Synthesis of small-molecule probes for chemical biology

MASARYK UNIVERSITY

FACULTY OF SCIENCE

DEPARTMENT OF CHEMISTRY

Synthesis of small-molecule probes for

chemical biology

Ph.D. Dissertation

Lukáš Maier

Thesis supervisor: Mgr. Kamil Paruch, PhD. Brno 2014

2

Bibliographic Entry

Author: Mgr. Lukáš Maier

Department of Chemistry, Faculty of Science,

Masaryk University

Title of Thesis: Synthesis of small-molecule probes for chemical

biology

Ph.D. Degree

programme: Chemistry

Field of Study: Organic Chemistry

Supervisor: Mgr. Kamil Paruch, Ph.D.

Academic Year: 2014/2015

Number of Pages: 196 pages + 9 of appendices, 313 pages on

supplementary CD

Keywords: chemical biology small-molecule probes

nucleosides; nucleoside analogs; pseudoisocytidine;

tubercidine fluorescent probes naphthalene

chromophore, diastereoselective organic synthesis

NMR spectroscopy biological activity

3

Bibliografický záznam

Autor: Mgr. Lukáš Maier

Ústav Chemie, Přírodovědecká fakulta, Masarykova

univerzita

Název práce: Syntéza nízkomolekulárních sond pro chemickou

biologii

Studijní program: Chemie

Studijní obor: Organická chemie

Vedoucí práce: Mgr. Kamil Paruch, Ph.D.

Akademický rok: 2014/2015

Počet stran: 196, + 9 stránek příloh, přiložené CD (313 stran)

Klíčová slova: chemická biologie nízkomolekulární sondy

nukleosidy nukleosidové analogy pseudoisocytidin

tubercidin fluorescentní sondy naftalenové

chromofory, diastereoselektivní organická syntéza

NMR spektroskopie biologická aktivita

4

Summary

My Ph.D thesis is focused particularly on the synthesis of new carbocyclic C-nucleosides.

This topic is divided into two individual parts.

First part deals with the synthesis of direct analogs of pseudoisocytidine. Pseudoisocytidine

was shown to be active against cytarabine-resistant leukemias, but its clinical progression had

to be halted due to hepatotoxicity of uknown origin. We carried out replacement of the sugar

moiety by the cyclopentane core and synthesized carbocyclic pseudoisocytidine and its

oxygen and sulfur analogs in 13 linear steps via -ketoester intermediate. This project also

included preparation of properly substituted cyclopentanone intermediate and its conversion

into a new carbocyclic C-nucleoside bearing tertiary hydroxyl group at 1’-position. This

served as a starting point for the development of more flexible synthetic route that would

allow synthesis of various carbocyclic C-nucleoside analogs, covered in the second part.

The second part describes synthesis of key cyclopentanone intermediates. Proper choice of

protecting groups proved to be crucial for successful synthesis of the cyclopentanone

precursor and its subsequent conversion into the desired target compounds. We developed

methodology that enables orthogonal manipulation of 1´-, 2´-, and 5´-positions of the

cyclopentane core. In addition, the synthesis is highly diastereoselective and can produce both

classes with alternative stereochemistry at 1´-position. The scope and limitations of the

methodology is demonstrated by the synthesis of selected carbocyclic C-nucleosides

containing pyrimidine and purine bases and their isosteres.

The last part of the thesis describes synthesis of small-molecule fluorescent probes and

their application in the studies of haloalkane dehalogenases. The probes contain properly

substituted naphthalene-based chromophore plus linkers of variable length that enabled

covalent interaction with the enzymes. These probes were used to study the behavior of

haloalkane dehalogenases by time-dependent fluorescence techniques.

5

Shrnutí

Tato dizertační práce je zameřena zejména na syntézu nových karbocyklických

nukleosidových analogů. V úvodu práce jsou stručně popsány principy a nástroje chemické

biologie a detailněji shrnuty poznatky v oblasti struktury, biologické aktivity a syntézy

nukleosidových analogů.

První část je věnována syntéze dosud nepopsaných karbocyklických nukleosidových

analogů pseudoisocytidinu. Pseudoisocytidin, patřící mezi C-nukleosidy, vykazuje

významnou inhibiční aktivitu vůči cytarabin-rezistentním buněčným leukemickým liniím.

Jeho hepatotoxicita, jejíž příčiny dosud nejsou objasněny, ale znemožnila detailnější klinické

studie. Metabolická degradace pseudoisocytidinu in vivo může být příčinou jeho nežádoucí

toxicity. Jeho přímý karbocyklický analog, vzniklý náhradou cukerné časti za cyklopentan,

může být chemicky i metabolicky více stabilní. Ve 13 lineárnich syntetických krocích jsme

připravili tři karbocycklické analogy pseudoisocytidinu. V této části je také zahrnuta syntéza

cyklopentanonového intermediátu, který byl použit pro přípravu nového typu

karbocyklického C-nukleosidu nesoucí terciární -OH skupinu v poloze 1´. Úspěšná příprava

této molekuly vedla k vývoji flexibilnější a obecnější syntetické metodologie, která je

detailněji diskutována v druhé části dizertační práce.

Syntéza cyklopentanonových intermediátů je podrobně popsána v druhé části. Volba

chránících skupin a jejich kombinace se ukázala jako klíčový faktor při syntéze samotných

cyklopentanonových intermediátů i při jejich následné konverzi na požadované cílové

sloučeniny. Námi vyvinutá diastereoselektivní metodologie umožňuje přípravu nových

karbocyklických analogů s definovanou stereochemii v pozici C-1´. Metodologie navíc

umožňuje selektivní manipulaci s pozicemi 1´-, 2´- a 5´-. Aspekty, výhody a nevýhody naší

metodologie jsou demonstrovány na příkladech konkrétních syntéz jednotlivých

karbocyklických nukleosidových analogů obsahujících pyrimidinové i purinové typy bází.

Třetí část je věnována syntéze fluorescentních sond pro studium haloalkan dehalogenas.

Fluorescentně modifikované chloroalkany byly použity jako substráty pro enzymatickou

dehalogenaci. Jejich interakce s dvěma typy haloalkan dehalogenas byla studována pomocí

fluorescentních technik.

6

© Lukáš Maier, Masaryk University, 2014

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Acknowledgement

First of all, I would like to thank Mgr. Kamil Paruch, Ph.D., for his guidance, useful

advices, fruitul discussions and overall support. For the possibility to work on multi-faceted

organic synthesis thus giving me chance to become more scientifically matured. I would like

to also thank my former supervisor Radek Marek for introduction to the world of science and

NMR spectroscopy in particular, which turned out to be essential for my work. I thank other

members of the group, namely Ondřej Hylse, Prashant Khirsariya and Soňa Krajčovičová for

beneficial collaboration on the project. My thanks also belong to the colleagues from the

group and the Department of Chemistry for creation of pleasant working atmosphere and

interesting discussions – Míša Petrůjová, Silvia Kováčová, Peter Šebej and Tomáš Šolomek. I

would like to thank doc. Mgr. Marek Nečas, Ph.D., for X-ray analysis and Mgr. Miroslava

Bittová, Ph.D., for HR-MS analysis. Special thanks belong to my brother for his multifaceted

support.

I would like to thank my parents for general support during my overall education process.

And last but not least I would like to deeply thank my wife and son for their love, endless

mental support, admirable forbearance and keeping me in touch with the real world.

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Table of contents

1. Tools and challenges of chemical biology.........................................................................10

2. Natural nucleosides and their analogs.............................................................................. 11

3. Biologically active nucleoside analogs used as drugs.......................................................13

4. Structural variations of nucleoside analogs and their biological activity......................17

4.1. Mechanism of action of nucleoside analogs.....................................................................19

4.2. Effect of C-2´, C-3´ modifications........................................................................................21

4.3. Conformational behaviour of sugar or pseudosugar part.........................................22

4.4. Effect of C-5´modifications.................................................................................................... 24

4.5. Modification of the base.......................................................................................................... 25

5. Nucleoside analogs with non-THF cores .....................................................................................28

5.1. Azanucleosides.............................................................................................................................28

5.2. Thionucleosides........................................................................................................................... 29

5.3 Carbocyclic nucleosides.............................................................................................................30

5.4. Synthetic approaches toward cyclopentane nucleosides............................................32

5.4.1. Carbocyclic C-N nucleosides – synthesis.....................................................................33

5.4.2. Carbocyclic C-N nucleosides – biology ....................................................................39

5.5.1. Carbocyclic C-C nucleosides – synthesis.....................................................................41

5.5.2. Carbocyclic C-C nucleosides – biology........................................................................43

6. Aims of the thesis............................................................................................................................. .....44

7.1. Synthesis of pseudoisocytidine analogs..............................................................................45

7.1.1. Introduction...........................................................................................................................45

9

7.1.2. Results and discussion........................................................................................................45

7.1.3. Concluding remarks............................................................................................................59

7.1.4. Experimental procedures..................................................................................................60

7.2. Development of general methodology for carbocyclic C-nucleosides

synthesis...........................................................................................................................................83

7.2.1 Introduction............................................................................................................................83

7.2.2 Results and discussion.........................................................................................................85

7.2.3. Experimental procedures................................................................................................107

7.3. Synthesis of naphthalene-based fluorescent probes...................................................173

7.3.1. Introduction – fluorescent probes................................................................................173

7.3.2. Synthesis of naphthalene based fluorescent probes................................................173

7.3.3. Results and discussion.....................................................................................................175

8. Conclusion..................................................................................................................................................178

9. Literature.................................................................................................................................................180

LIST OF ABBREVIATIONS

Appendix A - Curriculum Vitae

Appendix B - Amaro, M.; Brezovský, J.; Kováčová, S.; Maier, L.; Chaloupková, R.;

Sýkora, J.; Paruch, K.; Damborský, J.; Hof, M. J. Phys. Chem. B. 2013, 117,

7898.

10

1. Tools and challenges of chemical biology

Chemical biology is a relatively new scientific discipline that merges mainly chemistry and

biology.1 Typically, it applies chemical tools, methods and analyses to study and manipulate

biological systems. In order to investigate underlying biology or create new function,

chemical biologists often use small organic molecules with defined biological activity as

probes.2 Along this line, organic compounds can be used to modulate protein-protein

interactions, activate programmed cell death, induce of differentiation, pluripotency etc.

Conversely, methods of chemical biology can be also employed to carry out the target

identification for a biologically active organic substance with unknown mode of action.3

Chemical biology is therefore an important part of modern drug discovery.

The spectrum of the tools and methods used in chemical biology is very broad and their

comprehensive description is far beyond the scope of this thesis. Nevertheless, an itemized

list is given below.

Basic methods of chemical biology include:

affinity chromatography techniques; often targeting selected peptide with a biotin

label.4

mass spectrometry-based identification of protein by affinity tags or isotope labeling

(SILAC).5

application of DNA microarrays.6

synthesis of proteins and their modification, (e. g., by bioorthogonal reactions),

synthesis of “stapled” peptides, etc.7

In some methods, organic synthesis represents a central tool; for instance:

enzyme probes, i. e. reagents that can be used to identify the active state of an

enzyme.8

imaging techniques used to visualize function, location and dynamics of selected

protein; typically involving the use of green fluorescent protein (GFP) or small-

molecule fluorescent probes.9

identification of small molecules (typically by high-throughput phenotypic

screening)10

that can be used to manipulate cells,11

e. g., to influence differentiation

of stem cells, induce pluripotency, elicit synthetic lethal phenotype in cancer cells

etc.

11

My research efforts described in this thesis are related to the last two chemical biology areas

of research listed above.

It should be noted that, despite the fact that more than 60 million chemical substances have

been documented to date, identification of a bona fide chemical biology probe is a non-trivial

task.12

Expanding of chemical space, preferably around the subclasses with known (albeit not

completely defined) attractive biological activity is therefore one of the main tasks of organic

synthesis in chemical biology.

2. Natural nucleosides and their analogs

Discovery of structure and function of nucleic acids and their components enabled rapid

development of molecular biology and related field of chemical sciences.13

Due to their

multifaceted function nucleosides, nucleotides and their analogs remain attractive molecules

for scientist across almost all natural sciences.14

Nucleosides and nucleotides are evolutionary

well conserved molecules which are essential for living organisms. They are organized into

supramolecular structures: oligonucleotides, G-quadruplexes, nucleic acids, etc., and some of

them (e.g. ATP, cAMP, AdoMet) can serve individually as molecules responsible for

fundamental processes in cells – energy transport, signal transduction, catalysis of enzymatic

reactions etc.15

General structure of nucleosides and nucleotides is depicted in Fig. 1. They

consist of base (pyrimidine or purine type) and sugar (ribose or 2´-deoxyribose). Nucleotides

bear the mono-, bis- or triphosphate moiety attached to position 5´-.

Figure 1. General structure of nucleosides and nucleotides

12

Some nucleoside analogs have been isolated from bacteria, sponges and marine organisms.

Interestingly, in 1950´s Bergmann and co-workers isolated „spongothymidine 1 and

spongouridine “ from sponge Cryptotethia crypta.16

Structures of both molecules were solved

later, notably without the use of NMR spectroscopy and other modern analytical tools (see

thymidine 2 for comparison). Molecule 1 inspired chemists to modify not only the base

(keeping natural sugar part was considered to be necessary for biological activity at that time)

but also sugar part of nucleoside to get modulation of biological activity.17

In Fig. 3 are given

examples of nucleoside analogs isolated from various natural sources.18

Note that various

types of RNA contain relatively high amount of post-translationally modified nucleoside

analogs, e. g., pseudouridine (compound 3, commonly marked as Ψ), which is the most

abundant C-nucleoside.19

Pseudouridine was found in various types of RNA (significantly in

tRNA), but not in mRNA. Ψ is converted from uridine selectively only in RNA sequences

(not in DNA) by Ψ synthase.20

Its role is still not very clear, but various aspects of

pseudouridine have been reviewed.21

Figure 2. ˶Spongothymidine˝ (1) and thymidine (2)

Showdomycin 422

and tubercidines 523

have been subjects of several biological studies.

Neplanocin A24

(6, isolated from Ampullariella regularies) and aristeromycin25

(7, isolated

from Streptomyces citricolor), which possess antiviral and antitumor activities, represent

frequently studied naturally occuring carbocyclic nucleoside analogs.26

13

Figure 3. Examples of naturally occuring nucleoside analogs

Nucleic acids structures and functions would be hardly deciphered without detailed

understanding of their invidual parts. Correspondingly, modification of nucleosides in order

to probe their function and potential application in medicine has been of high interest for

decades, which is illustrated by the list of clinically used nucleoside analogs below.

3. Biologically active nucleoside analogs used as drugs

Historically oldest are nucleoside analogs with antitumor properties (summarized in Fig.

4). It should be noted that with the exception of capecitabine (which is in fact a prodrug of 5-

fluorouracil) all compounds retain main structural features of the natural nucleosides, i. e. the

tetrahydrofuran part with free primary hydroxyl and base attached via the C-N bond.

14

Figure 4. Anticancer nucleoside analogs

Structurally more diverse are clinically used nucleoside analog antivirals (depicted in Fig.

5). Nevertheless, the presence of free primary hydroxyl at the 5‘-position in those that contain

the THF core or analogous cycles (with the exception of prodrug sofosbuvir) is also typical

for this class.

15

Figure 5. Antiviral nucleoside analogs

16

Fig. 6 summarizes the few nucleoside analogs with different clinical use.

Figure 6. Other clinically used nucleoside analogs

In addition, a number of nucleoside analogs are currently undergoing clinical trials; some of

them are listed in Fig. 7).

17

Figure 7. Examples of investigational drugs

4. Structural variations of nucleoside analogs and their biological activity

Structural modifications of nucleosides can be relatively small, e. g., changes in

stereochemistry with respect to the natural configurations. On the other hand, some molecules

with structures very different from those of typical structure of nucleosides are still

18

considered to be nucleoside analogs (e. g., rather unusual allene 8) and they can still exhibit

biological activity.27

Structural variations of nucleosides and their analogs are shortly described below. General

structure of nucleoside analogs is depicted in Fig. 8.

Figure 8. General structure of nucleoside analogs

One can modify sugar ring, base and/or sugar-base linkage. Typically, X is O, C, NR, or S.

It should be noted that relatively small changes in the structure can result in different

chemical (stability, reactivity), physical (solubility) and biological properties. One of the most

illustrative examples of tight structure-activity relationship is exemplified by cytarabine (9).

Cytarabine differs only in stereochemistry at C-2´position compared to cytidine (10). This

minute altering of structure leads to dramatical turn in biological activity. Cytarabine is

efficiently converted into its triphosphate, which inhibits DNA polymerases, ribonucleotide

reductase (RNR) and RNA polymerases. It results in DNA damage of rapidly dividing cancer

cells. Since 1969 cytarabine (initially in combination with daunorubycin) has been used for

treatment of blood cancers such as acute myeloid leukaemia.28

19

On the other hand, another small modification of the system – methylation of C-2´hydroxyl

leads to non-cytotoxic derivative 11.29

Replacing oxygen by different atoms can dramatically affect metabolic stability, capability

to interact with biomolecules (by H-bond or other noncovalent interactions). Seemingly

negligible changes in bond lengths between C-1´ and C-4´ and electronic properties of certain

atoms can lead to different conformational behavior, which often plays a significant role in

the enzyme substrate recognition/interaction. Exploring of conformational motion of sugar or

pseudo sugar part and its biological relevancy has been also continuously studied over

decades. This effort resulted into establishing of new interesting field - conformationally

locked nucleosides, which will be discussed in greater detail in the section 4.3. Phosphate

groups in nucleotides can be replaced by proper isosteres (section 4.4.), modifications and/or

replacement of the base moiety is often exploited in the structure-activity relationship (SAR)

development.

4.1. Mechanism of action of nucleoside analogs

Chemistry of several classes of nucleoside analogs is relatively well explored and methods

of modern organic synthesis enable preparation of a wide variety of them. However, the

biology and the corresponding potential use of nucleosides in therapy are usually not

straightforward due to several factors: (i) many nucleoside analogs are not effectively

phosphorylated by the cellular kinases; (ii) metabolic stability toward deaminases, hydrolysis

of the nucleoside-base linkage, sugar ring opening are also limiting for many of analogs; and

(iii) the resulting metabolites of mostly unidentified structures can be potentially toxic. These

factors often complicate the testing and interpretation of data.

Mechanisms of action of both purine and pyrimidine nucleoside analogs are similar. After

entering the cells, they are converted into nucleotides by kinases or degraded (e. g.,

deaminated by deaminases), as shown in Fig. 9.30

20

Figure 9. Common metabolic pathways of pyrimidine nucleosides

Nucleoside analogs or their metabolites might interfere with important enzymes. Common

biological targets of antiviral nucleosides31

are reverse transcriptases, RNA and DNA

polymerases. Some cancers are of viral origin (human papillomaviruses family) too.32

Despite

the fact that drug resistance often emerges, some nucleoside analogs (Fig. 5) are used in

combination treatment of various viral and retroviral diseases (hepatitis, HIV etc.). Primary

targets for anticancer nucleosides are DNA polymerases and ribonucleotide reductase. On the

other hand, targeting the RNA and/or RNA polymerases often leads to undesired toxicity.

Nucleoside analogs and their anticancer properties are decribed in the reviews by Plunkett33

,

Gmeiner34

and current development of anticancer and antiviral nucleoside analogs has been

recently reviewed by Dumontet.35

It should be noted that the biological behavior of a particular nucleoside can be highly

specific and, correspondingly, the drug design and SAR development can be highly

unforseeable. The complexity of biological activity can be illustrated by 5-azacytidine and its

2´-deoxyanalog decitabine (Fig. 4). Azacytidine and decitabine are both used in the treatment

of solid tumors and leukemias.36

After getting into the cell, the nucleosides are converted into

the corresponding triphosphates. 5-azacytidine triphosphate is efficiently incorporated into

RNA and disrupts the RNA metabolism, which results in inhibition of protein synthesis. In

addition, 5-azacytidine diphosphate is reduced by RNR to its 2´-deoxy azacytidine

diphosphate, which is phosphorylated into triphosphate and incorporated into DNA. DNA

21

synthesis is then inhibited. Moreover, azacytidine itself inhibits DNA methyltransferases

(crucial enzymes for epigenetic modulation of genes) which results in hypomethylation of

DNA. Hypermethylated DNA has been found in some cancer cell lines (e.g., cells of accute

myelogenous leukemia); inhibition of such methylation processes has been therefore

considered therapeutically viable. In addition, the metabolic pathways described above are

highly dose-dependent. Elucidation of the exact modes of action of these drugs is further

complicated by their very rapid deamination by cytidine deaminase and instability in aqueous

solutions.

4.2. Effect of C-2´, C-3´ modifications

It is well known that 3´-OH is essential for creation of oligonucleotides and nucleic acid

chains. Substituents at 2´- and 3´- positions often interact with important cellular enzymes,

such as DNA polymerases37

or RNR.38

RNR catalyzes reduction of 2´-OH group of

ribonucleotides, which provides the corresponding 2-deoxyribonucleosides. Important

enzyme interactions with ribonucleotides with RNR are schematically shown in Fig. 10.

Figure 10. Interaction of RNR with ribonucleosides33

(hydrogen atoms in amino acid

residues are omitted for clarity)

Modulation (inhibition) of RNR and polymerases function could be achieved by proper

modification of positions C-2´ and C-3´. Frequent modifications include methylation,

oxidation followed by methylenation, addition of alkyl nucleophiles (e.g. Grignard reagents)

or replacement by other groups, e. g., H and N3.39

Replacement of OH by isosteric fluorine or

the difluoro moiety is of great importance due to the extraordinary chemical and metabolic

stability of the C-F bond and similar structural parameters of the C-F and C-OH moieties.40

An important example of such modified nucleoside analogs is gemcitabine. Gemcitabine is

used in treatment of various cancers41

(e. g., bladder, breast or pancreatic). Its mechanism of

action is complex, and one of the modes involves inhibition of RNR by gemcitabine 5´-

22

diphosphate.42

Triphosphorylated gemcitabine is efficiently incorporated into DNA chain,

DNA polymerases can then add only one more nucleotide and the chain is terminated.43

4.3. Conformational behaviour of sugar or pseudosugar part

Due to sugar ring puckering the 5-membered sugar ring can adopt two low energy

conformations: 2´-endo (S-type, predominantly occur in DNA duplexes) or 3´-endo (N-type,

predominantly occur in RNA duplexes) (Fig. 11).44

Figure 11. Important conformers of nucleosides

Several groups of conformationally locked nucleosides (LNA, general structure 12) have

been synthesized in order to probe the role of individual conformers on biological activity and

the stability of oligonucleotides duplexes.45

Incorporation of those molecules into oligonucleotides duplexes usually enhances their

chemical, metabolical and thermal stability and causes adoption of the conformation naturally

seen in RNA duplex sequences. Moreover, LNA can be efficiently (by automated synthesis)

incorporated into oligonucleotide chains which possess enhanced hybridization capability

with complementary strands as well as good water solubility.46

These properties make them

very attractive for cellular catching of mRNA molecules used in antisense therapy (whereas

classical oligoribonucleotides are usually quickly degraded by cellular enzymes).47

Other

derivatives were X = S or NH were also reported.48

Analogs with different lengths of the

bridge (including alkenes) have been also studied.49

Recently, structurally interesting

23

spirocyclic, bicyclic, tetracyclic and other modified conformationally locked nucleosides have

been synthesized and investigated in detail.50

Selected examples are depicted in Fig. 12.

Figure 12. Examples of conformationally restricted nucleosides

Another very interesting subgroup of conformationally locked nucleosides are

bicyclo[3.1.0]hexanes, generally known as methanocarba nucleosides (Fig. 13).51

Figure 13. Methanocarbanucleosides

Those two systems as N- and S-types allow study of two extreme conformations of

carbocyclic nucleosides in the absence of conformational equilibrium. Methanocarba analogs

were used as excelent probes for studying enzyme-nucleoside binding. Very interesting data

have been obtained with AZT (17) analogs 18 and 1952

Compound 18 is extremely potent

against HIV reverse transcriptase (HIV-RT) with IC50 = 1 nM. On the other hand, compound

19 is inactive. The mechanism of action consists of sequential phosphorylation and interaction

of the resulting triphosphate with HIV-RT. Interestingly, S-type analog (19) is efficiently

phosphorylated by cellular kinases, but the phosphorylated form is unable to interact with

HIV-RT. These facts additionally illustrate a non-trivial mode of action of nucleoside analogs

in general.

24

4.4. Effect of C-5´modifications

Modifications of 5´-OH are of great importance. It is worth mentioning that nucleoside

analogs are usually active only as phosphates, biphosphates or triphosphates. Since cellular

kinases are quite very substrate-specific and many nonnatural nucleosides are poor substrates

for them, significant effort has been invested into installation of proper bioisosteres of the

(unstable) triphosphate moiety.53

Among this phosphonates, posphorothioates, isolectronic

boranophosphates or phosphoroamidites have been studied.54

Some of those groups are

frequently used as protecting groups during the synthesis of oligonucleotides and modified

nucleid acids.55

Interestingly, synthetically challenging fluorinated phophinates (Fig. 14) and

their thioanalogs have been synthesized only recently and have been applied in synthesis of

dinucleotides.56

Figure 14. Phosphates and its isosteres

Adefovir dipivoxil discovered by A. Holý and co-workers57

is an illustrative example of

pronucleotide approach used in designing of new drugs.58

Adefovir dipivoxil is used for

treatment of chronic hepatitis type B.59

25

In some cases, even relatively simple motifs can be used to replace the phosphate, e.g., in

recently identified gemcitabine analogs 20 and 21, which are capable of RNR inhibition in

vivo.60

Effects of C-1ánd C-4´ modifications are not well understood yet, but some molecules

with modified C-1ánd C-4´positions were studied as adenosine A1 receptor agonists61

or

nucleoside reverse transcriptase inhibitors (NRTI).62

A recent example of such modified

analogs is 2´,4´-difluorouridine 22. Installation of fluorine at position C-4ésentially locked

the N-type conformation, which is generally seen in RNA strands. This is a rare example of

conformationally restricted analogs without bicyclic sugar moiety.63

4.5. Modification of the base

Replacing of natural bases by modified pyrimidines, purines and other heterocycles or

even by properly substituted aromatic rings is of great importance. The bases play (apart from

genetic function) crucial roles in hydrogen bonding, π-π stacking and hydrophobic

interactions in oligonucleotide sequences.64

Different barriers of rotation around the linkage

between sugar or pseudosugar moiety and modified bases should be also taken into account

with respect to overall structural behaviour of the studied molecules.65

Modification of bases

for potential application in pharmacology is of great interest and nucleosides with modified

base or base isosteres can eventually serve as very important probes for mechanistic studies of

DNA, RNA polymerases function.66

Several approaches have been applied: altering bases in

order to tune H-bonding modes and parameters, stacking ability, hydrophobic interactions,

size and shape effects, fluorescent properties.67

This field has been reviewed recently by Eric

T. Kool.68

Structural variety of such modified bases is demonstrated by examples in Fig. 15.

26

Figure 15. Examples of base analogs

Research in this area resulted in the concept of universal base analogs. Ideally, universal

base should pair with all natural bases equally well and enable DNA or RNA synthesis by

polymerases. Frequently studied universal bases are 3-nitropyrrole (27), 5-nitroindole (28)

and 5-nitrobenzimidazole (29).69

As illustrated in Fig. 16, stability, reactivity and potential tautomerism can complicate the

design of new bases and should be taken into account.

27

Figure 16. Aspects of base analogs design (blue colour refers to hydrogen donor group and

red to hydrogen acceptor group)

Recent report of a semi-synthetic Escherichia coli by Floyd E. Romesberg et al. represents

a culmination of the effort to expand genetic alphabet by non-natural nucleotides.70

Non-classical base to sugar connection via C-C bond leads to an establishment of the

extensively studied field of C-nucleosides (general structure 30), recently reviewed by M.

Hocek and co-workers.71

In general, C-nucleosides should be more both metabolically and chemically stable and

thus could also serve as appropriately robust biological probes. However, installation of the

C-C linkage is in some cases insufficient to ensure good stability of the analogs –

pseudoisocytidine is known to undergo opening of the furan ring in vivo72

and related C-2-

deoxyribonucleoside 31 is unstable.73

28

In addition to showdomycin (4) and pseudouridine (3) mentioned above, formycins (32)74

have been frequently studied as inhibitors of purine nucleoside phophorylase (PNP), which

catalyzes the reversible phosphorolysis of purine nucleosides to generate purine base and

ribose 1-phosphate.75

Therapeutically relevant results in the field of C-nucleosides have been

obtained very recently for the first C-nucleoside HCV polymerase inhibitor, clinical candidate

GS-6620 (33, with EC50 GT1-6 replicons = 68 – 427 nM).76

Unfortunately, the compound has

an unfavourable pharmacokinetic profile and the clinical profiling has been thus

discontinued.77

5. Nucleoside analogs with non-THF cores

Nucleoside analogs possessing non-THF cores form a vast group of structurally diverse

compounds encompassing, e. g., acyclic nucleosides analogs, L-nucleosides or analogs where

the sugar scaffold is modified by more than one heteroatom; these classes will not be

reviewed here for the sake of space. Instead, carbocyclic analogs will be described in greater

detail in the following chapters, along with brief comments on aza- and thionucleosides.

5.1. Azanucleosides

Forodesine.HCl (34, known also as immucillin) and related compounds are representative

examples of azanucleosides where pyrrolidine mimics the sugar moiety. These molecules

have been synthesized as model compounds for studying inhibition of PNP. Interestingly,

those compounds are oxonium transition state mimics78

of adenosine-PNP complex

29

(represented by structure 35) with extraordinary binding parameters (KD for 34 = 56 pM).79

Deficiency of PNP results in accumulation of deoxyguanosine in the blood. T-cells efficiently

convert deoxyguanosine into dGTP and the absence of PNP causes arrest of DNA synthesis

and subsequent apoptosis of the cell. Thus, PNP has been considered an interesting target for

the treatment of T-cell proliferative and autoimmune disorders, e. g., cutaneous T-cell

lymphomas or chronic lymphocytic leukemia (CLL).80

Unfortunately, phase II clinical trials

in patients with advanced-stage CLL (previously unsuccessfully treated with fludarabine)

failed due to poor clinical response.81

Molecules 36 and 37 represent next generation PNP

inhibitors based on azanucleosides with improved binding parameters (for 36 KD = 8 pM).82

Several other modified azanucleosides83

, oxaazanucleosides84

, including azetidines85

were

synthesized and evaluated as PNP inhibitors or antiretroviral agents.

5.2. Thionucleosides

Although two compounds (lamivudine and emtricitabine) particularly in combinations with

other drugs are used for treatment of HIV and/or HBV,86

the thio-, and oxathionucleosides

analogs remain relatively unexplored both synthetically and biologically.87

30

5.3 Carbocyclic nucleosides

Carbocyclic nucleosides represent an interesting group of nucleoside analogs, from

chemical as well as biological perspective. Replacing of oxygen atom by the methylene group

in the sugar moiety improves chemical and metabolical stability while biological activity

could be retained in some carbocyclic compounds. Of approximately 30 of approved

nucleoside analogs used as drugs88

, abacavir, entecavir and ticagrelor are carbocyclic (Fig.

17).

Figure 17. Clinically used carbocyclic nucleosides

Of additional compounds profiled in clinical trials (Fig. 18), compound CPE-C was

recently withdrawn because of adverse toxicity89

(see section 5.4.2.). Another, perhaps better,

analog (RX-3117) is still being profiled.90

31

Figure 18. Examples of investigational drugs with carbocyclic sugar moiety

According to the nature of pseudoglycosidic bond, carbocyclic analogs can be divided into

two distinct groups with: (i) C-C and (ii) C-N linkage (Fig. 19).

Figure 19. Important groups of modified nucleosides

Of the nucleoside classes depicted in Fig. 19, carbocyclic C-analogs represent the system

that is chemically most stable. For “classical” nucleoside analogs, synthetic methodology is

typically based on the facile glycosylation of position 1´ according to well established

methods (Vorbrüggen or Hilbert-Johnson couplings, Scheme 1).91

32

Scheme 1. Silicon induced glycosylation in nucleoside chemistry

Since the bond between the base and the carbocyclic scaffold is no more glycosidic,

synthetic routes used for construction of the molecules have to be different than those used in

clasical nucleoside chemistry. Cyclopropane92

, cyclobutane,93

cyclohexane94

as well as

cyclopentane95-97

analogs have been described in the literature. Since the latter group has been

the subject of my PhD research, general synthetic methodologies related to this class will be

described in greater detail below.

5.4. Synthetic approaches toward cyclopentane nucleosides

Synthetic methodologies including enantioselective95

variants and even biosynthesis96

have

been extensively reviewed in the past.95,97

Fig. 20 illustrates the versatility of norbornene-like

precursors frequently used in the chemistry of carbocyclic nucleosides. Their easy

preparations or commercial availability together with proper geometry, which dictates

subsequent creation of desired stereoisomers, make those intermediates quite popular. Other

methodologies rely on the use of naturally occuring sugars which are available as single

enantiomers.

33

Figure 20. Frequently used starting materials for construction of cyclopentane nucleoside

analogs

5.4.1. Carbocyclic C-N nucleosides - synthesis

Both linear and convergent approaches have been studied. Mitsunobu reaction, which is

stereoselective, very general and tolerates different functional groups, is often the method of

choice for attachment of the base to the carbocyclic core.98

One example is the synthesis

published by Strazewski99

(Scheme 2), which is the most efficient (10 steps from D-ribose)

preparation of (-)-neplanocin A (6) to date.

Scheme 2. Mitsunobu coupling as the key step in synthesis of neplanocin A (6)

Nucleophilic displacement of hydroxyl (via the corresponding mesylates, tosylates and

triflates100

), halide, epoxide ring opening101

(often with lack of regio- and stereoselectivity)

and transition metal-mediated coupling have been also applied.102

Pioneering work on Pd-

catalyzed allylic substitution for synthesis of carbocyclic nucleosides was published by B.

34

Trost (Scheme 3).103

Allylic substitution of epoxide 43 proceeded smoothly and 5´- methylene

installation was also carried out by allylic substitution – on carbonate 45. The nitrosulfone

moiety served as a methylene surrogate in compound 47, which upon ozonolytical oxidation

gave ester 48. Reduction and deprotection yielded racemic aristeromycin 7.

Scheme 3. Trost synthesis of racemic aristeromycin

Surprisingly, when intermediate 46 was dihydroxylated by standard catalytical cis-

dihydroxylation (OsO4/NMO), unexpected diastereomer 49 was produced (Scheme 4).

Perhaps, osmium tetraoxide coordination with nitrosulfone moiety dictates the unexpected

diastereoselectivity.

35

Scheme 4. Unexpected diastereoselectivity of dihydroxylation

Note that two regioisomers could in principle lead to the same π-allyl Pd complex and the

site of attack of nucleophile is determined by steric hindrance. (Scheme 5).

Scheme 5. Regioselectivity aspect of Pd-catalyzed allylic substitution

Another advantage of this reaction is the possibility of using C-nucleophiles (active

methylenes104

) for introduction of the C-C bond linkage between the carbocyclic core and

nucleobase. Enantioselective variants of this methodology were also developed.105

Interestingly, nosylated Vince lactam (50) is activated toward Pd-catalyzed transformation

(Scheme 6), as decribed by Katagiri and co-workers.106

Resulting π-allyl-Pd complex is

attacked by purine nucleophile, which results in diastereoselective formation of key

intermediate (51).

36

Scheme 6. Pd-catalyzed transformation of activated Vince´s lactam

Despite their relative inefficiency, linear approaches for construction of heterocyclic base

were also used in both carbocyclic N- and carbocyclic C-nucleosides. Cyclopentyl amines

were frequently used for construction of pyrimidine or purine bases. One example is elegant

enantioselective synthesis of cyclopentane intermediate 58 (Scheme 7) by J. W. Leahy.107

The

crucial step was enantioselective Diels-Alder reaction with Hawkins catalyst 53. The resulting

intermediate 54 was subjected to diastereoselectvive cis-dihydroxylation followed by

protection and ozonolytical cleavage, which produced unstable aldehyde 55 that was

selectively oxidized into methylester 56. Benzylation of the primary hydroxyl followed by

amide formation afforded amide 57. Subsequent borane reduction and acetylation gave

optically pure 58.

37

Scheme 7. Synthesis of non-racemic cyclopentyl amine intermediate 38

Several other elegant methodologies utilize Vince lactam 59 as the starting material.

Racemic 59 is readily obtained by the Diels-Alder reaction of cyclopentadiene with

tosylcyanide and it can be further enzymatically resolved to get both enantiomers.108

One of

the examples of using the Vince lactam is illustrated in Scheme 8.109

38

Scheme 8. Vince lactam in cyclopentyl amine intermediate synthesis

Cyclopentylamine intermediates can be used for construction of pyrimidine base, typically

by reactions with isocyanates 63 or acrylamides 64 under basic or acidic conditions.

Purines are usually synthesized by reaction of cyclopentylamines and 5-amino-4,6-

dichlopyrimidine via intermediate 65, which is typically condensed with

trimethylorthoformate. Other heterocyclic systems, e.g., imidazoles110

and azapurines were

synthesized as well.

Some key cyclopentane intermediates can be also prepared from sugars.111

D-ribose, D-

ribonolactone and other suitable sugars including their opposite enantiomers are commonly

used. The main advantage of these materials is the commercial availability of both

enantiomers. Efficient conversion of D-ribose 66, into cyclopentenone 69 in 3 steps in overall

yield of 41% is depicted in Scheme 9.112

The opposite enantiomer, prepared from D-xylose

has been used in the synthesis of L-carbocyclic nucleosides.113

A rather unusual

transformation is PCC-induced oxidative cleavage of the C-C bond in intermediate 67.

39

Scheme 9. Example of sugar based pathway in synthesis of cyclopentane intermediates

Very elegant is the conversion of D-ribose into D-(69) and L-cyclopentenone (73).114

Proper order of steps enables selective synthesis of both enantiomers (Scheme 10).

Application of ring-closing metathesis in the synthesis of various cyclopentane scaffolds has

been reviewed recently.115

Scheme 10. Synthesis of D and L-cyclopentenones

5.4.2. Carbocyclic C-N nucleosides – biology

Altough the mode of biological action of carbocyclic C-N nucleosides might be very

similar to the classical nucleosides (e.g. targeting DNA or RNA polymerases, RNR etc.),

some particular carbocyclic C-N analogs exhibit unique biological activity (e. g. ticagrelor,

40

Fig. 17). It is of note that systematic biological studies addressing comparison of common

nucleosides analogs with the corresponding carbocyclic counterparts are missing. Moreover,

the cyclopentane surrogate offers additional possible structural variations (e. g., installation of

extra substituent in position C-6´ and/or installation of double bond between C-1´ and C-6´,

Fig. 21), which are not accesible in clasical sugar-based series. Importance of these

modifications is demonstrated by identification of clinically used entacavir (Fig. 17) or

clinically tested RX-311790

and cyclopentenyl cytosin – CPE-C (Fig. 18).

Figure 21. Possible modifications of carbocyclic moiety

Another notable example of such modified analogs is cytidine analog carbodine.

Carbodine, unlike cytidine (10), shows potent antiviral activity against several strains of

influenza A and B viruses as well as antitumor properties (against lymphoid leukemia in

mice).116

Unsaturated analog, CPE-C, shows excellent antitumor properties even against ara-

C resistant leukemia cell lines, solid human A549 lung cell lines and metastatic LOX

melanoma, and antiviral activity against several virus types.117

Its mechanism of action

involves inhibition of conversion of UTP to CTP. De novo synthesis of pyrimidine

nucleosides is thus disrupted. Since CPE-C was about 100 times more potent in HL-60 cells

than carbodine, the presence of double bond is therefore very important. Unfortunately, the

cardiotoxicity of CPE-C (5 of 26 patients suffered from extreme hypotension) resulted in

discontinuation of the clinical trial.118

Additional example - carbocyclic analog of ribavirin demonstrated importance of

enhanced metabolic stability of carbocyclic C- nucleosides toward phosphorylase.

41

Ribavirin (Fig. 5) exhibits broad spectrum of antiviral properties via complex mechanism

of action (e. g. inhibition of RNA polymerase as triphosphate).119

However, ribavirin

monophosphate (which might be created due to ribavirin rapid phosphorylase degradation in

cells to triazole carboxamide moiety and recycling to its monophosphate trough action of

purine nucleoside phosphorylase), inhibits inosine-5´-monophosphate dehydrogenase

(IMPDH). IMPDH inhibition is probably not essential for antiviral activity of ribavirin,120

but

might be responsible for adverse toxicity, since the enzyme is important for de novo synthesis

of guanine nucleotides.121

Thus few carbocyclic analogs of ribavirin (general structure 75)

which are phosphorylase-stable were synthesized and biologically evaluated.

Interestingly, none of the analogs was efficiently phoshorylated by adenosine kinase while

ribivarin is as efficiently phosphorylated as adenosine itself. Inhibition of IMPDH was almost

negligible in all cases; however, synthetically prepared 5´-monophosphate of 75d inhibited

E.coli IMPDH with IC50 = 0.1 μM (ribavirin monophosphate with IC50 = 0.48 μM). In

addition, 5´-triphosphate of 75d is a more potent inhibitor of viral RNA polymerase

(influenza) in vitro (Ki = 100 μM) then 5´-triphosphate of ribavirin.

5.5.1. Carbocyclic C-C nucleosides - synthesis

Synthesis of carbocyclic C-nucleosides is relatively challenging and, correspondingly,

carbocyclic C-nucleosides are quite rare. The known syntheses (summarized below) are quite

lengthy, linear, and usually produce only one particular target compound.

G. Just et al. prepared intermediate 76 (Scheme 11) and utilized it in their synthesis of

some carbocyclic nucleosides analogs; namely carbocyclic showdomycin (80) and

pyrazomycin (82) (Scheme 12).122

42

Scheme 11. Part of showdomycin and pyrazinomycin analogs synthesis

Scheme 12. Transformation of α-ketoester intermediate into carbocyclic-C-nucleosides

A similar intermediate (differs in stereochemistry at C-2´) has been converted into 6-

azauracil nucleoside 83 by cyclization with thiosemicarbazide. It should be noted that

compound 83 is incompletely characterized (structure supported only by UV spectra and

melting point).123

43

Recent synthesis of carbocyclic-C nucleosides by Rao and co-workers is based on the

nucleophillic substitution of mesylate 84 by ethyl cyanoacetate, which produced 85 in

moderate yield.124

This intermediate was then used for preparation of 9-deazaneplanocin (86)

and its pyrimidine analogs 87 (Scheme. 13).

Scheme 13. Example of synthesis of some carbocyclic-C-nucleosides

5.5.2. Carbocyclic C-C nucleosides - biology

As noted above, these compounds are quite rare; in addition, data on their biological

activity of are reported only sporadically. Additionally, many compounds prepared in the 70s

and 80s of 20th

century are poorly structurally characterized.

Compounds 86 and 87 showed neither any promising anti-HIV activity nor cytotoxicity.

Marginal activity was observed for 86 and 87 against a particular strain of West Nile virus

and Punta toro virus. Carbocyclic analog of tiazofurin 88 was approximately 10 times less

potent against breast carcinoma cell line then tiazofurin itself.125

44

Nevertheless, it should be noted that this class of compound offers the possibility of

additional interactions with the biological systems as it offers are some structural subclasses

that would not be viable in the series of classical nucleosides as well as in C-nucleosides and

carbocyclic C-N nucleosides. Namely, installation of the double bond between C-1´and C-6´

is viable (Fig. 22), but analogous process in the other three series would result in oxonium

species and unstable enamines, respectively. Similarly, hydroxylation at C-1´ produces stable

tertiary alcohol (Fig. 22) while in the other three series it would result in unstable hemiketals

and aminals.

Figure 22. Possible alteration of carbocyclic-C nucleosides

Our experimental realization of the latter strategy is described in section 7.2.

6. Aims of the thesis

One of the tasks of my doctoral research was synthesis of carbocyclic pseudoisocytidine

and its analogs.

Another, and the main, task was the development of a convergent and general synthetic

route for carbocyclic C-nucleosides. Ideally, the methodology was envisioned to enable

preparation of selected subclasses of carbocyclic C-nucleoside analogs with the possibility of

selective manipulation of particular positions around the carbocyclic scaffold.

The third aim was synthesis of small-molecule organic fluorescent probes for enzymatic

studies which were performed in collaboration.

45

7. Results and discussion

This chapter is divided into two sections. First part describes our target-oriented synthesis

of pseudoisocytidine analogs and second part is focused on new flexible synthesis of

carbocyclic C-nucleoside analogs.

7.1. Synthesis of pseudoisocytidine analogs

Results of this project were partially published in: Maier, L.; Hylse, O.; Nečas, M.;

Trbušek, M.; Arne, M. Y.; Dalhus, B.; Bjoras, M.; Paruch, K. New carbocyclic nucleosides:

synthesis of carbocyclic pseudoisocytidine and its analogs. Tetrahedron Lett. 2014, 55, 3713-

3716.

7.1.1. Introduction

The main aim of this project was synthesis and biological evaluation of carbocyclic analog of

pseudoisocytidine (89). Pseudoisocytidine has been shown to be active against cytarabine-

resistant leukemias,126

but hepatotoxic in vivo,127

which may be caused by the opening of the

tetrahydrofuran core (Scheme 14).72

Scheme 14. Opening of pseudoisocytidine 89 and structures of its direct carbocyclic analog

90a and related compounds 90b and 90c.

The direct carbocyclic analog 90a cannot undergo such ring-opening process and its

toxicological profile might therefore be superior to that of pseudoisocytidine, while its

biological activity could be retained.

7.1.2. Results and discussion

The overall retrosynthetic strategy is depicted in Scheme 15. It includes the previously

described substituted norbornene intermediate 76,122

which was envisioned to yield ketoester

46

92 after oxidative cleavage. The desired stereochemistry of 92 is dictated by the geometry of

the bicyclo[2.2.1]heptene scaffold produced in a Diels-Alder reaction between

cyclopentadiene 93 and properly substituted dienophile 94, possessing a leaving group that is

utilized in a subsequent elimination-diastereoselective cis-dihydroxylation sequence.

Compound 92 was envisioned as a precursor to the heretofore unknown aldehyde-ester 91,

which upon reaction with urea, thiourea or guanidine, and global deprotection would provide

the target compounds 90a-c.

Scheme 15. Retrosynthetic analysis of pseudoisocytidine analogs (P = protecting group)

The synthesis started from commercially available methyl propiolate (95), which was

converted into methyl-(Z)-3-bromo-2-propenoate (94a) (Scheme 16).128

47

Scheme 16. Reagents and conditions: a) LiBr, CH3COOH, CH3CN, 90 °C, 80 % for 94a or

PhSO2Na, Bu4NHSO4, H3BO3, H2O:THF 1:1, rt, 58 % of 94b b) EtAlCl2, cyclopentadiene,

CH2Cl2, 0 °C; 80 % for 96a, 70-95% for 96b. b) OsO4, NMO, acetone:H2O 4:1, 40 °C; 80%

from 96a, 90% for 96b. c) Me2CH(OMe)2, cat. TsOH, acetone, rt, 99% for 97a and 97b. d)

DBU, Et2O, 0 °C to rt, for 97a; 95%, DBU, CH3CN, 90 °C for 97b; 86%. e) O3, CH2Cl2, -78

°C then Me2S -78° C to rt.

Since we found that the bromo compound 94a irritates skin (especially upon repeated

exposure), we utilized an alternative starting material for the Diels-Alder reaction, sulfone

94b.129

Thermal Diels-Alder reaction between cyclopentadiene 93 and 94a was very sluggish

- the conversion after 24 h in refluxing benzene or toluene was negligible and significant

dimerization of cyclopentadiene occured. On the other hand, the reaction proceeded smoothly

at low temperature (0 °C) upon catalysis with EtAlCl2 with very good diastereoselectivity (9:1

endo:exo). Sulfone 94b underwent even the uncatalyzed Diels-Alder reaction quite efficiently

(in 60% yield, room temp., 14 h), although higher conversion and yield have been obtained in

the presence of catalyst (EtAlCl2). The structure of the major endo diastereomer 96b was

confirmed by X-ray crystallography (Fig. 23).

48

Figure 23. X-ray crystal structures of 96b (left) and the corresponding cis-diol (S-2) after

dihydroxylation

Diastereoselective cis-dihydroxylation of both adducts 96a and 96b provided the

corresponding diols (see the crystal structure of sulfone intermediate S-2, Fig. 23) which were

subsequently protected as acetonides. Elimination of bromide or phenylsulfonyl group under

basic conditions afforded the key known intermediate 76, which underwent ozonolytic

cleavage to produce rather unstable aldehyde 98 that was immediately used in the next step

without purification.

It should be noted that, in principle, compound 76 could be prepared more directly by

dihydroxylation and protection of the Diels-Alder adduct of methyl propiolate and

cyclopentadiene 99, which we prepared in 60-80% yield.130

Scheme 17. Reagents and conditions: a) AlCl3, cyclopentadiene, PhH, 0 °C, 60-80%. b)

OsO4, NMO, acetone:H2O 4:1, 40 °C.

49

Attempted dihydroxylations of the adduct, however, yielded complex hydroxylation

mixtures that contained the desired product 100 together with the unwanted diastereomer, the

regioisomer with dihydroxylated double bond in the vicinity of the ester group 101 as well as

a tetrahydroxylated product 102.

In accordance with the published results,122

our attempts to selectively reduce aldehyde in

the presence of α-ketoester in 98 were not successful. On the other hand, reduction with

excess of LiAl(t-BuO)3H yielded inseparable mixture of epimeric diols 103 in which the

primary hydroxyl group could be selectively silylated to provide α-hydroxyester 104 (Scheme

18).

Scheme 18. Reagents and conditions: a) LiAl(t-BuO)3H, THF, 0 °C to rt, 50-80% from 76. b)

TBDPSCl, imidazole, CH2Cl2, rt, 70-92%.

Oxidation of the hydroxyl group in 104 proved challenging. Many standard methods (e.g.

MnO2, PDC, KMnO4, Swern oxidation) including RuO2 plus NaIO4 conditions that were

previously used for a structurally similar intermediate,122

failed to give the desired product

105 in acceptable yield. Fortunately, oxidation with Dess-Martin periodinane yielded α-

ketoester 105 in very good yield and purity (Scheme 19).

Scheme 19. Reagents and conditions: a) Dess-Martin periodinane, CH2Cl2, 0 °C to rt, 80-

90%. b) Ph3P+CH2OMeCl

-, LDA, THF, 0 °C to rt, 37-65% (Z:E 7:5).

50

One-carbon homologation of 105 was accomplished via the Wittig reaction with

methoxymethylenetriphenylphosphorane. The reaction produced enol ether 106 in 37-65%

yield as a separable mixture of Z and E isomers (Z:E ~7:5). It should be noted that the Wittig

olefination was rather sensitive to the type and quality of base. Generation of the

phosphonium ylide with LDA gave us the most consistent and reproducible results, while

reactions with LiHMDS, KHMDS, or t-BuOK afforded olefination product in substantially

lower yields. Attempts to selectively hydrolyze enol ether 106 into the desired aldehyde 91 in

the presence of acetonide and TBPDS group met only with a limited success. With PPTS or

acetic acid, partial cleavage of acetonide and/or TBDPS group was observed, while the enol

ether moiety remained intact.

We thus attempted direct transformation of 106 into pyrimidine 107b by reaction with urea

(Scheme 20). With sodium ethoxide in ethanol or NaH in THF, we observed mainly cleavage

of TBDPS group and the desired product 107b was formed in very low yield. However, when

we used t-BuOK in t-BuOH as a base, the desired product was formed in 30-45% yield.

Reactions of 106 with thiourea and guanidine under similar conditions yielded compounds

107b and 107c, respectively. Selective deprotection of TBDPS group in pyrimidines 107a-c

with TBAF revealed primary hydroxyl group, which can be utilized for further selective

derivatization, e. g., preparation of phosphates and its isosteres). Final hydrolysis of acetonide

under acidic conditions provided the target compounds 90a, 90b and 90c in good overall

yields.

51

Scheme 20. Reagents and conditions a) guanidinium.HCl for compound 107a (20-35%) , urea

for 107b (30-45%) and thiourea for 107c (40-50%), t-BuOK, t-BuOH, reflux. b) TBAF, wet

THF, rt, 90% for 108a, 96% for 108b, 90% for 108c. c) HCl:H2O:MeOH 1:1:1, rt, 69% for

90a, 76% for 90b, 71% for 90c.

The relative configurations of 90a-c have been confirmed by 2D NMR experiments (see

SI_1, p. 45). We were able to separate the enantiomers of intermediates 90b and 90c as well

as 107a by HPLC on chiral stationary phase (see SI_1, p. 51).

We tested the effect of compounds 90a, 90b and 90c on the viability of leukemia cell lines

that were available to us: SU-DHL-4 (diffuse large B-cell lymphoma, del/mut TP53), JEKO-1

(mantle cell lymphoma, del/mut TP53), and JVM-3 (mantle cell lymphoma, wt-TP53). The

viability of SU-DHL-4 and JEKO-1 was affected by neither 10 µM nor 100 µM concentration

of the compounds. However, JVM-3 was more sensitive: 90% viability was observed upon

treatment with 100 µM 2c; with 10 µM and 100 µM 90a we observed 90% and 83% viability,

respectively.

52

We then turned our attention on possible modifications of positions 2´-OH in the target

compounds 90a, 90b, and 90c. One of the most attractive modifications of pseudoisocytidine

would be introduction of the difluoro moiety into position C-2´.

We aimed to simultaneously protect 5´-OH and 3´-OH by standard siloxane-based

protecting group (Scheme 21).131

We have chosen analog 90b since is more readily obtained

than pseudoisocytidine analog 90a.

Unfortunately, silylation of 5´- and 3´-OH with 1,3-dichloro-1,1,3,3-

tetraisopropyldisiloxane under a variety of conditions gave only low yields (best one ca. 30%)

of the desired intermediate 109. The starting material was always consumed (as indicated by

TLC), but the reaction mixtures contained several components and the MS analysis indicated

some oligomeric structures. Correspondingly, performing the reaction under high dilution

conditions might afford better yield (not tried).

Scheme 21. Reagents and conditions: a) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane,

pyridine, DMF, rt, 33 %. b) Dess-Martin periodinane, CH2Cl2, 0 °C to rt.

With properly protected intermediate in hand we were able to oxidize 2´-OH to the

corresponding ketone 110 (obtained as crude mixture without further purification).

Unfortunately, treatment of ketone 110 with DAST or Deoxo-fluor

did not provide the

desired fluorinated compound 111 and most of the starting material was recovered.

53

Since pseudoisocytidine intermediate for this further manipulation is relatively difficult to

prepare and its protection was quite inefficient, we did not pursue this synthesis further.

Since we could prepare α-ketoester 105 on multigram scale, we attempted to transform it

into additional carbocyclic C-nucleosides analogs. We proposed two possible modifications

of intermediate 105 (Scheme 22). Pathway a) relies on keto functionalization via Z-selective

Wittig reaction with proper phosphorane. The resulting diester 112 would be cyclized with

hydrazine to give corresponding diazine 113.

Scheme 22. Proposed transformations of α-ketoester intermediate 105

The Wittig reaction of 105 with methyl (triphenylphosphoranylidene)acetate proceeded

with high yield (70-80 %) and exclusive selectivity (Scheme 23). The product stereochemistry

was confirmed by NOESY experiment (see SI_1, p. 56). Attempts to cyclize this intermediate

with hydrazine hydrate under neutral or acidic conditions (with AcOH) failed. In all cases

54

only the starting material was detected by TLC and 1H NMR. Similar cyclization with

analogous diacid, which is somewhat suprisingly a better intermediate for the cyclization then

the corresponding ester, has been described in the literature.132

Scheme 23. Reagents and conditions: a) methyl (triphenylphosphoranylidene)acetate, NaH,

THF, rt, 70-80 %.

Direct double olefination of the -ketoester moiety is synthetically quite interesting

(pathway b), Scheme 22), and if the resulting diene 114 reacted in Diels-Alder reaction, the

sequence could lead to substituted (hetero)arenes 115. We chose the Petasis reagent 116

(freshly prepared from titanocene dichloride and CH3Li)133

for the desired olefination

(Scheme 24). Under a variety of conditions (varying amount of Petasis reagent, different

solvents and temperatures) we only obtained complex mixtures containing decomposed

starting material together with some olefin-containing substances (as indicated by

characteristic signals in 1H NMR).

Scheme 24. Attempted double olefination of 105

The reactions were generaly quite difficult to analyze by TLC since the -ketoester moiety

is unstable on silica gel. Our attempts to purify and isolate some possible products failed due

to their instability. In some cases, we were able to obtain satisfactory 1H NMR spectra, which

55

indicated that both carbonyls were unselectively olefinated. Of note, solutions in CDCl3

rapidly decomposed.

We also tried to perform the olefinations in two separate steps: first Wittig olefination of

the ketone with methyltriphenylphosphonium bromide, then olefination of the ester carbonyl

with Petasis reagent (Scheme 25). Wittig olefination proceed smoothly in good yield (67%).

Compound 117 is relatively stable and can be isolated by column chromatography.

Scheme 25. Reagents and conditions: a) Ph3P+CH3Br

-, NaH, THF, rt, 67%.

However, the subsequent olefination did not work well and the stability of resulting

product was very limited. Instead of pursuing this strategy, we focused on the development of

a more general methodology, which is described in chapter 7.2.

Another possible synthetic route was proposed based on -ketonitrile 118 (Scheme 26).

This intermediate could be eventually used in the synthesis of aminopyrazole 120 and

corresponding fused heterocyclic ring systems.

56

Scheme 26. α-ketonitrile route to different carbocyclic-C analogs (P = protecting group)

We applied the methodology which we have successfully used in the preparation of -

ketoester 105. 2,3-dibromopropanenitrile 121 was converted into steroisomeric mixture of

sulfones 122134

with E/Z ratio aprox 4:1 with excellent overall yield (92%, Scheme 27).

Subsequent (uncatalyzed) Diels-Alder reaction of E-sulfone 122a and cyclopentadiene

produced norbornene 123135

as a separable mixture of endo 123a and exo 123b diastereomers

(ca. 5:1). Z-isomer 122b gave single single endo cycloadduct (not shown).

Scheme 27. Reagents and conditions: a) PhSO2Na, CH3COOH, CH3COONa, H2O:dioxane

1:1, rt, 74 % of 122a and 18 % of 122b b) cyclopentadiene, CH2Cl2, 0 °C; 81 % of 123a, 18%

of 123b.

Cis-dihydroxylation of both cycloadduct followed by acetonide formation and elimination

gave key norbornene intermediate 124. (Scheme 28).

Scheme 28. Reagents and conditions: a) i) OsO4, NMO, acetone:H2O 4:1, 40 °C; 95%, ii)

Me2CH(OMe)2, cat. TsOH, acetone, rt, 95% iii) DBU, CH3CN, 90 °C 79%.

57

Unfortunately, ozonolysis of alkene 124 did not provide the desired α-ketonitrile

intermediate 118 and only decomposition of starting material was observed.

Clearly, the strategy described above can be applied only to construct the (hetero)cyclic

bases by elaboration of the ketoester 105. In order to target compounds that are unaccessible

by the methodology described above, we envisioned a different and perhaps more general

route that utilizes a versatile cyclopentanone intermediate (126, Scheme 29).

Scheme 29. Reagents and conditions: a) LiHMDS, TBSOTf, THF, -78°C. b) O3, CH2Cl2, -

78°C, then Me2S -78 °C to rt, 52 % from 105.

In order to quickly access and evaluate potential of compound 126, we converted one of

the synthetic intermediates, ketoester 105, into the corresponding silyl enol ether 125.

Subsequent unprecedent ozonolysis of this, rather unusual, substrate afforded 126 in

acceptable yield (Scheme 29). The two-step sequence provided sufficient amounts of material

for the preliminary studies. We initially studied introduction of phenyl group using PhMgBr

or PhLi under a variety of conditions (e. g., variable temperature and solvent, presence or

58

absence of CeCl3) and found that the best results were obtained when PhLi was added to a

solution of 126 in THF at 0 °C. Under those conditions, a single diastereomer of addition

product 127, resulting from attack of the reagent from the less sterically hindered side of 126,

was obtained in 75% yield (Scheme 30). We were unable to detect the other diastereomer by

NMR spectroscopy.

Scheme 30. Reagents and conditions: a) PhLi, THF, 0 °C; 75% b) TBAF, wet THF, rt; then

PPTS, MeOH rt, 42% from 126. c) TBAF, wet THF, rt, then TsCl, TEA, DMAP, CH2Cl2, 0

°C; then NaH, BrPhCOCl, THF, 0 °C to rt, 42 % from 127.

The relative stereochemistry of the addition product 127 (supported also by 2D NMR; see

SI_1, p. 73) was unambiguously assigned by X-ray crystal structure of its p-bromobenzoyl

derivative 129 (Figure 24), which was prepared by my colleague Ondřej Hylse.

59

Figure 24. X-ray crystal structure of compound 129

Since the arrangement around the tertiary alcohol carbon of compound 128 mimics that of

the acetal product of glycosylase-mediated cleavage,136

the compound was tested against

glycosylases Neil1, Neil2, NTH1, and hOGG1 and was found to selectively inhibit Neil1 in a

dose-dependent manner: at 1 mM we observed 46% inhibition, at 0.5 mM and 0.125 mM

concentrations 22% and 2% inhibition, respectively.

7.1.3. Concluding remarks

In summary, the first synthesis of 3 new racemic carbocyclic nucleoside analogs of

pseudoisocytidine 90a-c, each in 13 steps was completed. The synthetic approach builds on a

user-friendly preparation of functionalized norbornene 76 developed herein and proceeds

through α-ketoester intermediate 105. While analogs of 105 have been previously used for the

buildup of the heterocyclic ring directly122

or after a two carbon homologation137

, our one-

carbon homologation enables synthesis of additional (hetero)cycles. Furthermore, we have

performed preliminary studies towards a potentially more versatile strategy for the

preparation of carbocyclic nucleoside analogs that utilizes highly diastereoselective additions

of organometallic reagents onto cyclopentanone 126, which itself is available in two-steps

from α-ketoester intermediate 105. The effort has already led to a novel carbocyclic

nucleoside analog (compound 128), which, to our knowledge, is the only carbocyclic C-

nucleoside where R1´

is an oxygenated substituent.

60

Along this line, we have tested the prepared carbocyclic analogs in three leukemia cell lines

and compounds 90a and 90c were found to be moderately active against wt-TP53 - mantle

cell lymphoma cell line JVM-3, which is generally the most sensitive to chemotherapeutic

treatment. The ability of compound 121 to inhibit glycosylase Neil 1 has served as the starting

point for more thorough exploration of the biological activity of this series of novel

carbocyclic nucleoside analogs.

7.1.4. Experimental procedures

General

All reagents and solvents were of reagent grade and were used without further purification.

Anhydrous solvents (THF, dichloromethane, CH3CN, toluene, DMF) were used from

commercial suppliers (Aldrich, Acros) or distilled and stored over 4Ǻ molecular sieves. All

reactions were carried out in oven-dried glasware and under N2 or argon atmosphere. Column

chromatography was carried on silica gel (230-400 mesh) unless otherwise stated. TLC plates

were visualized under UV and/or with phosphomolybdic acid, KMnO4, CAM or

H2SO4/MeOH solution.

NMR spectra were recorded on Bruker Avance 500 Mhz instruments, with operating

frequencies, 500.13 MHz for 1H, 125.77 MHz for

13C, 470.53 MHz for

19F and 160.46 Mhz

for 11

B. Bruker Avance 300 Mhz, with operating frequencies 300.13 MHz for 1H, 75.48 MHz

for 13

C The 1H, and

13C NMR chemical shifts (δ in ppm) were referenced to the residual

signals of solvents: CDCl3 [7.24 (1H) and 77.23 (

13C)], CD2Cl2 [5.32 (

1H) and 53.5 (

13C)],

CD3OD [3.35 (1H) and 49.3 (

13C)], acetone-d6 [2.09 (

1H) and 29.90, 206.7 (

13C)], and

DMSO-d6 [2.50 (1H) and 39.51 (

13C)].

19F NMR chemical shifts (δ in ppm) were referenced

to the signal of trifluorotoluene (-63.72). Structural assignment of resonances have been

performed with the help of 2D NMR gradients experiments (COSY, multiplicity edited 1H-

13C HSQC,

1H-

13C HMBC, NOESY,

1H-

15N HSQC and

1H-

15N HMBC).

The diffraction data were collected with a Rigaku partial χ geometry diffractometer equipped

with a Saturn 944+ HG CCD detector and a Cryostream Cooler (Oxford Cryosystems, UK).

Cu Kα radiation (λ= 1.54184 Å, MicroMax-007HF rotating anode source, multilayer optic

VariMax) was used. Data reduction and final cell refinement were carried out using the

CrysAlisPro software ([CrysAlisPro] CrysAlisPRO, Agilent Technologies UK Ltd).

61

High resolution mass spectra have been measured on Agilent 6224 Accurate-Mass TOF LC-

MS with dual electrospray/chemical ionization mode with mass accuracy greater than 2 ppm,

applied mass range from 25 to 20,000 Da.

IR spectra (4000-400 cm-1

) were collected on an EQUINOX 55/S/NIR FTIR spectrometer and

on Bruker Platinum ATR (4000-400 cm-1

). Solid samples were measured neat or as KBr

pellets and oily samples as a film evaporated from CH2Cl2 solutions.

Melting points were measured on SMP 40 Stuart® apparatus in capillary and are uncorrected.

(Z)-methyl 3-bromoacrylate (94a):128

The compound was prepared according to the literature procedure128

for synthesis of ethyl-

(Z)-3-bromo-2-propenoate. LiBr (1.303 g, 15.0 mmol) was dissolved in anhydrous CH3CN

(11 mL). Methyl propiolate (1.074 mL, 12.0 mmol) and AcOH (0.839 mL, 15 mmol) were

added under N2 and the resulting mixture was refluxed under N2 for 24 h. After cooling down

water (10 mL) was added and the mixture was carefully neutralized by 200 mg of solid

K2CO3. The aqueous phase was subsequently extracted with Et2O (3x 15 mL). Organic

extracts were dried with MgSO4, filtered and the solvents were evaporated under reduced

pressure to yield 94a as a colorless oil (1.58 g, 80%). The crude product was used without

further purification. GC-MS and 1H,

13C NMR analysis indicated the presence of (Z)-isomer

only. NMR data were consistent with the literature.128

GC-MS: m/z = 164 [M+], 166 [M

+],

133 [M+-OMe], 85 [M

+-Br].

1H NMR (300 MHz, CDCl3): δ = 6.97 (d, J = 8.3 Hz, 1H), 6.60

(d, J = 8.3 Hz, 1H), 3.74 (s, 3H) ppm. 13

C NMR (75 MHz, CDCl3): δ = 164.47 (-COOMe),

124.35, 121.63, 51.79 (-OMe) ppm.

(1R*,2R*,3R*,4S*)-methyl 3-bromobicyclo[2.2.1]hept-5-ene-2-carboxylate (96a):

Methyl-(Z)-3-bromo-2-propenoate 94a (1.789 g, 10.84 mmol) was dissolved in anhydrous

CH2Cl2 (15 mL). The resulting mixture was cooled with ice bath to 0 °C and EtAlCl2 (1.8M

solution in toluene, 3 mL, 5.42 mmol, 0.5 equiv) was added dropwise over period of 5

62

minutes. The reaction mixture was stirred for 30 min at 0 °C. Freshly distilled

cyclopentadiene (4.5 mL, 54.2 mmol) was then added in one portion. The resulting mixture

was stirred for an additional 1 hour while maintaining reaction temperature between 0-5°C.

The ratio of endo:exo isomers of the product in an aliquot was determined to be 9:1 by GC-

MS analysis. The reaction mixture was catiously poured into a mixture of 15 mL of 10% HCl,

50 g of ice and 50 mL of Et2O and stirred at 0 °C till a white solid precipitated from the

mixture. The precipitate was removed by filtration and the resulting filtrate was extracted with

Et2O (5x 30 mL). Combined organic extracts were dried over MgSO4, filtered and the solvent

was removed under vacuum to produce yellow viscous oil. The residue was purified by flash

column chromatography (hexane/EtOAc 10:1) to yield pure endo diastereomer as colorless oil

which solidified upon freezing to white solid (1.88 g, 75%); m.p.: 31-32 °C. GC-MS: m/z =

232 [M+], 230 [M

+], 199 [M

+-OMe], 133 [M

+-OMe], 165, 151, 66 [cyclopentadiene].

1H

NMR (300 MHz, CDCl3): δ = 6.53 (dd, J = 5.4, 3.0 Hz, 1H), 6.10 (dd, J = 5.4, 3.0 Hz, 1H),

4.61 (dd, J = 9.2, 3.0 Hz, 1H), 3.66 (s, 3H), 3.20 (dd, J = 9.2, 3.0 Hz, 1H,), 3.20 (br s, 1H),

3.09 (br s, 1H), 1.64 (d, J = 9.2 Hz, 1H), 1.37 (d, J = 9.2 Hz, 1H) ppm. 13

C NMR (126 MHz,

CDCl3): δ = 171.75, 136.94, 134.32, 51.71, 51.32, 50.15, 50.01, 47.70, 40.05 ppm. IR (KBr):

ν˜max = 3452, 2987, 1733, 1430, 1189, 1039, 835, 746 cm–1

. HR-MS (ESI): calcd for

C9H11BrO2 [M+H]+

: 231.0014. Found: 231.0018.

(1R*,2S*,3S*,4R*,5S*,6R*)-methyl 3-bromo-5,6-dihydroxybicyclo[2.2.1]heptane-2-

carboxylate (S-1):

NMO (0.5354 g, 4.57 mmol) and 4% solution of OsO4 in water (0.227 mL, 0.036 mmol)

were added to a solution of 96a (1.056 g, 4.57 mmol) in acetone/water (12 mL, 4:1). The

reaction mixture was stirred at 40 °C, after 14 h solid Na2S2O5 (0.5 g) was added and the

resulting black solution was stirred for additional 30 min at room temperature. Volatiles were

removed under reduced presssure and the black residue was preadsorbed on silica gel and

then purified by flash column chromatography (petrolether/EtOAc 2:1) to yield S-1 as a white

crystalline solid (969 mg, 80%). m.p. = 144-146 °C. 1H NMR (300 MHz, CDCl3): δ = 4.90-

4.87 (m, 1H), 4.49-4.44 (m, 2H), 3.68 (s, 3H), 3.09 (dd, J = 11.2, 3.2 Hz, 1H); 2.87 (d, J =

5.2Hz, -OH, 1H,), 2.63 (d, J = 5.5 Hz, -OH, 1H), 2.51 (d, 1H, J = 3.5 Hz), 2.37 (br s, 1H),

63

2.07 (dm, J = 11.1 Hz, 1H), 1.27 (dm, J = 11.1 Hz, 1H) ppm. 13

C NMR (75 MHz, CDCl3): δ =

170.93, 71.62, 68.53, 51.79, 50.82, 48.39, 48.02, 46.52, 33.13 ppm. IR (KBr): ν˜max = 3382,

3263, 2981, 1738, 1430, 1367, 1170, 1047, 860 cm–1

. HR-MS (APCI): calcd for C9H13BrO4

[M-H2O+H]+ = 246.9964. Found: 246.9964.

(3aR*,4R*,5S*,6S*,7R*,7aS*)-methyl 6-bromo-2,2-dimethylhexahydro-4,7-

methanobenzo[d][1,3]dioxole-5-carboxylate (97a):

Diol S-1(1.1302 g; 4.26 mmol) was dissolved in acetone (12 mL), 2,2-dimethoxypropane

(1.776 g, 4 eq., 17.05 mmol) and 3 mg of TsOH were added and the resulting mixture was

stirred at 25C. till the TLC indicated disappearance of the starting material (30-60 min). The

remaining 2,2-dimethoxypropane and acetone were evaporated under reduced pressure yield

crude acetonide 97a, whose purity (analyzed by GC-MS, 1H NMR and

13C NMR) was

satisfactory and it was therefore used without further purification directly in the next step.

GC-MS: m/z = 289 [M+ -Me], 275 [M

+-OMe], 229 [M

+-OOCMe2], 135, 79.

1H NMR (300

MHz, CDCl3): δ = 4.01-3.94 (m, 3H); 3.69 (s, 3H); 2.61-2.54 (m, 3H), 2.08-2.04 (dm, 1H);

1.89-1.85 (dm, 1H); 1.43 (s, 3H), 1.25 (s, 3H) ppm. 13

C NMR (75 MHz, CDCl3): δ = 171.02,

110.72, 80.79, 79.98, 51.96, 51.11, 48.80, 47.25, 43.54, 29.44, 25.56, 24.36 ppm.

(Z)-methyl 3-(phenylsulfonyl)acrylate (94b):

Compound 94b was prepared by a slightly modified literature procedure.129

Sodium

benzensulfinate (3.671g, 22.36 mmol) and Bu4NHSO4 (1.138 g 3.354 mmol) were added to a

solution of methylpropiolate (1.88 g, 22.36 mmol) in H2O/THF 1:1 (80 mL). H3BO3 (2.074

g, 33.54 mmol) was added and the reaction mixture was vigorously stirred at room temp. for

48 h. After that pH was adjusted by 1M HCl to pH = 4 and the mixture was extracted with

CH2Cl2 (4x 50 mL). The organic phase was washed with brine (1x 30 mL), dried with

MgSO4, filtered and the solvents were removed under reduced pressure. The resulting yellow

oil was purified by flash column chromatography (petrolether/EtOAc 3:2) to afford both

64

isomers as white crystals (101 mg, 2% of E isomer and 2.93 g, 58% of Z isomer 94b). E

isomer: m.p. = 99-100 °C. 1H NMR (300 MHz, CDCl3): δ = 7.91-7.89 (m, 2H), 7.69-7.54 (m,

3H), 7.32 (d, J = 15.2 Hz, 1H), 6.82 (d, J = 15.2 Hz, 1H), 3.78 (s, 3H) ppm. 13

C NMR (75

MHz, CDCl3): δ = 164.11, 143.71, 138.75, 134.60, 130.73, 129.86, 128.56, 52.98 ppm. Z

isomer: m.p. = 64-65 °C. 1H NMR (300 MHz, CDCl3): δ = 7.99-7.97 (m, 2H), 7.67-7.53 (m,

3H), 6.54 (d, J = 11.5 Hz, 1H), 6.48 (d, 1H, J = 11.5 Hz), 3.88 (s, 3H) ppm. 13

C NMR (75

MHz, CDCl3): δ = 164.53, 139.40, 135.60, 134.22, 131.73, 129.46, 128.24, 52.83 ppm. IR

(KBr): ν˜max = 3456, 3037, 2954, 1734, 1633, 1340, 1311, 1238, 1153, 763, 731 cm–1

.

(1R*,2R*,3R*,4S*)-methyl 3-(phenylsulfonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylate

(96b):

EtAlCl2 (15.92 mmol, 8.85 ml of 1.8M solution in toluene) was added dropwise over the

period of 5 min to a cooled (ice bath, 0 °C) solution of compound 94b (7.206 g, 31.85 mmol)

in anhydrous CH2Cl2 (30 mL). The reaction mixture was stirred for 30 min, then freshly

distilled cyclopentadiene (1.32 mL, 159.25 mmol) was added. The mixture was stirred for

additional 1 h at 0 °C, then it was poured onto 10% HCl (80 mL) with ice (200 g). The

mixture was filtered to remove white polymeric byproducts, to the filtrate was added brine

(200 mL) and it was extracted with CH2Cl2 (4x 100 ml). The organic extracts were dried with

MgSO4, filtered and concentrated in a vacuum to afford a yellow oil. The product was

precipitated by addition of Et2O (20 mL); the resulting white crystalline solid was filtered and

washed with Et2O (3x 15 mL). The product (pure endo diastereomer by NMR) was dried in a

vacuum and used directly without further purification. Analytically pure material was isolated

by flash column chromatography (petrolether/EtOAc 3:1) as white crystals (7.72 g, 83 %).

m.p. = 150-151 °C. 1H NMR (300 MHz, CDCl3): δ = 7.93-7.84 (m, 2H), 7.67-7.51 (m, 3H),

6.59 (dd, J = 5.3, 3.0 Hz. 1H), 6.26 (dd, J = 5.3, 3.0 Hz, 1H), 4.12 (dd, J = 10.0, 3.1Hz, 1H),

3.42 (dd, J = 10.0, 3.1 Hz, 1H), 3.21 (br s, 1H), 3.02 (br s, 1H), 1.47 (dm, J = 8.9 Hz, 1 H),

1.25 (dm, J = 8.9 Hz, 1 H) ppm. 13

C NMR (75 MHz, CDCl3): δ = 170.87, 141.40, 137.70,

133.40, 132.13, 129.07, 127.86, 69.17, 51.87, 49.16, 48.12, 47.07, 46.64 ppm. HR-MS

(APCI): calcd for C15H16O4S [M+H]+: 293.0842. Found: 293.0841.

65

Crystal data for 96b: CCDC ref. No. 929386. Crystallized from CH2Cl2, C15H16O4S, Mrel =

292.35, T = 120 K, space group P-1, a = 8.2134(4) Å, b = 8.3868(4) Å, c = 10.7465(6) Å, α =

108.251(5), β = 94.702(4), γ = 99.040(4), V = 687.466 Å3, R = 0.031.

(1R*,2S*,3S*,4R*,5S*,6R*)-methyl 5,6-dihydroxy-3-

(phenylsulfonyl)bicyclo[2.2.1]heptane-2-carboxylate (S-2):

NMO (1.8 g, 15.4 mmol) and OsO4 (700 μL, 4% wt. solution in H2O, 0.12 mmol) were added

to a stirred solution of alkene 96b (4.511 g, 15.4 mmol) in acetone/H2O (45 mL, 4:1). The

yellow solution was stirred for 14 h at 40 °C. Solid Na2S2O5 (0.5 g) was then added and the

resulting black mixture was stirred for 30 min. Volatiles were removed under reduced

pressure and the black residue was preadsorbed on silica gel. Flash column chromatography

(EtOAc) yielded S-2 as a white crystalline solid (4.52 g, 90 %). m.p.: 144-145 °C. 1H NMR

(500 MHz, CDCl3): δ = 7.93-7.85 (m, 2H), 7.66-7.51 (m, 3H), 4.96-4.89 (m, 1H), 4.78-4.70

(m,1H), 3.76 (dd, J =11.9, 4.1Hz, 1H), 3.18 (d, J = 4.9Hz, 1H), 3.11 (dd, J = 11.9, 4.1Hz,

1H), 3.06 (d, J = 4.9 Hz, 1H), 2.56 (br s, 1H), 2.43 (br s, 1H), 2.09 (d, J = 10.9 Hz, 1H), 1.14

(d, J = 10.9 Hz, 1H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 170.13, 141.25, 133.85, 129.39,

128.00, 77.48, 77.23, 76.98, 69.32, 68.39, 64.65, 52.31, 48.00, 47.90, 44.73, 33.24 ppm. IR

(KBr): ν˜max = 3456, 1747, 1637, 1385, 1144, 752, 721 cm–1


C15H18O6S [M+H]+: 327.0897. Found: 327.0892.

Crystal data for compound S-2: CCDC ref. No. 929387. Crystallized from CH2Cl2,

C15H18O6S, Mrel = 326.36, T = 120 K, space group P-1, a = 10.4471(4) Å, b = 11.4450(6) Å, c

= 13.4617(6) Å, α = 65.794(4), β = 87.155(3), γ = 82.891(4), V = 1456.77 Å3, R = 0.030.

(3aR*,4R*,5S*,6S*,7R*,7aS*)-methyl 2,2-dimethyl-6-(phenylsulfonyl)hexahydro-4,7-

methanobenzo[d][1,3]dioxole-5-carboxylate (97b):

66

2,2-dimethoxypropane (3.1 mL, 24.5 mmol) and TsOH (2 mg) were added to a solution of

diol S-2 (2.00 g, 6.13 mmol) in acetone (24 mL). The reaction mixture was stirred at 25C till

the starting material was not detected by TLC (petrolether/EtOAc 1:1); within 30 min. The

reaction mixture was evaporated to dryness (orange oil) and the crude product was used

without further purification directly in the next step. Pure product could be obtained by flash

column chromatography (petrolether/EtOAc 1:1) as colorless crystals (2.22 g, 99%). m.p.:

149-150 °C. 1H NMR (500 MHz, CDCl3): δ = 7.90 (m, 2H), 7.63 (m, 1H), 7.55 (m, 1H), 5.18

(d, J = 5.4 Hz, 1H), 4.97 (d, J = 5.4 Hz, 1H), 3.75 (dd, J = 11.7, 4.1 Hz, 1H), 3.71 s (3H),

3.17 (dd, J = 11.7, 4.1 Hz, 1H), 2.69 (m, 1H), 2.53 (m, 1H), 1.99 (m, 1H), 1.41 (s, 3H), 1.33

(s, 3H), 1.04 (m, 1H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 169.69, 141.35, 133.83, 129.41,

128.05, 108.88, 77.29, 76.06, 64.27, 52.35, 45.11, 45.07, 44.01, 32.86, 25.46, 24.52 ppm. IR

(KBr): ν˜max = 3456, 1747, 1637, 1385, 1144, 752, 721 cm–1


C18H22O6S [M+H]+

: 367.1210. Found: 367.1214.

(3aR*,4R*,7S*,7aS*)-methyl 2,2-dimethyl-3a,4,7,7a-tetrahydro-4,7-

methanobenzo[d][1,3]dioxole-5-carboxylate (76):

Method a) DBU (1.6 mL, 10.65 mmol) was added to cooled (0 °C, ice bath) solution of

acetonide 97a (1.299 g; 4.26 mmol) in anhydrous Et2O (10 mL). The reaction mixture was

allowed to warm to 25C and then it was stirred for 16 h. The resulting suspension was

filtered to remove the solid residue. The glass filter was rinsed by Et2O (3x 15 mL). The

filtrates were collected, the solvents were evaporated under reduced pressure and the resulting

yellow viscous liquid was purified by flash column chromatography (hexane/EtOAc 3:1) to

yield a white solid (908 mg, 95%, 2 steps from 96a).

Method b) DBU (2.8 mL, 18.4 mmol) was added to a stirred solution of crude acetonide 97b

(1.37 g, 6.13 mmol) in anhydrous MeCN (10 mL). The reaction mixture was then stirred at

90 °C for 1 h (monitored by TLC with hexane/EtOAc 3:1). The solvent was then evaporated

and the orange residue was purified by flash column chromatography (hexane/EtOAc 3:1) to

afford a colorless oil which upon freezing crystallized to white crystals (1.18 g, 86%, 2 steps

from 96b). m.p.: 61-62 °C. 1H NMR (300 MHz, CDCl3): δ = 6.91 (d, J = 3.1 Hz, 1H), 4.27

(d, J = 5.3 Hz, 1H), 4.21 (d, J = 5.3 Hz, 1H), 3.70 (s, 3H), 3.14 (br s, 1H), 2.93 (br s, 1H),

67

2.05 (m, 1H), 1.79 (m, 1H), 1.46 (s, 3H), 1.31 (s, 3H) ppm. 13


164.65, 147.55, 142.37, 114.40, 80.23, 80.15, 51.76, 47.40, 45.81, 42.99, 26.19, 24.59 ppm.

IR (KBr): ν˜max = 3452, 3000, 2931, 1712, 1643, 1597, 1267, 1062, 856, 756 cm–1

. HR-MS

(ESI): calcd for C12H16O4 [M+H]+

: 225.1121. Found: 225.1124.

(R*)-methyl 2-hydroxy-2-((3aR*,4R*,6S*,6aS*)-6-(hydroxymethyl)-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)acetate (103):

O3/O2 mixture (5mL/min oxygen flow, ozonolysis rate ~ 12 mmol/5min) was bubbled through

a cooled solution (-78 °C) of compound 76 (2.7447 g, 12.24 mmol) in CH2Cl2 (35 mL) till the

TLC (hexane/EtOAc 3:1) indicated disappearance of the starting material and blue colour of

the reaction mixture persisted. After that N2 was bubbled through the reaction mixture to

remove residual ozone and oxygen. Me2S (4.5 mL, 61.2 mmol) was added in one portion and

the reaction mixture was stirred for 4 h while allowed to warm to room temperature. Brine (20

mL) was then added and the mixture was extracted with CH2Cl2 (1x 30 mL). Organic phase

was washed with brine (2x 10 mL), dried with MgSO4, filtered and the solvent was

evaporated. The resulting colorless oil was immediately used in the next step without

additional purification. The purity of crude aldehyde 98 was satisfactory by 1H NMR (the

main impurity is DMSO as a product of Me2S oxidation, see Supporting information).

Attempts to purify the compound by standard flash column chromatography failed due to the

compound‘s instability.

Li(AlO-tBu)3H (3.98g, 15.64 mmol) was added portionwise to a cooled (ice bath, 0 °C)

solution of crude aldehyde 98 (1.742 g, 6.8 mmol) in anhydrous THF (15 mL). The reaction

mixture was then stirred overnight while allowed to warm to room temp. It was then poured

into 5% aqueous solution of NaHSO4 (50 mL) with crushed ice (50 g). The resulting white

liquid was then diluted with brine (30 mL) and extracted with EtOAc (4x 50 mL). The

organic phase was dried with MgSO4, filtered and concentrated in a vacuum to give a yellow

oil. Flash column chromatography (CH2Cl2/MeOH 15:1) yielded 103 as a colorless oil (620

mg, 65 % from compound 98) as a mixture of both epimers with the ratio of 5:2 based on 1H

68

NMR. 1H NMR (500 MHz, CDCl3): δ = 4.62 (dd, 1H major epimer), 4.43 dd, 0.4 H minor

epimer), 4.39 (d), 4.34 (m), 4.15 (d), 3.78 (s), 3.77 (s), 3.65 (m), 3.01 (br s), 2.46 (m), 2.23

(m), 2.02 (m), 1.76 (m), 1.48 (s), 1.44 (s), 1.29 (s), 1.26 (s) ppm. 13

C NMR (126 MHz,

CDCl3): δ = 174.98, 112.91, 83.53, 83.49, 82.24, 80.95, 70.97, 70.19, 64.85, 64.73, 53.61,

52.92, 52.80, 49.04, 48.50, 47.48, 30.66, 27.89, 27.86, 27.46, 25.56, 25.46 ppm. HR-MS

(ESI): calcd for C12H20O6 [M+H]+: 261.1333. Found 261.1330, calcd for C12H20O6 [M1+Na]

+:

283.1152. Found: 283.1156.

methyl 2-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-2-hydroxyacetate (104):

TBDPSCl (691 μL, 2.79 mmol) and imidazole (453 mg, 6.65 mmol) were added to a solution

of starting material 103 (692 mg, 2.66 mmol) in anhydrous CH2Cl2 (8 mL). The reaction

mixture was then stirred 14 at 25C. The solvent was evaporated and the viscous residue was

purified by flash column chromatography (hexane/EtOAc 3:1) to yield a colorless oil 104

(1.07 g, 81%) as a mixture of epimers with the ratio of 5:2 based on 1H NMR.

1H NMR (500

MHz, CDCl3): δ = 7.66-7.60 (m), 7.43-7.32 (m), 4.57-4.51 (m), 4.43-4.30 (m), 4.18-4.14 (m),

3.76 (s), 3.73-3.64 (m), 2.84 d (0.4 H, minor epimer), 2.74 (d, 1H, major epimer), 2.45-2.35

(m), 2.29-2.19 (m), 2.01-1.94 (m), 1.71-1.64 (m), 1.61-1.53 (m), 1.47 ((-C(CH3)2), major

epimer), 1.43 ((-C(CH3)2), minor epimer), 1.27 ((-C(CH3)2), major epimer), 1.24 ((-C(CH3)2),

minor epimer), 1.04 ppm. 13

C NMR (126 MHz, CDCl3): δ = 175.23, 175.15, 135.87, 133.92,

133.89, 129.86, 127.88, 112.67, 82.59, 82.48, 81.86, 80.76, 70.83, 70.71, 70.11, 69.99, 65.02,

64.94, 52.87, 52.71, 49.36, 48.87, 47.10, 46.62, 30.70, 28.00, 27.97, 27.67, 27.12, 25.66,

25.54, 19.55 ppm. IR (KBr): ν˜max = 3452, 2933, 2858, 1740, 1639, 1429, 1211, 1112, 704

cm–1

. HR-MS (ESI): calcd C28H38O6Si [M+Na]+: 521.2330. Found: 521.2334.

methyl 2-((3aR*,4S*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-2-oxoacetate (105):

69

Dess-Martin periodinane (581 mg, 1.37 mmol) was added to a cooled (0 °C, ice bath) solution

of starting material 104 (487 mg, 0.977 mmol) in anhydrous CH2Cl2 (8 mL). The reaction

mixture was then allowed to warm to 25C and then stirred for 14 h. The solvent was

evaporated, the residue was suspended in cold Et2O (50 mL) and the solid was removed by

filtration. The filtrate was washed with saturated aqueous NaHCO3 solution (2x 10 mL), dried

with MgSO4, filtered and concentrated in a vacuum to provide a colorless oil 105 (412 mg,

85%) which solidified upon freezing. Attempts to purify the compound by column

chromatography failed due to partial epimerization and decomposition on silica gel. m.p.: 76-

78 °C. 1H NMR (500 MHz, CDCl3): δ = 7.65-7.56 (m, 4H), 7.43-7.31 (m, 6H), 4.82-4.76 (m,

1H), 4.41 (dd, J = 6.0, 3.6Hz, 1H), 3.85 (s, 3H), 3.69-3.53 (m, 3H), 2.41-2.29 (m, 2H), 1.82-

1.75 (m, 1H), 1.47 (s, 3H), 1.26 (s, 3H), 1.02 (s, 9H) ppm. 13


194.60, 161.73, 135.81, 133.68, 129.94, 127.94, 112.66, 82.70, 81.74, 64.18, 54.18, 53.24,

47.69, 30.98, 27.68, 27.09, 25.27, 19.50 ppm. IR (KBr): ν˜max = 3448, 3415, 2956, 2933,

1738, 1714, 1259, 1114, 1032, 708 cm–1

. HR-MS (ESI): calcd for C28H36O6Si [M+Na]+

:

519.2173. Found: 519.2176.

methyl 2-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-3-methoxyacrylate (106):

LDA (2.63 mL of 2M solution in THF, 5.26 mmol) was added dropwise over the period of 10

min to a cooled (0 °C, ice bath) suspension of (methoxymethyl)triphenylphosphonium

chloride (1.929g, 5.63 mmol) in anhydrous THF (15 mL). The resulting orange mixture was

stirred at 0 °C for 30 min. and then a solution of ketone 105 (932 mg, 1.88 mmol) in

anhydrous THF (20 mL) was added. The yellow mixture was stirred for additional 3 h while

allowed to warm to 25C., then it was quenched with saturated aqueous solution of NH4Cl (15

70

mL). The aqueous phase was extracted with EtOAc (3x 20 mL), the combined organic parts

were dried with MgSO4, filtered and volatiles were evaporated. The dark brown residue was

purified by flash column chromatography (CH2Cl2/EtOAc 20:1) to afford two isomeric enol

ethers 106 in the overall yield of 503 mg (51%) with ratio of Z:E ~7:5. Z isomer (293 mg,

30%, yellow oil): 1H NMR (500 MHz, CDCl3): δ = 7.63 (m, 4H), 7.36 (m, 6H), 6.51 (s, 1H),

4.59 (m, 1H), 4.41 (m, 1H), 3.79 (s, 3H), 3.77 (m, 1H), 3.70 (s, 3H), 3.68 (m, 1H), 2.61 (m,

1H), 2.20 (m, 1H), 1.97 (m, 1H), 1.83 (dd, J = 12.24, 11.98 Hz, 1H), 1.47 (s, 3H), 1.27 (s,

3H), 1.04 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 166.60, 157.05, 135.85, 135.84,

134.01, 129.80, 127.83, 112.60, 108.56, 84.56, 82.37, 65.02, 62.21, 51.27, 47.78, 47.34,

34.85, 28.14, 27.11, 25.61, 19.56 ppm. HR-MS (ESI): calcd for C30H40O6Si [M+H]+

:

525.2667. Found: 525.2671.

E isomer (210 mg, 21%, yellow oil): 1H NMR (500 MHz, CDCl3): δ = 7.64 (m, 4H), 7.36 (m,

6H), 7.33 (s, 1H), 4.74 (m, 1H), 4.44 (m, 1H), 3.79 (s, 3H), 3.79 (m, 1H), 3.69 (s, 1H), 3.66

(s, 3H), 3.27 (m, 1H), 2.24 (m, 1H), 1.94 (m, 2H), 1.48 (s, 3H), 1.26 (s, 3H), 1.04 (s, 9H)

ppm.13

C NMR (126 MHz, CDCl3): δ = 168.30, 160.08, 135.87, 134.13, 134.11, 129.76,

127.82, 112.47, 110.71, 84.37, 82.52, 65.13, 61.76, 51.30, 48.20, 40.88, 33.65, 28.23, 27.11,

25.78, 19.59 ppm. IR both isomers (KBr): ν˜max = 3450, 2935, 2858, 1693, 1639, 1429,

1378, 1211, 1113, 704, 505 cm–1


: 547.2486.

Found: 547.2485.

General procedure for synthesis of compounds 107a-107c

Urea (or thiourea or guanidinium.HCl) (3 equiv.) and t-BuOK (6 equiv. for 107a and 4 equiv.

for 107b and 107c ) was added to anhydrous t-BuOH (0.05 M) solution of starting material

106 (mixture of Z+E enol ethers). The resulting solution was refluxed for 14 h. The reaction

mixture was cooled to 25C., diluted with H2O (15 mL) and pH was adjusted to 6-7 with 1M

HCl. The resulting solution was extracted with EtOAc (4x 20 mL). Combined organic

extracts were dried with Na2SO4, filtered and concentrated in a vacuum. The resulting brown

oil was purified by flash column chromatography (CH2Cl2/MeOH 15:1 for 107a and 20:1 for

107b and 107c).

2-amino-5-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-4(1H)-one (107a):

71

Colorless amorphous solid (166 mg, 43%). 1H NMR (500 MHz, DMSO-d6): δ = 10.83 (br s,

1H), 7.61 (m, 4H), 7.44 (m, 6H), 6.34 (br s, 2H), 4.64 (m, 1H), 4.38 (m, 1H), 3.68 (m, 2H),

2.83 (m, 1H), 2.18 (m, 1H), 1.95 (m, 1H), 1.80 (m, 1H), 1.39 (s, 3H), 1.18 (s, 3H), 1.00 (s,

9H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ = 161.80*, 155.08, 134.99, 133.15, 129.73,

127.79, 114.27*, 111.38, 83.20, 81.83, 64.89, 47.26, 44.07, 33.58, 27.72, 26.61, 25.21, 18.84

ppm.* - these resonances were indirectly detected by 1H-

13C HMBC experiment. HR-MS

(ESI): calcd for C29H37N3O4Si [M-H]- : 518.2481. Found: 518.2481.

5-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-

dimethyltetrahydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione

(107b):

Slightly yellow amorphous solid (85 mg, 38%). 1H NMR (500 MHz, CDCl3): δ = 9.23 (br s,

1H), 8.82 (s, 1H), 7.63 (m, 4H), 7.37 (m, 6H), 7.08 (d, J = 6.49 Hz, 1H), 4.63 (m, 1H), 4.46

(dd, J = 6.8, 5.2 Hz, 1H), 3.73 (ddd, J = 16.4, 10.2, 5.7 Hz, 2H), 2.87 (m, 1H), 2.31 (m, 1H),

2.13 (dd, J = 12.9, 6.8 Hz, 1H), 1.89 (dd, J = 24.3, 12.9 Hz, 1H), 1.48 (s, 3H), 1.26 (s, 3H),

1.05 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 163.50, 152.09, 136.83, 135.86, 133.93,

129.89, 127.90, 114.64, 113.09, 83.33, 82.37, 65.01, 47.45, 45.49, 33.68, 28.00, 27.14, 25.48,

19.57 ppm. HR-MS (ESI): calcd for C29H36N2O5Si [M-H]- : 519.2321. Found: 519.2303.

5-((3aR*,4R*,6S*,6aS*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-2-thioxo-2,3-dihydropyrimidin-

4(1H)-one (107c):

72

Yellow ammorphous solid (198 mg , 45%). 1H NMR (300 MHz, CDCl3): δ = 10.66 (br s, 1H),

10.21 (s, 1H), 7.63 (m, 4H), 7.38 (m, 6H), 7.07 (d, J = 5.2 Hz, 1H), 4.70 (dd, J = 6.60, 13.61

Hz, 1H), 4.48 (dd, J = 6.60, 11.77 Hz, 1H), 3.74 (m, 2H), 2.90 (m, 1H), 2.34 (m, 1H), 2.10

(m, 1H), 1.90 (m, 1H), 1.51 (s, 3H), 1.29 (s, 3H), 1.05 (s, 9H) ppm. 13

C NMR (75 MHz,

CDCl3): δ = 174.93, 160.80, 137.14, 135.84, 133.86, 129.92, 127.92, 119.13, 113.38, 83.01,

82.43, 64.97, 47.33, 45.72, 33.41, 28.02, 27.14, 25.54, 19.56 ppm. HR-MS (ESI): calcd for

C29H36N2O4SSi [M+Na]+

: 559.2057. Found: 559.2021.

General procedure for synthesis of compounds 108a-c

TBAF (1.1 equiv., 1 M solution in THF) was added to a THF (0.1 M) solution of starting

material 107a-c in wet THF was added) and the reaction mixture was stirred at room temp for

14h. THF was evaporated and the brown oily residue was purified on a short silica gel column

(CH2Cl2/MeOH 2:1 for 108a and 10:1 for 108b and 108c).

2-amino-5-((3aR*,4R*,6S*,6aS*)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-4(1H)-one (108a):

White crystalline solid (80 mg, 90%). m.p.: > 250 °C, decomp. 1H NMR (500 MHz, DMSO-

d6): δ = 10.80 (s, 1H), 7.43 (s, 1H), 6.33 (s, 2H), 4.63 (m, 1H), 4.54 (m, 1H), 4.32 (m, 1H),

3.40 (m, 2H), 2.79 (m, 1H), 2.04 (m, 1H), 1.89 (m, 1H), 1.65 (m, 1H), 1.40 (s, 3H), 1.19 (s,

3H) ppm.13

C NMR (126 MHz, DMSO-d6): δ = 162.04*, 155.03, 153.10*, 114.52*, 111.28,

83.10, 82.21, 62.57, 47.41, 44.31, 33.84, 27.72, 25.19 ppm. * - resonances were indirectly

detected through 1H-

13C HSQC or

1H-

13C HMBC experiments. IR (KBr): ν˜max = 3434,

3097, 2983, 2736, 1670, 1614, 1562, 1207, 1051, 867 cm–1


C13H19N3O4 [M+H]+

: 282.1448. Found: 282.1447.

73

5-((3aR*,4R*,6S*,6aS*)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (108b):

White crystalline solid (89 mg, 96%). m.p.: 246-247 °C, decomp. 1H NMR (500 MHz,

DMSO-d6): δ = 10.99 (s, 1H), 10.70 (br s, 1H), 7.30 (s, 1H), 4.58 (m, 2H), 4.33 (dd, J = 5.3,

12 Hz, 1H), 3.40 (m, 2H), 2.83 (m, 1H), 2.04 (m, 1H), 1.94 (m, 1H), 1.56 (m, 1H), 1.40 (s,

3H), 1.20 (s, 3H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ = 164.00, 151.06, 137.93, 111.99,

111.43, 82.94, 82.05, 62.40, 47.24, 43.43, 33.83, 27.66, 25.18 ppm. IR (KBr): ν˜max = 3490,

3228, 2933, 1720, 1666, 1384, 1377, 1065, 1040, 860, 791 cm–1


C13H18N2O5 [M-H]- : calcd for C13H18N2O5 [M-H]

- : 283.1288. Found: 283.1287.

5-((3aR*,4R*,6S*,6aS*)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (108c):

White crystalline solid (99 mg, 90%). m.p. 199-201 °C. 1H NMR (500 MHz, DMSO-d6): δ =

12.41 (s, 1H), 12.24 (br s, 1H), 7.35 (s, 1H), 4.59 (m, 1H), 4.58 (m, 1H), 4.34 (dd, J = 5.20,

12 Hz, 1H), 3.40 (m, 2H), 2.88 (m, 1H), 2.05 (m, 1H), 1.96 (m, 1H), 1.56 (dd, 1H, J = 12,

23.7 Hz), 1.40 (s, 3H), 1.20 (s, 3H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ = 174.59,

160.97, 137.86, 117.78, 111.53, 82.78, 82.06, 62.31, 47.18, 43.36, 33.44, 27.63, 25.19 ppm.

IR (KBr): ν˜max = 3450, 3263, 2927, 1658, 1541, 1456, 1383, 1371, 1213, 1155, 866 cm–1

.

HR-MS (ESI): calcd for C13H18N2O4S [M-H]- :297.0915. Found: 297.0904.

General procedure for synthesis of compounds 90a-c

35% HCl (1 mL) and H2O (1 mL) were added to MeOH (0.2 M) solution of starting material

108a-c.The reaction mixture was stirred at room temp for 30 min. The solvents were

74

evaporated in a vacuum and the resulting brown solid was purified by flash column

chromatography (CH2Cl2/7 N NH3 in MeOH 5:1 for compound 90a and CH2Cl2/MeOH 5:1 to

1:1 for compounds 90b and 90c.

2-amino-5-((1R*,2R*,3S*,4S*)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)pyrimidin-

4(1H)-one (90a):

White crystalline solid (46 mg, 69 %). m.p.: > 250 °C, decomp. 1H NMR (500 MHz, DMSO-

d6): δ = 10.94 (br s, 1H), 7.37 (s, 1H), 6.33 (br s 2H), 4.73 (br s –OH), 4.45 (m, 1H), 4.09 (br

s – OH), 3.81 (m, 1H), 3.67 (m, 1H), 3.36 (m, 2H), 2.76 (m, 1H), 1.91 (m, 1H), 1.84 (m, 1H),

1.25 (m, 1H).13

C NMR (126 MHz, DMSO-d6): δ = 164.10*, 155.40, 151.76*, 115.31, 75.14,

73.33, 63.39, 46.66, 42.42, 29.12.*- these resonances were indirectly detected through 1H-

13C

HSQC or 1H-

13C HMBC experiments. IR (KBr): ν˜max = 3421, 2921, 1652, 1606, 1486,

1118, 1043, 777 cm–1

. HR-MS (ESI): calcd for C10H15N3O4[M-H]-

: 240.0990. Found:

240.0969.

5-((1R*,2R*,3S*,4S*)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)pyrimidine-

2,4(1H,3H)-dione (90b):

White solid (60 mg, 76 %). m.p. > 300 °C, decomp. 1H NMR (500 MHz, DMSO-d6): δ =

10.94 (br s, 1H), 10.62 (br s, 1H), 7.15 (s, 1H), 4.46 (m, 1H), 4.37 (d, J = 6.40 Hz, 1H), 4.22

(d, J = 4.19 Hz, 1H), 3.79 (m, 1H), 3.68 (m, 1H), 3.34 (m, 2H), 2.76 (m, 1H), 1.91 (m, 2H),

1.11 (m, 1H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ = 164.52, 151.07, 137.32, 113.18,

74.41, 73.03, 63.13, 46.36, 41.35, 29.50. IR (KBr): ν˜max = 3448, 2926, 1709, 1659, 1446,

1225, 1113, 766, cm–1

. HR-MS (ESI): calcd for C10H14N2O5 [M-H]-: 241.0830. Found:

241.0833.

75

5-((1R*,2R*,3S*,4S*)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-2-thioxo-2,3-

dihydropyrimidin-4(1H)-one (90c):

White solid (60 mg, 71 %). m.p: 172-175 °C, decomp.1H NMR (500 MHz, DMSO-d6): δ =

12.34 (br s, 1H), 12.18 (br s, 1H), 7.20 (s, 1H), 4.49 (m, 1H), 4.43 (d, J = 6.44 Hz, 1H), 4.25

(d, J = 4.47 Hz, 1H), 3.81 (m, 1H), 3.69 (m, 1H), 3.34 (m, 2H), 2.81 (m, 1H), 1.93 (m, 2H),

1.12 (m, 1H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ = 174.30, 161.40, 137.33, 119.02,

74.24, 73.03, 63.09, 46.37, 41.40, 29.12 ppm. IR (KBr): ν˜max = 3437, 3162, 3070, 2931,

1654, 1587, 1463, 1232, 1033, 766 cm–1

. HR-MS (ESI): calcd for C10H14N2O4S [M-H]-

:

257.0602. Found: 257.0608.

5-((6aR,8S,9S,9aR)-9-hydroxy-2,2,4,4-

tetraisopropylhexahydrocyclopenta[f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidine-

2,4(1H,3H)-dione (109):

Pyridine (0.09 mL, 1.11 mmol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.035 mL,

0.111 mmol) were added to a solution of 90b (27 mg, 0.111 mmol) in anhydrous DMF

(0.135 mL). The reaction mixture was stirred for 14 h at room temperature. DMF and pyridine

were evaporated and the white solid residue was purified by flash column chromatography

(SiO2, CH2Cl2/MeOH 20:1) to afford product 109b as a colorless oil. 1H NMR (500 MHz,

CDCl3): δ = 9.84 (d, J = 4.0 Hz, 1H, -NH), 9.49 (s, 1H, -NH), 7.16 (d, J =5.5 Hz, 1H), 4.29

(m, 1H), 4.04 (dd, J =11.8, 6.1 Hz, 1H), 3.92 (dd, J =11.8, 3.6 Hz, 1H), 3.68 (dd, J = 11.8,

3.6 Hz, 1H), 3.05 (d, J = 5.4 Hz, 1H), 2.76 (m, 1H), 2.23 (m, J = 7.3, 11.83 Hz, 1H), 1.87 (m,

J =14.3, 7.3 Hz, 1H), 1.36 (dd, J = 24.1, 12.0 Hz, 1H), 1.08-1.00 (m, 30H) ppm.13

C APT

NMR (126 MHz, CDCl3): δ = 164.05, 152.60, 137.32, 114.79, 74.77, 73.88, 64.07, 48.17,

76

44.49, 28.45, 17.81, 17.69, 17.67, 17.61, 17.51, 17.41, 17.37, 17.32, 13.71, 13.69, 13.24,

12.93.ppm.

dimethyl 2-((3aS*,4S*,6R*,6aR9)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)maleate (112):

Methyl(triphenylphosphoranylidene)acetate (678 mg, 2.03 mmol, 1.2 eq.) was added to a

solution of -ketoester 105 (841 mg, 1.69 mmol) in anhydrous THF (10 ml). The reaction

mixture was stirred for 14 h at room temperature under N2 atmosphere. The solvent was

evaporated and the residue was directly purified by flash column chromatography (SiO2,

hexane/EtOAc 5:1) to afford product 112 (771 mg, 82 %, colorless oil). 1H NMR (500 MHz,

CDCl3): δ = 7.63-7.60 (m, 4H), 7.41-7.34 (m, 6H), 5.88 (d, J =1.5 Hz, 1H), 4.42 (m, 2H),

3.80 (s, 3H), 3.71 (s, 3H), 3.69 (m, 2H), 2.88 (dtd, J =7.9, 6.7, 1.4 Hz, 1H), 2.27 (m, 1H), 2.02

(m, 1H), 1.67 (app d, J =11.4 Hz), 1.47 (s, 3H), 1.26 (s, 3H), 1.04 (s, 9H) ppm. 13

C NMR (126

MHz, CDCl3): 168.70, 165.38, 151.27, 135.85, 133.74, 133.70, 129.96, 127.95, 119.10,

113.30, 83.64, 82.14, 64.50, 52.52, 52.06, 50.33, 46.47, 32.24, 27.99, 27.13, 25.52, 19.55

ppm. IR: ν˜max = 2952 (w), 1726 (s, carbonyl), 1457 (w), 1197 (m), 1168 (m), 739 (s), 504

(m) cm–1

. HR-MS (ESI): calcd for C31H40O7Si [M+Na]+: 575.2445. Found: 575.2451.

(E)-3-(phenylsulfonyl)acrylonitrile134

(122a):

2,3-dibromopropanenitrile (1.07 g, 5 mmol) was added to a solution of sodium

benzensulfinate (820 mg, 5 mmol) in H2O:dioxane (15+15 mL), followed by CH3COOH (286

L, 5 mmol) and CH3COONa (411 mg, 5 mmol). The reaction mixture was stirred for 21 h at

room temperature, then concentrated to half volume, and poured into sat. solution of NaHCO3

(15 mL) and extracted with CH2Cl2 (3 x 20 mL). Combined organic extracts were dried over

77

MgSO4, filtered, and the residual brown oil was purified by flash column chromatography

(SiO2, hexane/EtOAc 3:2) to afford E isomer (714 mg, 74%, white solid) and Z isomer (173

mg 18%, white solid). E isomer: 1

H NMR (300 MHz, CDCl3): δ = 7.94-7.91 (m, 2H), 7.78-

7.73 (m, 1H), 7.67-7.62 (m, 1H), 7.28 (d, J = 15.7 Hz, 1H), 6.57 (d, J = 15.7 Hz, 1H) ppm.

13C NMR (75 MHz, CDCl3): δ = 149.18, 137.41, 135.16, 130.06, 128.62, 113.50, 110.84

ppm. Z isomer: 1

H NMR (300 MHz, CDCl3): 8.00 (m, 1H), 7.75-7.70 (m, 1H), 7.63-7.58 (m,

1H), 7.08 (d, J = 11.3 Hz, 1H), 6.04 (d, J = 11.3 Hz) ppm. 13

C NMR (75 MHz, CDCl3) δ

148.42, 138.27, 135.29, 130.07, 128.77, 108.85 ppm. Spectral data for both isomers were

identical with those reported in the literature.135

endo-(phenylsulfonyl)bicyclo[2.2.1]hept-5-ene-2-carbonitrile (123a):

Freshly distilled cyclopentadiene (2.369 mL, 29.02 mmol) was added to a cooled (ice bath, 0

°C) solution of compound 122a (1.21 g, 5.8 mmol) in anhydrous CH2Cl2 (20 mL). The

reaction mixture was stirred for 3 h at 0 °C, then the volatiles were evaporated and the oily

residue was purified by flash column chromatography (SiO2, petrolether/EtOAc 3:2) to afford

exo 123b isomer (eluted first, 223 mg, 18 %, white solid) and endo 123a isomer (eluted

second, 1.21 g, 81%, white solid). Analytical data for end isomer: 1H NMR (500 MHz,

CDCl3): δ = 7.89-7.87 (m, 2H), 7.71-7.68 (m, 1H), 7.62-6.59 (m, 1H), 6.33 (ddd, J = 22.2,

5.6, 3.0 Hz, 2H), 3.81 (dd, J = 5.3, 3.2 Hz, 1H), 3.40 (m, 1H), 3.34 (m, 1H), 2.70 (dd, J =

22.2, 5.3, 2.0 Hz, 1H), 1.73 (ddd, J = 9.6, 3.7, 1.8 Hz, 1 H), 1.66 (dm, J = 9.6 Hz, 1 H) ppm.

13C NMR (126 MHz, CDCl3): δ = 139.58, 135.63, 134.62, 134.57, 129.99, 128.17, 120.20,

70.00, 49.37, 48.89, 45.67, 32.18. ppm. Spectral data were identical with those reported in the

literature.135

Acetonide (S-3):

78

NMO (82 mg, 0.7 mmol) and OsO4 (32 μL, 4% wt. solution in H2O, 0.0056 were added to a

stirred solution of alkene 123a (182 mg, 0.7 mmol) in acetone/H2O (10 mL, 4:1). The yellow

solution was stirred at 40 °C for 14 h. Solid Na2S2O5 (0.1 g) was then added and the resulting

black mixture was stirred for 30 min. Volatiles were removed under reduced pressure and the

black residue was pre-adsorbed on silica gel. Flash column chromatography (SiO2,

EtOAc/petrolether 10:1) yielded the diol as a white crystalline solid (194 mg, 95 %).

2,2-dimethoxypropane (456 μL, 3.68 mmol) and TsOH (2 mg) were added to a solution of the

previosuly prepared diol (270 mg, 0.92 mmol) in acetone (8 mL) were added. The reaction

mixture was stirred at room temp. for 1 h. The reaction mixture was evaporated to dryness

and the resulting yellow oil was purified by flash column chromatography (SiO2,

petrolether/EtOAc 3:2) to yield compound S-3 as a white crystalline solid (305 mg, 99 %). 1H

NMR (300 MHz, CDCl3): δ = 8.01 (m, 2H), 7.68-7.56 (m, 3H), 5.25 (d, J = 5.1 Hz, 1H), 4.75

(d, J = 5.1 Hz, 1H), 3.57 (dd, J =11.1, 3.7 Hz, 1H), 3.07 (dd, J = 11.1, 4.4 Hz, 1H), 2.73 (m,

overlapped, 2H), 2.03 (d, J = 11.3 Hz, 1H), 1.07 (d, J = 11.3 Hz, 1H), 2.43 (br s, 1H), 2.09 (d,

J = 10.9 Hz, 1H), 1.42 (s, 3H), 1.35 (s, 3H), 1.14 (d, J = 10.9 Hz, 1H) ppm. 13

C NMR (75

MHz, CDCl3): δ = 139.04, 134.64, 129.84, 128.89, 116.42, 109.71, 77.98, 76.04, 63.55,

45.44, 43.90, 32.67, 27.79, 25.40, 24.41 ppm.

Nitrile intermediate (124):

DBU (516 μL, 3.46 mmol) was added to a solution of the acetonide S-3 (384 mg, 1.15 mmol)

in 2 ml of anhydrous CH3CN and the reaction mixture was stirred at 90 °C for 2 h. All

volatiles were removed in a vacuum and the dark brown residue was purified by flash column

chromatography (SiO2, petrolether/EtOAc 2:1) to afford product 124 (173 mg, 79%, white

crystals). m.p: 74-78 °C. 1

H NMR (300 MHz, CDCl3): δ = 6.91 (d, J = 3.0 Hz, 1H), 4.33 (d, J

= 5.2 Hz, 1H), 4.23 (d, J = 5.2 Hz, 1H), 3.04 (brs,1H), 2.99 (br s, 1H), 2.12 (d, J = 9.7Hz,

1H), 1.87 (d, J = 9.7 Hz, 1H), 1.47 (s, 3H), 1.33 (s, 3H) ppm. 13

C NMR (75 MHz, CDCl3): δ

= 153.50, 121.05, 115.45, 114.93, 79.50, 79.32, 48.63, 47.22, 43.51, 26.06, 24.57 ppm. IR

(KBr): ν˜max = 2992, 2947, 2926, 2216 -(CN), 1454, 1373, 1267, 1200, 1056, 992, 581 cm–1

.

79

(3aR*,6R*,6aR*)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyldihydro-3aH-

cyclopenta[d][1,3]dioxol-4(5H)-one (126):

LiHMDS (280 μL of 1 M solution in THF, 0.28 mmol) followed by TBDMSOTf (0.28 mmol,

64 μL) were added dropwise to a cooled (-78 °C) solution of starting material 105 (140 mg,

0.28 mmol) in THF (2 mL) and the resulting mixture was stirred for additional 2 h at -78 °C.

All volatiles were evaporated and the crude mixture contains isomers of silyl enol ether 125

was immediately dissolved in anhydrous CH2Cl2 (6 mL) and cooled to -78 °C. O3/O2 mixture

(5 mL/min oxygen flow) was bubbled through the solution until TLC (hexane/EtOAc 10:1)

indicated disappearance of starting material and blue colour of the reaction mixture persisted.

Then N2 was bubbled through the reaction mixture to remove residual ozone and oxygen.

Me2S (120 μL, 1.4 mmol) was added in one portion and the reaction mixture was stirred for 4

h while allowed to warm to room temperature. Brine (10 mL) was then added and the mixture

was extracted with CH2Cl2 (2x 15 mL). The organic phase was washed with brine (2x 10

mL), dried with MgSO4, filtered, and the solvents were evaporated. The resulting yellow oil

was purified by flash column chromatography (hexane/EtOAc 5:1) to yield 126 as a white

crystalline solid (62 mg, 52 %). m.p: 96-97 °C . 1H NMR (500 MHz, CDCl3): δ = 7.60-7.57

(m, 4H), 7.45-7.36 (m, 6H), 4.61 (d, J = 5.4 Hz, 1H), 4.33 (d, J = 5.4 Hz, 1H), 3.78 (dd, J =

10.2, 2.8 Hz, 1H), 3.59 (dd, J = 10.2, 2.8 Hz, 1H), 2.73 (dd, J = 18.4, 9.0 Hz, 1H), 2.47 (m,

1H), 2.17 (m, 1H), 1.41 (s, 3H), 1.32 (s, 3H), 1.00 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3):

δ = 213.41, 135.94, 135.82, 132.97, 132.61, 130.24, 130.19, 128.09, 111.48, 81.79, 79.32,

77.48, 77.23, 76.98, 66.13, 39.31, 37.49, 27.05, 27.02, 24.89, 19.35. IR (KBr): ν˜max = 3486,

2933, 1755, 1589, 1429, 1108, 705.85 cm–1


:

447.1962. Found: 447.1962.

80

(3aR*,4R*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-4-

phenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (127):

PhLi (138 μL of 1.8 M solution in Bu2O, 0.25 mmol) was added dropwise to a cooled (0 °C,

ice bath) solution of ketone 126 (70 mg, 0.17 mmol) in THF (4 mL) and the resulting mixture

was stirred for 1 h at 0 °C. Then it was quenched with saturated aqueous solution of NH4Cl

(15 mL). The aqueous phase was extracted with EtOAc (3x 15 mL), the combined organic

parts were dried with MgSO4, filtered and volatiles were evaporated. The dark brown residue

was purified by flash column chromatography (hexane/EtOAc 10:1) to yield 127 as a

colorless oil (125 mg, 75%). 1H NMR (500 MHz, CDCl3): δ = 7.69-7.62 (m, 4H), 7.47-7.33

(m, 10H), 7.28-7.23 (m, 1H), 4.70-4.63 (m, 2H), 3.87 (dd, J = 10.3, 5.0 Hz, 1H), 3.79 (dd, J =

10.3, 4.4 Hz, 1H), 3.32 (d, J = 1.5 Hz, 1H), 2.73-2.66 (m, 1H), 2.31 (dd, J = 13.6, 6.8 Hz,

1H), 2.18 (dd, J = 13.6, 1.5 Hz, 1H), 1.59 (s, 3H), 1.35 (s, 3H), 1.08 (s, 9H) ppm. 13

C NMR

(126 MHz, CDCl3): δ = 145.25, 135.86, 135.84, 133.85, 133.81, 129.92, 128.53, 127.93,

127.92, 127.26, 125.32, 114.74, 87.11, 82.23, 78.57, 64.46, 46.14, 42.73, 27.16, 26.61, 24.89,

19.60 ppm.

(3aR*,4R*,6R*,6aR*)-6-(hydroxymethyl)-2,2-dimethyl-4-phenyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-ol (S-4):

TBAF (154 μL of 1 M solution in THF, 154 mmol) was added to a solution of compound 127

(72 mg, 0.14 mmol) in wet THF (3 mL). The reaction mixture was stirred at 25 °C for 14 h.

The solvent was evaporated and the brown oily residue was purified on a short silica gel

column (CH2Cl2/MeOH 20:1) to yield S-4 as a white crystalline solid (33 mg, 87%). 1H NMR

(500 MHz, CDCl3): δ = 7.480-7.42 (m, 2H), 7.36-7.31 (m, 2H), 7.26-7.22 (m, 1H), 4.70-4.63

(m, 1H), 3.83 (dd, J = 10.7, 5.0 Hz, 1H), 3.67 (dd, J = 10.7, 6.7 Hz, 1H), 3.38 (d, J = 1.5 Hz,

81

1H), 2.73-2.65 (m, 1H), 2.32 (dd, J = 13.5, 6.6 Hz, 1H), 1.97 (dd, J = 13.5, 1.5 Hz, 1H), 1.60

(s, 3H), 1.37 (s, 3H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 144.87, 128.52, 127.36, 125.32,

115.08, 86.83, 83.11, 78.29, 64.06, 46.33, 42.28, 26.57, 24.93 ppm.

(1R*,2R*,3R*,4R*)-4-(hydroxymethyl)-1-phenylcyclopentane-1,2,3-triol (128):

PPTS (156 mg, 0.620 mmol) was added to a solution (MeOH, 4 mL) of acetonide S-4 (33 mg,

0.124 mmol). The reaction mixture was stirred at 25 °C for 24 h. The solvent was evaporated

and the residue was purified by flash column chromatography (CH2Cl2/MeOH 20:1 to 2:1) to

yield 128 as a white crystalline solid (14 mg, 48 %). m.p: 141.3-143°C. 1H NMR (500 MHz,

DMSO-d6): δ = 7.49-7.44 (m, 2H), 7.33-7.27 (m, 2H), 7.22-7.17 (m, 1H), 4.66 (s, 1H), 4.58

(d, J = 6.09 Hz, 1H), 4.55 (m, 1H), 4.44 (d, J = 7.68 Hz, 1H), 3.82 (dd, J = 14.14, 6.83 Hz,

1H), 3.79 (m, 1H), 2.81 (m, 1H), 2.22 (m, 1H), 1.95 (dd, J = 13.5, 8.4 Hz, 1H), 1.66 (dd, J =

13.5, 10.07 Hz, 1H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ = 146.27, 127.55, 126.03,

125.33, 80.73, 77.89, 72.91, 62.70, 46.85 ppm. IR (KBr): ν˜max = 3465, 3272, 2960, 2933,

1645, 1417, 1130, 1022, 700 cm–1

. HR-MS (ESI): calcd for C12H6O4 [M+H]+

: 224.0976.

Found: 224.0970.

((3aR*,4R*,6R*,6aR*)-6-hydroxy-2,2-dimethyl-6-phenyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)methyl 4-methylbenzenesulfonate (S-5):

Et3N (154 μL, 1.1), DMAP (3 mg, 0.028 mmol) and TsCl (58 mg, 0.304 mmol) were added to

a cooled (0 °C, ice bath) solution of compound 127 (73 mg, 0.276 mmol) in CH2Cl2 (5 mL).

The mixture was stirred at 25 °C for 3 h. The solvent was evaporated and the residue was

purified by flash column chromatography (CH2Cl2/EtOAc 20:1) to yield S-5 as a colorless

solid (107 mg, 93 %). m.p: 141.3-143°C. 1H NMR (500 MHz, CDCl3): δ = 7.81-7.76 (m, 2H),

7.44-7.27 (m, 6H), 7.26-7.21 (m, 1H), 4.64 (d, J = 7.90 Hz, 1H), 4.53 (m, 1H), 4.22 (dd, J =

82

10.10, 4.74 Hz 1H), 4.10 (dd, J = 10.10, 4.84 Hz, 1H), 3.28 (brs, 1H), 2.79-2.72 (m, 1H), 2.43

(s, 3H), 2.24 (dd, J = 13.6, 6.6 Hz, 1H), 1.97 (m, J = 13.6 Hz, 1H), 1.55 (s, 3H), 1.31 (s, 3H)

ppm. 13

C NMR (126 MHz, CDCl3): δ = 145.19, 144.32, 130.16, 128.61, 128.20, 125.29,

115.39, 86.43, 81.60, 78.08, 70.71, 43.46, 42.08, 26.51, 24.92, 21.85 ppm.

(3aR*,4R*,6R*,6aR*)-2,2-dimethyl-4-phenyl-6-(tosyloxymethyl)tetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl 4-bromobenzoate (129)

Solution of tosylate S5 (47 mg, 0.113 mmol) in anhydrous THF (1 mL) was added dropwise

to a cooled (0 °C, ice bath) stirred suspension of NaH (60%, 5 mg, 0.135 mmol) in anhydrous

THF (2 mL). The resulting mixture was vigorously stirred at 25 °C for 2 h. Then, 4-

bromobenzoyl chloride (27 mg, 0.124 mmol) was added in one portion and the mixture was

stirred at 25 °C for additional 24 h. The reaction was quenched with saturated aqueous NH4Cl

(0.2 mL) and the solvent was evaporated. The residue was dissolved in CH2Cl2 (1 mL) and

purified using preparative TLC (silica gel, CH2Cl2) to give benzoate 129 as a white solid (35

mg, 51 % yield). 1H NMR (500 MHz, CDCl3): δ = 7.85 (m, 2H), 7.73 (m, 2H), 7.52 (m, 2H),

7.40 (m, 2H), 7.34-7.23 (m, 5H), 4.82 (d, J =7.00 Hz, 1H), 4.51 (dd, J = 7.1, 4.4 Hz, 1H),

3.96 (m, 2H), 3.04 (dd, J = 13.9, 7.1 Hz 1H), 2.56 (m, 1H), 2.43 (s, 3H), 2.19 (dd, J = 13.9,

10.2 Hz, 1H), 1.56 (s, 3H), 1.33 (s, 3H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 164.63,

145.24, 131.85, 131.42, 130.29, 130.16, 128.73, 128.36, 128.19, 125.96, 114.30, 86.83, 85.57,

81.11, 70.34, 42.33, 37.35, 26.67, 25.09, 21.85 ppm. Crystal data for 129: CCDC ref. No.

937690. Crystallized from toluene, C29H29BrO7S, Mrel = 601.51, T = 120 K, space group P-1,

a = 8.0723(4) Å, b = 12.4224(5) Å, c = 13.9348(6) Å, α = 92.267(4), β = 96.617(4), γ =

106.814(4), V = 1324.79 Å3, R = 0.026.

WST-1 assay of compounds 90a, 90b, and 90c

The following B-lymphoid cell lines were used: SU-DHL-4 (diffuse large B-cell lymphoma,

del/mut TP53), JEKO-1 (mantle cell lymphoma, del/mut TP53), and JVM-3 (mantle cell

lymphoma, wt-TP53). Cells were seeded in 96-well plates in duplicates (1 × 10(5) cells per

well, volume 200 µl) and subjected to 72 h exposure of the studied compounds at 10 µM and

83

100 µM concentrations (the concentrations used were obtained by dilution of 50 mM stock

solutions of the compounds in DMSO). DMSO was added to mock control. The cell viability

was assessed by the metabolic WST-1 assay (Roche) using spectrophotometer 1420

Multilabel Counter Victor (PerkinElmer).

Supporting Informations_1: Copies of 1H and

13C NMR spectra are available on attached

CD (SI_1). Assignment of 1H,

13C and

15N NMR resonances of important intermediates and

final compounds 90a-c based on 2D NMR experiments is also included as well as the IR

spectra of selected compounds.

7.2. Development of general methodology for carbocyclic C-nucleosides synthesis

Results of this project are currently being incorporated into a manuscript which will be

submitted to Journal of Organic Chemistry.

7.2.1 Introduction

As we noted above, the known syntheses of carbocyclic C-nucleosides often address

preparation of a single target compound. While more convergent, modular approach (i. e.,

attachment of base onto a properly functionalized cyclopentane scaffold) has been used in the

series of natural nucleosides,91

C-nucleosides71

and carbocyclic C-N nucleosides,95,97,99

we

have been unable to find reports of similar approach in the class of carbocyclic C-nucleosides.

Clearly, a flexible synthesis could enable sufficiently quick mapping of the chemical space

within this series (e. g., SAR development of glycosylase inhibitors based on the lead

compound 128 described above) and preparation of direct analogs of the nucleosides with

known biological activity.

One good candidate is carbocyclic C-nucleoside 132. This compound is the direct analog

of C-nucleoside 130, which is closely related to cytotoxic tubercidine23

(7-deazaadenosine).

Interestingly, replacement of C-N linkage by C-C linkage in compound 130 lead to almost no

detrimental effect on observed cytoxicity (ID50 values for HeLa cells = 0.64 nM for

tubercidine, 0.84 nM for 130).139

On the other hand, carbocyclic 7-deazadenosine 131 is a moderate inhibitor of S-adenosyl-

L-homocysteinase (in leukemia L-1210 cell lines)140

while tubercidine did not show any

84

activity. Both compounds tubercidine and 131 need to be phosphorylated in cells and are

stable toward adenosine deaminase. Compound 130 served as the lead in recent identification

of HCV polymerase inhibitor, clinical candidate GS-6620 33.76

Furthermore, compound 135

bearing double bond might be substituted (e. g., by fluorine) at 6´-position.

Figure 25. Tubercidine and its known analogs 130 and 131, and our proposed carbocyclic C

analogs (132-135)

Retrosynthetically, we envisioned two possible disconnections, both leading to the key

cyclopentanone intermediate 136 (Scheme 31). Disconnection a) is based on the possible

nucleophilic addition of (hetero)aryl organometallic species 137 to ketone. Disconnection b

suggests transition metal catalyzed C-C coupling between suitable partners (e. g., heretofore

unknown enol triflate 138 derived from cyclopentanone and heterocyclic boronate 139).

85

Scheme 31. Retrosynthetic analysis of carbocyclic C-nucleosides examplified on the

tubercidine analogs 132 and 133

We envisioned that the desired cyclopentanone 136 might be prepared from inexpensive

and commercially available norbornadiene 142 (Scheme 32). Norbornene cis-dihydroxylation

followed by oxidative cleavage could yield properly modified cyclopentane 141 which, upon

further manipulation, would provide the desired ketone 136.

Scheme 32. Retrosynthetic analysis of key cyclopentanone intermediate 136

7.2.2 Results and discussion

Synthesis of cyclopentanone intermediate

86

First, we focused on efficient preparation of the acetonide-protected cyclopentanone 126,

which we had succesfully used previously (see section 7.1). We modified the route reported

for analog of 126 (with R = Bn).141

The synthetic sequence starts with diastereoselective

dihydroxylation of norbornene 142 (only traces of the other diastereomer of 143 could be

detected and eventually isolated when the synthesis was carried out on large scale) followed

by acetonide formation (144). Subsequent ozonolytical cleavage with reductive work-up

followed by monosilylation afforded intermediate 146 (Scheme 33).

Scheme 33. Reagents and conditions: a) OsO4 or K2Os2O8.2H2O, NMO acetone:H2O 4:1, 40

°C then Na2S2O5, (40-55%); b) 2,2-DMP, TsOH, acetone, rt, (95%); ii) c) O3, CH2Cl2/MeOH,

-78 °C then NaBH4, -78 °C to rt, (67%); d) NaH, TBDPSiCl, THF, rt, (76 %); iii) a) PPh3, I2,

imidazole, CH2Cl2, 0 °C to rt, (85%); b) DBU, PhCH3, 110 °C, (75%); iv) O3, CH2Cl2/MeOH,

-78 °C then thiourea, -78 °C to rt, (92%) or OsO4, NaIO4, THF:H2O 1:1, rt, (65-85%); v) O3,

CH2Cl2, -78 °C then Me2S, -78 °C to rt (48%).

Two different routes to the desired cyclopentanone 126 were then examined: i)

substitution, elimination and ozonolytical cleavage (described in Scheme 34), and ii)

oxidation to aldehyde, silylenol ether formation and ozonolytical cleavage (not shown). Route

i) gave more reproducible results with generally higher yields and better purity of 126.

87

Scheme 34. Reagents and conditions: a) PPh3, I2, imidazole, CH2Cl2, 0 °C to rt, (85%); b)

DBU, PhCH3, 110 °C, (75%); c) O3, CH2Cl2/MeOH, -78 °C then thiourea, -78 °C to rt, (92%)

or OsO4, NaIO4, THF:H2O 1:1, rt, (65-85%).

Of note, rather unusual behaviour has been observed during the ozonolysis of exocyclic

alkene 148 in different solvents (Scheme 35).

Scheme 35. Unusual ozonolysis of alkene 148

When we carried out the ozonolysis in anhydrous CH2Cl2 or hexane (with reductive

workup with Me2S), we observed two products: cyclopentanone 126 (29% yield) along with

heretofore unknown enol ether 150 (48%, for structural elucidation see SI-2, 74-76).142

Reduction with Me2S was relatively sluggish, inefficient and the reactive Criegee

intermediate143

(as zwitterion 149a or singlet biradical 149b144

) perhaps colapsed to give 150.

88

A possible suggested mechanism involves protonation of 149c by formic acid (ozonolysis by-

product) and the resulting intermediate 149d would eliminate O2.

On the other hand, ozonolysis in a mixture CH2Cl2/wet MeOH mixtures or in wet MeOH

alone provided ketone 126 in excellent yields (80-95%) and no traces of undesired alkene 150

were observed (by TLC or 1H NMR). This is consistent with several studies of Dussault´s

group, where the presence of nucleophiles facilitated the formation of carbonyl compound and

suppressed formation of undesired side products.145

Since enol ether 150 is structurally

interesting, we plan to study the ozonolysis in greater detail in the future.

Alternatively, alkene 148 could be transformed into cyclopentanone 126 by oxidative

cleavage under standard conditions (OsO4, NaIO4) in good yield (65-85%).

Nucleophilic addition pathway a.

We had shown that ketone 126 could be easily modified by diasteroeselective nucleophilic

addition of organometallic species; e. g., PhLi.146

Then, we explored whether this method can

be applied to introduce also heterocyclic moieties, e. g., (substituted) pyrimidines (Scheme

36). This method could eventually provide platform for preparation of structurally novel

carbocyclic-C nucleosides analogs with hydroxylated C-1´position which might be further

modified.

89

Scheme 36. Reagents and conditions: a) 151, n-BuLi, THF, -78 °C then 126, -78 °C to rt, (65-

80%); b) Pd/C, H2, EtOH, 80C, (85-95%); c) TBAF, THF, rt, (90-98%).

Attempts to use unprotected 5-iodouracil under conditions for metallation, developed by

group of P. Knochel, failed.147

However, easily prepared bromopyrimidine148

151 was

quantitatively lithiated (according to TLC, and 1H NMR of reaction mixture) under standard

conditions (n-BuLi, THF, -78C, 10 min to 1h). Subsequent addition to the ketone 126 gave

adduct 152 in good yields (65-80%) and excellent diastereoselectivity (the other diastareomer

not detected by TLC and NMR). Debenzylation of 152 (followed by TBDPS deprotection)

gave intermediates 153 and 154, respectively, in very good yields.

Preparation of the other regioisomer (Scheme 37) proved to be very challenging since

lithiation of 155149

gave complex mixtures (presumably due to lone pair repulsion between

nitrogen and C-anion).150

The product 156 could be isolated after debenzylation (prior

purification was difficult) only in very low yields (10%).

90

Scheme 37. Reagents and conditions: a) 155, n-BuLi, THF, -78 °C then 156, -78 °C to rt; b)

Pd/C, H2, EtOH, 80C, (10%, over two steps).

Unfortunately, cleavage of the acetonide in 154 proved extraordinarily difficult. We were

not able to produce the desired compound 157 when we carried the deprotection using a

variety of standard conditions (aq. HCl in MeOH, CH3COOH, CF3COOH, CSA, PPTS151

, I2

in MeOH,152

FeCl3.6H2O,153

BCl3,154

In(OTf)3,155

Dowex® 50WX8 100-200 mesh156

,

including variations in temperature and additives, e.g., ethylene glycol or propan-1,3-dithiol

in order to promote transketalization).

Scheme 38. Reagents and conditions: a) CSA, CH2Cl2: MeOH 3:1, rt, (46%).

Typically, we observed either low conversion or predominant decomposition. Similarly,

when we tried to cleave the acetonide in intermediate 153, only desilylation and

91

decomposition was observed. This general failure can be rationalized by facile carbocation

formation under acidic conditions. Indeed, when intermediate 154 was treated with CSA in

dichloromethane/MeOH 3:1 mixture, elimination of C-1´- OH group was observed and alkene

158 was isolated in 46% yield (Scheme 38).

Interestingly, acetonide deprotection was somewhat successful in fully protected

intermediate 152. Compound 159 was isolated in 35 % yield in reaction with PPTS/MeOH

(Scheme 39).

Scheme 39. Reagents and conditions: a) PPTS, MeOH, rt, (35%).

Additional (unexpected) reactivity of the acetonide protecting group is demonstrated by the

following observation. Previously prepared model compound 127146

was subjected to

conditions for ionic reduction of C-1´-OH (Et3SiH/BF3.OEt2 in anhydrous CH2Cl2).

Surprisingly, product of migration (160) was isolated in 56% yield, whereas no reduction

product (161) was observed (Scheme 40). Structure of 160 was unambigously assigned based

on extensive 2D NMR experimentation and careful analysis of 13

C resonances (see Scheme 40

with important 13

C resonances and key 1H-

13C HMBC interaction).

92

Scheme 40. Reagents and conditions: a) Et3SiH, BF3.OEt2, CH2Cl2, 0C to rt, (56%).

Pd-catalyzed C-C coupling pathway b.

In general, enol triflates can serve as readily available precursors for transition metal

catalyzed introduction of (hetero)aryl base moiety.157

If necessary, triflates can be also

transformed into the corresponding boronates so that both variants of coupling are available.

Treatment of cyclopentanone 126 with LDA at -78C followed by addition of N-phenyl-

bis(trifluoromethansulfonimide) provided stable enol triflate 162 in good yield (60-80%,

Scheme 41). The Suzuki coupling between 162 and PhB(OH)2 proceeded smoothly and

afforded compound 163 in good yield (70-80%). Conversion of enoltriflate 162 to the

corresponding boronate 164 was also achieved by Pd-catalyzed borylation.158

Potentially

93

useful intermediate 165, whose triple bond might be further elaborated (e. g., by click

chemistry), could be obtained as well (Scheme 41).

Scheme 41. Reagents and conditions: a) LDA, THF, -78 °C then PhNTf2, -78 °C to rt, (60-

80%); b) pin2B2, Pd(Ph3P)2Cl2, Ph3P, KBr, KOPh, PhCH3, 50C (full conversion) c)

PhB(OH)2, Pd(dppf)Cl2, K3PO4, DME, H2O, 80C, (70-80%) d) TMS-acetylene,

Pd(Ph3P)2Cl2, CuI, 2,6-lutidine, DMF, 70 °C, (79%).

Hydrogenation of 163 followed by deprotection of TBDPS and acetonide (Scheme 42)

gave single diastereomer 168 (hydrogenation proceeds from the less hindered bottom face of

the cyclopentene scaffold), whose structure was confirmed by 2D NMR experiments.

Scheme 42. Reagents and conditions: a) Pd/C, H2, EtOH, 80C, (86%); b) TBAF, THF, rt,

(97%) c) HCl, H2O, MeOH, rt, (62%).

On the other hand, when the acetonide in intermediate 163 was cleaved prior to reduction

(albeit in low yield with PPTS in MeOH) the alternative diastereomer 171 was isolated in

94

60% yield (Scheme 43). For comparison of 1H and

13C NMR chemical shifts of epimers 168

and 171 see SI_2, p. 158-9).

Scheme 43. Reagents and conditions: a) PPTS, MeOH, rt, (38%, 41% of starting material

recovered); b) Pd/C, H2, EtOH, 80C, (60% of 170 and 27% of other epimer); c) TBAF,

THF, rt, (85% of 171).

These preliminary results clearly indicated that it might be possible to develop

stereospecific routes for both sub-series.

By the transition metal-catalyzed pathway, I prepared several new carbocyclic C-

nucleosides (Scheme 44). Namely, carbocyclic pseudouridine 175 (epimer to the previously

prepared compound 90b, for comparison of structural data see SI-2, p. 175-6).

Scheme 44. Reagents and conditions: a) 155, n-BuLi, THF, -78°C, 2-isopropoxy-4,4,5,5-

tetramethyl-1,3,2-dioxaborolane, then 162, Pd(dppf)Cl2, K3PO4, DME, H2O, 80C, (41%) b)

Pd/C, H2, EtOH, 80C, (82%); c) TBAF, THF, rt, (98%) d) HCl, H2O, MeOH, rt, (52%).

95

Similarly, compound 134 (Scheme 45) was prepared by reaction of known 7-

bromopyrrolo[1,2-f][1,2,4]triazin-4-amine159

176 (see Scheme 46 for its preparation). Of note,

it was not necessary to protect the amine in 176 prior to the Suzuki coupling.

Scheme 45. Reagents and conditions: a) Pd(dppf)Cl2, K3PO4, DME, H2O, 80C, (62%, based

on 176) b) i) TBAF, THF, rt, (64%); ii) Pd/C, H2, EtOH, 80C, (93%); iii) HCl, H2O, MeOH,

rt, (56%).

Scheme 46. Reagents and conditions: a) BocNH-NH2, 2M HCl, dioxane, 100 °C, (68%); b)

ClSO2NCO, DMF, CH3CN, -30 °C to rt, (62%); c) 4M HCl/dioxane, 0 °C to rt then

formamidine acetate, K3PO4, EtOH, 80 °C, (83% over 2 steps from 180); e) 1,3-dibromo-5,5-

dimethylhydantion, DMF, 0 °C to rt, (80%, contaminated by 5,5-dimethylhydantoin).

However, preparation of the epimer 132 proved to be challenging; mainly due to the

difficulties of selective cleavage of acetonide in 177 prior to the hydrogenation step (Scheme

47).

96

Scheme 47. Unsuccesful acetonide deprotection

Previously described conditions (PPTS, MeOH, H2O) did not provide the desired

compound. Harsher conditions (HCl) lead only to decomposition of starting material.

Therefore, we set to explore alternative protecting groups that could be used instead of

acetonide and whose final deprotection would be easier.

2´-OH and 3´-OH protecting groups optimization

The requirements for desirable protecting groups were i) compatibility with the conditions

of the synthetic sequence, ii) facile cleavage (preferably under non-acidic conditions) and

ideally iii) orthogonality to the TBDPS group.

Initially, we tested the benzophenone ketal protection group (in 183, Scheme 48), whose

chemical robustness is similar to that of acetonide, but it can be cleaved hydrogenolytically.160

We prepared intermediate 184 (by the methodology described above, see experimental

section), which underwent nucleophilic addition with excellent diastereoselectivity and in

very good yield (75%). TBDPS cleavage and debenzylation afforded 185. However,

deprotection of the benzophenone ketal failed under several conditions (H2, HCOONH4,

different metal catalysts, e. g., PtO2, Pd/C, Pd(OH)2/C), in different solvents at various

temperatures with or without additives (AcOH, TFA).

97

Scheme 48. Unsuccesful elaboration of diphenyl ketal variant to desired compound 157

With these rather frustrating results, we decided to prepare series of protected norbornene

diols 186-192. Somewhat surprisingly, only compound 186 have been recently described in

the literature161

although without any experimental details. Endo diastereomer of 186 has been

frequently used in ROMP studies.162

Yields of the protections of the corresponding diol 143 are given in parentheses.

Compounds 186 and 189 underwent undesired transformations (i. e. migrations of protecting

group and/or decomposition) during the ozonolyzis/NaBH4 reaction sequence.

98

Our attempts to use generally more stable TBDPS (in 190) failed due to very poor yield of the

protection (presumably due to the steric reason).

Most of the intermediates bearing cyclic di-t-butyl silyl group prepared from 191 underwent

decomposition during chromatography purification (described in the experimental section).

On the other hand, we were able to transform bis-benzylated compound 187, its PMB variant

188, and TIPS-protected 192 into the desired cyclopentanones in good yields (Scheme 49).

Scheme 49. Reagents and conditions: a) for 187: NaH, BnBr, TBAI, DMF, rt, (75-90%); for

188: PMB-Cl, NaH, BnBr, TBAI, DMF, rt, (80%); for 192: TIPSOTf, imidazole,DMAP,

DMF, 65C, (88%) b) i) O3, CH2Cl2/MeOH, -78 °C then NaBH4, -78 °C to rt; ii) NaH,

TBDPSiCl, THF, rt; (65% for 193 over two steps), (33% for 194, over two steps), (66 % for

195, over 2 steps); c) i) PPh3, I2, imidazole, CH2Cl2, 0 °C to rt, ii) DBU, PhCH3, 110 °C (70%

for 196 over two steps), (53% for 197 over two steps) d) Bu3P, 3-NO2PhSeCN, THF, rt, then

H2O2, 0 °C to rt (80%, over two steps) e) O3, CH2Cl2, -78 °C then thiourea, rt, (96% for 199),

(86% for 200), (92% for 201).

Protection of diol 143 with Bn or PMB proceeded under standard conditions in very good

yields. Similarly, bis-silylation with TIPSOTf produced compound 192 in excellent yield (88

%), even though the reaction is sluggish (7 days, at 65C). The reaction time was somewhat

shortened (to 5 days) by addition of DMAP.

99

Ozonolytical cleavage of 187, 188, and 192, followed by NaBH4 reduction provided the

corresponding diols (during the ozonolysis of 188, partial oxidative cleavage of PMB was

observed), which were then mono-silylated to yield compounds 193, 194, and 195,

respectively.

Intermediates 193 and 194 were efficiently transformed into alkenes 196 and 197,

respectively, via previously described iodination - elimination sequence. On the other hand,

elimination of HI from the TIPS-protected intermediate was very sluggish (we observed

10% conversion after 3h; at that time benzylated analog underwent full conversion).

Presumably, steric hinderance prevents the E2 elimination pathway. However, one-pot

selenation plus oxidation followed by intramolecular elimination163

proceeded smoothly

providing the desired exocyclic alkene 198, which upon ozonolysis afforded ketone 199 in

excellent yield (92%). Of note, the selenide intermediate could be isolated in good yields by

flash column chromatography and was fully characterized by NMR (see supporting

information). A remarkable shielding effect of Se atom was observed in the 13

C NMR

spectrum: chemical shift of -CH2O signal in hydroxylated 195 is 65 ppm, whereas -CH2Se-

resonates at 30 ppm.

On the other hand, ozonolysis of alkenes 196 and 197 was much less clean. We observed

several side reactions: epimerization at C-2´position, -elimination of benzyloxy group,

dimethylketal formation, and in case of 197 partial oxidative cleavage of the PMB protecting

group (as indicated from characteristic PMB aldehyde proton signal in 1H NMR spectrum).

We obtained significantly better results when we used thiourea for the reductive work-up,164

which enabled clean and rapid production of the desired ketones 199 and 200. Since ketone

200 was somewhat unstable and its overal production was far less efficient than that of

benzylated compound 199, subsequent transformations will be discussed only for ketone 199.

Reactivity of ketone 199

Nucleophilic addition of lithiated pyrimidine 155 to ketone 199 proceeded with good yield

and diastereoselectivity (traces of other diastereomer, partial epimerization of C-2´position as

well as -elimination of benzyloxy group were detected in NMR spectra of crude reaction

mixtures). Desilylation and subsequent debenzylation gave final product 157 (Scheme 50).

Even though this route enabled us to prepare compound 157, it should be noted that the final

100

deprotection was poorly reproducible and it was difficult to follow the conversion (both by

TLC and or by 1H NMR). Additionally, product 157 was often contaminated with several

impurities. Decomposition of the starting material or intermediates during the debenzylation

gave surprisingly carbocyclic pseudoridine 175 (as confirmed by 1H NMR of crude reaction

mixtures and isolated material, see supporting information). This side product was formed

presumably via E1 elimination of water followed by hydrogenation.

Scheme 50. Reagents and conditions: a) 151 or 202, n-BuLi, THF, -78 °C then 200, -78 °C to

rt, b) TBAF, THF, rt c) Pd/C, H2, EtOH, 80C, (53-65%, for 157 over 3 steps), (23% for 204

over 3 steps).

We then tried the same methodology to prepare the target compound 133 by employing

lithiated 202 (prepared from 176). Desilylation/debenzylation gave monobenzylated

compound 204. Thus far, we have not been able to remove the last benzyl group from 204.

Additionally similar problems with unwanted reactions during the hydrogenolytical cleavage

were also observed.

We also tried to use unprotected amine 176. While the lithiation of this compound was clean

and efficient (based on the 1H NMR spectra), its addition to ketone 199 failed and we

observed mainly -elimination of the benzyloxy group, which is discussed in greater detail

below. Temporary protection of amino group in 176 by 1,1,4,4-tetramethyl-1,4-

101

dichlorodisilylethylene according to the published protocol165

also failed and the elimination

was again observed.

In comparison to acetonide-protected ketone 126, the formation of enol triflate 205 from

benzyl-protected ketone 199 was considerably less efficient (Scheme 51). We used a variety

of conditions, varying the triflating agent (N-phenyl-bis(trifluoromethansulfonimide)),

Commins´ reagent, triflic anhydride), base (LDA, NaHMDS, KHMDS, DIPEA) solvents

(THF, toluene, DMF, NMP), and the temperature. Under best conditions, enolate was

generated with 1.3 eq. of LDA at -78 °C for 10 min and then quenched with 3 eq. of N-

phenyl-bis(triflouromethansulfonimide). After 3.5 h, nearly 100% conversion was observed

by 1H NMR, but upon quenching the reaction by sat. NH4Cl at -78C, rapid extraction and

flash column chromatography on the neutral and activated Al2O3, triflate 206 was isolated

only in 33% yield, together with epimerized starting material (206) and ,-unsaturated

ketone 207 (Scheme 51).

Scheme 51. Reagents and conditions: a) LDA, THF, -78 °C then PhNTf2, -78°C, (33%); b)

NaHMDS, THF, -78 °C then PhNTf2, -78 °C to rt, (36%).

The stability of enol triflate 205 in solution was further studied by 1H NMR. Isolated

compound was within 2 hours at room temp. in CDCl3 solution converted into a complex

mixture containing 206 and 207.

Of note, regioisomeric enol triflate 208 (for structural elucidation see SI_2, p. 131) , which we

isolated in 36 % yield when NaHMDS was used instead of LDA, was substantially more

stable in solution and on silica gel.

102

Nevertheles, even crude product 205 (isolated only by extraction) could be used in the model

Suzuki coupling, which provided intermediate 209 (Scheme 52).

With sufficient amount of cyclopentene 209 in hand, we envisioned modification of the

double bond via diastereoselective epoxidation. Epoxidation of fully protected intermediate

209 with mCPBA was sluggish and not diastereoselective (Scheme 52). However, notably

shorter reaction times and better diastereoselectivity (ca. 10:3, by 1H NMR) were achieved in

epoxidation of intermediate 211 with deprotected 5´-OH. The major diastereomer 213 was

isolated in 63% yield, isolation of pure minor diastereomer was not successful.

Scheme 52. Reagents and conditions: a) PhB(OH)2, Pd(dppf)Cl2, K3PO4, DME, H2O, 80C,

(54%); b) m-CPBA, CH2Cl2, solid NaHCO3, 0 °C to rt, (83%, diastereomeric mixture); c) i)

TBAF, THF, rt, (88%); ii) m-CPBA, CH2Cl2, solid NaHCO3, 0 °C to rt, (63% of desired

diastereomer) iii) Superhydride

, THF, 0 °C to rt, (59%).

The stereochemical analysis of both diastereomers was complicated due to overlapping of

analytically important 1H NMR resonances. Therefore, we tried to open the epoxide and

correlate the resulting products with 214 (prepared by diastereoselective addition of PhLi to

ketone 199, see experimental procedure), whose stereochemistry we knew.

103

The epoxide in 212 turned to be surprisingly robust: upon treatment with NaBH4, Li(Al-O-

tBu)3H or LiAlH4 it remained intact. However, Superhydride was effective and compound

213 was isolated. Its stereochemistry (and the diastereoselectivity of the epoxidation) was

verified by comparison to the other diastereomer 214 (see SI_2, p. 120-124).

As all nucleophillic additions of organometallics to ketone 199 were accompanied by the

formation of side products by epimerization and -elimination, we investigated

stability/reactivity of this ketone in greater detail. When ketone 199 was treated with

methanolic suspension of neutral Al2O3, ,-unsaturated ketone 207 was observed by 1H

NMR in the crude mixture as major component, together with other decomposition products.

On the other hand, when TEA in anhydrous CH2Cl2 was used, mainly epimerization at the C-

2´position occured and we isolated epimer 216 in 53% yield (Scheme 53). This might be

potentialy useful for preparation of otherwise challenging analogs with opposite

stereochemistry at 2´-position.

Scheme 53: Reagents and conditions: a) Al2O3, MeOH, rt. b) TEA, CH2Cl2 rt, (53%).

In contrast, TIPS-protected ketone 201 turned out to be much more robust: no

epimerization/elimination was detected when it was subjected to the conditions described

above. This substantially higher stability is likely due to the steric bulk of the TIPS protecting

groups.166

Optimization of protecting group for 5´-OH

As described above, TIPS proved to be optimal for protection of 2´-OH and 3´- OH. We

wanted to have an orthogonal protecting group at 5´-OH, which would allow flexible

manipulation of 5´-position. Of a relatively small number of groups that would be also

compatible with the conditions used in the synthetic route (e. g., oxidation, hydrogenation,

organometallics, Pd-catalyzed chemistry), we chose pivaloate and ethoxymethyl group. By

the methodology previously described for TBDPS-protected ketone 201, we prepared

intermediates 216 and 217. (First synthesis of these intermediates including synthesis of

104

model compound 171 was done by my colleague Prashant Khirsariya) All three ketones were

successfully converted to the corresponding enol triflates 218, 219, and 220 in high yields

(Scheme 54). We were pleased to observe that all triflates were stable and could be purified

by flash column chromatography and stored at -20oC under nitrogen for several days

(although enol triflate 218 notably decomposed at room temperature).

Scheme 54. Reagents and conditions: a) KHMDS, Commins reagent, THF, -78C to rt, for

218 (72 %), for 219 (97%), for 220 (79%) b) i) PhB(OH)2, Pd(dppf)Cl2, K3PO4, DME, H2O,

80C, (92% from 219); ii) TBAF, THF, rt, (89%); iii) H2, Crabtree catalyst, CH2Cl2, rt, (94

%); iv) PPTS, MeOH, 80C, (86%); c) i) Pd(dppf)Cl2, K3PO4, DME, H2O, 80C, (75% from

218); ii) TBAF, THF, rt, (70%).

Suzuki coupling of triflate 218 with PhB(OH)2 followed by global desilylation afforded triol

221 in very good yield.

To utilize orthogonality of the protecting groups at 5´-position, the product obtained by

Suzuki coupling of enol triflate 219 with PhB(OH)2 was subjected to desilylation by TBAF.

As depicted in Scheme 54 above, subsequent hydrogenation of this intermediate with Crabtree

catalyst proceeded with practically perfect diastereoselectivity and upon cleavage of the

CH2OEt group (with PPTS in MeOH) we isolated triol 171 in high overall yield (64%, 5 steps

from 216). Its structure was compared with 171 and 168 and unambiguously confirmed by X-

105

ray crystallography. This route is significantly superior to that descibed earlier (see Scheme

43, compound 171).

We then tried to apply this methodology in the synthesis of tubercidine C-analog 132.

Unfortunately, our attempts to prepare corresponding boronate from enol triflate 218 or 220

under the conditions desribed above for preparation of boronate 164 failed so far. Similarly,

preparation of pyrolotriazine boronate (or stannane) with unprotected amino group by Pd-

catalyzed coupling with bis(pinacolato)diboron failed and only the debrominated heterocycle

was typically observed.

On the other hand, Pd-catalyzed coupling of enol triflate 219 or 220 and heretofore unknown

boronic acid 223 (see Scheme 55 for its preparation, initially prepared by Prashant Khirsariya)

gave intermediates 224 and 225 in good yields (Scheme 56).

Scheme 55. Reagents and conditions: a) NaH, SEM-Cl, DMF, 0C to rt, (55 %), b) n-BuLi,

THF, -78 °C then 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, -78 °C to rt, (see

exp. section).

106

Scheme 56. Reagents and conditions: a) 225, Pd(dppf)Cl2, K3PO4, DME, H2O, 80C, (75%

for 226, 64% for 227); b) TBAF, THF, rt, (98% for 228, 81% for 229), c) Crabtree catalyst,

CH2Cl2, rt, (31 %, from 226); d) from 228, PPTS, MeOH, H2O, 80C, (132, contaminated by

tosylate salts). For 1H,

13C, and

15N resonances assignment of 132 and epimer 134 see SI_2, p.

189-191.

Intermediate 226 underwent diastereoselective hydrogenation with Crabtree catalyst followed

by simultaneous deprotection of bis-SEM and ethoxymethyl groups gave target compound

132 contaminated bytosylate salts. HPLC purification of 132 is currently in progress. On the

other hand, hydrogenation of 225 with Crabtree catalyst gave only low conversion (aprox. 30

%). Alternative hydrogenation with Pd(OH)2 afforded a mixture of diastereomers (aprox. 2:1

where the desired epimer 229 was major one) and moderate overall yield (55 %).

Difficulties were encountered also during the synthesis of unsaturated tubercidine analog 135.

Pivaloate in 227 was rapidly (10 min) cleaved by DIBAL-H at -78°C, which provided

intermediate 230 in excellent yield (Scheme 57).

Scheme 57. Reagents and conditions: a) DIBAL-H, CH2Cl2, -78 °C to room temp., (98%).

The SEM groups in 230 were resistant to treatments with TBAF, TEA-HF or NH4F.

Treatment of 230 with 2 M HCl at 25C yielded only 40% of impure mono-deprotected

product S-32 (interestingly TMS was also cleaved to afford the mono-methoxymethyl moiety,

see experimental section) along with unidentified decomposition products (in which the

double bond of the cyclopentenyl moiety was not retained) together with 10% of recovered

starting material. Similar cleavage at higher temperatures resulted in decomposition.

107

Clearly, the synthetic sequences including unsaturated intermediates and/or target compounds

should not include the use of protecting groups whose removal requires (strong) acids.

7.2.3. Experimental procedures

Experimental procedures for cyclopentanone preparation

(1R*,2R*,3S*,4S*)-bicyclo[2.2.1]hept-5-ene-2,3-diol (143):

NMO (12.71 g , 108.5 mmol) and OsO4 (4% wt. in H2O, 1.36 mL, 0.2 mol %) were added to

a solution of norbornadiene (10.0 g, 108.5 mmol) in acetone:H2O (200+50 mL). The reaction

mixture was stirred at 40 °C for 14 h. After cooling down to 25 °C, 0.5 g of Na2S2O5 was

added and the reaction mixture was stirred at 25°C for another 30 min. All volatiles were

removed under reduced pressure and the black residue was purified by flash column

chromatography (SiO2, hexane/EtOAc 2:1) to afford 143 as a white crystalline compound

(5.61 g, 41%). 1H NMR (500 MHz, CDCl3): δ = 6.02 (m, 2H), 3.69 (m, 2H), 2.93 (m, 2H),

2.68 m (2H), 1.87 (dm, 1H, J = 9.2 Hz), 1.61 (dm, 1H J = 9.2 Hz) ppm. 13

C NMR (126 MHz,

CDCl3): δ= 136.56, 69.18, 48.21, 42.37 ppm. Spectral data were consistent with reported.141

(exo, exo)-5,6-dimethylmethylendioxy-bicyclo[2.2.1]hept-2-ene (144):

2,2-dimethoxypropane (13.0 mL, 98.3 mmol) and TsOH (~ 5 mg) were added to a solution of

diol 143 (3.1 g, 24.6 mmol) in acetone (50 mL). The reaction mixture was stirred at 25 °C for

20 min., the solvent was evaporated and the residue was purified on a short pad of silica gel

(hexane/EtOAc 20:1) to afford 144 as a colorless oil which solidified upon standing at -20 °C

(3.86 g, 95 %). Note: the compound is quite volatile . 1H NMR (500 MHz, CDCl3): δ = 6.03

(m, 2H), 4.16 (d, J = 1.6 Hz, 2H), 2.74 (m, 2H), 1.95 (m, 1H), 1.65 (m, 1H), 1.45 (s, 3H), 1.30

(s, 3H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 136.89, 113.81, 80.63, 45.35, 43.02, 26.32,

24.58 ppm. Spectral data were consistent with reported.141

108

Diphenyl ketal (183):

Benzophenone dimethyl ketal (2.08 g, 9.15 mmol) was added to a solution of starting material

143 (0.800 g, 0.634 mmol) in anhydrous CH2Cl2 (8 mL), followed by addition of TsOH (1

mg). The reaction mixture was stirred at 25C for 18 h. The solvent was evaporated and the

residue was purified by flash column chromatography (SiO2, hexane/EtOAc 20:1) to get 183

in mixture with residual benzophenone and benzophenone dimethyl ketal as a white

crystalline solid. This material was used for a next step without further purification.

Analytical sample could be obtained by repeated flash column chromatography with slow

elution, gradient hexane to hexane/EtOAc 20:1. m.p. = 123-127°C. 1H NMR (500 MHz,

CDCl3): δ = 7.56-7.54 (m, 2H), 7.49-7.47 (m, 2H), 7.35-7.32 (m, 2H), 7.29-7.24 (m, 4H),

6.03 (m, 2H), 4.11 (d, J = 1.55 Hz, 2H), 2.95 (m, 2H), 2.19 (dm, J = 8.86 Hz, 1H), 1.75 (dm J

= 8.86 Hz, 1H). 13

C NMR (126 MHz, CDCl3): δ = 143.03, 141.71, 137.06, 128.46, 128.40,

128.28, 128.07, 126.73, 126.24, 114.41, 81.31, 45.43, 43.78 ppm. IR: ν˜max = 2979 (w), 2946

(w), 2924 (w), 1489 (m), 1270 (m), 1203 (m), 1068 (s), 1019 (s), 747 (s), 703 (s), 694 (s) cm–

1.

Carbonate (186):

1,1´-carbonyldiimidazole (0.250 g, 1.976 mmol) was added to a solution of starting material

143 (0.200 g, 1.58 mmol) in anhydrous toluene (6 mL). The reaction mixture was stirred at

55°C for 16 h. The reaction mixture was then cooled to 25 °C and the solvent was evaporated.

The solid residue was purified by column chromatography (SiO2, hexane/EtOAc 5:1) to

afford 186 as a white crystalline compound (0.191 g, 80%). m.p. = 86-88°C. 1H NMR (500

MHz, CDCl3): δ = 6.10 (m, 2H), 4.54 (d, J = 1.38 Hz, 2H), 3.12 (m, 2H), 1.88-1.80 (m, 2H)

ppm. 13

C NMR (126 MHz, CDCl3): δ = 156.53, 136.17, 78.92, 45.79, 41.15 ppm. IR: ν˜max =

3011 (w), 1777 (s), 1367 (m), 1160 (s), 1055 (s), 1014 (s), 742 (s), 697 (s) cm–1

. HR-MS

(ESI): calcd for C8H8O3 [M+Na]+ : 175.0371. Found: 175.0370.

109

(1R*,4S*,5S*,6R*)-5,6-bis(benzyloxy)bicyclo[2.2.1]hept-2-ene (187):

A solution of diol 143 (4.88 g, 38.7 mmol) in DMF (40 mL) was added dropwise to a

suspension of NaH (60% dispersion in mineral oil, 4.64 g, 116.11 mmol) in anhydrous DMF

(20 mL). The reaction mixture was stirred at 25 °C for 20 min, benzyl bromide (11.05 mL,

92.9 mmol) was slowly added and after additional 10 min. a solution of tetra-N-butyl

ammonium iodide (1.43 g, 3.87 mmol) in anhydrous DMF (10 mL). The reaction mixture was

stirred at 25 °C for additional 14 h and then quenched by dropwise addition of H2O (5 mL).

H2O (100 mL) was added and the mixture was extracted with Et2O (4 × 100 mL). Combined

organic extracts were dried over Na2SO4, filtered, and the solvent was evaporated. The

resulting yellow oil was purified by flash column chromatography (SiO2, hexane/EtOAc 20:1)

to afford 187 as a white crystalline solid (9.08 g, 77%). 13


139.23, 137.02, 128.48, 128.00, 127.59, 77.19, 72.50, 45.88, 44.18 ppm. IR: ν˜max = 3063

(w), 3029 (w), 1496 (w), 1453 (m), 1343 (w), 1114 (m), 733 (s), 696 (s) cm–1

. 1H NMR (500

MHz, CDCl3): δ = 7.35-7.24 (m, 10H), 5.99 (m, 2H), 4.67-4.61 (m, 4H), 3.51 (m, 2H), 2.83

(m, 2H), 2.18 (m, 1H), 1.65 (m, 1H) ppm. HR-MS (ESI) calcd for C21H22O2 [M+Na]+:

329.1517. Found: 329.1524.

(1R*,4S*,5S*,6R*)-5,6-bis(4-methoxybenzyloxy)bicyclo[2.2.1]hept-2-ene (188):

A solution of diol 143 (1.18g. 9.37 mmol), in anhydrous DMF (20 mL), was added dropwise

to a suspension of NaH (60% dispersion in mineral oil, 1.12 g, 28.11 mmol) in anhydrous

DMF (20 mL). The reaction mixture was stirred at 25°C for 20 min, 4-methoxybenzyl

chloride (2.8 mL, 20.62 mmol) was slowly added and after additional 10 min. a solution of

tetra-N-butyl ammonium iodide (0.346 g, 0.937 mmol) in anhydrous DMF (10 mL). The

reaction mixture was stirred at 25 °C for additional 14 h and then quenched by dropwise

addition of H2O (5 mL). H2O (100 mL) was added and the mixture was extracted with Et2O (4

× 100 mL). Combined organic extracts were dried over Na2SO4, filtered, and the solvent was

110

evaporated. The resulting yellow oil was purified by flash column chromatography (SiO2,

hexane/EtOAc 15:1) to afford 188 as a white semi-solid (3.2 g, 93%). 1H NMR (500 MHz,

CDCl3): δ = 7.25 (d, J = 8.62 Hz, 4H), 6.83 (d, J = 8.62 Hz, 4H), 5.97 (m, 2H), 4.56 (d, J =

11.65 Hz, 2H), 4.51 (d, J = 11.65 Hz, 2H), 3.78 (s, 6H), 3.46 (d, J = 1.76 Hz, 2H), 2.78 (m,

2H), 2.14-2.12 (m, 1H), 1.62-1.60 (m, 1H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 159.29,

137.01, 131.35, 129.59, 113.89, 76.98, 76.79, 72.10, 55.50, 45.87, 44.17 ppm.

(1R*,4S*,5S*,6R*)-5,6-bis(tert-butyldimethylsilyloxy)bicyclo[2.2.1]hept-2-ene (189):

Imidazole (558 mg, 8.21 mmol) and TBSCl (544 mg, 3.61 mmol) were added to a solution of

starting material 143 (0.207 g, 1.641 mmol) in anhydrous CH2Cl2 (15 mL). The reaction

mixture was stirred at 25C for 14 h. The solvent was evaporated and the semi-solid residue

was purified by flash column chromatography (SiO2, hexane/EtOAc 10:1) to afford 189 as a

white crystalline solid (0.442 g, 76%). 1H NMR (500 MHz, CDCl3): δ = 5.98 (m, 2H), 3.62

(m, 2H), 2.51 (m, 2H), 2.12 (dm, J = 8.16 Hz, 1H), 1.54 (dm, J = 8.16 Hz, 1H) 0.89 (s, 18H),

0.05 (s, 6H), 0.03 (s, 6H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 136.95, 70.98, 49.65, 43.41,

26.30, 18.60, -4.02, -4.65 ppm.

(1R*,4S*,5S*,6R*)-5,6-bis(tert-butyldiphenylsilyloxy)bicyclo[2.2.1]hept-2-ene (190):

TBDPSCl (1 mL, 3.96 mmol) and imidazole (433 mg, 6.36 mmol) were added to a solution of

starting material 143 (0.200 g, 1.59 mmol) in anhydrous CH2Cl2 (10 mL). The reaction

mixture was stirred at 25 °C for 14 h. The solvent was evaporated and the yellow residue was

purified by flash column chromatography (SiO2, hexane/EtOAc 20:1) to afford 190 as a white

crystalline solid (0.236 g, 25 %), along with monosilylated compound (0.417 g, 43%). m.p. =

133-136°C. 1H NMR (500 MHz, CDCl3): δ = 7.74-7.70 (m, 8H), 7.39-7.29 (m, 12H), 5.51

(m, 2H), 3.85 (d, J = 1.5 Hz, 2H), 2.30 (dm, J = 8.59 Hz, 1H), 2.22 (m, 2H), 1.36 (dm, J =

8.59 Hz, 1H), 1.08 (s, 18H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 136.63, 136.27, 136.15,

111

135.28, 134.70, 129.71, 129.60, 127.68, 127.67, 72.30, 48.87, 42.96, 27.43, 19.60 ppm. IR

(ν˜max): 1472 (w), 1102 (m), 1086 (m), 697 (s), 499 (s), 481 (s) cm–1

.

(1R*,4S*,5S*,6R*)-5,6-bis(triisopropylsilyloxy)bicyclo[2.2.1]hept-2-ene (191):

Imidazole (1.78 g, 26.16 mmol) was added to a cooled solution (0 °C, ice bath) of starting

material 143 (1 g, 7.93 mmol) in anhydrous DMF (10 mL) followed by dropwise additon of

di-tert-butylsilyl bis(trifluoromethansulfonate) (2.84 mL, 8.72 mmol). The reaction mixture

was stirred at 0 °C and allowed to warm to 25C and then stirred for additional 14 h. The

reaction mixture was quenched by slow addition of H2O (60 mL) and extracted with Et2O (3x

50 mL). Organic extracts were dried over Na2SO4, filtered, and the solvent was evaporated.

The yellow residue was purified by flash column chromatography (SiO2, hexane/EtOAc 20:1)

to afford 191 as a colorless oil (1.4179 g, 78%). 1H NMR (500 MHz, CDCl3): δ = 6.04 (m,

2H), 4.13 (d, J = 1.53 Hz, 2H), 2.77 (m, 2H), 2.18 (dm, J = 9.22 Hz, 1H), 1.62 (dm, J = 9.22

Hz, 1H), 1.10 (s, 9H), 1.03 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 137.51, 78.53,

47.20, 42.51, 28.30, 27.73, 22.88, 20.62 ppm. IR: ν˜max = 2933 (w), 2858 (w), 1475 (m),

1171 (w), 1036 (s), 1021 (s), 853 (s), 823 (s), 704 (m), 648 (m) cm–1


C15H27O2Si [M+H]+ : 267.1780. Found: 267.1779.

(1R*,4S*,5S*,6R*)-5,6-bis(triisopropylsilyloxy)bicyclo[2.2.1]hept-2-ene (192):

Imidazole (4.963 g, 72.91 mmol) and DMAP (290 mg, 2.38 mmol) were added to a solution

of starting material 143 (2.00 g, 15.85 mmol) in anhydrous DMF (16 mL), followed by

dropwise addition of TIPSOTf (9.8 mL, 36.46 mmol) at 25 °C. The reaction mixture was

stirred at 65°C for 5 days. The reaction mixture was cooled to 25 °C, quenched with H2O (30

mL), and extracted with Et2O (3x 50 mL). Organic extracts were dried over Na2SO4, filtered,

and the solvent was evaporated. The residual yellow oil was purified by flash column

chromatography (SiO2, hexane, visualization by KMnO4 or CAM stain) to afford 192 as a

112

colorless oil (6.7 g, 88 %). 1H NMR (500 MHz, CDCl3): δ = 5.98 (m, 2H), 3.82 (d, J = 1.74

Hz, 2H), 2.60 (m, 2H), 2.18 (dm, J = 8.40 Hz, 1H), 1.58 (dm, J = 8.40 Hz, 1H), 1.10-1.05 (m,

42H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 136.91, 71.40, 49.84, 43.45, 18.51, 18.44, 13.07

ppm. HR-MS (APCI): calcd for C25H50O2Si2 [M+H]+ : 439.3422. Found: 439.3427.

General procedure for ozonolytical cleavage of protected norbornene intermediates

Mixture of O3/O2 (O2 flow = 5mL/min, ozonolysis rate ~ 12 mmol/5 min) was bubbled to a

cooled (-78°C) solution of starting material in CH2Cl2:MeOH (1:3, ~ 15 mmol of starting

material/10 mL). After completion of ozonolysis (TLC, blue color persisted), excess of O3

was removed by bubbling of N2 into the reaction mixture. NaBH4 (0.25 eq.) was added in one

portion and the reaction mixture was stirred at -78 °C for 1 h. Another portion of NaBH4

(1.025 eq.) was added and the reaction mixture was stirred for additional 3 h while allowed to

warm to 25 °C. The reaction mixture was concentrated under reduced pressure and the

residual viscous oil was partitioned between dichloromethane and brine. Aqueous phase was

reextracted with CH2Cl2. Organic extracts were dried, filtered over Na2SO4, and the solvent

was evaporated. The residue was purified by flash column chromatography to afford the

corresponding diol.

((3aR*,4R*,6S*,6aS*)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4,6-

diyl)dimethanol (145):

Following general procedure using 6.2 g (37.32 mmol) of 144. Flash column chromatography

(SiO2, CH2Cl2/MeOH 20:1 to 10:1) afforded 145 as a colorless oil (5.11 g, 67%). 1H NMR

(500 MHz, CDCl3): δ = 4.39 (m, 2H), 3.67 (m, 4H), 2.26 (m, 2H), 2.06 (m, 1H), 1.66 (br s,

113

2H), 1.49 (s, 3H), 1.30 (s, 3H), 1.24 (m, 1H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 112.66,

83.67, 64.51, 47.63, 30.66, 27.75, 25.32 ppm. HR-MS (APCI): calcd for C10H18O4 [M+H]+ :

203.1278. Found: 203.1276. Spectral data were consistent with the literature.141

((3aR*,4R*,6S*,6aS*)-2,2-diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxole-4,6-

diyl)dimethanol (S-6):

Following general procedure using 2.585 g (7.92 mmol) of 183. Flash column

chromatography (SiO2, CH2Cl2/MeOH 20:1) afforded S-6 as a colorless oil (0.950 g, 37%).

1H NMR (500 MHz, CDCl3): δ = 7.52-7.50 (m, 2H), 7.45-7.43 (m, 2H), 7.34-7.24 (m, 6H),

4.39-4.36 (m, 2H), 3.71-3.64 (m, 4H), 2.47-2.43 (m, 2H), 2.15-2.10 (m, 1H), 1.65 (br s, 2H),

1.38-1.31 (m, 1H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 142.60, 142.29, 128.50, 128.39,

128.22, 126.70, 126.64, 113.25, 84.42, 64.78, 47.32, 31.01 ppm. IR: ν˜max = 3307 (m), 2924

(m), 1446 (m), 1261 (m), 1086 (m), 1067 (m), 1052 (m), 973 (m), 695 (s), 635 (m) cm–1

. HR-

MS (ESI): calcd for C20H22O4 [M+H]+

: 327.1591. Found: 327.1581.

((1R*,3S*,4S*,5R*)-4,5-bis(benzyloxy)cyclopentan-1,3-diyl)dimethanol (S-7):


(SiO2, CH2Cl2/MeOH 20:1) afforded S-7 as a colorless oil (8.95 g, 88%). 1H NMR (500 MHz,

CDCl3): δ = 7.34 – 7.26 (m, 2H), 4.57 (d, AB, J = 11.83 Hz, 2H), 4.50 (d, AB, J = 11.83 Hz,

2H), 3.69 (m, 2H), 3.62 (m, 2H), 3.50 (m, 2H), 2.43 (m, 2H), 1.94 (m, 1H), 1.75 (br s, 2H),

0.91 (m, 1H) ppm.13

C NMR (126 MHz, CDCl3): δ = 138.51, 128.62, 128.21, 127.93, 81.16,

71.39, 65.42, 43.99, 25.51 ppm. HR-MS (APCI): calcd for C21H26O4 [M+H]+ : 343.1904.

Found: 343.1900.

114

((1R*,3S*,4S*,5R*)-4,5-bis(4-methoxybenzyloxy)cyclopentane-1,3-diyl)dimethanol (S-8):


chromatography (SiO2, CH2Cl2/MeOH 20:1) afforded S-8 as a colorless oil (1.81 g, 56%). 1H

NMR (500 MHz, CDCl3): δ = 7.26 (m, 4H), 6.85 (m, 4H), 4.50 (d, AB, J = 11.76 Hz, 2H),

4.41 (d, AB, J = 11.76 Hz, 2H), 3.79 (s, 6H), 3.65-3.58 (m, 4H), 3.49-3.45 (m, 2H), 2.39 (m,

2H), 1.92 (m, 1H), 1.83 (br s, 2H), 0.88 (m, 1H) ppm.13


159.49, 130.60, 129.79, 129.61, 114.02, 80.78, 70.93, 65.49, 55.50, 43.97, 25.51. IR (ν˜max):

3367 (b), 2924 (w), 2858 (w), 1448 (m), 1203 (m), 1084 (s), 735 (s), 696 (s) cm–1

.

((3aR*,4R*,6S*,6aS*)-2,2-di-tert-butyltetrahydro-3aH-cyclopenta[d][1,3,2]dioxasilole-

4,6-diyl)dimethanol (S-9):


chromatography (SiO2, CH2Cl2/MeOH 20:1) afforded S-9 as a white solid (0.643 g, 62 %).

m.p.: 99-103°C. 1H NMR (500 MHz, CDCl3): δ = 4.27-4.23 (m, 2H), 3.74 (d, J = 6.05 Hz,

2H), 2.15 (m, 2H), 1.86 (m, 1H), 1.06 (s, 9H), 1.06 (overlapped, m, 1H), 1.00 (s, 9H) ppm.

13C NMR (126 MHz, CDCl3): δ = 81.86, 65.15, 49.47, 28.35, 27.70, 27.03, 22.11, 19.66 ppm.

IR: ν˜max = 3298 (br), 2886 (m), 2858 (m), 1472 (m), 1065 (s), 1032 (s), 849 (m), 819 (m),

651 (m) cm–1

. HR-MS (ESI) calcd for C15H30O4Si [M+Na]+ : 325.18056. Found: 325.18060.

((1R*,3S*,4S*,5R*)-4,5-bis(triisopropylsilyloxy)cyclopentane-1,3-diyl)dimethanol (S10):

115


chromatography (SiO2, CH2Cl2/MeOH 20:1) afforded S-10 as a white solid (1.026 g, 80%).

m.p. = 107 – 109 °C. 1H NMR (500 MHz, CDCl3): δ = 4.03 (dd, J = 2.71, 7.36 Hz, 2H), 3.61

(ddd, J = 27.0, 10,5, 5.7 Hz, 4H), 2.26 (m, 2H), 2.05 (m, 1H), 1.67 (br s,-OH, 2H), 1.14 (m,

1H), 1.07 (m, 42H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 77.73, 65.14, 45.02, 25.75, 18.52

ppm. HR-MS (ESI): calcd for C25H54O4Si[M+Na]+: 497.34528. Found: 497.34518.

General procedure for TBDPS protection of cyclopantane intermediate

Oven-dried flask was charged with NaH (1.2 eq, 60 % dispersion in mineral oil) and

anhydrous THF (1 mL). Solution of starting material in anhydrous THF (10 mmol of starting

material/20 mL) was added to a suspension of NaH in THF. The reaction mixture was stirred

at 25 °C for 20 min and TBDPSCl (1.05 eq.) was added to the reaction mixture. The reaction

mixture was stirred under N2 atmosphere for 14 h. The reaction mixture was quenched by

addition of silica gel and adsorbed to a silica gel. Flash column chromatography afforded the

desired intermediate.

((3aS*,4S*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-

3aH-cyclopenta[d][1,3]dioxol-4-yl)methanol (146):


(SiO2, hexane/EtOAc 3:1) afforded 146 as a colorless oil (3.24 g, 76 %). 1H NMR (500 MHz,

CDCl3): δ = 7.64-7.62 (m, 4H), 7.42-7.34 (m, 6H), 4.39-4.36 (m, 1H), 4.31-4.29 (m, 1H),

3.73-3.58 (m, 4H), 2.29-2.20 (m, 4H), 2.06-2.01 (m, 1H), 1.47 (s, 3H), 1.37 (dd, J = 23.7,

10.9 Hz, 1H), 1.27 (s, 3H), 1.04 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 135.86,

135.85, 133.89, 129.88, 127.89, 112.62, 83.90, 82.91, 65.25, 65.14, 48.12, 47.36, 30.87,

116

27.91, 27.13, 25.43, 19.55. ppm. IR: ν˜max = 3456 (br), 2930 (w), 2857 (w), 1471 (w), 1427

(w), 1110 (s), 1060 (s), 701 (s), 504 (s) cm–1

. HR-MS (ESI): calcd for: C26H36O4Si [M+Na]+:

463.22571. Found: 463.22752.

((3aS*,4S*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-diphenyltetrahydro-

3aH-cyclopenta[d][1,3]dioxol-4-yl)methanol (S11):

Following general procedure using 0.571 g (1.75 mmol) of S-6. Flash column

chromatography (SiO2, hexane/EtOAc 5:1) afforded S-11 as a colorless oil (0.742 g, 75%). 1H

NMR (500 MHz, CDCl3): δ = 7.63-7.61 (m, 4H), 7.51-7.49 (m, 2H), 7.46-7.44 (m, 2H), 7.40-

7.38 (m, 2H), 7.36-7.25 (m, 10H), 4.38 (dd, J = 7.1, 4.9 Hz, 1H), 4.29 (dd, J = 7.1, 5.6 Hz,

1H), 3.68 (dd, J =6. 25, 1.92 Hz, 2H), 3.64 (m, 2H), 2.49 (m, 1H), 2.40 (m, 1H), 1.03 (s, 9H)

ppm. 13

C NMR (126 MHz, CDCl3): δ = 142.87, 142.48, 135.86, 135.84, 133.89, 129.87,

128.39, 128.35, 128.19, 128.12, 127.89, 126.67, 126.59, 113.14, 84.25, 83.93, 65.26, 65.01,

47.60, 47.00, 31.15, 27.10, 19.54. IR: ν˜max = 2930 (w), 1449 (w), 1427 (w), 1264 (m), 1105

(m), 1064 (m), 733 (s), 698 (s) cm–1

. HR-MS (ESI): calcd for C36H40O4Si [M+H]+

: 565.2769.

Found: 565.2762.

((1S*,2S*,3R*,4R*)-2,3-bis(benzyloxy)-4-((tert

butyldiphenylsilyloxy)methyl)cyclopentyl)methanol (193):

Following general procedure using 2.5 g (7.3 mmol) of S-7. Flash column chromatography

(SiO2, hexane/EtOAc 3:1) afforded 193 as a white solid (3.15 g, 74 %). m.p.: 91-95C. 1H

NMR (500 MHz, CDCl3): δ = 7.62-7.60 (m, 4H), 7.41-7.24 (m, 16H), 4.56 (dd, J = 11.9, 4.4

Hz, 2H), 4.43 (dd, J = 28.7, 11.9 Hz, 2H), 3.83 (m, 1H), 3.66-3.55 (m, 4H), 3.48 (dd, J = 10.2,

6.0 Hz, 1H), 2.50-2.46 (m, 1H), 2.38-2.36 (m, 1H), 1.93-1.86 (m, 2H), 1.03 (s, 9H) ppm. 13

C

NMR (126 MHz, CDCl3): δ = 138.86, 138.47, 135.86, 135.84, 133.75, 129.94, 128.63,

117

128.50, 128.10, 128.04, 127.94, 127.91, 127.69, 82.95, 79.35, 71.70, 71.15, 65.96, 65.34,

44.34, 43.77, 27.16, 25.44, 19.52 ppm. IR: ν˜max = 3486 (w), 2950 (w), 1469 (w), 1105 (m),

1064 (m), 698 (s), 493 (s) cm–1

. HR-MS (APCI): calcd for C37H44O4Si [M+Na]+: 603.29015.

Found: 603.29011.

((1S*,2S*,3R*,4R*)-4-((tert-butyldiphenylsilyloxy)methyl)-2,3-bis(4-

methoxybenzyloxy)cyclopentyl)methanol (194):

Following general procedure using 1.793 g (6.17 mmol) of starting material S-8. Flash

column chromatography (SiO2, hexane/EtOAc 5:1) afforded 194 as a yellowish wax (1.76 g,

58 %). 1H NMR (500 MHz, CDCl3): δ = 7.62-7.60 (m, 4H), 7.45-7.33 (m, 6H), 7.22-7.20 (m,

4H), 6.83-6.80 (m, 4H), 4.49 (dd, J =11.6, 2.8 Hz, 1H), 4.39 (d, J = 11.6 Hz, 1H), 4.32 (d, J =

11.6 Hz, 1H), 3.80 (m, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.66-3.58 (m, 2H), 3.56-3.45 (m, 3H),

2.45 (m, 1H), 2.35 (m, 1H), 1.87 (m, 1H), 1.03 (s, 9H), 1.03 (m, 1H) ppm. 13

C NMR (126

MHz, CDCl3): δ = 159.46, 159.34, 135.85, 135.83, 133.77, 130.96, 130.57, 129.94, 129.93,

129.66, 129.63, 127.93, 114.04, 113.93, 82.73, 78.88, 71.26, 70.73, 66.08, 65.41, 55.50,

55.47, 44.35, 43.72, 27.15, 25.43, 19.52 ppm. HR-MS (ESI): calcd for C39H48O6Si [M+Na]+:

663.3112. Found: 663.3313.

((3aS*,4S*,6R*,6aR*)-2,2-di-tert-butyl-6-((tert-

butyldiphenylsilyloxy)methyl)tetrahydro-3aH-cyclopenta[d][1,3,2]dioxasilol-4-

yl)methanol (S-12):


chromatography (SiO2, hexane/EtOAc 3:1) afforded S-12 as a colorless oil (0.107 g, 21%). 1H

NMR (500 MHz, CDCl3): δ = 7.66 (m, 4H), 7.40-7.34 (m, 6H), 4.26 (m, 1H), 4.20 (m, 1H),

3.85 (dd, J = 10.05, 4.76, 1H), 3.73 (m, 3H), 2.14 (m, 2H), 1.89 (m, 1H), 1.05 (s, 18H), 1.00

118

(s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 135.87, 135.85, 134.10, 129.80, 127.84,

82.28, 80.65, 65.58, 65.40, 49.66, 49.32, 28.79, 27.70, 27.12, 27.05, 22.11, 19.63, 19.56 ppm.

HR-MS (ESI): calcd for C31H48O4Si2[M+Na]+: 565.29833. Found: 563.29825.

((1S*,2S*,3R*,4R*)-4-((tert-butyldiphenylsilyloxy)methyl)-2,3-

bis(triisopropylsilyloxy)cyclopentyl)methanol (195):

Following general procedure with slight modification using 1.026 g (1.83 mmol) of S-10 and

3 eq. of NaH (5.49 mmol). Flash column chromatography (SiO2, hexane/EtOAc 15:1)

afforded 195 as a colorless oil (1.07 g, 82 %). 1H NMR (500 MHz, CDCl3): δ = 7.62 (m, 4H),

7.42-7.33 (m, 6H), 4.14 (m 1H), 3.92 (dd, J = 7.3, 3.5 Hz, 1H), 3.66 (m, 1H), 3.54 (m, 1H),

3.52 (m, 2H), 2.33 (m, 1H), 2.22 (m, 1H), 1.96 (m, 1H), 1.08-1.01 (m, 51H) ppm. 13

C NMR

(126 MHz, CDCl3): δ = 135.89, 135.86, 135.03, 133.85, 133.76, 129.87, 129.83, 127.95,

127.85, 77.84, 65.95, 65.57, 45.55, 44.76, 31.81, 27.14, 26.79, 25.83, 22.87, 19.41, 18.59,

18.53, 18.52, 18.47, 14.32, 13.41, 13.16 ppm. IR: ν˜max = 2941 (m), 2864 (m), 1463 (m),

1106 (s), 1065 (s), 882 (m), 700 (m) cm–1

. HR-MS (ESI): calcd for C41H72O4Si3 [M+Na]+

735.46306. Found: 735.46314.

((1R*, 2R*, 3S*, 4S*)-4-(hydroxymethyl)-2, 3-

bis((triisopropylsilyl)oxy)cyclopentyl)methyl pivalate (S-13):

DMAP (0.051 g, 0.421 mmol) and DIPEA (0.711 mL, 8.42 mmol) were added to a solution of

S-10 (2.00 g, 4.21 mmol) in anhydrous CH2Cl2 (40 mL). Pivaloyl chloride (0.518 mL, 4.21

mmol) was added dropwise and the reaction mixture was stirred at 25 °C for 24 h. The

reaction mixture was quenched with water (25 mL) and extracted with CH2Cl2 (2 x 50 mL).

The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure to

yield the crude product, which was purified by flash column chromatography on silica gel

(hexane/EtOAc 10:1) to afford S-13 as white crystals (1.29 g, 71 %). NMR (500 MHz,

119

CDCl3): δ = 4.08 (dd, J = 11.2, 6.0 Hz, 1H), 4.03 (app d, overlapped, 1H), 3.93 (dd, J = 11.2,

6.0 Hz, 1H), 3.62 (dd, J = 10.5, 6.2, Hz, 1H), 3.54 (dd, J = 10.5, 6.2 Hz, 1H), 2.38 (m, 1H),

2.27 (m, 1H), 2.05 (m, 1H), 1.18(s, 9H), 1.07 (m, 1H, overlapped, resolved by 1H-

13C HSQC

experiment), 1.07 (m, 42H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 178.78, 77.24

(overlapped with CDCl3 signal, resolved by 1H-

13C HSQC experiment), 76.65, 65.79, 65.14,

44.96, 42.10, 39.06, 27.44, 25.95, 18.53, 18.50, 18.48, 18.44, 13.2 ppm. IR (ν˜max): 2942

(m), 2865 (m), 1731 (m), 1713 (m), 1463 (m), 1143 (s), 881 (s), 677 (s) cm-1

. HR-MS (ESI):

calcd for C30H62O5Si2 [M+Na]+ 581.4033. Found: 581.4033.

General procedure for iodination of protected intermediates (without TIPS protecting

groups)

Iodine (1.1 eq.) was added to a cooled (0 °C, ice bath) solution of PPh3 (1.2 eq.) and

imidazole (3 eq.) in anhydrous CH2Cl2 (1 mmol of I2/5 mL). The reaction mixture was stirred

at 0 °C for 20 min. Solution of starting material (1.0 eq.) in anhydrous CH2Cl2 (1 mmol/8 mL)

was added to above mentioned mixture and the reaction mixture was stirred while allowed to

warm to 25°C for 3 h-14 h (followed by TLC). The solvent was removed under reduced

pressure and yellow residue was purified by flash column chromatography to afford the

corresponding iodide.

tert-butyl(((3aR*,4R*,6R*,6aS*)-6-(iodomethyl)-2,2-dimethyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane (147):


(SiO2, hexane/EtOAc 20:1 to 15:1) afforded 147 as a colorless oil (3.29 g, 85%). 1H NMR

120

(500 MHz, CDCl3): δ = 7.65-7.62 (m, 4H), 7.43-7.35 (m, 6H), 4.43 (dd, J = 6.9, 4.9 Hz, 1H),

4.15 (dd, J = 6.9 Hz, 1H), 3.68 (d, J = 5.59 Hz, 2H), 3.32 (dd, J = 9.9, 5.3 Hz, 1H), 3.22 (dd, J

= 9.9, 6.9 Hz, 1H), 2.28 (m, 1H), 2.16 (m, 1H), 2.08 (m, 1H), 1.46 (s, 3H), 1.46 (m, 1H), 1.27

(s, 3H), 1.05 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 135.88, 133.83, 133.81, 129.92,

127.92, 112.79, 85.46, 83.11, 64.81, 47.54, 46.92, 35.54, 27.89, 27.17, 25.49, 19.56, 10.37

ppm. IR: ν˜max = 2930 (m), 2857 (m), 1471 (w), 1210 (w), 1111 (m), 1069 (m), 702 (s) cm–1

.

tert-butyl(((3aR*,4R*,6R*,6aS*)-6-(iodomethyl)-2,2-diphenyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane (S-14)


chromatography (SiO2, hexane/EtOAc 40:1) afforded S-14 as a colorless oil (0.685 g, 78%).

1H NMR (500 MHz, CDCl3): δ = 7.63-7.61 (m, 4H), 7.50-7.48 (m, 2H), 7.45-7.39 (m, 4H),

7.36-7.26 (m, 10H), 4.42 (dd, J = 7.2, 5.04 Hz, 1H), 4.16 (dd, J = 7.2, 6.28 Hz, 1H), 3.72-3.63

(m, 2H), 3.33 (dd, J = 9.9, 5.6 Hz 1H), 3.23 (dd, J = 9.9, 7.0 Hz), 2.49 (m, 1H), 2.35 (m, 1H),

2.09 (m, 1H), 1.38 (m, 1H), 1.04 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 142.78,

135.87, 135.85, 133.80, 129.91, 128.43, 128.41, 128.23, 128.19, 127.91, 126.57, 126.49,

113.34, 86.00, 84.06, 64.95, 46.96, 46.65, 35.88, 27.12, 19.55, 10.06 ppm. IR (ν˜max): 2929

(w), 2856 (w), 1489 (w), 1471 (w), 1109 (m), 737 (m), 698 (s), 503 (m) cm-1

. HR-MS (ESI)

calcd for C39H39IO3Si[M+Na]+: 697.1603. Found: 697.1606.

(((1R*,2R*,3S*,4R*)-2,3-bis(benzyloxy)-4-(iodomethyl)cyclopentyl)methoxy)(tert-

butyl)diphenylsilane (S-15):


(SiO2, hexane/EtOAc 30:1 to 20:1) afforded S-15 as a colorless oil (2.97 g, 97%). 1H NMR

(500 MHz, CDCl3): δ = 7.63-7.61 (m, 4H), 7.42-7.26 (m, 16H), 4.54 (dd, J = 11.9, 8.3 Hz,

121

2H), 4.44 (dd, J = 16.1, 11.9 Hz, 2H), 3.86 (dd, J = 4.7, 3.5 Hz, 1H), 3.62 (dd, J = 10.3, 4.9

Hz 1H), 3.48 (dd, J =10.3, 6.2 Hz, 1H), 3.41-3.35 (m, 2H), 3.21 (dd, J = 9.8, 6.9 Hz), 2.37-

2.35 (m, 1H), 2.37-2.35 (m, 1H), 2.27-2.25 (m, 1H), 1.99-1.93 (m, 1H), 1.12-1.04 (m, 1H),

1.04 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 138.84, 138.56, 135.88, 133.72, 129.95,

128.58, 128.51, 128.15, 128.02, 127.95, 127.88, 127.70, 83.48, 79.32, 72.04, 71.39, 65.16,

44.11, 43.49, 30.02, 27.20, 19.52, 12.28 ppm. HR-MS (ESI): calcd for C26H35O3ISi [M+Na]+

573.12924. Found: 573.12920.

tert-butyl(((1R*,2R*,3S*,4R*)-4-(iodomethyl)-2,3-bis(4-

methoxybenzyloxy)cyclopentyl)methoxy)diphenylsilane (S-16)


chromatography (SiO2, hexane/EtOAc 30:1 to 20:1) afforded S-16 as a colorless oil (1.534 g,

74%). 1H NMR (500 MHz, CDCl3): δ = 7.63-7.61 (m, 4H), 7.42-7.33 (m, 6H), 7.24-7.19 (m,

4H), 4.47 (m, 2H), 4.40-4.34 (m, 2H), 3.83 (dd, J = 4.7, 3.5 Hz, 1H), 3.48 (dd, J = 6.09,

10.28, 1H), 3.35 (m, 1H), 3.19 (dd, J = 9.8, 6.9 Hz), 2.33 (m, 1H), 2.23 (m, 1H), 1.08 (m,

1H), 1.04 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 159.46, 159.34, 135.88, 135.86,

133.75, 130.97, 130.72, 129.94, 129.70, 129.60, 127.93, 113.99, 113.93, 83.11, 78.91, 71.63,

71.01, 65.19, 55.49, 44.12, 43.52, 30.01, 27.20, 19.52, 12.39 ppm. IR (ν˜max): 2918 (m),

2852 (m), 2360 (m), 1513 (s), 1248 (s), 1111 (s), 823 (m), 703 (m) cm–1

. HR-MS (ESI):

calcd for C39H47O3Si [M+Na]+ :773.2135. Found: 773.2136.

(3aR*,4R*,6R*,6aS*)-2,2-di-tert-butyl-4-((tert-butyldiphenylsilyloxy)methyl)-6-

(iodomethyl)tetrahydro-3aH-cyclopenta[d][1,3,2]dioxasilole (S-17):


chromatography (SiO2, hexane/EtOAc 10:1) afforded S-17 as a colorless oil (0.151 g, 54%

122

and 20% of starting material was recovered). 1H NMR (500 MHz, CDCl3): δ = 7.67-7.64 (m,

4H), 7.42-7.33 (m, 6H), 4.30 (m, 1H), 4.03 (m, 1H), 3.81 (dd, J =10.2, 4.6 Hz, 1H), 3.74 (dd,

J = 10.2, 5.9 Hz, 1H), 3.52 (dd, J = 9.8, 3.7 Hz, 1H), 3.26 (dd, J = 9.8, 7.1 Hz, 1H), 2.13 (m,

1H), 1.95 (m, 1H), 1.18 (m, 1H), 1.05 (s, 9H), 1.03 (s, 9H), 0.99 (s, 9H) ppm. 13

C NMR (126

MHz, CDCl3): δ = 135.91, 135.89, 134.07, 129.83, 127.87, 83.36, 81.15, 65.04, 49.16, 48.80,

33.40, 27.65, 27.15, 27.05, 22.12, 19.63, 19.58, 11.06 ppm. HR-MS (ESI): calcd for

C31H47O3ISi2 [M+Na]+

: 673.20006. Found: 673.20011.

General procedure for preparation of alkenes by HI elimination

DBU (2.7 eq.) was added to a solution of starting material in anhydrous toluene (0.5

mmol/mL). The reaction mixture was heated to 90C and stirred for 4 h under N2 (complete

conversion determined by 1H NMR and/or by TLC). The reaction mixture was cooled to 25

°C. The solvent was evaporated under reduced pressure and the brown residue was purified

by flash column chromatography to afford the desired alkene.

tert-butyl(((3aR*,4R*,6aS*)-2,2-dimethyl-6-methylenetetrahydro-3aH-


Following general procedure using 1.539 g (2.795 mmol) 147. Flash column chromatography

(SiO2, hexane/EtOAc 20:1) afforded 148 as a colorless oil (0.855 g, 72 %). 1H NMR (500

MHz, CDCl3): δ = 7.63-7.61 (m, 4H), 7.43-7.34 (m, 6H), 5.15 (m, 1H), 5.05 (m, 1H), 4.61 (d,

J = 5.62 Hz, 1H), 4.48 (d, J = 5.62 Hz, 1H), 3.46 (m, 2H), 2.76-2.71 (m, 1H), 2.36 (m, 1H),

2.13 (dm, J = 15.91, Hz, 1H), 1.45 (s, 3H), 1.30 (s, 3H), 1.03 (s, 9H) ppm. 13

C NMR (126

123

MHz, CDCl3): δ = 149.83, 135.83, 133.77, 133.75, 129.91, 129.90, 127.90, 112.65, 110.56,

82.74, 81.96, 64.62, 45.85, 32.74, 27.07, 26.99, 24.69, 19.46 ppm.

tert-butyl(((3aR*,4R*,6aS*)-6-methylene-2,2-diphenyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane (S-18):


chromatography (SiO2, hexane/EtOAc 20:1) afforded S-18 as a colorless oil (0.433 g, 78 %).

1H NMR (500 MHz, CDCl3): δ = 7.57-7.53 (m, 6H), 7.40-7.29 (m, 14H), 5.19 (m, 1H), 4.45

(d, J = 5.90 Hz, 1H), 4.39 (d, J = 5.9 Hz, 1H), 3.42 (m, 2H), 2.83 (m, 1H), 2.55 (m, 1H), 2.16

(m, 1H), 1.01 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 147.24, 138.94, 138.82, 135.85,

135.82, 134.02, 129.81, 129.79, 128.51, 128.45, 128.15, 128.00, 127.86, 127.65, 112.32,

110.93, 80.39, 79.53, 71.77, 69.65, 64.04, 43.66, 30.15, 27.11, 19.60 ppm. HR-MS (APCI):

calcd for C36H38O3Si [M+H]+

: 547.2663 Found: 547.2664.

(((1R*,2R*,3S*)-2,3-bis(benzyloxy)-4-methylenecyclopentyl)methoxy)(tert-

butyl)diphenylsilane (196):

Following general procedure using 2.97 g (4.3 mmol) of S15. Flash column chromatography

(SiO2, hexane/EtOAc 20:1) afforded 196 as a colorless oil (1.73 g, 72 %). 1H NMR (500

MHz, CDCl3): δ = 7.62-7.60 (m, 4H), 7.40-7.24 (m, 16H), 5.15 (m, 1H), 5.10 (m, 1H), 4.64

(d, J = 12.3 Hz, 1H), 4.56 (d, J = 12.3 Hz, 1H), 4.48-4.41 (m, 2H), 4.07 (d, J = 4.20 Hz, 1H),

3.78 (dd, J = 7.7, 4.3 Hz, 1H), 3.71 (ddd, J = 24.2, 10.2, 4.1 Hz, 2H), 2.64-2.57 (m, 2H),

2.26-2.24 (m, 1H), 1.01 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 147.24, 138.94,

138.82, 135.85, 135.82, 134.02, 129.81, 129.79, 128.51, 128.45, 128.15, 128.00, 127.86,

127.65, 112.32, 80.39, 79.53, 71.77, 69.65, 64.04, 43.66, 30.15, 27.11, 19.60 ppm. IR

124

(ν˜max): 1454 (w), 1427 (w), 1108 (m), 822 (w), 735 (m), 696 (s), 613 (m), 502 (s) cm-1

. HR-

MS (ESI) calcd for C37H42O3Si [M+Na]+: 585.27954. Found: 585.27949.

(((1R*,2R*,3S*)-2,3-bis(4-methoxybenzyloxy)-4-methylenecyclopentyl)methoxy)(tert-



chromatography (SiO2, hexane/EtOAc 20:1) afforded 197 as a colorless oil (0.900 g, 71 %).

1H NMR (500 MHz, CDCl3): δ = 7.61-7.59 (m, 4H), 7.40-7.31 (m, 6H), 7.27 (m, 2H), 7.19

(m, 2H), 6.85 (m, 2H), 6.78 (m, 2H), 5.12 (app s, 1H), 5.08 (app s, 1H), 4.57 (d, J = 11.72,

1H), 4.47 (d, J = 11.72, 1H), 4.40 (d, J = 12.19 Hz), 4.33 (d, J = 12.19 Hz), 4.03 (d, J = 4.28

Hz), 3.79 (s, 3H), 3.76 (s, 3H), 3.73 (m, 1H), 3.67 (m, 2H), 2.57 (m, 2H), 2.22 (m, 1H), 1.01

(m, 1H), 1.00 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 159.30, 147.44, 135.85, 135.83,

134.05, 134.01, 131.04, 130.98, 129.80, 129.69, 129.55, 127.85, 113.94, 113.88, 112.15,

79.98, 79.08, 71.34, 69.23, 64.09, 55.51, 55.48, 43.65, 30.18, 27.10, 19.60 ppm. IR: ν˜max =

2930 (m), 2857 (m), 1612 (w), 1512 (s), 1247 (s), 1111 (s), 703 (s) cm–1

. HR-MS (ESI) calcd

for C39H46O5Si [M+Na]+: 645.3012. Found: 645.3012.

(3aR*,4R*,6aS*)-2,2-di-tert-butyl-4-((tert-butyldiphenylsilyloxy)methyl)-6-

methylenetetrahydro-3aH-cyclopenta[d][1,3,2]dioxasilole (S-19):

Following general procedure using 0.151 g (0.232 mmol) of S17. Flash column

chromatography (SiO2, hexane/EtOAc 10:1) afforded S19 as a colorless oil (39 mg, 32 %). 1H

NMR (500 MHz, CDCl3): δ = 7.65-7.63 (m, 4H), 7.42-7.34 (m, 6H), 5.19 (app s, 1H), 5.06

(app s, 1H), 4.70 (d, J = 7.3 Hz, 1H), 4.41 (dd, J = 7.31, 4.01 Hz, 1H), 3.66 (m, 2H), 2.58 (m,

1H), 2.23-2.13 (m, 2H), 1.04 (s, 9H), 1.03 (s, 9H), 1.01 (s, 9H) ppm. 13

C NMR (126 MHz,

125

CDCl3): δ = 151.47, 135.87, 133.95, 129.85, 127.87, 110.05, 81.35, 79.98, 65.39, 48.54,

33.61, 29.92, 27.59, 27.22, 27.10, 22.14, 19.89, 19.54 ppm.

General procedure for preparation of alkenes via selenation-elimination sequence

2-nitrophenyl selenocyanate (1.8 eq., related to the starting material) and Bu3P (2.8 eq.) were

added to a solution of starting material in anhydrous THF (5 mmol of starting material/10

mL). Reaction mixture was stirred for 20 min to 12 h at 25 °C under N2 atmosphere (control

by TLC). After full conversion of selenation step (the selenide could be also isolated by flash

column chromatography in case of S-20, see the following text) was added aq. H2O2 30%

solution (3 eq.) to a cooled (0 °C, ice bath) reaction mixture. The reaction mixture was stirred

at 25°C for 14 h and quenched by sat. NaHCO3 solution, extracted by EtOAc. Organic

extracts were dried over Na2SO4 and the brown residue was purified by flash column

chromatography (SiO2, hexane) to afford the desired alkene.

((1S*,2R*,3R*)-3-((tert-butyldiphenylsilyloxy)methyl)-5-methylenecyclopentane-1,2-

diyl)bis(oxy)bis(triisopropylsilane) (198):


chromatography (SiO2, hexane) afforded 198 as a colorless oil (0.660 g, 80% over two steps).

1H NMR (500 MHz, CDCl3): δ = 7.65-7.63 (m, 4H), 7.42-7.33 (m, 6H), 5.08 (m 1H), 4.89

(m, 1H), 4.42 (app s, 1H), 4.15 (m, 1H), 3.59 (d, J = 6.61 Hz, 2H), 2.56 (dd, J = 16.6, 10 Hz,

1H), 2.33 (m, 1H), 2.06-2.02 (m, 1H), 1.12-1.01 (m, 52H) ppm. 13

C NMR (126 MHz, CDCl3):

δ = 150.60, 135.89, 135.83, 133.93, 133.79, 129.86, 129.80, 127.85, 107.81, 77.74, 76.31,

65.96, 45.48, 29.77, 27.11, 19.45, 18.51, 18.49, 18.46, 13.17, 13.01 ppm. HR-MS (ESI): calcd

for. C41H70O3Si3 [M+Na]+: 717.4520. Found: 717.4529.

126

((1S*,2R*,3R*)-3-((tert-butyldiphenylsilyloxy)methyl)-5-((2-

nitrophenylselanyl)methyl)cyclopentane-1,2-diyl)bis(oxy)bis(triisopropylsilane) (S-20):

Isolated by flash column chromatography (SiO2, hexane/EtOAc 30:1), yellowish oil. 1H NMR

(500 MHz, CDCl3): δ = 8.26 (dd, J = 8.3, 1.4 Hz, 1H), 7.65-7.60 (m, 4H), 7.54 (app d, J = 8.3

Hz, 1H), 7.46-7.33 (m, 7H), 7.29-7.25 (m, 1H), 4.27 (m, 1H), 3.86 (dd, J = 7.3, 3.4 Hz, 1H),

3.56 (dd, J = 10.3, 4.7 Hz, 1H), 3.50 (dd, J = 10.3, 5.7 Hz, 1H), 3.34 (dm, J = 7.9 Hz, 1H),

2.54 (m, spectral overlap, 2H), 2.18 (m, spectral overlap, 2H), 1.12-1.01 (m, 52H) ppm. 13

C

NMR (126 MHz, CDCl3): δ = 147.29, 135.89, 135.84, 133.78, 133.66, 133.60, 133.57,

129.93, 129.89, 129.45, 127.90, 126.61, 125.41, 80.36, 77.01, 65.78, 45.59, 41.40, 30.95,

29.71, 27.16, 22.87, 19.40, 18.57, 18.56, 18.54, 18.49, 13.41, 13.15 ppm.

((1R*,2R*,3S*)-4-methylene-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl pivalate (S-

21):


chromatography (SiO2, hexane/EtOAc 50:1) afforded S-21 as a pale yellow oil (0.820 g, 59 %

over two steps). 1H NMR (500 MHz, CDCl3): δ = 5.10 (m, 1H), 4.90 (m, 1H), 4.38 (d, J =

2.7 Hz, 1H), 4.15 (dd, J = 11.2, 5.9 Hz, 1H), 3.98 (dd, J = 5.0, 2.7 Hz, 1H), 3.95 (dd, J = 11.2,

5.9 Hz, 1H), 2.61 (app dd, J = 16.7, 10.2 Hz, 1H), 2.47 (m, 1H), 1.98 (dm, J = 16.7 Hz, 1H),

1.17 (s, 9H), 1.09-1.04 (m, 42 H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 178.75, 149.52,

109.37, 77.94, 76.41, 65.57, 42.19, 39.10, 29.94, 27.42, 18.50, 18.47, 18.45, 18.40, 13.07

ppm. IR: ν˜max = 2942 (m), 2865 (m), 1732 (m), 1463 (w), 1282 (w), 1139 (s), 881 (s), 678

(s) cm–1

. HR-MS (ESI): calcd for C30H60O4Si [M+H]+: 563.3930. Found: 563.3929.

127

General procedure for ozonolysis of exocyclic alkenes intermediates


cooled (-78°C) solution of starting material in CH2Cl2:MeOH (1:3, 3 mmol of starting

material/25 mL). After full conversion of ozonolysis (TLC, blue color persisted) was excess

of O3 removed by bubbling of N2 into the reaction mixture. Thiourea (2 eq.) was added into

reaction mixture and the reaction mixture was stirred for another 2-3 h at 25 °C (white

precipitate of thiourea oxidation byproducts appeared). The solvents were removed and the

solid residue was purified by flash column chromatography to afford the desired ketone.

(3aR*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyldihydro-3aH-


Following general procedure using 3.25 g (7.69 mmol) of alkene 148. Flash column

chromatography (SiO2, hexane/EtOAC 5:1) afforded 126 as a white crystalline solid (3 g,

92%). Yield 92%. See experimental section 7.1.4 for analytical data.

(3aR*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-diphenyldihydro-3aH-


128


chromatography (SiO2, hexane/EtOAC 5:1) afforded 184 as a white crystalline solid (0.398 g,

92%). m.p.: > 250 °C. 1H NMR (500 MHz, CDCl3): δ = 7.49-7.47 (m, 4H), 7.44 (m, 2H),

7.34-7.20 (m, 14H), 4.50 (d, J = 6.03, 1H), 4.29 (d, J = 5.6, 1H), 3.68 (dd, J = 10.08, 2.77 Hz,

1H), 3.51 (dd, J = 10.08, 3.26 Hz, 1H), 2.69 (m, 1H), 2.60 (m, 1H), 2.11 (d, J = 18.18 Hz,

1H), 0.91 (s, 9H) ppm. 13

C NMR (125 MHz): δ = 212.05, 141.74, 141.09, 135.93, 135.73,

132.84, 132.53, 130.21, 130.13, 128.69, 128.57, 128.48, 128.29, 128.06, 128.00, 126.58,

126.45, 112.05, 82.70, 79.51, 66.17, 39.64, 38.13, 27.04, 19.30 ppm. IR (ν˜max): 2961 (w),

1756 (s), 1104 (m), 1068 (s), 699 (s), 502 (m) cm-1

. HR-MS (ESI) calcd for:

C35H36O4Si[M+Na]+: 571.22571. Found: 571.2274.

(2R*,3R*,4R*)-2,3-bis(benzyloxy)-4-((tert-butyldiphenylsilyloxy)methyl)cyclopentanone

(199):

Following general procedure using 0.961 g (1.2 mmol) of alkene 196. Flash column

chromatography (SiO2, hexane/EtOAC 10:1) afforded 199 as a colorless oil (0.926 g, 96%).

1H NMR (500 MHz, CDCl3): δ = 7.57-7.53 (m, 4H), 7.43-7.24 (m, 16H), 4.79 (d, AB, J =

12.2 Hz, 1H), 4.60 (d, AB, J = 12.2 Hz, 1H), 4.59 (d, AB, J = 12.1 Hz, 1H), 4.52 (d, AB, J =

12.1 Hz, 1H), 4.03 (m, 2H), 3.76 (dd, J = 10.5, 4.1 Hz, 1H), 3.59 (dd, J = 10.5, 5.4 Hz, 1H),

2.63-2.59 (m, 1H), 2.50 (dd, J = 19.0, 9.8 Hz, 1H), 2.16 (dd, J = 19.0, 5.1 Hz, 1H), 0.98 (s,

9H) ppm. 13

C NMR (126 MHz): δ = 212.96, 138.30, 137.79, 135.79, 135.72, 133.27, 133.09,

130.12, 130.07, 128.62, 128.58, 128.19, 128.05, 128.04, 128.02, 127.99, 127.90, 80.46, 72.25,

71.94, 64.16, 39.98, 35.89, 27.07, 19.43 ppm. IR: ν˜max = 1717 (s), 1622 (w), 1105 (s), 736

(s), 502 (m) cm–1

. HR-MS (ESI): calcd for C36H40O4Si[M+Na]+ : 587.25881. Found:

587.25889.

(2R*,3R*,4R*)-4-((tert-butyldiphenylsilyloxy)methyl)-2,3-bis(4-

methoxybenzyloxy)cyclopentanone (200):

129


chromatography (SiO2, hexane/EtOAC 3:1) afforded 200 as a colorless oil (0.546 g, 86%). 1H

NMR (500 MHz, CDCl3): δ = 7.45-7.42 (m, 4H), 7.32-7.28 (m, 2H), 7.26-7.21 (m, 4H), 7.14-

7.12 (m, 2H), 7.09-7.07 (m, 2H), 6.72-6.99 (m, 4H), 4.57 (d, J = 11.74, 1H), 4.42 (d, J =

11.74, 1H), 4.39 (d, J = 11.74, 1H), 4.32 (d, J = 11.74, 1H), 3.89-3.85 (m, 2H), 3.66 (s, 3H),

3.65 (s, 3H), 3.63-3.60 (m, 1H), 3.45 (dd, J = 5.54, 10.71 Hz), 2.46 (m, 1H), 2.36 (dd, J =

19.0, 9.8 Hz), 2.03 (m, 1H), 0.87 (s, 9H) ppm. 13

C NMR (126 MHz): δ = 213.14, 159.60,

159.48, 135.78, 135.72, 133.32, 133.14, 130.38, 130.11, 130.06, 129.90, 129.81, 129.62,

128.03, 128.00, 114.04, 114.00, 71.78, 71.51, 64.13, 55.48, 55.45, 39.97, 35.94, 27.07, 19.44

ppm. IR: ν˜max = 2930 (w), 2856 (w), 1753 (s), 1611 (m), 1512 (s), 1246 (s), 1107 (s), 1034

(m), 821 (m), 702 (s) cm–1


: 647.27994. Found:

647.27991.

(2R*,3R*,4R*)-4-((tert-butyldiphenylsilyloxy)methyl)-2,3-

bis(triisopropylsilyloxy)cyclopentanone (201):


chromatography (SiO2, hexane/EtOAc 20:1) afforded 201 as a colorless oil (0.605 g, 92%).

1H NMR (500 MHz, CDCl3): δ = 7.62-7.60 (m, 4H), 7.43-7.34 (m, 6H), 4.53 (dm, J = 4.23

Hz, 1H), 4.43 (dd, J = 4.23, 1.48 Hz, 1H), 3.69 (dd, J = 10.6, 5.6 Hz, 1H), 3.56 (m, 1H), 2.46

(m, 1H), 2.39 (dd, J = 18.6, 10.1 Hz, 1H), 1.95 (m, 1H), 1.14-1.03 (m, 51H) ppm. 13

C NMR

(126 MHz, CDCl3): δ = 213.40, 135.79, 135.71, 133.23, 133.11, 130.14, 130.06, 128.04,

128.02, 79.07, 74.55, 65.51, 42.66, 34.05, 27.05, 19.37, 18.38, 18.35, 18.22, 12.86, 12.80

ppm. IR: ν˜max = 2942 (s), 2864 (s), 1762 (m), 1464 (m), 1104 (s), 1062 (s), 883 (s), 824 (s),

701 (s), 679 (s) cm–1

. HR-MS (ESI): calcd for: C40H68O4Si3[M+Na]+: 719.4323. Found:

719.4322.

((1R*,2R*,3R*)-4-oxo-2,3-bis(triisopropylsilyloxy)cyclopentyl)methyl pivalate (217)

130



1H NMR (500 MHz, CDCl3): δ = 4.39 (dd, J = 3.9, 1.5 Hz, 1H), 4.35 (m, J = 3.9 Hz, 1H),

4.11 (dd, J = 11.6, 8.6 Hz, 1H), 4.03 (dd, J = 11.6, 6.2 Hz, 1H), 2.60 (m, 1H), 2.49 (dd, J =

18.9, 10.1 Hz, 1H), 1.94 (dm, J = 18.9, 1H), 1.18 (s, 1H), 1.08-1.03 (m, 42H) ppm. 13

C NMR

(126 MHz, CDCl3): δ = 212.01, 178.52, 78.76, 74.25, 65.40, 39.64, 39.05, 34.29, 27.36,

18.33, 18.31, 18.29, 18.17, 12.81, 12.78 ppm. IR: ν˜max = 2942 (m), 2866, 1763 (m), 173 (s),

1463 (m), 1132 (s), 881 (s), 676 (s) cm–1

. HR-MS (APCI): calcd for: C29H58O5Si2 [M+H]+

543.3896. Found: 543.3892.

tert-butyl(((3aR*,4R*)-2,2-dimethyl-4,5-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-

yl)methoxy)diphenylsilane (150):


cooled (-78°C) solution of starting material 148 (0.304, 0.719 mmol) in anhydrous CH2Cl2

(30 mL). After full conversion of ozonolysis (TLC, blue colour persisted), excess of O3 was

removed by bubbling of N2 into the reaction mixture. Me2S (0.211 mL, 2.87 mmol, 4 eq.) was

added and the reaction mixture was allowed to warm to 25 °C and stirred for additional 3 h.

The reaction mixture was diluted with CH2Cl2 (20 mL), washed with brine (3x 30 mL), dried

over MgSO4, filtered, and the solvent was evaporated. The oily residue was purified by flash

column chromatography SiO2 (hexane/EA 5:1) to afford ketone 126 (eluted first) followed by

product 150 (eluted second), which was isolated as a colorless oil (0.140 g, 48%). 1H NMR

(500 MHz, CDCl3): δ = 7.62-7.59 (m, 4H), 7.44-7.37 (m, 6H), 5.75 (d, J = 3.6 Hz, 1H), 4.26

(m, 1H), 3.74 (dd, J = 10.6, 3.9 Hz, 1H), 3.67 (dd, J = 10.6, 4.9 Hz, 1H), 2.75 (dd, J = 16.9,

6.2 Hz 1H), 2.44 (m, 1H), 2.38 (dd, J = 16.9, 3.6 Hz, 1H), 1.49 (s, 3H), 1.39 (s, 3H) ppm. 13

C

131

NMR (126 MHz, CDCl3): δ = 168.90, 135.91, 135.79, 132.84, 132.55, 130.31, 130.25,

128.16, 128.14, 111.72, 100.24, 74.86, 64.67, 36.91, 28.98, 27.89, 27.00, 26.23, 19.37.

(2S*,3R*,4R*)-2,3-bis(benzyloxy)-4-(((tert-

butyldiphenylsilyl)oxy)methyl)cyclopentanone (216):

TEA (50µL, 0.355 mmol) was added to a solution of ketone 199 (0.134 g, 0.237 mmol) in

anhydrous CH2Cl2 (4 mL). The reaction mixture was stirred under N2 atmosphere at 25 °C for

48 h. Aprox. 70 % conversion was observed by 1H NMR. The reaction mixture contained also

approx. 10 % of product of elimination (207). The solvent was evaporated and the yellow

residue was purified by flash column chromatography (SiO2, hexane/EtOAc 10:1) to afford

compound 206 as a white semi-solid (0.070 g, 53 %). 1H NMR (500 MHz, CDCl3): δ = 7.63-

7.57 (m, 4H), 7.43-7.25 (m, 14H), 7.22-7.19 (m, 2H), 5.06 (d, J = 11.5 Hz, 1H), 4.74 (m, 2H),

4.54 (d, J = 11.5 Hz, 1H), 4.13-4.04 (m, 2H), 3.84 (dd, J = 10.4, 4.2 Hz, 1H), 3.72 (dd, J =

10.4, 3.3 Hz, 1H), 2.54-2.38 (m, 2H), 1.03 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3) : δ =

213.24, 138.38, 137.80, 135.81, 133.46, 133.43, 130.02, 128.69, 128.59, 128.48, 128.17,

127.99, 127.91, 87.28, 80.55, 77.48, 73.18, 73.09, 61.99, 39.62, 37.86, 27.12, 19.55 ppm. IR:

ν˜max = 1749 (s), 1452 (w), 1426 (w), 1105 (s), 698 (s), 503 (s) cm–1

. HR-MS (ESI): calcd

for C36H40O4Si[M+Na]+ : 587.25881. Found: 587.25890.

(±)-2-(benzyloxy)-4-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopent-2-enone (207):

Side product, which could be isolated from the most of the nucleophilic addition on ketone

199 or enol triflate formation. TLC hexane/EA 5:1, Rf = 0.5 (UV, CAM). 1H NMR (500

MHz, CDCl3): δ = 7.61-7.59 (m, 4H), 7.43-7.25 (m, 11H), 6.34 (d, J = 3 Hz, 1H), 4.90 (s,

2H), 3.65 (dd, J = 9.9, 5.7 Hz, 1H), 3.59 (dd, J = 9.9, 6.4 Hz, 1H), 2.97 (m, 1H), 2.51 (dd, J =

132

19.2, 6.3 Hz, 1H), 2.21 (dd, J = 19.2, 1.8 Hz, 1H), 1.03 (s, 9H) ppm. 13

C NMR (126 MHz,

CDCl3): δ =

201.77, 156.95, 135.96, 135.77, 135.75, 133.56, 130.02, 129.78, 128.80, 128.42,

127.97, 127.74, 71.98, 66.85, 37.51, 36.89, 27.03, 19.47 ppm. HR-MS (ESI): calcd for

C29H32O3Si [M+Na]+ : 479.20129. Found: 479.20130.

Experimental procedures for nucleophilic addition pathway

General procedure for nucleophilic addition of lithiated heterocycles

n-BuLi (1.5 eq., 1.6M in hexanes) was added dropwise to a cooled (-78C) solution of

heteroarylbromide (1.5 eq.) in anhydrous THF (1 mmol of starting material/1mL). The

reaction mixture was stirred at -78 °C for 10 min to 1 h (lithiation progress monitored by TLC

and/or by 1H NMR). Solution of ketone (1 eq.) in anhydrous THF (1 mmol/10 mL) was added

dropwise. The reaction mixture was stirred for 1 h at -78 °C and then allowed to warm to 25

°C, quenched with sat. aq. NH4Cl, and extracted with EtOAc. The combined organic extracts

were dried over Na2SO4, filtered, and concentrated in a vacuum. The residue was purified by

flash column chromatography to give the desired adduct.

(3aR*,4R*,6R*,6aR*)-4-(2,4-bis(benzyloxy)pyrimidin-5-yl)-6-((tert-

butyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol

(152):

133

Following general procedure using bromide 148 (0.256 g, 0.690 mmol) and ketone 126

(0.195 g, 0.460 mmol). Flash column chromatography (SiO2, hexane/EtOAc, 5:1) afforded

152 as a white wax (0.250 g, 76 %). 1H NMR (500 MHz, CDCl3): δ = 8.48 (s, 1H), 7.63-7.61

(m, 4H), 7.46-7.23 (m, 16H), 5.46 (AB d, J = 12.2 Hz, 1H), 5.40 (m, 2H), 5.32 (d, J = 12.2

Hz, 1H), 4.77 (d, J =7.7 Hz, 1H), 4.35 (dd, J = 7.7, 5.9 Hz, 1H), 3.71 (dd, J = 10.2, 5.3 Hz,

1H), 3.61 (dd, J = 10.2, 6.4 Hz, 1H), 3.47 (d, J = 1.4 Hz, 1H, -OH), 2.61 (m, 1H), 2.26 (m,

1H), 2.14 (m, 1H), 1.49 (s, 3H), 1.29 (s, 3H), 1.02 (s, 9H) ppm. 13

C NMR (126 MHz,

CDCl3): δ =

167.35, 164.37, 157.13, 136.96, 136.10, 135.84, 133.93, 133.89, 129.87, 128.77,

128.65, 128.50, 128.32, 128.26, 128.18, 127.88, 117.89, 114.69, 83.67, 82.62, 75.87, 69.31,

68.78, 65.17, 46.28, 41.21, 27.10, 26.69, 25.07, 19.54 ppm.

6-((3aR*,4R*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-4-hydroxy-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione

(156):

Following general procedure using 126 (0.300 g, 0.808 mmol) and 155 (0.228 g, 0.538

mmol). The crude product isolated as a mixture with a starting material (1:1) and was used in

the next step without further purification (separation of the product was at this stage

extraordinary difficult due to very similar polarity). Pd/C (10%, 2 mg, 0.0173 mmol) was

added to a solution of the crude adduct (120 mg) in EtOH (5 mL). The reaction mixture was

thoroughly purged with H2 and heated to 80 °C. The resulting mixture was stirred at 80 °C for

2 h under H2 atmosphere. The reaction mixture was cooled down, filtered through Celite and

washed with CH2Cl2/MeOH mixture (10:1). The solvents were evaporated and the residue

was purified by flash column chromatography (SiO2, CH2Cl2/MeOH, 20:1) to give product

156 as a yelowish solid (36 mg, 12 %). 1H NMR (500 MHz, DMSO-d6): δ = 10.98 (s, 1H, -

NH), 10.36 (s, 1H, -NH), 7.64-7.62 (m, 4H), 7.47-7.42 (m, 6H), 5.59 (s, 1H), 4.81 (d, J = 1.4

134

Hz, 1H, 1´-OH), 4.57 (d, J = 7.6 Hz, 1H), 4.39 (dd, J = 7.6, 4.7 Hz, 1H), 3.71 (ddd, J = 21.0,

10.0, 7.2 Hz, 1H), 2.57 (m, 1H), 2.05 (dd, J = 13.2, 6.9 Hz, 1H), 1.92 (m, 1H), 1.48 (s, 3H),

1.25 (s, 3H), 1.01 (s, 9H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ =

163.88, 158.31, 151.49,

134.99, 133.10, 129.79, 127.82, 113.94, 96.54, 83.88, 82.22, 76.41, 65.33, 45.50, 26.59,

26.03, 25.04, 18.82 ppm. IR (ν˜max): 3507 (w), 2954 (w), 1710 (s), 1660 (s), 1071 (s), 502 (s)

cm-1

. HR-MS (ESI) calcd for C29H36O6N2Si [M+Na]+: 559.22348. Found: 559.22350.


diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (S-

22):

Following general procedure using bromide 151 (0.152 mg, 0.409 mmol) and ketone 126

(0.150 g, 0.273 mmol). Flash column chromatography (SiO2, hexane/EtOAc 10:1) afforded

compound S-22 as a yellow glassy solid (0.170 g, 75 %). 1H NMR (500 MHz, CDCl3): δ =

8.40 (s, 1H), 7.57-7.54 (m, 4H), 7.42-7.21 (m, 20H), 7.16-7.05 (m, 6H), 5.39 (AB d, J = 12.2

Hz, 1H), 5.34 (m, 2H), 5.34 (d, J = 12.2 Hz, 1H), 4.78 (d, J =7.8 Hz, 1H), 4.26 (dd, J = 7.8,

4.3 Hz, 1H), 3.61 (dd, J = 10.1, 6.0 Hz, 1H), 3.55 (m, 1H), 3.37 (m, 1H, -OH), 2.85 (m, 1H),

2.12 (m, 2H) 2.05 (m, 1H), 1.80 (m, 1H) ppm. 13


167.36,

164.40, 157.28, 142.03, 141.09, 136.93, 135.98, 135.84, 135.80, 133.93, 133.87, 129.88,

128.82, 128.75, 128.66, 128.49, 128.47, 128.42, 128.38, 128.31, 128.22, 127.88, 126.43,

126.40, 117.73, 114.94, 84.31, 83.54, 76.33, 69.33, 68.71, 65.27, 45.45, 41.24, 27.06, 19.54

ppm. IR (ν˜max): 3507 (w), 1591 (m), 1558 (m), 1450 (m), 1064 (m), 694 (s) cm-1

. HR-MS

(ESI) calcd for C53H52O6N2Si [M+H]+ : 841.36674. Found: 841.36724.

1R*,2R*,3R*,4R*)-2,3-bis(benzyloxy)-1-(2,4-bis(benzyloxy)pyrimidin-5-yl)-4-((tert-

butyldiphenylsilyloxy)methyl)cyclopentanol (S-23):

135

Following general procedure using bromide 151 (0.116 g, 0.205 mmol) and 199 (0.114 g,

0.307 mmol). Flash column chromatography (SiO2, hexanes/EtOAc, 3:1) afforded S-23 as a

white wax (0.137 g, 78 %). 1

H NMR (500 MHz, CDCl3): δ = 8.61 (s,1H), 7.58-7.54 (m, 4H),

7.50-7.46 (m, 2H), 7.43-7.39 (m, 2H), 7.37-7.22 (m, 12H), 7.19-6.97 (m, 10H), 5.41 (s, 2H),

5.19 (d, AB, J = 11.9 Hz, 1H), 4.98 (d, AB, J = 11.9 Hz, 1H), 4.65 (s, 1H), 4.55 (d, AB, J =

11.9 Hz, 1H), 4.44 (m, 2H), 4.21 (d, J = 12.6 Hz, 1H), 4.18 (d, J = 6.0 Hz, 1H), 4.13 (s, 1H),

3.80 (dd, J = 5.8, 3.1 Hz, 1H), 3.40 (dd, J = 10.3, 5.8 Hz, 1H), 3.40 (dd, J = 10.3, 8.4 Hz, 1H),

2.55 (m, 1H), 1.91 (dd, J = 13.9, 9.1 Hz, 1H), 1.82 (dd, J = 13.9, 8.7 Hz, 1H), 1.01 (s, 9H)

ppm. 13

C NMR (126 MHz, CDCl3): δ = 166.50, 164.06, 158.21, 138.43, 137.89, 137.01,

135.82, 135.77, 133.82, 133.68, 129.94, 128.71, 128.63, 128.61, 128.49, 128.45, 128.30,

128.14, 128.04, 127.95, 127.90, 127.76, 127.72, 127.13, 116.77, 79.67, 79.48, 79.11, 72.36,

72.07, 69.22, 68.53, 65.48, 44.66, 37.31, 27.11, 19.39 ppm. IR (ν˜max): 2928 (w), 2858 (w),

1591 (m), 1560 (m), 1454 (m), 1109 (m), 1060 (m), 733 (s), 696 (s) cm-1

. HRMS (ESI):

calculated for C54H56N2O6Si [M+H]+: 857.3980. Found: 857.3992.

(1R*,2R*,3R*,4R*)-2,3-bis(benzyloxy)-4-((tert-butyldiphenylsilyloxy)methyl)-1-(4-

(dibenzylamino)pyrrolo[1,2-f][1,2,4]triazin-7-yl)cyclopentanol (S-24)

Following general procedure using bromide 202 (0.191 g, 0.485 mmol) and 199 (0.210 g,

0.373 mmol). Flash column chromatography (SiO2, hexanes/EtOAc, 5:1) afforded S-24 as a

yellow wax (0.078 g, 55 %). 1

H NMR (500 MHz, CDCl3): δ = 7.68 (s, 1H), 7.63-7.60 (m,

4H), 7.41-7.24 (m, 21H), 7.14-7.12 (m, 3H), 7.02-7.00 (m, 2H), 6.68 (d, J = 4.7 Hz, 1H), 6.48

136

(d, J = 4.7 Hz, 1H), 4.99 (m, 4H), 4.79 (d, J = 5.9 Hz, 1H), 4.60 (AB d, J = 12.0 Hz, 2H), 4.39

(m, 3H), 4.10 (m, 1H), 3.70 (m, 2H), 2.66 (m, 1H), 2.47 (dd, J = 13.7, 9.0 Hz, 1H), 2.05 (m,

1H), 1.03 (s, 9H) ppm. HR-MS (ESI) calcd for C56H58O4N4[M+H]+: 879.43001. Found:

879.43059.

General procedure for debenzylation

Pd/C (10%, 10 mol %) was added to a solution of starting material in EtOH (0.1 mmol/5 mL).

The reaction mixture was thoroughly purged with H2 and heated to 80C under H2 atmosphere

for 2-14 h (monitored by TLC and or by 1H NMR). The reaction mixture was cooled to 25C,

filtered through Celite and concentrated under reduced pressure. Crude product was purified

by flash column chromatography to afford the desired product.


dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione

(153):


chromatography (SiO2, CH2Cl2/MeOH, 10:1) afforded 153 as a yellowish solid (0.140 g, 96

%). 1H NMR (500 MHz, CDCl3): δ = 10.20 (d, J = 4.9 Hz, 1H, -NH), 9.77 (s, 1H, -NH), 7.66-

7.63 (m, 4H), 7.40-7.32 (m, 6H), 5.03 (d, J = 7.7 Hz, 1H), 4.62 (dd, J = 7.6, 5.4 Hz, 1H), 3.76

(m, 2H), 3.40 (d, J = 0.7 Hz, 1H, -OH), 2.59 (m, 1H), 2.49 (m, 1H), 1.90 (dd, J = 13.0, 6.05

Hz, 1H), 1.52 (s, 3H), 1.31 (s, 3H), 1.05 (s, 9H) ppm. 13


163.31, 153.09, 138.99, 135.85, 133.96, 133.89, 129.81, 127.85, 116.46, 114.66, 82.60, 82.03,

76.05, 64.83, 46.21, 40.67, 27.08, 26.62, 24.96, 19.53 ppm.

5-((3aR*,4R*,6R*,6aR*)-4-hydroxy-6-(hydroxymethyl)-2,2-diphenyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (185)

137

Following general procedure using 67 mg (0.111 mmol) of S-25. Flash column

chromatography (SiO2, CH2Cl2/MeOH 5:1) afforded 185 as colorless semi-solid (38 mg,

81%). 1H NMR (500 MHz, DMSO-d6): δ = 11.06 (br s, 1H, N-H), 10.85 (br s, 1H, N-H),

7.47-7.32 (m, 10H), 4.75 (d, J = 8.0 Hz, 1H), 4.64 (m, 1H), 4.32 (d, J = 4.2 Hz, 1H, -OH),

4.28 (dd, J = 7.9, 5.5 Hz, 1H), 3.46 (m, 2H), 2.62 (m, 1H), 2.27 (m, 1H), 1.74 (dd, J = 13.1,

6.8 Hz, 1H), 1.68 (dd, J = 12.2, 6.0 Hz, 1H) ppm. 13


163.37, 151.11, 142.06, 141.23, 138.77, 128.25, 128.02, 127.77, 126.53, 125.96, 114.12,

113.52, 82.83, 82.74, 76.04, 62.04, 44.87, 40.64 ppm. HR-MS (ESI) calcd for

C23H22N2O6[M+Na]+: 445.1359. Found: 445.1436.

5-((1R*,2R*,3R*,4R*)-1,2,3-trihydroxy-4-(hydroxymethyl)cyclopentyl)pyrimidine-

2,4(1H,3H)-dione (157):

Pd(OH)2 on charcoal (~15 % Pd, 9 mg, 0.013 mmol) was added to a solution of S-26 (82 mg,

0.133 mmol) in EtOH (8 mL). The reaction mixture was heated at 85 °C while H2 was

bubbled through the reaction mixture for 25 min. The hot suspension (no less than 60 °C,

otherwise the product may precipitate) was then filtered through PTFE syringe filter (0.2 μm)

and concentrated in a vacuum. The residue was rinsed with cold EtOH (0 °C, 3 × 1 mL) to

afford pure product 157 as a white solid (0.029 g, 84 % yield). m.p.: > 250 °C. 1H NMR (500

MHz, DMSO-d6): δ = 10.96 (bs, 1H, -NH), 10.71 (bs, 1H, -NH), 7.29 (s, 1H), 4.54 (s, 1H, -

OH), 4.42-4.50 (m, 3H, -3 x OH), 4.11 (m, 1H), 3.74 (dd, J = 10.5, 6.0 Hz, 1H), 3.43 (m, 1H),

3.34 (m, 1H), 2.13 (m, 1H), 1.89 (dd, J = 13.3, 9.7 Hz, 1H), 1.63 (dd, J = 13.3, 8.4 Hz, 1H)

138

ppm. 13

C NMR (126 MHz, DMSO-d6): δ = 163.32, 151.29, 138.64, 114.46, 78.29, 72.91,

72.60, 63.17, 46.74, 36.25 ppm. For 1H,

13C and

15N assignment see supporting information.

HRMS (ESI): calcd for C10H14N2O6 [M+H]+ = 257.0779. Found: 257.0775.

(1R*,2R*,3R*,4R*)-1-(4-(benzylamino)pyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-

(hydroxymethyl)cyclopentane-1,2,3-triol (203):


chromatography (SiO2, CH2Cl2/MeOH 5:1) afforded 203 as a yellow solid (53 mg, 80%). 1H

NMR (500 MHz, DMSO-d6): δ = 8.83 (br s, 1H, N-H), 7.86 (s, 1H), 7.35-7.29 (m, 4H), 7.23

(m, 1H), 6.94 (d, J = 4.4 Hz, 1H), 6.61 (d, J = 4.4 Hz, 1H), 4.94 (s, 1H, -OH), 4.72 (m, 3H,

overlapped), 4.55 (m, 2H, 2x - OH), 4.35 (m, 1H), 3.79 (dd, J = 11.4, 6.3 Hz, 1H), 3.51 (m,

1H), 3.41 (m, 1H), 2.24 (m, 1H), 2.08-1.98 (m, 2H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ

= 153.95, 146.57, 139.34, 133.72, 128.24, 127.16, 126.75, 114.87, 109.16, 99.90, 77.95,

74.02, 72.32, 62.90, 46.66, 42.58, 36.16 ppm. HR-MS (ESI): calcd for C19H22O4N4 [M+Na]+:

393.15333. Found: 393.15338.

(1R,2R,3R,4R)-1-(2,4-bis(benzyloxy)pyrimidin-5-yl)-4-((tert-

butyldiphenylsilyloxy)methyl)cyclopentane-1,2,3-triol (159):

.

PPTS (32 mg, 0.128 mmol) was added to a solution of starting material 152 (30 mg, 0.0428

mmol) in MeOH (1 mL). The reaction mixture was stirred at 25 °C for 72 h. The solvent was

139

evaporated and the residue was purified by flash column chromatography (SiO2,

CH2Cl2/MeOH 20:1) to afford product 159 as a white crystalline solid (10 mg, 35 %). 1H

NMR (500 MHz, DMSO-d6): δ = 8.44 (s, 1H), 7.59-7.57 (m, 4H), 7.46-7.31 (m, 13H), 7.26-

7.20 (m, 3H), 5.36 (d, J = 4.6 Hz, 1H), 4.81 (s, 1H), 4.71 (d, J = 7.5 Hz, 1H), 4.56 (d, J = 6.6

Hz, 1H), 4.14 (m, 1H), 3.70 (dd, J = 12.3, 6.6 Hz, 1H) 3.63 (dd, J = 9.9, 5.7 Hz, 1H), 3.48

(dd, J = 9.9, 7.1 Hz, 1H), 2.32 (m, 1H), 1.91 (dd, J = 9.1, 4.4 Hz, 1H), 0.97 (s, 9H) ppm. 13

C

NMR (126 MHz, DMSO-d6): δ = 166.42, 163.22, 157.01, 136.76, 136.02, 134.96, 134.92,

133.15, 129.72, 128.33, 128.24, 127.88, 127.82, 127.77, 118.14, 77.73, 73.67, 71.97, 68.19,

67.69, 65.55, 46.80, 36.79, 26.57, 18.78 ppm.

General procedure for TBDPS deprotection of nucleophilic addition products

TBAF (1M THF, 1.1-1.3 eq.) was added to a stirred solution of starting material in THF (0.3

mmol of starting material/3mL) reaction mixture was stirred for 14 h at 25 C. All volatiles

were removed under reduced pressure. Brown residue was purified by flash column

chromatography to afford the product.

5-((3aR*,4R*,6R*,6aR*)-4-hydroxy-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (154):

140

Following general procedure using 60 mg (0.111 mmol) of 153. Flash column

chromatography (SiO2, CH2Cl2/MeOH 15:1 to 10:1) afforded compound 154 as a colorless

solid (31 mg, 94%). m.p.: > 250C decomp. 1H NMR (500 MHz, DMSO-d6): δ = 11.03 (br s,

1H, -NH), 10.79 (br s, 1H, -NH), 7.36 (s, 1H), 4.79 (d, J = 7.7 Hz, 1H), 4.57 (m, 1H, -OH),

4.41 (dd, J = 7.6, 5.1 Hz, 1H), 4.10 (d, J = 1.6 Hz), 3.40 (m, 2H), 2.36 (m, 1H), 2.19 (m, 1H),

1.66 (dd, J = 12.9, 6.9 Hz, 1H), 1.45 (s, 3H), 1.23 (s, 3H) ppm. 13

C NMR (126 MHz, DMSO-

d6): δ =

163.30, 151.12, 138.59, 114.29, 113.17, 82.17, 82.03, 75.72, 62.53, 45.32, 40.04

(CH2-6´ - overlapped with DMSO, detected through 1H-

13C HSQC), 26.19, 25.11 ppm.

(3aR*,4R*,6R*,6aR*)-4-(2,4-bis(benzyloxy)pyrimidin-5-yl)-6-(hydroxymethyl)-2,2-

diphenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (S-25):


chromatography (SiO2, CH2Cl2/EtOAc 1:1) afforded compound S-25 as a colorless glass (85

mg, 73%). 1H NMR (500 MHz, CDCl3): δ = 8.48 (s, 1H), 7.46-7.42 (m, 6H), 7.36-7.20 (m,

14H), 5.41 (AB dd, J = 12.2, 0.8 Hz, 2H), 5.38 (m, 2H), 4.48 (d, J = 7.8 Hz, 1H), 4.58 (dd, J

= 7.8, 5.5 Hz), 3.67 (m, 1H), 3.53 (m, 1H), 3.44 (d, J = 1Hz, 1H, -OH), 2.83 (m, 1H), 2.24 (m,

1H), 2.13 (dd, J = 13.5, 7.2 Hz, 1H) ppm. 13


167.31, 164.47,

157.20, 141.71, 140.90, 136.89, 135.88, 128.96, 128.83, 128.67, 128.60, 128.52, 128.44,

128.36, 128.23, 126.51, 126.49, 117.58, 115.22, 84.50, 83.57, 76.39, 69.38, 68.98, 64.07,

45.60, 40.89 ppm. IR (ν˜max): 2929 (w), 1592 (m), 1560 (m), 1422 (s), 1066 (s), 695 (s) cm-

1. HR-MS calcd for C37H34O6N2[M+H]

+: 603.24896. Found: 603.24908.

(1R*,2R*,3R*,4R*)-2,3-bis(benzyloxy)-1-(2,4-bis(benzyloxy)pyrimidin-5-yl)-4-

(hydroxymethyl)cyclopentanol (S-26):

141

Following general procedure using substrate S-23 (188 mg, 0.219 mmol). Flash column

chromatography (SiO2, CH2Cl2/MeOH, 30:1) afforded product S-26 as a colorless glassy

solid (132 mg, 97 % yield). 1H NMR (500 MHz, CDCl3): δ = 8.62 (s, 1H), 7.51-7.49 (m, 2H),

7.41-7.25 (m, 11H), 7.23-7.11 (m, 5H), 7.01-6.98 (m, 2H), 5.46 (s, 1H), 5.26 (d, AB, J = 11.6

Hz, 1H), 4.96 (d, AB, J = 11.6 Hz, 1H), 4.56 (d, AB, J = 11.9 Hz, 1H), 4.49 (overlapped d,

2H), 4.25 (d, AB, J = 12.4 Hz, 1H), 4.16 (d, J = 5.9 Hz, 1H), 4.14 (br s, 1H), 3.74 (dd, J =

5.9, 4.0 Hz, 1H), 3.27 (dd, J = 10.7, 7.5 Hz, 1H), 3.06 (dd, J = 10.7, 5.3 Hz, 1H), 2.44 (m,

1H), 1.83 (dd, J = 8.9, 4.9 Hz, 1H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 166.63, 163.86,

157.50, 138.24, 137.73, 136.86, 135.61, 129.16, 128.92, 128.71, 128.66, 128.56, 128.43,

128.11, 128.10, 117.16, 80.12, 79.53, 78.88, 72.63, 72.37, 69.52, 69.22, 64.53, 44.17, 37.19,

27.15 ppm. HRMS (ESI): calculated for C38H38N2O6 [M+H]+: 619.2803. Found: 619.2798.

(1R*,2R*,3R*,4R*)-2,3-bis(benzyloxy)-1-(4-(dibenzylamino)pyrrolo[1,2-f][1,2,4]triazin-

7-yl)-4-(hydroxymethyl)cyclopentanol (S-27):


chromatography (SiO2, CH2Cl2/MeOH, 20:1) afforded product S-27 as a yellow glassy solid

(120 mg, 67 % yield). 1H NMR (500 MHz, CDCl3): δ = 7.74 (s, 1H), 7.37-7.27 (m, 15H),

7.11-7.08 (m, 3H), 6.99-6.96 (m, 2H), 6.74 (d, J = 4.7 Hz, 1H), 6.52 (d, J = 4.7 Hz, 1H), 5.00

(m, 4H, overlapped), 4.67 (m, 3H, overlapped), 4.47 (AB d, J = 12.2 Hz, 1H), 4.36 (s, 1´-OH,

1H), 4.36 (AB d, J = 12.2 Hz), 4.07 (dd, J = 5.2, 3.0 Hz, 1H), 3.68 (m, 2H), 2.64 (m, 1H),

142

2.38 (dd, J = 14.6, 7.0 Hz, 1H), 2.22 (dd, J = 14.6, 10.2 Hz, 1H) ppm. 13

C NMR (126 MHz,

CDCl3): 156.31, 146.33, 138.60, 138.03, 136.88, 133.60, 129.09, 128.62, 128.17, 128.07,

127.99, 127.89, 127.84, 127.72, 127.54, 115.04, 110.60, 105.07, 81.56, 79.82, 79.41, 72.61,

72.48, 65.69, 51.50, 43.53, 36.99 ppm. IR: ν˜max = 2928 (w), 2857 (w), 1587 (m), 1527 (m),

1110 (m), 728 (s), 695 (s) cm–1

.

(1R*,2R*,3R*,4R*)-2,3-bis(benzyloxy)-4-((tert-butyldiphenylsilyloxy)methyl)-1-

phenylcyclopentanol (S-28):

Following the experimental procedure described for synthesis of compound 128 using (0.134

g, 0.339 mmol) of starting material 199 and PhLi (1.8 M Bu2O, 283 L, 0.509 mmol). Flash

column chromatography (SiO2, hexane/EtOAc 15:1) afforded S-28 (151 mg) as a colorless oil

as impure product (we were not able not separate it from enone 207 and epimeric ketone

impurities). Only important and characteristic resonances of 13

C are listed. 13

C NMR (126

MHz, CDCl3): δ = 85.07 (C-2´or C-3´), 82.05 (C-quaternary – OH), 80.18 (C-2´or C-3´),

72.53 (CH2- Bn), 72.27 (CH2-Bn), 65.40 (C-5´- CH2), 44.69 (C-4´- CH), 40.63 (C-0´-CH2)

ppm. HRMS (ESI): calculated for C42H46O4 [M+Na]+: 665.30576. Found: 665.30576.

(1R*,2R*,3R*,4R*)-2,3-bis(benzyloxy)-4-(hydroxymethyl)-1-phenylcyclopentanol (214):

Following general procedure using crude starting material S-28 (151 mg) and TBAF (1M

THF, 0.282 mL, 0.282 mmol). Flash column chromatography (SiO2, hexane/EtOAc 2:1)

afforded product 214 as a colorless oil (0.060 g, 44 % over 2 steps). 1H NMR (500 MHz,

CDCl3): δ = 7.50-7.48 (m, 2H), 7.40-7.21 (m, 11H), 7.10-7.05 (m, 2H), 4.71 (d, AB, J = 11.9

143

Hz, 1H), 4.63 (d, AB, J = 11.9 Hz, 1H), 4.39 (d, AB, J = 12.1 Hz, 1H), 4.36 (d, AB, J = 12.1

Hz, 1H), 3.97 (m, 3H, overlapped, C-H2´, C-H3´, -OH), 4.00 (m, 1H), 3.71 (dd, J = 10.5, 4.8

Hz, 1H), 3.56 (dd, J = 10.5, 6.7 Hz, 1H), 2.63 (m, 1H), 2.22 (dd, J = 14.1, 8.9, 1H), 2.22 (dd,

J = 14.1, 9.5, Hz, 1H), 1.57-1.46 (br s, 1H, -OH, overlapped with solvent H2O) ppm. 13

C

NMR (126 MHz, CDCl3) : 144.97, 138.41, 137.80, 128.64, 128.50, 128.35, 128.21, 128.08,

127.99, 127.97, 126.99, 125.35, 84.75, 81.40, 80.39, 72.74, 72.37, 64.77, 44.71, 40.64 ppm.

HR-MS (ESI): calcd for C26H28O4 [M+Na]+ : 427.1885. Found: 427.1890.

Experimental procedures via triflate pathway

(3aR*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-6,6a-dihydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl trifluoromethanesulfonate (162):

LDA (2M solution in THF, 1.21 mL, 2.42 mmol) was added dropwise to a cooled solution (-

78 °C) of starting material 126 (0.792 g, 1.86 mmol) in anhydrous THF (6 mL). The reaction

mixture was stirred at -78 °C for 2 h. Solution of N-phenyl-bis(trifluoromethansulfonimide

(0.793 g, 2.22 mmol) in THF (5 mL) was added and the reaction mixture was stirred for 14 h

while allowed to warm to 25 °C. The reaction mixture was quenched by sat. NH4Cl (20 mL),

diluted with water (30 mL), and extracted with EtOAc (3 x 50 mL). Organic extracts were

dried over Na2SO4, filtered, and the solvent was evaporated. The dark brown residue was

purified by flash column chromatography (SiO2, hexane/EtOAc 20:1) to afford 162 as a

colorless oil (0.770 g, 74%). 1H NMR (500 MHz, CDCl3): δ = 7.62-7.58 (m, 4H), 7.43-7.36

(m, 6H), 5.62 (d, J = 2.5 Hz, 1H), 5.03 (dd, J = 5.8, 1.8 Hz, 1H), 4.54 (d, J =1.8 Hz, 1H), 3.75

(dd, J = 10.3, 4.9 Hz, 1H), 3.64 (dd, J = 10.3, 4.4 Hz, 1H), 2.92 (m, 1H), 1.43 (s, 3H), 1.33 (s,

3H), 1.03 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 149.15, 135.78, 133.22, 133.07,

130.16, 128.05, 128.04, 118.92, 118.90 (q, C-F

J = 320.7 Hz), 111.91, 80.99, 79.68, 64.22,

49.57, 27.27, 27.00, 25.87, 19.35. 19

F (470 MHz, CDCl3): δ = -73.30 ppm. IR: ν˜max = 2933

(w), 2860 (w), 1443 (m), 1424 (s), 1209 (s), 1129 (m), 1111 (m), 702 (m), 601 (m), 504 (m)

cm–. HR-MS (APCI): calcd for C26H31F3O6SSi [M+H]

+: 557.1635. Found: 557.1636.

144

(3R*,4R*,5R*)-4,5-bis(benzyloxy)-3-((tert-butyldiphenylsilyloxy)methyl)cyclopent-1-

enyl trifluoromethanesulfonate (205):

LDA (2M solution in THF, 108 L, 0.216 mmol) was added dropwise to a cooled solution (-

78°C) of starting material 199 (0.094 g, 0.166 mmol) in anhydrous THF (3 mL) and the

reaction mixture was stirred for 10 min at -78 °C. Solution of N-phenyl-

bis(trifluoromethansulfonimide) (0.178 g, 0.498 mmol) in THF (2 mL) was added and the

reaction mixture was stirred for 3.5 h (nearly 100% conversion by 1H NMR). The reaction

mixture was quenched (at -78 °C) by dropwise addition of sat. solution of NaHCO3 (10 mL)

and immediately extracted with Et2O (3x 10 mL). Organic extracts were dried over Na2SO4,

filtered, and the solvent was evaporated. The brown residue was purified by flash column

chromatography (freshly oven-dried neutral Al2O3, hexane/EtOAc 20:1) to afford 205 as a

colorless oil (0.038 g, 33%, followed by epimeric mixture of starting material – 43 mg, 46%).

1H NMR (500 MHz, CDCl3): δ = 7.62-7.59 (m, 4H), 7.45-7.24 (m, 16H), 5.87 (d, J = 2.1 Hz,

1H), 4.72 (d, AB, J = 11.3 Hz, 1H), 4.64 (d, AB, J =11.3 Hz, 1H), 4.61 (d, AB, J = 11.5 Hz,

1H), 4.56 (dd, J = 6.2, 1.7 Hz, 1H), 4.40 (d, AB, J = 11.5 Hz, 1H), 4.00 (m, 1H), 3.80 (dd, J

= 10.4, 3.8 Hz, 1H), 3.69 (dd, J = 10.4, 5.1 Hz, 1H), 3.07 (m, 1H), 1.02 (s, 9H) ppm. 13

C

NMR (126 MHz, CDCl3): δ = 147.72, 137.89, 137.84, 135.81, 135.78, 133.42, 133.31,

130.08, 130.04, 128.64, 128.62, 128.37, 128.11, 128.05, 127.99, 123.07, 118.79 (q, C-F

J =

320.7 Hz), 77.79, 76.98, 72.67, 71.88, 63.01, 49.56, 27.00, 19.42 ppm.19

F (470 MHz, CDCl3):

δ = -73.40 ppm.

(3R*,4R*)-2,3-bis(benzyloxy)-4-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopent-1-en-1-yl

trifluoromethanesulfonate (208):

NaHMDS (1M THF, 181 L, 0.181 mmol) was added dropwise to a cooled solution (-78°C)

of starting material 199 (0.085 g, 0.150 mmol) in anhydrous THF (3 mL). The reaction

145

mixture was stirred at -78 °C for 10 min. Solution of N-phenyl-

bis(trifluoromethansulfonimide) (0.070 g, 0.195 mmol) in THF (2 mL) was added and the

reaction mixture was stirred at -78 °C for 3.5 h (nearly 100% conversion by 1H NMR). The

reaction mixture was quenched by dropwise addition of sat. solution of NaHCO3 (10 mL) and

immediately extracted with Et2O (3 x 10 mL). Organic extracts were dried over Na2SO4,

filtered, and the solvent was evaporated. The brown residue was purified by column

chromatography (SiO2, hexane/EtOAc 20:1) to afford 208 as a colorless oil (0.038 g, 36%).

1H NMR (500 MHz, CDCl3): δ = 7.63-7.61 (m, 4H), 7.45-7.24 (m, 16H), 5.11 (d, AB, J =

11.8 Hz, 1H), 4.96 (d, AB, J = 11.8 Hz, 1H), 4.56 (d, AB, J =11.5 Hz, 1H), 4.49 (m, 1H),

4.45 (d, AB, J =11.5 Hz, 1H), 3.61 (dd, J = 10.3, 6.1 Hz, 1H), 3.52 (dd, J = 10.3, 7.0 Hz,

1H), 2.78 (ddd, J = 15.2, 8.6, 2.8 Hz, 1H), 2.43 (m, 1H), 2.25 (d, J = 15.2, 3.2 Hz, 1H), 1.05

(s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 144.04, 138.12, 136.77, 135.82, 133.44,

130.10, 130.09, 128.73, 128.65, 128.37, 128.03, 128.01, 127.98, 127.85, 127.59, 118.65 (q, C-

FJ = 320.6 Hz), 79.43, 71.91, 70.57, 65.29, 40.92, 29.19, 27.10, 19.48 ppm.

19F (470 MHz,

CDCl3): δ = -74.13 ppm.

General procedure for enol triflate formation from TIPS protected ketones

KHMDS (1 M THF solution, 1.2 eq.) was added to a solution of starting material and

Commins’ reagent (1.2 eq.) in THF (1 mmol of starting material/5 mL) at -78 °C and stirred

at -78 °C for 1 h. The reaction mixture was then allowed to warm to 25 °C, stirred for 1 h,

then quenched with saturated aqueous solution of NH4Cl, and extracted with EtOAc, dried

over MgSO4, filtered and concentrated under reduced pressure to yield the crude product,

which was purified by flash column chromatography to afford the desired enol triflate.

146

(3R*,4R*,5R*)-3-((tert-butyldiphenylsilyloxy)methyl)-4,5-

bis(triisopropylsilyloxy)cyclopent-1-enyl trifluoromethanesulfonate (218)

Following general procedure using 180 mg (0.257 mmol) of 201. Flash column

chromatography (SiO2, hexane/CH2Cl2, 10:1) afforded product 218 as a colorless oil (154 mg,

72 % yield). 1H NMR (500 MHz, CDCl3): δ = 7.62-7.59 (m, 4H), 7.44-7.34 (m, 6H), 5.71 (d,

J = 2.1 Hz), 4.65 (dd, J = 4.88, 0.54 Hz, 1H), 4.20 (app t, J = 5.15 Hz, 1H), 3.80 (dd, J =

10.4, 3.8 Hz, 1H), 3.67 (dd, J = 10.4, 3.6 Hz, 1H), 2.88 (m, 1H), 1.09-0.95 (51H) ppm. 13

C

NMR (126 MHz, CDCl3): δ = 150.41, 135.93, 135.83, 133.47, 133.27, 130.06, 129.98,

127.95, 120.29, 118.86 (q, C-F

J = 320.9 Hz), 75.55, 74.10, 62.81, 50.38, 27.08, 19.34, 18.40,

18.39, 18.32, 13.22, 13.18 ppm. 19

F (470 MHz, CDCl3): δ = -73.29 ppm. IR: ν˜max = 3326

(m), 2866 (m), 1426 (m), 1602 (s), 1246 (s), 1140 (s), 1111 (s), 882 (s), 739 (s) cm–1

. HR-MS

(ESI): calcd for: C41H67O6Si3SF3 [M+Na]+: 851.3810. Found: 851.3810.

(3R*,4R*,5R*)-3-((ethoxymethoxy)methyl)-4,5-bis(triisopropylsilyloxy)cyclopent-1-enyl

trifluoromethanesulfonate (219):

Following general procedure using 0.360 g, (0.696 mmol) of 216. Flash column


HR-MS (ESI): calcd for C28H55O7Si2SF3 [M+Na]+: 671.3061. Found: 671.3057.

147

((1R*,4R*,5R*)-3-(trifluoromethylsulfonyloxy)-4,5-bis(triisopropylsilyloxy)cyclopent-2-

enyl)methyl pivalate (220):

Following general procedure using 0.550 g, (1.01 mmol) of 217. Flash column

chromatography (SiO2, hexane/EtOAc 50:1) afforded 220 as a colorless wax (0.540 g, 79 %).

1H NMR (500 MHz, CDCl3): δ = 5.69 (d, J = 1.7 Hz), 4.59 (app d, J = 4.8 Hz, 1H), 4.45 (dd,

J = 11.6, 3.4 Hz, 1H), 4.16 (dd, J = 6.3, 4.9 Hz, 1H), 4.04 (dd, J = 11.6, 4.1 Hz, 1H), 3.06 (m,

1H), 1.15 (s, 9H), 1.10-1.02 (m, 42H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 178.50, 150.91,

120.91, 118.79 (q, C-F

J = 319.8 Hz), 75.51, 74.24, 61.76, 47.29, 39.09, 27.27, 18.39, 18.36,

18.33, 18.30, 13.27, 13.08 ppm. 19

F (470 MHz, CDCl3): δ = -73.35 ppm. IR (ν˜max): 2944

(m), 2867 (m), 1735 (m), 1427 (m), 1211 (s), 1138 (s), 882 (m), 825 (m), 682 (m), 609 (m)

cm-1

.

General procedure for Suzuki coupling of enol triflates and (hetero)aryl boronic acids

Hetero(aryl) boronic acid or boronate (1.5 eq.) and K3PO4 (3 eq.) were added to a solution of

triflate in DME/H2O 4:1 (0.1 mmol of starting material/mL). Reaction mixture was

thoroughly flushed with N2 or Ar for 5 minutes and Pd(dppf)Cl2.CH3CN (10 mol%) was added

to the reaction mixture. The reaction mixture was then heated to 80 °C and stirred for 2-14 h

under N2. After cooling down to 25C was the reaction mixture partitioned between H2O and

EtOAc. Organic extracts were dried over Na2SO4, evaporated, and the brown residue was

purified by flash column chromatography to afford the product.

148

tert-butyl(((3aR*,4R*,6aS*)-2,2-dimethyl-6-phenyl-4,6a-dihydro-3aH-


Following general procedure using 0.441 g (0.792 mmol) of triflate 162 and PhB(OH)2 (145

mg, 1.18 mmol). Flash column chromatography (SiO2, hexane/EtOAc 20:1) afforded 163 as a

colorless oil (0.326 g, 85%). 1H NMR (500 MHz, CDCl3): δ = 7.63-7.58 (m, 6H), 7.40-7.27

(m, 9H), 6.03 (d, J = 2.62 Hz, 1H), 5.48 (dd, J = 5.8, 1.8 Hz, 1H), 4.69 (dm, J =5.8 Hz, 1H),

3.82 (dd, J = 10.3, 4.9 Hz, 1H), 3.67 (dd, J = 10.3, 4.4 Hz, 1H), 3.07 (m, 1H), 1.40 (s, 3H),

1.37 (s, 3H), 1.00 (s, 9H) ppm. 13

C NMR (125 MHz, CDCl3): δ = 143.82, 135.86, 135.81,

134.81, 133.78, 133.62, 129.92, 128.65, 128.11, 127.91, 127.88, 126.56, 110.57, 85.40, 81.89,

65.23, 53.80, 27.73, 27.06, 26.23, 19.45 ppm. HR-MS (ESI) calcd for C31H36O3Si[M+Na]+:

507.23259. Found: 507.23253.

(((1R*,4S*,5R*)-4,5-bis(benzyloxy)-3-phenylcyclopent-2-enyl)methoxy)(tert-


Following general procedure using crude triflate 205 (0.295 g, 0.423 mmol) and PhB(OH)2

(77 mg, 0.634 mmol). Flash column chromatography (SiO2, hexane/EtOAc 20:1) afforded

209 as a colorless oil. (0.170 g, 64%). 1H NMR (500 MHz, CDCl3): δ = 7.64-7.62 (m, 4H),

7.53-7.51 (m, 2H), 7.41-7.22 (m, 19H), 6.32 (d, J =2.1 Hz, 1H), 5.09 (dd, J = 6.1, 1.4 Hz,

1H), 4.81 (d, AB, J = 11.6 Hz, 1H), 4.61 (d, AB, J =10.7 Hz, 1H), 4.56 (d, AB, J =11.6 Hz,

1H), 4.55 (d, AB, J = 10.7 Hz, 1H), 4.04 (m, 1H), 3.92 (dd, J = 10.1, 4.3 Hz, 1H), 3.73 (dd, J

= 10.1, 6.0 Hz, 1H), 3.22 (m, 1H), 1.02 (s, 9H) ppm. 13


140.50, 139.07, 138.72, 135.90, 135.86, 135.51, 133.95, 133.87, 131.53, 129.92, 129.90,

128.64, 128.54, 128.52, 128.42, 128.12, 127.93, 127.86, 127.79, 127.62, 126.22, 80.27, 80.16,

72.65, 69.50, 64.63, 53.36, 27.13, 19.55 ppm.

149

((1R,2S,5R)-5-((tert-butyldiphenylsilyloxy)methyl)-3-phenylcyclopent-3-ene-1,2-

diyl)bis(oxy)bis(triisopropylsilane) (S-29):

Following general procedure using 0.108 g (0.130 mmol) of triflate 201 and Ph(BOH)2 (32

mg, 0.260 mmol). Flash column chromatography (SiO2, hexane to hexane/EtOAc 50:1)

afforded impure (S-29) as a colorless oil (0.074 g, 75 %). 1H NMR (500 MHz, CDCl3): Only

important resonances are listed: δ = 6.01 (d, J = 1.7 Hz, 1H), 5.06 (d, J = 4.4 Hz, 1H), 4.10

(dd, J = 6.8, 4.4 Hz, 1H), 3.95 (dd, J = 10.1, 3.9 Hz, 1H), 3.69 (dd, J = 10.1, 7.0 Hz, 1H), 3.08

(m, 1H) ppm. HR-MS (ESI): calcd for C46H72O3Si3[M+Na]+: 779.46815. Found: 779.46812.

2,4-bis(benzyloxy)-5-((3aS*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-

dimethyl-6,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine (164):

Following general procedure using triflate 162 (0.380 g, 0.683 mmol) and crude 2,4-

bis(benzyloxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine S-30 (0.754 mmol,

see text below for its preparation). Flash column chromatography (SiO2, hexane/EtOAc 10:1

to 5:1) afforded 164 as a colorless oil (0.196 g, 41%). 1H NMR (500 MHz, CDCl3): δ = 8.54

(s, 1H), 7.59-7.52 (m, 4H), 7.48-7.44 (m, 2H), 7.40-7.25 (m, 14H), 6.29 (d, J =2.7 Hz, 1H),

5.49-5.45 (m, 3H, spectral overlap), 5.43 (s, 2H), 4.59 (d, J =5.8 Hz, 1H), 3.76 (dd, J = 10.1,

4.8 Hz, 1H), 3.61 (dd, J = 10.1, 4.8 Hz, 1H), 3.04 (m, 1H), 1.36 (s, 3H), 1.33 (s, 3H), 1.27 (s,

6H), 0.95 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 168.47, 163.41, 157.79, 136.93,

136.36, 135.97, 135.81, 135.78, 133.74, 133.59, 131.93, 129.89, 128.77, 128.67, 128.33,

128.22, 128.11, 127.89, 127.85, 110.56, 85.67, 81.09, 69.42, 68.98, 65.14, 54.19, 27.69,

27.00, 26.09, 19.42 ppm. IR: ν˜max = 2930 (w), 1586 (m), 1552 (m), 1427 (s), 1335 (w),

150

1239 (w), 1111 (m), 700 (s), 504 (s) cm–1

. HR-MS (APCI): calcd for C43H46N2O5Si:

699.3249. Found: 699.3246.

2,4-bis(benzyloxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (S-30):

n-BuLi (1.6 M hexanes, 0.565 mL, 0.905 mmol) was added to a cooled (-78C) solution of

151 (0.280 g, 0.754 mmol) in anhydrous THF (5 mL). The reaction mixture was stirred at -78

°C for 10 min. (lithiation complete according to the TLC and 1H NMR). Solution of 2-

isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.140 mL, 1.0556 mmo) in THF (2 mL)

was added dropwise and the reaction mixture was stirred for 40 min at -78 °C. The reaction

mixture was quenched with sat. aq. NH4Cl (20 mL) and extracted with CH2Cl2 (3 x 10 mL).

The organic extracts were dried over Na2SO4, filtered, and concentrated in a vacuum to afford

crude product S-30 (mixture of boronate esters and boronic acid) as a yellow oil. This

material was used without further purification.

6-((3R,4R,5S)-3-((ethoxymethoxy)methyl)-4,5-bis(triisopropylsilyloxy)cyclopent-1-enyl)-

N,N-bis((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[1,2-a]pyrazin-1-amine (224):

Following general procedure using triflate 219 (0.184 g, 0.284 mmol) and boronic acid 223

(0.155 g, 0.354 mmol). Flash column chromatography (SiO2, hexane/EtOAc 30:1 to 20:1)

afforded 224 as a yellow semi-solid (0.191 g, 75%).1H NMR (500 MHz, CDCl3): δ = 8.00 (s,

1H), 6.98 (d, J = 4.7 Hz, 1H), 6.91 (app s, 1H), 6.74 (d, J = 4.7 Hz, 1H), 5.25-5.16

(overlapped m, 6H, -OCH2-), 4.68 (q, J = 6.7 Hz, 2H), 4.09 (dd, J = 7.4, 4.3 Hz, 1H), 3.99

(dd, J = 9.5, 3.4 Hz, 1H), 3.71-3.50 (m, 8H), 3.28 (m, 1H), 1.19 (t, J = 6.7 Hz, 3H), 1.12 (m,

21H), 1.02-0.80 (m, 21H), -0.01 (s, 18H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 156.11,

151

146.38, 134.90, 134.72, 127.52, 115.68, 111.82, 105.70, 95.58, 78.17, 77.87, 76.82, 67.45,

66.18, 63.42, 49.82, 18.60, 18.51, 18.47, 18.43, 18.30, 18.22, 15.36, 13.73, 13.18, 12.87, -

1.15 ppm. IR: ν˜max = 2944 (w), 2865 (w), 1582 (w), 1512 (m), 1373 (w), 1248 (m), 1012

(s), 833 (s) cm–1

. HR-MS (APCI): calcd for C45H88N4O6Si4[M+H]+ : 893.5854. Found:

893.5851.

((1R,4S,5R)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-

f][1,2,4]triazin-7-yl)-4,5-bis(triisopropylsilyloxy)cyclopent-2-enyl)methyl pivalate (225):

Following general procedure using triflate 220 (0.162 g, 0.240 mmol) and boronic acid 223

(0.137 g, 0.312 mmol). Flash column chromatography (SiO2, hexane/EtOAc 20:1) afforded

225 as a yellow semi-solid (0.164 g, 75%). 1H NMR (500 MHz, CDCl3): δ = 7.98 (s, 1H),

6.98 (d, J = 4.8 Hz, 1H), 6.76 (app s, 1H), 6.74 (d, J = 4.8 Hz, 1H), 5.24-5.17 (m, 5H), 4.64

(dd, J = 11.3, 3.4 Hz, 1H), 4.12 (dd, J = 7.5, 4.3 Hz, 1H), 4.05 (dd, J = 11.3, 5.4 Hz, 1H),

3.67 (m, 4H), 1.12 (m, 30H), 1.07-0.79 (m, 29H), -0.01 (s, 18H) ppm. 13

C NMR (126 MHz,

CDCl3): δ = 178.67, 156.18, 146.49, 135.40, 133.27, 127.22, 115.79, 111.69, 105.51, 78.01,

77.85, 76.80, 66.17, 63.07, 49.09, 39.09, 27.41, 18.56, 18.49, 18.43, 18.41, 13.71, 13.12, -

1.17 ppm. IR: ν˜max = 2945 (w), 2858 (w), 1732 (s), 1582 (s), 1513 (s), 1248 (w), 1142 (s),

1079 (s), 855 (s), 681 (m) cm-1

. HR-MS (ESI): calcd for C47H91O6Si4 [M+H]+: 919.6012.

Found: 919.6012.

General procedure for hydrogenation of fully protected intermediates

152

Pd/C (10%, 10 mol %) or Pd(OH)2 (10% Pd basis, 10 mol %) was added to a solution of

starting material in EtOH (1 mmmol of starting material /5 mL). Ammonium formate was in

some cases employed as source of H2. The reaction mixture was thoroughly flushed with H2

and reaction mixture was heated to 80 °C and stirred under H2 atmosphere. Reaction progress

was monitored by TLC and/ or by 1H NMR. After completion of hydrogenation was reaction

mixture cooled to 25 °C, filtered through Celite, and washed with CH2Cl2. The solvents were

evaporated and the residue was purified by flash column chromatography to afford the

product.

tert-butyl(((3aR*,4R*,6R*,6aS*)-2,2-dimethyl-6-phenyltetrahydro-3aH-


Following general procedure using 163 (0.140 g, 0.288 mmol) and 10% Pd/C (30 mg, 0.028

mmol). Flash column chromatography (SiO2, hexane/EtOAc 20:1) afforded 166 as a colorless

oil (0.120 g, 86 %).1H NMR (500 MHz, CDCl3): δ = 7.68-7.63 (m, 6H), 7.44-7.36 (m, 6H),

7.32-7.26 (m, 4H), 7.23-7.18 (m, 1H), 4.60 (m, 1H), 4.54 (m, 1H), 3.64 (m, 2H), 3.21 (m,

1H), 2.35 (m, 2H), 1.85 (dd, J = 11.8, 6.3 Hz, 1H), 1.45 (s, 3H), 1.25 (s, 3H), 1.07 (s, 9H)

ppm. 13

C NMR (126 MHz, CDCl3): δ = 139.65, 135.87, 135.83, 133.66, 129.96, 129.06,

128.21, 127.97, 126.73, 110.10, 83.93, 82.90, 65.18, 48.00, 47.14, 31.45, 27.14, 26.52, 24.24,

19.47 ppm. HR-MS (ESI): calcd for C31H38O3Si [M+Na]+ : 509.2492. Found: 509.2491.

153

((3aS*,4R*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-

3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (173):

10% Pd/C (0.030 g, 0.028 mmol, 10 mol%) and ammonium formate (0.106 g, 1.68 mmol)

were added to a solution of compound 172 (0.196 g, 0.28 mmol) in EtOH (10 mL). The

reaction mixture was thoroughly flushed with H2 and heated up to 80 C and stirred under N2

atmosphere for 6 h. After cooling down was reaction mixture diluted with CH2Cl2 (30 mL)

and filtered through a pad of Cellite. Celite was further washed with CH2Cl2 (30 mL). The

filtrate was evaporated to dryness and the brown residue was purified by flash column

chromatography (SiO2, CH2Cl2/MeOH 20:1) to afford 173 as a colorless semi-solid (0.120 g,

82%). 1H NMR (500 MHz, CDCl3): δ = 9.54 (br s, 1H, N-H), 8.97 (br s, 1H, N-H), 7.65-7.61

(m, 4H), 7.43-7.34 (m, 6H), 7.10 (d, J = 5.5 Hz, 1H), 4.69 (m, 1H), 4.46 (d, J = 5.5 Hz, 1H),

3.56 (m, 2H), 3.18 (m, 1H), 2.29 (m, 1H), 1.91 (m, 1H), 1.68 (dd, J = 12.2, 6.0 Hz, 1H), 1.39

(s, 3H), 1.25 (s, 3H), 1.04 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 164.10, 152.29,

137.52, 135.84, 135.81, 133.64, 129.97, 127.98, 113.01, 110.02, 83.32, 80.39, 64.42, 46.64,

38.43, 29.04, 27.12, 26.51, 24.28, 19.43 ppm.

5-((3aS*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-6,6a-dihydro-

3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (158):

Note if the reaction was stopped after 40 min (not full conversion according to the TLC) and

besides desired 173 product 158 could be in small amount isolated from reaction mixture by

flash column chromatography (SiO2, CH2Cl2/MeOH 20:1) and was fully characterized by

NMR. 1H NMR (500 MHz, CDCl3): δ = 9.63 (d, J = 5.3 Hz, 1H, N-H), 8.88 (br s, 1H, N-H),

7.65 (d, J = 5.8 Hz, 1H), 7.63-7.58 (m, 4H), 7.42-7.32 (m, 6H), 6.76 (d, J = 2.6 Hz, 1H), 5.22

154

(d, J = 5.8 Hz, 1H), 4.63 (app d, J = 5.7 Hz, 1H), 3.82 (dd, J = 10.2, 4.4 Hz, 1H), 3.59 (dd, J

= 10.2, 5.0 Hz), 3.07 (m, 1H), 1.36 (s, 3H), 1.35 (s, 3H), 0.99 (s, 9H) ppm. 13

C NMR (126

MHz, CDCl3): δ = 162.61, 151.54, 137.63, 135.89, 135.81, 134.32, 133.71, 133.47, 132.25,

129.94, 127.94, 127.92, 110.85, 109.82, 85.73, 81.05, 64.89, 54.33, 27.71, 27.03, 26.16, 19.44

ppm. mp. = 223-228C. IR: ν˜max = 1707 (s), 1679 (s), 1447 (w), 1109 (m), 1047 (m), 698

(s), 503 (s) cm–1

. HR-MS (ESI): calc for C29H34O5N2Si[M+Na]+ : 541.21292. Found:

541.21291. Spectral properties were fully in agreement with the molecule 158 prepared by

elimination of OH group from 154 with CSA.

General procedures for TBDPS protection of Suzuki coupling products

TBAF (1M THF, 1.1-1.3 eq.) was added to a stirred solution of starting material in THF (0.3

mmol of starting material/3mL) reaction mixture was stirred at 25 C for 14 h. All volatiles

were removed under reduced pressure. The brown residue was purified by flash column

chromatography to afford the product.

((3aR*,4R*,6R*,6aS*)-2,2-dimethyl-6-phenyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-

yl)methanol (167):

Following general procedures using 166 (54 mg, 0.111 mmol) and TBAF (1M THF, 0.123

mL, 0.123 mmol). Flash column chromatography (SiO2, hexane/EtOAc 1:1) afforded product

167 as a white semi-solid (27 mg, 97 %). 1H NMR (500 MHz, CDCl3): δ = 7.32-7.28 (m, 4H),

7.21 (m, 1H), 4.70 (m, 1H), 4.58 (d, J = 5.2 Hz, 1H), 3.59 (m, 2H), 3.14 (m, 1H), 2.39-2.26

155

(m, overlapped, 2H), 1.78 (dd, J = 12.3, 6.2 Hz, 1H), 1.45 (s, 3H), 1.26 (s, 3H) ppm. 13

C

NMR (126 MHz, CDCl3): δ = 139.65, 135.87, 135.83, 133.66, 129.96, 129.06, 128.21,

127.97, 126.73, 110.10, 83.93, 82.90, 65.18, 48.00, 47.14, 31.45, 27.14, 26.52, 24.24, 19.47

ppm.

((1R*,4S*,5R*)-4,5-bis(benzyloxy)-3-phenylcyclopent-2-enyl)methanol (210):

Following general procedures using 209 (0.103 g, 0.165 mmol) and TBAF (1M THF, 182 μL,

0.182 mmol). Flash column chromatography (SiO2, hexane/EtOAc 20:1) afforded product

210 as a colorless oil (0.056 g, 88 %). 1H NMR (500 MHz, CDCl3): δ = 7.57-7.55 (m, 2H),

7.42-7.23 (m, 13H), 6.33 (d, J = 2.0 Hz, 1H), 5.14 (dd, J = 6.0, 1.2 Hz, 1H), 4.92 (d, AB, J =

11.5 Hz, 1H), 4.65 (d, AB, J = 11.5 Hz, 1H), 4.64 (d, AB, J = 10.6 Hz, 1H), 4.56 (d, AB, J =

10.6 Hz, 1H), 4.00 (m, 1H), 3.82 (dd, J = 10.6, 5.7 Hz, 1H), 3.71 (dd, J = 10.6, 5.7 Hz, 1H),

3.25 (m, 1H) 1.40 (s, 3H), 1.37 (s, 3H) ppm. 13

C NMR (126 MHz, CDCl3) : δ 141.26, 138.87,

138.41, 135.07, 129.89, 128.70, 128.64, 128.44, 128.43, 128.28, 128.11, 128.01, 127.66,

126.16, 81.08, 79.72, 72.70, 69.40, 64.33, 53.03 ppm. HR-MS (ESI): calcd for

C26H26O3[M+Na]+ : 409.17742. Found: 409.17753.

(1S*,2R*,3R*,5S*)-3-(hydroxymethyl)-5-phenylcyclopentane-1,2-diol (171):

Following general procedures using 170 (0.074 g, 0.098 mmol) and TBAF (1M solution in

THF, 0.123 mL, 0.123 mmol. Flash column chromatography (SiO2, hexane/EtOAc 1:1) to

afford product 171 as a white semi-solid (0.027 g, 97 %). 1H NMR (500 MHz, CDCl3): δ =

7.32-7.28 (m, 4H), 7.21 (m, 1H), 4.70 (m, 1H), 4.58 (d, J = 5.2 Hz, 1H), 3.59 (m, 2H), 3.14

(m, 1H), 2.39-2.26 (m, overlapped, 2H), 1.78 (dd, J = 12.3, 6.2 Hz, 1H), 1.45 (s, 3H), 1.26 (s,

156

3H) ppm. 13


139.65, 135.87, 135.83, 133.66, 129.96, 129.06,

128.21, 127.97, 126.73, 110.10, 83.93, 82.90, 65.18, 48.00, 47.14, 31.45, 27.14, 26.52, 24.24,

19.47 ppm. IR: ν˜max = 3290 (m), 2938 (w), 1396 (w), 1213 (s), 1111 (s), 1178 (s), 759 (s),

697 (s) cm-1

. HR-MS (APCI): calcd for C12H16O3[M+NH4]+: 226.1438. Found 226.1436.

Crystal data for 171: Crystallized from CH3OH, C12H16O3, Mrel = 601.51, T = 120 K, space

group Pbca, a = 9.8445(4) Å, b = 6.9522(5) Å, c = 30.4659(12) Å, α = 90.00, β = 90.00, γ =

90.00, V = 2085.11 Å3.

5-((3aS*,4R*,6R*,6aR*)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aH-

cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (174):

Following a general procedure using 173 (0.094 g, 0.181 mmol) and TBAF (1M THF, 0.199

mL, 0.199 mmol). Flash column chromatography (SiO2, CH2Cl2/MeOH 10:1) afforded

product 174 as a white solid (0.050 g, 98 %). M. p. > 250C decomp. 1H NMR (500 MHz,

DMSO-d6): δ = 11.01 (s, 1H, N-H), 10.63 (d, J = 4.6 Hz, 1H, N-H), 7.06 (d, J = 5.5 Hz, 1H),

4.65 (m, 1H, O-H), 4.59 (m, 1H), 4.41 (d, J = 5.5 Hz, 1H), 3.30 (m, 2H, overlapped with H2O

signal), 2.97 (m, 1H), 2.01 (m, 1H), 1.86 (m, 1H), 1.49 (dd, 12.2, 6.0 Hz), 1.39 (s, 3H), 1.18

(s, 3H) ppm. 13

C NMR (126 MHz, DMSO-d6) : δ =

164.30, 151.00, 137.71, 110.27, 108.64,

82.53, 79.55, 61.38, 46.08, 37.73, 27.93, 26.07, 23.94 ppm. IR: ν˜max = 3477 (m), 1703 (m),

1658 (s), 1213 (m), 1021 (s), 863 (s), 522 (m), 449 (m) cm–1

. HR-MS (ESI) calcd for

C13H18O5N2 [M+Na]+: 305.11079. Found: 305.11085.

157

((3aR*,4R*,6R*,6aS*)-6-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2,2-

dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methanol (S-31):

TBAF (1M solution in THF, 0.432 mL, 0.432 mmol) was added to a solution of starting

material 177 (0.180 g, 0.332 mmol) in anhydrous THF (6 mL). The reaction mixture was

stirred at 25 °C for 48 h. The solvent was evaporated and the brown residue was filtered

through a short plug of silica gel with CH2Cl2/MeOH 10:1 mixture to afford crude

intermediate, which was dissolved in 10 mL of EtOH. 10% Pd/C (0.0360 g, 0.0332 mmol)

was added into the solution and the reaction mixture was thoroughly purged with H2. The

reaction mixture was heated to 80 C and stirred under H2 atmosphere for 1 h. Reaction

mixture was cooled down to 25 °C and filtered through a pad of Celite with CH2Cl2/EtOAc

1:1 mixture (50 mL). Solvents were removed under vacuum and the yellow residue was

purified by flash column chromatography (SiO2, CH2Cl2/MeOH 10:1) to afford S-31 as a

yellow glassy solid (0.056 g, 55% over 2 steps). 1H NMR (500 MHz, DMSO-d6): δ = 7.81 (s,

1H), 7.53 (br s, 2H, -NH2), 6.79 (d, J = 4.4 Hz, 1H), 6.49 (d, J = 4.4 Hz, 1H), 4.83 (m, 1H),

4.70 (m, 1H, -OH), 4.52 (app d, J = 5.4 Hz), 3.65 (m, 1H), 3.38 (m, 2H), 2.21 (m. 1H), 2.11

(dd, J = 7.4, 14.8 Hz, 1H), 1.78 (dd, J = 12.2, 6.4 Hz), 1.27 (s, 3H), 1.15 (s, 3H) ppm. 13

C

NMR (126 MHz, DMSO-d6): δ =

155.43, 147.45, 129.02, 113.71, 109.49, 108.74, 100.40,

82.89, 79.66, 61.54, 46.27, 38.05, 29.22, 26.10, 23.97 ppm. HR-MS (ESI) calcd for

C15H21O3N4 [M+Na]+: 305.16082. Found: 305.16088.

(1R,2S,5R)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-f][1,2,4]triazin-

7-yl)-5-((ethoxymethoxy)methyl)cyclopent-3-ene-1,2-diol (226):

158

TBAF (1M in THF, 0.398 mL) was added to a solution of starting material 224 (161 mg,

0.181 mmol) in THF (3 mL). The reaction mixture was stirred at 25 °C for 16 h. THF was

evaporated and the dark brown residue was purified by flash column chromatography (SiO2,

CH2Cl2 to CH2Cl2/MeOH 20:1) to afford product 226 as a yellow semi-solid (0.104 g, 98 %).

1H NMR (500 MHz, CDCl3): δ = 7.97 (s, 1H), 7.04 (d, J = 4.8 Hz, 1H), 6.85 (d, J = 4.8 Hz,

1H), 6.47 (d, J = 2.2 Hz, 1H), 5.24 (m, 4H), 4.97 (dd, J = 6.0, 0.9 Hz, 1H), 4.71 (s, 2H), 4.18

(m, 1H), 3.79-3.57 (m, 9H), 3.11 (m, 1H), 1.18 (m, 3H), 0.97 (m, 4H), - 0.01 (s, 18H) ppm.

13C NMR (126 MHz, CDCl3): δ

=

156.40, 146.82, 134.80, 132.62, 128.10, 115.14, 111.89,

107.04, 95.57, 77.85, 74.63, 74.57, 68.78, 66.32, 63.55, 52.13, 18.43, 15.40, -1.16 ppm. IR:

ν˜max = 3728 (m br), 2953 (m), 2918 (m), 1725 (s, carbonyl), 1578 (m), 1513 (m), 1395 (w),

1160 (m), 858 (m), 853 (m) cm–1

. HR-MS (APCI): calcd for C27H48N4O6Si2[M+H]+:

581.3185. Found: 581.3186.

((1R,4S,5R)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-

f][1,2,4]triazin-7-yl)-4,5-dihydroxycyclopent-2-enyl)methyl pivalate (227):

According to the procedure desribed for 226 using starting material 225 (140 mg, 0.153

mmol) and TBAF (1M THF, 0.337 mL). Flash column chromatography (SiO2, CH2Cl2 to

CH2Cl2/MeOH 20:1) afforded product 227 as a yellow semi-solid (0.075 g, 81 %). 1H NMR

(500 MHz, CD2Cl2): δ = 7.99 (s, 1H), 7.08 (d, J = 4.8 Hz, 1H), 6.86 (d, J = 4.8 Hz, 1H), 6.44

(d, J = 2.2 Hz, 1H), 5.22 (m, 4H), 4.97 (dd, J = 6.0, 0.9 Hz, 1H), 4.40 (dd, J = 11.0, 5.2 Hz),

4.15 (m, 2H), 3.69 (m, 4H), 3.13 (m, 1H), 1.19 (s, 9H), 0.99 (m, 4H), -0.02 (s, 18H) ppm. 13

C

NMR (126 MHz, CD2Cl2): δ = 178.32, 156.40, 146.88, 135.21, 131.32, 127.61, 115.21,

111.54, 106.78, 77.79, 74.46, 74.09, 66.08, 64.28, 51.49, 38.82, 29.78, 27.07, 18.22, -1.41, -

1.62 ppm. IR: ν˜max = 3278 (w), 2954 (w), 2918 (w), 1725 (m), 1578 (m), 1160 (w), 1084

(s), 1008 (m), 858 (s), 833 (s) cm–1

. HR-MS (ESI): calcd for C29H50N4O6Si2 [M+Na]+ :

607.3352. Found: 607.3352.

159

(1R,2S,3S,5R)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-

f][1,2,4]triazin-7-yl)-5-((ethoxymethoxy)methyl)cyclopentane-1,2-diol (228):

Note: this experiment was performed by Prashant Khirsariya. Crabtree catalyst (2.6 mg, 0.032

mmol) was added to CH2Cl2 solution (2 mL) of starting material 226 (95 mg, 0.163 mmol).

The reaction mixture was thoroughly flushed with H2 and stirred at 40 °C for 24 h. The

reaction mixture was analyzed by 1H NMR and next two portions of the catalyst (6 mol% in

total) were added in 3h intervals in order to get approx. 80% conversion. The reaction mixture

was cooled down to 25 °C and it was purified by PTLC (repeated elution, CH2Cl2/MeOH 3:2)

to yield product 228 as a white solid. 1H NMR (300 MHz, CDCl3): δ = 7.92 (s, 1H), 6.97 (d, J

= 4.7 Hz, 1H), 6.53 (d, J = 4.7 Hz, 1H), 5.21 (s, 4H), 5.22 (m, 4H), 4.69 (s, 2H), 3.65 (m,

10H), 2.44 (m, 1H), 2.35 (m, 2H), 1.80 (m, 1H), 0.97 (m, 4H), -0.01 (s, 18H) ppm.

(1R,2S,3S,5R)-3-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-

(hydroxymethyl)cyclopentane-1,2-diol (132):

Note: this experiment was performed by Prashant Khirsariya. PPTS (60 mg, 0.24 mmol) was

added into a solution of 228 (35 mg, 0.06 mmol) in (MeOH/H2O 10:1, 2.2 mL). The reaction

mixture was heated to 70 °C and stirred for 24 h. The reaction mixture was cooled to 25 °C

and purified by PTLC (repeated elution, CH2Cl2/MeOH 3:2) to yielded impure product 132

(contaminated by tosylate salts) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ = 7.78 (s,

1H), 7.51 (br s, 2H, -NH2), 6.81 (d, J = 4.1 Hz, 1H), 6.47 (d, J = 4.1 Hz, 1H), 4.49 (m, 2H, -

OH), 4.36 (m, 1H, -OH), 3.99 (dm, J = 6.1 Hz), 3.75 (dm, J = 4.5 Hz, 1H), 3.55 (dd, J = 17.6,

8.3 Hz, 1H), 3.45 (m, 2H), 2.18 (m, 2H), 2.03 (m, 1H), 1.26 (m, 1H). ppm. 13

C NMR (126

MHz, DMSO-d6): δ =

155.38, 147.14, 133.01, 113.78, 107.19, 100.45, 75.93, 73.15, 62.87,

46.34, 39.46 (overlapped with DMSO-d6, detected through 1H-

13C HSQC experiment), 29.82,

ppm. HR-MS (ESI) calcd for C15H21O3N4 [M+Na]+: 305.16082. Found: 305.16088.

160

(1R,2S,5R)-3-(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[1,2-f][1,2,4]triazin-

7-yl)-5-(hydroxymethyl)cyclopent-3-ene-1,2-diol (230):

DIBAL-H (1M hexanes, 0.348 mmol) was dropwise added to a cooled (-78°C) solution of

227 (66 mg, 0.108 mmol) in anhydrous CH2Cl2 (2 mL). The reaction mixture was stirred at -

78 °C for 10 min, quenched by sat. solution of NH4Cl (10 mL), then diluted with sat. solution

of potassium sodium tartrate (20 mL) and extracted with CH2Cl2 (3x 30 mL). The residue

was purified by flash column chromatography (SiO2, CH2Cl2 to CH2Cl2/MeOH 10:1) afford

product 230 as a yellow semi-solid (0.055 g, 98 %). 1H NMR (500 MHz, CD3OD): δ = 8.10

(s, 1H), 7.18 (d, J = 4.8 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 7.03 (d, J = 4.8 Hz, 1H), 5.32 (app

s, 4H), 5.01 (dd, J = 5.8, 0.6 Hz, 1H), 4.11 (m, 1H), 3.96 (dd, J = 10.8, 4.8 Hz, 1H), 3.80 (m,

4H), 3.73 (dd, J = 10.8, 6.6 Hz, 1H), 3.09 m (1H), 1.06 (m, 4H), -0.08 (s, 18H) ppm. 13

C

NMR (126 MHz, CD3OD): δ = 157.47, 147.69, 134.83, 133.46, 128.61, 116.93, 113.42,

107.78, 79.63, 77.39, 74.56, 67.21, 63.99, 55.20, 19.29, -0.98 ppm. HR-MS (ESI): calcd for

C24H43N4O5Si2 [M+Na]+ : 523.2772. Found: 523.2776.

(1R,2S,5R)-3-(4-(ethoxymethylamino)pyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-

(hydroxymethyl)cyclopent-3-ene-1,2-diol (S-32):

2M HCl (0.158 mmol) was added to a solution of 230 (33 mg, 0.063 mmol) in CH3OH:H2O

2:1 (3 mL) mixture. The reaction mixture was stirred at 25 °C for 24 h. The solvents were

evaporated and the yellow solid residue was purified by flash column chromatography (SiO2,

CH2Cl2 to CH2Cl2/MeOH 2:1) to yield product impure product S-32 as a white solid (8 mg, ~

161

40%). 1H NMR (500 MHz, DMSO-d6): δ = 8.95 (app t, J = 6.4 Hz, 1H, -NH), 8.06 (s, 1H),

7.04 (d, J = 4.6 Hz, 1H), 6.91 (d, J = 2.2 Hz, 1H), 6.83 (d, J = 4.6 Hz, 1H), 4.93 (d, J = 6.4

Hz, 2H), 4.72 (d, J = 5.7 Hz, 1H), 3.79 (m, 1H), 3.66 (dd, J = 10.5, 4.8 Hz, 1H), 3.38 (dd, J =

10.5, 7.1 Hz, 1H), 3.28 (s, 3H), 2.81 m (1H), ppm. 13


154.13, 147.30, 132.93, 130.63, 126.25, 115.31, 111.09, 101.42, 75.14, 72.15, 71.24 (br),

61.99, 55.16, 53.95 ppm.

(1R*,2S*,5R*)-5-((tert-butyldiphenylsilyloxy)methyl)-3-phenylcyclopent-3-ene-1,2-diol

(169):

Freshly prepared PPTS (0.111 g, 0.443 mmol) was added to a solution of starting material 163

(0.043 g, 0.089 mmol) in MeOH (3 mL). The reaction mixture was stirred at 25 °C for 96 h

(70% conversion according to the 1H NMR). The solvent was evaporated and the residue was

purified by flash column chromatography (SiO2, gradient elution from hexane/EtOAc to

initially elute starting material - 18 mg, 33% recovered to CH2Cl2/EtOAc 10:1) to afford

product 169 as a white semi-solid. Yield: 0.015 g, 38 %. 1H NMR (500 MHz, CDCl3): δ =

7.67-7.62 (m, 4H), 7.54-7.50 (m, 2H), 7.44-7.31 (m, 8H), 7.26 (m, 1H), 6.09 (d, J = 2.2 Hz,

1H), 4.96 (m, 1H), 4.22 (dd, J = 9.9, 4.9 Hz, 1H), 3.86 (dd, J = 10.0, 5.5 Hz, 1H), 3.79 (dd, J

= 10.0, 5.5 Hz, 1H), 2.99 (m, 1H), 2.69 (d, J = 5.1 Hz, 1H, -OH), 2.43 (d, J = 4.7 Hz, 1H, -

OH), 1.04 (s, 9H) ppm. 13

C NMR (126 MHz, CDCl3) : δ = 143.55, 135.83, 135.79, 134.69,

133.60, 133.54, 129.99, 129.31, 128.78, 128.05, 127.98, 127.96, 126.26, 75.75, 74.77, 65.09,

54.00, 27.09, 19.45 ppm.

(1S*,2R*,3R*,5R*)-3-(hydroxymethyl)-5-phenylcyclopentane-1,2-diol (168):

0.5 mL of concentrated aq. HCl was added to a solution of starting material 167 (0.027 g,

0.108 mmol) in MeOH:H2O 2:1 (3 mL). The reaction mixture was stirred at 25 °C for 30 min.

162

All volatiles were evaporated and the yellow residue was purified by flash column

chromatography (SiO2, CH2Cl2/CH3OH 10:1) to afford product 167 as a white glassy solid

(0.014 g, 62 %). 1H NMR (500 MHz, DMSO-d6): δ = 7.31-7.23 (m, 4H), 7.18-7.13 (m, 1H),

4.46 (m, 2H), 4.15 (d, J = 3.6 Hz, 1H), 3.88 (m, 1H), 3.77 (m, 1H), 3.56 (m, 1H), 3.41 (m,

1H), 2.97 (m, 1H), 2.10 (m, 1H), 2.06 (m, 1H), 1.74 (m, 1H) ppm. 13

C NMR (126 MHz,

DMSO-d6) : δ =

141.66, 128.59, 127.53, 125.52, 75.95, 75.72, 63.05, 45.47, 45.32, 30.49

ppm. HR-MS (APCI): calcd for C12H16O3[M+Na]+: 231.1337. Found: 231.1339.

(1S*,2R*,3R*,5S*)-3-((tert-butyldiphenylsilyloxy)methyl)-5-phenylcyclopentane-1,2-diol

(162)

10% Pd/C (0.007 g, 0.0067 mmol, 10 mol%) was added to a solution of starting material 161

(0.030 g, 0.067 mmol) in EtOH (3 mL). The reaction mixture was thoroughly flushed with H2

and heated to 80 C under H2 atmosphere (balloon) for additional 4 h. The reaction mixture

was allowed to cool to 25 °C and filtered through a pad of Celite and washed with

CH2Cl2/EtOAc 1:1 mixture (30 mL). The crude mixture was purified by PTLC (SiO2,

CH2Cl2/EtOAc 40:1) to afford 162 as a colorless oil (0.018 g, 60 %), 0.008 g (27%) of the

epimer was also isolated). 1H NMR (500 MHz, CDCl3): δ = 7.69-7.65 (m, 4H), 7.45-7.36 (m,

6H), 7.32-7.27 (m, 2H), 7.24-7.19 (m, 3H), 4.11 (m, 1H), 4.01 (m, 1H), 3.84 (dd, J = 10.1,

4.8 Hz, 1H), 3.69 (dd, J = 10.1, 6.4 Hz, 1H), 3.15 (m, 1H), 2.70 (br s, 1H, -OH), 2.43 (br s,

1H, -OH), 2.34-2.27 (m, 1H), 2.18-2.11 (m, 1H), 1.55-1.45 (m, 1H), 1.07 (s, 9H) ppm. 13

C

NMR (126 MHz, CDCl3) : δ = 142.96, 135.84, 135.83, 133.55, 133.50, 130.04, 130.03,

128.81, 128.01, 127.61, 126.75, 79.20, 75.90, 66.07, 50.53, 46.64, 31.53, 27.15, 19.49 ppm.

(1R*,2S*,5R*)-5-(hydroxymethyl)-3-phenylcyclopent-3-ene-1,2-diol (221):

163

TBAF (1M THF, 0.323 mL, 0.323 mmol) was added to a solution of starting material S-32

(0.074 g, 0.098 mmol) in anhydrous THF (2 mL). The reaction mixture was stirred at 25 °C

for 14 h. The solvent was evaporated and the brown residue was purified by flash column

chromatography (SiO2, CH2Cl2/MeOH 20:1 to 10:1) to afford product 221 as a white semi-

solid (0.014 g, 70 %). 1H NMR (500 MHz, acetone-d6): δ = 7.66 (m, 2H), 7.36 (m, 2H), 7.27

(m, 1H), 6.34 (d, J = 2.1 Hz), 4.90 (m, 1H), 4.11 (m, 1H), 4.00 (d, J = 5.17 Hz, 1H, -OH),

3.97 (d, J = 6.85 Hz, 1H, -OH), 3.85 (dd, J = 9.6, 4.7 Hz, 1H), 3.70 (m, 1H, -OH), 3.85 (dd, J

= 9.6, 5.3 Hz, 1H), 2.91 (m, 1H) ppm. 13

C NMR (126 MHz, acetone-d6): δ = 144.30, 136.69,

130.32, 129.16, 128.11, 127.00, 89.90, 75.85, 74.43, 63.71, 55.38. IR: ν˜max = 3442 (m),

3141 (m), 2924 (m), 2854 (m), 1635 (s), 1520 (w), 1469 (w), 1265 (w), 1075 (s), 727 (s).

tert-butyl(((3aR*,4R*,6aS*)-2,2-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methoxy)diphenylsilane (164):

Bis(pinacolato)diboron (0.140 g, 0.549 mmol), KBr (0.090 mg, 0.749 mmol), and KOPh

(0.099 mg, 0.749 mmol) were added to a solution of starting material 162 (0.278 g, 0.499

mmol) in anhydrous toluene (5 mL). The solution was evacuated and backfilled with Ar (3

cycles). Pd(Ph3P)2Cl2 (0.011 mg, 0.0149 mmol, 3 mol %) and Ph3P (0.008 g, 0.0298 mmol, 6

mol %) were added and the reaction mixture was heated up to 60C and stirred for 3 h. The

reaction mixture was cooled down, diluted with EtOAc (30 mL) and sat. NaHCO3 (30 mL).

The aqueous phase was re-extracted with EtOAc (3 x 10 mL). Organic extracts were dried

over Na2SO4, the solvent was evaporated and the brown residue was purified by flash column

chromatography (SiO2, hexane/EtOAc 20:1 to 10:1) to afford 164 containing phenol as a

colorless oil (0.280 g). 1H NMR (500 MHz, CDCl3): δ = 7.63-7.57 (m, 4H), 7.43-7.33 (m,

6H), 6.43 (d, J = 2.8 Hz, 1H), 5.28 (app d, J = 5.8 Hz, 1H), 4.58 (d, J =6.2 Hz, 1H), 3.75 (dd,

J = 10.1, 4.6 Hz, 1H), 3.57 (dd, J = 10.1, 4.6 Hz, 1H), 3.00 (m, 1H), 1.35 (s, 3H), 1.33 (s,

3H), 1.27 (s, 6H), 1.26 (s, 6H) ppm. 13

C NMR (126 MHz, CDCl3): δ = (only important

164

resonances) - 155.78 - (C-H double bond), 137.62 (C-B observed indirectly by 1H-

13C

HMBC), 83.70 - C-quarternary-pinacolate fragment, 24.95- CH3 – pinacolate fragment, 24.94

- CH3 – pinacolate fragment. LR-MS calcd for C31H43BO5Si [M+Na]+: 557.4. Found: 557.4.

tert-butyl(((3aR,4R,6aS)-2,2-dimethyl-6-((trimethylsilyl)ethynyl)-4,6a-dihydro-3aH-


Copper (I) iodide (5 mg, 0.02877), Pd(PPh3)2Cl2 (10 mg, 0.01436 mmol), 2,6-lutidine (71 μL,

0.6109 mmol) was added into degassed solution of triflate 162 (200 mg, 0.359 mmol) and

TMS-acetylene ( 66 μL, 0.467 mmol) in DMF (3 mL). The reaction mixture was stirred at 70

°C for 4 h under N2 atmosphere. After cooling to 25 °C, the reaction mixture was filtered

through Celite which was then washed with EtOAc (30 mL). The solvents were evaporated

and the brown residue was purified by flash column chromatography (SiO2, hexane/EtOAc

20:1) to afford product 165 as a pale yellow oil (143 mg, 79 %). 1H NMR (500 MHz, CDCl3):

δ = 7.62-7.60 (m, 4H), 7.43-7.34 (m, 6H), 5.94 (d, J = 2.74 Hz, 1H), 5.04 (app d, J = 5.19 Hz,

1H), 4.53 (d, J = 5.57 Hz, 1H), 3.72 (dd, J = 10.1, 5.0 Hz, 1H), 3.56 (dd, J = 10.1, 5.1 Hz,

1H), 2.96 (m, 1H), 1.42 (s, 3H), 1.34 (s, 3H), 1.02 (s, 9H), 0.20 (s, 9H) ppm. 13

C NMR (126

MHz, CDCl3): δ = 140.30, 135.93, 135.81, 133.66, 133.44, 130.01, 129.97, 128.02, 127.95,

127.94, 110.83, 100.26, 97.43, 86.72, 81.56, 64.62, 54.13, 27.64, 27.07, 26.11, 19.44, 0.21

ppm. IR: ν˜max = 2957 (w), 2929 (w), 2150 (w, alkyne), 1471 (w), 1249 (m), 1111 (s), 857

(s), 702 (s) cm-1

. HR-MS (ESI): calcd for C30H40NO3Si2 [M+Na]+ : 527.24082. Found:

527.24083.

7-((3aS*,6R*,6aR*)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-6,6a-dihydro-

3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrrolo[1,2-f][1,2,4]triazin-4-amine (177):

165

Bromide 176 (0.057 g, 0.218 mmol), K3PO4 (0.152 g, 0.72 mmol) and Pd(dppf)Cl2 (0.018 g,

0.0218 mmol, 10 mol %) were added into a solution of crude boronate 164 (0.161 g, 0.301

mmol, contained ca. 20% of PhOH) in DME/H2O 4:1 (5 mL). Reaction flask was evacuated

and backfilled with N2 (3 cycles), heated up to 80 C and stirred for 16 h. The reaction

mixture was cooled to 25 °C, diluted with EtOAc (30 mL) and sat. NaHCO3 (30 mL).

Aqueous phase was re-extracted with EtOAc (3 x 20 mL). Organic extracts were dried over

Na2SO4, the solvents were evaporated, and the brown residue was purified by flash column

chromatography (SiO2, CH2Cl2/EtOAc 1:1) to afford 177 as a yellow glassy solid. Yield:

0.080 g, 62% (based on 176). 1H NMR (500 MHz, DMSO-d6): δ = 7.96 (s, 1H), 7.71 (br s,

2H, -NH2), 7.63-7.56 (m, 4H), 7.47-7.33 (m, 6H), 6.92 (d, J = 4.5 Hz, 1H), 6.81 (d, J = 2.6

Hz, 1H), 6.76 (d, J = 4.5 Hz), 5.46 (app d, J = 5.4 Hz, 1H), 4.62 (d, J = 5.7 Hz, 1H), 3.84

(dd, J = 10.0, 4.8 Hz, 1H), 3.65 (dd, J = 10.0, 5.5 Hz, 1H), 3.10 (m, 1H), 1.33 (s, 3H), 1.25 (s,

3H), 0.94 (s, 9H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ =155.50, 148.09, 135.04, 133.31,

132.95, 132.82, 129.77, 129.75, 127.79, 127.70, 124.87, 115.82, 110.91, 109.45, 101.48,

85.50, 80.03, 64.85, 53.73, 27.24, 26.51, 25.58, 18.71 ppm. IR: ν˜max = 3364 (w), 1659 (m),

1594 (m), 1521 (m), 1469 (m), 1055 (s), 1005 (s), 698 (s), 619 (m), 490 (w) cm–1

. HR-MS

(ESI): calcd for C31H37O3N4Si [M+H]+ : 541.26294. Found: 541.26278.

5-((1R*,2S*,3R*,4R*)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)pyrimidine-

2,4(1H,3H)-dione (175):

35% HCl (1 mL) and H2O (1 mL) were added to a solution of starting material 166 (0.045 g,

0.159 mmol) in MeOH (3 mL). The reaction mixture was stirred at 25 °C for 30 min. The

solvents were evaporated and the resulting yellow solid was purified by flash column

chromatography (SiO2, CH2Cl2/MeOH 5:1 to 1:1) to afford compound 175 as a white solid

(0.020 g, 52 %). m.p. = 238-246C. 1H NMR (500 MHz, DMSO-d6): δ = 10.92 (s, 1H, N-H),

166

10.64 (br s, 1H, N-H), 7.15 (s, 1H), 4.45 (m, 1H, O-H), 4.40 (d, J = 7.4 Hz, 1H, O-H), 4.32

(d, J = 3.9 Hz, 1H, O-H), 3.76 (m, 1H), 3.67 (m, 1H), 3.50 (m 1H), 3.35 (m, 1H), 2.84 (m,

1H), 1.93 (m, 1H), 1.80 (dd, 23.3, 11.6 Hz), 1.53 (m, 1H) ppm. 13

C NMR (126 MHz, DMSO-

d6): δ =

164.66, 151.00, 138.06, 111.24, 75.72, 73.52, 62.94, 44.77, 36.39, 28.76 ppm. IR

(ν˜max): 3087 (w), 3038 (w), 1704 (s), 1654 (m), 1015 (m), 849 (m), 542 (m) cm-1

. HR-MS

(ESI): calcd for C10H34N2O5 [M-H]-: 241.0830. Found: 241.0832.

(1R*,2S*,3R*,5R*)-3-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-

(hydroxymethyl)cyclopentane-1,2-diol (134):

35% HCl (1 mL) and H2O (2 mL) were added to a solution of starting material S-31 (0.055 g,

0.181 mmol) in MeOH (2 mL). The reaction mixture was stirred at 25 °C for 2.5 h. The

solvents were evaporated in a vacuum and the resulting yellow solid was purified by flash

column chromatography (SiO2, CH2Cl2/NH3 in MeOH 10:1) to afford compound 134 as a

white solid (0.027 g, 56 %). mp. = 205-208 °C (decomp). IR (ν˜max): 2953 (br w), 2916 (s),

1585 (m), 1517 (w), 1370 (w), 1319 (w), 1248 (m), 1075 (s), 832 (s) cm–1

. 1H NMR (500

MHz, DMSO-d6): δ = 7.78 (s, 1H), 7.50 (br s, 2H, -NH2), 6.80 (d, J = 4.3 Hz, 1H), 6.55 (d, J

= 4.3 Hz, 1H), 4.48 (m, 1H, -OH), 4.44 (d, J = 6.5 Hz, 1H, -OH), 4.16 (m, 1H, -OH), 4.05 (m,

1H), 3.79 (m 1H), 3.57 (m, 1H), 3.47 (m, 1H), 3.42 (m, 1H), 2.17 (dd, J = 11.0, 23.2 Hz, 1H),

2.05 (m, 1H), 1.80 (m, 1H) ppm. 13


155.36, 147.12,

130.71, 113.61, 109.65, 100.42, 75.71, 73.45, 62.93, 44.58, 36.84, 29.35 ppm. HRMS

(APCI): calculated for C12H16N4O3 [M+H]+: 265.1295. Found: 265.1297.

((1R*,2R*,3R*,4R*,5R*)-3,4-bis(benzyloxy)-5-phenyl-6-oxabicyclo[3.1.0]hexan-2-

yl)methanol (212):

167

mCPBA (77% purity, 0.039 g, 0.173 mmol) was added to a cooled solution (0C, ice bath) of

210 (0.056 g, 0.144 mmol) in anhydrous CH2Cl2 (3mL). The reaction mixture was stirred for

5 h under N2 atmosphere while allowed to warm to 25 °C. Crude NMR showed 10:3

diastereomeric ratio of epoxides. The reaction mixture was diluted with CH2Cl2 (10 mL) and

washed with sat. NaHCO3 solution (3x 10 mL). Organic extracts were dried over Na2SO4,

evaporated, and the brown residue was purified by flash column chromatography (SiO2,

hexane/EtOAc 2:1) to afford 212 as a colorless oil (25 mg, 43%). IR: ν˜max = 3436 (m), 1497

(m), 1144 (m), 1113 (m), 1086 (m), 1052 (m), 1026 (m), 885 (m), 694 (s) cm–1

. 1H NMR (500

MHz, CDCl3): δ = 7.50-7.48 (m, 2H), 7.35-7.30 (m, 8H), 7.23-7.21 (m, 3H), 7.11-7.09 (m,

2H), 4.62 (d, AB, J = 11.6 Hz, 1H), 4.33 (d, AB, J =11.1 Hz, 1H), 4.42 (d, AB, J =11.6 Hz,

1H), 4.41 (d, AB, J = 11.1 Hz, 1H), 4.33 (d, J = 4.9 Hz, 1H), 3.96 (dd, J = 10.7, 5.3 Hz, 1H),

3.84 (dd, J = 10.7, 7.3 Hz, 1H), 3.81 (s, 1H), 3.76 (dd, J = 8.4, 4.9 Hz, 1H), 2.66 (m, 1H)

ppm. 13

C NMR (126 MHz, CDCl3): δ = 138.05, 137.98, 135.04, 128.68, 128.49, 128.45,

128.43, 128.35, 128.12, 128.10, 127.89, 80.45, 77.94, 73.74, 73.40, 65.49, 63.57, 62.13, 46.92

ppm. HR-MS (ESI): calcd for C26H26O4 [M+Na]+ : 425.1729. Found: 425.1738.

(1S*,2R*,3R*,4R*)-2,3-bis(benzyloxy)-4-(hydroxymethyl)-1-phenylcyclopentanol (213):

LiEt3BH (0.918 mL, 0.918 mmol) was added dropwise to a cooled (0C, ice bath) solution of

212 (0.053 g, 0.131 mmol) in anhydrous THF (2 mL). The reaction mixture was stirred under

N2 atmosphere for 30 h and allowed to warm to 25 °C. The reaction mixture was then

carefully quenched by sat. NH4Cl (10 mL) and diluted with EtOAc (20 mL). Saturated

solution of potassium sodium tartrate (20 mL) was added and reaction mixture was poured

into a separatory funnel. Aqueous phase was reextracted by EtOAc (3 x 10 mL) and organic

extracts were dried over Na2SO4, evaporated, and the brown residue was purified by flash

column chromatography (SiO2, hexane/EtOAc 2:1) to afford 213 as a colorless oil (0.031 g,

59%, (contains 10% of unreacted starting material). 1H NMR (500 MHz, CDCl3): δ = 7.57-

7.55 (m, 2H), 7.37-7.21 (m, 8H), 7.20-7.13 (m, 3H), 7.01-6.94 (m, 2H), 4.54 (d, AB, J = 11.8

Hz, 1H), 4.41 (d, AB, J =11.8 Hz, 1H), 4.33 (dd, J =7.8, 3.9 Hz, 1H), 4.16 (d, AB, J = 11.6

168

Hz, 1H), 4.06 (d, AB, J = 11.6 Hz, 1H), 3.83 (d, J = 3.3 Hz, 1H), 3.74 (dd, J = 10.1, 3.5 Hz,

1H), 3.68 (dd, J = 10.1, 4.1 Hz, 1H), 2.99 (dd, J = 14.0, 11.8 Hz, 1H), 2.76 (br s, 2H, 2X –

OH), 2.59 (m, 1H), 1.65 (dd, J = 14.0, 2.8 Hz, 1H), ppm. 13


142.54, 138.67, 138.48, 128.57, 128.23, 128.19, 127.85, 127.83, 127.55, 127.00, 85.94, 83.14,

82.30, 73.61, 72.58, 64.03, 43.43, 37.57 ppm. IR (ν˜max): 3382 (br w), 2916 (s), 2849 (m),

1559 (w), 1105 (w), 732 (m) cm–1

.

Experimental procedures for pyrrolotriazine base synthesis

tert-butyl 1-H-pyrrol-1-ylcarbamate (179):

Aqueous 2 N HCl (0.2 mL) was added to a solution of tert-butyl carbazate (2.00 g, 7.57

mmol) and 2,5-dimethoxytetrahydrofuran (2.17 mL,16.64 mmol) in 1,4-dioxane (14 mL) and

the resulting mixture was stirred at 100 °C for 48 h. The reaction mixture was quenched with

saturated aqueous solution of NaHCO3 (20 mL) and extracted with EtOAc (3 x 20 mL). The

organic phase was washed with brine (15 mL), dried over MgSO4, filtered, and concentrated

under reduced pressure to yield the crude product, which was purified by flash column

chromatography (SiO2, hexane/EtOAc 5:1 to 3:1) to afford 179 (1.88 g, 68 %) as a pale

yellow solid. 1H NMR (500 MHz, CDCl3): δ = 7.11 (br s, 1H, N-H), 6.65 (m, 2H), 6.11 (m,

2H), 1.47 (s, 9H) ppm. Spectral data were in accordance with the literature.159

tert-butyl (2-cyano-1H-pyrrol-1-yl)carbamate (180):

Chlorosulfonyl isocyanate (0.752 mL, 8.64 mmol) was added to a solution of 179 (1.5 g, 8.23

mmol) in acetonitrile (12 mL) at -20 °C and the resulting suspension was stirred for 45 min. at

-20 °C. Then DMF (1.9 mL) was added at -20 °C and the mixture was stirred at 0 °C for 45

min and then at 25 °C overnight. Ice cold water (30 mL) was added and the mixture was

extracted with EtOAc (3 × 50 mL). The organic phase was washed with saturated aqueous

169

solution of NaHCO3 (50 mL), brine (50 mL), dried over MgSO4, filtered, and concentrated

under reduced pressure to yield the crude product, which was purified by flash column

chromatography (SiO2, hexane/EtOAc 5:1) to afford 180 as yellowish crystals (1.06 g, 62%).

1H NMR (500 MHz, CDCl3): δ = 7.39 (br s, 1H, N-H), 6.87 (dd, J = 2.8, 1.7 Hz, 1H), 6.11

(dd, J = 4.3, 1.7 Hz, 1H), 6.15 (dd, J = 4.3, 3.0 Hz, 1H), 1.48 (s, 9H) ppm. 13

C NMR (126

MHz, CDCl3): δ = 153.81, 128.27, 119.20, 112.23, 108.55, 106.40, 83.85, 28.23 ppm.

pyrrolo[2,1-f][1,2,4]triazin-4-amine (181):

4M HCl solution in dioxane (13 mL, 51 mmol) was slowly added to a cooled (0C, ice bath)

solution of nitrile 180 (1.05 g, 5.01 mmol) in anhydrous dioxane (8 mL). The reaction mixture

was stirred for 14 h and allowed to warm to 25 °C. All volatiles were removed under vacuum

and to this crude hydrochloride was added formamidine acetate (2.61 g, 25.14 mmol), K3PO4

(5.3 g, 25.14 mmol) and anhydrous EtOH (15 mL). The resulting mixture was stirred at 80 C

for 14 h. After cooling down, the reaction mixture was diluted with H2O (50 mL) and

extracted with CH2Cl2 (3 x 50 mL). Organic extracts were dried over Na2SO4, evaporated and

the brown residue was purified by flash column chromatography (SiO2, CH2Cl2 to CH2Cl2

20:1) to afford 181 as a yellow solid (0.561 g, 83% , over 2 steps). 1H NMR (500 MHz,

DMSO-d6): δ = 7.78 (s, 1H), 7.66 (br s, 2H, -NH2), 7.58 (dd, J = 2.5, 1.6 Hz, 1H), 6.85 (dd, J

= 4.3, 1.6 Hz,1H), 6.59 (dd, J = 4.3, 2.5 Hz) ppm. 13


155.51, 147.92, 118.04, 114.34, 110.01, 101.17 ppm. IR: ν˜max = 3324 (w), 3117 (w), 1667

(s), 1602 (s), 1528 (m), 1489 (m), 1069 (w), 725 (s) cm–1

. HR-MS (APCI): calcd for C6H6N4

[M+H]+: 135.0665. Found: 135.0668.

7-bromopyrrolo[2,1-f][1,2,4]triazin-4-amine (176):

170

1,3-dibromo-5,5-dimethylhydantoin (0.597 mg, 2.09 mmol) was added in one portion to a

cooled (-20 C) solution of starting material 181 (0.561 g, 4.18 mmol) in anhydrous DMF (4

mL). The reaction mixture was stirred under N2 atmosphere for 16 h while allowed to warm

to 25 °C. The reaction mixture was quenched by H2O (10 mL) and extracted by EtOAc (3 x

20 mL). Organic extracts were dried over Na2SO4, evaporated, and the brown residue was

purified by flash column chromatography (SiO2,CH2Cl2/MeOH 20:1 to 10:1) to afford 176 as

a yellow solid (0.731 g, 80%, 70% purity (contaminated by dimethylhydantoin). 1H NMR

(500 MHz, DMSO-d6): δ = 7.92 (s, 1H), 7.85 (br s, 2H, -NH2), 7.00 (dd, J = 4.6 Hz, 1H), 6.77

(dd, J = 4.6 Hz, 1H) ppm. 13

C NMR (126 MHz, DMSO-d6): δ = 155.21, 148.73, 115.75,

112.31, 102.51, 99.44 ppm.

N,N-dibenzyl-7-bromopyrrolo[1,2-f][1,2,4]triazin-4-amine (202):

Solution of starting material 176 (0.713 g, 3.35 mmol) in anhydrous DMF (15 mL) was

dropwise added to a cooled (0C, ice bath) suspension of NaH (0.535 g, 13.39 mmol, 60%

dispersion in mineral oil, washed with pentane) in DMF (2 mL). The reaction mixture was

stirred at 0° for 30 min. BnBr (1.4 mL, 11.725 mmol) was added dropwise and the reaction

mixture was stirred for additional 14 h under N2 atmosphere while allowed to warm to 25 °C.

The reaction mixture was cooled to 0C (ice bath) and H2O (3 mL) was added dropwise, then

Et2O (30 mL) and H2O (30 mL) were added, and the layers were separated. Aqueous phase

was reextracted with Et2O (3 x 20 mL). Organic extracts were dried over Na2SO4, filtered,

concentrated, and the brown residue was purified by flash column chromatography (SiO2,

hexane/EtOAc 5:1) to afford initially 202 (410 mg, 31 %) as a yellow glassy solid followed

by monobenzylated compound (100 mg, 10%). 1H NMR (500 MHz, CDCl3): δ = 8.10 (s, 1H),

7.35-7.25 (m, 10H), 6.57 (d, AB, J = 4.8 Hz, 1H), 6.54 (d, AB, J = 4.8 Hz, 1H), 4.99 (s, 4H)

ppm. 13

C NMR (126 MHz, CDCl3): δ = 155.99, 148.18, 136.60, 129.14, 127.89, 127.43,

171

115.85, 113.18, 106.29, 102.04, 51.63 ppm. HR-MS (APCI): calcd for C20H17BrN4 [M+H]+:

393.0709. Found: 393.0704 and 395.0686.

7-bromo-N,N-bis((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine

(222):

A solution of 176 (0.500 g, 2.34 mmol) in DMF (4 mL) was added to a suspension of NaH

(60 % in mineral oil, 0.234 g, 5.86 mmol) in DMF (1.5 mL) and the mixture was stirred at 25

°C for 30 min. SEMCl (0.872 mL, 4.92 mmol) was then added dropwise and the mixture was

stirred at 25 °C for 5 h. The reaction mixture was quenched with water (10 mL) and extracted

with EtOAc (3 x 25 mL). The organic phase was washed with brine (15 mL), dried over

MgSO4, filtered, and concentrated under reduced pressure to yield the crude product, which

was purified by flash column chromatography (SiO2, hexane/EtOAc 20 : 1) to afford 222

(0.650 g, 58 %) as a colorless oil. 1

H NMR (500 MHz, CDCl3): δ = 8.06 (s, 1H), 7.06 (d, AB,

J = 4.8 Hz, 1H), 6.74 (d, AB, J = 4.8 Hz, 1H), 5.19 (s, 4H), 3.66 (m, 4H), 0.96 (m, 4H), -0.01

(s, 9H) ppm. 13

C NMR (126 MHz, CDCl3): δ = 156.09, 147.52, 115.99, 113.96, 106.86,

102.09, 77.82, 66.32, 18.41, -1.18 ppm. IR: ν˜max = 2954 (w), 1582 (w), 1517 (w), 1264 (m),

1074 (m), 858 (m), 732 (s) cm–1

. HR-MS (ESI): calcd for C18H34N4O2Si2Br [M+H]+:

473.1403. Found: 473.1402.

(4-(bis((2-(trimethylsilyl)ethoxy)methyl)amino)pyrrolo[2,1-f][1,2,4]triazin-7-yl)boronic

acid (223):

n-BuLi (1.6 M in hexanes, 2.4 mL, 3.84 mmol) was added to a solution of 222 (0.909 g, 1.919

mmol) in THF (6 mL) at -78 °C and the mixture was stirred at -78 °C for 30 min (lithiation

172

was followed by TLC). 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.515 mL,

2.303 mmol) was added dropwise to the reaction mixture at -78 °C. The reaction mixture was

then allowed to warm to 25 °C and stirred for 1 h. Saturated aqueous solution of NH4Cl (10

mL) was added and the mixture was extracted with EtOAc (3 × 30 mL). The organic phase

was washed with brine (15 mL), dried over MgSO4, filtered, and concentrated under reduced

pressure to yield the crude boronate (might be eventually used without column

chromatography purification), which partially hydrolyzed to the boronic acid during the flash

column chromatography on silica gel (CH2Cl2/EtOAc 9:1) to afford 223 (0.418 g, 50 %) as

yellow oil together with 400 mg of boronate/boronic acid mixture with approx. 2:1 ratio. as a

light yellow wax (the crude mixture could be used in the Suzuki coupling without further

purification. 1H NMR (500 MHz, DMSO-d6): δ = 8.35 (s, 2H, -B(OH)2), 8.16 (s, 1H), 7.20

(d, AB, J = 4.5 Hz, 1H), 7.01 (d, AB, J = 4.8 Hz, 1H), 5.22 (s, 4H), 3.65 (app t, J = 8.13 Hz,

4H), 0.91 (app t, J = 8.13 Hz, 4H), -0.03 (s, 18H) ppm. 13


155.74, 146.08, 126.25 (br, C-B(OH)2, detected through 1H-

13C HMBC), 120.16, 116.92,

106.21, 77.62, 65.07, 17.52, -1.45 ppm. 11B NMR (160.5 MHz, DMSO-d6): δ = 25.90 (br)

ppm. IR: ν˜max = 2952 (m), 1585 (m), 1517 (w), 1370 (m), 1248 (m), 1075 (s), 856 (s), 832

(s) cm–1

.

Supporting Informations_2: Copies of 1H and

13C NMR spectra are available on attached

CD (SI_2). Assignment of 1H,

13C and

15N NMR resonances of important intermediates and

final compounds (e. g. 132, 134, 157 and 175) based on 2D NMR experiments is also

included as well as the IR spectra of selected compounds.

173

7.3. Synthesis of naphthalene-based fluorescent probes

Part of the results of this project were published in Amaro, M.; Brezovský, J.; Kováčová, S.;

Maier, L.; Chaloupková, R.; Sýkora, J.; Paruch, K.; Damborský, J.; Hof, M. J. Phys. Chem. B.

2013, 117, 7898.

7.3.1. Introduction – fluorescent probes

Fluorescent organic molecules are frequently used in biological studies.9 Typically, the

chromophore is covalently linked to the substance of interest and the whole system is studied

by fluorescence-based analytical techniques.167

By proper tuning of the chromophore

structure, one can modulate absorption/emission properties, life-time, limit of detection as

well as physicochemical properties of the probe. Since the photophysical properties of

molecules are typically dependent on the particular surrounding environment, non-covalently

linked fluorescent probes can be very valuable tools for studying non-covalent interactions,

hydration (solvation) of proteins etc.168

Upon excitation, the dipole moment of the fluorescent

molecule is changed and reorientation of the surrounding solvent molecules occurs

accordingly. These changes are typically coupled with characteristic red shift of time-resolved

emmision spectra.169

7.3.2. Synthesis of naphthalene based fluorescent probes

Our collaborators (group of Prof. Jiří Damborský) have been involved in study of

haloalkane dehalogenases.170

These enzymes can hydrolyze the C-halogen bond in

halogenalkanes, which yields the corresponding hydroxylated alkanes. Detailed knowledge of

enzyme hydration and dynamics is important in order to design more efficient and robust

enzymes. Coumarin-120 fluorescent probe (231) was recently used for mapping the dynamic

behavior and hydration of several haloalkane dehalogenases.171

In order to expand this effort

beyond the use of commercially available probes, we have designed new naphthalene-based

probes for time-dependent fluorescence studies (232a-232d).

174

Properly substituted naphtalene (sensitive solvatofluorochromic moiety)172

was attached to

chloroalkane chains of variable length via polar amide linker. The overall synthetic route is

depicted in Scheme 58.

Scheme 58: Reagents and conditions: a) aq. HCHO, NaBH3CN, MeOH, rt. (69%) b) i)

SO2Cl2, CH2Cl2, ii) TEA, 2-(2-aminoethoxy)ethanol, CHCl3, 0 °C to rt. 14 h, (56%, over 2

steps), c) NaH, THF, for n = 1, chloro-5-iodopentane, rt., 14 h, (33%); for n = 2, chloro-6-

iodohexane, room temp., 14 h, (56%); for n = 3, 1-bromo-7-chloroheptane, rt., 14 h, (41%);

for n = 4, chloro-8-iodooctane, rt., 14 h, (22%).

175

Reductive amination of commercially available 6-amino-2-napthoic acid 233 proceeded

well and the resulting acid 234 was transformed into the corresponding acyl chloride. Acid

chloride was then treated with 2-(2-aminoethoxy)ethanol in the presence of triethylamine to

produce amide 235. We realized that chloroform is a proper solvent for this reaction; running

the reaction in dichloromethane produced an untractable gummy residue. Amide 235 was then

selectively alkylated to afford the target fluorescent probes 232a-d.

It should be noted that the purification of many intermediates and the probes was non-

trivial as they were often contaminated by highly fluorescencent (TLC with UV detection)

impurities of similar polarity. Running the reactions in the dark reduced the content of these

impuruties. Nevertheless, the probes had to be purified using repeated preparative HPLC

(SiO2, hexane:propan-2-ol 60:40 to 40:60). In addition to probes 232a-d, we have also

prepared N-methylated C6 probe (236).

7.3.3. Results and discussion

The probes described above were then combined with dehalogenases DbjA and DhaA

which were mutated in such a way that the hydrolysis stopped at the stage of ester formation;

i. e. the fluorescent probe remained covalently attached to the enzyme. We found that probe

232a bearing C-5 linker was not a good substrate and we observed only non-specific binding

to the enzymes. C-5 linker is very likely too short to reach the catalytic region of the

dehalogenases through the tunnel mouth. On the other hand, probes 232b and 232c formed

covalent complexes with the enzymes whose fluorescence properties (e.g., longer lifetime,

higher Stokes shifts and less complex photophysics) were superior to those observed with

coumarin-based probe.

On the other hand, the behavior of probe 232d in aqueous environment was more complex.

The dynamics of 236 is complicated by the restricted rotation around the amide bond (as seen

in temperature-dependent NMR study, Fig. 25). 1H NMR signals of atoms 3´ and 4´ were

broadened and 13

C NMR resonances of the N-CH3 group and of the amide carbonyl were

substantially broad and we detected them only through 1H-

13C HSQC and

1H-

13C HMBC

176

experiments. Particular 1H signals were resolved at 0 °C. On the other hand, the N-CH3

resonance gave two sets of signals (two distinct carbonyl signals, for instance), which

indicated the presence of two isomeric forms (Fig. 26). Probe 236 was therefore not used for

the time-dependent fluorescence studies.

Fig. 25 Portion of temperature dependent 1H NMR spectra (500 MHz) of 236

30 °C

5°C

0 °C

177

Fig. 26 Portion of temperature-dependent 13

C NMR spectra (126 Mhz) of 236

The overall results of this project and the experimental details of time-dependent fluorescence

studies are provided in Appendix 1. Synthetic procedures for all fluorescent probes and the

corresponding NMR spectra are provided in Supporting Informations (SI_3).

30 °C

0 °C

178

8. Conclusion

During my doctoral research, I synthesized cyclopentane-containing nucleoside analogs

with the C-C connection between the (heterocyclic) base and the carbocyclic scaffold.

Namely, I have carried out the following:

1. Target-oriented synthesis of of previously unknown racemic carbocyclic pseudoisocytidine

90a and its analogs 90b and 90c, which were prepared in 13 steps from commercially

available materials.

90a and its sulfur analog 90c were moderately active against the mantle cell lymphoma cell

line, JVM-3.

We also prepared a versatile cyclopentanone intermediate 126, which we converted into novel

carbocyclic nucleosides via highly stereoselective addition of organometallic nucleophiles;

the phenyllithium adduct 128, whose stereochemistry was unambiguously confirmed by X-

ray crystallography, inhibits glycosylase NEIL1 in a dose-dependent manner.

2. Development of a flexible synthesis of carbocyclic C-nucleoside analogs utilizing properly

protected cyclopentanones 201, 216, 217, which I prepared in gram quantities from

norbornadiene.

179

I utilized these cyclopentanones to prepare four classes of new carbocyclic C-nucleosides.

Some of the compounds were moderately active against several human glycosylases.

This methodology will be used in our group to prepare a library of differently susbtituted

carbocyclic C-nucleosides, whose biological activity will be tested in the future.

In addition, I prepared new naphthalene-based fluorescent probes 232b-d that were

successfully used in time-resolved fluorescence studies of haloalkane dehalogenases DbjA

and DhaA.

180

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LIST OF ABBREVIATION

1H-

13C HMBC – 2D NMR experiment, heteronuclear multiple bond correlation (long range

coupling between 1H and

13C nuclei)

1H-

13C HSQC – 2D NMR experiment, heteronuclear single quantum coherence (correlation

between 1H and

13C nuclei via direct C-H coupling)

AdoMet- (S)-adenosyl methionine

APCI-MS – atmospheric pressure chemical ionization mass spectrometry

APT – attached proton test (NMR experiment for resolution of CH,CH3 and C,CH2 groups)

ATP – adenosine triphosphate

br – broad resonance in NMR spectra

cAMP – cyclic adenosine monophosphate

CMV – cytomegalovirus

CMP – cytidine monophosphate

COSY – correlated spectroscopy (through bond connectivity between 1H nuclei)

d – dublet (signals multiplicity in NMR spectra)

dCMP – deoxycitidine monophosphate

dd – dublet of dublet (signals multiplicity in NMR spectra)

DCM – dichloromethane

DME – dimethoxyethane

DMF – N,N-dimethylformamide

DMSO – dimethylsulfoxide

DNA – deoxyribonucleic acid

dUMP – deoxyuridine monophosphate

ESI-MS – electro spray ionization mass spectrometry

HBV – hepatitis type B viruse

HCV – hepatitis type C virus

HIV – human immunodeficiency virus

IC50 – inhibition concentration

ID50 – ideal dose

196

IR – infrared spectroscpy

m – multiplet (signals multiplicity in NMR)

m.p.: – melting point

mRNA – messenger RNA

NMR – nuclear magnetic resonance

NOESY – 2D NMR experiment with using Nuclear Overhauser Effect to investigated through

space 1H-

1H interactions

PNP – purine nucleoside phosphorylase

RNA – ribonucleic acid

RP-HPLC – reversed phase high performance liquid chromatography

rt – room temperature

ROMP – ring-opening metathesis polymerization

s – singlet (signals multiplicity in NMR)

SILAC – stable isotope labelling by amino acid in cell culture

t – triplet (signals multiplicity in NMR)

tRNA- transfer RNA

TEA – triethylamine

TLC – thin layer chromatography

Synthesis of small-molecule probes for chemical biology

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