However, there have been no reports that such compounds are biologically active. Figure 18. Racemic Carbocyclic C-nucleosides. Figure 17. Examples of synthesized carbocyclic C-nucleosides. 23 Therefore, enantiomeric synthesis of the carbocyclic C-nucleosides offers a synthetic challenge but avails biologically interesting targets. In the latter required, no significant biological activities have been reported. It was of interest to explore further the carbocyclic C-nucleosides because most of the hitherto known carbocyclic C- nucleosides possess only unnatural heterocyclic moieties. In 1997, Leahy and coworkers reported their progress toward the synthesis of novel carbocyclic formycin (Figure 19). 129 They synthesized a common intermediate, which was expected to be used to access carbocyclic nucleoside analogs such as carbocyclic formycin. To date, carbocyclic formycin has not been well explored. Figure 19. Exploring for the synthesis of Carbocyclic Formycin. 24 Beginning in 2004, when the Schneller group started to pursue the design and syntheses of novel carbocyclic C-nucleosides, a convenient access to carbocyclic C- nucleosides based on the formycin framework arose. 141-143 Their synthesis of novel carbocyclic formycin analogs were reported as the following (Figure 20). 1) A model study to carbocyclic formycin A and B analogs. Zhou, J. et al . Tetrahedron Lett. 2004, 45, 8233-8234. 2) C-4' Truncated carbocyclic formycin derivatives. Zhou, J. et al Tetrahedron 2006, 62, 7009-7013 . 3) Carbocyclic 4?-epi-formycin Zhou, J. et al Tetrahedron 2008, 64, 433-438. Figure 20. Synthesis of carbocyclic formycin analogs. 25 When considering formycin derivatives, it should be kept in mind that formycin, like aristeromycin, is limited by its toxicity, which, in some instances, resides in its 5'- nucleotide derivatives. Based on the previously described non-cytotoxic antiviral properties of 5?-noraristeromycin, this dissertation research focuses on the combination of the structural components of formycin and 5'-noraristeromycin resulting in carbocyclic 5'-norformycin and their N-1 and N-2 methylated derivatives (Figure 21). Figure 21. Carbocyclic 5'-norformycin and its methylated versions. 26 A MODEL STUDY TO N-METHYL CARBOCYCLIC FORMYCIN ANALOGS As was pointed out in the introduction, 1-methyl and 2-methylformycin, which assumed anti- and syn- conformation respectively, have displayed interesting biological activities, 121 such as 2-methylformycin being active towards vaccinia virus. The methylated version of carbocyclic formycin has never been reported in the literature. Based on the previous work reported in the Schneller group, a model study to N-methyl carbocyclic formycin analogs 1 and 2 (Figure 22) was selected as the beginning point of this dissertation. Figure 22. (?)-1 and (?)-2. The route to 1 and 2 was foreseen as accessible from a readily available epoxide. To move in that direction, the plan was to first introduce a functionally useful group to the ?anomeric position? of the requisite cyclopentyl ring through a C-C linkage that 27 would, concurrently, produce the vicinal-trans hydroxyl of epoxide origin. This arrangement was then to be elaborated to a heterocyclic ring of the targets. Most of the biologically active C-nucleosides have been synthesized by this general method, including the synthesis of formycin B 144 and of oxoformycin. 145 Synthesis of pyrazole derivative. The synthesis started with the addition of lithiated 3,3-diethoxy-1-propyne to cyclopentene oxide at -78 ?C in the presence of boron trifluoride diethyl etherate, 146,147 affording (?)-3 (Scheme 4). Hydrolysis of (?)-3 with a mixture of acetic acid and 10% aqueous hydrochloric acid resulted in acetylenic aldehyde (from step b in Scheme 4). Although an unstable compound, this aldehyde is a versatile intermediate in the synthesis of the target C-nucleosides. 148-150 Treatment of the non- isolated aldehyde with hydrazine monohydrate gave a pyrazole derivative, 151,152 which was acetylated to provide the key intermediate (?)-4. O O H n-BuLi/hexanes O O Li O a O O OH H 3 b O H OH H c, d OAc H N N Ac 4 Reagents and conditions: a) BF 3 EtO 2 , 64%; b)10% HCl, AcOH; c) hydrazine monohydrate, AcOH; d). Ac 2 O, pyridine, DMAP, 73% for three steps. Scheme 4 Synthesis of pyrazole ring 28 Synthesis of the key nitro nitrile intermediate. Nitration of pyrazole (?)-4 with ammonium nitrate and trifluoroacetic anhydride in trifluroacetic acid following literature conditions 153 led directly to the 1,4-dinitropyrazole (?)-5 154 (Scheme 5). Habraken and Poels 155 have shown that 1,4-dinitropyrazoles react with secondary amines at C-5 in a cine substitution with expulsion of the N-nitro group as nitrite. Similarly when (?)-5 was treated with excess potassium cyanide in aqueous ethanol at room temperature, the desired cine substitution 156,157 of the N-nitro with a cyano occurred, and the key nitro nitrile intermediate (?)-6 was obtained in a pure crystalline form. Scheme 5 Synthesis of key nitro nitrile compound In general, the occurrence of a ?cine? substitution (i.e., the entering group comes in ortho to the leaving group) is evidence for a 1, 2-addition-elimination mechanism. The actual molecule initially formed is a 3H-pyrazole; the ultimate product obtained then arises in a subsequent fast hydrogen rearrangement reaction (Scheme 6). 29 Scheme 6 mechanism of cine substitution Synthesis of the final N-methyl carbocyclic formycin analogs. It was envisioned that the methylation of unsubstituted (?)-1 may be complex since there are several possible methylation sites. For this reason introduction of the methyl substituent was considered at this stage. Treatment of (?)-6 with sodium hydride followed by quenching with methyl iodide gave the 1-methyl product ((?)-7) and 2-methyl product ((?)-8), which could be separated by column chromatography (Scheme 7). Scheme 7 Introducing methyl group to pyrazole ring To confirm the site of methylation and the stereochemical orientations of the two cyclopentyl ring substituents, an X-ray structural analysis of what was assumed to be the 2-methyl product (?)-8 (Figure 23) was conducted. This analysis of (?)-8 not only 30 confirmed the structure but also showed it possessed a high degree of syn character (that is, cyclopentyl ring lies under pyrazole). Figure 23. X-ray structure for compound (?)-8. Deacetylation of (?)-8 to (?)-9 was conducted in a solution of saturated ammonia in methanol. Hydrogenation of (?)-9 in the presence of palladium/carbon afforded the reduced product (?)-10. Treatment of (?)-10 with formamidine acetate in refluxing ethanol proceeded with ring annulation to (?)-(1?,2?)-2-(3-[7-amino-2-methyl-1H- pyrazolo [4, 3-d] pyrimidyl])cyclopentanol, ((?)-2) (Scheme 8). 31 Scheme 8 Synthesis of (?)-2 N 1 -methyl derivative (?)-1 was also obtained by treatment of (?)-7 in the same route as discussed above (Scheme 9). However this was complicated because when (?)-7 was subjected to the deacetylation, an inseparable mixture containing the desired unprotected secondary alcohol was produced. Subjecting this mixture to steps b and c of Scheme 8 gave (?)-(1?,2?)-2-(3-[7-amino-1-methyl-1H-pyrazolo[4,3-d]pyrimidyl]) cyclopentanol ((?)-1). OAc H N N CH 3 NO 2 CN 7 OH H N N CH 3 NO 2 CN unknown 1 N N N N NH 2 OH H 3 C Mix 1 Deacetylation Scheme 9 Synthesis of (?)-1 Normally, the deacetylation reaction (Scheme 9) occurs under alkaline conditions, such as catalytic sodium methoxide in methanol, a mixture of potassium carbonate and methanol, or saturated ammonia in methanol, etc. It was assumed N-1 methylation decreased the pyrazole ring?s stability (possibly nucleophilic causing attack at C-4), probably affording ring opened or functional group changed products. A possible mechanism for this is proposed in Scheme 10. 32 Scheme 10 Methylated pyrazole ring?s stability 33 Structural assignments for N 1 -methyl- and N 2 -methylated products Townsend et al. used magnetic circular dichroism (MCD) for assigning the structures to the N-methylated C-nucleosides. 158 The MCD spectra of formycin A and 1- methylformycin are very similar but they are quite different from 2-methylformycin. In the model study of this dissertation, similar results were observed, which were based on the comparisons of NMR spectral data ( 1 H and 13 C) of the two methylated isomers and the C-nucleoside version lacking the methyl group. 143 Selected 1 H NMR spectral data for the two isomeric methylated products are compiled in Table 1. The methyl signal for the 2-methylated product is upfield from the signal observed for 1-methylated derivative. There was a similar relationship with the H 5 signals. It is of some interest that the signal for the ?anomeric (H 1? ) proton? of the 2- methylated isomers wherein rotation about the glycosidic bond (C3-C1?) is restricted is observed approximately 0.1 ppm downfield from the ?anomeric? signal observed for the other methyl isomer. In Table 1, the selected 13 C NMR data for the two methylated versions were also collected. The signals for C 7 and C 7a of 2-methylated product are largely downfield from those of the other two products; however, the signals for C 3 and C 3a of the 2-methylated version are upfield by comparison. As with Townsend, the 2- methylated derivative is dissimilar to the 1-methyl and the non methyl group versions. Comparative TLC mobilities of the methylated isomers were presented in Table 2. Table 1 Selected 1 H NMR and 13 C NMR Spectral Data a 1 H NMR NCH 3 H 5 H 1 ? (?)-11 b 8.16 3.26-3.18 (?)-1 (1-methyl) 4.15 8.12 3.23-3.14 (?)-2 (2-methyl) 4.10 8.03 3.32-3.22 13 C NMR c NCH 3 C 3 C 3a C 5 C 7 C 7a C 1? (?)-11 b 147.1 139.6 150.5 151.0 121.8 46.0 1-methyl-(?)-1 38.8 145.5 141.0 150.8 151.2 121.8 45.9 2-methyl-(?)-2 38.6 136.8 136.1 151.2 155.6 129.9 45.0 a Me 2 SO-d 6 was used as a solvent and chemical shifts are in parts per million from an internal standard. b Data from ref. 143. c 13 C assignment for 11, 1 and 2 is determined by comparison to formycin and methylated formycins. 158 Table 2 Comparative TLC (Silica gel) Data for the N-methylated isomers Solvent (CH 2 Cl 2 : MeOH=10:1 v/v) 1-methyl-(?)-1 R=0.22 2-methyl-(?)-2 R=0.08 34 Comparisons of NMR spectral data ( 1 H and 13 C) and TLC mobilities of the two isomers allow us to make structural assignments for the subsequent methylated derivatives. For example, after obtaining the two racemic methylated carbocyclic formycin analogs (?)-12 and (?)-13 (Figure 24) under the same synthetic route (Scheme 11) discussed above, their structural assignments were confirmed by comparisons of their NMR spectral data and TLC mobilities. Figure 24. Compounds (?)-12 and (?)-13. In summary, in the first stage of this dissertation, an efficient means to N- methylated carbocyclic formycin analogs has been developed. Application of this pathway employing more functionalized epoxides now allows for access to a comprehensive and diverse library of formycin and formycin-like carbocyclic C- nucleosides as antiviral candidates. 35 Scheme 11 Synthesis of Compounds (?)-12 and (?)-13 36 ENANTIOSELECTIVE SYNTHESIS OF CARBOCYCLIC 5'- NORFORMYCIN Exploring the key step toward target compound 22. As mentioned in the introduction, the synthesis of carbocyclic C-nucleosides has been challenging. 129,136 With that as back drop, the enantioselective synthesis of carbocyclic 5'-norformycin (22) (Figure 25) was undertaken in this dissertation research. Figure 25. Attempts to synthesize the key epoxide. Based on former work developed in the Schneller group, the functionalized epoxide 24 was seen as central to obtaining carbocyclic 5'-norformycin (22). A retrosynthetic analysis (Scheme 12) of 24 and previous experience in this project revealed that the base part of target compound 22 could be built from a cyclopentyl alkyne or acetylenic aldehyde (see 23), which could be 37 achieved from the epoxide 24. This key epoxide 24 was foreseen from protected cyclopentene 25 that, in turn, could be produced from diene 26 through a ring closing metathesis (RCM) procedure; and diene 26 could be accessed from chiral diol 28. Scheme 12 Retrosynthesis of epoxide 24 The starting material, (+) 2, 3-O-isopropylidene-L-threitol (28), is accessible by improving upon a literature route from dimethyl L-tartrate (29). 159 While expensive, 28 is also commercially available. The first stage of the synthesis focused on how to selectively introduce the carbon needed for obtaining 27 (Scheme 13). Treatment of diol 28 with excess sodium hydride and one equivalent tert-butyldimethylsilyl chloride produced compound 30, and diol 28 underwent a highly selective mono-silylation in excellent yield. 160 Oxidation of the primary hydroxyl group of 30 with sulfur trioxide- pyridine complex and dimethyl sulfoxide 161 furnished aldehyde 31, which was converted in good yield to olefin 32 by a Wittig reaction using potassium tert-butoxide and 38 methyltriphenylphosphonium bromide. Submitting olefin 32 to regioselective hydroboration with 9-borabicyclo[3,3,1]nonane (9-BBN) followed by oxidative hydrolysis, smoothly provided 33 in high yield. Removing the silyl group with a 1 M solution of tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) provided the desired diol 27. Scheme 13 Synthesis of diol 27 With diol 27 in hand, the plan was to oxidize the two hydroxyl groups of 27 to the dialdehyde 34 that was to be subjected to Wittig olefination to the diene 26, which was envisioned as a precursor of the synthesis of epoxide 24 (Scheme 14). Unfortunately, the oxidation of the two primary hydroxyl groups of 27 could not be accomplished. Examples of oxidation conditions attempted are: 1-(3-dimethyl aminopropyl)-3- ethylcarbodiimide (EDC), 162 or a Swern-like oxidation (with sulfur trioxide pyridine complex and dimethyl sulfoxide) 161 and a Dess-Martin oxidation. 163 Considering the likely sensitivity of the dialdehyde, a backup plan considered introducing two carbonyl groups asynchronously. This called for 33 with only one free 39 hydroxyl group. Oxidation of the primary hydroxyl group of 33 under Swern-like oxidation conditions yielded aldehyde 35, which was subjected to a Wittig reaction using potassium tert-butoxide and methyltriphenylphosphonium bromide to produce olefin 36. Deprotection of 36 with a 1 M solution of tetrabutylammonium fluoride (TBAF) in tetrahydrofuran afforded 37. Oxidation of 37 successfully provided aldehyde 38. However, the following Wittig olefination was disappointing; the desired diene 26 was not furnished, possibly due to the high instability of both aldehyde and diene (Scheme 14). Scheme 14 Attempted synthesis of diene 26 Although much work has been done on the synthesis of desired epoxide 24, it should be admitted that the route just outlined was proving impractical. More obstacles were foreseen, such as the difficulty with a requisite ring closing metathesis (RCM) of 40 diene 26; enantioselectivity of alkene epoxidation (prior to 24); and, the stereochemistry of the nucleophilic addition to the epoxide that was to produce the alkyne precursor for heterocyclic ring construction (that is, pyrazolo[4, 3-d] pyrimidine). Therefore this route was abandoned in order to explore another pathway to synthesize the target compound not requiring a cyclopentyl epoxide. For this, a procedure to cyclopentyl alkynes was brought forth. 141,143,148-150,157 Attempts to synthesize the cyclopentyl alkyne from allylic monoacetate 39. In 2003 the Schneller group reported the synthesis of 9-deaza-5?-noraristeromycin (42) (Scheme 15). 137 They prepared this carbocylic C-nucleoside from the readily available (+)-(1R, 4S)-4-hydroxy-2-cyclopentyl monoacetate (39). 137 Using Deardorff?s method for coupling allylic acetates, 164,165 treatment of the protected monoacetate 40 with ethyl cyanoacetate in the presence of palladium (0) to give the cis oriented carbocylic C- nucleoside precursor 41. Scheme 15 Synthesis of 9-deaza-5?-noraristeromycin 41 This research work sparked an idea to further study the synthesis of carbocylic 5?- norformycin from (+)-monoacetate 39. A retrosynthetic analysis was devised (Scheme 16) with the critical alkyne shown as the middle structure. Scheme 16 Retrosynthesis from monoacetate (+)-Monoacetate 39 is one of the most important enantiopure precursors for carbocyclic nucleoside synthesis. Access to this material called for enzyme-catalyzed reactions, which themselves are powerful and offer convenient procedures to enantiopure compounds. 166 For the purposes of this project, the enzymatic hydrolysis of prochiral diacetate 43, reported by Laumen and Schneider, 167 provided a means to (+)-monoacetate 39. 42 Diacetate 43 was obtained from cyclopentadiene following a literature procedure (Scheme 17). 168 This began with epoxidation of freshly cracked cyclopentadiene, which was followed by tetrakis(triphenylphosphine)palladium(0) catalyzed epoxide ring opening in the presence of acetic anhydride. In this latter reaction the presumed palladium intermediate reacted with acetic anhydride to give the meso-diacetate 43. Using the optimized procedure developed in Schneller laboratory, 82 treatment of meso- diacetate 43 with Pseudomonas cepacia lipase (PCL) afforded the allylic monoacetate 39. Although PCL normally displays a pro-R-hydrolytic preference, 169 it showed a pro-S- preference for this substrate. Scheme 17 Synthesis of (+)-monoacetate 39 Attention then was drawn to palladium (0)-catalyzed allylations that are a common tool in organic synthesis. 170,171 The literature is rife with the use of carbon, oxygen, and nitrogen nucleophiles in this reaction. The use of acetylenic nucleophiles is not so common. To investigate this idea, the secondary hydroxyl group of 39 was protected with tert-butyldimethylsilyl chloride. The reaction of 44 using freshly prepared palladium catalyst and trimethylsilylacetylene under the conditions reported 164,165 failed to produce the desired coupling product 45 (Scheme 18). Scheme 18 Attempted synthesis of compound 45 43 In order to investigate whether the reaction failed due to the alkalinity of sodium hydride, n-butyllithium was considered in this reaction instead of sodium hydride. Unfortunately, the coupling also was unsuccessful. A possible reason for this result may lie with the carbon anions lacking sufficient nucleophilicity to attack the ?-allyl palladium (II) intermediate to give the desired compound. Synthesis of the cyclopentyl alkyne from cyclopentenone. (-)-(4R, 5R)-4,5-O- Isopropylidene-2-cyclopentenone (D-like 2-cyclopentenone (46)) is another important versatile chiral synthon in carbocyclic nucleosides. 172 The utility of 46 is demonstrated by its application for the preparation of the potential antiviral agents 5?- methylaristeromycin 173 and 5?-homoneplanocin A 174 (Scheme 19) beginning with a 1,4- addition to introduce a modified side chain. O OO 46 O OO Me HO OH Me HO H N N N N H 2 N (5'R)-5'-methylaristeromycin Kharasch Conjugative Addition O OO 46 O OO MeOOC SPh HO OH N N N N H 2 N 5'-homoneplanocin A Conjugative Addition HO Scheme 19 Examples using conjugative addition to 2-cyclopentenone 46 With the promise of the conjugative addition for the goals of this research, a retrosynthetic analysis was designed, and attention focused on the cyclopentyl unit that would lend itself to this purpose (Scheme 20). This suggested that target compound 22 44 could be made from the (+)-L-like 2-cyclopentenone 47. A 1,4-addition reaction could then be performed to give the important intermediate cyclopentyl alkyne. Using a series of procedures developed in the former work, the target synthesis was foreseen. Scheme 20 Retrosynthesis from L-like-2-cyclopentenone 47 L-Like 2-cyclopentenone 47, which is the enantiomer of previously descried D- like cyclopentenone 46, was easily prepared from the (+)-monoacetate 39 (Scheme 21). Glycolization with 4-methylmorpholine N-oxide (NMO) in the presence of a catalytic amount of osmium tetraoxide followed by protection of the vicinal-diol with 2,2- dimethoxypropane afforded 48. Pyridinium chlorochromate oxidation of 48 proceeded with the fortuitous ?-elimination of acetic acid to afford the conjugated L-like 2- cyclopentenone 47. 175 Scheme 21 Synthesis of L-like-2-Cyclopentenone 47 from monoacetate 39 45 Another route to 47 considered natural sugars with predefined stereochemistry. This route has been found some success from D-ribose in 45% (8 steps) 172 and 38% (6 steps) 176 overall yield, respectively. More recently, Snape et al. 177 and Smith et al. 178 also developed more scalable routes to both enantiomers of cyclopentenone. For this research, a new synthetic route to 47 was developed that was guided by the method of Chu and his group (Scheme 22). 172 In that direction, D-ribose was converted to the isopropylidene protected derivative 49 with 2,2-dimethoxypropane in the presence of a catalytic amount of sulfuric acid. This was followed by silylation of the primary hydroxyl group using tert-butyldimethylsilyl chloride to give lactol 50 in 85% yield. Compound 50 was converted to an olefin 51 by the Wittig reaction. The yield of this step was very low due to the appearance of side product 52 where the silyl group was transferred from a primary position to a secondary position. Chu and his group 172 did not mention this phenomenon. Scheme 22 Route from Chu?s paper 172 46 Finally, a concise and more economic route from D-ribose to the differently protected cyclopentenone (that is (+)-(4S, 5S)-4,5-O-cyclopentyl-2-cyclopentenone (53)) was accomplished (Scheme 23) in 6 steps (40% yield). OH OH OO O OO HO OO HO OO O OO O OH HO OO O OH HO HO OH a b c Reagents and conditions: a) cyclopentenone, H + ,75%;b)CH 3 PPh 3 Br, t-BuOK, THF, overnight; c) NaIO 4 ,CH 2 Cl 2 ,H 2 O, rt, 30 min, 85% for two steps; d) vinylmagnesium bromide, THF, -30 o C, 1 h, 85%; e) 1st genertion Grubbs catalyst, CH 2 Cl 2 ,rt,12h;f)CH 2 Cl 2 , DMSO, DIPEA, SO 3 -Py, rt, overnight , 78% for two steps. d ef 54 55 56 57 58 53 D-ribose Scheme 23 Synthesis of cyclopentyl protected L-like-2-cyclocyclopentenone 53 The cyclopentyl vicinal diol protecting group was chosen for 53 since the isopropylidene protected derivatives were found to be highly volatile under some conditions. Thus, in presence of a catalytic amount of sulfuric acid, cyclopentyl protected D-ribose 54 was afforded from ribose. A Wittig reaction of 54 with triphenylphosphonium methylidene followed by oxidative cleavage of the vicinal diol 55 179-181 with sodium metaperiodate afforded the vinyl aldehyde 56 179,182 in 85% yield (two steps). Treatment of 56 with vinylmagnesium bromide gave the diene 57 (85%), 47 which was subjected to ring-closing methathesis (RCM) reaction using the Grubbs 1 st generation catalyst 183,184 (Figure 26) to give 58 in 88% yield. Cyclopentenone 53 was then directly obtained by oxidation of the secondary alcohol under sulfur trioxide- pyridine complex and dimethyl sulfoxide 161 oxidation conditions without isolation of the cyclopentenol 58 in 40% overall yield and six steps from D-ribose. This procedure turned out to be much improved in view of overall yield and number of steps compared to any previously reported procedure. Figure 26. Grubbs Catalyst for RCM. Since the Grubbs? catalyst was important to this process, a comment about it is in order. Ruthenium-based Grubbs? catalysts (Figure 26) are among the most important catalysts for olefin metathesis (the metal-catalyzed carbon-carbon double bond redistribution 185 ). They show great functional tolerance, high catalytic reactivity and moderate air and moisture stability. These advantages make them widely used in many organic syntheses. 185-187 The generally accepted mechanism of their use in ring closure metathesis was proposed by Chauvin 188 and is generalized in Scheme 24. In this process, a metal carbene I is first formed followed by a [2+2]-cycloaddition to afford a metallocyclobutane intermediate II. The subsequent retro [2+2]-cycloaddition releases olefin (ethylene in this case) and gives a new metal carbene III, which leads to a second 48 metallocyclobutane IV. Subsequent retro ring opening gives a new olefin and the original catalytic metal carbene I to complete the cycle. The key intermediate is the metallacyclobutane II, which can undergo cycloreversion either towards products or back to starting materials. When the olefins of the substrate are terminal, the driving force for RCM is the removal of ethene from the reaction mixture. H 2 C CH 2 M CH 2 H 2 C M H 2 C M M CH 2 =CH 2 I II III IV Scheme 24 General mechanism of Ring Closure Metathesis 49 50 Key step for the synthesis of target compound 22. With cyclopentenone 53 in hand, exploration of a way to introduce the alkynyl unit through 1,4-conjugative addition for the important intermediate cyclopentyl alkyne was investigated. Conjugate addition of organometallic compounds to ?, ?-unsaturated ketones is a widely employed reaction in organic synthesis. 189 Organocuprates are commonly employed for the 1,4-addition of alkyl and alkenyl groups to ?, ?-unsaturated ketones; 190 however, cuprates cannot be used in alkynylation reactions owing, presumably, to the strength of the alkynylcopper (I) ligand bond. 191,192 . Several alternatives have been reported that achieve conjugative addition to ?, ?- unsaturated enones, but each suffers from limitations for the purposes here. However, a brief overview of contextualization is in order. Acetylenic alanes 193-195 conjugately add their alkynyl units to ?, ?-unsaturated enones only under certain circumstances. If the ?, ?-unsaturated ketone is able to achieve an S-cis conformation, it has been found that 1, 4-addition (Scheme 25) will then proceed in good yield. Cyclic ketones in which the enone system is rigidly constrained to a transoid geometry, such as 2-cyclohexenone, react with the alane reagent to give the tertiary carbinol derived from 1, 2- rather than l, 4-addition desired here of the acetylenic unit. A reasonable explanation for this reactivity mode involves the necessity for a six- membered transition state for conjugate addition. 193 Scheme 25 Conjugate addition of acetylenic alane. Alkynylboron reagents 196-198 also add only to ?, ? -enones that assume an S-cis conformation. As in the case of alkynylalanes, transoid ketones gave no indication of the desired 1, 4- reaction. This observation suggested a cyclic transition state analogous to the one proposed for the 1, 4-addition reactions of alkynylalanes (Scheme 26). Scheme 26 Conjugate addition of alkynylboron reagents. In the investigations into the application of 1,4-addition reactions to the synthesis of prostaglandins, Pappo et al. 194 were able to perform this reaction on a fixed S-trans enone (such as cyclopentenone). Two 1,4-addition products were obtained in approximately a 1:2 ratio (Scheme 27). The fact that the entering octynyl group added cis to the hydroxy function indicated participation of that group in the 1,4-addition process by way of a five-membered cyclic intermediate analogous to the structures discussed above. Blockage of the hydroxy function by a tetrahydropyranyl group prevented reaction with the aluminum reagent. 51 Scheme 27 Conjugate addition of trioctynylaluminum reagent An indirect method of conjugate addition of an alkynyl group to a fixed S-trans enone without neighboring-group participation had been developed by Corey and Wollenburg. 192,199 This procedure involves addition of bis(tri-n-butylstanny1)ethylene by cuprate addition and subsequent oxidative elimination of a stannyl group to give, overall, conjugate addition of acetylide to the enone (Scheme 28). Scheme 28 Conjugate addition of mixed cuprate reagent 52 Each of the sequences which achieve conjugate addition of alkynyl units suffers from severe limitations. The enone substrate must be capable of an S-cis conformation or, alternatively, must have a conveniently located functional group for direction of an intramolecular attack by the organometallic reagent. For those enones which fit neither one of these requirements, it is only possible to conjugately add the ethynyl group, and this must be done by an indirect method. Schwartz et al. 189,200 found that the nickel (II) acetylacetonate (Ni(acac) 2 )/ diisobutylaluminum hydride (DIBAL) system would indeed catalyze the conjugate addition of terminal alkynyl units from dialkylaluminum acetylides to both S-cis and S- trans enones (Scheme 29). The latter observation was pertinent to this project. Furthermore, the reaction was found to proceed with high stereospecificity. Scheme 29 Nickel-catalyzed 1,4-addition of organoaluminum acetylide to S-trans enones From the above discussion, it was believed that through the above procedure, a nickel-catalyzed conjugate addition of alkynyl groups to 53 could be affected. Fortunately, this addition was successful and the desired cyclopentyl alkyne precursor 59 was obtained (Scheme 30), and the product formed possessed the anti stereochemistry. 53 Scheme 30 Key step for the synthesis of target compound The stereochemical assignment for 59 is based on 1 H NMR (Figure 27). From the NMR spectral data, ?anomeric? hydrogen ( H-1) has no resonance with any one of the hydrogens at positions 2 and 3; in the other hand, the resonance for H-2 with H-3 appears at ? 4.3 as a doublet with coupling constants equal to 5.0 Hz, so does for H-3 with the same coupling constants. All these 1 H NMR data confirmed that the two hydrogens with fixed stereochemistry are syn with alkynyl group. Figure 27. 1 H NMR spectral data for compound 59. The typical procedure for effecting conjugate addition by this route is illustrated as follows: To nickel(II) acetylacetonate (Ni(acac) 2 ) in ether at -5 ?C is added 1 M diisobutylaluminum hydride in hexane solution. The reaction mixture rapidly turned dark red-brown and it was then cooled to -25 ?C. To the resultant red-brown solution was added fresh dialkylaluminum acetylide (prepared in the usual manner from the lithium 54 acetylide and dialkylaluminum chloride) as a solution in ether. 2-Cyclopentenone 53 in ethereal solution was then added dropwise over a period of time. The reaction mixture was allowed to stir at -25 ?C for several hours. After hydrolysis, the conjugate adduct 59 was afforded. It was found that optimal yields of the conjugate adduct were obtained when an excess of dialkylaluminum acetylide (2.2 eq) was employed. Using a smaller excess of the aluminum acetylide resulted in a decreased yield of the desired product. It was important to employ an excess of the aluminum acetylide, probably, because the initial product of conjugate addition was an aluminum enolate, which could react with additional unsaturated ketone to give the aldol adduct (Scheme 31). In the presence of excess aluminum acetylide, the added 2-cyclopentenone 53 in the presence of the nickel catalyst preferentially reacted with it. If less aluminum acetylide was employed, oligomers would be generated as by-products. Ni (acac) 2 / DIBAL Me 2 AlO OO TMS CCSi Me Me Me AlMe 2 53 O O O TMS OH O O O O O 53 Scheme 31 Possible unwanted adol side reaction 55 Finally, the exploration of the key step towards target compound 22 ended with the successful nickel-catalyzed conjugate addition. This represents the first time 53 (an important enantiopure precursor) was called forth for synthesizing carbocyclic C- nucleosides. Also, the 1,4-conjugate addition to a cyclopentenone process is another route towards carbocyclic C-nucleosides avoiding functionalized expoxide openings. Enantioselective synthesis of carbocyclic 5?-norformycin (22). With cyclopentyl alkyne precursor 59 in hand and calling upon reaction conditions developed earlier in this work, target compound carbocyclic 5?-norformycin (22) was believed obtainable. The synthesis of 22 began with the reduction of the cyclopentyl alkyne precursor 59 using sodium borohydride and cerium chloride (Luche reagent) 201 to afford the ?- alcohol 60 (Scheme 32) as the only isomer. The Luche reagent is a prominent reagent for the stereoselective conversion of ketone to secondary alcohols. In this process, Ce(III) acts as a good Lewis acid and is strongly oxophilic, and lead to the chelation intermediate 202 (Figure 28) that promotes the reduction. The existence of the bicyclic cyclopentanone arrangement in 59 guides the borohydride attack from the less hindered ? face. Figure 28. Chelation intermediate to 59. 56 O OO 59 TMS a HO OO 60 TMS b Reagents and conditions: a) NaBH 4 /CeCl 3 7H 2 O, MeOH, 75%; b) benzoic acid, DIAD, PPh 3 ,THF, rt, 70%; c) TBAF, THF, 92%; d) i.BuLi / hexanes, MTBE followed by DMF; ii. AcOH, hydrazine monohydrate; iii. Ac 2 O,pyridine, DMAP, 73% for three steps; e) NH 3, MeOH, 98% BzO OO 61 TMS c BzO OO 62 H d(i) HO OO 64a,R=Bz; 64b,R=H H e AcO AcO OAc N N Ac 65 RO OO C O H 63 d(i) d(ii, iii) Scheme 32 Synthesis of compound 65 Inversion of the C-4? center of 60 was accomplished by a Mitsunobu reaction with benzoic acid to give 61. Removing the trimethylsilyl group of 61 with 1 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) gave the cyclopentyl alkyne 62. Directly formylation of 62 (step i of (d)) failed to afford the corresponding propargylic aldehyde 64a probably due to the instability of benzoic group under the reaction conditions. Thus, treatment of 62 with methanolic ammonia at room temperature produced the unprotected cyclopentyl alkyne 63. Formylation (step i of (d)) of 63 with subsequent 141-143 reaction of the resultant substituted propargylic aldehyde 64b with hydrazine monohydrate and followed by acetylation with acetic anhydride catalyzed by 57 4-dimethylaminopyridine (DMAP) provided the key synthetic intermediate pyrazole 65 (Scheme 32). In moving forward towards 22, the key nitronitrile derivative 67, was achieved by, first, nitration 153 of 65 with ammonium nitrate and trifluoroacetic anhydride in trifluoroacetic acid to the 1, 4-dinitropyrazole 66 (Scheme 33). This was followed by a cine-substitution reaction of 66 with potassium cyanide to afford 67. 156,157 Scheme 33 Synthesis of nitronitrile compound 67 In exploring the conditions for the cine-substitution reaction, often an undesired side product 68 was observed (Scheme 34). This is believed to have arisen via a nucleophilic substitution reaction consisting of a displacement of the N-nitro group by the nitrile anion and/or ethanol. 155 To limit the amount of 68, the optimum reaction conditions were explored: for example, (1) reaction time was an important factor in this reaction, requiring quenching within five minutes; (2) a solvent volume ratio of ethanol and ethyl acetate should be nearly 1:1 and, (3) the reaction should be conducted at room temperature. 58 Scheme 34 Displacement on the N-nitro group Three steps remained to the target compound 22 (Scheme 35). This began with catalytic (Pd) hydrogenation of the nitronitrile derivative 67. Deacetylation of the resulting aminonitrile derivative 69 with a solution of saturated ammonia in methanol (to 70) followed by formamidine acetate in refluxing ethanol produced the desired fused pyrimidine target (1S,2R,3S,4S)-4-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl) cyclopentane-1,2,3-triol (22). 59 Scheme 35 Synthesis of target compound 22 Enatioselective Synthesis of N-1 and N-2 Methylated Carbocyclic 5'- Norformycin Analogs As mentioned in the introduction section, N-1-methylformycin was highly resistant to enzymatic deamination; N-2-methylformycin exhibited high activity against vaccinia virus while not affecting cellular DNA and RNA synthesis. 122 Both N- methylated formycin analogs were non-cytotoxic. The project for this dissertation research sought to extend these observations to the N-methyl carbocyclic 5?-norformycin analogs 71 and 72 (Figure 29). Figure 29. N-methyl carbocyclic 5?-norformycin analogs. The methylated formycin derivatives 73 and 74 (Scheme 36) have been reported by Townsend and his group. 158 In that regard, methylation of formycin (75) with methyl iodide in the presence of base afforded 1-methylformycin (71) and 2-methylformycin (72). 60 O N N H N N NH 2 HO OH 75 O N N N N NH 2 HO OH 73 H 3 C O N N N N NH 2 HO OH 74 H 3 C HO HO HO Reaction conditions: a) NaOEt, EtOH, MeI, 3.8% for 73, 24% for 74 a Scheme 36 Synthesis of methylated formycin derivatives With carbocyclic 5?-norformycin (22) in hand, application of the Townsend conditions for achieving 71 and 72 were evaluated. In view of the low yields in this approach (Scheme 36), it was conceived that methylation of carbocyclic 5?-norfomycin (22) could result in lower yields of the desired products since there are several possible competing methylation sites (Figure 30). Figure 30. Possible methylation sites of 22. 61 Thus, it was decided to introduce the methyl group at the pyrazole stage using the nitronitrile intermediate 67 (Scheme 37). Treatment of 67 with sodium hydride followed by quenching with methyl iodide gave a major product (N-1-methyl 76), a minor one (N- 2-methyl 77) and an undesired side product 78, which were separated by column chromatography. Scheme 37 Methylation of 67 Formation of the side-product 78 could be explained from a possible impurity of 68 with 67. During the synthesis of nitronitrile compound 67, a displacement reaction (Scheme 34), in addition to the cine substitution, took place with by-product 68 being obtained. By-product 68 and compound 67 have identical R f values. Only 13 C DEPT NMR data can distinguish 68 from 67. Thus, it is reasonable to believe that the methylation took place with a mixture of compound 67 and 68 (Scheme 38) leading to only three of the maximum four compounds (compound 79 being only in trace amounts). 62 Scheme 38 Possible reason for side product 79 63 Based on the model study discussed in the first part of this dissertation, structural assignments for the above methylated derivatives was confirmed by the comparisons of their TLC mobilities and their NMR spectral data ( 1 H and 13 C). The comparative TLC mobilities of the methylated isomers were the easiest way to distinguish the different isomers and the data are presented in Table 3. Table 3 Comparative TLC Data for the N-methylated isomers Solvent (hexane/ethyl acetate =3:1 v/v) 1-methyl (76) R=0.52 2-methyl (77) R=0.34 1-methyl (78) R=0.45 Selected 1 H NMR and 13 C NMR spectral data for the three isomeric methylated products are compiled in Table 4. The 1 H methyl signal for the 1-methyl product 76 is downfield from the signal observed for the 2-methyl derivative 77; the 1 H methyl signal for the by-product 78 was expected to be close with that signal of 76, but, possibly, because of the absence of the cyano group in 78, that peak is close to the signal of 77. The 1 H signal for the ?anomeric (H 1? ) proton? of the 2-methylated isomer 77 wherein rotation about the glycosidic bond (C3-C1?) is restricted is observed downfield from the ?anomeric? signals observed for the 1-methyl isomer 76 and by-product 78. From some NMR spectral data, by-product 78 can be distinguished from the 1-methyl isomer 76: from the 1 H NMR spectrum, a singlet peak (belonging to H 5 in 78) is lacking in 76; from the 13 C NMR spectrum, there has no CN signal in 78, but this signal exists in 76; from the 13 C DEPT NMR spectral data, a positive peak at C 5 in 78 is observed which means C 5 is connected with a hydrogen atom instead of cyano group. Table 4 Selected 1 H NMR and 13 C NMR Spectral Data a 1 H NMR NCH 3 H 5 H 1 ? 76 (1-methyl) 4.10 -- 4.02-4.08 77 (2-methyl) 4.01 -- 4.06-4.14 78 (1-methyl) 3.90 8.11 4.01-4.08 13 C NMR b NCH 3 CN C 5 (DEPT) 76 (1-methyl) 39.90 107.26 115.68 (no peak) 77 (2-methyl) 39.84 107.31 113.92 78 (1-methyl) 40.11 -- 131.15 (positive) a CDCl 3 was used as a solvent and chemical shifts are in parts per million from an internal standard. b 13 C assignment for 76, 77 and 78 is determined by comparison to methylated formycins 16 and methylated carbocyclicformycin analogs. With the N-1-methylated nitro nitrile compound 76 in hand, steps towards target 71 compound began with hydrogenation of 76 in the presence of palladium/carbon to the 64 reduced product 80. Deacetylation of 80 to 81 was conducted in a solution of saturated ammonia in methanol. Treatment of 81 with formamidine acetate in refluxing ethanol produced the desired fused pyrimidine target (1S,2R,3S,4S)-4-(7-amino-1-methyl-1H- pyrazolo[4,3-d]pyrimidin-3-yl)cyclopentane-1,2,3-triol (71) (Scheme 39). Scheme 39 Synthesis of N-1 methylated 5?-norcarboformycin 71 Since the regioselectivity of Scheme 35 gave low yields of the N-2-methylated derivative 77, proceeding to 72 in the same manner, as just described for 71, was no longer considered to be appropriate. Consequently, it was decided that the synthesis of 72 had to return to the original plan of methylation of 22 to get 72. (Scheme 40) Scheme 40 Alternate route to synthesize 72 65 Attempted Synthesis of N-2 methylated 5?-norcarboformycin 72 It was obvious that the methylation of free 5?-norcarboformycin (22) would require treatment of 22 with freshly made sodium ethylate (Scheme 41). Following stirring to effect a clear solution, methyl iodide was then added and the solution was stirred until the starting material was no longer present (TLC). After three additional quantities of methyl iodide were added, the solution was stirred for three days. TLC (R f ) analysis showed the absence of N-1 methyl 5?-norcarboformycin (71). By contrast, a TLC spot with strong UV absorbency was seen and its R f value was lower than that of compound 71. The reaction residue was then analyzed by NMR spectroscopy. Signals ( 1 H methyl signal and H 5 signal) were found. These peaks were different from the N-1 methyl derivative. It is speculated that these are due to the presence of N-2 methylated 5?- norcarbofomycin (72). However, this mixture was complicated and the yield was unsatisfactory. These conditions were not considered further for the methylated derivatives. Scheme 41 Synthesis of compound 72 66 67 Future Direction: Proposed synthesis of another carbocylic C- nucleoside-Pyrazomycin analogs As mentioned in the introduction, pyrazomycin was isolated from the culture filtrates of Streptomyces candidus 102 and has been shown to be an inhibitor of a variety of viruses and tumors. Pyrazomycin exhibited activity against the vaccinia, herpes simplex, and measles viruses in vitro. 102,103 The Friend leukemia virus was also inhibited by pyrazomycin as reported by DeLong et al. 203 In seeking structural analogs of pyrazomycin for improved bioactivity, the 5?- norcarbocyclic system 84 is a worthy target. In that regards, during the synthesis of the 5?-norcarboformycin (22), a relevant intermediate 4-aminopyrazole derivative 69, arose. To achieve 84, replacement of the C-4 amino group of 69 with a hydroxyl group (or a hydrogen) could lead to the synthesis of 5?-norcarbopyrazomycin 85 and its derivative 87 (Scheme 42). A classical method 66 exists in aromatic systems to carry out the aforedescribed conversions: that is the 4-aminopyrazole derivative 69 can be diazotized and the diazonium salt solution rendered alkaline to provide a neutral zwitterionic diazopyrazole 82. On photolysis in aqueous acetone with a medium-pressure mercury lamp and Pyrex filter, nitrogen is evolved and the 4-hydroxypyrazole derivative 83 is obtained. On the other hand, on photolysis in aqueous dioxane (catalyzed by trifluoroacetic acid) using visible light on 82 will give the deaminated nitrile derivative 86. Hydrolysis of the nitrile group in 83 and 86 using alkaline hydrogen peroxide, will afford the amides. After deacetylation, the carbopyrazomycin analogs could be accomplished. OAc H H N N NH 2 AcO AcO CN Diazotization Photolysis OAc H H N N OH AcO AcO CN 69 Nitrile hydrolysis OAc H H N N OH AcO AcO CNH 2 O Deacetylation OH H H N N OH HO HO CNH 2 O 83 84 85 Photolysis Condition 2 OH H N HN HO HO CNH 2 O 87 Deacetylation OAc H N N N 2 AcO AcO CN 82 Condition 1 OAc H H N N H AcO AcO CN Nitrile hydrolysis 86 Neutralization Condition 1: a medium-pressure mercury lamp and Pyrex filter; acetone-water (3 : 1 v/v); Condition 2: visible light; aqueous dioxane containing trifluoroacetic acid. Scheme 42 Proposed synthesis of 85 and 87 68 69 BIOLOGICAL RESULTS Target compounds were designed as antiviral agents and compound 1 was evaluated against a wide variety of viruses. The spectrum of viruses used is shown in Table 5. Table 5 The spectrum of viruses to be assayed Virus family Individual viruses Adenoviridae Adenovirus Arenaviridae Pichinde virus Bunyaviridae Punta toro virus Coronaviridae Human coronavirus, Severe acute respiratory syndrome (SARS) Filoviridae Ebola virus Flavivridae Hepatitis C virus (HCV), West Nile virus, Yellow fever virus Hepadnaviridae Hepatitis B virus (HBV) Heroesviridae Epstein-Barr virus (EBV), Human Cytomegalovirus (HCMV), Varicella-Zoster virus (VZV), Herpes simplex virus (HSV) Orthomyxoviridae Influenza A virus, Influenza B virus Paramyxoviridae Parainfluenza virus, Measles virus, Respiratory syncytial virus (RSV) Picornaviridae Rhinovirus Poxviridae Cowpox virus, Vaccinia virus (VV) Reoviridae Reovirus Rhabdoviridae Vesicular stomatitis virus (VSV) Togaviridae Venezuelan Equine Encephalitis virus (VEE), Sindbis virus 70 The detailed results were showed in Table 6-Table 13. No activity was found. It was also found to have no toxicity effects on the viral host cells. Table 6 Antiviral Activity of Compounds against HSV-1, HSV-2, HCMV and VZV Based on Cytopathogenic Effect (CPE) Inhibition Assay a, b Compound 1 EC50 a >300 EC90 b >300 HSV-1 d CC50 c >300 EC50 >300 EC90 >300 HSV-2 d CC50 >300 EC50 >300 EC90 >300 HCMV d CC50 >300 EC50 >300 EC90 >300 VZV d CC50 >300 a Effective concentration (?M) required to reduce virus plaque formation by 50%. b Effective concentration (?M) required to reduce virus plaque formation by 90%. c Cytotoxic concentration (?M) required to reduce cell growth by 50% d Tested on human foreskin fibroblasts (HFF) cells. 71 Table 7. Antiviral Activity of Compounds against RSV A, Parainfluenza and SARS CoV Based on Neutral Red Visual Inhibition Assay Compound RSV A a Parainfluenza b SARS CoV c EC50 IC50 d EC50 IC50 EC50 CC50 1 >100 >100 >100 >100 >100 >100 Units = ?M a RSV A was tested on MA-104 cells. b Parainfluenza was tested on MA-104 cells. c SARS CoV was tested on vero cells. Table 8. Antiviral Activity of Compounds 1 against Vaccinia Virus and Cowpox Virus Based on Cytopathogenic Effect (CPE) Inhibition Assay Compound Vaccinia Virus a Cowpox Virus b EC50 CC50 EC50 CC50 1 >300 >300 >300 >300 Units = ?M a Vaccinia Virus was tested on HFF cells. b Cowpox Virus was tested on HFF cells. 72 Table 9. Antiviral Activity of Compound 1 in Vero Cell Cultures Minimum inhibitory concentration b (?M) Compound Minimum cytotoxic concen-tration a (?M) Para- Influenza- 3 virus Reovirus- 1 Sindbis virus Coxsackie virus B4 Punta Toro virus 1 >200 >200 >200 >200 >200 >200 Brivudin >250 >250 >250 >250 >250 >250 (S)-DHPA >250 50 250 >250 >250 >250 Ribavirin >250 150 250 >250 >250 150 a Required to cause a microscopically detectable alteration of normal cell morphology. b Required to reduce virus-induced cytopathogenicity by 50%. Table 10. Antiviral Activity of Compound 1 in HEL Cell Cultures Minimum inhibitory concentration b (?M) Compound Minimum cytotoxic concentr- ation a (?M) Herpes simplex virus-1 (KOS) Herpes simplex virus (G) Vaccinia virus Vesicular stomatitis virus Herpes simplex virus-1 (TK - KOS ACV) 1 >200 >200 >200 >200 >200 >200 Brivudin >250 0.08 0.8 6 >250 250 Ribavirin >250 250 250 50 150 250 Acyclovir >250 0.4 0.16 >250 >250 150 Ganciclovir >100 0.032 0.096 >100 >100 4 a Required to cause a microscopically detectable alteration of normal cell morphology. b Required to reduce virus-induced cytopathogenicity by 50%. 73 Table 11. Antiviral Activity of Compound 1 against Cytomegalovirus in Human Embryonic Lung (HEL) Cells Antiviral activity EC 50 (?M) a Cytotoxicity (?M) Compound AD-169 strain Davis strain Cell morphology (MCC) b Cell growth (CC 50 ) c 1 N. D. d >100 >100 >100 Ganciclovir 7.1 9.0 >400 138 Cidofovir 1.3 3.2 >400 89 a Effective concentration (?M) required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU). b Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. c cytotoxic concentration required to reduce cell growth by 50%. d Not determined. e No complete inhibition at higher drug concentration. 74 Table 12. Antiviral Activity of Compound 1 against Varicella-zoster in Human Embryonic Lung (HEL) Cells Antiviral activity EC 50 (?M) a Cytotoxicity (?M) TK + VZV TK - VZV Compound OKA strain 07/1 strain Cell morphology (MCC) b Cell growth (CC 50 ) c 1 >100 >100 >100 >100 Acyclovir 3.4 42 >1778 356 Brivudin 0.013 >240 1200 452 a Effective concentration (?M) required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU). b Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. c Cytotoxic concentration required to reduce cell growth by 50%. Table 13. Antiviral Activity of Compound 1 against HBV Compound HBV (Assay: VIR) a EC50 (3TC) EC90 (3TC) CC50 (3TC) SI b (3TC) 1 >10 (0.047) >10 (0.14) >300 (2260) 16900 Units = ?M a VIR data are based on extracellular virion HBV DNA. b SI = CC50/EC90 75 CONCLUSION Interest in carbocyclic formycin analog stems from the fact that the C-nucleoside formycin has shown potentially significant antiviral properties and that formycin can replace adenosine in a variety of biochemical reactions (for example, enzymes of nucleotide metabolism, RNA polymerase, polynucleotide phosphorylase, the pyrophosphorylase of tRNA and adenosine kinase). However, the usefulness of formycin is limited by its toxicity, which, in some instances, resides in its 5'-nucleotide derivatives. This dissertation reports a combination of the structural components of formycin and the carbocyclic nucleoside 5'-noraristeromycin resulting in carbocyclic 5'- norformycin 22. The N-methylated derivatives are also investigated. Arising in this research was a facile procedure to the enantiopure precursor, L-like 2-cyclopentenone 44 that permitted a convenient means to the desired carbocyclic C-nucleosides through a conjugate addition process. This scheme circumvented the previous, cumbersome approach to carbocyclic C-nucleosides that employed functionalized expoxide ring openings. The synthetic process elaborated herein opens an accessible means to carbocyclic 5'-nor C-nucleosides of potential therapeutic usefulness and a number of other similar analogs. 76 The formycin literature suggested this investigation be extended to the synthesis of the N-1 and N-2 methylated carbocyclic 5'-norformycin (62 and 63). Thus, synthetic routes to these compounds ((?)-1, (?)-2, (?)-12, (?)-13) were developed and a thorough requisite structural analysis (N-1 versus N-2) carried out. Furthmore, it is hypothesize that the N-2-methylated carbocyclic formycins possess a syn character. Application of the result of this research allows for access to a comprehensive and diverse library of formycin and formycin-like carbocyclic C- nucleosides as antiviral candidates. 77 EXPERIMENTAL SECTION Materials and methods. Melting points were recorded on a Meltemp II point apparatus and are uncorrected. 1 H and 13 C NMR spectra were recorded on a Bruker AV 250 Spectrometer (operated at 250 or 62.9 MHz, respectively) or AV 400 Spectrometer (operated at 400 or 100 MHz, respectively). All 1 H chemical shifts are reported in ? relative to the internal standard tetramethylsilane (TMS, ? 0.00). 13 C chemical shifts are reported in ? relative to CDCl 3 (center of triplet, ? 77.23) or relative to DMSO-d (center of septet, ? 39.51). The spin multiplicities are indicated by the symbols s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). Elemental analyses were performed by the Atlantic Microlabs, Atlanta, Georgia. Reactions were monitored by thin-layer chromatography (TLC) using 0.25 mm E. Merck silica gel 60-F 254 coated silica gel plates with visualization by the irradiation with Mineralight UVGL-25 lamp or exposure to iodine vapor. Column chromatography was performed on Whatman silica gel (average particle size 2-25 ?m, 60 ?) and elution with the indicated solvent system. Yields refer to chromatographically and spectroscopically ( 1 H and 13 C NMR) homogeneous materials. 78 (?)-(1?, 2?)-2-(3, 3-Diethoxy-prop-1-ynyl) cyclopentanol (3). To a solution of propiolaldehyde diethyl acetal (97%) (10.7 mL, 75 mmol) in anhydrous tetrahydrofuran (225 mL) at ?78 ?C under an atmosphere of nitrogen was added n-butyllithium (31.5 mL, 79 mmol, 2.5 M solution in hexanes) over 5 min. The reaction was stirred for ca. 30 minutes then cyclopentene oxide (98%) (6.5mL, 75 mmol) in anhydrous tetrahydrofuran (75 mL) was added, followed by neat BF 3 ?OEt 2 (9.5 mL, 75 mmol,) over 15 min. The reaction mixture was stirred at -78 ?C for 2 h. The reaction was quenched by addition of saturated aqueous sodium bicarbonate solution and partitioned between ethyl acetate and water. The combined organic phases were washed with brine, dried (Na 2 SO 4 ), filtered and evaporated in vacuo to afford a liquid residue. Purification by flash column chromatography (eluting with 25-33% ethyl acetate/hexanes) furnished the secondary alcohol (10.2 g, 64.1%) as a light yellow oil; 1 H NMR (400 MHz, CDCl 3 ) ? 1.23 (t, 6H, J=7.1 Hz), 1.56 (m, 1H), 1.72 (m, 3H), 2.08 (m, 2H), 2.32 (br s, 1H), 2.64 (m, 1H), 3.57 (m, 2H), 3.71 (m, 2H), 4.23 (m, 1H), 5.26 (d, 1H, J=1.5 Hz); 13 C NMR (100 MHz, CDCl 3 ) ? 15.4, 18.6, 31.0, 33.8, 39.6, 58.5, 80.3, 88.5, 91.4, 91.8; Anal. calcd for C 12 H 20 O 3 ?0.1H 2 O: C, 67.29; H, 9.44. Found: C, 67.20; H, 9.18. (?)-(1?, 2?)-2-(1-acetyl-1H-pyrazol-3-yl)cyclopentanol (4). Acetal 3 (8.5 g, 40 mmol) was dissolved in glacial acetic acid (395 mL) and 10% HCl (95 mL), and the mixture was stirred at room temperature for 1 h. To this, a solution of hydrazine monohydrate (24.7 g, 0.493 mol) in glacial acetic acid (190 mL) was added dropwise over 20 min. The resulting solution was heated at reflux overnight and concentrated in vacuo to afford dark-brown oil. The crude product was redissolved in pyridine (188 mL), acetic anhydride (94.8 mL, 1.02 mol) and DMAP were added, and the resulting solution 79 was stirred for 16 h at room temperature. Solvent was removed in vacuo, and the crude residue was redissolved in ethyl acetate (394 mL), washed with 10% HCl, water, and brine, dried, concentrated, and chromatographed to afford 4 (6.9 g, 29 mmol, 73%) as a light yellow oil; 1 H NMR (400 MHz, CDCl 3 ) ? 1.75 (m, 1H), 1.87 (m, 3H), 2.07 (s, 3H), 2.20 (m, 2H), 2.69 (s, 3H), 3.27 (m, 1H), 5.31 (m, 1H), 6.35 (d, 1H, J=2.8 Hz), 8.19 (d, 1H, J=2.8 Hz); 13 C NMR (100 MHz, CDCl 3 ) ? 15.2, 22.0, 30.8, 33.6, 39.6, 60.6, 79.1, 88.0, 91.3, 91.6, 171.4, 177.4; Anal. calcd for C 12 H 16 N 2 O 3 : C, 61.00; H, 6.83; N, 11.86. Found: C, 61.11; H, 6.80; N, 11.98. (?)-(1?, 2?)-2-(1-acetyl-4-nitro-5-cyano-1H-pyrazol-3-yl) cyclopentanol (6). Trifluoroacetic anhydride (29.4 mL) was added dropwise to a stirred solution of 4 (5.00 g, 21.1 mmol) and ammonium nitrate (16.8 g) in trifluoroacetic acid (248 mL) at 0 ?C. The resulting solution was allowed to warm to room temperature and stirred overnight. The solvent was evaporated on rotavapor at room temperature, then was diluted with methylene chloride, washed with water, satd. sodium bicarbonate and brine, dried, and concentrated in vacuo to give the dinitro compound 5 (6.00 g, 21.1mmol >100%) as a colorless liquid. The crude product thus isolated was committed to the next step without further purification. An analytically pure 5 was prepared by chromatography (hexane:ethyl acetate =5: 1). Colorless oil; 1 H NMR (400 MHz, CDCl 3 ) ? 1.89 (m, 4H), 2.02 (s, 3H), 2.25 (m, 1H), 2.34 (m, 1H), 3.86 (m, 1H), 5.42 (m, 1H), 9.04 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) ? 21.4, 24.1, 31.7, 32.8, 44.3, 78.0, 125.2, 134.4, 150.7, 171.2; Anal. calcd for C 10 H 12 N 4 O 6 : C, 42.26; H, 4.26; N, 19.71. Found: C, 42.33, H, 4.35; N, 19.54. 80 A solution of the 1,4-dinitro compound (6.00 g, 21.1 mmol) in ethanol (8 mL) and ethyl acetate (8 mL) was added dropwise over 1 min to a stirred solution of potassium cyanide (1.20 g, 18.4 mmol) in ethanol (22 mL) and water (6 mL). Following an additional 5 min at room temperature, the reaction mixture was neutralized with acetic acid (1 mL) and evaporated on the rotavapor, then diluted with ethyl acetate (100 mL), washed with water and brine, dried, concentrated in vacuo to afford 6 (3.81 g, 14.4 mmol, 68.2%) as a dark syrup. Purification by chromatography (hexane:ethyl acetate:methanol=5:2:1) to afford 6 as a white solid, mp 132.5-133.8 ?C; 1 H NMR (250 MHz, CDCl 3 ) ? 1.76 (m, 1H), 1.92 (m, 3H), 2.06 (s, 3H), 2.27 (m, 1H), 3.38 (m, 1H), 3.53 (s, 1H), 3.85 (m, 1H), 5.38 (m, 1H); 13 C NMR (62.5 MHz, CDCl 3 ) ? 20.9, 22.1, 30.6, 31.4, 42.3, 76.2, 110.5, 122.8, 133.4, 145.2, 172.9; Anal. calcd for C 11 H 12 N 4 O 4 : C, 50.00; H, 4.58; N, 21.20. Found: C, 50.13; H, 4.79; N, 20.98. (?)-(1? ,2?)-2-(5-cyano-1-methyl-4-nitro-1H-pyrazol-3-yl)cyclopentyl acetate (7) and (?)-(1? ,2?)-2-(5-cyano-2-methyl-4-nitro-1H-pyrazol-3-yl)cyclopentyl acetate (8). To a stirred solution of 6 (1.85 g, 7.01 mmol) in anhydrous THF (150 mL), was added portion wise 60% sodium hydride in mineral oil (0.36 g, 9.0 mmol) at 0 ?C. The mixture was stirred at room temperature for 30 min. and then iodomethane (0.64 mL, 1.4 g, 9.5 mmol) was added dropwise. After a period of 24 h the reaction mixture was absorbed by silica gel, and then purified by silica gel column chromatography. Compound 7 (1.16 g, 4.17 mmol, 60.0%) and 8 (0.55 g, 1.98 mmol, 28.4%) was obtained after eluting with a mixture of hexane/ethyl acetate in a ratio of 1:1. Compound 7 was a white syrup; 1 H NMR (400 MHz, CDCl 3 ) ? 1.72 (m, 1H), 1.88 (m, 3H), 2.01 (s, 3H), 2.23 (m, 1H), 2.29 (m, 1H), 3.83 (m, 1H), 4.06 (s, 3H), 5.41 (m, 1H); 13 C NMR (100.6 81 MHz, CDCl 3 ) ? 21.2, 23.7, 31.5, 32.5, 39.7, 43.5, 79.5, 111.2, 115.3, 135.3, 151.0, 170.8; Compound 8 was a white solid, mp 103-104 ?C; 1 H NMR (400 MHz, CDCl 3 ) ? 1.93 (m, 3H), 2.03 (s, 3H), 2.05 (m, 1H), 2.15 (m, 1H), 2.42 (m, 1H), 3.42 (m, 1H), 3.96 (s, 3H), 5.30 (m, 1H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 21.0, 24.9, 30.8, 32.9, 39.3, 45.0, 80.1, 110.7, 122.0, 133.4, 145.2, 171.2; Anal. calcd for C 12 H 14 N 4 O 4 : C, 51.80; H, 5.07; N, 20.13. Found: C, 52.02; H, 5.11; N, 20.34. (?)-(1? ,2?)-2-(5-cyano-2-methyl-4-nitro-1H-pyrazol-3-yl)cyclopentanol (9). Ammonia gas was introduced to a solution of compound 8 (2.0 g, 7.2 mmol) in MeOH (100 mL). This reaction mixture was allowed to stir at room temperature until TLC analysis indicated starting material was no longer present. The solvent was then removed in vacuo and the residue purified by chromatography (CH 2 Cl 2 /MeOH, 20:1) to afford 9 (1.4 g, 5.9 mmol, 83%) as a light yellow solid, mp 129-130 ?C; 1 H NMR (400 MHz, CD 3 OD) ? 1.85 (m, 4H), 2.13 (m, 1H), 2.32 (m, 1H), 3.74 (m, 1H), 4.26 (m, 1H), 5.00 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) ? 23.2, 28.6, 35.8, 39.7, 45.8, 77.5, 110.8, 121.8, 134.1, 145.9; Anal. calcd for C 10 H 12 N 4 O 3 : C, 50.84; H, 5.12; N, 23.72. Found: C, 50.98; H, 5.14; N, 23.72. (?)-(1?,2?)-2-(5-cyano-2-methyl-4-amino-1H-pyrazol-3-yl)cyclopentanol (10). A catalytic amount of Pd/C (1%) was added to a solution of 9 (1.4 g, 5.9 mmol) in methanol (30 mL) at room temperature, the resulting mixture was allowed to shake under 20 psi of hydrogen overnight. After the reaction was complete, concentrated in vacuo to give compound 10 (1.4 g, 6.7 mmol >100%) as a colorless liquid. The crude product thus isolated was used in the next step without further purification. 82 (?)-(1?,2?)-2-(3-[7-amino--2-methyl-1H-pyrazolo[4,3d]pyrimidyl]) cyclopentanol (2). A solution of 10 (0.70 g, 3.4 mmol) in ethanol (15 mL) was stirred with formamidine acetate (0.68 g, 6.7 mmol) under reflux for 1 h. The reaction mixture was cooled to room temperature. The solvent was removed in vacuo and the residue purified by chromatography (CH 2 Cl 2 /MeOH, 10:1) to afford 2 (0.47 g, 2.0 mmol, 60%) as a light yellow solid, mp = 209 ?C (dec); 1 H NMR (250 MHz, DMSO-d 6 ): ? 1.61 (m, 1H), 1.86 (m, 2H), 2.07 (m, 2H), 2.32 (m, 1H), 3.26 (m, 1H), 4.10 (s, 3H), 4.47 (m, 1H), 7.43 (br s, 2H), 8.03 (s, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) ? 22.3, 28.7, 34.7, 39.1, 45.5, 77.5, 130.3, 136.6, 137.2, 151.6, 156.0; Anal. calcd for C 11 H 15 N 5 O: C, 56.64; H, 6.48; N, 30.02. Found: C, 56.55; H, 6.56; N, 29.86. (?)-(1?,2?)-2-(3-[7-amino--1-methyl-1H-pyrazolo[4,3d]pyrimidyl]) cyclopentanol (1). Ammonia gas was introduced to a solution of compound 7 (2.0 g, 7.2 mmol) in MeOH (100 mL). This reaction mixture was allowed to stir at room temperature until TLC analysis indicated starting material was no longer present. The solvent was removed in vacuo and the residue purified by chromatography (CH 2 Cl 2 /MeOH, 10:1) to afford mixtures including deacetylated product and unknown compounds. Take these mixtures (around 1.5g >100%) followed hydrogenation (A catalytic amount of Pd/C (1%) was added to a solution of the mixtures (1.5 g, 6.3 mmol) in methanol (30 mL) at room temperature, the resulting mixture was allowed to shake under 20 psi of hydrogen overnight. Concentrated in vacuo to give another mixture (1.5 g, >100%) as a yellow liquid. The crude product thus isolated was used in the next step without further purification. A solution of this mixture (1.5 g) in ethanol (30 mL) was stirred with formamidine acetate (1.36 g, 13.4 mmol) under reflux for 3 h. The reaction 83 mixture was cooled to room temperature. The solvent was removed in vacuo and the residue purified by chromatography (CH 2 Cl 2 /MeOH, 10:1) to afford 1 (0.24 g, 1.0 mmol, 20% for three steps)as a white solid, mp = 215 ?C (dec); 1 H NMR (250 MHz, DMSO-d 6 ): ? 1.62 (m, 1H), 1.75 (m, 2H), 1.92 (m, 2H), 2.07 (m, 1H), 3.18 (m, 1H), 4.15 (s, 3H), 4.37 (m, 1H), 7.26 (br s, 2H), 8.12 (s, 1H); 13 C NMR (62.9 MHz, DMSO-d 6 ) ? 22.1, 29.4, 34.1, 38.8, 45.9, 76.8, 121.8, 141.0, 145.5, 150.8, 151.2; Anal. calcd for C 11 H 15 N 5 O: C, 56.64; H, 6.48; N, 30.02. Found: C, 56.46; H, 6.51; N, 29.75. (?)-(1?, 2?)-2-(3, 3-Diethoxy-prop-1-ynyl) cyclohexanol (14). To a solution of propiolaldehyde diethyl acetal (97%) (10.7 mL, 75 mmol) in anhydrous tetrahydrofuran (225 mL) at ?78 ?C under an atmosphere of nitrogen was added n-butyllithium (31.5 mL, 79 mmol, 2.5 M solution in hexanes) over 5 min. The reaction was stirred for ca. 30 minutes then cyclohexane oxide (98%) (7.4 g, 75 mmol) in anhydrous tetrahydrofuran (75 mL) was added, followed by neat BF 3 ?OEt 2 (9.5 mL, 75 mmol,) over 15 min. The reaction mixture was stirred at -78 ?C for 2 h. The reaction was quenched by addition of saturated aqueous sodium bicarbonate solution and partitioned between ethyl acetate and water. The combined organic phases were washed with brine, dried (Na 2 SO 4 ), filtered and evaporated in vacuo to afford a liquid residue. Purification by flash column chromatography (eluting with 25-33% ethyl acetate/hexanes) furnished the secondary alcohol (10.9 g, 64.2%) as a light yellow oil; 1 H NMR (400 MHz, CDCl 3 ) ? 1.21 (m, 9H, ), 1.35 (m, 1H), 1.62 (m, 1H), 1.70 (m, 1H), 1.97 (m, 2H), 2.27(m, 1H), 2.72 (br s, 1H), 3.48 (m, 1H), 3.57 (m, 2H), 3.71 (m, 2H), 5.26 (d, 1H, J=1.5 Hz); 13 C NMR (100 MHz, CDCl 3 ) ? 15.4, 24.4, 25.0, 31.9, 33.5, 39.0, 61.0, 73.3, 78.0, 87.8, 91.8; Anal. calcd for C 13 H 22 O 3 ?0.2H 2 O: C, 67.91; H, 9.84. Found: C, 67.71; H, 9.73. 84 (?)-(1?, 2?)-2-(1-acetyl-1H-pyrazol-3-yl) cyclohexanol (15). Acetal 14 (9.1 g, 40 mmol) was dissolved in glacial acetic acid (395 mL) and 10% HCl (95 mL), and the mixture was stirred at room temperature for 1 h. To this, a solution of hydrazine monohydrate (24.7 g, 0.493 mol) in glacial acetic acid (190 mL) was added dropwise over 20 min. The resulting solution was heated at reflux overnight and concentrated in vacuo to afford dark-brown oil. The crude product was redissolved in pyridine (188 mL), acetic anhydride (94.8 mL, 1.02 mol) and DMAP were added, and the resulting solution was stirred for 16 h at room temperature. Solvent was removed in vacuo, and the crude residue was redissolved in ethyl acetate (394 mL), washed with 10% HCl, water, and brine, dried, concentrated, and chromatographed to afford 15 (7.0 g, 28 mmol, 70%) as a light yellow oil; 1 H NMR (400 MHz, CDCl 3 ) ? 1.42 (m, 4H), 1.58 (m, 1H), 1.78 (m, 1H), 1.89 (s, 3H), 2.00 (m, 1H), 2.11 (m, 1H), 2.64 (s, 3H), 2.86 (m, 1H), 4.97 (m, 1H), 6.27 (d, 1H, J = 2.8 Hz), 8.11 (d, 1H, J = 2.8 Hz); 13 C NMR (100 MHz, CDCl 3 ) ? 21.5, 22.1, 24.7, 25.4, 32.0, 32.2, 43.1, 75.1, 108.6, 128.9, 159.4, 169.8, 170.6; Anal. calcd for C 13 H 18 N 2 O 3 : C, 62.38; H, 7.25; N, 11.19. Found: C, 62.34; H, 7.26; N, 11.15. (?)-(1?, 2?)-2-(1-acetyl-4-nitro-5-cyano-1H-pyrazol-3-yl) cyclohexanol (17). Trifluoroacetic anhydride (29.4 mL) was added dropwise to a stirred solution of 15 (5.28 g, 21.1 mmol) and ammonium nitrate (16.8 g) in trifluoroacetic acid (248 mL) at 0 ?C. The resulting solution was allowed to warm to room temperature and stirred overnight. The solvent was evaporated on rotavapor at room temperature, then was diluted with methylene chloride, washed with water, satd. sodium bicarbonate and brine, dried, and concentrated in vacuo to give the dinitro compound 16 (6.30 g, 21.1mmol >100%) as a 85 colorless liquid. The crude product thus isolated was committed to the next step without further purification. An analytically pure 16 was prepared by chromatography (hexane:ethyl acetate =4: 1). Yellow oil; 1 H NMR (400 MHz, CDCl 3 ) ? 1.43 (m, 3H), 1.79 (m, 3H), 1.88 (s, 3H), 2.08 (m, 1H), 2.23 (m, 1H), 3.64 (m, 1H), 5.07 (m, 1H), 9.02 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) ? 21.4, 24.5, 25.4, 30.6, 32.1, 41.0, 75.3, 124.7, 135.0, 150.0, 170.4; Anal. calcd for C 11 H 14 N 4 O 6 : C, 44.30; H, 4.73; N, 18.79. Found: C, 44.53, H, 4.75; N, 18.79. A solution of the 1,4-dinitro compound (6.30 g, 21.1 mmol) in ethanol (8 mL) and ethyl acetate (8 mL) was added dropwise over 1 min to a stirred solution of potassium cyanide (1.20 g, 18.4 mmol) in ethanol (22 mL) and water (6 mL). Following an additional 5 min at room temperature, the reaction mixture was neutralized with acetic acid (1 mL) and evaporated on the rotavapor, then diluted with ethyl acetate (100 mL), washed with water and brine, dried, concentrated in vacuo to afford 17 (3.81 g, 13.7 mmol, 65.0%) as a dark syrup. Purification by chromatography (hexane:ethyl acetate:methanol=5:2:1) to afford 17 as a white solid, mp 133.5-134.8 ?C; 1 H NMR (400 MHz, CDCl 3 ) ? 1.39 (m, 3H), 1.78 (s, 3H), 1.80 (m, 3H), 1.98 (m, 1H), 3.60 (m, 1H), 4.97 (m, 1H), 14.96 (brs, 1H); 13 C NMR (100 MHz, CDCl 3 ) ? 20.5, 23.8, 24.5, 29.5, 31.5, 39.7, 73.8, 111.9, 121.5, 133.5, 145.4, 169.5; Anal. calcd for C 12 H 14 N 4 O 4 : C, 51.80; H, 5.07; N, 20.13. Found: C, 51.62; H, 5.21; N, 19.93. (?)-(1? ,2?)-2-(5-cyano-1-methyl-4-nitro-1H-pyrazol-3-yl)cyclohexyl acetate (18) and (?)-(1? ,2?)-2-(5-cyano-2-methyl-4-nitro-1H-pyrazol-3-yl)cyclohexyl acetate (19). To a stirred solution of 17 (1.9 g, 7.0 mmol) in anhydrous THF (150 mL), was added portion wise 60% sodium hydride in mineral oil (0.36 g, 9.0 mmol) at 0 ?C. The 86 mixture was stirred at room temperature for 30 min. and then iodomethane (0.64 mL, 1.4 g, 9.5 mmol) was added dropwise. After a period of 24 h the reaction mixture was absorbed by silica gel, and then purified by silica gel column chromatography. Compound 18 (1.2 g, 4.3 mmol, 60%) and 19 (0.50 g, 1.7 mmol, 25%) was obtained after eluting with a mixture of hexane/ethyl acetate in a ratio of 2:1. Compound 18 was a yellow solid; mp: 108-110 o C; 1 H NMR (250 MHz, CDCl 3 ) ? 1.49 (m, 5H), 1. 85 (m, 1H), 1.89 (s, 3H), 2.05 (m, 1H), 2.20 (m, 1H), 3.61 (m, 1H), 4.09 (s, 3H), 5.06 (m, 1H); 13 C NMR (62.5 MHz, CDCl 3 ) ? 20.5, 23.8, 24.5, 29.5, 31.5, 39.7, 43.5, 73.8, 111.9, 121.5, 133.5, 145.4, 169.5; Compound 19 was a yellow solid, mp 155-156 ?C; 1 H NMR (400 MHz, CDCl 3 ) ? 1.52 (m, 4H), 1.88 (s, 3H), 1.95 (m, 2H), 2.03 (s, 3H), 2.26 (m, 2H), 2.15 (m, 1H), 4.08 (s, 3H), 5.43 (m, 1H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 21.0, 24.9, 30.8, 32.9, 39.3, 45.0, 80.1, 110.7, 122.0, 133.4, 145.2, 171.2. (?)-(1? ,2?)-2-(5-cyano-1-methyl-4-nitro-1H-pyrazol-3-yl) cyclohexanol (20). Ammonia gas was introduced to a solution of compound 18 (1.2 g, 4.3 mmol) in MeOH (100 mL). This reaction mixture was allowed to stir at room temperature until TLC analysis indicated starting material was no longer present. The solvent was then removed in vacuo and the residue purified by chromatography (CH 2 Cl 2 /MeOH, 10:1) to afford 20 (0.51 g, 2.0 mmol, 50%) as a light yellow solid; 1 H NMR (400 MHz, CD 3 OD) ? 1.48 (m, 2H), 1.85 (m, 4H), 2.13 (m, 1H), 2.32 (m, 1H), 3.74 (m, 1H), 4.09 (s, 3H), 4.26 (m, 1H), 5.00 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) ? 23.2, 24.5, 28.6, 35.8, 39.7, 45.8, 77.5, 110.8, 121.8, 134.1, 145.9. (?)-(1?,2?)-2-(3-[7-amino--1-methyl-1H-pyrazolo[4,3d]pyrimidyl]) cyclohexanol (12). A catalytic amount of Pd/C (1%) was added to a solution of 20 (0.51 87 g, 2.0 mmol) in methanol (30 mL) at room temperature, the resulting mixture was allowed to shake under 20 psi of hydrogen overnight. After the reaction was complete, concentrated in vacuo to give reduced compound (0.50 g, 2.0 mmol >100%) as a colorless liquid. The crude product thus isolated was used in the next step without further purification. A solution of reduced compound (0.50 g, 2.0 mmol) in ethanol (15 mL) was stirred with formamidine acetate (0.29 g, 2.9 mmol) under reflux for 3 h. The reaction mixture was cooled to room temperature. The solvent was removed in vacuo and the residue purified by chromatography (CH 2 Cl 2 /MeOH, 6:1) to afford 12 (0.30 g, 1.2 mmol, 60%) as a light yellow solid, mp : 234-235 ?C; 1 H NMR (400 MHz, DMSO-d 6 ): ? 1.46 (m, 3H), 1.77 (m, 4H), 1.96 (m, 1H), 3.95 (td, 1H, J = 5.2, 9.6 Hz), 4.17 (s, 3H), 4.52 (d, 1H, J = 4.8 Hz), 7.20 (br s, 2H), 8.11 (s, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) ? 25.1, 25.9, 31.6, 36.1, 39.2, 44.8, 71.4, 122.0, 141.7, 146.6, 151.0, 151.4. (?)-(1? ,2?)-2-(5-cyano-2-methyl-4-nitro-1H-pyrazol-3-yl) cyclohexanol (21). Ammonia gas was introduced to a solution of compound 19 (0.5 g, 1.7 mmol) in MeOH (50 mL). This reaction mixture was allowed to stir at room temperature until TLC analysis indicated starting material was no longer present. The solvent was then removed in vacuo and the residue purified by chromatography (CH 2 Cl 2 /MeOH, 10:1) to afford 21 (0.34 g, 1.4 mmol, 80%) as a light yellow solid; ?C 1 H NMR (400 MHz, CD 3 OD) ? 1.48 (m, 2H), 1.85 (m, 4H), 2.13 (m, 1H), 2.32 (m, 1H), 3.74 (m, 1H), 4. 08 (s, 3H), 4.25 (m, 1H), 5.00 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) ? 22.2, 24.5, 30.6, 34.5, 39.7, 45.8, 77.3, 113.8, 121.8, 134.1, 145.9. 88 (?)-(1?,2?)-2-(3-[7-amino--2-methyl-1H-pyrazolo[4,3d]pyrimidyl]) cyclohexanol (13). A catalytic amount of Pd/C (1%) was added to a solution of 21 (0.34 g, 1.3 mmol) in methanol (15 mL) at room temperature, the resulting mixture was allowed to shake under 20 psi of hydrogen overnight. After the reaction was complete, concentrated in vacuo to give reduced compound (0.34 g, 1.3 mmol >100%) as a colorless liquid. The crude product thus isolated was used in the next step without further purification. A solution of reduced compound (0.34 g, 1.3 mmol) in ethanol (10 mL) was stirred with formamidine acetate (0.2 g, 2 mmol) under reflux for 3 h. The reaction mixture was cooled to room temperature. The solvent was removed in vacuo and the residue purified by chromatography (CH 2 Cl 2 /MeOH, 6:1) to afford 13 (0.17 g, 0.67 mmol, 50%) as a light yellow solid, mp : 172-174; 1 H NMR (400 MHz, DMSO-d 6 ): ? 1.49-1.95 (m, 6H), 2.07 (m, 1H), 3.15 (m, 1H), 4.13 (s, 3H), 4.37 (d, 1H, J = 5.2 Hz), 4.82 (m, 1H), 7.83 (s, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) ? 21.8, 22.5, 30.4, 34.7, 38.4, 46.1, 77.2, 126.3, 137.5, 142.7, 147.3, 154.2. ((4S, 5S)-5-((tert-butyldimethylsilyloxy) methyl)-2, 2-dimethyl-1, 3-dioxolan- 4-yl) methanol (30). To a well stirred 60% sodium hydride in mineral oil suspension (1.56 g, 65.1 mmol) in 30 mL THF at room temperature under N 2 was added the solution of the diol 28 (5.00 g, 31.0 mmol) in 30 mL THF dropwise. The reaction mixture was stirred at room temperature for 45 min, during which time a large amount of opaque white precipitate had formed. TBSCl (4.67 g, 31.0 mmol) in 30 mL THF was added and the stirring was continued for additional 45 min. The reaction mixture was diluted with 100 mL EtOAc and washed with 10% aqueous K 2 CO 3 , H 2 O, brine and dried with MgSO 4 . 89 The solvent was removed in vacuo and the residue was purified with flash chromatography (silica, 1:3 EtOAc:hexane) to give 7.3 g (85%) of 30 as a colorless oil. 1 H-NMR (400 MHz, CDCl 3 /TMS): ? 0.06 (m, 6H), 0.90 (m, 9H), 1.39 (m, 6H), 3.66 (m, 2H), 3.68 (m, 1H), 3.74 (m, 2H), 3.82 (m, 2H). 13 C-NMR (100 MHz, CDCl 3 /TMS): ? = - 5.5, 18.3, 25.8, 26.9, 27.0, 62.7, 63.7, 78.0, 80.0, 109.1ppm. The spectroscopic data were in accord with those reported in the literature. 168 (4R, 5S)-5-((tert-butyldimethylsilyloxy) methyl)-2, 2-dimethyl-1, 3-dioxolane- 4-carbaldehyde (31). To a -5 o C solution of 30 (4.01 g, 14.5 mmol) in CH 2 Cl 2 (85 mL) and DMSO (12 mL) was added DIPEA (6.20 ml, 35.4 mmol), followed by slow addition of a solution of SO 3 ? Pyridine (5.9 g, 29 mmol) in DMSO (18 mL). The reaction mixture was stirred at this temperature for 2 h, at which point it was diluted with Et 2 O (200 mL) and rinsed with water, 5% NaHCO 3 , 10% CuSO 4 and brine. The organic solution was dried over anhydrous MgSO 4 . The solvent was removed under vacuum and the residue purified by column chromatography (EtOAc/hexanes = 1:3) to afford the aldehyde 31 as a colorless oil (3.5 g, 87 %); 1 H-NMR (400 MHz, CDCl 3 /TMS): ? 0.08 (m, 6H), 0.89 (m, 9H), 1.39 (d, 3H, J = 0.8 Hz), 1.46 (d, 3H, J = 0.8 Hz); 3.79 (m, 2H), 4.09 (m, 1H), 4.31 (dd, 1H, J = 1.6, 7.2 Hz), 9.75 (d, 1H, J = 2.0Hz) ppm. 13 C-NMR (100 MHz, CDCl 3 /TMS): ? -5.5, 18.3, 25.8, 26.3, 26.8, 62.8, 77.5, 81.9, 111.4, 200.7 ppm. The spectroscopic data were in accord with those reported in the literature. 168 Tert-butyl (((4S, 5S)-2, 2-dimethyl-5-vinyl-1, 3-dioxolan-4-yl) methoxy) di- methylsilane (32). Methyltriphenylphosphonium bromide (4.5 g, 15 mmol) was added in portions to a suspension of t-BuOK (1.56 g, 15.9 mmol) in anhydrous ether (50 mL) at 0 o C. After the resulting suspension was stirred at 0 o C for 1 h and rt for 1.5 h, it was cooled 90 to 0 o C and treated dropwise with a solution of aldehyde 31 (3.40 g, 12.4 mmol) in anhydrous ether (20 mL). The reaction was then stirred overnight at room temperature and filtered to remove a solid. The filtrate was concentrated under reduced pressure and the residue purified by column chromatography (EtOAc/hexanes = 1:7) to give 32 as a colorless oil (2.6 g, 80%). 1 H-NMR (400 MHz, CDCl 3 /TMS): ? 0.02 (m, 6H), 0.85 (m, 9H), 1.35 (d, 6H, J = 1.2 Hz), 3.69 (m, 3H), 4.29 (m, 1H), 5.16 (m, 1H), 5.27 (m, 1H), 5.85 (m, 1H) ppm. 13 C-NMR(100 MHz, CDCl 3 /TMS): ? = -5.4, 18.3, 25.9, 26.9, 27.0, 79.3, 81.3, 109.1, 118.0, 135.8 ppm. 2-((4S, 5S)-5-((tert-butyldimethylsilyloxy) methyl)-2, 2-dimethyl-1, 3- dioxolan-4-yl) ethanol (33). To 32 (1.0 g, 3.7 mmol) in THF (20 mL) at 0 o C under N 2 was added 9-BBN-H (0.5 M in THF, 9.3 mL, 4.6 mmol), and the mixture stirred for 3 hours. Sodium hydroxide (1 M, 5.6 mL) and then H 2 O 2 (33% in water, 2.8 mL) were added and the stirring continued for a further 30 mins. The reaction mixture was diluted with EtOAc and then washed with saturated NaHCO 3 . The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give crude product, which was purified by flash column chromatography (EtOAc/hexanes = 1:5) to yield 33 as a colorless oil (1.00 g, 93.5%). 1 H-NMR (400 MHz, CDCl 3 /TMS): ? 0.09 (m, 6H), 0.91(m, 9H), 1.40 (s, 3H), 1.44 (s, 3H), 1.84-1.98 (m, 2H), 3.69-3.73 (m, 1H), 3.79-3.83 (m, 4H), 4.08 (m, 1H) ppm; 13 C-NMR (100 MHz, CDCl 3 /TMS): ? -5.4, 18.4, 25.9, 26.9, 27.3, 35.6, 60.8, 63.7, 78.6, 80.8, 108.8 ppm. 2-((4S, 5S)-5-(hydroxymethyl)-2, 2-dimethyl-1, 3-dioxolan-4-yl) ethanol (27). To a solution of 33 (1.00 g, 3.45 mmol) in dry THF (40 mL) was added 1.0 M TBAF in THF (6.9 mL, 6.9 mmol) at 0 o C. The reaction mixture was stirred at rt overnight and the 91 solvent was evaporated under reduced pressure. The residue was dissolved in CH 2 Cl 2 and the resulted solution was washed with brine. After drying over anhydrous Na 2 SO 4 and filtration, the residue was purified by column chromatography (EtOAc/hexanes = 1:2) to give 27 as a colorless oil (0.39 g, 65%). 1 H-NMR (400 MHz, CDCl 3 /TMS): ? 1.41 (m, 6H), 1.84-1.87 (m, 2H), 3.03 (brs, 2H), 3.61-3.69 (m, 1H), 3.76-3.82 (m, 4H), 4.06 (m, 1H). 13 C NMR (100 MHz, CDCl 3 /TMS): ? 27.1, 27.4, 35.4, 60.3, 62.0, 76.3, 81.4, 109.1. (((4S,5S)-5-allyl-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)(tert-butyl) dimethylsilane (36). To a -5 o C solution of 33 (3.4 g, 11 mmol) in CH 2 Cl 2 (70 mL) and DMSO (10 mL) was added DIPEA (4.80 mL, 27.4 mmol), followed by slow addition of a solution of SO 3 ? pyridine (4.5 g, 22 mmol) in DMSO (15 ml). The reaction mixture was stirred at this temperature for 2 hour, at which point it was diluted with CH 2 Cl 2 (200 mL) and rinsed with water, 5% NaHCO 3 and brine. The organic solution was dried over anhydrous MgSO 4 . The solvent was removed under vacuum and the residue purified by column chromatography (EtOAc/hexanes = 1:3) to afford the unpure aldehyde 35 as a colorless oil (3.40 g, 100%), The crude product thus isolated was used in the next step without further purification. Methyltriphenylphosphonium bromide (4.30 g, 14.1 mmol) was added in portions to a suspension of t-BuOK (1.47 g, 15.0 mmol) in anhydrous ether (50 mL) at 0 o C. After the resulting suspension was stirred at 0 o C for 1 h and rt for 1.5 h, it was cooled to 0 o C and treated dropwise with a solution of the above aldehyde 35 (3.40 g, 12.4 mmol) in anhydrous ether (20 mL). The reaction was then stirred overnight at room temperature and filtered to remove a solid. The filtrate was concentrated under reduced pressure and the residue purified by column chromatography (EtOAc/hexanes = 1:7) to give 36 as a 92 colorless oil (1.7 g, 50% for two steps). 1 H-NMR (400 MHz, CDCl 3 /TMS): ? 0.09 (m, 6H), 0.93 (s, 9H), 1.41 (s, 3H), 1.44 (s, 3H), 2.39-2.47 (m, 2H), 3.72-3.79 (m, 3H), 3.80- 4.01 (m, 1H), 5.11-5.18 (m, 2H), 5.86-5.93 (m, 1H). 13 C-NMR (100 MHz, CDCl 3 /TMS): ? -5.4, 18.4, 25.9, 27.0, 27.4, 37.6, 63.6, 77.8, 80.6, 108.6, 117.4 133.7. ((4S, 5S)-5-allyl-2, 2-dimethyl-1, 3-dioxolan-4-yl) methanol (37). To a solution of 36 (1.2 g, 4.2 mmol) in dry THF (80 mL) was added 1.0 M TBAF in THF (8.4 mL, 8.4 mmol) at 0 o C. The mixture was stirred at room temperature overnight and the solvent was evaporated under reduced pressure. The residue was dissolved in CH 2 Cl 2 and the resulting solution was washed with brine. After drying over anhydrous Na 2 SO 4 and filtration, the residue was purified by column chromatography (EtOAc/hexanes = 1:2) to give 37 as a colorless oil (0.60 g, 83%). 1 H-NMR (250 MHz, CDCl 3 /TMS): ? 1.43 (s, 6H), 2.21 (br, 1H), 2.38-2.44 (m, 2H), 3.58-3.65 (m, 1H), 3.77-3.86 (m, 2H), 3.95-4.03 (m, 1H), 5.11-5.20 (m, 2H), 5.77-5.91 (m, 1H). 13 C-NMR (100 MHz, CDCl 3 /TMS): ? 27.0, 27.3, 37.3, 61.9, 75.8, 81.1, 108.8, 117.9, 133.5. (4R, 5S)-5-allyl-2, 2-dimethyl-1, 3-dioxolane-4-carbaldehyde (38). To a -5 o C solution of 37 (0.17 g, 1.0 mmol) in CH 2 Cl 2 (6 mL) and DMSO (0.8 mL) was added DIPEA (0.4 mL), followed by slow addition of a solution of SO 3 ? pyridine (0.4 g, 2 mmol) in DMSO (1 mL). The reaction mixture was stirred at this temperature for 2 h, at which point it was diluted with CH 2 Cl 2 (20 mL) and rinsed with water, 5% NaHCO 3 and brine. The organic solution was dried over anhydrous MgSO 4 . The solvent was removed under vacuum and the residue purified by column chromatography (EtOAc/hexanes = 1:3) to afford the aldehyde 38 as a colorless oil (0.10 g, 60%). 1 H-NMR (400 MHz, CDCl 3 /TMS): ? 1.51 (m, 6H), 2.39-2.57 (m, 2H), 3.62-3.79 (m, 1H), 4.03-4.11 (m, 1H), 93 5.13-5.21 (m, 2H), 5.84-5.88 (m, 1H), 9.76 (d, 1H, J = 2Hz). 13 C-NMR (100 MHz, CDCl 3 /TMS): ? 26.2, 27.0, 37.3, 76.2, 84.0, 111.1, 118.7, 132.7, 201.1. (Z)-Cyclopentene-3, 5-diol diacetate (42). Dicyclopentadiene was cracked by distillation maintaining the distillation head at around 40 ?C by controlling the temperature of heating mantle at 160~180 ?C. Cyclopentadiene (254 g, 3.80 mol) was obtained which was immediately dissolved in 2200 mL CH 2 Cl 2 . Sodium carbonate (1000 g, 9.420 mol) was then added portionwise and a suspension was obtained. The suspension was cooled down to -10 ?C and then treated with a solution of sodium acetate (20 g, 0.30 mol) in 500mL of peracetic acid (32% in acetic acid) dropwise. The temperature was maintained at -10 ?C to -5 ?C during the addition. After the addition, the resulting mixture was stirred at room temperture overnight. The mixture was filtered and the filtrate was evaporated to give a pale yellow liquid (250 g) which was the crude monoepoxide (6-Oxabicyclo [3.1.0] hex-2-ene). This crude product was used directly in the next step. Fresh tetrakis(triphenylphosphine)palladium(0) 82 was prepared at first. PdCl 2 (5.07 g, 28.6 mmol) and triphenylphosphine (38.01 g, 145.0 mmol) were added to 340 mL anhydrous dimethyl sulfoxide. The mixture was heated to about 160?C under a nitrogen atmosphere until complete solution occurred. The heat was taken away and stirring continued for 5 min. Hydrazine hydrate (4.05 g, 81.0 mmol) was added dropwise in 1 min with rapid stirring. The solution was cooled to room temperture with water bath and yellow crystals appeared. Solid was collected by filtration and washed with 2 ? 30 mL ethanol and 2 ? 30 mL ether. Product as a light yellow solid (30.5 g) was dried and kept under nitrogen, whose 1 H and 13 C NMR spectral data agreed with literature. 168 94 A solution of crude monoepoxide from last step in 200 mL THF was added dropwise to a dry ice acetone cooled solution of tetrakis(triphenylphosphine) palladium(0) (7.0 g) in 600 mL dry THF and acetic anhydride (450 g, 4.41 mol) at 0 ?C to 5 ?C. After addition, the resulting mixture was stirred at rt overnight. Filtration of the resulting mixture through a pad of silica gel removed the catalyst. Ethyl ether (2 ? 100 mL) was used to wash it. Evaporation of the solvent afforded a dark residue. Fractional distillation afforded 42 (150 g, 85.0% for three steps) as a pale yellow oil, whose 1 H and 13 C NMR spectral data agreed with literature. 82 (+)-(1R, 4S)-4-Hydroxy-2-cyclopenten-1-yl acetate ((+)-39). Compound 42 (280 g, 1.52 mol) was added to 0.1 M phosphate buffer (1000 mL). The pH of the resulting suspension was adjusted to 7 by addition of 6 N NaOH dropwise. Pseudomonas cepacia lipase (20 g, Amano International Enzyme Corporation) was added to the mixture. The mixture was stirred and the pH of the mixture was kept constant around 7 during the hydrolysis by the continuous addition of 1 N NaOH. After the addition of 1.5 L of NaOH solution, the reaction mixture was filtered through a celite pad. The filtrate was extracted with 3 ? 800 mL ethyl acetate. The combined organic phases were dried over anhydrous MgSO 4 . Evaporation of the solvent under reduced pressure afforded yellow oil. The residual oil was fractionally distilled (bp is around 80 ?C under reduced pressure to give (+)-39 (172 g, 80.2%) as a light yellow solid, whose 1 H and 13 C NMR spectral data agreed with literature. 167 (1R, 4S)-4-(tert-butyldimethylsilyl) cyclopent-2-enyl acetate (44). To 1.42 g (10 mmol) of monoacetate 39 dissolved in 20 mL of anhydrous CH 2 Cl 2 , was added 1.8 g (12 mmol) of tert-butyldimethylchlorosilane and 0.8 g (12 mmol) of imidazole. The 95 mixture was stirred for 4 h and was subsequently added to 20 mL of 1:l ether/hexanes and 10 mL of water. The organic layer was separated, and the aqueous layer was extracted repeatedly with ether. The combined ether layer was washed with saturated sodium dihydrogen phosphate and dried. Concentration and evaporation of the volatiles gave 2.30 g (90%) of the desired product which was used for the subsequent reaction. The 1 H and 13 C NMR spectral data agreed with literature. 179 2,3-O-Isopropylidene-D-ribose (49). To a stirred suspension of D-ribose (8.0 g, 53 mmol) in acetone (100 mL) was added dropwise conc. H 2 SO 4 (1 mL) at room temperature and the reaction mixture was stirred at room temperature for 2.5 h. The mixture was neutralized with solid NaHCO 3 , filtered and evaporated under reduced pressure to give colorless syrup. The residue was purified by silica gel column chromatography using hexane and ethyl acetate (1:2) as the eluent to afford 49 as colorless syrup (9.4 g, 93%): 1 H NMR (MeOH-d 4 ), ? = 1.31 (s, 3H, CH 3 ), 1.44 (s, 3H, CH 3 ), 3.59 (dd, 1H, J=5.6, 12.0 Hz), 3.63 (dd, 1H, J=4.8, 12.0 Hz), 4.19 (irregular t, 1H, J=4.4, 5.2 Hz), 4.52 (d, 1H, J=6.0 Hz), 4.77 (d, 1H, J=6.0 Hz), 5.26 (s, 1H). All the spectral data were identical to the literature. 172,179 (3aR,6R,6aR)-6-((tert-butyldimethylsilyloxy)methyl)-2,2 dimethyltetrahydro -furo [3, 4-d][1, 3] dioxol-4-ol (50). To 16 g (84 mmol) of 40 dissolved in 100 mL of anhydrous CH 2 Cl 2 was added 13.9 g (92.4 mmol) of tert-butyldimethylchlorosilane, 8.56 g (125 mmol) of imidazole and a catalytic amount of DMAP. The mixture was stirred for 4 h and was subsequently added to 200 mL of 1:l ether/hexane and 100 mL of water. The organic layer was separated, and the aqueous layer was extracted repeatedly with ether. The combined organic layer was washed with saturated sodium dihydrogen phosphate 96 and dried. Concentration and evaporation of the volatiles gave 21.9 g (85.1%) of the desired product which was used for the subsequent reaction. The 1 H and 13 C NMR spectral data agreed with literature. 172 2-(tert-Butyldimethylsilyloxy)-1-((4R,5S)-2,2-dimethyl-5-vinyl-1,3-dioxolan- 4-yl)ethanol (51) and 2-(tert-butyldimethylsilyloxy)-2-((4S,5S)-2,2-dimethyl-5-vinyl- 1,3-dioxolan-4-yl)ethanol (52). A suspension of NaH (2.5 g, 0.062 mol, 60% dispersion in mineral oil) in tetrahydrofuran (250 mL) was cooled to 0 ?C, and DMSO (7.3 mL, 0.10 mol) was added. After being stirred at room temperature for 0.5 h, the resulting white suspension was cooled to 0 ?C and treated with methyltriphenylphosphonium bromide (22.2 g, 62.2 mmol). The reaction mixture was stirred at room temperature for 1 h and then recooled to 0 ?C. A solution of lactol 50 (12.8 g, 42.0 mmol) in tetrahydrofuran (80 mL) was added to the resulting reaction mixture at 0 ?C. After being heated at reflux for 3 h, the reaction mixture was cooled to room temperature. Diethyl ether (300 mL) was added to the reaction mixture and washed with H 2 O (100 mL) and brine (100 mL). The organic layer was dried over MgSO 4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane=1/10), giving compound 51 (3.2 g, 26%) as a colorless oil, and 52 (3.1 g, 24%) as a colorless oil. For 51 and 52 all the spectral data were identical to the literature. 172,179 (3a'R,6'R,6a'R)-6'-(hydroxymethyl)tetrahydrospiro[cyclopentane-1,2'-furo [3,4-d][1,3]dioxol]-4'-ol (54). To a stirred suspension of D-ribose (15.0 g, 100 mmol) in cyclopentanone (47.5 g, 565 mmol, 50.0 mL) was added dropwise conc. H 2 SO 4 (1 mL) at room temperature and the reaction mixture was stirred at room temperature for 2.5 h. The mixture was neutralized with NH 3 ?H 2 O, evaporated under reduced pressure to give 97 brown syrup, which was added with CH 2 Cl 2 (500 mL) to the reaction mixture and washed with H 2 O (100 mL) and brine (100 mL). The organic layer was dried over MgSO 4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography using hexane and ethyl acetate (1:2) as the eluent to afford 54 as yellow syrup (16.2 g, 75.0 %): 1 H NMR (400 MHz, CDCl 3 /TMS), ? 1.61-1.80 (m, 6H), 1.94-2.07 (m, 2H), 3.71-3.83 (m, 2H), 4.33 (s, 1H), 4.47 (d, 1H, J = 6.0 Hz), 4.80 (d, 1H, J = 6.0 Hz), 5.09 (s, 1H). 13 C NMR (100 MHz, CDCl 3 /TMS): ? 23.2, 23.6, 35.7, 35.8, 65.3, 81.7, 86.4, 87.4, 106.5, 121.5. 1-((2R,3S)-3-vinyl-1,4-dioxaspiro[4.4]nonan-2-yl)ethane-1,2-diol (55). To a stirred suspension of methyl triphenylphosphonium bromide (104 g, 289 mmol) in THF (1.0 L) was added potassium t-butoxide (37.9 g, 310 mmol, purity of reagent: 95%) at 0 ?C. The reaction mixture was stirred at 0 ?C for 20 mins and then at rt for 1 h. After the mixture was allowed to cool to 0 ?C, a solution of 54 (21.6 g, 100 mmol) in THF (300 mL) was added and the mixture was stirred at rt overnight. To this mixture was carefully added water (200 mL) and the mixture was extracted with ethyl acetate (4 * 500 mL), dried, filtered and evaporated under reduced pressure to give yellow syrup. This syrup was purified by silica gel column chromatography using hexane and ethyl acetate (1:2? 1:2.5) as the eluent to give 55 (23 g) with the contamination of triphenylphosphine oxide, which was used for next reaction without further purification: 1 H NMR (400 MHz, CDCl 3 /TMS), ? 1.65-1.77 (m, 6H), 1.78-1.97 (m, 2H), 2.87 (t, 1H, J = 5.6 Hz), 3.01 (d, 1H, J = 5.2 Hz), 3.70?3.76 (m, 2H), 3.81-3.87 (m, 1H), 4.05 (dd, J = 6.4, 1H, 8.4 Hz), 4.63 (t, 1H, J = 6.4 Hz), 5.32 (td, 1H, 1H, J = 1.2, 10.4 Hz), 5.46 (td, 1H, J = 1.6, 17.2 98 Hz), 6.03 (ddd, 1H, J = 6.8, 10.4, 17.6); 13 C NMR (100 MHz, CDCl 3 /TMS): ? 23.2, 23.7, 36.7, 37.1, 64.5, 70.0, 78.1, 78.4, 118.9, 132.1, 132.8. (2S,3S)-3-vinyl-1,4-dioxaspiro[4.4]nonane-2-carbaldehyde (56). To a solution of crude 55 (23 g) in methylene chloride (320 mL) was added aqueous NaIO 4 (160 mL, 22.2 g, 0.650 M solution) at rt and the reaction mixture was stirred at the same temperature for 30 mins. After methylene chloride (300 mL) and water (300 mL) were added, the organic layer was filtered, dried and evaporated under reduced pressure. The residue was purified by short column silica gel chromatography using hexane and ethyl acetate (5:1) as the eluent to give 56 as colorless oil (15.5 g, 85.1 % from 54). 1 H NMR (400 MHz, CDCl 3 /TMS), ? 1.74-1.81 (m, 6H), 2.04-2.10 (m, 2H), 4.34 (dd, 1H, J = 3.2, 7.6 Hz), 4.75 (t, 1H, J = 7.6 Hz), 5.34 (td, 1H, J = 1.2, 10.4 Hz), 5.48 (td, 1H, J = 1.2, 17.2 Hz), 5.77 (ddd, 1H, J = 6.8, 10.4, 17.2 Hz), 9.55 (d, 1H, J = 3.6 Hz). 13 C NMR (100 MHz, CDCl 3 /TMS): ? 23.1, 24.0, 36.6, 36.8, 79.2, 81.8, 119.8, 121.1, 131.2, 200.7. 1-((2R,3S)-3-vinyl-1,4-dioxaspiro[4.4]nonan-2-yl)prop-2-en-1-ol (57). To a stirred solution of aldehyde (20.0 g, 110 mmol) in tetrahydrofuran (800 mL) was added vinylmagnesium bromide (1 M solution in tetrahydrofuran, 220 mL, 220 mmol) at -78 ?C, and the reaction mixture was stirred at -40 ?C for 1 h. After the mixture was allowed to warm to room temperature, the mixture was quenched with saturated ammonium chloride solution and brine, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, concentrated to dryness. The resulting oil was purified by silica gel column chromatography (hexanes/ethyl acetate=7:1) to give diene 57 (19.6 g, 85.2%) as an inseparable mixture of diastereomers. 1 H NMR (400 MHz, CDCl 3 /TMS) for separated isomers : ? 1.72-1.79 (m, 6H), 1.97-2.02 (m, 2H), 4.00-4.03 (m, 1H), 4.04-4.15 (m, 1H), 99 4.46-4.58 (m, 1H), 5.22-5.49 (m, 4H), 5.81-6.15 (m, 1H); the other isomer: ? 1.67-1.74 (m, 6H), 1.91-1.94 (m, 2H), 3.91-3.97 (m, 1H), 4.09-4.18 (m, 1H), 4.56-4.61 (m, 1H), 5.19-5.46 (m, 4H), 5.97-6.08 (m, 1H). (+) - (4S, 5S)-4, 5-O-Cyclopentyl-2-cyclopentenone (53). To a solution of diene 57 (22.0 g, 105 mmol) in anhydrous CH 2 Cl 2 (500 mL) was added the first generation Grubbs catalyst (432 mg, 1 mol %) after the solution was flushed with N 2 for 20 min. After stirring at room temperature for 12 h, DMSO (130 mL) DIPEA (17.5 mL, 105 mmol) and SO 3 ? pyridine (19 g, 93 mmol) were added to the dark brown solution. The reaction mixture was stirred at room temperature overnight and filtered over silical gel pad with CH 2 Cl 2 . The filtrate was concentrated in vacuum and residue was purified by column chromatography (hexanes/ethyl acetate=5:1) to afford 53 as a white crystal (13.9 g, 74.0% from 57). [?] 20 D ?69.4 (c = 0.60, CHCl 3 ); mp = 69-70 ? C; 1 H NMR (400 MHz, CDCl 3 ): ? 1.62-1.78 (m, 6H), 1.81-1.90 (m, 2H), 4.43 (d, 1H, J = 5.6 Hz), 5.25 (ddd, 1H, J = 0.8, 2.4, 5.6 Hz), 6.31(d, 1H, J = 6.0 Hz), 7.65 (dd, 1H, J = 2.4, 6.0 Hz); 13 C NMR (100 MHz, CDCl 3 ) ? 23.2, 24.0, 37.3, 37.9, 76.1, 78.1, 124.2, 135.4, 159.8, 203.9; Anal. calcd for C 10 H 12 O 3 : C, 66.65; H, 6.71. Found: C, 66.77; H, 6.66. Analytic pure 58 was prepared by silica gel column chromatography (hexanes/ethyl acetate=5:1). ?-Alcohol of 58, colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 1.61-1.71 (m, 6H), 1.72-1.88 (m, 2H), 2.80 (d, 1H, J = 9.2 Hz), 4.52 (dd, 1H, J =6.4, 9.2 Hz), 4.64 (t, 1H, J = 5.6 Hz), 4.98 (dt, 1H, J = 0.8, 5.6 Hz), 5.85-5.89 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) ? 23.3, 23.4, 37.2, 37.6, 74.1, 77.1, 83.4, 121.7, 131.7, 136.9; 100 ?-Alcohol of 58, colorless oil: 1 H NMR (250 MHz, CDCl 3 ) ? 1.61-1.75 (m, 6H), 1.75-1.88 (m, 2H), 2.25 (d, 1H, J = 5.25 Hz), 4.47 (d, 1H, J =5.75 Hz), 4.83 (s, 1H), 5.27 (dt, 1H, J = 0.75, 5.75 Hz), 5.96 (m, 1H), 6.04 (m, 1H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 23.5, 23.7, 36.8, 37.2, 81.1, 84.3, 85.9, 121.4, 135.4, 135.6. 4-[2-(trirnethylsilyl)ethynyl]-2, 3-O-Cyclopentylcyclopentone (59). To 5.46 g (7.70 mL, 55.6 mmol) of trimethylsily1 acetylene in 120 mL of ether at 0 ?C was added 22.2 mL (55.6 mmol) of a 1.6 M solution of n-BuLi in hexane. The reaction mixture was allowed to stir at -40 ?C for 1.5 h and was then added dropwise to an ether solution of 55.6 mL (55.6 mmol) of 1.0 M solution of dimethylaluminum chloride in hexane at room temperature. The reaction mixture was allowed to stir at this temperature for 3.5 h. To 1.43 g (5.56 mmol) of Ni(acac) 2 in 50 mL of ether at -3 ?C was added 5.56 mL (5.56 mmol) of a 1.0 M solution of DIBAH in hexane. The reaction mixture was allowed to stir at 0 ?C for 10 min and was then cooled to -25 ?C, after which the above solution of dimethyl[2-(trimethylsilyl)ethynyl]aluminum was added. Then 5.00 g (27.8 mmol) of cyclopentenone 53 in 100 mL of ether was added dropwise to the reaction mixture over 2.5 h. The reaction mixture was stirred at -30 ?C for another 6 h. The reaction mixture was hydrolyzed with saturated aqueous KH 2 PO 4 (100 mL) for overnight. The organic layer was extracted with ether, and the ether layer was then washed with brine, dried over Na 2 SO 4 , and concentrated. The residue was purified by column chromatography (hexanes/ethyl acetate=20:1) to afford the conjugate adduct 59 as a yellow crystal (3.2 g, 42%). mp = 64-65 ? C; 1 H NMR (400 MHz, CDCl 3 ): ? 0.15 (s, 9H), 1.68-1.88 (m, 8H), 2.42 (dt, 1H, J = 1.75, 18.0 Hz), 2.88 (dd, 1H, J = 8.75, 18.0 Hz), 3.32 (dd, 1H, J = 0.75, 8.75 Hz), 4.35 (d, 1H, J = 5.0 Hz), 4.73 (d, 1H, J = 5.0 Hz). 13 C NMR (62.9 MHz, 101 CDCl 3 /TMS): ? -0.13, 23.0, 23.8, 29.7, 36.1, 36.3, 40.5, 78.0, 81.4, 89.2, 104.5, 122.7, 212.3 ppm. Anal. calcd for C 15 H 22 O 3 Si: C, 64.71; H, 7.96. Found: C, 64.90; H, 7.99. (1R, 2R, 3S, 4S)-4-[2-(trirnethylsilyl)ethynyl]-2, 3-O-Cyclopentylcyclopentan- 1-ol (60). Sodium borohydride (0.30 g, 7.1 mmol) was added portionwise to a solution of 59 (1.3 g, 4.7 mmol) and cerium(III)chloride heptahydrate (1.5 g, 4.0 mmol) in methanol (50 mL) at 0 ?C. After 1.5 hr, the solvent was removed and the residue was neutralized by saturated NH 4 Cl solution. Extracted by CH 2 Cl 2 (3*50 mL) and the organic layer was washed with brine, dried over anhydrous Na 2 SO 4 and concentrated. The residue was purified by column chromatography (hexanes/ethyl acetate=10:1) to afford 60 as a white crystal (0.98 g, 75%). mp = 44-45 ? C; 1 H NMR (250 MHz, CDCl 3 ): ? 0.15 (s, 9H), 1.65- 1.81 (m, 6H), 1.87-1.98 (m, 3H), 2.02 (dd, 1H, J = 2.3, 6.0 Hz), 2.33 (d, 1H, J = 9.5 Hz), 2.87 (d, 1H, J = 6.8 Hz), 4.29 (ddd, 1H, J = 6.0, 10.0, 15.3 Hz), 4.49 (m, 2H). 13 C NMR (100 MHz, CDCl 3 /TMS): ? -0.01, 22.8, 24.0, 33.3, 35.1, 35.5, 36.5, 71.9, 78.4, 84.0, 87.2, 106.0, 121.1 ppm. Anal. calcd for C 15 H 24 O 3 Si: C, 64.24; H, 8.63 Found: C, 64.12; H, 8.69. (1S, 2R, 3S, 4S)-4-[2-(trimethylsilyl)ethynyl]-2, 3-O-Cyclopentylcyclopentan- 1-benzoate (61). A solution of triphenylphosphine (1.8 g, 6.8 mmol) in dry THF (50 mL) was cooled to -20 ? C, and diisopropyl azodicarboxylate (1.4 mL, 6.8 mmol) was added over a period of 10 min. This mixture was stirred at -20 ? C for 20 min to yield a white precipitate of triphenylphosphine-diisopropyl azodicarboxylate complex. To this latter complex as a suspension were added a solution of 60 (1.6 g, 5.7 mmol) in dry THF (10 mL) and benzoic acid (0.84 g, 6.8 mmol). The cooling bath was removed, and the reaction mixture was stirred at room temperature for 2 h. After evaporation of the 102 reaction mixture to dryness, the residue was purified by column chromatography (hexanes/ethyl acetate=10:1) to afford as a white crystal 61 (1.54 g, 69.8%). mp = 92-93 ? C; 1 H NMR (400 MHz, CDCl 3 ): ? 0.08 (s, 9H), 1.68-1.78 (m, 6H), 1.89-1.94 (m, 2H), 2.14 (d, 1H, J = 14.0 Hz), 2.51 (ddd, 1H, J = 4.8, 8.0, 14.0 Hz), 3.15 (d, 1H, J = 8.0 Hz), 4.68 (d, 1H, J = 5.8 Hz), 4.75 (d, 1H, J = 5.8 Hz), 5.33 (d, 1H, J = 4.8 Hz ), 7.41-7.55 (m, 2H), 7.55-7.58 (m, 1H), 8.08-8.10 (m, 2H). 13 C NMR (100 MHz, CDCl 3 /TMS): ? -0.03, 21.6, 22.9, 23.0, 35.4, 35.5, 37.0, 74.4, 79.7, 84.9, 85.6, 107.1, 120.8, 128.3, 129.9, 130.1, 133.0, 165.8. Anal. calcd for C 22 H 28 O 4 Si: C, 68.71; H, 7.34. Found: C, 68.96; H, 7.38. (1S, 2R, 3S, 4S)-4-Ethynyl-2, 3-O-Cyclopentylcyclopentan-1-benzoate (62). To a solution of 61 (1.4 g, 3.6 mmol) in dry THF (100 mL) was added 1.0 M TBAF in THF (3.6 mL, 3.6 mmol) at 0 o C. The mixture was stirred at room temperature overnight and the solvent was evaporated under reduced pressure. The residue was dissolved in CH 2 Cl 2 and the resulting solution was washed with brine. After drying (anhydrous Na 2 SO 4 ) and filtration, the residue was purified by column chromatography (EtOAc/hexanes = 1:9) to give 62 as colorless oil (1.0 g, 92%). 1 H NMR (400 MHz, CDCl 3 ): ? 1.68-1.78 (m, 6H), 1.89-1.98 (m, 2H), 2.17 (d, 1H, J = 14.0 Hz), 2.21 (d, 1H, J = 2.8 Hz), 2.54 (ddd, 1H, J = 4.6, 7.8, 14.0 Hz), 3.15 (d, 1H, J = 7.8 Hz), 4.68 (d, 1H, J = 5.8 Hz), 4.75 (d, 1H, J = 5.8 Hz), 5.33 (d, 1H, J = 4.6 Hz ), 7.44-7.48 (m, 2H), 7.57-7.61 (m, 1H), 8.10-8.13 (m, 2H). 13 C NMR (62.9 MHz, CDCl 3 /TMS): ? 22.9, 24.0, 35.1, 35.3, 35.4, 35.7, 70.6, 79.5, 84.8, 84.9, 85.4, 120.9, 128.3, 129.9, 130.1, 133.1, 165.7. Anal. calcd for C 19 H 20 O 4 : C, 73.06; H, 6.45. Found: C, 73.16; H, 6.50. (1S, 2R, 3S, 4S)-4-ethynyl-2, 3-O-Cyclopentylcyclopentan-1-ol (63). Ammonia gas was introduced to a solution of compound 62 (0.32 g, 1.0 mmol) in MeOH (100 mL). 103 This reaction mixture was allowed to stir at room temperature until TLC analysis indicated starting material was no longer present. The solvent was then removed in vacuo and the residue purified by chromatography (EtOAc/hexanes = 1:6) to afford 63 (0.20 g, 98%) as a white solid, mp = 65-66 ? C; 1 H NMR (400 MHz, CDCl 3 ): ? 1.62-1.71 (m, 6H), 1.84-1.88 (m, 2H), 1.94 (dt, 1H, J = 1.6, 13.6 Hz), 2.22 (d, 1H, J = 8.0 Hz), 2.28 (d, 1H, J = 2.8 Hz), 2.32 (ddd, 1H, J = 4.8, 7.6, 13.6 Hz), 3.00 (d, 1H, J = 7.6 Hz), 4.21 (td, 1H, J = 0.8, 4.8 Hz), 4.55 (dd, 1H, J = 0.4, 6.0 Hz), 4.73 (d, 1H, J = 6.0 Hz ). 13 C NMR (62.9 MHz, CDCl 3 /TMS): ? 22.9, 23.9, 35.2, 35.3, 35.4, 37.2, 71.8, 78.0, 85.0, 85.9, 86.9, 120.5. Anal. calcd for C 12 H 16 O 3 : C, 69.21; H, 7.74. Found: C, 69.30; H, 7.68. (1S,2R,3S,4S)-4-(1-acetyl-1H-pyrazol-3-yl)cyclopentane-1,2,3-triyl triacetate (65). To a solution of 63 (1.0 g, 4.8 mmol) in anhydrous t-butyl methyl ether (90 mL) with stirring at -78 o C under N 2 was added n-butyllithium (4.8 mL, 12.0 mmol, 2.5 M solution in hexanes), and the reaction mixture stirred at the same temperature for 30 min. An excess of DMF (1.8 mL, 24 mmol) was added in one portion and the cold bath removed. The reaction mixture was allowed to warm to room temperature and aged for 30 min. The TBME solution was poured into a vigorously stirred, biphasic mixture prepared from a 10% aqueous solution of KH 2 PO 4 (40 mL) and TBME (40 mL) cooled over ice to ca. +5 o C. The resulting layers were separated and the organic extract was washed with H 2 O. The combined aqueous layers were back extracted with TBME. The combined organic layers were dried (anhydrous Na 2 SO 4 ), filtered, and the filtrate concentrated to give the crude acetylenic aldehyde as an oil. The crude product thus isolated was used in the next step without further purification. 104 To a solution of crude aldehyde (from the previous process) in glacial AcOH (60 mL) was added a solution of hydrazine monohydrate (2.6 g, 25 mmol) in glacial AcOH (18 mL). The resulting solution was heated at reflux for 24 h and then concentrated in vacuo to afford a dark brown oil. This crude product was dissolved in pyridine (20 mL), and Ac 2 O (11.6 mL, 122.4 mmol) and DMAP were added. The resulting solution was stirred for 24 h at room temperature. The solvent was removed in vacuo, and the crude residue dissolved in EtOAc (200 mL), washed with 10% HCl and brine, dried (anhydrous Na 2 SO 4 ), concentrated, and purified by silica gel column chromatography to afford 65 (1.3 g, 73% over three steps) as a light yellow oil. 1 H NMR (250 MHz, CDCl 3 ): ? 1.78- 1.92 (m, 1H), 1.93 (s, 3H), 1.97 (s, 3H), 1.99 (s, 3H), 2.55 (s, 3H), 2.62-2.82 (m, 1H), 3.32-3.43 (m, 1H), 5.08-5.15 (m, 1H), 5.24-5.35 (m, 2H), 6.26 (d, 1H, J = 2.75 Hz ), 8.07 (d, 1H, J = 2.75 Hz ). 13 C NMR (62.9 MHz, CDCl 3 /TMS): ? 20.6, 20.7, 20.9, 21.6, 32.8, 39.4, 75.0, 75.1, 75.2, 108.4, 129.2, 156.5, 169.2, 169.6, 169.8, 170.1 ppm. Anal. calcd for C 16 H 20 N 2 O 7 : C, 54.54; H, 5.72; N, 7.95. Found: C, 54.49; H, 5.82; N, 7.82. (1S,2R,3S,4S)-4-(5-cyano-4-nitro-1H-pyrazol-3-yl)cyclopentane-1,2,3-triyl triacetate (67). Trifluoroacetic anhydride (2.1 mL, 3.1 g, 15 mmol) was added dropwise to a stirred solution of 65 (0.90 g, 2.6 mmol) and ammonium nitrate (1.9 g, 24 mmol) in TFA (30 mL) at 0 o C. The resulting solution was allowed to warm to rt and stirred overnight. The solvent was evaporated in vacuo at room temperature and then diluted with CH 2 Cl 2 , washed with H 2 O, saturated aqueous NaHCO 3 solution and brine, dried over anhydrous Na 2 SO 4 , and the organic phase concentrated in vacuo to give the 1,4- dinitro pyrazole derivative 66 (1.1 g) as a syrup. The crude product thus isolated was used to the next step without further purification. 105 A purifided 57 was prepared by silica gel column chromatography (EtOAc/hexanes = 1:4). A colorless oil; 1 H NMR (400 MHz, CDCl 3 ): ? 1.92-2.05 (m, 1H), 2.01 (s, 3H), 2.09 (s, 3H), 2.11 (s, 3H), 2.86-2.94 (ddd, 1H, J = 7.2, 9.6, 14.0 Hz), 4.07-4.12 (m, 1H), 5.19-5.23 (ddd, 1H, J = 4.4, 5.6, 7.2 Hz), 5.37-5.39 (t, 1H, J = 4.4 Hz), 5.58-5.61 (dd, 1H, J = 5.6, 7.2 Hz ), 9.08 (s, 1H). 13 C NMR (100 MHz, CDCl 3 /TMS): ? 20.5, 20.6, 20.9, 32.6, 38.3, 74.2, 74.6, 74.9, 125.0, 134.1, 147.9, 169.6, 169.9, 170.2. At room temperature, a solution of the 1,4-dinitro compound in EtOH (9.3 mL) and EtOAc (9.3 mL) was added dropwise over 5 min to a stirred solution of KCN (1.3 g, 20 mmol) in EtOH (23.0 mL) and H 2 O (5.5 mL). Following an additional 5 min at room temperature, the reaction mixture was neutralized with AcOH (2.0 mL). After evaporation of the solvent in vacuo, the residue was diluted with EtOAc (110 mL), washed with H 2 O and brine, dried (anhydrous Na 2 SO 4 ), and concentrated in vacuo to a residue that was subjected to chromatographic purification (silica gel, CH 2 Cl 2 /MeOH, 20/1) to afford 67 (0.77 g, 80% over two steps) as a light yellow syrup. 1 H NMR (400 MHz, CDCl 3 ): ? 1.78-1.84 (ddd, 1H, J = 4.4, 9.6, 14.4 Hz), 2.05 (s, 3H), 2.10 (s, 3H), 2.16 (s, 3H), 2.99-3.07 (ddd, 1H, J = 7.2, 9.6, 14.4 Hz), 4.18-4.25 (dd, 1H, J = 9.6, 18.0 Hz), 5.20-5.24 (ddd, 1H, J = 3.6, 5.6, 7.2 Hz), 5.37-5.39 (dd, 1H, J = 3.6, 5.6 Hz), 5.68- 5.71 (dd, 1H, J = 5.6, 8.8 Hz ). 13 C NMR (100 MHz, CDCl 3 /TMS): ? 20.7, 20.7, 20.9, 33.3, 37.4, 73.1, 74.5, 74.6, 110.5, 122.6, 134.0, 143.9, 170.3, 170.4, 171.8. (1S,2R,3S,4S)-4-(4-amino-5-cyano-1H-pyrazol-3-yl)cyclopentane-1,2,3-triyl triacetate (69). A catalytic amount of Pd/C (1%) was added to a solution of 67 (0.70 g, 1.8 mmol) in MeOH (30 mL). The resulting mixture was shaken under 30 psi of H 2 overnight. After the reaction was complete, the solvent was evaporated in vacuo and the 106 product purified by silica gel chromatography (CH 2 Cl 2 /EtOAc/MeOH, 8:1:0.5) to afford 69 (0.55 g, 85%) as a syrup. 1 H NMR (400 MHz, CDCl 3 ) ? 2.08-2.11 (m, 9H), 2.72-2.91 (m, 10H), 3.37-3.43 (m, 1H), 4.12 (m, 2H), 5.23 (t, 1H, J = 4.8 Hz), 5.29-5.38 (m, 2H). 13 C NMR (100 MHz, CDCl 3 ) ? 20.8, 20.9, 21.0, 30.6, 36.6, 74.8, 75.0, 75.3, 112.3, 113.9, 134.3, 135.0, 170.2, 170.7, 170.9. 4-Amino-3-((1S,2S,3R,4S)-2,3,4-trihydroxycyclopentyl)-1H-pyrazole-5- carbonitrile (70). Anhydrous NH 3 was introduced to a solution of compound 69 (0.50 g, 1.4 mmol) in MeOH (50 mL) at 0 o C. The reaction mixture was stirred at room temperature. After the starting material was no longer present (TLC), the solvent was removed in vacuo and the residue purified by silica gel chromatography (CH 2 Cl 2 /MeOH, 6:1) to afford 61 (0.26 g, 80%) as a light yellow solid, mp 174?176 ?C. 1 H NMR (400 MHz, CDCl 3 ) ? 1.68 (m, 1H), 2.58 (m, 1H), 3.22 (m, 1H), 3.87 (m, 1H), 4.09 (m, 1H), 4.29 (m, 1H), 13.17 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ? 33.0, 36.5, 73.6, 74.8, 76.1, 112.2, 114.0, 129.9, 130.2. (1S,2R,3S,4S)-4-(7-Amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl) cyclopentane- 1,2,3-triol (22). A solution of 70 (0.22 g, 1.0 mmol) in EtOH (30 mL) was stirred with formamidine acetate (0.16 g, 1.5 mmol) under reflux for 50 min. The resulting white precipitate was isolated by filtration, washed with EtOH, and dried to afford analytically pure 14 (0.13 g, 54%) as a dark white solid, mp 242?243 o C (dec), 1 H NMR (400 MHz, DMSO-d 6 ) ? 1.72-1.79 (m, 1H), 2.38-2.45 (m, 1H), 3.37(dd, 1H, J = 8.0, 17.6 Hz), 3.74 (dd, 1H, J = 2.4, 4.4 Hz), 3.91 (dq, 1H, J = 2.8, 5.2, 7.6 Hz), 4.33 (dd, 1H, J = 5.2, 8.0 Hz ), 4.57 (m, 1H), 4.91 (brs, 1H), 5.19 (brs, 1H), 7.28 (brs, 2H), 8.15 (s, 1H), 12.46 (m, 1H). 13 C NMR (100 MHz, DMSO-d 6 ) ? 36.6, 41.0, 75.6, 76.4, 78.6, 122.4, 139.7, 147.6, 107 151.1, 151.4. Anal. Calcd for C 10 H 13 N 5 O 3 ?0.25MeOH: C, 47.49; H, 5.44; N, 27.01. Found: C, 47.41; H, 5.35; N, 26.92. (1S,2R,3S,4S)-4-(5-cyano-1-methyl-4-nitro-1H-pyrazol-3-yl)cyclopentane- 1,2,3-triyl triacetate (76)and (1S,2R,3S,4S)-4-(5-cyano-2-methyl-4-nitro-1H-pyrazol- 3-yl)cyclopentane-1,2,3-triyl triacetate (77). To a stirred solution of 67 (100 mg, 0.270 mmol) in anhydrous THF (10 mL), was added portion wise 60% sodium hydride in mineral oil (14 mg, 0.32 mmol) at 0 o C. The mixture was stirred at room temperature for 30 min. and then iodomethane (0.02 mL, 0.4 mmol) was added dropwise. After a period of 24 h the reaction mixture was absorbed by silica gel, and then purified by silica gel column chromatography. Compound 76 (52 mg, 0.13 mmol, 50.0%) and 77 (~5 mg, 5%) was obtained after eluting with a mixture of hexane/ethyl acetate in a ratio of 1:1. Compound 76 was a yellow syrup; 1 H NMR (400 MHz, CDCl 3 ) ? 1.82 (m, 1H), 2.00 (s, 3H), 2.07 (s, 3H), 2.10 (s, 1H), 2.90 (ddd, 1H, J = 8, 9.6, 14.4 Hz), 4.06 (td, 1H, J = 6.8, 9.2, 14.4 Hz), 4.10 (s, 3H), 5.22 (ddd, 1H, J = 4.8, 6.0, 8.0 Hz), 5.38 (t, 1H, J = 5.2 Hz), 5.60 (dd, 1H, J = 5.2, 6.8 Hz); 13 C NMR (100.6 MHz, CDCl 3 ) ? 20.6, 20.7, 21.0, 33.0, 38.0, 40.0, 74.0, 74.8, 74.9, 107.3, 115.7, 135.4, 148.6, 169.8, 169.9, 170.1; Compound 77 was a yellow syrup; 1 H NMR (400 MHz, CDCl 3 ) ? 1 H NMR (400 MHz, CDCl 3 ) ? 1.81 (m, 1H), 2.02 (s, 3H), 2.06 (s, 3H), 2.12 (s, 1H), 2.87 (m, 1H), 4.01 (s, 3H), 4.06 (m, 1H), 5.22 (m, 1H), 5.46 (t, 1H, J = 5.2 Hz), 5.63 (dd, 1H, J = 5.2, 6.8 Hz); 13 C NMR (100.6 MHz, CDCl 3 ) ? 20.7, 20.9, 21.0, 32.9, 37.8, 39.8, 74.5, 74.8, 74.9, 107.3, 115.7, 135.4, 149.2, 169.4, 170.0, 170.3. 4-amino-1-methyl-3-((1S,2S,3R,4S)-2,3,4-trihydroxycyclopentyl)-1H- pyrazole-5-carbonitrile (81). A catalytic amount of Pd/C (1%) was added to a solution 108 of 76 (1.4 g, 5.9 mmol) in methanol (30 mL) at room temperature, the resulting mixture was allowed to shake under 25 PSI of hydrogen overnight. After the reaction was complete, concentrated in vacuo to give (1S,2R,3S,4S)-4-(4-amino-5-cyano-1-methyl- 1H-pyrazol-3-yl)cyclopentane-1,2,3-triyl triacetate (80) (1.4 g, 6.7 mmol >100%) as a colorless liquid, The crude product thus isolated was committed to the next step without further purification. Anhydrous NH 3 was introduced to a solution of compound 71 (0.50 g, 1.4 mmol) in MeOH (50 mL) at 0 o C. The reaction mixture was stirred at room temperature. After the starting material was no longer present (TLC), the solvent was removed in vacuo and the residue purified by silica gel chromatography (CH 2 Cl 2 /MeOH, 6:1) to afford 81 (0.26 g, 80%) as a light yellow solid, 1 H NMR (400 MHz, CDCl3) ? 1.68 (m, 1H), 2.58 (m, 1H), 3.22 (m, 1H), 3.87 (m, 1H), 4.09 (m, 1H), 4.12 (s, 3H), 4.29 (m, 1H), 13.17 (s, 1H). 13 C NMR (100 MHz, CDCl3) ? 33.0, 36.5, 73.6, 74.8, 76.1, 112.2, 114.0, 129.9, 130.2. (1S,2R,3S,4S)-4-(7-Amino-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-3- yl)cyclopentane-1,2,3-triol (71) A solution of 81 (0.22 g, 1.0 mmol) in EtOH (30 mL) was stirred with formamidine acetate (0.16 g, 1.5 mmol) under reflux for 50 min. The resulting white precipitate was isolated by filtration, washed with EtOH, and dried to afford analytically pure 62 (0.13 g, 54%) as a gray solid, mp 242?243 o C (dec), 1 H NMR (400 MHz, DMSO-d 6 ) ? 1.72-1.79 (m, 1H), 2.38-2.45 (m, 1H), 3.37(dd, 1H, J = 8.0, 17.6 Hz), 3.74 (dd, 1H, J = 2.4, 4.4 Hz), 3.91 (dq, 1H, J = 2.8, 5.2, 7.6 Hz), 4.00 (s, 3H), 4.33 (dd, 1H, J = 5.2, 8.0 Hz ), 4.57 (m, 1H), 4.91 (brs, 1H), 5.19 (brs, 1H), 7.28 (brs, 2H), 8.15 (s, 1H), 109 12.46 (m, 1H). 13C NMR (100 MHz, DMSO) ? 36.4, 40.7, 75.4, 76.3, 78.5, 122.2, 139.8, 147.5, 150.8, 151.1. 110 REFERENCES (1) Orgel, L. Science (Washington, D. C.) 2000, 290, 1306-1307. (2) Voet, D. V. Biochemistry 1990, chapter V. (3) Porkka-Heiskanen, T.; Strecker, R. E.; Thakkar, M.; Bjorkum, A. A.; Greene, R. W.; McCarley, R. W. Science (Washington, D. C.) 1997, 276, 1265-1268. (4) Robins, R. K. Chem. Eng. News 1986, 64, 28-40. (5) Isono, K. Pharmacol. Ther. 1991, 52, 269-286. (6) Isono, K. J. 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