Purine Carbocyclic Nucleside Analogues as Antiviral Agents by Qi Chen 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 13, 2010 Copyright 2010 by Qi Chen Approved by Stewart W. Schneller, Chair, Professor of Chemistry and Biochemistry Edward J. Parish, Professor of Chemistry and Biochemistry Susanne Striegler, Associate Professor of Chemistry and Biochemistry Evert C. Duin, Associate Professor of Chemistry and Biochemistry Abstract The carbocyclic nucleosides aristeromycin and neplanocin A are two of the most potent inhibitors of S-adenosylhomocysteine hydrolase, an enzyme that plays a critical role in methyl transfer reactions during a viral replication cycle. As a consequence, these two carbocyclic nucleosides have been found to have significant broad-spectrum antiviral activities. However, their clinical applications are limited due to the toxicity associated with their 5'-hydroxyl metabolites. To develop new antiviral agents that retain the antiviral activities of aristermycin and neplanocin A while avoiding their toxicities, homoaristeromycin (1) has been synthesized. Also, in order to detect the relationship between the syn-anti conformations of nucleoside analogues and antiviral activites, a series of 8-methyl carbocyclic nucleosides derivatives have been prepared. Among the analogues in this series 8-methylhomoaristeromycin (2) displayed a significant activity towards human cytomegalovirus (HCMV) and moderate activity against yellow fever virus. 3-Deazapurine carbocyclic nucleosides (that is 3-deazaaristeromycin and 3- deazaneplanocin A) have been discovered to possess remarkable antiviral activites, especially towards Ebola virus, with limited toxicity. Therefore, in this dissertation research, the 3-deaza structural element was introduced to supplement the library of 8- methylcarbocyclic nucleoside analogues that included 8-methyl-3-deaza-5?- ii norneplanocin A (3), 8-methyl-3-deazaneplanocin A (4), 8-methyl-3-deaza-5?- homoneplanocin A (5) and 8-methyl-3-deazaaristeromycin (6). Aristeromycin analogues modified at C-4? (4?-isoaristeromycin 8a, 3-deaza-4?- isoaristeromycin 8b, 4?-isohomoaristeromycin 9a, 3-deaza-4?-isohomoaristeromycin 9b) have been also investigated as part of a program to further understand the structure- activity relationships in the adenine derived purine carbocyclic nucleosides. Although anologues 4, 9a and 9b were found to be inactive towards most viruses, the antiviral data still provide valuable information for structure-activity relationships. A final feature of this dissertation arose from considering S-adenosyl-L- methionine (AdoMet) dependant methyl transferations. AdoMet plays a pivotal role in viral mRNA capping process and inhibition of this process offers another pathway to antiviral drug design. Sinefungin is one of the most potent inhibitors of AdoMet dependant methyltransferases and has shown potential in antiviral, antifungal, antiparasitic drug discovery. However, its toxicity due to inhibition of cellular methyltransferases limits its usefulness. In this direction, deamino-carbocyclic sinefungin (10) has been prepared and targeted for antiviral analysis. The bioassay data for compounds 3, 5, 6, 10 will be forthcoming and under study in our laboratories. iii Acknowledgments It is a pleasure to thank those who made this dissertation research possible. Though only my name appears on the cover, a great many people have contributed to its production. First, I owe my deepest gratitude to my advisor Dr. Stewart W. Schneller. It has been a great fortune to have an advisor who encouraged me not only grow as an experimentalist but also as an independent thinker. His guidance and support helped me overcome many difficult situations and complete this dissertation. I sincerely thank you, Dr. Schneller, for all you have done for me. I am also graduateful to Drs. Edward Parish, Susanne Striegler, Evert Duin and Peter Livant for their constructive suggestions and lectures on related topics that helped me improve my knowledge in the area. I like to thank Drs. Wei Ye, Xueqiang Yin, Yan Zhang, Haisheng Wang, Mingzhu He and other members in my research group for their inspiring discussion, advice and friendship. I like to thank Drs. Michael Meadows and Yonnie Wu for their assistance in obtaining Nuclear Magnetic Resonance (NMR) spectroscopic and Mass Spectrometric (MS) data. Gratitude is also extended to the Department of Chemistry and Biochemistry and the National Institutes of Health for their financial support. Finally, and most importantly, I would like to thank my husband, Chong Liu, for his unending encouragement, support and patience. iv Table of Contents Abstract............................................................................................................................... ii Acknowledgments.............................................................................................................. iv List of Tables .................................................................................................................... vii List of Figures ................................................................................................................... ix List of Schemes ................................................................................................................. xi Introduction..........................................................................................................................1 Introduction of Viruses ..........................................................................................1 Perspective for Antiviral Drug Design ..................................................................2 Nucleoside Analogues ...........................................................................................6 Adenosylhomocysteine(AdoHcy) Hydrolase as A Key Enzyme for Antiviral Activity ....................................................................................................................9 The Inhibitors of AdcHcy Hydrolase as Antiviral Agents.....................................15 Target Design Based on Inhibition of AdoHcy Hydrolase ..................................21 Target Design Based on Direct Inhibition of Methyltransferase...........................27 Chapter 1 Synthesis of Important Precursors.....................................................................30 Chapter 2 Synthesis of 5?-Homoaristeromycin and 8-Methyl-5?-Homoaristeromycin......36 Chapter 3 Synthesis of 8-Methyl-3-Deazaneplanocin Analogues.....................................45 Chapter 4 Synthesis of 8-Methyl-3-Deazaaristeromycin Analogues.................................67 Chapter 5 Synthesis of 4?-Isoaristeromycin and Its 3-Deaza Analogues...........................81 v Chapter 6 Synthesis of Deamino-Carbocyclic Sinefungin ................................................87 Biological Results ............................................................................................................96 Conclusion .....................................................................................................................111 Experimental Section ......................................................................................................113 References ......................................................................................................................158 vi List of Tables Table 1 Viruses affected by AdoHcy hydrolase inhibitors................................................13 Table 2 Mitsunobu reaction between 3-deaza-6-chloropurine and cyclopentanols...........51 Table 3 Mitsunobu reaction for coupling (Part A) ............................................................79 Table 3 Mitsunobu reaction for coupling (Part B) ............................................................80 Table 4 The spectrum of viruses to be assayed..................................................................96 Table 5 Antiviral activity of compound 2, 4 against HSV-1, HSV-2 and HCMV, based on cytopathogenic effect (CPE) inhibition assay...........................................................98 Table 6 Antiviral activity of compound 2, 4 and 9a against adenovirus, influenza A (H1N1), influenza A (H3N2), influenza B inhibition assay .....................................99 Table 7 Antiviral activity of compounds 2, 4 and 9a against measles, PIV, rhinovirus, rift valley fever inhibition assay ..................................................................................100 Table 8 Antiviral activity of compounds 2, 4 and 9a against yellow fever, RSVA, and inhibition assay .......................................................................................................101 Table 9 Antiviral activity of compounds 2, 4 and 9a against dengue, tacaribe, VEE and WNV inhibition assay.............................................................................................102 Table 10 Antiviral activity of compounds 2, 4 and 9a against EBV virus based on DNA hybridization inhibition assay.................................................................................103 Table 11 Antiviral activity of compounds 2, 4 and 9a against vaccinia virus and cowpox virus based on cytopathogenic effect (CPE) inhibition assay.................................104 Table 12 Antiviral activity of compounds 2, 9a in vero cell cultures..............................105 Table 13 Antiviral activity of compounds 2, 9a in HEL cell cultures.............................106 Table 14 Antiviral activity of compounds 2, 9a against cytomegalovirus in Human Embryonic Lung (HEL) cells..................................................................................107 vii Table 15 Antiviral activity of compounds 2, and 9a against varicella-zoster in Human Embryonic Lung (HEL) Cells.................................................................................108 Table 16 Cytoxity and antiviral activity of compounds 2 and 9a in hela cell cultures....109 Table 17 Antiviral activity of compounds 2, 4 and 9a against HCV and HBV ..............110 viii List of Figures Figure 1 Examples of NRTIs and NNRTIs..........................................................................5 Figure 2 Naturally occuring nucleosides ............................................................................6 Figure 3 Structure of IDU and Brivudin .............................................................................7 Figure 4 Aristeromycin and neplanocin A ..........................................................................8 Figure 5 Structure of mRNA 5?-terminal cap ....................................................................10 Figure 6 S-Adenosylmethionine (AdoMet) .......................................................................11 Figure 7 Metablic pathway of AdoMet .............................................................................14 Figure 8 Metabolites of Ari ...............................................................................................17 Figure 9 Phosphate derivatives of Ari and Npc A ............................................................18 Figure 10 3-Deazaaristeromycin and 3-deazaneplanocin .................................................19 Figure 11 Structure of DHCeA, DHCaA and their 3-deaza analogues ............................20 Figure 12 5?-Deoxyaristeromycin .....................................................................................20 Figure 13 5?-Homoanalogues of Ari and Npc A ...............................................................21 Figure 14 syn and anti conformation for nucleosides .......................................................22 Figure 15 8-Methyl-5?-noraristeromycin ..........................................................................23 Figure 16 8-Methyl-5?-homoaristeromycin .......................................................................23 Figure 17 Analogues of 3-deazaAri and 3-deazaNcp A ...................................................24 Figure 18 3-Deaza-5?-norAri and its 8-methyl analogues .................................................25 Figure 19 8-Methyl analogue of C 3 -DHCeA ....................................................................25 ix Figure 20 D, L-like nucleosides .........................................................................................26 Figure 21 5?-Methylneplanocin A .....................................................................................27 Figure 22 Target compounds for 4?-isoAri analogues ......................................................27 Figure 23 Analogues of AdoHcy ......................................................................................28 Figure 24 Deamino-carbocyclic sinfungine ......................................................................29 Figure 25 Precursors for carbocyclic nucleosides synthesis .............................................30 Figure 26 First generation of Grubbs catalysts .................................................................34 Figure 27 X-ray crystal structure of compound 43 ...........................................................49 Figure 28 NOESY analysis for two isomers of 94 ...........................................................78 Figure 29 NOESY analysis for 100 ..................................................................................86 x List of Schemes Scheme 1 Acyclic Nucleosides ...........................................................................................7 Scheme 2 Stability of carbocyclic nucleosides ...................................................................8 Scheme 3 AdoMet-dependent methyl-transferation .........................................................12 Scheme 4 Mechanism of AdoHcy hydrolase.....................................................................15 Scheme 5 Metabolic pathways of Npc A...........................................................................16 Scheme 6 Enzymatic hydrolysis of 16...............................................................................31 Scheme 7 Synthesis of diacetate 16...................................................................................31 Scheme 8 Synthesis of enone12 from monoacetate 11......................................................32 Scheme 9 Synthesis of enone 12 from D-ribose ................................................................33 Scheme 10 Mechanism of ring closure metathesis............................................................35 Scheme 11 Jones method for homoaristeromycin synthesis..............................................37 Scheme 12 Synthesis of homoaristeromycin from enone..................................................38 Scheme 13 Retrosynthesis for homoaristeromycin and 8-methyl-homoaristeromycin.....39 Scheme 14 Synthesis of the intermediate 27 .....................................................................40 Scheme 15 Synthesis of (-)-homoAri ................................................................................41 Scheme 16 Hydroboration reaction for compound 29.......................................................42 Scheme 17 Synthesis of compound 34 ..............................................................................42 Scheme 18 Synthesis of 2 from intermediate 27 ...............................................................43 Scheme 19 Strategy for target compounds 3, 4, 5 from nucleobase 38.............................46 xi Scheme 20 Retrosynthetic analysis of 38 .........................................................................47 Scheme 21 Synthesis of 6-chloro-3-deazapurine...............................................................48 Scheme 22 Mechanism for the ring closing reaction of 42 ...............................................49 Scheme 23 di-Boc protected 3-deazaadenine as nucleophile in the Mitsunobu reaction..52 Scheme 24 Synthesis of 8-methyl-3-deazaadenine............................................................53 Scheme 25 Protection for 8-methyl-3-deazaadenine .........................................................54 Scheme 26 Selective deprotection of compound 46..........................................................54 Scheme 27 Postulated mechanism for Boc-deprotection by TABF ..................................55 Scheme 28 Retrosynthesis of 8-methyl-3-deaza-5?-norneplanocin A (3)..........................56 Scheme 29 Synthesis of 8-methyl-3-deaza-5?-norneplanocin A........................................57 Scheme 30 Retrosynthesis of 8-methyl-3-deaza-neplanocin 4..........................................58 Scheme 31 Previous synthesis for cyclopentenone 49 ......................................................59 Scheme 32 New strategy for synthesis for cyclopentenone 49..........................................60 Scheme 33 Synthesis of cyclopentenol 49a.......................................................................61 Scheme 34 Synthesis of 8-methyl-3-deazaneplanocin ......................................................62 Scheme 35 Retrosynthesis of 8-methyl-3-deaza-homoneplanocin 5.................................62 Scheme 36 Shtuo?s method for synthesis of cyclopentenol 72..........................................63 Scheme 37 Synthesis of compound 75 ..............................................................................64 Scheme 38 Proposed mechanism for the reaction of 75 with 38.......................................65 Scheme 39 Synthesis of precursor 72 ................................................................................66 Scheme 40 Synthesis of compound 5 ................................................................................66 Scheme 41 Retrosynthesis of compound 6, 7 ....................................................................68 Scheme 42 Mitsunobu reaction of nucleobase with compound 27....................................69 xii Scheme 43 Reaction and general mechanism for Hendrickson reagent............................70 Scheme 44 S N 2 reaction for the compound 27 ..................................................................71 Scheme 45 Reaction and mechanism for the palladium catalyzed substitution ................72 Scheme 46 Mitsunobu reaction for compound 11 with 3-deaza-8-methyl nucleobase .....73 Scheme 47 Proposed mechanism for Mitsunobu reaction.................................................74 Scheme 48 Synthesis and Mitsunobu reaction for 85........................................................75 Scheme 49 Synthesis and Mitsunobu reaction for the isomer of 85.................................76 Scheme 50 Synthesis of Grignard reagent 90....................................................................77 Scheme 51 Synthesis of 3-deaza-8-methylaristeromycin..................................................78 Scheme 52 Retrosynthetic analysis of 4?-isoaristeromycin and 3-deaza-4? isoaristeromycin........................................................................................................82 Scheme 53 Retrosynthetic analysis of 4?-isohomoaristeromycin and 3-deaza-4?- isohomoaristeromycin...............................................................................................83 Scheme 54 Old method for synthesis of compound 8 and 10............................................84 Scheme 55 Synthesis of bis-Boc protected adenine and 3-deazaadenine..........................85 Scheme 56 Synthesis of 8 and 9 ........................................................................................86 Scheme 57 Retrosynthetic analysis of deamino-carbocyclic sinfungine 10......................88 Scheme 58 Synthesis of phosphonium salt 12...................................................................89 Scheme 59 First attempt for synthesis of aldehyde 127 ....................................................90 Scheme 60 Revised synthesis for target compound 10......................................................91 Scheme 61 Synthesis of amino acid olefin 140 .................................................................92 Scheme 62 First attempt of metathesis ..............................................................................94 Scheme 63 Second attempt of metathesis..........................................................................95 xiii 1 Introduction There are about 2,200 viral species, which have been recognized since the discovery of the tobacco mosaic virus in 1892, and the list is continuing to grow. 1 These highly infectious intracellular parasites comprising either DNA or RNA genomic material sometimes can lead to life-threatening diseases including smallpox, yellow fever, poliomyelitis, influenza and measles, 2-5 which could cause damages in the central nervous system and various vital organs, such as lung, liver and intestine. Approximately 20% of the human cancers are due to viral infections. Viruses also can cross species boundaries. The acquired immune deficiency syndrome (AIDS), 6 Ebola hemorrhagic fever, 7 severe acute respiratory syndrome (SARS), 8 and more recently H5N1 avian and H1N1 swine influenza 9-11 are examples of cross-species (zoonotic) infections between humans and other hosts. 12 Introduction of Viruses Viruses are unique particles. They are extremely small, often made up of nothing but more than a nucleic acid molecule within a protein shell; they are ultimate, intracellular parasites, must depend on a living host for reproduction. All viral genomes are synthesized by cellular machinery and packed into progeny infectious particles called virions, which are ready to start the next infectious cycle. 13 2 There are four characteristics which can be used for classification of all viruses: 1. The nature of the nucleic acid in the virion (DNA or RNA); 2. The symmetry of the protein shell (capsid); 3. The presence or absence of a lipid membrane (envelope); 4. Dimensions of the virion and capsid. Since the nucleic acids of viruses carry all the information for virus propagation, no doubts they are considered as the most important property. The current classification system, which is based on the genome type of the virus and the replication pathway, was established by David Baltimore, a Nobel prize-winning biologist. All viruses fall into one of the following seven groups: 14 1. Double-stranded DNA viruses; 2. Single-stranded DNA viruses; 3. Double-stranded RNA viruses; 4. Positive-sense single-stranded RNA viruses; 5. Negative-sense single-stranded RNA viruses; 6. Positive-sense single-stranded RNA viruses that use reverse transcription; 7. Circular double-stranded DNA viruses that use reverse transcription. Viruses attach to and enter host cells to start the infectious cycle. Several crucial steps like translation of viral mRNA by the host ribosome, replication of viral genome, assembly and release of the progeny virions from the host cell are involved in all viruses life cycle. Each stage in the viral life cycle has been studied extensively as potential targets for antiviral drug design. 15 3 Perspective for Antiviral Drug Design Vaccines and drugs are two effective tools for fighting virus infections. Many commercially successful vaccines have been used to diminish viral infections, and in a few cases lead to eradication of some viral diseases, like smallpox, 16 polio, 17 measles, 18 mumps and rubella. 19 However, some other diseases especially caused by human immunodeficiency (HIV), hepatitis C (HCV), Ebola and SARS virus still remain untouched by vaccine approaches. 20-23 Side effects are often observed with many vaccines especially when applied to immunosuppressed individuals. Live-attenuated vaccines can inadvertently cause the spread of viruses to vaccinees and may also be able to infect unvaccinated individuals. 24-27 Finally, since vaccine act as a prophylactic measure, the development of antiviral drugs to treat people who are already infected is also needed. Viruses are infectious particles with high replication and mutation rates. The mutation rate for RNA viruses is one mutation per genome per replication compared to DNA viruses? one mutation per genome per 1000 replications. 28 Because the latter use the host cell?s DNA polymerase for viral replication, which has proofreading capability, they are often larger and evolve much more slowly than RNA viruses. 29 The high mutation rates along with using the host cell?s molecular machinery makes viruses more difficult to fight with drugs than bacteria and other infections. It has been 50 years since 5-iodo-2?-deoxyuridine (IDU) was described as the first antiviral drug (and still) marketed. 30 Now, after five decades, 50 compounds are licensed for clinical use to treat viral infections, including 25 compounds as anti-HIV drugs and others approved for treating herpes simplex virus (HSV), varicella-zoster virus (VZV), 4 cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), and influenza virus infections. 31 Antiviral drug design could target any stage of the viral replicative circle. Taking retroviruses (HIV) as an example, infusion to the host cell is the first step for its replication. The HIV virus employs chemokine receptors as co-receptors for cellular entry, such as CCR5 and CXCR4, which are naturally expressed on the surfaces of immune cells promoting their activation or migration by chemaxis. 32,33 UK-427857 (Maraviroc) 34-36 is a potent inhibitor of CCR5 that prevents HIV entry into T-cells, and thus, possesses good pharmacological properties. Other compounds like AMD3100, 37,38 work as antagonists of CXCR4 with similar results. The replication of viral RNA can be prevented by reverse transcriptase (RT) inhibitors, including NRTIs (nucleoside reverse transcriptase inhibitors) 39 like AZT (azidothymidine, Retrovir ? ) 40 ddI (Didanosine, videx ? ) 41 or (-)-3TC (Lamivudine, Epivir ? ) 42 (Figure 1) and NNRTIs (Non-nucleoside reverse transcriptase inhibitors) 43 like nevirapine (Viramune ? ) 44-46 or efavirenz (Sustiva ? ) (Figure 1). 47-49 The former type acts as alternative substrates of natural nucleosides, which, when incorporated into viral DNA strands, results in termination of chain elongation. 39,50-53 The latter NNRTIs specifically bind to a non-substrate binding (i.e., allosteric) site of HIV-1 RT, which results in a change of enzyme conformation from catalytically active into fixed inactive form. 54,55 5 Figure 1. Examples of NRTIs and NNRTIs The cleavage of newly expressed viral protein precursor into smaller and mature pieces and assembly with viral RNA into newly formed viruses can be inhibited by protease inhibitors 56-59 like ritonavir (Norvir ? ) or/and lopinavir (Kaletra ? ). 60 These drugs were rationally designed containing a peptidomimetic hydroxyethylene bond instead of the normal peptide linkage to block the action of viral protease. 61 Lastly, the release of progeny viruses from host cells can be halted by the administration of neuraminidase inhibitors. 62-64 A well known example is oseltamivir (Tamiflu ? ), which is widely used for the treatment of influenza A and B viral infections, 65 including avian flu. 66 Nucleoside Analogues Nucleosides are naturally occurring biological molecules which are fundamental building blocks of DNA and RNA (Figure 2). They also participate as essential intermediates in virtually all aspects of cellular metabolism. 6 N N N N NH 2 NH N N N O NH 2 N N NH 2 O NH N O O Me O HO OH HO O HO OH HO O HO OH HO O HO HO NH N O O O HO HO OH Adenosine Guanosine Cytidine Uridine Thymidine Figure 2. Naturally Occuring Nucleosides For example, adenosine is a major component of adenosine triphosphate (ATP), coenzyme A and nicotinamide adenosine dinucleotide (NAD + ) and is implicated in sleep regulation. 67,68 Because of their pervasiveness, nucleoside analogues produce a variety of biological effects, 69 including functioning as non-toxic, selective inhibitors of viral kinases and polymerases. 70-73 IDU and its 5 position modified analogue Brivudin (Figure 3) were first recognized as selective antiviral agents against HSV and VZV. 74 Later on, acyclic guanosine analogs acyclovir, penciclovir, ganciclovir and their oral prodrug forms valaciclovir, famciclovir and valganciclovir (Scheme 1) were approved for the treatment of herpesvirus (HSV, VZV and/or CMV) infections. 75,76 All of these compounds target the viral DNA polymerase and, consequently, interrupt viral DNA synthesis. As a result, they only effect DNA-containing viruses and leave the RNA viruses untouched. 77 On the other hand, many 2?, 3?-dideoxynucleoside analogues, such as azidothymidine (AZT), have been licensed as anti-HIV agents. After triphosphorylation by cellular kinases, they 7 are incorporated by the HIV reverse transcriptase into the viral DNA chain and result in chain termination since they lack the 3? hydroxyl group for further chain elongation. 78 NH O ON O OHOH HO I IDU NH O ON O OHOH HO Br H H Brivudin Figure 3. Structure of IDU and Brivudin Scheme 1. Acyclic Nucleosides Among the modified nucleosides, one of the most important classes is the carbocyclic nucleoside in which the oxygen in the sugar portion of traditional nucleosides is replaced 8 by a methylene unit. 79,80 This characteristic makes the carbocyclic nucleoside more resistant to phosphorylases due to the absence of the glycosidic bond (Scheme 2). 70,71,81,82 Scheme 2. Stability of Carbocyclic Nucleosides Two of the most important carbocyclic nucleosides are aristeromycin (Ari) and Neplanocin A (NpcA) (Figure 4), which show prominent broad-spectrum antiviral activity. Both compounds are naturally occurring carbocyclic analogues of adenosine. Ari was first synthesized by Shealy 83 in 1966 before it was isolated from a metabolite of Streptomyces cirtricolor 84 in 1968, while NpcA was isolated from the culture broth of Ampullariella regulars in 1981. 85,86 Their antiviral potency has been proven to be due to the inhibition of AdoMet dependant biomethyltransferation, a pivotal step in viral replication. 87,88 9 N N N N NH 2 HO HO OH N N N N NH 2 HO HO OH Aristeromycin (Ari) Neplanocin A (NpcA) Figure 4. Aristeromycin and Neplanocin A Adenosylhomocysteine (AdoHcy) Hydrolase as a Key Enzyme for Antiviral Activity Generally speaking, there are two strategies for anti-viral drug design: one, is to target on the viral proteins. This approach could develop many very specific, low toxic antiviral agents with high selectivity but, will more likely, lead to high drug-resistance by viral adaptation. Another option is to target host proteins. This approach opens the door for broad spectrum antiviral compound discovery with a low chance of drug-resistance development but high toxicity. 77 IMP dehydrogenase is an example of the latter spposed, which is a host target enzyme that is responsible for the NAD-dependent oxidation of IMP to xanthosine 5?-monophosphate (XMP). 89 Inhibition of this cellular enzyme upon viral metabolic demand could have anti-viral effectiveness preferentially since there is an increasing demand for RNA and/or DNA synthesis in virus-infected cells. A well known anti-viral agent acting as an IMP dehydrogenase inhibitor is ribavirin, 90 which is clinically used for the treatment of rous sarcoma virus (RSV) infections. 91-93 In our laboratories we have been focusing on methylation of the capped structure of viral m-RNA as the subject for searching anti-viral agents. A relevant enzyme in modulating the process is AdoHcy hydrolase, which is a cellular enzyme that is also 10 employed for viral protein translation and has been studied as a potential target responsible for the inhibition of this capping process. N HN N N O H 2 N CH 3 O OH OH O P O O O P O O O P O O O O Base O O CH 3 PO O O O Base O O CH 3 PO O O mRNA Guanosine N 7 methyl cap 2'-O methyl cap 5' 5' Figure 5. Structure of mRNA 5?-Terminal Cap Viruses and eukaryotic cells both require capping structures at the 5?-end of m-RNA for successful ribosomal translation (Figure 5). These capping structures consist of a 7- methylguanosine linked to the 5?-end of m-RNA by a 5?-5? triphosphate bridge. 94 Most 5? -capped RNA structures are also methylated at the 2?-hydroxyl site of one or more of the penultimate nucleotides. The importance of the capped structures in mRNA synthesis has been shown to be manifold: 95,96 1) facilitating post transcriptional processing, nucleocytoplasmic transport; 2) recognition of mature mRNA by the translation machinery; 97-99 3) improving affinity for binding to the ribosome in the translational initiation complex; 100 and, 4) preventing the mRNA degradation by 5?-end nuclease. 101 The capping process consists of three enzymatic reactions. First, the 5? triphosphate end of primary RNA is cleaved to a diphosphate terminus by RNA triphosphatase. Then, 11 capping with a GMP group by RNA guanylyltransferase follows. Finally, RNA (guanine- 7)methyltransferase catalyzes the methylation reaction at the N7 position of guanine. 102 (i) pppN(pN) n ? ppN(pN) n + P i (ii) ppN(pN) n + pppG G(5?)pppN(pN) n + PP i (iii) G(5?)pppN(pN) n + AdoMet ? m 7 G(5?)pppN(pN) n + AdoHcy Figure 6. S-Adenosylmethionine (AdoMet) AdoMet (S-adenosyl-L-methionine) (Figure 6) serves as the methyl donor in step (iii) of the capped mRNA. AdoMet is also a participant in many other biological methylation processes such as methylation of histamine, 103 serotonin, 104 membrane phospholipids 105 and DNA, 106-108 RNA or protein. 109,110 Besides acting as a universal methyl donor, AdoMet is also involved in many other group transfer reactions, such as, (1) after decarboxylation, an aminopropyl donor in the biosynthesis of polyamines spermidine and spermine, 111 (2) a 3-amino-3-carboxypropyl donor in tRNA modification 112 and (3) an adenosyl donor in the modification of the enzyme pyruvate formate-lyase. 113 12 Scheme 3. AdoMet-dependent Methyl-Transferation However, in the methyl transfer pathways, AdoMet is converted into adenosylhomocysteine (AdoHcy) by different methyltransferases. 114-116 It is to be noted that the metabolite AdoHcy is a potent biofeed back inhibitor of AdoMet-dependent methyltransferases in vivo (Scheme 3). 117-122 The methylase enzyme affinity for AdoHcy is the same or even higher than for AdoMet. 123 This inhibition is relieved by hydrolysis of AdoHcy to adenosine and L-homocysteine (Figure 7). This reversible reaction is catalyzed by the enzyme AdoHcy hydrolase, which makes this enzyme an interesting target for antiviral drug design. 124,125 By inhibiting this enzyme, the concentration of AdoHcy builds up, and increases the intracellular ratio of AdoHcy/AdoMet, which will lead to the suppression of the viral mRNA methylation and, consequently, prevending the capping process from occurring. Viruses, like vesicular stomatitis virus (VZV), that rely on the mRNA capping process for their replication, are particularly sensitive to inhibition by AdoHcy hydrolase inhibitors. 126 Many other negative-sense single-stranded RNA viruses, double strand RNA viruses or even some of DNA viruses are also affected by AdoHcy hydrolase inhibitors (Table 1). 13 Virus type Virus classification specific example (-) RNA Rhabdovirus Rabies (-) RNA Filovirus Ebola, Marburg (-) RNA Arenavirus Junin, Tacarobe ds RNA Reovirus Rota (-) RNA Paramyxovirus Parainfluenza, Respiratory Syncytial virus (+) RNA (RT) Retrovirus HIV ds DNA Herpesvirus Cytomegalovirus ds DNA Poxvirus Vaccinia, Monkeypox (-) RNA: Negative-sense single-stranded RNA virus ds RNA: Double-standed RNA virus (+) RNA (RT): Positive-sense single-stranded RNA virus that use reverse transcription ds DNA: Double-stranded DNA virus Table 1. Viruses affected by AdoHcy hydrolase inhibitors 14 Figure 7. Metabolic pathways of AdoMet The mechanism of AdoHcy hydrolysis has first been proposed by Palmer and Abeles in 1976. 127 They used enzyme purified from beef liver, which contains tightly bound NAD + , to oxidize the 3?-hydroxyl group to a ketone form in first step (Scheme 4). This change increases the acidity of 4? hydrogen and makes it labile and easily removed by enzymatic base (II? III). Following proton removal, L-homocysteine is eliminated (III? IV) and 3?- ketoadenosine is produced by adding a water molecule through a Michael addition (IV? V? VI). Finally the catalytic cycle is completed by forming the adenosine via reduction on the C-3? keto group (VI? VII). 127,128 15 Scheme 4. Mechanism of AdoHcy Hydrolase The reaction favors the synthetic direction (VII? VI) with an equilibrium constant of 10 -6 M, quick removal of adenosine and homocysteine in the cell facilitates the reaction towards the hydrolytic direction (I? VII). Therefore, adenosine analogues capable of serving as substrates for the AdcHcy hydrolase become attractive target compounds for inhibition of this enzyme. 129 The Inhibitors of AdcHcy Hydrolase as Antiviral Agents Ari 130,131 and NpcA 85,132,133 are considered as the first generation inhibitors of AdoHcy hydrolase. Both compounds show antiviral activity against, for example, poxvirus, reovirus, and smallpox virus ect.. Despite the high potency, the cytotoxicity of these compounds precluded them as clinical antiviral agents. This toxicity has been attributed to formation of their corresponding 5?-triphosphates (Figure 9) by adenosine kinase, followed by adenylate kinase and subsequently, nucleoside diphosphokinase. These nucleotides can interfere with a number of fundamental cellular processes. 134-137 16 5?-Triphosphate NpcA (NpcTP) also could serve as a substrate of AdoMet synthetase forming the AdoMet counterpart of NpcA that is NpcMet 135,136,138-140 which may be responsible for the antiviral activity and/or cytotoxicity of NpcA by direct inhibition of viral or cellular methyltransferase. 141,142 NpcA?s effect could be reduced by adenosine deaminase and converted to its inactive form: neplanocin D, a carbocyclic analog of inosine (Scheme 5). The mechanism for NpcA inhibition of AdcHcy hydrolase has been classified as an ?enzyme specific cofactor depletion mechanism?. 143,144 The study shows that NpcA will be oxidized to its 3?-keto form by NAD + and no futher transformation occurs because this latter product lacks the C-4' proton. The effect of this oxidation is depletion of NAD + for the I? II step of Scheme 4. Hence, the concentration of AdoHcy remains high and biofeedback inhibition of the methyltransferases occurs. 132,145 Scheme 5. Metabolic pathways of NpcA 17 A possible reason for the toxicity of Ari is due to the metabolite 5?-monophosphated Ari, which is transformed into the inosine monophosphate (IMP) analogue by adenosine monophosphate (AMP) deaminase 146,147 followed by conversion to the carbocyclic analog of guanosine monophosphate (GMP) (Figure 8). This latter metabolite is a good inhibitor of hypoxanthine (guanine) phosphoribosyltransferase, 146 an important enzyme in the normal cell purine salvage pathway. 88 Figure 8. Metablites of Ari Therefore, it is apparent that the 5?-phosphorylation of Ari and NpcA (Figure 9) is, for most part, the cause of the unwanted cytotoxicity. Thus, a great number of Ari and NpcA analogues have been designed and synthesized by several groups, including the Schneller laboratory, to avoid the undesired nucleotide metabolism process while retaining the promising antiviral activity. 18 N N N N NH 2 OP O O O HO OH N N N N NH 2 OP O O O HO OH P O O O N N N N NH 2 OP O O O HO OH P O O O P O O O N N N N NH 2 OP O O O HO OH N N N N NH 2 OP O O O HO OH P O O O N N N N NH 2 OP O O O HO OH P O O O P O O O Ari monophosphate Ari diphosphate Ari triphosphate NpcA monophosphate NpcA diphosphate NpcA triphosphate Figure 9. Phosphate Derivatives of Ari and NpcA Among various modifications, 3-deazaaristerimycin (3-deazaAri) and 3- deazaneplanocin A (3-deazaNpcA) (Figure 10) have drawn considerable attention. Evidences showed that both 3-deazaAri and 3-deazaNpcA could fully protect mice against a lethal challenge with Ebola virus. 148 In fact, when mice were treated with 3- deazaNcpA, increased amounts of interferon ? were found in Ebola infected mice, but not in the uninfected mices. 149 It was suggested that 3-deazaNpcA restrained the methylation of viral mRNA through inhibiting the AdoHcy hydrolase, hereby preventing the mature mRNA from the (-) RNA template. This results in the accumulation of double-stranded RNA, which is an excellent inducer for interferon. 150 Filovirus including Ebola and Marburg virus are highly infectious and have no vaccine or antiviral drug available in clinical use. 151 Thus, an AdoHcy hydrolase inhibitor based on 3-deazaAri and 3- 19 deazaNpcA, may provide the most promising chemotherapeutic strategy for treating this dreadful viral disease. Besides the higher activity of 3-deazaNcpA and 3-dezazAri, they also possess less cytotoxicity than their parent compounds. 152 Figure 10. 3-Deazazaristeromycin and 3-Deazaneplanocin Based on the fact that neither of these two compounds are substrates for adenosine kinase and adenosine deaminase, 153,154 additional adenine carbocyclic nucleosides were designed and synthesized. For example, since it was clear that the 5?-hydroxyl group was essential for substrate activity of adenosine kinase and deaminase, 155 Borchardt et al. designed and synthesized the 5?-truncated analogues of NpcA, 3-deazaNcpA (DHCeA and C 3 -DHCeA) (Figure 11). 156 They also found that both compounds had much lower cytotoxicity than their parent compounds while still retaining antiviral activity 157,158 (DHCeA is 34 times less cytotoxic comparing with NpcA, measured as the ID 50 value). 88 Similar results have been found for their Ari counterparts, DHCaA and C 3 -DHCaA (Figure 11). 159 20 Figure 11. Structure of DHCeA , DHCaA and their 3-deaza analogues Similarly 5?-deoxyAri (Figure 12) was designed and synthesized by Schneller and coworkers. 160 Without the hydroxyl group at C-5? position, this compound displayed moderate antiviral activity against vaccinia virus (VV) and vesicular stomatitis virusVSV with little toxicity. 161 Figure 12. 5?-Deoxyaristeromycin Another reasonable strategy for diminishing 5?-hydroxyl interference via its phosphorylation was extension of the C-5? side chain, rendering the 5?-hydroxyl group displaced to a site possibly less susceptible to phosphorylation by adenosine kinase. In that direction, 5?-homoaristeromycin (5?-homoAri) (Figure 13) was synthesized by Schneller and other research groups. 162-164 An improved stereoselective synthesis approach was reported in 2005 from the Schneller laboratories and notable antiviral 21 activities toward vaccinia, cowpox and monkeypox viruses were found with low cytotoxicity (CC 50 >100 ?g/mL). 165 A 5?-Homoneplanocin A (5?-homoNpcA) (Figure 13) analog was first described by Shuto in 1996, 166 and synthesized later by the Schneller group via a more efficient and practical route. 5?-HomoNpcA also showed activities against CMV, vaccinia virus, parpinfluenza virus, VSV, arenaviruses, HBV and HCV with no interfering toxicity. 167 Figure 13. 5?-Homoanaolgues of Ari and NpcA Target Design Based on Inhibition of AdoHcy Hydrolase Since the discovery of AdoHcy hydrolase as a potent target for antiviral drugs discovery, many adenosine analogues, including acyclic, carbocyclic or adenine base modified, have been designed, synthesized and tested for their activities. Due to their unique stability against phosphorylase metabolism, the aforementioned carbocyclic nucleosides arose as a prominent group of compounds. 22 Figure 14. syn and anti conformation for nucleosides In considering structural variations for adenine derived antiviral candidates, nucleoside conformation offers an option. In the case of purine nucleosides rotation around the glycosidic bond results in two conformational extremes: a syn or an anti conformation (Figure 14). 168 Concerning nucleoside-enzyme interactions, syn-anti conformation is one of the most important conformational aspects. 169 Adding a bulky substituent on a nucleobase or a sugar portion are both feasible ways for fixing the nucleoside conformation. But like syn/anti conformation around the glycosyl bond, the sugar puker is also an important conformational aspect in nucleoside-enzyme interactions. 170,171 Since that, introducing substituent only on a nucleobase is sufficient to control the syn/anti conformation without greatly affecting its sugar puckering. For example, 8-bromoadenosine and 8-bromoguanosine have been found to adopt a syn conformations in the solid state as well as in solution. The steric repulsion between the 8- bromo group and the sugar portion is the only reasonable explanation for this. Therefore the 8-bromoguanosine 5?-triphosphate does not interact with Q? replicase or Escherichia coli transcriptase which prefers an anti rotational isomer either during or after the polymerization reaction. 172,173 Modification of the purine base on carbonucleosides by adding a methyl group at the C-8 position favor of the syn conformation. In fact, previous 23 research in our laboratory showed 8-methyl-5?-noraristeromycin (Figure 15) possesses moderate inhibitory activity towards AdoHcy hydrolase. 174 In this dissertation, a series of 8-methyl carbocyclic adenosine analogs as probes for the conformational role in AdoHcy hydrolase inhibitory effect will be pursued. Figure 15. 8-Methyl-5?-noraristeromycin To use the 8-alkylated aristeromycin framework for drug design must mention the high cytoxicity 131,137 that is associated with aristeromycin. Recognizing that this toxicity is due to C-5? phosphorylations, perhaps by adding a methylene at this center (hence, 5?- homoaristeromycin) (Figure 16) would decrease this possibility. Figure 16. 8-Methyl-5?-homoaristeromycin As mentioned above, 3-deazaAri and 3-deazaNpcA are two of the most potent inhibitors for AdoHcy hydrolase, with a K i =2 ? 10 -9 M and 5 ? 10 -11 M, respectively (5? 10 -9 M for Ari). 130,141 The 3-deazaAri prototype has good antiviral activity against 24 vaccinia, vesicular stomatitis, measles, parinfluenza, and reo viruses, 153 while 3- dezazNpcA showed excellent activity against VSV, parainfluenza, vaccinia and yellow fever viruses. 154 In addition to their antiviral activities, both compounds display much lower cytotoxicity than the parent compounds Ari and NpcA. 88 Research shows that 3- deazaAri, unlike Ari, is not deaminated by adenosine deaminase nor phosphorylated by adenosine kinase. These properties are also seen with 3-deazaadenosine. 175 However 3- deazaAri undergoes further anabolism to the corresponding S-AdoHcy analogues, which may cause some unwanted side effects. 137,153,175 3-deazaNpcA is neither phosphorylated nor converted to its S-AdoHcy analogue metabolites, which makes its cytotoxicity much lower. 141,176 So another project in this dissertation is to modify 3-dezazAri and 3- deazaNpcA with a methyl group at the C-8 position with and without extension of the 5?- hydroxyl group by a methylene group (Figure 17) rendering it less cytotoxic. Figure 17. Analogues of 3-deazaAri and 3-deazaNcp A In our laboratories, 3-deaza-5?-noraristeromycin (Figure 18) has been prepared and proven to possess antiviral activities against a series of DNA and RNA viruses. 177 Based on that, the 8-methyl analogue of 3-deaza-5?-norAri (Figure 18) was set as a target agent. 25 N N N NH 2 HO HO OH 3-deaza-5'-norAri N N N NH 2 HO HO OH 3-deaza-8-methyl-5'-norAri Me Figure 18. 3-Deaza-5?-norAri and its 8-methyl analogue Additionally, c 3 -DHCeA is known to have potent antiviral activity with less associated toxicity, 156 so the 8-methyl analogue of c 3 -DHCeA (Figure 19) is another valuable target for 8-methyl modification. Figure 19. 8-Methyl analogue of C 3 -DHCeA Stereochemistry is also considered in the design of the target compounds for this dissertation. Natural nucleosides are D-nucleosides (being derived from D-ribose) in which the heterocyclic base connects with the sugar ring in the ? configuration via an N- glycosidic bond. To facilitate correlations with natural nucleosides the D-designation has carried over to carbocyclic nucleosides where stereo similar structures of D-nucleosides 26 are referred to as D-like carbocyclic nucleosides and their enantiomers are recognized as L-like carbocyclic nucleosides (Figure 20). Figure 20. D, L -like Nucleosides The real world is asymmetric, where, for example, two enantiomers will likely have different or even opposite activity. For example, (-)-5?-norAri (the D-like enantiomer) is 100-fold more potent against cytomegalovirus (CMV) than (+)-5?-norAri (the L-like enantiomer); however, the latter is more active against HBV. 161,178 Therefore, pharmacologic drug design often requires compounds to be optically pure (only one enantiomer). Not only do enantiomers have different pharmaceutical properties, changing a stereocenter in a chiral molecule (to a diastereomer) may affect the biological activity as well. A pronounced example of this is between two disastereoisomers of 5?- methylneplancin A (Figure 21). In this case only the 5?R isomer was found to be an excellent AdoHcy hydrolase inhibitor (K i =0.086?M for murine L929 cell) and to display significant activity against a variety of DNA and RNA viruses, whereas the 5?S isomer lacked hydrolase activity (K i =55.7?M) and antiviral effects. 179,180 27 Figure 21. 5?-Methylneplanocin A A study showed that modification at the C-4? position of Ari and NpcA offered a possibility for interesting biological activities. 159 Since the 5?-hydroxyl group is necessary for phosphorylation and deamination, development of C-4? modified analogues may lead to nucleotides or inosine analogues, which may cause biological inactivity and/or cytotoxicity. 155 As a consequence, a change of the configuration at the 4?-position was investigated for impact on biological effects and toxicity. In this direction, this dissertation project sought inverting C-4? stereocenter of Ari, homoAri and their 3-deaza analogues (Figure 22. designated as iso-analogues). Figure 22. Target compounds for 4?-isoAri analogues Target Design Based on Direct Inhibition of Methyltransferase As mentioned, AdoHcy is the metabolite of AdoMet following methyl transfer in the capping of mRNA and is a potent feedback inhibitor for the transmethylation reaction. 28 However, AdoHcy itself is not a long lasting inhibitor of the AdoMet process due to its rapid catabolic breakdown. 119,181,182 Moreover, extracellular AdoHcy can not cross the cellular membrane as an intact molecule, 183 It is with this in mind that many AdoHcy analogues have been synthesized with the modification in the sugar ring, base portion (C 7 AdoHcy, Figure 23), or even amino acid part (SIBA, Figure 23) to overcome the disadvantages of AdoHcy. 184-193 O HO OH S N N N NH 2 HOOC NH 2 O HO OH S N N N N NH 2 H 3 C O HO OH N N N N NH 2 H 3 C 5'-Deoxy-5'-isobutyl-thioadenosine (SIBA) A9145C NH 2 H 2 N HOOC O HO OH N N N N NH 2 H 2 N HOOC Sinefungin S-Tubercidinyl-L-homocysteine (C 7 AdoHcy) NH 2 Figure 23. Analogues of AdoHcy In the area of AdoHcy analogue discovery, sinefungin (Figure 23), which was first isolated from Streptomyces griseolus at Lilly Research Laboratories, 194 was found to possess antifungal and antiparasitic activities. 195 These biological effects have been attributed to the inhibition of many AdoMet depended methylation reactions. The structure of sinefungin can envisioned as a sulfurless analogue of AdoHcy. 196-200 Both 29 sinefungin and its related metabolite, A9145C (Figure 23) are extremely active as inhibitors of vaccinia virion mRNA (guanine-7-)methyltransferase and nucleoside-2?- methyltransferase. 198,199 For example, the inhibition constant K i of A9145C for mRNA (guanine-7-)methyltransferase is 1.34 nM compared to AdoHcy (K i =1040 nM). 199,201 Unfortunately, both compounds possess significant toxicity for host cells at concentrations higher than 10 ?M. 199 This is due to the associated potent inhibition of cellular methyltransferase, including, for example, tRNA methyltransferase or protein methylase, by sinefungin and A9145C. 196,197 However, biological potential of sinefungin has stimulated many research groups, including the Schneller group, to pursue the synthesis of sinefungin and related analogues where modification at the side chain, heterocyclic base and ribofuranose unit is considered. 193,202,203 In that regard, this dissertation research sought the synthesis of the deamino- carbocyclic analogue of sinefungin (Figure 24). It was believed that this target compound would have improved activity by removing the side chain amino, while simultaneously making the compound more lipophilic to easily penetrate through the cellular membrane. Figure 24. Deamino-carbocyclic sinefungin 30 Chapter 1. Synthesis of Important Precursors A variety of compounds have been recognized as versatile precursors for the carbocyclic nucleosides synthesis. Among them (1R, 4S)-4-hydroxy-2-cyclopentyl monoacetate ((+)-monoacetate, 11) and (-)-(4R,5R)-4,5-O-isopropylidene-2- cyclopentenone (12) (Figure 25) are considered as most important intermediates for the D-like configuration carbocyclic nucleosides in this research. O OO OAc HO D-Cyclopentenone(+)-Monoacetate 11 12 Figure 25. Precursors for carbocyclic nucleosides synthesis In this direction, the past decades have seen enzymatic- and microbial- catalysts become powerful tools for organic synthesis, especially for enantioselective synthesis. These catalysts not only provide a simple and highly efficient approach for organic chemists but also are economical and environmentally friendly. Relevant here is the transformation of allylic diacetate 16 to the allylic monoacetate 11 and its enantiomer 13 by choosing different enzymatic conditions, pseudomonas cepacia lipase (PCL) to 11 or pig liver esterase (PLE) to 13 respectively (Scheme 6). 160,204,205 31 Scheme 6. Enzymatic hydrolysis of 16 To achieve 11 and 13 in this way, meso-diacetate 16 was synthesized in 3 steps from cyclopentadiene following a literature procedure (Scheme 7). 206 First, epoxidation of freshly cracked cyclopentadiene by peroxidic acid, the Pd-catalyst tetrakis- (triphenylphosphine)palladium(0) was used to open the oxidation product vinyl epoxide 14. The ?-allylpalladium complex 15 that formed and is used for construction of various stereo- and regiospecific substituted cyclopentanoids. In this case, acetic anhydride produced the cis-1,4-addition product 16. The reaction with acetic anhydride is presumed to be initiated by a nucleophilic attack of the basic oxygen anion in 15 on acetic anhydride. The resultant acetoxy anion subsequently trans attacked the distal end of the ?-allylpalladium complex through the less hindered face to realize the cis stereochemical product 16. AcO OAc 14 O Pd Ph 3 P PPh 3 O Na 2 CO 3 ,AcOOH,CH 2 Cl 2 15 16 Pd(PPh 3 ) 4 ,THF Ac 2 O Scheme 7. Synthesis of diacetate 16 32 These results are relevant to the centrality of cyclopentenone 12, as well as its different protecting group analogues, as a versatile synthon in carbocyclic nucleosides synthesis. There are several stereoselective procedures available to 12, 156 one of which is used in our laboratory from (+)-monoacetate 11 (Scheme 8). 207 This synthesis begins with the conversion of 11 into its phosphate derivative 17, followed by dihydroxylation with osmium tetraoxide to give diol 18. After protection of 18 with 2,2- dimethoxypropane, the acetate group was removed by treating with lithium hydroxide. The synthesis was completed to 12 by an oxidative elimination of 20 with pyridinium chlorochromate (PCC). Scheme 8. Synthesis of enone 12 from monoacetate 11 Another preparative route to 12 was developed in our laboratory in 2004 (Scheme 9). 208 This approach is considered more economical, by including the inexpensive natural 33 sugar D-ribose as the starting material, and being more efficient (5 steps start from D- ribose with 42% yield). Scheme 9. Synthesis of enone 12 from D-Ribose This synthesis started with the protection of D-ribose with 2,2-dimethoxypropane and simultaneous methylation of the anomeric hydroxyl group. The primary hydroxyl group in 22 was transformed into an iodo substituent (compound 23) by treating with triphenylphosphine and iodine. 209 Reductive cleavage of 23 with activated zinc powder in hot methanol afforded aldehyde 24. 210 Reaction of 24 with the Grignard reagent vinyl magnesium bromide provided diene 25, which was followed by the crucial ring closing step utilizing a 1 st generation Grubbs catalyst (Figure 26), for a ring closure metathesis 34 (RCM) process that was completed easily. After the oxidation of the intermediate allylic alcohol by pyridinium dichromate 12 was obtained in high yield. Metathesis is one of the most important methods available for ring formation. The extraordinary functional group tolerance and high catalytic reactivity of the ruthenium based Grubbs catalysts enable various efficient inter- or intamolecular metathesis. 211 Figure 26. First Generation of Grubbs Catalysts The mechanism of the Grubbs reaction was first proposed by Chauvin 212,213 (Scheme 10) with initiation at one of the olefins coordinated to the metal center. This intermediate then undergoes a [2+2] cycloaddition to afford a metallocyclobutane a. Followed by cleavage of the cyclobutane to release an olefin, a metal carbene b is formed, poised to react with the other side olefin. After a second [2+2] cycloaddition and cleavage of cyclobutane c, a new carbon-carbon bond is formed and the catalyst is restored for another cycle. 35 Scheme 10. Mechanism of Ring Closure Metathesis 36 Chapter 2. Synthesis of 5?-Homoaristeromycin and 8-Methyl-5?- Homoaristeromycin As an inhibitor of S-adenosyl-L-homocysteine hydrolase, the carbocyclic nucleoside aristeromycin, shows broad-spectrum antiviral activity. However, significant toxicity excludes it from clinic use. 87,147 One useful strategy to lower its toxicity is extension of the C-5? hydroxymethyl side chain by a methylene group to provide 5?- homoaristeromycin. 166 A previously reported method 163 leading to 5?-homoaristeromycin involved a radical cyclization of an ?, ?-unsaturated ester derived from a halo sugar (see 137 in scheme 11). 37 O O O O OH i,PPh 3 ,NBS,CH 2 Cl 2 ii,DIBAL,-70 O O O HO Br Ph 3 PCHCO 2 Et, PhCO 2 H DMF Br OH EtO 2 C O O OHEtO 2 C O O OHEtO 2 C O O OH O O TBDMSO O O TBDMSO N N N N O O TBDMSO N N N N Cl Cl + + HO OH HO N N N N NH 2 Bu 3 SnH, AIBN (TfO) 2 O, pyridium, DMAP OTf O O TBDMSO i, DIBAL, ether, ii, DMAP,TBDMSCl,CH 2 Cl 2 6-chloropurine,LiH,DMF, 135 136 137 138-a 139 140 138-b 141-a 141-b 1 Scheme 11. Jones Method for Homoaristeromycin synthesis A problem associated with the radical cyclization (137? 138) is that two isomers result in the ratio of 6:1. A further difficulty is in the SN 2 reaction for coupling of the purine base with the carbocyclic ring produces two products (that is, the N9 and N7 isomers). Furthermore, this route suffers from too many steps, limited scale-up and low yields. Thus, a new stereoselective synthesis for 5?-homoaristeromycin was developed in our lab. 165 This began with the 1,4-addition of ethyl trimethylsilylacetate to enone 12 followed by in situ cleavage of the trimethylsilyl group that established the ethylacetate 38 group at the C4 position. The ? (up) side chain ester was proven to be the only product. 214 A selective reduction of the ketone group of 142 afforded the precursor for coupling with 6-chloropurine. The reduction and amination was followed to achieve the target compound 1. (Scheme 12) Scheme 12. Synthesis of Homoaristeromycin from enone An alterative method for introducing the homo side chain at the C4 position is via the 1,4-addition of a vinyl organocuprate reagent to enone 12. This reaction occurs with good stereochemical selectivity. Based on this, the synthetic strategy for 5?-homoaristeromycin (1) and 8-methyl-5?-homoaristeromycin (2) is shown below. (Scheme 13) 39 Scheme 13. Retrosynthesis for Homoaristeromycin and 8-methyl- homoaristeromycin Both compounds were synthesized through the same intermediate 27. The Mitsunobu reaction was considered for coupling of cyclopentyl moiety with the 6-chloropurine base portion in both pathways. The requisite 5?-homohydroxyl unit was to be achieved by 40 hydroboration of the vinyl group introduced early in the scheme 14 via the Gilman reagent. O O O O O O OH O O Vinylmagnesium bromide, TMSCl, HMPA, CuBr Me 2 S, THF, 92% LiAlH 4 ,THF,96% 12 26 27 Scheme 14. Synthesis of the intermediate 27 In this research a successful synthesis of 27 involves two steps by starting with the incorporation of a vinyl group into compound 12 by the 1,4-enone addition to give the vinyl ketone 26 in 92% yield. Reduction of 26 with lithium aluminum hydride provided alcohol 27 in 96% yield. (Scheme 14) 41 O O N N N N Cl O O N N N N NH 2 O O N N N N N Boc Boc O O N N N N N Boc Boc HO OH HO N N N N NH 2 HO OH O O 6-Chloropurine, DIAD, NH 3 , MeOH, 100?C, O O N N N N NH 2 HO (Boc) 2 O, Et 3 N,DMAP, THF,87% 9-BBN, NaOH,1N, HCl, MeOH 67% in two steps 27 28 29 30 31 32 1 Ph 3 P,THF; 73% in two steps; H 2 O 2 ,THF; Scheme 15. Synthesis of (-)-HomoAri The cyclopentyl alcohol 27 was coupled with 6-chloropurine under Mitsunobu conditions to give 28, which, treated with methanolic ammonia at 100 ?C overnight, afforded 29 as a single isomer with some inseparable DIAD derived side-products (Scheme 15). The reaction of crude olefin 29 with 9-BBN generated not only the desired target alcohol 30, but also 1, 5-cyclooctanediol (Scheme 16). Since both compounds have very similar polarity, the separation of the desired alcohol from the diol byproduct was extremely difficult. 42 Scheme 16. Hydroboration Reaction for Compound 29 To overcome this problem, we switched to the bis-Boc protected compound 31, which was obtained by treatment of compound 29 with tert-butyl dicarbonate and a catalytic amount of 4-(dimethylamino)pyridine, for another attempt at the hydroboration- oxidation sequence. By adding two Boc protectiong groups, the molecular weight and hydrophobic character of the desired alcohol product were increased, rendering any products from it much easier to separate by column chromatography. Deprotection of 32 with hydrochloric acid led to the target compound 1 (Scheme 15). Scheme 17. Synthesis of Compound 34 43 The synthesis of the 8-methyl-5?-homoaristeromycine (2) started with the preparation of the 8-methyl modified 6-chloropurine base, which was obtained by following a reported procedure. 215 Commercially available 4, 5-diamino-6-hydroxylpyrimidine hemisulfate was treated with acetoamide to furnish the bicyclic heterocyclic ring 33. The hydroxyl functional group of 33 was converted to chloro by refluxing 33 in phosphorus oxychloride to afford 34 (Scheme 17). OH O O O O N N N N Me Cl O O N N N N Me Cl HO O O N N N N Me NH 2 HO HO OH N N N N Me NH 2 HO 27 34,Ph 3 P, DIAD, THF, 68% 9-BBN, THF, 85% NH 3 , MeOH, 100?C, 94% HCl, MeOH, 80% 35 36 372 Scheme 18. Synthesis of 2 from intermediate 27 Drawing from the hydroboration difficulties descrided previously (Scheme 16) a different procedure was used for the synthesis of 8-methyl-5?-homoaristeromycin (2). In this regard the synthesis was carried out by starting with a Mitsunobu reaction coupling of 34 with 27. The product 35 was subjected to hydroboration and followed by oxidation 44 with hydrogen peroxide to give the desired alcohol 36. Ammonation of 36 by treating with methanolic ammonia provided compound 37, which was easily converted to the final product 2 after a deprotection step under an acidic conditions (Scheme 18). 45 Chapter 3. Synthesis of 8-Methyl-3-Deazaneplanocin Analogues Due to the efficiency and apparent versatility of the previously described synthetic schemes to carbocyclic nucleoside that demonstrated that the convergent synthetic strategy is preferred over a linear one, 8-methyl-3-deaza carbocyclic nucleoside analogues were sought with this in mind. In this chapter neplanocin A was chosen as the framework for 8-methyl-3-deaza nucleobase series. Compounds 3, 4, 5, were set as target compounds. All three were envisioned as being assembled by coupling the same nucleobase, bis-Boc protected 8-methyl-3-deazaadenine (38) with the cyclopentyl portions of neplanocin A, 5?-norneplanocin A, and 5?-homoneplanocin A, respectively (Scheme 19). 46 Scheme 19. Strategy for target compounds 3, 4, 5 from nucleobase 38 The direct synthesis of nucleobase bis-Boc protected 8-methyl-3-deazaadenine is not available in published literature. As a consequence, a practical and efficient procedure leading to 6-chloro-3-deazapurine was developed for this research. After careful analysis, it was determined to subject 42 with methyl possessing ring closing it would be possible to realize the 6-chloro-8-methyl-3deazapurine, needed for the nucleobase 38 in few steps (Scheme 20). 47 Scheme 20. Retrosynthetic analysis of 38 Thus, 4-hydroxypyridine, which is commercially available and inexpensive, was chosen as the starting material for the synthesis. After nitration (Scheme 21), a nitro group was installed adjacent to the hydroxyl group (39). The nitration product 39 was treated with phosphorus pentachloride and then, without purification, ethanol, whereby hydroxyl group of 39 was substituted by an ethoxyl group through a chloride intermediate. By introducing the ethoxy leaving group, compound 40 was converted to 41 through an ammonia nucleophylic substitution process. Reduction of the nitro group of 41 with tin chloride in concentrated hydrochloric acid was accompanied by a chloration on the 2 position to give 42. 48 39 N OH N OH NO 2 N OEt NO 2 HNO 3 ,H 2 SO 4 ,72%; i) PCl 5 ,ClCH 2 CH 2 Cl; ii) EtOH, 99% N NH 2 NO 2 N NH 2 NH 2 Cl NH 4 OAc, H 2 O, 81% SnCl 2 , HCl, 71% N N H N Cl Me i) CH 3 C(OMe) 3 , formic acid, ii) NaOH, 96% 40 41 42 43 Scheme 21. Synthesis of 6-chloro-3-deazapurine Ring closure reaction of 42 at 100?C with trimethyl orthoacetate and added formic acid as catalyst failed to conveniently give the 8-methyl derivative in contrast to previously described 3-deaza-6-chloropurine. Instead of getting the desired ring closure product 43, a di-imino substituted intermediate 44 was separated from the reaction. Treating the intermediate 44 with 1.5N sodium hydroxide and refluxing for one more day, 43 was isolated and its structure proven by X-ray crystallography (Figure 27). Acid condition also promoted the ring closing reaction of 44 and proved to be equally effective. Below is the proposed mechanism for this ring closing reaction process (Scheme 22). 49 Figure 27. X-ray crystal structure of compound 43 N NH 2 NH 2 Cl N N H N Cl Me CH 3 C(OMe) 3 ,formicacid, NaOH, 96% 42 43 N N N Cl C OCH 3 CH 3 C OCH 3 CH 3 N N N Cl C OCH 3 CH 3 C OCH 3 CH 3 OH N N N CH 3 Cl O OCH 3 H 3 C H N N NH CH 3 Cl +COCH 3 O H 3 C OH+ - OCH 3 43 44 44 N N N Cl C OCH 3 CH 3 C OCH 3 CH 3 H N N N CH 3 Cl OCH 3 H 3 C H+ - HOCH 3 44 OH N N N CH 3 Cl OCH 3 H 3 C O H 43 + COCH 3 O H 3 C Scheme 22. Mechanism for the ring closing reaction of 42 With 43 in hand its use in the Mitsunobu reaction, which has been studied in our laboratories for 3-deaza carbocyclic nucleosides, was considered. 216 As background, in our laboratory we have shown that the 3-deaza-6-chloropurine reacted with saturated 50 cyclopentanol under Mitsunobu conditions, and only one isomer, N-1 (purine N-9), was obtained. However, if the substrates were more reactive allylic alcohols, two regioisomers were observed, with the N-3 (purine N-7) isomer as the major product along with the minor N-1 (purine N-9) isomer. (See table 2) 51 Table 2. Mitsunobu reaction between 3-deaza-6-chloropurine and cyclopentanols Based on this, an improved method was developed to overcome this unwanted N-3 (purine N-7) coupled by-product. In this regard, tert-butoxycarbonyl protected 3- 52 deazaadenine derivative was chosen as the substrate for the Mitsunobu coupling reaction and found no N-3 isomer. The high regioselectivity (see Scheme 23) may be due to the steric hindrance from two bulky Boc protecting groups on the 4-nitrogen, which blocked the nucleophilic substitution at the N-3 position. In addition to steric control, the reaction occurred faster and was achieved in higher yield than 6-chloro-3-deazaadenine, due to improvement of the solubility for the tert-butoxycarbonyl protected 3-deazaadenine. Therefore, the di-Boc protected 8-methyl-3-deazaadenine 38 was considered as a better candidate for the desired Mitsunobu coupling reactions than 6-chloro derivative 43. Scheme 23. di-Boc protected 3-deazaadenine as nucleophile in Mitsunobu reaction The desired precursor to 38 was 45. The literature 153,177 suggested that standard conditions (ammonia/methanol) would not be successful to displace the chloro substract of 43. Therefore, a procedure using anhydrous hydrazine, followed by treatment with Raney nickel to the resultant hydrazino derivative was chosen and afforded 8-methyl- 3- deazaadenosine (45) (Scheme 24). 53 Scheme 24. Synthesis of 8-methyl-3-deazaadenine Compound 45 was next treated with excess tert-butyl dicarbonate and a catalytic amount of 4-(dimethylamino)pyridine in tetrahydrofuran at room temperature for 2 days and the tris-Boc-protected 46 along with a by-product bearing high polarity observed in TLC (Scheme 25). After the separation and NMR spectroscopic analysis, the by-product 46-b was determined to be another bis-Boc-protected compound isomeric to but different from 38. The 1 H-NMR data of 46-b showed two different peaks at ?=1.71 and 1.55 respectively, for tert-butyl groups of the Boc protecting moiety, which indicated that the two Boc groups were installed in different chemical environment. Based on that, it was suggested that the N-6 amino group was deactivated by the first attached Boc group, along with, perhaps, increased steric crowding at the newly formed NHBoc center, reduced the N-9 in the heterocyclic center more accessible, this suggested the bis-Boc- protected 38 could not be obtained by controling the amount of tert-butyl dicarbonate. A more successful approach began by forming the tris-Boc-protection followed with selectively deprotecting of N-9 site. Thus, after recovery of 46-b, it was treated with tert- butyl dicarbonate and a catalytic amount of 4-(dimethylamino)pyridine for one day, to provide 46 in high yield. (Scheme 25) 54 Scheme 25. Protection for 8-methyl-3-deazaadenine Attempts to remove the N-9 Boc group were seen as possible by hydrogenolysis, or under acidic or basic conditions. But none of these were effective without also removing the N-6 amino Boc groups. A recent study 217 has shown that tetra-n-butylammonium fluoride (TBAF) was a mild and selective deprotection approach, which had been known for the removal of a silyl ether group. Applying TABF to compound 46 for two days, the N-6 bis-Boc protected 8-methyl-3-deazaadenine 38 was obtained as a single isomer. (Scheme 26) Scheme 26. Selective deprotection of Compound 46 The mechanism for this selective reaction can be envisioned as proceeding in two different ways (Scheme 27). In the first case 218,219 (a), the fluoride can act as an base and promote a ?-elimination to result in the free amino group or (b) the fluoride is acting as a 55 nucleophile and directly attacks the Boc carbonyl group leading to an amino anion and Boc-F, which, upon hydrolysis, becomes tert-butanol, carbon dioxide and hydrofluoric acid. 220 Studies have shown that the reaction more likely follows pathway b, since there were several substrates without a susceptible proton that still underwent the reaction. 217 Scheme 27. Postulated mechanism for Boc-deprotection by TABF The selective deprotection of the N-9 Boc group of compound 46 can be considered as another piece of evidence to support the theory. The selectivity for the leaving amide anion generally followed the order: aromatic > benzylic > aliphatic. In this case, the leaving group was hetero aromatic amide anion comparing with the alternative tert- butoxycarbonyl amide anion from C-6. The activities for those two were quite close. If the reaction occured through pathway a, the likelihood for cleavage at the N-6 Boc group would increase, since there are more hydrogen atoms available at that center. The results indicated that the fluoride anion acted as a nucleophile attacking the less crowded N-9 carbonyl group, by pathway b, and giving the N-9 deprotected product 38. 56 Synthesis of 8-Methyl-3-Deaza-5?-Norneplanocin A With the important building block 38 in hand, the synthesis of 8-methyl-3-deaza-5?- norneplanocin A (3) calling on the retrosynthetic designed below. Thus, target compound 3 was to be assembled through a coupling reaction of 38 with 47, which was accessible from the crucial precursor, cyclopentenone 12 (Scheme 28). Scheme 28. Retrosynthesis of 8-methyl-3-deaza-5?-norneplanocin A (3) Beginning with conversion of cyclopentenone 12 to allylic alcohol 47 called on a highly stereospecific 1, 2 -reduction under Luche conditions of sodium borohydride and cerium (?) chloride heptahydrate (Scheme 29), 221 this was followed by a Mitsunobu coupling reaction to join 47 with nucleobase 38. After cleavage of the protecting groups under acidic conditions, 8-methyl-3-deaza-5?-norneplanocin A (3) was afforded in good yield. 57 Scheme 29. The synthesis of 8-methyl-3-deaza-5?-norneplanocin A Synthesis of 8-Methyl-3-Deaza-Neplanocin A A straightforward approach to compound 4 is outlined in Scheme 30 by coupling the allylic cyclopentenol 49 with 38. Chiral cyclopentenol 49 with various protecting groups for example, trityl for compound 49a, benzoyl for 49b has been used as a key intermediate for the synthesis of various neplanocin A related compounds. The first synthesis for 49 was in 1983 by Marquez. 222 Since then, many new or modified synthetic routes have been developed. The following is a brief review and analysis of diverse synthesis methods for this pivotal molecule. 58 Scheme 30. Retrosynthesis of 8-methyl-3-deaza-neplanocin 4 The synthesis of the desired cyclopentenol rings start with D-ribose. In the early investigations, the strategy was based on a Wittig reaction to afford a cyclopentenone 54 (55 to 54) 223 or substituted cyclopentenone 50. 224 The key step for building up two important carbocyclic intermediates (50 and 54) was realized by employing the Wittig type reaction after the installation of the methyl phosphonate either on C-1(route on top for 50) or C-4 (route on bottom for 54) of D-ribose (Scheme 31). Compound 50 was selectively reduced to the benzoyl group protected 49b and 53 was transferred to the methoxy methyl group protected 49c by a palladium catalyzed migration of tertiary allylic alcohol. This old methodology usually suffered from long synthetic steps, and low yield. 59 Scheme 31. Previous synthesis for cyclopentenone 49 New synthetic approaches to 49 have been developed by utilizing a ring closing metathesis (RCM) to build the carbocyclic ring of 49. Based on this, in 2005, Schneller et al. discovered a new synthetic route to 49 (Scheme 32 on the top) by applying a Wittig reaction at the C-4 position of D-ribose and later a Grignard reaction to install a vinyl group at C-1 to realize the compound 57, the precursor for RCM. 225 After the RCM ring closure, an oxidative rearrangement of tertiary allylic alcohol of 56 was employed to complete the process to cyclopentenone 49. Two yeas later, Chu?s group used a similar strategy to obtain diene 65 for the RCM reaction (Scheme 32 on the bottom). 226 60 Scheme 32. New strategy for synthesis for cyclopentenone 49 Since the efficiency and relatively high yield, Chu?s approach was chosen for the synthesis of 49 for this research (Scheme 33). Thus, D-Ribose was transferred to the isopropylidene protected 59 by treatment with 2, 2-dimethypropane in the presence of a catalytic amount of p-toluenesulfonic acid. This was followed by selective protection of the primary hydroxyl group by triphenylmethyl chloride gave 60. Reaction of 60 with vinylmagnesium bromide opened the furanose ring of 60 to provide the single diastereoisomer 61. The newly produced allylic hydroxyl group of 61 was protected with a tert-butyldimethylsilyl (TBDMS) group. The unprotected secondary hydroxyl group of 62 was oxidized under Swern condition to afford the ketone 63, which in turn underwent a Wittig reaction with methyltriphenylphosphonium bromide and butylithium (n-BuLi) in tetrahydrofuran to result in the diene 64. Subjecting on 64 to the RCM reaction, with either first or second generation Grubbs of catalysts failed. This may be due to steric 61 interference from the bulky protecting groups of 64. To circumvent this, the TBDMS protecting group was removed by TBAF to provid dienol 65. The RCM reaction was then successfully carried out on 65 with a second-generation of Grubbs catalyst to form 49a. OO TrO OH OO TrO O OTBS OO TrO OH OH D-ribose O OO HO OH O OO TrO OH OO TrO OH OTBS OO TrO OTBS OO TrO OH 2,2-dimethypropane, TsOH H 2 O,acetone, rt, 88%; TrCl, Et 3 N, DMAP, DMF, rt, 85% vinylmagnesium bromide,THF, -78 ?C to rt, 98%; TBSCl, imidazole, CH 2 Cl 2 /DMF, rt, 79%; (COCl) 2 ,Et 3 N,DMSO, CH 2 Cl 2 ,-60?C,94%; Ph 3 PCH 3 Br, n-BuLi, THF, rt, 91%; TBAF,THF,95% second-generation Grubbs catalyst, CH 2 Cl 2 ,rt,93% 59 60 6162 63 64 65 49a Scheme 33. Synthesis of cyclopentenol 49a After finishing the synthesis of 49a, a Mitsunobu reaction was conducted with nucleobase 38 (Scheme 34). The coupling product 66 was achieved in good yield. Hydrochloride acid was used to remove the protecting groups to give the final product 8- methyl-3deazaneplanocin A (4). 62 Scheme 34. Synthesis of 8-methyl-3-deazaneplanocin Synthesis of 8-Methyl-3-Deaza-Homoneplanocin A Based on the success of synthesis of the previously described 8-methyl-3-deaza neplanocin analogues, a similar synthetic route for 8-methyl-3-deaza-homoeplanocin (5) was designed as illustratede below by coupling of cyclopentenol 72 with nucleobase 38. Scheme 35. Retrosynthesis of 8-methyl-3-deaza-homoneplanocin 5 63 To provide access to the homoneplanocin cyclopentyl portion 72 the 1996 method of Shuto?s research 166 was considered (Scheme 36). The approach was derived from the synthesis of the neplanocin building block 49c reported by Hill (Scheme 31). 223 From the same intermediate 54, a 1,2-addition of the carbanion of ethyl acetate was used instead of methoxymethyl for 49c in Scheme 31. After the ester reduction and two different subsequent protection steps for the primary and tertiary alcohols, the same palladium- catalysed allylic rearrangement was employed. A selective deprotection was followed to this important intermediate 72. Scheme 36. Shtuo?s method for synthesis of cyclopentenol 72 A few years later, a more efficient approach to cyclopentenol 72 was discovered in our laboratories (Scheme 37). 167 This route started with the important precursor cyclopentenone 12, which is the enantiomer of 54. The synthesis began with a regio and stereoselective 1,4-addition on 12 with methyl phenylthioacetate to yield 73. Oxidation of the thioether with m-chloroperoxybenzoic acid (m-CPBA) to the sulfoxide was followed 64 by dehydrosulfenylation providing a new double bond that quickly rearranged to the thermodynamically more stable conjugated system 74. After a Luche reduction on 74, 75 was cleanly afforded in high yield. Scheme 37. The Synthesis of compound 75 The initial plan was to use the compound 75 as a substrate for the Mitsunubo reaction, but, instead of getting the desired coupling product, 75 underwent an elimination reaction to give the product 76. A Mechanism for this outcome is given in Scheme 38. Appearance of by-product 76 was believed to be due to the deprotonation of cyclopentenol 75 (instead of 38) by the triphenylphosphine-diisopropyl azodicarboxylate (TPP-DIAD) complex. The driving force for this carbanion formation from 75 was large delocalized conjugated system as a consequence. 65 NN CO 2 iPr iPrO 2 C Ph 3 P NN CO 2 iPr Ph 3 P iPrO 2 C OH O O MeO 2 C H OH O O O MeO NNH CO 2 iPr Ph 3 P iPrO 2 C O O O O MeO NNH CO 2 iPr Ph 3 P iPrO 2 C H O O O O MeO PPh 3 O O MeO 2 C 76 75 Scheme 38. The proposed mechanism for the reaction of 75 with 38 A solution for avoiding this was reduction of both ester and ketone functionalities in 74 by diisobutylaluminum hydride (DIBAL). The coupling precursor 72 was obtained after selective protection of the primary alcohol of 77. The large protecting reagent tert- butyldimethylsilyl chloride (TBDMSCl) was used to assure the primary alcohol selectivity, since the activities for secondary allylic alcohol and primary alcohol would have been competitive (Scheme 39). 66 Scheme 39. The synthesis of precursor 72 This synthesis method for 72 was preferred from the previous one (Scheme 36) for 3 reasons: 1) fewer steps from a similar intermediate (12); 2) higer overall yield (27% versus 9.5%) and 3) less expensive reagents particularly with palladium reagent of the old method. 167 Compound 72 was subjected to Mitsunubo reaction conditions with nucleobase 38 to give compound 78, which was treated with hydrochloric acid to remove the isopropylidene and TBDMS simultaneously to obtain the final product 8-methyl-3-deaza- homoneplanocin (5) (Scheme 40). Scheme 40. The synthesis of compound 5 67 Chapter 4. Synthesis of 8-Methyl-3-Deazaaristeromycin Analogues The initial plan for synthesis of 8-methyl-3-deaza aristeromycin analogues was based upon the success of the synthesis of neplanocin analogues with the same nucleobase. Thus, the retrosynthetic analysis was shown in Scheme 41 was established. Compound 27 was chosen as the coupling component for the cyclopentyl portion, since the terminal alkene provided a versatile framework for various C-5? aristeromycin modifications. 68 Scheme 41. Retrosynthesis of compound 6, 7 The bis-Boc protected nucleobase 38 was reacted with cyclopentanol 27 under Mitsunobu condition. In that direction, alcohol 27, nucleobase 38 and triphenylphosphine (TPP) were suspended at 0 ?C for 10 minutes and diisopropyl azodicarboxylate (DIAD) was added dropwise over 20 minutes. After 12 hours at room temperature, no reaction occurred. Upon continuing the reaction for another 12 hours at 50 ?C, an unknown product was detected by thin layer chromatography (TLC). Instead of the expected 79, the compound confirmed by NMR spectral analysis was 80a, a product of nucleophilic substitution of nucleobase 38 to DIAD (Scheme 42). This result may be due to the slow formation of the desired alkoxyphosphonium salt between alcohol 27 and TPP or perhaps, the sluggishness of the reaction of the deprotoned nucleobase with this salt. Based on these suggestions, we switched the nucleobase 38 to 43 and modified the conditions to improve the formation of phosphonium salt before other side reactions occured. Two 69 conditions were tried: first, TPP and 27 were dissolved in tetrahydrofuran (THF) at -40 ?C. After stirring at this temperature for 20 minutes, a solution of DIAD in THF was added over an hour. The unexpected side product 80b, as before, was obtained. In the second approach, the TPP-DIAD complex was preformed first in THF at 0 ?C, followed by the addition of a mixture of 27 with 43 in THF at -20 ?C. No reaction occurred even after heating the reaction mixture to 70 ?C for more than 12 hours. Scheme 42. Mitsunobu reaction of nucleobase with compound 27 The consistent appearance of side products was intriguing us to seek coupling reagents other than DIAD. Recently, a study showed an alternative protocol by using the Herdrickson ?POP? (triphenylphosphonium anhydride trifluoromethanesulfonate) reagent for the Mitsunobu reaction. 227 In the reported case, the POP reagent was used for dehydration in the formation of esters, amides and other similar compounds in a manner similar to the Mitsunobu reaction. A key intermediate, an alkoxyphosphonium salt, was apparent in this procedure. Since azodicarboxylates are not involved in the POP process, it was believed this would avoid the steps leading to 80 (Scheme 43). Thus, the reaction 70 was carried out by addition of triflic anhydride to an ice-chilled solution of triphenylphosphine oxide in dry dichloromethane (DCM). To this 27, 43, and diisopropylethylamine (DIEA) were added in succession. Unfortunately, no reaction occurred except for recovery the starting materials, which indicated that use of the Hendrickson reagent for secondary alcohols (as in 27) was not amenable to success, perhaps, due to steric interference. Scheme 43. The reaction and general mechanism for Hendrickson reagent Because of the failure of the Mitsunobu type reactions, a direct S N 2 reaction seemed to be a reasonable alternative for the enantioselective coupling reaction. Similar conditions 71 have been called on before by treating an appropriate nucleobase under basic circumstance, with the corresponding cyclopentyl tosylate 224 or epoxide 228 for the desired carbocyclic nucleosides synthesis. In this regard, compound 27 was readily transferred to tosylate 81 by treating with p-toluenesulfonyl chloride. However, no product could be found after the addition of the sodium salt of 43 to a solution of 81 in N, N- dimethylformamide (DMF) for over 24 hours (Scheme 44). Scheme 44. The S N 2 reaction for the compound 27 Even though similar reactions met no difficulty for cyclopentenol 47, 49a and 72, attempts at coupling cyclopentanol 27 to the 8-methyl-3-deazapurine base did not succeed. Previous experiences caused us to replace 27 with an unsaturated cyclopentene ring for the coupling reaction. For that purpose, monoacetate 11 became an option for several reasons: it was amenable to scale up; it provided the simplest template for the coupling reaction and itself also could serve as the precursor for noraristeromycin analogues. From studies that have shown that an enantioselective substitution promoted by tetrakis(triphenylphosphine)palladium catalyst was very effective for building up both the 3-deaza 177 and 8-methyl-noraristeromycin. 174 Therefore, it was reasonable to believe that the similar outcome would occur with a base combining two synthetic elements that 72 is, 3-deaza, and 8-methyl. The stereochemistry was insured by the nucleophilic attack to the less congested ?-face of the early formatted carbocation palladium complex. 229 (Scheme 45) OAcHO N N H N X Me + 11 N N N X Me HO 82a, X= N(Boc) 2 ; 82b, X= Cl; a 38, X= N(Boc) 2 ; 43, X= Cl; a) NaH, (Ph 3 P) 4 Pd, THF, 10% for 82a, OAcHO OAc HO Pd L L N N N X Me N N N X Me HO Scheme 45. Reaction and mechanism for the palladium catalyzed substitution Both 3-deaza-8-methyl-6-chloropurine 43 and bis-Boc protected 38 were used. After reacted with sodium hydride, the nucleobases were treated with a solution of tetrakis(triphenylphosphine) palladium, TPP and 11. The reaction produced only a trace amount of 82a (less than 10%) after 24 hours at 55 ?C. This result was due to the nucleophilic activity for acetate anion competing with the deprotonated nucleobase 43 or 38 for the palladium complex, this consequence led to a return to further investigation of the Mistunobu reaction. 73 Study of the Mitsunobu Reaction Based on the experience with the secondary allylic alcohols (47, 49a, 72) as good substrates for the Mitsunobu reaction with 3-deaza-8-methyl purine bases (Table 3 entry 1~3), monoaceated 11 was subjected to the Mitsunobu reaction with nucleobase 43 and 38. However, byproduct 80 was obtained rather than the desired coupling product 82 (Scheme 46). Scheme 46. Mitsunobu reaction for compound 11 with 3-deaza-8-methyl nucleobase The Mitsunobu reactions conducted with various cyclopentanols or cyclopentenols and a 3-deaza-8-methyl nucleobase are summarized in Table 3. Based on the trend of reactivity towards alcohol substrates and the accompanying complicating materials (triphenylphosphine oxide and compound 80), a hypothetical mechanism is presented here (Scheme 47). The first two steps are the same as the conventional Mitsunobu reaction: that is, deprotonation of the nucleobase after a TPP-DIAD complex is formed. The steps that follow proceed in two directions. A competition arises between the modified purine anion (A in Scheme 47), which is more active than adenine due to the 74 electron-donating methyl group on C-8 position and the absence of the electron- withdrawing N-3 leads to reaction in the complex at the isobutyl carbonyl (path b), this occurs before formation of more common alkoxyphosphonium salt (B). The kinetic controlled compound 80 is afforded as well as triphenylphosphine oxide. Increasing the reactivity of the cyclopentyl, allylic substrate 47, 49a and 72 for example, facilitates the reaction towards the conventional Mitsunobu reaction direction (pathway a). Scheme 47. Proposed mechanism for Mitsunobu reaction To test this hypothesis, monoacetate 11 bearing an allylic alcohol functional group was subjected to the Mitsunobu reaction but also yielded 80. It was believed that the electron-withdrawing acetate group adjacent to the double bond in 11, which could lower 75 the activity of the allylic alcohol acting as the Mitsunobu nucleophile, could be the reason for this result. To verify this assumption, the Mitsunobu reaction was tested with the TBDMS protected compound 85, which was easily obtained from 11 in two steps: protection of the C-4 hydroxyl of 11 with tert-butyldimethylsilyl chloride followed by selective removal of the acetate under basic conditions. This time Mitsunobu reaction of 38 with 85 produced 86, this opened an effective pathway to many based modified aristeromycin analogues synthesis (Scheme 48). OAcHO N N H N N(Boc) 2 Me 11 OAcTBSO 84 OHTBSO TBSCl, imidazole, CH 2 Cl 2 ,80% 85 K 2 CO 3 , MeOH, 74% + 38 Ph 3 P, DIAD, THF, 78% TBSO 86 N N N N(Boc) 2 Me Scheme 48. Synthesis and Mitsunobu reaction for 85 Due to the success in realizing compound 85, the most straightforward scheme to the preferred ?-isomer of 86 would be to manipulate the hydroxyl group in 85 to an ?- configuration prior to Mitsunobu conditions (Scheme 49). Thus, the conversion of the hydroxyl stereocenter of 85 was accomplished by applying a Mistunobu reaction with benzoic acid to afford an ?-benzoyl group that was removed in strong basic conditions to give 88. This diastereomer of compound 85 was subjected to the Mitsunobu conditions, but the result was complicated by an inseparable mixture: 89a (N-9 isomer) and 89b (N- 76 7). The ratio of these two products was 1:1 by TLC in a combined yield of less than 14%. Disappointingly 80a was also separated from the reaction mixture, this suggested that the bulky TBS protecting group on the same side of the cyclopentyl moiety as the incoming nucleobase was affecting the reaction. To evaluate this assumption, compound 93 (See Table 3) with a methylene group between the cyclopentenol ring and the TBS protected primary alcohol was sought with the hope the longer side chain of 93 would alleviate the congestion in the transition state. Scheme 49. Synthesis and Mitsunobu reaction for the isomer of 85 Compound 93 was achieved from the monoacetate 11 (Scheme 51) by adapting a published procedure. 230,231 Thus, five equivalents of freshly made (isopropoxydimethylsilyl)methyl Grignard reagent (90) (Scheme 50) was added slowly to a solution of compound 11 in the presence of copper(I) iodide in THF to afford 91a in high regioselectivity (91a : 91b= 9:1). Conversion 91a to the useful intermediate 92 was carried out by the Tamao oxidation conditions to 92. After the selective protection of the primary alcohol of 92, the desired coupling precursor 93 was obtained in good yield. The 77 Mitsunobu reaction with 93 processed nicely to give the N-9 isomer 94a in 48% yield. The N-7 isomer 94b was also found and separated from 94a by chromatography column. The ratio of two isomers (94a:94b) was around 4:1.The structures of the two isomers were distinguished by a NOESY spectral analysis (Figure 28). The methyl group on the C-8 position of 94a can be expected adapt to a syn conformation while the N-7 isomer 94b would be in the more preferred anti conformation due to the bulky Boc protecting group on the C-6 position. A consequence of these conformations would be a strong NOESY relationship between hydrogen at C-3 with the C-6? hydrogens in 94a, and a NOESY consequence between the C-8 methyl group and the C-6? protons of 94b. After these predictions were observed, the dihydroxylation of 94b by osmium tetroxide, followed desilylation under acid conditions and removal the Boc protecting groups, compound 94 was smoothly transferred to the final product 6, 3-deaza-8- methylaristeromycin. Scheme 50. Synthesis of Grignard reagent 90 78 HO OAc HO Si O HO OH TBSO OH TBSO N N N N(Boc) 2 Me TBSO N N N N(Boc) 2 Me HO OH HO N N N NH 2 Me HO OH 90,CuI,THF,0?C,80% 11 91a 92 KHCO3, KF, H 2 O 2 , THF,MeOH, 71% 93 TBSCl, imidazole, CH 2 Cl 2 ,68% 94a Ph 3 P, DIAD, THF, 30% NMO, OsO 4 ,CH 2 Cl 2 , 83% + N N H N N(Boc) 2 Me 38 95 HCl, MeOH,70% 6 HO OSi 91b :=1:9 + TBSO N N N N(Boc) 2 Me 94b + Scheme 51. Synthesis of 3-deaza-8-methylaristeromycin Figure 28. NOESY analysis for two isomers of 94 79 Table 3. Mitsunobu reaction for coupling (Part A) Entry Alcohol Purine Base Product 1) 2) 3) 4) N N H N Cl Me 43 N N N X Me OO 80a, X= N(Boc) 2 ; 80b,X=Cl; 5) 80 Table 3. Mitsunobu reaction for coupling (Part B) Entry Alcohol Purine Base Product 6) OAcHO 11 N N H N Cl Me 43 7) 8) OH TBSO 88 TBSO 89a N N N N(Boc) 2 Me TBSO 89b N N N N(Boc) 2 Me 9) 81 Chapter 5. Synthesis of 4?-Isoaristeromycin and Its 3-Deaza Analogues The 4?-isoaristeromycin (8a) and its 5?-homo analogue (9a) have been studied and synthesized before in our laboratories, but the synthetic routes suffered from long steps, and low yields. Investigations in this project produced two important cyclopentyl building blocks 49a and 72 as good scaffolds for isomeric carbocyclic nucleoside analogues including 4?-isoaristeromycin and its 5?-congener. Therefore, new synthetic procedures were explored for 4?-isoaristeromycin (8a) and 4?-isohomoaristeromycin (9a) as well as their 3-deaza analogues (8b and 9b, respectively). For the 4?-isoaristeromycin, two synthetic approaches were analyzed (Scheme 52). Both methods shared the same intermediate 100 as the precursor for a Mitsunobu reaction. Compound 100 coupling with the 6-chloropurine was the old method; use of bis-Boc protected adenine in the coupling presented a new approach. The intermediate 100 was achieved in different pathways derived from the same starting material, D-ribose. 225,226 Total synthetic steps for 8a were same (11steps) for two procedures, but the yield was improved significantly for the new one (25.3% Vs 17.5%). 82 HO OH N N N N NH 2 HO OH O O O O O O TrO O OH O O TrO O O N N N N Cl TrO TrO O TrO O O N N N N N(Boc) 2 TrO HO OH N N N NH 2 HO O O N N N N(Boc) 2 TrO Old method: New method: 8a 102 100 98 96 49a 108 8b D-ribose Total yield: 17.5% Total yield: 25.3% 107 Scheme 52. Retrosynthetic analysis of 4?-isoaristeromycin and 3-deaza-4?- isoaristeromycin. The two pathways envisioned for the 4?-isohomoaristeromycin (9a) started with the intermediate 77, which was the precursor of allylic alcohol 72. The way to 9a in old method was involved a tedious oxidation then reduction of C-1? hydroxyl group (77? 97? 99? 101). However, the new synthetic route was designed to be more efficient 83 by eliminating unnecessary steps. This resulted in 3 fewer steps and an improved yield. Moreover, these routes were amenable to the 3-deaza analogues (Scheme 53). Scheme 53. Retrosynthetic analysis of 4?-isohomoaristeromycin and 3-deaza-4?- isohomoaristeromycin. The old synthestic routes for 8a and 9a started with the cyclopentenone 96 and 97 (Scheme 54). The former one was derived directly from the D-ribose (Scheme 32 on the top) 225 and the later from diol 77 (Scheme 54). For 97, after selectively protecting the primary alcohol of 77, compound 111 was subjected to a Dess-Martin oxidation to provide 97. Hydrogenation to the top face was applied to both 96 and 97 to ensure the ?- 7 steps, total yield 55.6% 4 steps, total yield 59.5% 84 stereochemistry of the C-4 position (structure proof vide infra for Figure 29). This was followed by a stereospecific reduction of the ketone functional group of 98 and 99 to furnish the coupling precursors 100 and 101. Both compounds were treated with 6- chloropurine, TPP and DIAD to afford 102 and 103, which went through the ammonolysis and deprotection to give final products 8a and 9a, respectively. Scheme 54. Old method for synthesis of compound 8 and 10 Before discussing the new synthetic approach developed here for the 4?- isoaristeromycin analogues, the synthesis of two requisite nucleobases, bis-Boc protected 85 adenine and 3-deazaadenine is described (Scheme 55). Protected adenine 113 was accomplished by generating the tris-Boc protected adenine 112 then selectively removing N-9 Boc group with aqueous sodium bicarbonate (NaHCO 3 ). For the synthesis of the 3- deazaadenine derivatives (115), 2-chloro-3, 4-diaminopyridine (41) was used as a starting material, the same reagent used for the synthesis of 3-deaza-8-methyladenine and triethyl orthoformate to give 114. Displacement of the chloro substituent in 114 was carried out by treatment with hydrazine followed by Raney nickel reduction. The newly formed amine group in 3-deazaadenine 115 was then protected with Boc groups in a two-step sequence: tris-Boc protection by tert-butyl dicarbonate; followed by selective deprotection of the N-9 Boc group (Scheme 55). Scheme 55. The synthesis of bis-Boc protected adenine and 3-deazaadenine For the cyclopentyl portion, a new chiral center on the C-4 position was established by employing a catalytic hydrogenation to cyclopentenone 49a or 72. The stereochemistry of 86 the products 100 and 106 were determined by a NOESY spectral analysis (Figure 29). The strong NOESY relationship between C-4 with C-2, C-3 and one of the C-6 protons indicates the ?-configuration at C-4 postion. With these two coupling precursors available, Mitsunobu reactions with both nucleobase 113 and 117 were conducted. After removing protecting groups for all coupling products by hydrochloric acid, final products 8a, 8b, 9a, and 9b were achieved in high yields (Scheme 56). O O N X N N N(Boc) 2 PO HO OH N X N N NH 2 HO Ph 3 P, DIAD, 113 or 117, THF; HCl, MeOH; 107,n=1,P=Tr,X=N,76%; 108,n=1,P=Tr,X=C,71%; 109, n=2, P=TBS, X=N, 79%; 110, n=2, P=TBS, X=C, 70%; OH O O PO OH O O PO H 2 ,Pd/C,MeOH; 49a,n=1,P=Tr; 72, n=2, P=TBS; 100,n=1,P=Tr,94%; 106,n=2,P=TBS,90%; n n n n 8a, n=1, X=N, 81%; 8b,n=1, X=C,77%; 9a, n=2, X=N, 90%; 9b,n=2, X=C,87%; Scheme 56. The synthesis for 8 and 9 OO OH TrO H H H H H NOE Figure 29. NOSEY for 100 87 Chapter 6. Synthesis of Deamino-Carbocyclic Sinefungin After careful analysis, a practical convergent synthetic strategy was designed for deamino-carbocylic sinefungin 10. It was envisioned the target 10 could be prepared from an alaninol synthon (122) and an appropriate carbocyclic nucleoside, the latter being accessed by utilizing a Mitsunobu reaction to connect a functionalized cyclopentenol with a protected adenine. An initial attempt (pathway a in Scheme 57) to the carbocyclic nucleoside aldehyde 127 following conversion of the coupled product of 124 with 113 was not encouraging. Thus, an alternative pathway was explored by using the n-butenyl side chain of 129 to achieve the aldehyde functional group (pathway b in Scheme 57). 88 NHO O PPh 3 I O O N N N N N(Boc) 2 O N N N N H N(Boc) 2 O O OH O O OH + + O O O a b HO OH N N N N NH 2 H 2 N HOOC 10 122 127 124 113 129 12 113 N N N N H N(Boc) 2 + Scheme 57. Retrosynthetic analysis of deamino-carbocyclic sinefungine 10 In that direction, naturally occurring D-serine bearing a chiral center was seen as a good source for building a nucleophilic alanine synthon. Therefore, a precursor of this species 122 (actually a Wittig precursor) was prepared from L-serine methyl ester hydrochloride following a methodology developed by Sibi and coworkers. 232 The synthesis commenced with the conversion of starting reagent to the oxazolidinone 118 by treating with triphosgene and triethylamine. Reduction of the ester functionality with sodium borohydride provided a highly water-soluble primary alcohol 119, which was converted to a corresponding iodo compound 121 in two steps: tosylation of the primary 89 alcohol, followed by refluxing with sodium iodide. The phosphonium salt 122 was then achieved by stirring the iodo compound 121 with excess TPP in DMF for over 24 hours (Scheme 58). Scheme 58. The Synthesis of phosphonium salt 12 The first attempt for the synthesis of the carbocyclic nucleoside aldehyde 127 started with a 1, 4-Michael addition to cyclopentenol 12 by applying a copper salt along with Grignard reagent and allylic magnesiumbromide to 123. An enantioselective reduction with lithium aluminum hydride generated the substrate 124 for the Mitsunobu reaction. The resulting coupled product 125 was then subjected to hydroboration conditions, but the purification for the product 126 was difficult, due to a formation of the byproduct derived from 9-BBN and the apparent loss of the Boc protecting groups under the reaction condition or during purification. An attempt to carry-out a Swern oxidation in this mixture was inconsistent most likely due to complexity of the mixture including 126 but 1 H NMR spectrum does show some evidences for existence of 127 (Scheme 59). 90 Scheme 59. First attempt for synthesis of aldehyde 127 Based on that, the synthetic route was revised by changing the Grignard reagent from allylic magnesiumbromide to the 3-butenylmagnesium bromide in the copper catalytic Michael addition to provide 128. After reduction of ketone 128 to the secondary alcohol 129 and coupling with the protected adenine 113, the resultant 130 was directly converted to the aldehyde 127 through an oxidative cleavage of the terminal alkene. In comparing to the previous approach, the yield by this method was improved and the reaction steps were shortened as well. The alaninol synthon 122 was then introduced by a Wittig reaction to afford 131a along with 131b, which was an unexpected product arising by losing one Boc protecting group. Its reprotection formed 132. Traditional hydrogenation of 132 produced 133 in high yield. By treating 133 with strong base, the oxazolidinone was readily converted to the amino alcohol 134. With 134 in hand, it will be necessary to conduct the proper oxidation and deprotection to restore the amino acid 91 and other functional groups. The final compound and its biological activity results are expected in the future study of our labs (Scheme 60). O O O O O O 12 128 O O OH 129 O O 130 N N N N N(Boc) 2 O O 127 N N N N N(Boc) 2 O O O 132 N N N N N(Boc) 2 NBoc O O O O N N N N N(Boc) 2 BocHN HOOC O O N N N N N(Boc) 2 NH O O O O N N N N NHBoc NH O O 131a O O N N N N N(Boc) 2 Boc N O O O O N N N N N(Boc) 2 BocHN HOH 2 C HO OH N N N N NH 2 H 2 N HOOC 134 135 N N N N H N(Boc) 2 CuBr,Me 2 S, MgBr TMSCl, HMPA, THF, 88% LiAlH 4 , THF,91% Ph 3 P, DIAD, THF, 70% + 113 NaIO 4 ,OsO 4 ,MeOH,H 2 O, 68% 122,n-BuLi,THF,45% DMAP, (Boc) 2 O, 87% Pd/C, H 2 , 50Pis, 8hr, 98% LiOH, THF, H 2 O; 99% TEMPO, MeCN, buffer (PH=6.7), NaClO 2 HCl, MeOH 133 10 131b + Scheme 60. The revised synthesis for target compound 10 92 New Synthetic Approach for Deamino-Carbocyclic Sinefungin Since previous methods for the synthesis of compound 10 suffered from harsh reaction conditions (toxicity for synthesis of compound 118 and 127; losing protecting group caused by strong basic condition for coupling of 122 with 127), long reaction time (synthesis of phosphonium salt 122) and a low yield for the Wittig reaction (127 to 131), a new synthetic approach was also explored based on the idea of coupling amino acid block with carbonucleoside piece by exerting olefin metathesis. Advantages of this method are including mild reaction conditions, easily prepared precursors and normally stable and high reaction yields. The amino acid olefin portion began with D-serine methyl ester. 233 After protecting of the amino group by using di-tert-butyl dicarbonate, the oxazolidine 137 was then obtained by using dimethoxypropane (DMP) with trifluoride etherate as catalyst. Reduction of ester 137 to aldehyde 138 was realized by using diisobutylaluminum hydride and the aldehyde function group was smoothly transformed to the terminal olefin by utilizing a Wittig methylenation reaction (Scheme 61). Scheme 61. Synthesis of amino acid olefin 140 93 The first attempt of olefin metathesis was carried out between 139 and 128. The reaction gave a good yield of coupling product 141 along with some 128 self-coupling byproduct 142. The 1 H NMR coupling constant (J H-H ) of hydrogen installed on the double bond is 15.5, ensure the trans configuration of 141. After catalytic hydrogenation, 143 was treated with acid for the selective hydrolysis of the acetonide, but unfortunately the fully deprotected product 144b was also produced, which significantly lowered the yield of 144a (Scheme 62). 94 N N N N N(Boc) 2 O O 128 , Grubbs catalyst, 61% N N N N N(Boc) 2 O O O NBoc 141 H 2 , Pd/C, 100% N N N N N(Boc) 2 O O O NBoc 143 p-tolunenesulfonic acid,MeOH, 23% for 144a O NBoc 139 N N N N N(Boc) 2 O O HO NHBoc N N N N N(Boc) 2 HO OH HO NHBoc 144a 144b + 142 Scheme 62. First attempt of metathesis A better way to avoid this inconvenient situation is to use the deprotected 140 as the metathesis precursor. The free hydroxyl group doesn?t affect the reaction yield at all and by using 1.5 equivalents of 140 no self-coupling by-product 142 was found. The 1 H NMR coupling constant (J H-H = 15.5) of double bond hydrogens confirmed the E conformation 95 of 145. After going through the catalytic hydrogenation and oxditation 146 was obtained in high yield. Cleavage of all the protection groups in 146 gave the final product 10 (Scheme 63). N N N N N(Boc) 2 O O 128 , Grubbs catalyst, 65% N N N N N(Boc) 2 O O HO NHBoc 145 H 2 , Pd/C, 100% N N N N N(Boc) 2 O O HO NHBoc 144a HO NHBoc 140 N N N N N(Boc) 2 O O HO NHBoc 146 O N N N N NH 2 HO OH HO NH 2 10 O TEMPO, TCCA, NaBr, NaHCO 3 , aceton, 81% 1M HCl, MeOH,72% Scheme 63. Second attempt of metathesis 96 Biological Results Target compounds were evaluated against a wide variety of DNA viruses and RNA viruses. The spectrum of viruses used is shown in Table 4. Table 4. 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) Flavivridae Hepatitis C virus (HCV), West Nile virus, Yellow fever virus Hepadnaviridae Hepatitis B virus (HBV) Herpesviridae 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 97 Compound 2 showed significant activity against human cytomegalovirus (HCMV) and moderate activity against yellow fever virus. Compound 2 also showed slight activity against Flu A (H1N1) virus. Compound 4, 9a and 9b were found to be not active against most of the test viruses. The bioassay data for compounds 3, 5, 6, 10 will be forthcoming and under study in our laboratories. Antiviral Activity Assays 161 : The antiviral assays were based on an inhibition of virus induced cytopathicity in either HeLa, Vero, MDCK, or HEL cell cultures, following previously established procedures 234-236 . Briefly, confluent cell cultures in microtiter trays were inoculated with100 CCID 50 of virus, 1 CCID 50 being the virus dose required to infect 50% of the cell cultures. After a 1h virus adsorption period, residual virus was removed and the cell cultures were incubated in the presence of varying concentrations (400, 200, 100, ... ?g/mL) of the test compounds. Viral cytopathicity was recorded as soon as it reached completion in the control virus infected cell cultures. Cytostatic Activity Assays 161 : The cytostatic assays were performed as previously described 237 . Briefly, 100-pL aliquots of the cell suspensions (5 X 10 5 murine leukemia L1210 or 7.5 X 10 5 human T-lymphocyte Molt-4 or CEM cells/mL) were added to the wells of a microtiter plate containing 100 ?L of varying concentrations of the test compounds. After a 2-day (L1210) or 3-day (Molt-4 and CEM) incubation period at 37 ?C in a humidified CO 2 -controlled incubator, the number of viable cells was determined using a Coulter Counter. Cytostatic activity is expressed as the compound concentration that reduced the number of viable cells by 50% (CC 50 ). The cytotoxicity measurements were based on microscopically visible alteration of normal cell morphology HeLa, Vero, and MDCK) or inhibition of normal cell growth (HEL), as previously described. 98 Table 5. Antiviral Activity of Compounds 2, 4 against HSV-1, HSV-2 and HCMV, Based on Cytopathogenic Effect (CPE) Inhibition Assay Compound 2 4 EC50 a >300 >300 EC90 b >300 >300 HSV-1 d CC50 c >300 >300 EC50 >300 >300 EC90 >300 >300 HSV-2 d CC50 >300 >300 EC50 0.27 EC90 0.42 HCMV 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. 99 Table 6. Antiviral Activity of Compounds 2, 4 and 9a against Adenovirus, Influenza A (H1N1), Influenza A (H3N2), Influenza B Inhibition Assay Virus Compound Assay Cell Line EC50 IC50 SI Neutral Red A-549 >100 >100 0 4 Visual A-549 >100 >100 0 Neutral Red A-549 >100 >100 0 9a Visual A-549 >100 >100 0 Neutral Red A-549 >100 >100 0 Adeno 9b Visual A-549 >100 >100 0 Neutral Red MDCK 33 >100 >3 2 Visual MDCK 32 >100 >3.1 Neutral Red MDCK >100 >100 0 4 Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 Flu A (H1N1) 9a Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 2 Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 4 Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 Flu A (H3N2) 9a Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 2 Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 4 Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 9a Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 Flu A (H5N1) 9b Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 2 Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 4 Visual MDCK >100 >100 0 Neutral Red MDCK >100 >100 0 9a Visual MDCK >100 >100 0 Neutral Red MDCK 71 >100 >1.4 Flu B 9b Visual MDCK 32 >100 >3.1 Units = ?M 100 Table 7. Antiviral Activity of Compounds 2, 4 and 9a against measles, PIV, Rhinovirus, Rift Valley Fever Inhibition Assay Virus Compound Assay Cell Line EC50 IC50 SI Neutral Red vero 76 >100 >100 0 4 Visual vero 76 >100 >100 0 Neutral Red CV-1 >100 >100 0 9a Visual CV-1 >100 >100 0 Neutral Red vero 76 >100 >100 0 measles 9b Visual vero 76 >100 >100 0 Neutral Red 2 Visual Neutral Red MA-104 >100 >100 0 4 Visual MA-104 >100 >100 0 Neutral Red MA-104 >1.8 1.8 0 PIV 9b Visual MA-104 32 32 1 Neutral Red 2 Visual Neutral Red Hela Ohio-1 61 >100 >1.6 4 Visual Hela Ohio-1 >100 >100 0 Neutral Red Hela Ohio-1 >100 100 0 9a Visual Hela Ohio-1 >100 24 0 Neutral Red Hela Ohio-1 >100 >100 0 Rhinovirus 9b Visual Hela Ohio-1 >100 >100 0 Neutral Red vero 76 >100 >100 0 2 Visual vero 76 90 >100 >1.1 Neutral Red vero 76 >100 >100 0 4 Visual vero 76 >100 >100 0 Neutral Red Rift Valley Fever Visual a Measles was tested on Vero 76 cells. b PIV was tested MA-104 cells. c Rhinovirus was tested on Hela ohio-1 cells. d Rift Valley Virus was test on Vero 76 cells. 101 Table 8. Antiviral Activity of Compounds 2, 4 and 9a Against Yellow Fever, RSVA, and Inhibition Assay Virus Compound Assay Cell Line EC50 IC50 SI Neutral Red 2 Visual Neutral Red MA-104 >100 >100 0 4 Visual MA-104 >100 >100 0 Neutral Red MA-104 >100 >100 0 RSVA 9a Visual MA-104 >100 >100 0 Neutral Red 2 Visual Neutral Red vero 76 >100 >100 0 4 Visual vero 76 >100 >100 0 Neutral Red vero >100 15 0 SARS 9a Visual Neutral Red vero 6.2 >100 >16 2 Visual vero 100 >100 >1 Neutral Red 4 Visual Neutral Red vero >100 >100 0 Yellow Fever 9a Visual vero >100 >100 0 Units = ?M RSVA was tested on MA-104 cells. SARS was tested on vero 76 cells. Yellow Fever was tested on vero cells. 102 Table 9. Antiviral Activity of Compounds 2, 4 and 9a against Dengue, Tacaribe, VEE and WNV Inhibition Assay Virus Compound Assay Cell Line EC50 IC50 SI Neutral Red vero 77 >100 >1.3 2 Visual Neutral Red vero 76 >100 >100 0 4 Visual Neutral Red vero >100 >100 0 Dengue 9a Visual vero >100 >100 0 Neutral Red vero 76 >100 >100 0 2 Visual vero 76 >100 >100 0 Neutral Red 4 Visual Neutral Red Tacaribe 9a Visual Neutral Red vero 81 >100 >1.2 2 Visual vero >100 >100 0 Neutral Red vero >100 >100 0 4 Visual vero >100 >100 0 Neutral Red vero >100 >100 0 VEE 9a Visual vero Neutral Red vero >100 >100 0 2 Visual vero >100 >100 0 Neutral Red 4 Visual Neutral Red vero >100 >100 0 WNV 9a Visual vero >100 >100 0 Units = ?M Dengue was tested on Vero cells. Tacaribe was tested on Vero 76 cells. VEE was tested on Vero cells WNV was tested on Vero cells. 103 Table 10. Antiviral Activity of Compounds 2, 4 against EBV virus based on DNA hybridization Inhibition Assay Compound Virus Cell Line EC50 E90 CC50 SI 2 EBV Akata Cells >20 >20 53.7 <2.7 4 EBV Akata Cells >20 >20 76.9 3.8 104 Table 11. Antiviral Activity of Compounds 2, 4 and 9a Against Vaccinia Virus and Cowpox Virus Based on Cytopathogenic Effect (CPE) Inhibition Assay Compound 2 4 9a EC50 >300 >300 237 Vaccinia Virus a CC50 >300 >300 >300 EC50 >300 >300 293 Cowpox Virus b CC50 >300 >300 >300 Units = ?M a Vaccinia Virus was tested on HFF cells. b Cowpox Virus was tested on HFF cells. 105 Table 12. Antiviral Activity of Compounds 2, 9a 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 2 >100 >100 >100 >100 >100 >100 9a >200 >200 >200 >200 >200 >200 DS-5000 (?g/mL) >100 >100 >100 20 >100 20 (S)-DHPA >250 250 >250 >250 >250 >250 Ribavirin >250 50 146 250 >250 50 a Required to cause a microscopically detectable alteration of normal cell morphology. b Required to reduce virus-induced cytopathogenicity by 50%. 106 Table 13. Antiviral Activity of Compounds 2, 9a in HEL Cell Cultures Minimum inhibitory concentration b (?M) Compound Minimum cytotoxic concentr- ation a (?M) Herpes simplex virus-1 (KOS) Herpes simplex virus-2 (G) Vaccinia virus Vesicular stomatitis virus Herpes simplex virus-1 (TK - KOS ACV) 2 >100 >100 >100 >100 >100 >100 9a >200 >200 >200 >200 >200 >200 Brivudin >250 0.02 29 4 >250 50 Ribavirin >250 >250 250 112 125 >250 Cidofovir >250 2 1 20 >250 1 Ganciclovir >100 0.02 0.2 >100 >100 9 a Required to cause a microscopically detectable alteration of normal cell morphology. b Required to reduce virus-induced cytopathogenicity by 50%. 107 Table 14. Antiviral Activity of Compounds 2, 9a 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 2 >100 >100 >100 >100 9a >400 >400 >400 200 Ganciclovir 12.6 8.3 1575 175.6 209 Cidofovir 1.5 1.2 1270 87.3 122 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%. e No complete inhibition at higher drug concentration. 108 Table 15. Antiviral Activity of Compounds 2 and 9a 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 2 >100 >100 >100 >100 9a >400 >400 >400 200 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%. 109 Table 16. Cytoxity and Antiviral Activity of Compounds 2 and 9a in Hela Cell Cultures Minimum inhibitory concentration b (?M) Compound Minimum cytotoxic concentration a (?M) Vesicular stomatitis virus Coxsackie virus B4 Respiratory syncytial virus 2 >100 >100 >100 >100 9a >200 >200 >200 >200 DS-5000 (?g/mL) >100 20 9 0.8 (S)-DHPA >250 >250 >250 >250 Ribavirin >250 22 50 10 a Required to cause a microscopically detectable alteration of normal cell morphology. b Required to reduce virus-induced cytopathogenicity by 50%. 110 Table 17. Antiviral Activity of Compounds 2, 4 and 9a against HCV and HBV 2 4 9a 9b Compound (?M) (?M) (?M) (?M) EC50 59.7 >100 EC90 >100 >100 CC50 >100 >100 HCV SI 1.7 0 EC50 >100 >100 >100 >100 EC90 >100 >100 >100 >100 CC50 >100 >100 >100 >100 HBV Sib >100 >100 >100 >100 a SI = CC50/EC90 111 Conclusion S-Adenosyl-L-homocysteine (AdoHcy) hydrolase is a pivotal enzyme involved in a viral replication cycle, which makes it a unique target for antiviral drug design. The consequence by inhibition of AdoHcy hydrolase is elevation of intracellular level of AdoHcy, which acts as a feedback inhibitor of S-adenosylmethionine (AdoMet) dependent methyltransferase. AdoMet dependent biomethylation reaction is an essential step for viral m-RNA 5?-capping process. So AdoHcy hydrolase inhibitors can lead to significant antiviral activities. Aristeromycin (Ari) and neplanocin A (NpcA) are two of the most potent inhibitors of AdoHcy hydrolase. Besides showing remarkable antiviral properties, Ari and NpcA are also associated with high toxicity relevant to their 5?-nucleoside metabolites. Compound 1 was synthesized based on the modification of the 5?-hydroxyl group by adding a methylene group. 8-Methyl group was also introduced in order to examine the relationship between the anti-viral activities with syn-anti conformations. Compound 2 was prepared following this direction, which showed significant activity against human cytomegalovirus (HCMV) , moderate activity against yellow fever virus and slight activity against Flu A (H1N1) virus. Since 3-deaza has been identified as another valuable structural feature, compound 3, 4, 5, 6 have been synthesized as derivatives of Ari and Npc A. The configuration of 4?-stereocenter also provides an interesting aspect to explore new antiviral agents, since these changes may affect the phosphorylation and 112 deamination of 5?-hydroxyl group, which is essential for biological activity and/or cytotoxicity. Compound 8a, 8b, 9a, 9b were designed and prepared following this lead. Even though compounds 4, 9a and 9b show no antiviral activities, they still provide valuable structure-activity relationship information for future design of antiviral agents. At the end, for direct inhibition of methyltransferase, sinefungin provides a great model to study with. Its biological effects were significant, but cytotoxicity was associated as well. The relationship between the inhibitions of methyltransferase and its biological activities intrigued the study for design and synthesis of deamino-carbocyclic sinfungine 10. Two different synthetic approaches have been developed and evaluated. The antiviral data will be forthcoming as part of a ongoing study. 113 Experimental Section General Melting points were recorded on a Meltemp II melting point apparatus and the values are uncorrected. Elemental analyses were performed at Atlantic Microlab, Norcross, GA. 1 H and 13 C NMR spectra were recorded on either a Bruker AC 250 spectrometer (250 MHz for proton and 62.9 MHz for carbon) or a Bruker AC 400 spectrometer (400 MHz for proton and 101 MHz for carbon). All 1 H chemical shifts are referenced to internal tetramethylsilane (TMS) at 0.0ppm. 13 C chemical shift are reported in ? relative to CDCl 3 (center of triplet, ? 77.2), or relative to DMSO-d 6 (center of septet, ? 39.5). The spin multiplicities are indicated by the symbols: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). The reactions were monitored by thin-layer chromatography (TLC) using 0.25 mm Whatman Diamond silica gel 60-F254 precoated plates with visualization by irradiation with a Mineralight UVGL-25 lamp or exposure to iodine. Column chromatography was performed on Whatman silica, 230?400 mesh and 60 ? using elution with the indicated solvent system. Yields refer to chromatographically and spectroscopically ( 1 H and 13 C NMR) homogeneous material. 114 6-Oxabicyclo[3.1.0]hex-2-ene (14). Dicyclopentadiene was freshly cracked to cyclopentadiene (181 g, 2.75mol) and immediately dissolved in 1500 mL CH 2 Cl 2 . Sodium carbonate (350 g, 3.31 mol) was then added portionwise at -10 ?C and the suspension then treated with a solution of sodium acetate (14 g, 0.21 mol) in 350 mL of peracetic acid (39% in acetic acid) dropwise. The temperature was maintained -10 ?C to - 5 ?C during the addition. After the addition, resulting mixture was stirred at rt for 12 hrs. The mixture was filtered and the filtrate was evaporated to give a pale yellow liquid. The crude monoepoxide 14 was used directly in the next step. 162 (Z)-Cyclopentene-3,5-diol diacetate (16). Tetrakis(triphenylphosphine)palladium (0) was prepared at first. PdCl 2 (1.69 g, 9.53 mmol) and triphenylphosphine (12.67 g, 48.33 mmol) were added to 113 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 (1.35g, 27.0 mmol) was added dropwise in 1 min with rapid stirring. The solution was cooled to rt with water bath and yellow crystals appeared. Solid was collected by filtration and washed with 2 ? 10 mL ethanol and 2 ? 10 mL ether. A light yellow solid (10.1 g) was dried up and kept under nitrogen, whose 1 H and 13 C NMR spectral data agreed with literature. 206 A solution of crude 14 from last step in 100 mL THF was added dropwise to a dry ice acetone cooled solution of tetrakis(triphenylphosphine)palladium(0) (4.0 g) in 300 mL dry THF and acetic anhydride (225 g, 2.20 mol) at 0 ?C to -5 ?C. After addition, resulting mixture was stirred at room temperature overnight. Filtration of the resulting mixture was passed through a pad of silica gel to remove the catalyst. Ethyl ether (2 ? 50 mL) was used to wash it. Evaporation of the solvent and fractional distillation afforded 115 18 (120 g) as a pale yellow oil, whose 1 H and 13 C NMR spectral data agreed with literature. 162 (+)-(1R, 4S)-4-Hydroxy-2-cyclopenten-1-yl acetate ((+)-11). Compound 16 (120 g, 0.761 mol) was added to 0.1 M phosphate buffer (400 mL). The pH of the resulting suspension was adjusted to 7 by addition of 6 N NaOH dropwise. Pseudomonas cepacia lipase (12 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 760 mL of NaOH solution, the reaction mixture was filtered through a celite pad. The filtrate was extracted with 3 ? 400 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 under reduced pressure to give (+)-11 (85.2g, 78.8%) as a light yellow solid, whose 1 H and 13 C NMR spectral data agreed with literature. 238 Methyl-2,3-O-isopropylidene-?-D-ribofuranoside (22). Concentrated hydrochloric acid (10 mL) was added to a suspension of D-ribose (100 g, 0.666 mol) in acetone (420 mL), methanol (420 mL) and 2,2-dimethoxypropane (200 mL) at rt. The mixture was refluxed for 6 hr and then cooled to room temperature. After neutralized with pyridine, the reaction mixture was concentrated and followed by addation of water (1000 mL) and ether (300 mL). The separated aqueous phase was extracted with ether (2 x 200 mL) and ethyl acetate (2 x 200 mL). Then the organic layer was washed with water, and brine. The combined organic extracts were dried over anhydrous sodium sulfate overnight. The 116 solvent was evaporated and the residue was distilled to give 100.5 g (73.9%) of 22 as colorless oil. The NMR spectral data agreed with literature. 209 Methyl-5-deoxy-5-iodo-2,3-O-isopropylidene-?-D-ribofuranoside (23). Iodine (150.7 g, 0.590 mol) was treated portionwise to a solution of 22 (100.5 g, 0.490 mol), imidazole (50.7 g, 0.738 mol), and triphenylphosphine (156.8 g, 0.598 mol) in toluene (2.0 L) and acetonitrile (502 mL). After addition the reaction mixture was refluxed for 30 min, and then cooled to rt. Diluted with ether and repeated washing of the organic extracts sequentially with 10% sodium thiosulfate solution, water, and brine, the solution was dried over anhydrous MgSO 4 and concentrated in vacuo to give a residue which was filtered through a short plug of silica gel by flushing with hexanes/EtOAc = 15:1 to give 96 (132.4 g, 86.1%) as a colorless oil of the mixture of anomers. The NMR spectral data agreed with literature. 209 (2R,4R)-2-Dimethyl-5-vinyl-1,3-dioxolane-4-carboxaldehyde (24). Activated zinc powder (Aldrich, dust, <10 micron, 5.50 g, 84.6 mmol) was added in portion to a stirred solution of iodide 96 (5.00 g, 15.9 mmol) in methanol (35 mL) at room temperature. The reaction mixture was heated to reflux for 2 h and then cooled to room temperature, filtered through a short plug of celite, and washed with a 200 mL of a 1:1 mixture of THF/pentane. The filtrate was concentrated by evaporation around 20 ?C ~30 ?C, under reduced pressure to provide a colorless oil, which was purified by silica gel column chromatography (hexanes/EtOAc = 2:1) to afford the product 97 (2.41 g, 95.7%) as a colorless oil. The NMR spectral data agreed with literature. 210 (4S,5R)-1-(2,2-Dimethyl-5-vinyl-[1,3]dioxolan-4-yl))-prop-2-en-1-ol (25). To a solution of 24 (45.6 g, 0.290 mol) in anhydrous THF (1000 mL) was added dropwise a 117 solution of vinylmagnesium bromide (1.0 M in THF, 377 mL, 377 mmol) at -30 ?C. The reaction was allowed to warm to 0 ?C over 1 h and remained in this temperature for 2 h. the reaction was quenched with saturated NH 4 Cl (150 mL). The organic layer was separated, washed with brine, and dried by anhydrous MgSO 4 . The solvent was removed by evaporation under reduced pressure and the residue purified by silica gel column chromatography (EtOAc/hexanes = 1:4) to afford 25 as a mixture of two isomers (42.0 g, 78.3%) as a colorless oil. The NMR spectral data agreed with literature. 208 (3aR,6aR)-2,2-Dimethyl-3a,6a-dihydrocyclopenta[1,3]-dioxol-4-one ((4R,5R)-4,5- O-isopropylidene-2-cyclopentenone) (12). To a solution of the diene 25 (31.26 g, 168 mmol) in anhydrous CH 2 Cl 2 (600 mL), the Grubbs? catalyst benzylidene- bis(tricyclohexylphosphine)dichlororuthenium (1.50 g, 1.81 mmol, flushed with N 2 three times) was added. After stirred at room temperature for 4h, 4 ? molecular sieve (50 g) and pyridinium dichromate (63.5 g, 291 mmol) were added to the resulting dark brown mixture. The reaction was kept at the same temperature for 12 h and filtered over a silica gel pad with EtOAc. The filtrate was concentrated in vacuo, and the residue was purified by column chromatography on silica gel (EtOAc/hexanes = 1:9), giving compound 12 (23.0 g, 90.2%) as a white crystal. The NMR spectral data agreed with literature. 239 (3aR,6R,6aR)-2,2-Dimethyl-6-vinyltetrahydrocyclopenta[1,3]dioxol-4-one (26). To a suspension of CuBr?Me 2 S (0.12 g, 0.58 mmol) in THF (40 mL) at -78 ?C was added vinylmagnesium bromide (8.14 mL, 8.14 mmol) in dropwise and the mixture was stirred for 10 min before a solution of 12 (1.00 g, 6.58 mmol), TMSCl (1.70 mL, 13.3 mmol), and HMPA (3.00 mL, 17.1 mmol) in THF (10 mL) was added in dropwise. After stirring at -78 ?C for 3 h, the reaction mixture was warmed to 0 ?C followed by adding a 118 saturated NH 4 Cl (10 mL) and the resulting mixture stirred for 30 min. EtOAc (100 mL) was then added afterwards. The organic layer was separated and washed with brine (15 mL), then dried (MgSO 4 ). The solvent was removed under reduced pressure and the residue purified by silica gel column chromatography (EtOAc/hexanes = 1:4) to give 26 as a colorless liquid (1.10 g, 91.8%). The NMR spectral data agreed with literature. 240 (3aS,4S,6R,6aR)-2,2-Dimethyl-6-vinyltetrahydrocyclopenta[1,3]dioxol-4-ol (27). To a suspension of LiAlH 4 (0.6 g, 15.36 mmol) in dry THF (30 mL) at 0 ?C was added dropwise a solution of 26 (1.62 g, 8.88 mmol) in THF (10 mL). The reaction mixture was then stirred at room temperature for 3 h before it was quenched sequentially with H 2 O (0.6 mL), aqueous NaOH (15%, 0.6 mL), and H 2 O (1.8 mL). The resulting solid was removed by filtration and the filtrate evaporated in vacuo to afford sufficiently pure 27 as a colorless liquid (1.58 g, 95.7%) The NMR spectral data agreed with literature. 208 6-Chloro-9-[(3aS,4R,6R,6aR)-2,2-dimethyl-6-vinyltetrahydro-cyclopenta[1,3] dioxol-4-yl]-purine (28). To a solution of 6-chloropurine (1.69 g, 10.83 mmol), Ph 3 P (4.03 g, 15.4 mmol), and 27 (1.41 g, 7.66 mmol) in dry THF (50 mL) at 0 ?C, DIAD (3.18 mL, 15.4 mmol) was added dropwise. The mixture was then stirred at the same temperature for 30 min and warmed to room temperature. After stirring at room temperature for 12 h, the reaction was heated to 50 ?C and stirred for another 8 h. The solvent was removed under the reduced pressure, and the residue was purified by silica gel column chromatography (EtOAc/hexanes = 1:4) to afford 28 contaminated with the azadicarboxylate byproduct. 6-Amine-9-[(3aS,4R,6R,6aR)-2,2-dimethyl-6-vinyltetrahydro-cyclopenta[1,3] dioxol-4-yl]-purin (29). A solution of crude 28 (2.30 g, 7.17mmol) in MeOH (100 mL) 119 was cooled to 0 ?C for 30 min before saturated with NH 3 at same temperature for 1 hr. The solution was heated to 100 ?C for 24 hr in a Parr stainless steel, sealed reaction vessel. The solvent was removed under reduced pressure, and residue was purified by column chromatography (EtOAc/Methanol = 10:1) to give 29 as a white solid (1.68 g, 73.1% in two steps). 1 H NMR (250 MHz, CDCl 3 ) ? 8.33 (s, 1H), 7.84 (s, 1H), 6.10 (s, 2H), 5.95 (ddd, J = 17.1, 10.2, 6.8 Hz, 1H), 5.23-5.5.07 (m, 3H), 4.77 (m, 1H), 4.65 (t, J = 6.8Hz, 1H), 2.79 (m, 1H), 2.60-2.44 (m, 2H), 1.57 (s, 3H), 1.31 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 155.9, 153.0, 150.1, 140.0, 137.8, 120.6, 116.3, 114.3, 84.0, 83.6, 61.8, 48.3, 36.8, 27.6, 25.3. Compound 31. Triethylamine (99.5%, 3.00 mL, 21.5 mmol) was added to a solution of 29 (1.86 g, 7.17 mmol) in THF (100 mL) at room temperature, followed by addition of tert-butyl carbamate (6.25 g, 28.7 mmol) and DMAP (0.122 g, 1.00 mmol). The reaction mixture was stirred for 8 hr at same temperature under N 2 atmosphere. The solvent was removed under reduced pressure. The crude product was purified by chromatography (EtOAc/Hexane = 1:4) on silica gel to give 31 (3.13 g, 87.1%) as a white foam. 1 H NMR (400 MHz, CDCl 3 ) ? 8.86 (s, 1H), 8.10 (s, 1H), 5.96 (ddd, J = 17.2, 10.3, 6.9 Hz, 1H), 5.24-5.14 (m, 2H), 5.11 (dd, J = 7.4, 5.1, 1H), 4.85 (ddd, J = 11.5, 6.9, 5.1 Hz, 1H), 4.67 (t, J = 6.8 Hz, 1H), 2.84 (m, 1H), 2.61-2.48 (m, 2H), 1.59 (s, 3H), 1.48 (s, 18H), 1.32 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 151.9, 150.7, 144.2, 137.4, 116.3, 114.2, 83.9, 83.4, 61.9, 60.4, 48.1, 36.4, 27.9, 27.5, 25.1, 21.1, 14.2. Compound 32. To a solution of 31 (0.95 g, 1.90 mmol) in THF (200 mL) at 0 ?C under N 2 atmosphere was added 9-BBN-H (0.5 M in THF, 7.60 mL, 3.80 mmol). The resultant mixture was brought up to room temperature and stirred at same temperature for 120 3 hr. NaOH aqueous solution (1M, 3.60 mL) was added, followed by addition of H 2 O 2 solution (50% in H 2 O, 2.4 mL) and the stirring continued an addition 30 min. The reaction mixture was diluted with CH 2 Cl 2 (10 mL) and washed with saturated NaHCO 3 solution (3 x 10 mL). The organic layer was dried (MgSO 4 ) and concentrated later in vacuo to give the crude product which was purified by chromatography (EtOAc/Hexane = 1:5) to afford 32 as white foam combined with 9-BBN hydrolyzed byproduct. (1R,2S,3R)-3-(6-amino-purin-9-yl)-5-(2-hydroxyethyl)cyclopentane-1,2-diol (1). Crude compound 32 was dissolved in a mixture of 1N HCl (18 mL) and MeOH (18 mL). The reaction mixture was stirred at room temperature for 3 hr. The solution was then neuturalized with weekly basic exchange resin (Amberlite IRA-67). After filtration and evaporation of the solvent, the crude product was purified by chromatography (EtOAc/ MeOH/NH 3 ?H 2 O = 8:2:1) to give 1 (0.359, 67.6%) as white solid. 1 H NMR (400 MHz, MeOD) ? 8.19 (s, 1H), 8.18 (s, 1H), 4.76 (td, J = 9.4, 7.6 Hz, 1H), 4.50 (dd, J = 7.8, 6.4 Hz, 1H), 3.93 (t, J = 6.0 Hz, 1H), 3.69-3.63 (m, 2H), 2.42 (m, 1H), 2.14 (m, 1H), 1.92-1.82 (m, 2H), 1.71 (m, 1H); 13 C NMR (100 MHz, MeOD) ? 157.4, 153.5, 151.1, 142.2, 120.8, 76.8, 76.2, 62.6, 61.6, 41.9, 38.3, 33.7. 6-Hydroxy-8-methylpurine (33). 4,5-Diamino-6-hydroxypyrimidine hemisulfate salt (5.00 g, 14.3mmol) was refluxed in acetoamide (60.0 g, 1.02 mol) at 230 ?C. After a clear yelIow solution resulted, the reaction mixture was continued to reflex for 4hr. The excess acetoamide was distilled under reduced pressure. The residue was diluted with ice water (10 mL) and filtrated. The filtrate was washed with another ice water (10 mL). After second time filtration, the residue was dried in the oven to give a grey solid 33. 121 8-Methyl-6-chloropurine (34). 215 To a solution of phosphorus oxychloride (30 mL), containing N, N-dimethylaniline (3 mL) was added crude compound 33 portion wise and slowly. The mixture is heated to reflux for 7 hr. The excess phosphorus oxychloride was evaporated under reduced pressure. The residue was adjusted to strongly basic (PH > 10) by adding NH 3 ?H 2 O (30%, 100mL). After extracted by EtOAc (2 x 100 mL), the mixture was continued to extract by Et 2 O. The water phase was evaporated and adjusted the PH value around 7 by diluted with water. The aqueous solution was extracted with EtOAc (5 x 100 mL) and the combined organic layer was dried (Na 2 SO 4 ). After filtration and evaporation of the solvent, 34 was obtained as yellow solide 1.30, 54.0%). 1 H NMR (400 MHz, MeOD) ? 8.61 (s, 1H), 2.66 (s, 3H). 6-Chloro-9-[(3aS,4R,6R,6aR)-2,2-dimethyl-6-vinyltetrahydro-cyclopenta[1,3] dioxol-4-yl]-8-methyl-purine (35). To a solution of 34 (0.66 g, 3.9 mmol), Ph 3 P (1.60 g, 6.10 mmol), and 27 (0.60g, 3.2 mmol) in dry THF (50 mL) at 0 ?C, DIAD (1.33 mL, 6.40 mmol) was added dropwise. The mixture was then stirred at the same temperature for 30 min and warmed to room temperature. After stirred at room temperature for overnight, the reaction was heated to 50 ?C and stirred for another 8 h. The solvent was evaporated under the reduced pressure, and the residue was purified by silica gel column chromatography (EtOAc/hexanes = 1:2) to afford 35 (0.73 g) contaminated with the azadicarboxylate byproduct. 1 H NMR (400 MHz, CDCl 3 ) ? 8.62 (s, 1H), 5.95 (ddd, J = 17.2, 10.3, 6.8 Hz, 1H), 5.20-5.10 (m, 3H), 4.70-4.6 (m, 2H), 2.90-2.74 (m, 2H), 2.71 (s, 3H), 2.26 (dt, J = 6.1, 12.2 Hz, 1H), 1.54 (s, 3H), 1.26 (s, 3H). 2-[(3aR,4R,6R,6aS)-6-(6-chloro-8-methyl-9H-purin-9-yl)-2,2-dimethyltetrahydro- 3-cyclopenta[1,3]dioxol-4-yl]ethanol (36). To a solution of 35 (0.54 g, 1.61 mmol) in 122 THF (200 mL) at 0 ?C under N 2 atmosphere was added 9-BBN-H (0.5 M in THF, 6.44 mL, 3.22 mmol). The resultant mixture was brought up to room temperature and stirred at same temperature for 12 hr. a NaOH aqueous solution (1M, 3.0 mL) was added, followed by H 2 O 2 solution (50% in H2O, 3.22 mL) and the stirring continued an addition 30 min. The reaction mixture was diluted with CH 2 Cl 2 (10 mL) and washed with saturated NaHCO 3 solution (3 x 10 mL). The organic layer was dried (MgSO 4 ) and concentrated later in vacuo to give the crude product which was purified by chromatography (EtOAc/Hexanes = 1:2) to afford 36 (0.48 g, 84.7%) as white foam. 1 H NMR (400 MHz, MeOD) ? 8.63 (s, 1H), 5.21 (dd, J = 7.4, 5.5 Hz, 1H), 4.82 (m, 1H), 4.56 (m, 1H), 3.66 (t, J = 6.8 Hz, 2H), 2.73 (s, 3H), 2.68 (q, J = 12.0 Hz, 1H), 2.38 (m, 1H), 2.29 (m, 1H), 1.88 (dt, J = 13.6, 6.8 Hz, 1H), 1.74 (dt, J = 13.6, 6.8 Hz, 1H), 1.54 (s, 3H), 1.28 (s, 3H) ; 13 C NMR (100 MHz, MeOD) ? 158.3, 154.3, 152.0, 149.6, 132.0, 115.2, 86.3, 84.4, 63.6, 61.5, 37.6, 36.5, 28.0, 25.6, 14.8. 2-[(3aR,4R,6R,6aS)-6-(6-amino-8-methyl-9H-purin-9-yl)-2,2-dimethyltetrahydro- 3-cyclopenta[1,3]dioxol-4-yl]ethanol (37). A solution of 36 (0.48 g, 1.4 mmol) in MeOH (100 mL) was cooled to 0 ?C for 30 min before saturated with NH 3 at same temperature for 1 hr. The solution was heated to 100 ?C for 24 hr in a Parr stainless steel, sealed reaction vessel. The solvent was removed under reduced pressure, and residue was purified by column chromatography (EtOAc/Methanol = 7:1) to give 37 (0.43 g, 94%) as a white solid. 1 H NMR (400 MHz, MeOD) ? 8.10 (s, 1H), 5.22 (dd, J = 7.5, 5.5 Hz, 1H), 4.74 (m, 1H), 4.58 (m, 1H), 3.68 (t, J = 6.8 Hz, 2H), 2.91 (m, 1H), 2.64 (s, 3H), 2.38-2.23 (m, 2H), 1.91 (dt, J = 13.5, 6.8 Hz, 1H), 1.75 (dt, J = 13.5, 6.8 Hz, 1H), 1.55 (s, 3H), 1.30 (s, 3H); 13 C NMR (100 MHz, MeOD) ? 156.4, 152.8, 151.8, 151.7, 119.4, 115.1, 86.3, 123 84.4, 63.1, 61.6, 42.5, 37.6, 36.8, 28.0, 25.6, 14.5. Anal. Calcd for C 16 H 23 N 5 O 3 . 0.2H 2 O: C, 57.03; H, 7.00; N, 20.78. Found: C, 57.28; H, 7.15; N, 20.48. (1R,2S,3R,5R)-3-(6-amino-8-methyl-9H-purin-9-yl)-5-(2-hydroxyethyl) cyclopentane-1,2-diol (2). Compound 37 (0.43 g, 1.3 mmol) was dissolved in a mixture of 1N HCl (20 mL) and MeOH (20 mL). The reaction mixture was stirred at room temperature for 3 hr. After distilled the solvent, the residue was redissolved in distilled H 2 O (20 mL) and MeOH (20 mL). The solution was then neuturalized with weekly basic exchange resin (Amberlite IRA-67). After filtration and evaporation of the solvent, the crude product was purified by chromatography (EtOAc/ MeOH/NH 3 ?H 2 O = 8:2:1) to give 2 (0.30 g, 80%) as white solid. 1 H NMR (400 MHz, MeOD) ? 8.00 (s, 1H), 4.78 (dd, J = 7.2, 6.0 Hz, 1H), 4.58 (m, 1H), 3.96 (dd, J = 5.6, 5.2 Hz, 1H), 3.65-3.61 (m, 2H), 2.55 (s, 3H), 2.20-2.07 (m, 3H), 1.89 (m, 1H), 1.72 (m, 1H); 13 C NMR (100 MHz, MeOD) ? 156.1, 152.5, 152.3, 151.9, 119.3, 76.9, 74.9, 63.1, 61.6, 41.9, 38.1, 31.8, 14.5. Anal. Calcd for C 12 H 16 N 4 O 3 . 0.3H 2 O: C, 52.27; H, 6.61; N, 23.44. Found: C, 52.36; H, 6.41; N, 23.48. 4-Hydroxy-3-nitropyridine (39). To mechanically stirred concentrated HNO 3 (122 g) in 0 ?C was added fuming H 2 SO 4 (140 g, d = 1.94) slowly. 4-Hydroxypyridine (30 g, 95%, 0.30 mol) was then added portion-wise in 20 min afterwards. The temperature was warmed up to around 30 ?C during the addition. The reaction mixture was brought to reflux for 1hr. The reaction was cooled to room temperature with water bath and then pouring the solution over ice slowly with continuously stirring. Treatment of the resulting suspension with saturated NH 4 OH and then aqueous Na 2 CO 3 (30%) adjusted the pH to 7. Precipitate was collected by filtration and washed with H 2 O (60 mL ? 3) to afford 124 compound 39 (30.2 g, 72.0%) as a pale yellow solid after drying in oven at 100 ?C under vacuum. The NMR spectral data agreed with literature. 241,242 4-Ethoxy-3-nitropyridine (40). Compound 39 (30.2 g, 22.6 mmol) and PCl 5 (56.6 g, 95%, 2.58 mmol) were added to ClCH 2 CH 2 Cl (200 mL) sequentially. The resulting suspension was heated to reflux for about 3.5 h until slurry turned into clear solution. Temperature was lowered to 15 ?C by ice-water bath. Absolute ethanol (130 mL) was added to the reaction dropwise and the temperature was controlled below 50 ?C. After the addition, the mixture was brought to reflux for 1hr. After removal of heating, the reaction mixture was cooled to 10 ?C and stood for 1 hr to form precipitate. The solid was collected by filtration and washed with ethanol (2 ? 150 mL). Compound 40 (43.6 g, 99.0%) was obtained as white solid after drying in oven at 35 ?C under vacuum: mp 46- 48 ?C (lit. 243 mp 46.5-48 ?C) 4-Amino-3-nitropyridine (41). To 100 mL of water was added Compound 40 (43.6 g, 31.2 mmol) and ammonium acetate (65.6 g, 85.0 mmol). The resulting slurry was heated to reflux for 6 hours. After TLC showing the disappearance of the starting material 40, the heating was removed and the reaction mixture was cooled to room temperature. After adjusted the pH to 8 by approximately 35 mL of concentrated aq ammonium hydroxide , the slurry was chilled by an ice-water bath for 1 h to form precipitate. The solid was collected by filtration and washed with cold water (30 mL ? 2). Compound 41 (23.8 g, 81.2%) was afforded as a yellow solid after drying in oven at 100 ?C under vacuum. mp 198-200 ?C (lit. 244 mp 200 ?C) 2-Chloro-3,4-diaminepyridine (42). SnCl 2 (58.0 g, 30.5 mmol) was added to 240 mL HCl and the resulting suspension was heated to 60-70 ?C. The reaction mixture 125 became clear solution. Compound 41 (23.8 g, 17.2 mmol) was then added portionwise slowly at this temperature. After the addition, the reaction mixture was brought to reflux and allowed to react for another 5 h. After TLC showed the disappearance of the starting material 41, the heating was removed and the reaction was cooled to rt with an ice-water bath. The cooled mixture was poured over 250 g of crushed ice. 10 M NaOH and then saturated ammonium hydroxide solution were added to adjust the acidic solution to pH 8. The neutralized solution was extracted with ethyl acetate (160 mL ? 4). The combined organic layers were dried over anhydrous Na 2 SO 4 . Evaporation of the solvent afforded 42 (17.6 g, 71.2%) as a yellow solid. 1 H NMR (250 MHz, DMSO-d 6 ) ? 7.29 (d, J = 5.3 Hz, 1H), 6.43 (d, J = 5.3 Hz, 1H), 5.78 (br, 2H), 4.67 (br, 1H); 13 C NMR (62.9 MHz, DMSO- d 6 ) ? 142.9, 137.6, 135.1, 126.2, 108.3. (1E,1'E)-Dimethyl N',N''-2-chloropyridine-3,4-diyldiacetimidate (44). 42 (10.0 g, 69.6 mmol) was added to trimethyl orthoacetate (161 mL) and the mixture was heated to 80 ?C. After the solution was mostly clear, formic acid (4.2 mL) was added dropwise and the solution was continued to heat to 100 ?C for 8 hr. Removal the heat before the solvent was evaporated and the crude product was purified by chromatography ( EtOAc/Hexane = 1:1) to give 44 as white solid. 1 H NMR (400 MHz, CDCl 3 ) ? 7.87 (d, J = 5.2 Hz, 1H), 6.56 (d, J = 5.2 Hz, 1H), 3.71 (s, 3H), 3.64 (s, 3H), 1.74 (s, 3H), 1.70 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 164.0, 162.2, 148.9, 143.7, 133.8, 116.4, 53.8, 53.7, 17.3, 17.0. 4-Chloro-2-methyl-1H-imidazo[4,5-c]pyridine (43). 44, obtained from the last step was dissolved in aqueous NaOH solution (1.5 N, 110 mL) and the reaction mixture was heated to reflux for 10 min. After cooled down to the room temperature the solution was extracted by EtOAc (4 ? 100 mL). The combined organic phase was dried by Na 2 SO 4 . 126 The solvent was evaporated afterwards and the crude product was purified by chromatography (EtOAc/MeOH = 8:1) to give 43 (11.2 g, 96.0%) as white solid. 1 H NMR (400 MHz, MeOD) ? 8.07 (d, J = 5.6 Hz, 1H), 7.49 (d, J = 5.6 Hz, 1H), 2.64 (s, 3H). 2-Methyl-1H-imidazo[4,5-c]pyridin-4-amine (45). 44 (2.42 g, 14.4 mmol) was dissolved to a mixture of anhydrous hydrazine (99%, 18.8 mL) and propan-1-ol (14.0 mL). The solution was brought to reflux overnight. After the reaction mixture was cooled to rt the residual hydrazine and propan-1-ol was evaporated under reduced pressure. Water (38 mL) was added to dissolve the residues, followed by addition of Raney nickel (1.91 g) portionwisely. The mixture was heated to reflux for 1 and the reaction mixture was filtered through a Celite pad. The filtrate was evaporated under reduced pressure to afford 45 (1.96 g, 91.6%). 1 H NMR (250 MHz, MeOD) ? 8.00 (d, J = 5.8 Hz, 1H), 7.42 (d, J = 5.8 Hz, 1H), 2.57 (s, 3H). Tert-butyl-4-(bis(tert-butoxycarbonyl)amino)-2-methyl-1H-imidazo[4,5- c]pyridine-1-carboxylate (46). Compound 45 (1.7 g, 11.3 mmol) and 4- (dimethylamino)pyridine DMAP (0.134 g, 1.1 mmol) were dissolved in dry THF(50 mL) at room temperature, followed by addation of Boc 2 O (10 mL, 44 mmol) in the same temperature. The resulting suspension was stirred for 2 days under a nitrogen atmosphere. TLC analysis (EtOAc) showed 46 was obtained (R f = 0.9) with appearance of a little 46b (R f = 0.6). The ratio was about 9:1 (detected by 1 H NMR). After evaporation of the solvent, the mixture was separated by chromatography (EtOAc/Hextane = 2:1) to give 46 (4.22 g, 83.0%) as a white foam. 1 H NMR (400 MHz, CDCl 3 ) ? 8.37 (d, J = 5.6 Hz, 1H), 127 7.75 (d, J = 5.6 Hz, 1H), 2.84 (s, 3H), 1.73 (s, 9H), 1.42 (s, 18H); 13 C NMR (100 MHz, CDCl 3 ) ? 154.6, 151.4, 146.4, 142.9, 140.4, 110.2, 86.8, 83.1, 77.4, 28.3, 28.1, 18.6, 0.21. Byproduct 46b: 1 H NMR (250 MHz, CDCl 3 ) ? 8.29 (d, J = 6.0 Hz, 1H), 7.43 (d, J = 6.0 Hz, 1H), 2.82 (s, 3H), 1.71 (s, 9H), 1.55 (s, 9H). 4-(bis(tert-butoxycarbonyl)amino)-2-methyl-1H-imidazo[4,5-c]pyridine-1- carboxylate (38). Compound 46 (2.84 g, 6.34 mmol) was dissolved under argon in 100 mL of dry THF. A 1 M solution of Bu 4 NF (1M in THF, 19.7 mL, 19.7 mmol) in THF was added and the reaction mixture was stirred for 12 h. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography (hexanes/AcOEt = 1/2) on silica gel to afford 38 (1.79 g, 81.1%) as a white foam. 1 H NMR NMR (250 MHz, MeOD) ? 8.16 (d, J = 5.8 Hz, 1H), 7.54 (d, J = 5.8 Hz, 1H), 2.64 (s, 3H), 1.33 (s, 18H). (+)-2,3-(Isopropylidenedioxy)-4-cyclopenten-1-ol (47). To a stirred solution of cyclopentenone 12 (1.40 g, 9.08 mmol) and CeCl 3 ?7H 2 O (3.40 g, 9.12 mmol) in MeOH (60 mL) at 0 ?C was added NaBH 4 (0.68 g, 18.1 mmol) in small portions. After stirring at rt for 1 h the mixture was neutralized with conc. HCl, reduced to 2/3 volume, extracted with brine and ether, and the organic layers combined, dried (MgSO 4 ), and concentrated to give 47 as a colorless syrup (1.40 g, 99%) which was used directly in the next step. 1 H NMR (250 MHz, CDCl 3 ) ? 5.89 (s, 2H), 5.02 (m, 1H), 4.74 (m, 1H), 4.57 (m, 1H), 3.14 (br, 1H), 1.43 (s, 3H), 1.40 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 135.9, 131.5, 111.9, 83.1, 76.7, 73.7, 27.2, 26.1. Compound 48. To a solution of 38 (2.24 g, 6.44 mmol), Ph 3 P (1.48 g, 5.66 mmol), and 47 (0.81 g, 5.19 mmol) in dry THF (100 mL) at 0 ?C, DIAD (1.14 mL, 5.65 mmol) 128 was added dropwise. The mixture was then stirred at the same temperature for 30 min and warmed to room temperature for 12 hr. The solvent was removed under the reduced pressure, and the residue was purified by silica gel column chromatography (EtOAc/hexanes = 1:2) to afford 48 (1.94 g, 77%). 1 H NMR (400 MHz, CDCl 3 ) ? 8.12 (d, J = 5.6 Hz, 1H), 7.04 (d, J = 5.6 Hz, 1H), 6.29 (dt, J = 5.6, 2.0 Hz, 1H), 5.95 (dd, J = 6.0, 2.4 Hz, 1H), 5.50-5.48 (m, 2H), 4.64 (dd, J = 6.0, 1.2 Hz, 1H), 2.63 (s, 3H), 1.48 (s, 3H), 1.39 (s, 18H), 1.31 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 153.4, 151.4, 143.4, 140.9, 140.1, 136.1, 135.7, 131.0, 112.6, 106.0, 84.4, 84.0, 82.6, 68.2, 27.8, 27.0, 25.1, 14.7. (1S,2R,5R)-5-(4-amino-2-methyl-1H-imidazo[4,5-c]pyridin-1-yl)cyclopent-3-ene- 1,2-diol (3). Compound 48 (1.95 g, 4.00 mmol) was dissolved in a mixture of MeOH (42 mL) and 1N HCl (42 mL). The resulting solution was stirred at rt. for 12 hr and brought to reflux for another 3 hr. Basic resin (Amberlite IR67) was added to neutralize the solution, followed by filtration. The filtrate was removed under baccum and residue was purified by chromatography (EtOAc/ MeOH/NH 3 ?H 2 O = 8:2:1) to give 3 (0.64 g, 65%) as white solid. 1 H NMR (400 MHz, CDCl 3 ) ? 7.44 (d, J = 6.0 Hz, 1H), 6.61 (d, J = 6.0 Hz, 1H), 6.17 (td, J = 2.8, 6.4 Hz, 1H), 6.09 (dd, J = 1.6, 6.4 Hz, 1H), 5.34 (m, 1H), 4.49 (ddd, J = 1.2, 3.0, 6.0 Hz, 1H), 4.21 (dd, J = 5.6, 6.4 Hz, 1H), 2.52 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 153.2, 152.5, 140.5, 140.3, 136.4, 135.2, 126.8, 99.9, 78.1, 74.0, 67.5, 14.3; Anal. Calcd for C 12 H 14 N 4 O 2 : C, 58.53; H, 5.73; N, 22.75. Found: C, 58.37; H, 5.83; N, 22.52. 6-Hydroxymethyl-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-ol (59). 226 To a solution of d-ribose (25.0 g, 0.17 mol) and a catalytic amount of p-toluenesulfonic acid monohydrate (TsOH?H 2 O, 0.80 g, 0.5 mmol) in 500 mL of acetone was added 2,2 129 dimethoxypropane (19.1 g, 0.18 mol) at 0 ?C. The suspension was stirred for 1 hr at rt. until a clear solution was achieved. The solution was then treated with NaHCO 3 (0.05 g, 0.60 mmol) and was stirred for an additional 30 min at rt. The solid was filtered and the filtrate purified by silica gel column chromatography (hexane/EtOAc = 3:1) to give compound 59 as a mixture of ?- and ?-isomers (28.4 g, 88%). The NMR spectral data agreed with literature. 226 2,2-Dimethyl-6-(trityloxymethyl)tetrahydrofuro[3,4-d][1,3]-dioxol-4-ol (60). 226 To a solution of compound 5 (28.4 g, 0.15 mol) in 200 mL of anhydrous DMF (372 mL), a catalytic amount of DMAP (0.56 g, 4.48 mmol), trityl chloride (50.1 g, 0.180 mol) and Et 3 N (22.7 g, 0.223 mol) were added at rt. under nitrogen atmosphere. The resulting solution was stirred for 48 h at room temperature and poured into ice water (200 mL). The organic layer was extracted with CH 2 Cl 2 (300 mL ? 3), washed with saturated aqueous NH 4 Cl (200 mL ? 2) and water (200 mL), and then dried over Na 2 SO 4 anhydrous. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (hexane/EtOAc = 10:1 to 2:1) to give compound 60 (55.1 g, 84.9%) as a mixture of ?- and ?-isomers. The NMR spectral data agreed with literature. 226 1-[5-(1-Hydroxy-2-trityloxyethyl)-2,2-dimethyl-[1,3]dioxolan-4-yl]prop-2-en-1-ol (61). 226 To a solution of compound 60 (47.5 g, 0.110 mol) in 400 mL of anhydrous THF was added 330 mL of vinylmagnesium bromide (0.330 mol, 1.0 M in THF) dropwise at - 78 ?C under nitrogen atmosphere. After stirred at same temperature for 1 hr, the reaction mixture was brought up to rt. and was stirred for an additional 6 hr. NH 4 Cl aqueous solution (200 mL) was added slowly to quench the reaction at 0 ?C, and the resulting 130 solution was poured into ice (200 mL ). The organic layer was separated and the aqueous layer was washed with ether (200 mL ? 2). The combined organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography (hexane/EtOAc = 10:1 to 3:1) to give compound 61 (49.6 g, 98%). The NMR spectral data agreed with literature. 226 1-{5-[1-((tert-Butyldimethylsilanyl)oxyl)allyl]-2,2-dimethyl-[1,3]dioxolan-4-yl}-2 (trityloxy)ethanol (62). 226 To a solution of compound 61 (37.9 g, 82.3 mmol) in anhydrous CH 2 Cl 2 (400 mL) and DMF (50 mL) were added imidazole (16.8 g, 24.8 mmol) and TBDMSCl (15.5 g, 0.103 mmol) at 0 ?C under nitrogen atmosphere. The reaction mixture was stirred for 8 h at room temperature and then poured into 600 mL of ether-water solution (1:1). The organic layer was separated and the aqueous layer was washed with ether (150 mL ? 2). The combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column (hexane/EtOAc = 30:1) to give compound 62 (37.4 g, 79%). The NMR spectral data agreed with literature. 226 1-{5-[1-((tert-Butyldimethylsilanyl)oxyl)allyl]-2,2-dimethyl-[1,3]dioxolan-4-yl}-2- (trityloxy)ethanone (63). 226 To a solution of oxalyl chloride (4.67 g, 53.3 mmol) in 100 mL of anhydrous CH 2 Cl 2 was added DMSO (7.57 g, 106 mmol) at -60 ?C under nitrogen atmosphere, and then the resulting solution was stirred for 10 min. A solution of compound 62 (24.7 g, 42.7 mmol) in 250 mL of anhydrous CH 2 Cl 2 was added to the reaction mixture dropwise over 20 min at -60 ?C. After 30 min, Et 3 N (21.6 g, 0.213 mol) was added dropwise over 20 min to the reaction mixture at -60 ?C. The mixture was stirred for 1 h at -60 ?C and then stirred for 30 min at room temperature. The reaction 131 mixture was treated with water (200 mL) dropwise at 0 ?C. The organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 (200 mL ? 3). The combined organic layer was washed with brine (200 mL ? 2), dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column (hexane/EtOAc = 20:1 to 10:1) to give compound 63 (28.7 g, 94%). The NMR spectral data agreed with literature. 226 tert-Butyl-{1-[2,2-dimethyl-5-(1-(trityloxymethyl)vinyl)-[1,3]-dioxolan 4yl]allyloxy}dimethylsilane (64). 226 To a suspension of Ph 3 PCH 3 Br (12.8 g, 73.7 mmol) in 50 mL of THF was added 45 mL of n-BuLi (70.0 mmol, 1.6 M in hexane) at 0 ?C under N 2 atmosphere. After 30 min, a solution of compound 63 (8.50 g, 14.7 mmol) in 100 mL of THF was added to the reaction mixture at 0 ?C. The resulting mixture was stirred for 12 h at room temperature, treated with 25 mL of MeOH and 50 mL of water, and then poured into 200 mL of ether-water solution (2:1). The organic layer was separated and the aqueous layer was extracted with ether (100 mL ? 2). The combined organic layer was washed with brine (10 mL ? 2), dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column (hexane/EtOAc = 50:1 to 10:1) to give compound 64 (7.64 g, 91.0%). The NMR spectral data agreed with literature. 226 1-[2,2-Dimethyl-5-(1-(trityloxymethyl)vinyl)-[1,3]dioxolan-4-yl]prop-2-en-1-ol (65). 226 A solution of compound 64 (7.68 g, 13.4 mmol) in 50 mL of THF was treated with 35 mL of TBAF (16.8 mmol, 1.0 M in THF) at room temperature. After being stirred for 2 h, the reaction mixture was adsorbed on silica gel and purified on a silica gel 132 column (hexane/EtOAc = 30:1) to give compound 65 (5.81 g, 95%). The NMR spectral data agreed with literature. 226 2,2-Dimethyl-6-trityloxymethyl-4,6a-dihydro-3aH-cyclopenta-[1,3]dioxol-4-ol (49a). 226 To a solution of compound 65 (5.81 g, 12.7 mmol) in 200 mL of anhydrous CH 2 Cl 2 was added second-generation Grubbs catalyst (0.23 g, 0.25 mmol) at room temperature under argon atmosphere. After being stirred for 24 h, the reaction mixture was adsorbed on silica gel and purified on a silica gel column (hexane/EtOAc = 10:1 to 5:1) to give compound 49a (5.06 g, 93%). The NMR spectral data agreed with literature. 226 Compound 66. To a solution of 49a (0.80 g, 1.86 mmol), Ph 3 P (0.53 g, 2.02 mmol), and 38 (0.80 g, 2.30 mmol) in dry THF (40 mL) at 0 ?C was added dropwise a solution of DIAD (0.41 mL, 2.02 mmol) in THF (5 mL). The mixture was then stirred at the same temperature for 30 min and warmed to room temperature for 12 hr. The solvent was removed under the reduced pressure, and the residue was purified by silica gel column chromatography (EtOAc/hexanes = 1:4) to afford 66 (0.96 g, 68%). 1 H NMR (250 MHz, CDCl 3 ) ? 8.19 (d, J = 5.7 Hz, 1H), 7.48 (d, J = 7.0, 6H), 7.31-7.15 (m, 10H), 6.08 (s, 1H), 5.48 (s, 1H), 5.31 (d, J = 6.0 Hz, 1H), 4.69 (d, J = 6.0 Hz, 1H), 3.97 (s, 2H), 2.69 (s, 3H), 1.45 (s, 18H), 1.30 (s, 3H), 1.24 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 170.6, 151.3, 147.8, 143.4, 140.8, 140.0, 135.7, 128.2, 127.7, 127.1, 123.6, 112.6, 106.2, 87.1, 84.5, 83.8, 82.4, 61.1, 61.0, 60.0, 27.7, 20.7, 14.6. Anal. Calcd for C 45 H 50 N 4 O 7 . 0.6H 2 O: C, 70.49; H, 6.84; N, 7.15. Found: C, 70.47; H, 6.75; N, 7.20. (1S,2R,5R)-5-(4-amino-2-methyl-1H-imidazo[4,5-c]pyridin-1-yl)-3- (hydroxymethyl)cyclopent-3-ene-1,2-diol (4). Compound 66 (1.31 g, 2.34 mmol) was 133 dissolved in a mixture of MeOH (23.4 mL) and 1 N HCl (23.4 mL) and the resulting solution was stirred at rt. for 5 hr. After addition of basic resin (Amberlite IR67) for neutralization of the solution and filtration, the solvent was removed under vacuum and the residue was purified by column chromatography to give 4 (0.50 g, 78%) as white solid. 1 H NMR (400 MHz, MeOD) ? 7.57 (d, J = 6.0, 1H), 6.80 (d, J = 6.0, 1H), 5.98 (dd, J = 3.3, 1.5 Hz, 1H), 5.44 (d, J = 5.4 Hz, 1H), 4.54 (d, J = 5.8, 1H), 4.34-4.31 (m, 3H), 2.65 (s, 3H). 13 C NMR (62.9 MHz, MeOD) ? 153.1, 150.2, 140.6, 140.2, 126.8, 100.2, 78.4, 73.6, 67.5, 60.4, 14.3. Anal. Calcd for C 13 H 16 N 4 O 3 : C, 56.51; H, 5.84; N, 20.28. Found: C, 56.07; H, 5.82; N, 19.94. Methyl 2-((3aR,6S,6aR)-Tetrahydro-2,2-dimethyl-4-oxo-3aH-cyclopenta[d][1,3] dioxol-6-yl)-2-(phenylthio)-acetate (73). 167 To a solution of diisopropylamine (4.25 mL, 30.5 mmol) in THF (75 mL) was added dropwise n-BuLi (13 mL, 2.5 M in hexanes, 32.5 mmol) at 0 ?C. The solution was stirred at the same temperature for 30 min before it was cooled to -70 ?C. Methyl phenylthioacetate (5.00 g, 27.5 mmol) was then added dropwise. After the resulting pale-yellow solution was stirred at the same temperature for 1 h, hexamethylphosphoramide (HMPA) (15.0 mL) was added followed by the dropwise addition of 12 (3.85 g, 25.0 mmol) in THF (25.0 mL). This mixture was kept at -70 ?C for 1 h, and saturated NH 4 Cl solution (50 mL) was added to quench the reaction. The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 ? 150 mL). The combined organic phase was dried (anhydrous MgSO 4 ), and the solvent was removed. The residue was purified by column chromatography to afford 73 as a colorless liquid mixture of two diastereoisomers (6.73g, 80%). The NMR spectral data agreed with literature. 167 134 Methyl 2-((3aR,6aR)-4,6a-Dihydro-2,2-dimethyl-4-oxo-3aH-cyclopenta[d][1,3] dioxol-6-yl)acetate (74). 167 To a solution of 73 (2.24 g, 6.72 mmol) in CH 2 Cl 2 (70 mL) at -78 ?C was added m-CPBA (1.75 g, 77%, 7.77 mmol). The mixture was gradually warmed to -40 ?C and stirred at this temperature for 2.5 h. A solution of NaHSO 3 (0.52 g in 21 mL of H 2 O) was added to quench the reaction. The organic layer was separated, washed with saturated Na 2 CO 3 (2 ? 30 mL), and dried (anhydrous Na 2 SO 4 ). The crud product was purified by chromatography (EtOAc/Hextane = 1:2) to give the intermediate as white foam. This white foam was dissolved in toluene (84 mL), and calcium carbonate (0.70 g, 7 mmol) was added. The mixture was brought to reflux for 12 h. The solvent was removed and the residue was purified by column chromatography (hexane/EtOAc = 4:1 to 2:1) to afford 74 as colorless liquid (1.26 g, 83%). 1 H NMR (400 MHz, CDCl 3 ) ? 6.14 (s, 1H), 5.26 (d, J = 5.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 3.75 (s, 3H), 3.67-3.53 (m, 2H), 1.40 (s, 1H), 1.36 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) ? 201.7, 168.9, 168.5, 131.7, 115.6, 80.0, 77.7, 52.5, 35.6, 27.5, 26.4. Methyl 2-((3aS,4S,6aR)-4,6a-Dihydro-4-hydroxy-2,2-dimethyl-3aH-cyclopenta[d] [1,3]dioxol-6-yl)acetate (75). To a stirred solution of 74 (0.23 g, 1.0 mmol) and CeCl 3 ?7H 2 O (0.40 g, 1.0 mmol) in MeOH (10 mL) at 0 ?C was added in small portions of NaBH 4 (0.15 g, 3.9 mmol). The mixture was stirred at the same temperature for 1.5 h before the reaction was quenched with H 2 O (10 mL). CH 2 Cl 2 (30 mL) was added to the mixture, and the organic phase was separated. The aqueous phase was extracted with CH 2 Cl 2 (2 ? 15 mL). The combined organic phases were washed with brine and dried (anhydrous Na 2 SO 4 ). Evaporation of the solvent afforded 75 as a clean product (0.20 g, 135 86%). 1 H NMR (400 MHz, CDCl 3 ) ? 5.65 (s, 1H), 4.97 (d, J = 5.5 Hz, 1H), 4.72 (t, J = 5.5 Hz, 1H), 4.53 (br, 1H), 3.65 (s, 3H), 3.19 (s, 2H), 2.77 (d, J = 10 Hz), 1.36 (s, 6H); 13 C NMR (100 MHz, CDCl 3 ) ? 170.9, 138.3, 133.3, 112.6, 84.3, 78.0, 73.5, 52.1, 33.4, 27.7, 26.8. (2Z)-Methyl 2-((3aS,6aR)-2,2-Dimethyl-3aH-cyclopenta-[d][1,3]dioxol-6(6aH)- ylidene)acetate (76). To a suspension of 75 (0.14 g, 0.70 mmol), Ph 3 P (0.27 g, 1.05 mmol), and 38 (0.36 g, 1.05 mmol) in THF (5 mL) at 0 ?C was added a solution of DIAD (0.21 g, 1.05 mmol). The mixture was warmed to room temperature and then stirred for 2 h. The solvent was removed and the residue was purified by column chromatography (EtOAc/Hextane = 1:4) to afford 76 as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) ? 7.35 (d, J = 5.6 Hz, 1H), 6.5 (m, 1H), 5.95 (m, 1H), 5.14 (dd, J = 5.6, 2.0 Hz, 1H), 5.00 (d, J = 5.6 Hz, 1H), 3.13 (s, 3H), 1.40 (s, 3H), 1.38 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 166.7, 158.2, 143.6, 132.9, 114.0, 112.8, 81.9, 79.8, 51.4, 27.5, 26.3. (3aS,4S,6aR)-4,6a-Dihydro-6-(2-hydroxyethyl)-2,2-dimethyl-3aH- cyclopenta[d][1,3]dioxol-4-ol (77). 167 To a solution of 74 (1.00 g, 4.40 mmol) in anhydrous CH 2 Cl 2 (43 mL) at -50 ?C was added diisobutylaluminum hydride (DIBAL) (17.2 mL, 1 M in CH 2 Cl 2 , 17.2 mmol) dropwise. The mixture was stirred at the same temperature for 4 h before the reaction was quenched by saturated solution of NH 4 Cl (10 mL), and the mixture was warmed to room temperature and then stirred for 1 h. The organic phase was separated, and the aqueous solution was extracted with CH 2 Cl 2 (3 ? 10 mL). The combined organic phases were dried (anhydrous Na 2 SO 4 ). The solvent was removed under vacuum to cleanly afford 77 as a colorless liquid (0.77 g, 87%). The NMR spectral data agreed with literature. 167 136 (3aS,4S,6aR)-6-[2-(tert-Butyldimethylsilanyloxy)ethyl]-4,6a-dihydro-2,2- dimethyl-3aH-cyclopenta[d][1,3]dioxol-4-ol (72). 167 To an ice-chilled solution of 77 (0.66 g, 3.30 mmol) in CH 2 Cl 2 (40 mL) was added imidazole (0.36 g, 5.3 mmol) and tert- butyldimethylsilyl chloride (TBDMSCl) (0.54 g, 3.4 mmol). The mixture was stirred at room temperature for 12 h. The resulting precipitate was removed by filtration, and the filtrate was evaporated. The residue was purified by column chromatography (EtOAc/hexanes = 1:4) to afford 72 as a colorless oil (0.93 g, 90%). The NMR spectral data agreed with literature. 167 Compound 78. To a suspension of 72 (0.97 g, 3.1 mmol), Ph 3 P (0.98 g, 3.72 mmol), and 38 (1.29 g, 3.72 mmol) in THF (40 mL) at 0 ?C was added a solution of DIAD (0.75 g, 3.72 mmol). The mixture was warmed to room temperature and then stirred for 2 h. The solvent was removed and the residue was purified by column chromatography (EtOAc/Hextane = 1:4) to afford 76 (0.99 g, 48%) as a white foam. 1 H NMR (400 MHz, CDCl 3 ) ? 8.11 (d, J = 5.6 Hz, 1H), 7.13 (d, J = 6.0 Hz, 1H), 5.62 (s, 1H), 5.34 (s, 1H), 5.29 (d, J = 6.0 Hz, 1H), 4.56 (d, J = 6.0 Hz, 1H), 3.97-3.84 (m, 2H), 2.68 (s,3H), 2.51 (t, J = 5.6 Hz, 2H), 1.44 (s, 3H), 1.38 (s, 18H), 1.30 (s, 3H), 0.85 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 153.5, 151.5, 148.4, 143.5, 141.2, 140.2, 135.8, 124.1, 112.7, 106.3, 85.8, 84.5, 82.7, 67.7, 60.9, 32.3, 28.0, 27.3, 26.0, 25.9, 15.0, -5.2. (1S,2R,5R)-5-(4-amino-2-methyl-1H-imidazo[4,5-c]pyridin-1-yl)-3-(2- hydroxyethyl)cyclopent-3-ene-1,2-diol (5). Compound 78 (0.43 g, 0.65 mmol) was dissolved in a mixture of MeOH (6.5 mL) and 1 N HCl (6.5 mL) and the resulting solution was stirred at rt. for 5 hr. After addition of basic resin (Amberlite IR67) for neutralization of the solution and filtration, the solvent was removed under vacuum and 137 the residue was purified by column chromatography to give 5 (0.15 g, 80%) as white solid. 1 H NMR (400 MHz, MeOD) ? 7.47 (d, J = 6.4 Hz, 1H), 6.78 (d, J = 6.4 Hz, 1H), 5.78 (d, J = 1.6 Hz, 1H), 5.38 (dd, J = 6.0, 1.2 Hz, 1H), 4.46 ( d, J = 5.6 Hz, 1H), 4.26 (t, J = 6.0 Hz, 1H), 3.78 (td, J = 6.2, 1.6 Hz, 2H), 2.57 (s, 3H), 2.50-2.47 (m, 2H); 13 C NMR (100 MHz, MeOD) ? 153.4, 152.1, 148.0, 140.7, 138.9, 128.5, 126.8, 100.3, 78.4, 75.9, 68.0, 61.0, 34.0, 14.4. Anal. Calcd for C 14 H 18 N 4 O 3 . 0.57HCl: C, 53.74; H, 6.00; N, 17.90. Found: C, 53.83; H, 6.32; N, 17.83. Isopropyl 4-(bis(tert-butoxycarbonyl)amino)-2-methyl-1H-imidazo[4,5-c]pyridine- 1-carboxylate (80a). 1 H NMR (400 MHz, CDCl 3 ) ? 8.37 (d, J = 5.6 Hz, 1H), 7.78 (d, J = 5.6 Hz, 1H), 5.35 (sep, J = 6.4 Hz, 1H), 2.86 (s, 3H), 1.54 (d, J = 6.4 Hz, 6H), 1.42 (s, 18H); 13 C NMR (100 MHz, CDCl 3 ) ? 154.5, 151.3, 149.5, 143.9, 143.0, 140.3, 135.1, 110.2, 83.1, 73.8, 28.1, 22.1, 18.4. (1R,4S)-(+)-4-tert-Butyldimethylsilyloxy-2-cyclopentenyl acetate (84). To a solution of 11 (9.10 g, 63.5 mmol) in anhydrous CH 2 Cl 2 (200 mL) was treated with imidazole (11.2 g, 0.164 mol) and TBDMSCl (12.0 g, 80.0 mmol). The reaction mixture was stirred at rt. for 6 hr, followed by adding ice water (120 mL) and ether (120 mL). The organic layer was separated and washed with brine (3 ? 100 mL), dried with NaSO 4 . The solvent was evaporated under reduced pressure. The residue was purified by chromatography (EtOAc/Hextane = 1:4) to give 84 (13.0 g, 80%) as a colorless liquid. The NMR spectral data agreed with literature. 245,246 (1R,4S)-4-(tert-butyldimethylsilyloxy)cyclopent-2-enol (85). To a suspension of K 2 CO 3 (6.44 g, 46.0 mmol) in MeOH (250 mL) was added 84 (11.6 g, 45.2 mmol). The resulting mixture was stirred at rt. for 30 min. The solvent was removed under reduced 138 pressure and the residue was dissolved in ether (100 mL). After washed with brine (100 mL), the combined organic layers were dried with Na 2 SO 4 . The solvent was evaporated and residue was purified by chromatography (EtOAc/Hextane = 1:4) to give 85 (7.17 g, 74%) as a colorless liquid. The NMR spectral data agreed with literature. 245,246 Compound 86. To a suspension of 85 (0.08 g, 0.37 mmol), Ph 3 P (0.147 g, 0.56 mmol), and 38 (0.129 g, 0.372 mmol) in THF (5 mL) at 0 ?C was added a solution of DIAD (0.113 g, 0.560 mmol). The mixture was warmed to room temperature and then stirred for 2 h. The solvent was removed and the residue was purified by column chromatography (EtOAc/Hextane = 1:4) to afford 86 (0.16 g, 78%) as a white foam. 1 H NMR (400 MHz, CDCl 3 ) ? 8.17 (d, J = 5.6 Hz, 1H), 7.14 (d, J = 5.6 Hz, 1H), 6.24-6.21 (m, 1H), 6.07-6.05 (m, 1H), 5.78-5.74 (m, 1H), 5.22- 5.20 (m, 1H), 2.64 (s, 3H), 2.39- 2.26 (m, 2H), 1.45 (s, 18H), 0.93 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 153.4, 151.8, 143.6, 140.8, 140.1, 138.5, 136.1, 132.9, 106.6, 82.9, 76.4, 61.2, 40.6, 28.0, 26.0, 18.4, 14.9, 4.5. (1S,4S)-4-(tert-butyldimethylsilyloxy)cyclopent-2-enyl benzoate (87). To a solution of Ph 3 P (1.18 g, 9.93 mmol) and DIAD (2.05 g, 9.93 mmol) in anhydrous THF (40 mL) were added benzoic acid (1.18 g, 9.98 mmol) and a solution of 85 (1.42 g, 6.62 mmol) in THF (20 mL) at 0 ?C under nitrogen atmosphere. Then the reaction was brought to rt. and stirred at same temperature for 12 hr. The solvent was evaporated under reduced pressure and resulting residue was purified by chromatography (EtOAc/Hextane = 1:4 to 2:1) to give 87 contaminated with the azadicarboxylate byproduct. 1 H NMR (400 MHz, CDCl 3 ) ? 8.02-7.99 (m, 2H), 7.53 (tt, J = 7.6, 1.6 Hz, 1H), 7.41 (tt, J = 7.6, 1.6 Hz, 2H), 6.10- 6.06 (m, 2H), 6.03 (m, 1H), 5.12 (m, 1H), 2.32 (ddd, J = 14.5, 6.8, 2.4 Hz, 1H), 2.18 (ddd, 139 J = 14.5, 7.0, 4 Hz, 1H), 0.91 (s, 9H), 0.11 (s, 6H); 13 C NMR (100 MHz, CDCl 3 ) ? 166.6, 141.1, 133.0, 131.6, 130.5, 129.7, 128.4, 79.8, 76.5, 41.3, 21.7, 18.3, -4.54. (1S,4S)-4-(tert-butyldimethylsilyloxy)cyclopent-2-enol (88). The crude compound 87 was treated with LiOH (0.87 g, 19.9 mmol) in THF-H 2 O solution (100 mL) for 12 hr at rt.. The product was extracted by EtOAc (100 mL ? 3 ). The combined organic layer was washed with brine (100 mL) and dried by MgSO 4 . After removal of solvent, the residue was purified by chromatography (EtOAc/Hextane = 1:2) to give the clean 88 (1.02 g, 72%) as colorless liquid. The NMR spectral data agreed with literature. 247 Compound 94a and 94b. To a suspension of 93 231 (0.32 g, 1.40 mmol), Ph 3 P (0.55 g, 2.10 mmol), and 38 (0.57 g, 1.64 mmol) in THF (40 mL) at 0 ?C was added a solution of DIAD (0.42 g, 2.10 mmol). The mixture was warmed to room temperature and then stirred for 2 h. The solvent was removed and the residue was purified by column chromatography (EtOAc/Hextane = 1:4) to afford 94a (0.37g, 48%) and 94b (93.9 mg, 12%) as white foams. 94a: 1 H NMR (400 MHz, CDCl 3 ) ? 8.17 (d, J = 5.6 Hz, 1H), 7.23 (d, J = 5.6 Hz, 1H), 6.15 (m, 1H), 5.74 (dt, J = 5.6, 2.0 Hz, 1H), 5.51 (dt, J = 6.0, 2.4), 3.72 (dd, J = 10, 4.4 Hz, 1H), 3.63 (dd, J = 10, 6.0), 2.80 (m, 1H), 2.68 (s, 3H), 2.64 (m, 1H), 2.38 (m, 1H), 1.44 (s, 18H), 0.88 (s, 9H), 0.05 (s, 3H), 0.03 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 153.7, 151.8, 143.5, 141.4, 139.9,136.2, 135.2, 129.0, 106.9, 82.8, 64.1, 63.7, 47.0, 34.9, 28.1, 26.1, 18.5, 14.9, -5.1, -5.3. 94b: 1 H NMR (400 MHz, CDCl 3 ) ? 8.19 (d, J = 5.6 Hz, 1H), 7.28 (d, J = 5.6 Hz, 1H), 6.19 (td, J = 5.6, 2.4 Hz, 1H), 5.92 (td, J = 5.6, 2 Hz, 1H), 5.60 (m, 1H), 3.69 (d, J = 5.2, 1.2 Hz, 2H), 3.28 (m, 1H), 2.65 (s, 3H), 2.37 (m, 1H), 2.13 (m, 1H), 1.45 (s, 18H), 0.95 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H); 13 C 140 NMR (100 MHz, CDCl 3 ) ? 153.3, 151.8, 143.5, 141.0, 139.9, 137.9, 136.1, 130.6, 106.9, 82.8, 65.9, 62.2, 48.3, 33.6, 28.1, 26.0, 22.1, 14.9, -5.2. Compound 95. 4-methyl morpholine N-oxide NMO (31.7 mg, 0.27 mmol) was added to a solution of 94 (50.0 mg, 0.089 mmol) in CH 2 Cl 2 (5 mL) which contained a small amount of H 2 O (0.05 mL). After the solution was cooled to 0 ?C, a catalytic amount of solid OsO 4 (0.9 mg, 0.003 mmol) was added and the solution was stirred for 4 hr at rt. The reaction was quenched by addition of NaHSO 3 . The solvent was removed and the residue was purified by column chromatography (EtOAc/Hexane = 1:2) to give the 95 (43.8 mg, 83%) as white solid. 1 H NMR (400 MHz, CDCl 3 ) ? 7.94 (d, J = 5.6 Hz, 1H), 7.09 (d, J = 5.6 Hz, 1H), 4.60 (m, 1H), 4.50 (m, 1H), 4.17 (m, 1H), 3.60 (dd, J = 10.0, 4.8 Hz, 1H), 3.52 (dd, J = 10, 4.0 Hz, 1H0, 3.48 (m, 1H), 2.62 (m, 1H), 2.49 (s, 3H), 2.38 (m, 1H), 2.00 (br, 1H), 1.80 (m, 1H), 1.47 (s, 18H), 0.87 (s, 9H), 0.04 (s, 3H), 0.00 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 155.2, 152.1, 151.9, 143.4, 139.8, 106.4, 83.32, 83.28, 75.9, 71.1, 64.4, 63.5, 40.1, 32.5, 28.1, 26.0, 18.5, 14.8, -5.27, -5.34. (1R,2S,3R,5R)-3-(4-amino-2-methyl-1H-imidazo[4,5-c]pyridin-1-yl)-5- (hydroxymethyl)cyclopentane-1,2-diol (6). Compound 95 (40.0 mg, 0.089 mmol) was dissolved in a mixture of MeOH (5.0 mL) and 1 N HCl (5.0 mL) and the resulting solution was stirred at rt. for 5 hr. After addition of basic resin (Amberlite IR67) for neutralization of the solution and filtration, the solvent was removed under vacuum and the residue was purified by column chromatography to give 6 (17.3 mg, 70.0%) as white solid. 1 H NMR (400 MHz, MeOD) ? 7.62 (d, J = 6.0 Hz, 1H), 6.86 (d, J = 6.0 Hz, 1H), 4.62 (m, 1H), 4.55 (dd, J = 9.6, 4.4 Hz, 1H), 4.15 (m, 1H), 3.63-3.55 (m, 2H), 2.69 (m, 1H),2.64 (s, 3H), 2.40 (m, 1H), 1.63 (ddd, J = 14.8, 4, 1.6 Hz, 1H); 13 C NMR (100 MHz, 141 MeOD) ? 153.9, 152.8, 140.0, 139.7, 127.0, 99.9, 77.3, 72.6, 66.2, 65.9, 41.2, 33.4, 14.5. EI-MS calcd for C 13 H 18 N 4 O 3 : [(M + H) + ]: 279.1412, found: 279.1574. (3S,4S,6R)-2,2-dimethyl-6-(trityloxymethyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3] ioxol-4-ol (100). Similar procedure as 106, white solid: 1 H NMR (400 MHz, CDCl 3 ) ? 7.47-7.44 (m, 6H), 7.31-7.20 (m, 9H), 4.66 (t, J = 4.8 Hz, 1H), 4.44 (t, J = 5.6 Hz, 1H), 3.84 (s, 1H), 3.37 (dd, J = 8.8, 7.6 Hz, 1H), 3.09 (dd, J = 8.8, 6.4 Hz, 1H), 2.36 (s, 1H), 1.96-1.82 (m, 2H), 1.38 (s, 3H), 1.35 (s, 3H), 1.30-1.24 (m, 1H); 13 C NMR (100.6 MHz, DMSO-d 6 ) ? 144.3, 128.8, 127.7, 126.9, 79.4, 78.70, 72.19, 62.1, 39.3, 33.6, 25.7, 24.3. (3S,4S,6R)-6-(2-(tert-butyldimethylsilyloxy)ethyl)-2,2-dimethyl-4,6a-dihydro- 3aH-cyclopenta[d][1,3]dioxol-4-ol (106). A mixture of 72 (2.52 g, 8.01 mmol) and 10% Pd/C (0.86 g, 0.80 mmol) in 20 mL of anhydrous ethyl alcohol was hydrogenated at 50 psi in a Parr hydrogenation apparatus for 2 h when TLC showed that the starting material had disappeared and a new spot was detected (hexanes/EtOAc, 4:1), The catalyst was removed by filtration with celite and washed carefully with ethyl alcohol. The filtrate and washings were combined and evaporated in vacuo to give 2.28 g (90.0%) of product as an off-white solid. . 1 H NMR (400 MHz, CDCl 3 ) ? 4.44 (t, J = 4.8 Hz, 1H), 4.38 (t, J = 5.6 Hz, 1H), 3.80-3.70 (m, 1H), 3.64-3.58 (m, 2H), 1.87-1.83 (m, 1H), 1.69-1.75 (m, 2H), 1.57-1.53 (m, 1H), 1.47 (s, 3H) 1.30 (s, 3H), 1.50-1.24 (m, 2H), 0.79 (s, 9H), 0.05(s, 6H); 13 C NMR (100.6 MHz, DMSO-d 6 ) ? 110.1, 80.6, 78.77, 72.2, 61.6, 35.6, 35.0, 31.4, 25.9, 25.6, 24.1, 18.2, -5.4. EI-MS calcd for C 16 H 33 O 4 Si: [(M + H) + ]:317.2143, found:317.2148. Compound 107. Similar procedure as 109, white solid: 1 H NMR (400 MHz, CDCl 3 ) ? 8.88 (s, 1H), 7.98 (s, 1H), 7.48 -7.45 (m, 6H), 7.30-7.20 (m, 9H), 4.99-4.92 (m, 2H), 4.87 142 (d, J = 6.4 Hz, 1H), 3.57 (dd, J = 9.2, 7.2 Hz, 1H), 3.23 (dd, J = 9.2, 7.2 Hz, 1H), 2.56 (m, 1H), 2.22- 2.07 (m, 2H), 1.48 (s, 18H), 1.44 (s, 3H), 1.33 (s, 3H); Compound 109. To a suspension of Ph 3 P (0.95 g, 3.8 mmol), 106 (0.79 g, 2.5 mmol), and 113 (1.0 g, 3.0 mmol) in 50 mL of anhydrous THF was added dropwise a solution of DIAD (0.76 g, 3.8 mmol) in THF (10 mL) in 0 ?C. After stirred at room temperature for 12 hr, the mixture was heated to 60 ?C for another 12 hr. The solvent was removed and residue was purified by column chromatography (EtOAc/hexanes = 1:4) to give 109 as a white foam. (1.25 g, 79.0%) 1 H NMR (400 MHz, CDCl 3 ) ? 8.76 (s, 1H), 7.86 (s, 1H), 4.97 (d, J = 5.2, 1H), 4.87-4.81 (m, 2H), 3.77-3.67 (m, 2H), 2.65-2.60 (m, 1H), 2.28-2.19 (m, 1H), 2.13-2.08 (m, 1H), 1.94-1.88 (m, 1H), 1.71-1.68 (m, 1H), 1.56 (s, 18H), 1.52 (s, 3H), 1.35 (s, 3H), 0.86 (s, 9H), 0.037 (s, 3H), 0.027 (s, 3H); 13 C NMR (100.6 MHz, DMSO-d 6 ) ? 153.2, 151.2, 150.0, 159.9, 122.1, 111.0, 85.6, 82.4, 82.1, 61.9, 61.7, 39.8, 34.7, 31.9, 28.3, 26.5, 26.1, 24.3, 21.3, 18.4, -5.1. Compound 108. Similar procedure as 109, white solid: 1 H NMR (400 MHz, CDCl 3 ) ? 8.36 (d, J = 5.6 Hz, 1H), 7.82 (s, 1H), 7.48-7.45 (m, 6H), 7.39 (d, J = 5.6 Hz, 1H), 7.33- 7.23 (m, 9H), 4.78 (m, 1H), 4.68 (d, J = 5.2 Hz, 1H), 4.60 (m, 1H), 3.59 (dd, J = 9.6, 6.8 Hz, 1H), 3.25 (dd, J = 9.6, 7.2 Hz, 1H), 2.39 (m, 1H), 2.22-2.18 (m,2H), 1.45 (s, 18 H), 1.44 (s, 3H), 1.31 (s, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 151.6, 144.2, 141.3, 140.7, 137.0, 128.9, 128.0, 127.3, 111.5, 106.3, 87.0, 85.7, 83.2, 80.4, 62.4, 61.8, 43.7, 31.4, 26.3, 24.3. Compound 110. To a suspension of Ph 3 P (1.77 g, 6.75 mmol), 106 (1.42 g, 4.50 mmol), and 117 (1.5 g, 4.5 mmol) in 30 mL of anhydrous THF was added dropwise DIAD (1.37 g, 6.75 mmol) in THF (10 mL) at 0 ?C. Stirred at room temperature for 12 hr, 143 and then the mixture were heated to 50 ?C for 8 hr. The solvent was removed and residue was purified by column chromatography (EtOAc/hexanes = 1:4) to afford 110 as a white foam. (2.0 g, 70%). 1 H NMR (400 MHz, CDCl 3 ) ? 8.29 (d, J = 5.6 Hz, 1H), 7.84 (s, 1H), 7.37 (d, J = 5.6 Hz, 1H), 4.67-4.63 (m, 2H), 4.60-4.58 (m, 1H), 3.73-3.63 (m, 2H), 2.45- 2.41 (m, 1H), 2.24-2.20 (m, 2H), 1.91-1.83 (m, 1H), 1.72-1.63 (m, 1H), 1.47 (s, 3H), 1.37 (s, 18H), 1.28 (s, 3H), 0.83 (s, 9H), 0.05 (s, 6H); 13 C NMR (100.6 MHz, DMSO-d 6 ) ? 151.4, 144.6, 141.1, 141.0, 140.6, 136.7, 111.0, 106.0, 85.5, 82.8, 81.4, 61.8, 61.4, 39.7, 33.1, 31.6, 27.9, 27.8, 26.2, 25.9, 24.0, 22.0, 18.2, -5.3. (1R,2S,3R,5S)-3-(6-amino-9H-purin-9-yl)-5-(2-hydroxyethyl)cyclopentane-1,2- diol (9a). Compound 109 (0.58 g, 1.09 mmol) was treat with a mixture of 1N HCl (5 mL), MeOH (10 mL). The reaction mixture was stirred at room temperature overnight and then brought to reflux for 1 hr. The solution was neuturalized with basic ion exchange resin (Amberlite IRA-67). After filtration, the solvent was evaporated under reduced pressure. Purification of the residue by column chromatography afforded 1 as white solid. (0.27 g, 90%). 1 H NMR (400 MHz, DMSO-d 6 ) ? 8.16 (s, 1H), 8.11 (s, 1H), 7.15 (s, 2H), 4.97 (d, J = 6.4 Hz, 1H), 4.73-4.69 (m, 1H), 4.66 (d, J = 3.2 Hz, 1H), 4.51-4.49 (m, 1H), 4.39 (t, J = 5.2 Hz, 1H), 3.84-3.83 (m, 1H), 3.45-3.40 (m, 2H), 2.45- 2.41 (m, 1H), 2.10-2.01 (m, 1H), 1.95-1.87 (m, 1H), 1.69(td, J = 13.6, 6.4 Hz, 1H), 1.45 (td, J = 13.6, 6.4 Hz, 1H); 13 C NMR (100.6 MHz, DMSO-d 6 ) ? 156.0, 152.1, 149.5, 140.6, 119.5, 77.2, 73.0, 59.5, 59.1, 35.2, 33.5, 33.0. Anal. Calcd for C 12 H 17 N 5 O 3 : C, 51.60; H, 6.14; N, 25.08. Found: C, 51.43; H, 6.22; N, 24.83. EI-MS calcd for C 12 H 17 N 5 O 3 : [(M + H) + ]: 280.1410, found: 280.1427. 144 (1R,2S,3R,5S)-3-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-5-(2-hydroxyethyl) cyclopentane-1,2-diol (9b). 1 H NMR (400 MHz, MeOD) ? 8.17 (s, 1H), 7.66 (d, J = 6.0 Hz, 1H), 6.89 (d, J = 6.0 Hz, 1H), 4.82-4.75 (m, 1H), 4.39 (dd, J = 12.0, 3.6 Hz, 1H), 4.03 (t, J = 3.6 Hz, 1H), 3.67-3.58 (m, 2H), 2.55-2.46 (m, 1H), 2.19-2.14 (m, 2H), 1.86 (td, J = 13.6, 6.4 Hz, 1H), 1.64 (td, J=13.6, 6.4 Hz, 1H); 13 C NMR (100.6 MHz, MeOD) ? 153.5, 142.7, 140.0, 128.4, 99.3, 79.9, 75.4, 62.6, 61.3, 50.0, 37.4, 34.3, 34.0. Anal. Calcd for C 13 H 18 N 4 O 3 . 0.1H 2 O: C, 55.74; H, 6.55; N, 20.00. Found: C, 55.67; H, 6.62; N, 19.63. EI-MS calcd for C 13 H 18 N 4 O 3 : [(M + H) + ]: 279.1457, found: 279.1456. (1R,2S,3R,5S)-3-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)cyclopentane-1,2- diol (8a).White solid. 1 H NMR (400 MHz, DMSO-d 6 ) ? 8.17 (s, 1H), 8.11 (s, 1H), 7.17 (s, 2H), 5.00 (d, 1H, J = 6.4 Hz, 1H), 4.73-4.68 (m, 2H), 4.50-4.45 (m, 1H), 4.36 (t, J = 4.2 Hz, 1H), 3.97 (q, J = 3.6 Hz, 1H), 3.64-3.58 (m, 1H), 3.40-3.29 (m, 1H), 2.47-2.44 (m, 1H), 2.00-1.93 (m, 2H); 13 C NMR (100.6 MHz, DMSO-d 6 ) ? 156.0, 152.0, 149.5, 140.7, 119.6, 77.1, 72.0, 60.8, 59.4, 41.4, 30.3. Anal. Calcd for C 11 H 15 N 5 O 3 . 0.6H 2 O: C, 47.86; H, 5.91; N, 25.37. Found: C, 47.94; H, 5.91; N, 25.15. EI-MS calcd for C 11 H 15 N 5 O 3 : [(M + H) + ]: 266.1253, found: 266.1246. (1R,2S,3R,5S)-3-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-5-(hydroxymethyl) cyclopentane-1,2-diol (8b). White solid. 1 H NMR (400 MHz, DMSO-d 6 ) ? 8.16 (s, 1H), 7.64 (d, J = 6.0 Hz, 1H), 6.76 (d, J = 6.0 Hz, 1H), 6.17 (s, 2H), 5.08 (d, J = 6.8 Hz, 1H), 4.76 (d, J = 4.4 Hz, 1H), 4.65-4.60 (m, 1H), 4.40 (t, J = 4.8 Hz, 1H), 4.29-4.24 (m, 1H), 3.96 (q, J = 3.6 Hz, 1H), 3.64-3.60(m, 1H), 3.42-3.36 (m, 1H), 2.45-2.41 (m, 1H), 2.03- 1.90 (m, 2H); 13 C NMR (100.6 MHz, DMSO-d 6 ) ? 152.4, 140.9, 139.7, 137.8, 127.1, 97.2, 77.7, 72.0, 60.9, 60.0, 41.2, 30.0. Anal. Calcd for C 12 H 16 N 4 O 3 . 0.6H 2 O: C, 52.39; H, 6.30; 145 N, 20.37. Found: C, 52.20; H, 6.25; N, 20.26. EI-MS calcd for C 12 H 16 N 4 O 3 : [(M + H) + ]: 265.1301, found: 265.1304. (R)-(-)-4-Carbomethoxy-2-oxazolidinone (118). D-serine methyl ester hydrochloride (25.0 g, 0.162 mol) was in charge with CH 2 Cl 2 (380 mL). The suspension was cooled to 0 ?C before triethylamine (68 mL, 0.48 mol) was added dropwise. The reaction was stirred for 15 min. A solution of triphosgene (16.2 g, 54.0 mmol) in CH2Cl2 (165 mL) was transferred into the addition funnel and added to the reaction mixture over 5 hr. The reaction was stirred for 0.5 hr. Dry ether (500 mL) was added and the reaction was cooled to -78 ?C to precipitate triethylamine hydrochloride. After filtration and wash the precipitate by EtOAc (2 X 150 mL), the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (hexane:EtOAc = 1:9) to give 118 (22.5 g, 95.7%) as a pale yellow solid. The NMR spectral data agreed with literature. 232 (S)-(+)-4-Hydroxymethyl-2-oxaolidinone (119). To a solution of 118 (4.25 g, 29.3 mmol) in dry ethanol (70 mL) at 0 ?C was added sodium borohydride (1.23 g, 35.7 mmol) in portions. The reaction was stirred for 3 hr and then treated with saturated NH 4 Cl (5.8mL). The reaction mixture was stirred for additional 30 min and white solids were filtered through a pad of celide. The filter cake was washed with dry ethanol and the filtrate was concentrated. The resiude was purified through column chromatography (EtOAc:MeOH = 9:1) to give 119 (2.67 g, 77.5%) as white solid. The NMR spectral data agreed with literature. 232 (R)-(+)-4-(4?-Toluenesulfonyloxymethyl)-2-oxazolidinone (120). To a solution of 119 (16.5 g, 0.141 mol) in dry pyridine (53.7 mL), pure TsCl (42.5g, 0.223 mol) was added at 0 ?C. The reaction was stirred at rt. for 6 hr. Pyridine was removed under 146 reduced pressure. The residue was dissolved in CH 2 Cl 2 (300 mL). The organic layer was washed with 2.0 M HCl (3 X 50 mL). The acid layer was back extracted with CH 2 Cl 2 two times and combined organic layer was dried by MgSO 4 . The solvent was removed and crude product was purified through column chromatography (hexane:EtOAc = 1:1) to give 120 (27.5g, 71.9%). The NMR spectral data agreed with literature. 232 (R)-(-)-4-Iodomethyl-2-oxazolidinone (121). A mixture of 120 (2.60 g, 9.60 mmol) and sodium iodide (7.28 g, 44.8 mmol) in 60 mL of freshly distilled dry acetone was refluxed under dry nitrogen for 5 hr. The reaction was cooled to room temperature, the excess sodium iodide was filtered off, and the solids were washed with ethyl acetate. The combined filtrate was concentrated. The crude yellow solid was dissolved in EtOAc and washed with a saturated sodium sulfite solution until the layers became colorless. The aqueous layer was extracted with EtOAc (5 X 50 mL). The combined organic layers were dried over anhydrous MgSO 4 and filtered, and the solvent was removed under reduced pressure. The crude product was purified through column chromatography (EtOAc:hexanes = 3:1) to give 121 (2.13 g, 97.7%). The NMR spectral data agreed with literature. 232 (R)-(+)-4-(2-Oxazolidonyl)-methyltriphenylphosphonyl Iodide (122). A solution of the iodide 121 (2.39 g, 10.53 mmol) and triphenylphosphine (28.0 g, 106.9 mmol) in 30 mL of dry DMF was stirred at 100 ?C for 61 h. The DMF was removed under vacuum, and the resulting residue was triturated with dry THF to remove excess triphenylphosphine, followed by repeated washings with ether to afford a yellow solid. This solid was crystallized from MeOH/EtOAc and dried in an Abderhalden, giving 4.40 g (85%) of a white solid. The NMR spectral data agreed with literature. 232 147 (3aR,6S,6aR)-6-allyl-2,2-dimethyldihydro-3aH-cyclopenta[d][1,3]dioxol-4(5H)- one (123). A solution of CuI (8.17 g, 42.9 mmol), LiCl (1.81 g, 42.2 mmol) in THF (60 mL) was stirred fpr 5 min in rt before cooled down to -78 ?C. A solution of allylic magnesium bromide (1 M in THF, 40.87 mL, 40.87 mmol) was added dropwise. After stirred additional 30 min in the same temperature, TMSCl (5.56 mL) and HMPA (7.9 mL) was added, followed by addition of 12 (2.4 g, 15.6 mol) dropwise. After stirred at same temperature for 2 hr, the reaction mixture was warmed up to 0 ?C and quenched by saturated NH 4 Cl (10 mL). The solvent was evaporated under reduced pressure and the residue was purified by chromatography (EtOAc/Hexane = 1:4) to give 123 (2.30 g, 75.1%) as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) ? 5.71 (m, 1H), 5.12-5.06 (m, 2H), 4.55 (d, J = 5.2 Hz, 1H), 4.20 (d, J = 5.2 Hz, 1H), 2.74 (m, 1H), 2.50 (m, 1H), 2.22- 2.00 (m, 3H), 1.42 (s, 3H), 1.33 (s, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 214.2, 134.7, 118.2, 112.2, 81.6, 78.3, 39.5, 37.8, 36.2, 27.0, 25.0. (3aS,4S,6S,6aR)-6-allyl-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (124). To a suspension of LiAlH 4 (0.345 g, 8.83 mmol) in dry THF (20 mL) at 0 ?C was added dropwise a solution of 123 (1.00 g, 5.10 mmol) in THF (10 mL). After stirred at rt. for 3 hr, the reaction was quenched sequentially with H 2 O (0.30 mL), aqueous NaOH (15%, 0.30 mL) and H 2 O (1.00 mL). The solvent was evaporated to afforded 124 (0.96 g, 95%) as colorless liquid. Compound 125. To a suspension of Ph 3 P (2.72 g, 10.7 mmol), 124 (1.04 g, 5.24 mmol), and 113 (2.55 g, 7.60 mmol) in 50 mL of anhydrous THF was added dropwise a solution of DIAD (2.21 mL, 10.7 mmol) in THF (10 mL) in 0 ?C. After stirred at room temperature for 12 hr, the mixture was heated to 60 ?C for another 12 hr. The solvent was 148 removed and residue was purified by column chromatography (EtOAc/hexanes = 1:4) to give 125 as a white foam. (1.89 g, 70.0%) 1 H NMR (400 MHz, CDCl 3 ) ? 8.82 (s, 1H), 8.08 (s, 1H), 5.82 (m,1H), 5.13-5.04 (m,3H), 4.77 (m,1H), 4.51 (dd, J = 7.3, 4.6 Hz, 1H), 2.49-2.41 (m,2H), 2.30-2.22 (m, 3H), 1.55 (s, 3H), 1.44 (s, 18H), 1.29 (s, 3H). Compound 126. To a solution of 125 (1.50 g, 2.90 mmol) in THF (50 mL) at 0 ?C under N2, was added 9-BBN-H (0.5M in THF, 12 mL, 6 mmol) and resulting mixture stirred for 3hr at rt. NaOH solution (1M, 5.00 mL) was added and followed by adding H 2 O 2 (50% in H 2 O, 3.30 mL). The reaction mixure was stirred for another 2 hr and solvent was removed under reduced pressure. The residue was purified by chromatography (EtOAc/Hexane = 1:2) to give 126 (0.96 g, 62%) as a white foam. 1 H NMR (400 MHz, CDCl 3 ) ? 8.75 (s, 1H), 7.96 (s, 1H), 5.81 (m, 1H), 5.68 (m, 1H), 5.18- 5.08 (m, 2H), 4.98 (dd, J = 18.6, 8.6 H, 1H), 2.53-2.46 (m, 2H), 2.43-2.39 (m,2H), 2.29- 2.11 (m, 2H), 1.56 (s, 9H), 1.51 (s, 9H), 1.46 (m, 1H), 1.39 (s, 6H). (3aR,6S,6aR)-6-(but-3-enyl)-2,2-dimethyldihydro-3aH-cyclopenta[d][1,3]dioxol- 4(5H)-one (128). To a suspension of CuBr?Me 2 S (0.18 g, 0.87 mmol) in THF (40 mL) was added dropwise a solution of 3-butenyl magnesium bromide (1 M in THF, 26.00 mL, 13.00 mmol) at 78 ?C. After stirred additional 30 min in the same temperature, TMSCl (2.60 mL, 20.3 mmol) and HMPA (4.5 mL, 25.7 mmol) was added, followed by addition of 12 (1.54 g, 10.0 mol) dropwise. After stirred at same temperature for 2 hr, the reaction mixture was warmed up to 0 ?C and quenched by saturated NH 4 Cl (10 mL). The solvent was evaporated under reduced pressure and the residue was purified by chromatography (EtOAc/Hexane = 1:4) to give 128 (1.85 g, 88.0%) as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) ? 5.78 (m, 1H), 5.07-4.99 (m,2H), 4.55 (d, J = 5.2 Hz, 1H), 4.23 (d, J = 5.2 149 Hz, 1H), 2.76 (m, 1H), 2.40 (m, 1H), 2.16-2.05 (m, 3H), 1.55 (m, 1H), 1.43 (s, 3H), 1.33 (m, 1H), 1.35 (s, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 213.9, 137.3, 115.7, 112.2, 82.0, 78.2, 40.0, 36.4, 32.8, 31.4, 26.9, 25.0. Anal. Calcd for C 12 H 18 O 3 . 0.1H 2 O: C, 67.96; H, 8.65; Found: C, 67.96; H, 8.65. EI-MS calcd for C 12 H 18 O 3 : [(M + H) + ]: 211.1289, found: 211.1271. (3aS,4S,6S,6aR)-6-(but-3-enyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3] dioxol-4-ol (129). Similar produce as 124; 1 H NMR (400 MHz, CDCl 3 ) ? 5.73 (m, 1H), 5.00-4.90 (m,2H), 4.43 (t, J = 5.6 Hz, 1H), 4.27 (dd, J = 6.2, 2 Hz, 1H), 4.01 (m, 1H), 2.51 (d, J = 7.6 Hz, 1H), 2.08-1.99 (m, 3H), 1.84 (m, 1H), 1.61 (m, 1H), 1.45 (s, 3H), 1.30 (s, 3H), 1.38-1.82 (m, 2H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 138.1, 115.0, 111.7, 85.1, 79.3, 71.2, 41.0, 36.8, 32.2, 31.7, 26.2, 24.4. Anal. Calcd for C 12 H 20 O 3 : C, 67.89; H, 9.50; Found: C, 67.70; H, 9.58. EI-MS calcd for C 12 H 20 O 3 : [(M + H) + ]: 213.1446, found: 213.1403. Compound 130. Similar procedure as 125; 1 H NMR (400 MHz, CDCl 3 ) ? 8.83 (s, 1H), 8.10 (s, 1H), 5.83 (m, 1H), 5.10-4.93 (m, 3H), 4.78 (m, 1H), 4.51 (t, J = 6.4 Hz, 1H), 2.48 (m, 1H), 2.29 (q, J = 13.3 Hz, 1H), 2.23-2.14 (m, 3H), 1.76 (m, 1H), 1.62 (m, 1H), 1.56 (s, 3H), 1.46 (s, 18H), 1.31 (s, 3H). Compound 127. Compound 130 (4.21 g, 7.95 mmol) was dissolved in MeOH (21.0 mL) and H 2 O (10 mL), and NaIO 4 (2.54 g, 12 mmol) was added slowly. After the reaction mixture was cooled to 0 ?C, OsO 4 (17.5 mg) was added. The reaction was stirred at the same temperature for 1hr and then warmed to room temperature for 2 hr. The white solid that resulted was removed by filtration and the filtrate was concentrated in vacuo. CH 2 Cl 2 (117 mL) was added to the residue and washed with H 2 O (17.5 mL) and brine 150 (17.5 mL), and dried with MgSO 4 . The solvent was removed under reduced pressure to give 127 (2.87 g, 68%) as white foam. 1 H NMR (400 MHz, CDCl 3 ) ? 9.84 (d, J = 1.6 Hz, 1H), 8.84 (d, J = 1.6 Hz, 1H), 8.09 (s, 1H), 5.10 (m, 1H), 4.77 (m, 1H), 4.51 (m, 1H), 2.65 (td, J = 7.4, 1.2 Hz, 2H), 2.48 (m, 1H), 2.37 (q, J = 12.6 Hz, 1H), 2.18 (m, 1H), 1.91 (q, J = 7.2 Hz, 2H), 1.55 (s, 3H), 1.48 (s, 18 H), 1.31(s, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 201.7, 153.3, 152.1, 150.8, 144.4, 129.8, 114.4, 84.6, 84.1, 83.6, 62.1, 43.8, 42.2, 37.1, 28.0, 27.6, 25.6, 25.3. Compound 132. A solution of 122 232 (2.23 g, 4.61 mmol) in THF (70 mL) was cooled down to -78 ?C, before the n-BuLi (5.69 mL, 13.7 mmol) was added over a 10 min period. The resulting orange-color solution was stirred at same temperature for 1 hr, followed by adding a solution of 127 (2.30 g, 4.33 mmol) in THF (20 mL) slowly. After stirred at same temperature for 3 hr, the reaction was brought up to rt.. A saturated NH 4 Cl solution was added and stirred for 10 min. The THF was removed under reduced pressure. The aqueous solution was extracted with EtOAc (3 x 60 mL) and dried with MgSO 4 . After evaporated the solvent, the resulting residue was purified by chromatography (EtOAc/Hextane = 1:4) to give mixture of 131a and 131b (1.01 g, 45.3%). The mixture of 131 was dissolved in THF (1.01 g, 1.96 mmol), followed by addition of DMAP (0.024 g, 0.2 mmol), Boc 2 O (1.38 mL, 6.00 mmol) at room temperature. The reaction mixture was stirred at same temperature for 8 hr. After removal of solvent, the crude product was purified by chromatography (EtOAc/Hexane = 1:2) to afford 132 (1.18 g, 86.9%) as white solid. Compound 133. A parr hydrogenation apparatus was charged with 132 (0.79 g, 1.1 mmol), 10% Pd/C and 45 psi hydrogen in MeOH (20 mL) for 5 hr. The catalyst was 151 removed by filtration and washed with EtOH. Evaporated the combined solvent to give 143 (0.77 g, 97.6%) as a white foam. Compound 134. To a solution of 133 (0.74 g, 1.03 mmol) in THF:H 2 O (1:1) was added LiOH (0.12 g, 5.20 mmol) at room temperature. The reaction mixture was stirred at same temperature for 2hr. The solvent was evaporated under reduced pressure and the crude residue was purified by columgraphy (EtOAc: Hexane = 1:1) to give 134 (0.70 g, 98.5%) as white solid. (R)-methyl 2-(tert-butoxycarbonylamino)-3-hydroxypropanoate (136). Et 3 N (14.0 g, 138 mmol) was added to a suspended serine methyl ester hydrochloride (10 g, 64.3 mmol) in THF (200 mL). After the resulting white suspension was cooled to 0 ?C, a solution of (Boc) 2 O (14.3 g, 97%, 63.6 mmol) in THF (100 mL) was added dropwise over 45 min. The mixture was allowed to warm to room temperature and stirred for 6 hr, then warmed to 50 ?C for another 2hr. The solvent was removed in vacuo and the residue partitioned between Et 2 O (200 mL) and H 2 O (200 mL). The aqueous phase was extracted with Et 2 O (2 x 160 mL). The combined organic layer was washed with 3% HCl (100 mL), 5% NaHCO 3 (100 mL) and brine (200 mL). After drying by MgSO 4 , the solvent was evaporated to afford 136 ( 13.53 g, 96.0%) as a colourless oil. 1 H NMR (400 MHz, CDCl 3 ) ? 5.49 (d, J = 5.6 Hz, 1H), 4.34 (d, J = 3.2 Hz, 1H), 3.95-3.83 (m, 2H), 3.75 (s, 3H), 2.69 (m, 2.69, 1H), 1.42 (s, 9H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 171.6, 156.0, 80.5, 63.7, 55.9, 52.8, 28.5. (R)-3-tert-butyl 4-methyl 2,2-dimethyloxazolidine-3,4-dicarboxylate (137). To a solution of 136 (13.5 g, 61.7 mmol) in acetone (200 mL) and 2, 2-dimethyloxylpropane (66.7 mL) was added BF 3 ? OEt 2 (0.4 mL). The resulting solution was stirred at room 152 temperature for 2 hr. The solvent was removed under pressure and the residual was dissolved in CH 2 Cl 2 (200 mL) and washed with a mixture of saturated NaHCO 3 and H 2 O (1:1, 133 mL), then brine (133 mL).The solvent was evaporated in vacuo after dried by MgSO 4 to give 137 (15.02 g, 93.9%) as a pale yellow oil. 1 H NMR (400 MHz, DMSO, 70 ?C) ? 4.40 (dd, J = 7.2, 2.8 Hz, 1H), 4.16 (dd, J = 9.2, 7.2 Hz, 1H), 3.94 (dd, J = 9.2, 2.8 Hz, 1H), 3.69 (s, 3H), 1.56 (s, 3H), 1.46 (s, 3H), 1.40 (s, 9H); (R)-tert-butyl 4-formyl-2,2-dimethyloxazolidine-3-carboxylate (138). A solution of compound 137 (15.12 g, 58.3 mmol) in anhydrous toluene (120 mL) was cooled down to -78 ?C followed by addition of DIBALH (100 mL, 1M in hexane, 100 mmol) dropwise. Stirring was continued for 2 hr before careful addition of anhydrous MeOH (23 mL) and then the reaction mixture was warmed to room temperature. The mixture was poured into a solution of potassium sodium tartrate (100.8 g) in H 2 O (280 mL). The biphasic mixture stirred vigorously for 2hr and then separated into two phase. The aqueous layer extracted with Et 2 O (2 x 150 mL) and combined organic extracts was drievd by MgSO 4 . Evaporated the solvent in vacuo to give 138 (10.5 g, 78.2%) as colorless oil. (S)-tert-butyl 2,2-dimethyl-4-vinyloxazolidine-3-carboxylate (139). Methyltriphenylphosphonium bromide ( 26.2 g, 73.6 mmol) was suspended in THF (300 mL) under N 2 at room temperature and KHMDS (0.5 M in toluene, 140.8 mL, 70.4 mmol) was added. The resulting yellow suspension was stirred at room temperature for 60 min, then cooled to -78 ?C and a solution of the 138 (9.61 g, 41.9 mmol) in THF (128 mL) was added dropwise. The cooling bath was removed and the mixture allowed to reach room temperature over 2 hr. The reaction was quenched with MeOH (60 mL) and the resulting mixture was poured into a mixture of saturated potassium sodium tartrate and 153 H 2 O (1:1, 200 mL). Extraction with ether (2 x 100 mL), drying with MgSO 4 and evaporation of the solvent. The residue was purified by columatography (4:1 = Hexane : EtOAc) to give 139 (8.18 g, 85.9%) as colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) ? 5.81 (m, 1H), 5.25-5.12 (m, 2H), 4.33 (m, 1H), 4.04 (dd, J = 6.4, 8.8 Hz, 1H), 3.73 (dd, J = 2.0, 8.8 Hz, 1H), 1.60 (s, 3H), 1.51-1.43 (m, 12H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 151.7, 137.3, 115.5, 93.7, 79.3, 67.9, 59.5, 28.0, 26.4, 23.5. (S)-tert-butyl 1-hydroxybut-3-en-2-ylcarbamate (140). To a solution of 139 (6.65 g, 29.2 mmol) in MeOH (200 mL) was added p-tolunenesulfonic acid monohydrate (2.78 g, 14.6 mmol) at 0 ?C. The mixture was slowly warmed to room temperature and stirred for 12 hr. The solvent was evaporated and the residue was diluted with EtOAc (88 mL). The orangic layer was washed with saturated sodium bicarbonate solution (3 x 50 mL). The organic phase was combined and dried over MgSO 4 . Evaporating the solvent to give the desired 140 (3.75 g, 68.6%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl 3 ) ? 5.82 (ddd, J = 5.2, 10.6, 16.8 Hz, 1H), 5.33 (d, J = 8.0 Hz, 1H), 5.27-5.18 (m, 2H), 4.20 (br, 1H), 3.69-3.57 (m, 3H), 1.45 (s, 9H). (S)-tert-butyl 4-((E)-4-((3aR,4S,6R,6aS)-6-(6-(bis(tert-butoxycarbonyl)amino)-9H- purin-9-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)but-1-enyl)- 2,2-dimethyloxazolidine-3-carboxylate (141). To a solution of 128 (3.08 g, 5.81 mmol) and 139 (1.32 g, 5.81 mmol) in dry CH 2 CL 2 (150 mL) was added 2 nd generation of Grubbs catalyst (0.1g, 0.12 mmol) at room temperature under N 2 atmosphere. The reaction mixture was stirred 8 hr at room temperature and heated to reflux for overnight. The mixture was filtrated through celite and the solvent was removed under reduced pressure. The residue was purified by columatography (2:1 = Hexane : EtOAc) to give 154 141 (2.58 g, 60.9%) as white foam with 142 (1.11 g, 36.8%) as byproduct. 141 1 H NMR (400 MHz, CDCl 3 ) ? 8.78 (s, 1H), 8.04 (s, 1H), 5.52 (m, 1H), 5.43 (dd, J = 7.2, 15.4 Mz, 1H), 5.03 (t, J = 6.0 Hz, 1H), 4.71 (td, J = 11.6, 6.0 Hz, 1H), 4.45 (t, J = 6.4 Hz, 1H), 4.24 (m, 1H), 3.96 (dd, J = 6, 8.8 Hz, 1H), 3.66 (dd, J = 2.0, 8.8 Hz, 1H), 2.42 (m, 1H), 2.25 (m, 1H), 2.15-2.08 (m, 3H), 1.69 (m, 1H), 1.53 (m, 1H), 1.49 (s, 3H), 1.44 (s, 3H), 1.406 (s, 30 H), 1.25 (s, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 171.2, 153.3, 152.0, 151.9, 150.7, 150.5, 144.4, 131.5, 129.7, 114.1, 93.9, 84.6, 83.9, 83.4, 79.4, 68.6, 65.9, 62.3, 60.5, 59.2, 37.0, 30.4, 28.6, 27.9, 27.6, 25.2, 21.2, 14.3; EI-MS calcd for C 37 H 56 N 6 O 9 : [(M + H) + ]: 729.4174, found: 729.4180. 142 1 H NMR (400 MHz, CDCl 3 ) ? 8.79 (s, 1H), 8.06 (s, 1H), 5.42 (m, 1H), 5.04 (t, J = 6.4 Hz, 1H), 4.73 (td, J = 11.6, 6.0 Hz, 1H), 4.45 (t, J = 6.4 Hz, 1H), 2.44 (m, 1H), 2.24 (m, 1H), 2.14-2.06 (m, 4H), 1.68 (m, 1H), 1.51 (s, 3H), 1.41 (s, 18H), 1.26 (s, 3H); EI- MS calcd for C 52 H 74 N 10 O 12 : [(M + H) + ]: 1031.2497, found: 1031.2487. (S)-tert-butyl 4-(4-((3aR,4S,6R,6aS)-6-(6-(bis(tert-butoxycarbonyl)amino)-9H- purin-9-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)butyl)-2,2- dimethyloxazolidine-3-carboxylate (143). A parr hydrogenation apparatus was charged with 141 (1.15 g, 1.58 mmol), 10% Pd/C and 45 psi hydrogen in EtOH (20 mL) for 3 hr. The catalyst was removed by filtration and washed with EtOH. Evaporated the combined solvent to give 143 (1.15 g, 99.7%) as a white foam. 1 H NMR (400 MHz, CDCl 3 ) ? 8.77 (s, 1H), 8.05 (s, 1H), 5.02 (br, 1H), 4.71 (td, J = 6.0, 11.6 Hz, 1H), 4.41 (t, J = 6.0 Hz, 1H), 3.86-3.65 (m, 3H), 2.41 (m, 1H), 2.21 (td, J = 11.6, 22.8 Hz, 1H), 2.08 (m, 1H), 1.59-1.46 (m, 7H), 1.49 (s, 3H), 1.39 (s, 33H), 1.27 (m, 1H), 1.23 (s, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 153.3, 151.9, 150.7, 150.5, 144.3, 129.7, 114.0, 93.4, 84.6, 83.8, 155 83.4, 79.7, 66.9, 62.3, 57.5, 43.9, 37.1, 33.7, 32.8, 28.6, 27.9, 27.8, 27.6, 26.9, 26.4, 25.2, 24.6, 23.3; EI-MS calcd for C 37 H 58 N 6 O 9 : [(M + H) + ]: 731.4344, found: 731.4352. Compound 144a and 144b. To a solution of 143 (1.15 g, 1.58 mmol) in MeOH (50 mL) was added p-tolunenesulfonic acid monohydrate (0.15 g, 0.79 mmol) for 6 hr at room temperature. A basic resin (Amberlite IR67) was added to neutralize the reaction mixture. After filtration, the solvent was removed under reduced pressure. The residue was purified by columatography (Hextane: EtOAc =1:1) to give 144a (0.25 g, 23.0%) and 144b (0.23 g, 22.5%). 144a 1 H NMR (400 MHz, CDCl 3 ) ? 8.77 (s, 1H), 8.06 (s, 1H), 5.02 (dd, J = 6.0, 7.2 Hz, 1H), 4.74-4.68 (m, 2H), 4.41 (dd, J = 6.0, 7.4 Hz, 1H), 3.55-3.47 (m, 3H), 2.96 (br, 1H), 2.41 (td, J = 6.4, 12.4 Hz, 1H), 2.08 (td, J = 6.0, 12.4 Hz, 1H), 1.49 (s, 3H), 1.43-1.29 (m, 9H), 1.39 (s, 18H), 1.37 (s, 9H), 1.24 (s, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 156.5, 153.3, 151.9, 150.7, 150.5, 144.4, 129.7, 114.0, 84.7, 83.9, 83.4, 65.9, 62.3, 43.8, 37.0, 33.6, 31.5, 28.5, 27.9, 27.8, 27.6, 26.1, 25.2, 15.4; EI-MS calcd for C 34 H 54 N 6 O 9 : [(M + H) + ]: 691.4031, found: 691.4033. 144b 1 H NMR (400 MHz, CDCl 3 ) ? 8.76 (s, 1H), 8.06 (s, 1H), 5.11 (d, J = 4.0 Hz, 1H), 4.84 9d, J = 7.2 Hz, 1H), 4.68 (m, 1H), 4.41 (m, 1H), 3.89 (br, 1H), 3.76 (br, 1H), 3.57- 3.47 (m, 3H), 3.18 (br, 1H), 2.49 (m, 1H), 2.376 (m, 1H), 2.04 (m, 1H), 1.57 (m, 1H), 1.50 (m, 1H), 1.45-1.31 (m, 4H), 1.41 (s, 18H), 1.38 (s, 9H), 1.25 (m, 1H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 156.7, 153.6, 151.6, 150.8, 150.5, 144.6, 129.6, 84.7, 75.3, 75.2, 65.6, 62.0, 43.5, 34.3, 32.6, 31.5, 28.5, 28.3, 28.0, 27.6, 26.0, 25.2; EI-MS calcd for C 31 H 50 N 6 O 9 : [(M + H) + ]: 651.3718, found: 651.3719. 156 Compound 145. To a solution of 128 (1.49 g, 2.82 mmol) and 140 (0.79 g, 4.23 mmol) in dry CH 2 CL 2 (100 mL) was added 2 nd generation of Grubbs catalyst (0.1g, 0.12 mmol) at room temperature under N 2 atmosphere. The reaction mixture was stirred 8 hr at room temperature and heated to reflux for overnight. The mixture was filtrated through celite and the solvent was removed under reduced pressure. The residue was purified by columatography (1:1 = Hexane : EtOAc) to give 145 (1.26 g, 64.8%) as white foam. 1 H NMR (250 MHz, CDCl 3 ) ? 8.70 (s, 1H), 8.04 (s, 1H), 5.56 9(dt, J = 6.50, 15.5 Hz, 1H), 5.35 (dd, J = 5.75, 15.5 Hz, 1H), 5.07-4.95 (m, 2H), 4.67 (m, 1H), 4.38 (m, 1H), 4.02 (br, 1H), 3.52-3.46 (m, 2H), 3.24 (m, 1H), 2.34 (m, 1H), 2.23-2.02 (m, 4H), 1.58-1.46 (m, 2H), 1.43 (s, 3H), 1.32 (s, 18H), 1.30 (s, 9H), 1.18 (s, 3H); 13 C NMR (62.5 MHz, CDCl 3 ) ? 155.9, 153.2, 151.7, 150.5, 150.3, 144.4, 131.5, 129.5, 128.3, 113.8, 84.4, 83.7, 83.3, 79.4, 65.2, 62.1, 43.1, 36.8, 32.8, 30.4, 28.4, 27.7, 27.4, 25.1; EI-MS calcd for C 34 H 52 N 6 O 9 : [(M + H) + ]: 689.3874, found: 689.3876. Compound 144a. A parr hydrogenation apparatus was charged with 145 (0.83 g, 1.20 mmol), 10% Pd/C and 45 psi hydrogen in EtOH (20 mL) for 3 hr. The catalyst was removed by filtration and washed with EtOH. Evaporated the combined solvent to give 143 (0.81 g, 97.5%) as a white foam. (S)-6-((3aR,4S,6R,6aS)-6-(6-(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)-2,2- dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-2-(tert- butoxycarbonylamino)hexanoic acid (146). To a solution of 144a (0.70 g, 1.01 mmol) in acetone (25 mL), an aqueous 15% solution of NaHCO 3 (3.5 mL) was added. The mixture was stirred at 0 ?C, followed by adding NaBr (0.03 g, 0.29 mmol), TEMPO (4.0 mg, 0.03 mmol) into solution, then trichloroisocyanuric acid (0.50 g, 2.1 mmol) was 157 slowly added within 20 min. After addition, the mixture was warmed to rt. and stirred for overnight. Then 2-propanol (1 mL) was added. The mixture was filtered on celite and water was added. The mixure was extracted twice with EtOAc (20 mL X 2). The organic layers were dried with Na 2 SO 4. The solvent was evaporated and the residue was purified by column (EtOAc: Methanol = 10:1) to gave a white solid (0.58 g, 81%). 1 H NMR (400 MHz, MeOD) ? 8.78 (s, 1H), 8.55 (s, 1H), 5.03 (dd, J = 6.0, 7.2 Hz, 1H), 4.95-4.89 (m, 2H), 4.44 (dd, J = 5.6, 7.2 Hz, 1H), 2.42 (dt, J = 6.4, 12.4 Hz, 1H), 2.26-2.09 (m, 2H), 1.94-1.84 (m, 2H), 1.50-1.35 (m, 3H), 1.34-1.22 (m, 3H), 1.47 (s, 3H), 1.39 (s, 9H), 1.30 (s, 18H), 1.17 (s, 3H); 13 C NMR (100.6 MHz, MeOD) ? 173.2, 157.1, 154.9, 152.9, 151.7, 151.6, 151.1, 147.5, 130.7, 115.4, 86.1, 85.6, 85.1, 84.9, 63.3, 45.0, 38.1, 34.6, 29.8, 28.4, 28.2, 28.1, 27.9, 27.0, 25.5; EI-MS calcd for C 159 H 247 N 30 O 43 : [(M + H) + ]: 705.3778, found: 705.3820. (S)-2-amino-6-((1S,2R,3S,4R)-4-(6-amino-9H-purin-9-yl)-2,3- dihydroxycyclopentyl)hexanoic acid (10). To a solution of 146 (0.58 g, 0.83 mmol) in MeOH (10 mL) added 1 N HCl (5 mL). The mixture stirred at rt. for overnight. The solution was then neuturalized with weekly basic exchange resin (Amberlite IRA-67). After filtration and evaporation of the solvent, the crude product was purified by chromatography (EtOAc/ MeOH/NH 3 ?H 2 O = 3:1:1) to give 10 (0.22, 72%) as white solid. 1 H NMR (400 MHz, D 2 O) ? 8.22 (s, 1H), 8.18 (s, 1H), 4.75 (m, 1H), 4.47 (dd, J = 6.0, 9.0 Hz, 1H), 3.95 (dd, J = 4.0, 6.0 Hz, 1H), 3.74 (t, J = 6.0 Hz, 1H), 2.48 (dt, J = 7.8, 12.9 Hz, 1H), 2.03 (m,1H), 1.92-1,87 (m, 2H), 1.70-1.61 (m, 1H), 1.45-1.44 (m, 5H); 13 C NMR (100.6 MHz, D 2 O) ? 174.9, 154.7, 151.2, 149.0, 140.8, 118.6, 75.0, 74.6, 59.6, 54.8, 158 42.5, 33.2, 32.2, 30.3, 26.5, 24.2; Anal. Calcd for C 16 H 24 N 6 O 4 ?HCl?0.5H 2 O: C, 46.89; H, 6.39; N, 20.50. Found: C, 46.89; H, 6.66; N, 20.58. 159 References (1) Lustig, A.; Levine, A. J. J. Virol. 1992, 66, 4629. (2) Puissant, B.; Combadiere, B. Cell. Mol. Life Sci. 2006, 63, 2249. (3) Hovi, T.; Roivainen, M.; Blomqvist, S. Perspect. Med. Virol. 2007, 17, 69. 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