Synthesis and biological evaluation of 2′,4′- and 3′,4′-bridged nucleoside analogues

Bioorganic & Medicinal Chemistry 19 (2011) 5648–5669

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Synthesis and biological evaluation of 2⁰,4⁰- and 3⁰,4⁰-bridged

nucleoside analogues

K. C. Nicolaou ^a,b,c,, Shelby P. Ellery ^a, Fatima Rivas ^a, Karen Saye ^d, Eric Rogers ^d, Tyler J. Workinger ^e,

⇑

Mark Schallenberger ^a, Rommel Tawatao ^e, Ana Montero ^a, Ann Hessell ^d, Floyd Romesberg ^a,

Dennis Carson ^e, Dennis Burton ^d

^aDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

^bSkaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

^cDepartment of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

^dDepartment of Immunology and Microbial Science and International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute,

10550 North Torrey Pines Road, La Jolla, CA 92037, USA

^eMoores UCSD Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

a r t i c l e i n f o

a b s t r a c t

Article history:

Most nucleosides in solution typically exist in equilibrium between two major sugar pucker forms, N-

type and S-type, but bridged nucleosides can be locked into one of these conformations depending on

their speciﬁc structure. While many groups have researched these bridged nucleosides for the purpose

of determining their binding afﬁnity for antisense applications, we opted to look into the potential for

biological activity within these conformationally-locked structures. A small library of 2⁰,4⁰- and 3⁰,4⁰-

bridged nucleoside analogues was synthesized, including a novel 3⁰,4⁰-carbocyclic bridged system. The

synthesized compounds were tested for antibacterial, antitumor, and antiviral activities, leading to the

identiﬁcation of nucleosides possessing such biological activities. To the best of our knowledge, these bio-

logically active compounds represent the ﬁrst example of 2⁰,4⁰-bridged nucleosides to demonstrate such

properties. The most potent compound, nucleoside 33, exhibited signiﬁcant antiviral activity against

Received 20 May 2011

Revised 12 July 2011

Accepted 13 July 2011

Available online 23 July 2011

Keywords:

Nucleosides

Synthesis

Antiviral

Cytotoxic

pseudoviruses SF162 (IC₅₀= 7.0

lM) and HxB2 (IC₅₀= 2.4 lM). These ﬁndings render bridged nucleosides

as credible leads for drug discovery in the anti-HIV area of research.

Ó 2011 Published by Elsevier Ltd.

1. Introduction

a high binding afﬁnity for complementary single-stranded RNA

and DNA, respectively, giving the 2⁰,4⁰-BNAs a special advantage

as promising antisense drug candidates.

The ﬁrst antiviral antisense oligodeoxynucleotides were discov-

ered in 1978 by Zamecnik and Stephenson.¹Since then, interest in

antisense technologies has been on the rise due to these molecules

having the potential to treat diseases that small molecule drugs are

incapable of targeting. Perhaps the most promising candidates for

antisense drug therapy have been the 2⁰,4⁰-bridged nucleic acids

(BNAs), as they have recently entered human clinical trials.²Their

success is due to their more rigid structures, which translate into

higher afﬁnities for their biological targets. Most sugars and

nucleosides exist in solution in a fast equilibrium between two ma-

jor sugar pucker forms, North (N-type) and South (S-type),³but

bridged nucleosides are locked into one conformation: 2⁰,4⁰-

bridged systems such as 1a exist in the C3⁰-endo (N-type) form

1b, while 3⁰,4⁰-systems such as 2a exist in the C2⁰-endo (S-type)

conformation 2b (Fig. 1). Therefore, 2⁰,4⁰-BNAs with the N-type

conformation and 3⁰,4⁰-BNAs with the S-type conformation have

Due to this fact, several groups have directed their research ef-

forts toward the synthesis of novel bridged nucleosides in an at-

tempt to determine the best bridged nucleic acid for antisense

drug therapy. Following the synthesis and evaluation of the

2⁰-O,4⁰-C-⁴and 3⁰-O,4⁰-C-⁵bridged nucleic acids, there have been

reports on the synthesis of 2⁰-NO,4⁰-C systems,⁶2⁰-N,4⁰-C-bridged

systems,⁷2⁰,4⁰-propylene-BNAs,⁸carbocyclic-BNAs,⁹and more.¹⁰

However, only a few groups have explored the biological activities

of bridged nucleoside analogues themselves (see Fig. 2 for selected

Figure 1. Locked conformations of 2⁰,4⁰-bridged system 1a/1b and 3⁰,4⁰-bridged

system 2a/2b.

⇑

Corresponding author. Tel.: +1 858 284 2400; fax: +1 858 284 2469.

E-mail address: kcn@scripps.edu (K.C. Nicolaou).

0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.

doi:10.1016/j.bmc.2011.07.022

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5649

Additionally, a new type of 3⁰,4⁰-bridged nucleoside was synthe-

sized through a novel [2+2] cycloaddition pathway (see com-

pounds 52 and 53, Fig. 3). All compounds were tested for

antibacterial, antitumor, and antiviral activities, with some com-

pounds displaying signiﬁcant biological activities.

2. Results and discussion

2.1. Synthesis of 2⁰,4⁰-bridged nucleoside analogues

Beginning from commercially available diacetone

D-glucose,

oxidation, then reduction to invert the secondary alcohol, and

subsequent benzylation afforded literature-known diacetonide

54 (Scheme 1).²⁰One-pot selective acetonide removal and oxida-

tive cleavage with periodic acid furnished aldehyde 55 in 98%

yield, from which differentially protected sugar 56 was prepared

through a known sequence of steps.^5c,21Vorbrüggen coupling²²

with 2,6-diaminopurine then installed the base onto diacetate

56; however, two different products resulted, depending on the

speciﬁc conditions employed. The use of only 2 equiv of N,O-

bis(trimethylsilyl)acetamide (BSA) resulted in a quantitative yield

of the thermodynamic product N9-isomer 57. Increasing the

amount of BSA to 5 equiv caused the formation of compound

57 plus the kinetic product unnatural N7-isomer 58 in a 1.6:1 ra-

tio and 95% total yield. This appears to be due to the fact that ex-

cess BSA further silylates the 2,6-diaminopurine, resulting in a

species that can attack the diacetate with either the N9 or N7

purine positions.²³

2⁰,4⁰-Bridged compound 59 was constructed through base-in-

duced cyclization of N9-isomer 57 (aq 2 M NaOH, 94% yield,

Scheme 2). From this point, the synthesis diverged to produce a

number of substituted amino analogues as shown in Scheme 2.

Cyclization product 59 was peracylated with benzoyl chloride to

afford tetrabenzoate 60 in 52% yield, along with a number of less

substituted side products which were not isolated or characterized.

Desilylation of compound 60 (HFꢀpy) then led to the targeted

nucleoside 15 in 50% yield. Compound 59 was also deprotected di-

rectly with HFꢀpy to give diamino compound 16 in 85% yield. The

use of boron trichloride to remove the benzyl ether from this inter-

mediate furnished the expected b-nucleoside 17 in 85% yield, along

Figure 2. Previously synthesized bridged nucleoside analogues 3–14 and their

biological activities. A₃AR = adenosine A₃receptor. CC₅₀= Concentration at which

compound is cytotoxic to half the amount of cells. MT-4 = metallothionein 4, a

human T cell line. LA1 = lymphocytotropic strain of HIV-1.

with a 10% yield of the corresponding a-nucleoside (18), the mix-

ture being a result of oxonium formation at the anomeric position.

Additionally, the difference in the nucleophilicity of the amine

groups in intermediate 59 was exploited to furnish, through reduc-

tive alkylation, mono-substituted derivatives 61–64 with ethyl,

n-octyl, n-butyl, and isobutyl groups, respectively (see Scheme 2).²⁴

The yields in this step were moderate (48%, 29%, 57%, and 52%,

respectively) due to incomplete reactions and decomposition of

the alkyl aldehyde components. Desilylation (TBAF) of these prod-

ucts afforded compounds 19–21 and 23 in good yields. Further

debenzylation of n-butylamine 21 and isobutylamine 23 with

BCl₃furnished butylamine diol 22 and isobutylamine diol 24 in

71% and 64% yield, respectively. Due to the lack of biological activ-

ity of nucleosides 22 and 24, it was decided not to proceed with the

deprotection of compounds 19 and 20.

With N7-isomer 58 in hand, bridged nucleoside analogues with

unnatural bases attached became synthetically accessible. Thus, in

a similar fashion to the N9-isomer 57, N7-isomer 58 was cyclized

with aqueous sodium hydroxide to produce 2⁰,4⁰-bridged tetrahy-

drofuran ring system 65 in 85% yield (Scheme 3). Reductive alkyl-

ation of the latter compound with isobutyraldehyde formed

isobutylamine 66, which furnished targeted nucleoside 25 upon

desilylation (TBAF) in 34% yield over the two steps. Alternatively,

intermediate 65 could be desilylated directly with HFꢀpy to gener-

ate analogue 26. Debenzylation (BCl₃) then furnished nucleoside

27 in 46% yield over the two steps. Further explorations into these

examples). This appears to be a research area that warrants further

attention, for what has been published does show potential for

medicinal applications. Thus, and as shown in Figure 2, select

epoxide- and cyclopropane-bridged systems have been shown to

have signiﬁcant antiviral activity (3–6),¹¹and act as adenosine A₃

receptor inhibitors (7 and 8)¹²and DNA polymerase modulating

agents (9 and 10).¹³Also reported were antiviral 3⁰,4⁰-oxetane

bridged nucleoside analogue 11,¹⁴cytotoxic 3⁰,4⁰-tetrahydrofuran

bridged nucleoside derivative 12,¹⁵and antiviral 2⁰,3⁰-hexa-

hydroisobenzofuran bridged nucleosides 13.¹⁶Additionally,

a

number of 2⁰,4⁰-bridged nucleoside analogues such as compounds

14 have demonstrated the ability to act as adenosine A₃receptor

antagonists (see Fig. 2),¹⁷while other 2⁰,4⁰-bridged derivatives

have shown no signiﬁcant antiviral¹⁸or anticancer¹⁹activity.

Since there are a number of nucleoside analogue drugs cur-

rently in clinical use and in clinical trials, we felt it was important

to further probe the biological activity of these bridged nucleoside

systems while simultaneously developing new synthetic strategies

for their continued development. Herein, we describe the synthesis

of a library of 2⁰,4⁰- and 3⁰,4⁰-bridged nucleoside analogues which

not only differ by the structure of the bridge, but also by the

substitution on the adenine base (see compounds 15–51, Fig. 3).

5650

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

Figure 3. Synthesized compound library of bridged nucleoside analogues (15–53).

unnatural N7-isomeric analogues were not pursued due to instabil-

ity problems with the nucleoside base during chemical transforma-

tions, which suggested that these compounds may also be too

labile for biological applications.

Bridged nucleoside analogues were also synthesized using

nucleobases other than 2,6-diaminopurine (see Scheme 4). Vor-

brüggen reaction of 2-chloroadenine with diacetate 56 produced

nucleoside 67 as a single isomer in 85% yield. Deacetylation

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5651

Scheme 1. Synthesis of compounds 57 and 58. Reagents and conditions: (a) H₅IO₆

(1.2 equiv), EtOAc, 25 °C, 1 h, 98%; (b) 2,6-diaminopurine (1.5 equiv), BSA (2.3 or 5.0 equiv),

MeCN, 65 °C, 1.5 h; then TMSOTf (2.0 or 2.8 equiv), 65 °C, 3 h, 100% of 57 or 59% of 57 + 36%

of 58. H₅IO₆= periodic acid; EtOAc = ethyl acetate; MeCN = acetonitrile; Bn = benzyl;

TBDPS = tert-butyldiphenylsilyl; Ts = para-toluenesulfonyl; Ac = acetyl; BSA = N,O-bis(tri-

methylsilyl)acetamide; TMSOTf = trimethylsilyl triﬂuoromethylsulfonate.

Scheme 3. Synthesis of compounds 25–27. Reagents and conditions: (a) aq 2 M

NaOH, THF, 25 °C, 2 h, 85%; (b) isobutyraldehyde (8.0 equiv), NaBH₃CN (6.0 equiv),

MeOH, 25 °C, 48 h, 44%; (c) TBAF (2.0 equiv), THF, 25 °C, 16 h, 77%; (d) HFꢀpy

(5.0 equiv), THF, 25 °C, 12 h; (e) BCl₃(2.0 equiv), CH₂Cl₂, 25 °C, 1 h, 46% over the two

steps.

Scheme 2. Synthesis of compounds 15–24. Reagents and conditions: (a) aq 2 M

NaOH, THF, 25 °C, 2 h, 94%; (b) BzCl (4.0 equiv), py, 25 °C, 18 h, 52%; (c) HFꢀpy

(5.0 equiv), THF, 25 °C, 12 h, 50% for 15, 85% for 16; (d) BCl₃(2.0 equiv), CH₂Cl₂,

25 °C, 1 h, 85% of 17 + 10% of 18, 71% for 22, 64% for 24; (e) alkyl aldehyde

(8.0 equiv), NaBH₃CN (6.0 equiv), MeOH, 25 °C, 48 h, 48% for 61, 29% for 62, 57% for

63, 52% for 64; (f) TBAF (2.0 equiv), THF, 25 °C, 16 h, 79% for 19, 77% for 20, 77% for

21, 98% for 23. Bz = benzoyl; py = pyridine; THF = tetrahydrofuran; TBAF = tetra-n-

butylammonium ﬂuoride.

Scheme 4. Synthesis of compounds 28–32. Reagents and conditions: (a) 2-

chloroadenine (1.5 equiv), BSA (2.3 equiv), MeCN, 65 °C, 1.5 h; then TMSOTf

(2.0 equiv), 65 °C, 3 h, 85%; (b) aq 2 M NaOH, THF, 25 °C, 15 h, 87% for 68, 81% for

70; (c) HFꢀpy (5.0 equiv), THF, 25 °C, 12 h, 61% for 28, 85% for 30; (d) MsOH

(78 equiv), CH₂Cl₂, 0 °C, 1.5 h, 46%; (e) 2-ﬂuoroadenine (2.0 equiv), BSA (2.5 equiv),

MeCN, 65 °C, 1.5 h; then TMSOTf (2.0 equiv), 65 °C, 3 h, 78%; (f) BCl₃(2.0 equiv),

CH₂Cl₂, 25 °C, 1 h, 69%; (g) morpholine (2.0 equiv), DMSO, 95 °C, 24 h, 76% over the

two steps. MsOH = methanesulfonic acid; DMSO = dimethyl sulfoxide.

followed by cyclization to bridged intermediate 68 was effected

with aq NaOH; desilylation with HFꢀpy then gave nucleoside 28

in 53% overall yield from compound 67. The use of methanesulfon-

ic acid as a debenzylation agent was crucial to the successful gen-

eration of aminochloride diol 29 (46% yield),¹⁷as the typical boron

trichloride conditions proved to be too harsh for this analogue.

5652

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

Amino ﬂuoride derivative 31 could also be prepared in a similar

manner (see Scheme 4). Vorbrüggen coupling with diacetate 56

and 2-ﬂuoroadenine led to nucleoside 69 (78% yield), which could

then be deacetylated and cyclized with NaOH to produce interme-

diate 70 in 81% overall yield. Desilylation (HFꢀpy), followed by deb-

enzylation (BCl₃) under the standard conditions, then furnished,

sequentially, hydroxy benzyl ether 30 and diol 31 in 59% yield over

the two steps. Intermediate 70 was converted into bridged ana-

logue 32 through morpholine displacement of the ﬂuoride moiety

and desilylation (HFꢀpy) in 76% yield over the two steps (see

Scheme 4).

2,6-Dichloropurine nucleoside 71 was synthesized in 64% yield

through Vorbrüggen reaction of diacetate 56 and 2,6-dichloro-9H-

purine as shown in Scheme 5. This compound proved to be one of

the most derivatizable intermediates in this study (see Scheme 5).

The difference in the electrophilicity between the C2 and C6 car-

bons of nucleoside 71 was exploited to synthesize aminochloride

72, in 93% yield, via displacement of the C6 chlorine residue with

benzylamine.²⁵Cyclization induced by NaOH then afforded locked

nucleoside 73 in 68% yield. Desilylation of the latter compound

produced alcohol 33 (HFꢀpy, 96% yield), and subsequent debenzy-

lation of compound 33 (MsOH) furnished diol analogue 34 (61%

yield). A similar sequence of steps could also be employed to

synthesize nucleoside 36. Thus, compound 71 was reacted with

N-methylbenzylamine to afford intermediate 74 lacking the acet-

oxy group in 82% yield (see Scheme 5). Cyclization of compound

74 through the action of NaOH then produced the bridged interme-

diate 75 (98% yield), followed by desilylation (HFꢀpy) to form alco-

hol 35 (75% yield). Debenzylation (MsOH) of the latter compound

then generated dihydroxy derivative 36 (75% yield). Displacement

of both chlorine residues of intermediate 71 and concurrent ring

closure were effected by exposure to NaOH in MeOH to afford

locked nucleoside derivative 37 upon desilylation (HFꢀpy), in 69%

overall yield. Finally, debenzylation of the latter compound with

BCl₃furnished dimethoxy nucleoside diol 38 (77% yield).

Stille coupling reactions were employed to synthesize addi-

tional derivatives from dichloro compound 71 as shown in

Schemes 5 and 6.²⁶Reaction of intermediate 71 with tri-n-

butyl(1-ethoxyvinyl)tin and bis(triphenylphosphine)palladium(II)

chloride, followed immediately by base-induced deacetylation/

Scheme 6. Synthesis of compounds 40–47. Reagents and conditions: (a) allyl(tri-n-

butyl)tin (3.0 equiv), Pd(PPh₃)₂Cl₂(0.10 equiv), DMF, 95 °C, 6 h; (b) aq 2 M NaOH,

THF, 25 °C, 12 h, 42% over the two steps for 76, 76% for 77, 98% for 78; (c) HFꢀpy

(5.0 equiv), THF, 25 °C, 12 h, 87% for 40, 100% for 42, 99% for 44; (d) H₂, Pd(OH)₂

(10% w/w), EtOH, 50 °C, 12 h, 50%; (e) 2-(tri-n-butylstannyl)furan (2.0 equiv),

Pd(PPh₃)₂Cl₂(0.075 equiv), DMF, 95 °C, 7 h, 99%; (f) BCl₃(2.0 equiv), CH₂Cl₂, 25 °C,

1 h, 90% for 43, 40% of 45 + 33% of 46; (g) 2-(tri-n-butylstannyl)thiophene

(2.0 equiv), Pd(PPh₃)₂Cl₂(0.12 equiv), DMF, 80 °C, 7 h, 72%; (h) chlorosulfonyl

isocyanate (4.3 equiv), formic acid (4.3 equiv), py (5.8 equiv), CH₂Cl₂, 25 °C, 24 h,

35%.

Scheme 5. Synthesis of compounds 33–39. Reagents and conditions: (a) 2,6-

dichloro-9H-purine (2.0 equiv), BSA (2.5 equiv), MeCN, 95 °C, 1.5 h; then TMSOTf

(2.0 equiv), 80 °C, 3 h, 64%; (b) benzylamine (5.0 equiv) or N-methylbenzylamine

(5.0 equiv), MeOH, 55 °C, 12 h, 93% for 72, 82% for 74; (c) aq 2 M NaOH, THF, 25 °C,

15 h, 68% for 73, 98% for 75; (d) HFꢀpy (5.0 equiv), THF, 25 °C, 12 h, 96% for 33, 75%

for 35, 77% for 37, 31% over the three steps for 39; (e) MsOH (78 equiv), CH₂Cl₂, 0 °C,

1.5 h, 61% for 34, 75% for 36; (f) NaOH (15 equiv), MeOH/THF (1:1), 25 °C, 12 h, 90%;

(g) BCl₃(2.0 equiv), CH₂Cl₂, 25 °C, 1 h, 77%; (h) tri-n-butyl(1-ethoxyvinyl)tin

(2.0 equiv), Pd(PPh₃)₂Cl₂(0.10 equiv), DMF, 95 °C, 18 h. DMF = dimethylformamide.

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5653

cyclization and desilylation (HFꢀpy), generated diketone nucleoside

39 in 31% yield over the three steps (Scheme 5). Intermediate 71

also underwent reaction with allyl(tri-n-butyl)tin to afford, upon

subsequent cyclization with NaOH, compound 76 in 42% overall

yield for the two steps (see Scheme 6). Formation of the conjugated

oleﬁn at the C6 position of the purine within compound 76 was

presumed to have occurred as a result of palladium- and base-in-

duced isomerization of the coupled allyl moiety. Desilylation of

compound 76 (HFꢀpy) furnished intermediate 40 (87% yield),

which was converted to dipropyl derivative 41 (50% yield) by

reduction and concomitant debenzylation with hydrogen and

Pearlman’s catalyst. Stille reaction of dichloride 71 with 2-(tri-n-

butylstannyl)furan, followed by ring closure (NaOH), afforded

bridged product 77 (75% over the two steps) (Scheme 6). Sequen-

tial desilylation (HFꢀpy) and debenzylation (BCl₃) of compound

77 then produced, through the intermediacy of alcohol 42, difuran

diol 43 in 90% yield over the two steps. Dichloride 71 was also re-

acted with 2-(tri-n-butylstannyl)thiophene to furnish the corre-

sponding bis-thiophene derivative, which was converted to the

bridged nucleoside 78 by reaction with NaOH in 71% overall yield

(Scheme 6). Desilylation of the latter with HFꢀpy gave intermediate

44 (99% yield), which was debenzylated (BCl₃) to afford b-nucleo-

side 45 (40% yield), along with its a-anomer 46 (33% yield). Inter-

mediate 44 was also converted to sulfamoyl derivative 47 in 35%

yield by reaction with freshly prepared sulfamoyl chloride in the

presence of pyridine.²⁷

2.2. Synthesis of 3⁰,4⁰-bridged nucleoside analogues

Scheme 7. Synthesis of compounds 48–51. Reagents and conditions: (a) 2,6-

dichloro-9H-purine (2.0 equiv), BSA (3.5 equiv), MeCN, ꢁ40 °C, 5 min; then TMSOTf

The synthesis of the 3⁰-O,4⁰-C-bridged nucleoside analogues

began with conversion of literature-known dimesylate 79^4eto

dichloro nucleoside 80 through Vorbrüggen reaction with

2,6-dichloro-9H-purine (Scheme 7). The standard Vorbrüggen

coupling procedure that had been employed in the synthesis of the

other nucleosides mentioned above led to nucleoside 80 in low

yield; however, an 84% yield of this nucleoside was achieved by

the use of microwave irradiation instead of moderate heating.²⁸

Debenzylation and concomitant deacetylation of compound 80 with

boron trichloride led to a 90% yield of intermediate 81. Treatment of

the latter compound with potassium carbonate in THF then caused

smooth cyclization to afford compound 48 in 79% yield. At this point,

all that remained to reach the targeted dichloro-locked nucleoside

was manipulation of the mesyl group to form the 5⁰-hydroxyl

functionality. However, attempts to accomplish this goal by reaction

with NaOBz (followed by benzoate cleavage) resulted in additional

displacement of the C6 chloride residue, producing dibenzoate 49

in 82% yield (see Scheme 7). The 5⁰-benzoate could not be cleaved

without causing decomposition, thus an alternate route to these

3⁰,4⁰-bridged systems that did not involve chloride residues on the

nucleobase was implemented as described below.

Dichloride 80 was converted to difuran compound 82 via a Stille

reaction with 2-(tri-n-butylstannyl)furan (84% yield), and the latter

compound was deprotected (BCl₃) to give diol 83 in 82% yield (see

Scheme 7). Treatment of compound 83 with potassium carbonate

in THF afforded intermediate 50 in 91% yield. This compound

was successfully converted into diol 51 through the intermediacy

of the corresponding 5⁰-benzoate (NaOBz; then NaOMe, 69% yield

over the two steps). Other studies directed toward a number of dif-

ferent bridged nucleosides were thwarted, primarily due to difﬁ-

culties in bringing about the desired oxetane formation. The

ability of the nucleoside to undergo ring closure to the oxetane

derivative was found to be strongly dependent on the actual sub-

stituents on the nucleobase. Additionally, after cyclization of the

intermediate, the oxetane ring was found to be unstable to typical

reaction conditions as well as general handling, causing concern

about its stability during biological evaluation.

(1.5 equiv), l-waves, 80 °C, 5 min, 84%; (b) BCl₃(2.0 equiv), CH₂Cl₂, 25 °C, 1 h, 90%

for 81, 82% for 83; (c) K₂CO₃(4.0 equiv), THF, 25 °C, 18 h, 79% for 48, 91% for 50; (d)

NaOBz (2.0 equiv), DMF, 90 °C, 4.5 h, 82% for 49; (e) 2-(tri-n-butylstannyl)furan

(4.0 equiv), Pd(PPh₃)₂Cl₂(0.091 equiv), DMF, 95 °C, 3 h, 84%; (f) aq NaOMe, MeOH,

25 °C , 30 min, 69% over the two steps for 51.

Faced with these difﬁculties, we opted to synthesize analogues

with an alternate 3⁰,4⁰-bridged ring system which would be more

stable than those containing the 3⁰,4⁰-oxetane bridge, and could

be constructed through a ﬂexible route to afford a variety of ana-

logues. These analogues were synthesized via a [2+2] cycloaddition

reaction as shown in Scheme 8. Esteriﬁcation of known compound

84²⁹with trimethylsilyl diazomethane formed methyl ester 85

(88% yield). Elimination of the tosyl group (DBU) to form the

a,b-

unsaturated ester, followed by [2+2] cycloaddition of the resulting

product with 1,2-cis-dichloroethylene under photoirradiation con-

ditions, generated compound 86 in 18% yield over the two steps as

the major product, in addition to two other diastereomers (ca.

4:2:1) differing in the orientation of the chlorine residues. In all

three diastereomers of compound 86, the cyclobutane ring occup-

pied the position opposite the acetonide moiety as expected on

steric grounds. The trans relationship of the chlorine atoms within

isomer 86, proven by NMR spectroscopy (ROESY), conﬁrmed the

radical nature of the [2+2] photocycloaddition. The stereochemis-

try of the two diastereomers of compound 86 was not determined.

Following chromatographic separation of compound 86, its aceto-

nide moiety was cleaved and the resulting compound peracetylat-

ed in a one-pot reaction (AcOH, concd H₂SO₄, Ac₂O) to afford the

corresponding bis-acetate. Subsequent Vorbrüggen reaction of

the latter product with 2,6-diaminopurine and BSA provided

bridged nucleoside 52 in 36% yield over the two steps. Deacetyla-

tion with potassium carbonate in methanol then provided the de-

sired alcohol 53 in 83% yield.

Although the targeted product 53 was accessed by this method,

the yield of the key [2+2] cycloaddition reaction was disappointing.

It was reasoned that the inefﬁciency of this process was, in part,

due to the volatility of both the elimination product from

5654

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

Table 1

Antibacterial, anticancer, and antiviral activities of bridged nucleoside analogues

Compound

Antibacterial

activity MIC^a

Anticancer activity

Antiviral activity

b

c

(

l

M)

IC₅₀

(lM)

IC₅₀

(lM)

E. coli

S. aureus

CCRF-CEM

Raji

SF162

HxB2

15

19

21

33

36

40

NA

16

NA

—

NA

8

NA

—

0.36

NA

7.6

0.0005

—

0.25

NA

5.8

0.009

—

NA

60.0

68.0

7.0

28.0

2.4

24.9

2.4^e

—

27.9

d

—

Cladribine³⁰

AZT

—

0.078

—

0.037

a

b

c

Minimum inhibitory concentration.

Concentration that causes 50% of cell growth inhibition.

Concentration that causes 50% neutralization of virus.

d

While an IC₅₀below 100 lM was calculated, this was determined to be from

toxicity to the TZM-bl cells, not neutralization of the virus.

e

Compound 40 is toxic to the TZM-bl cells, however, below the toxicity limit it

did display antiviral activity. If the activities due to toxicity at higher concentration

of 40 are ignored, an IC₅₀of 2.4 lM can be determined. CCRF-CEM = Human T

leukemic lymphoblasts derived from acute lymphoblastic leukemia. Raji = Human B

lymphocytes derived from Burkitt’s lymphoma. SF162 and HxB2 = HIV-1 pseud-

oviruses. NA = not active at the highest concentration tested (64 lM for the anti-

bacterial assay, 10

lM for the cytotoxicity assay, and 100

lM for the antiviral

assay).

Scheme 8. Synthesis of compound 53. Reagents and conditions: (a) TMSCHN₂

(1.2 equiv), Et₂O/MeOH (1:1), 0 °C, 30 min, 88%; (b) DBU (1.2 equiv), benzene, 25 °C,

5 h; (c) 1,2-cis-dichloroethylene (15.9 equiv), MeCN, h

m

, 25 °C, 3.5 d, 18% over the

N(Bz)₂

NH₂

two steps; (d) concd H₂SO₄, Ac₂O (12 equiv), AcOH, 25 °C, 16 h; (e) 2,6-diaminop-

urine (1.5 equiv), BSA (2.3 equiv), MeCN, 65 °C, 1.5 h; then TMSOTf (2.0 equiv),

65 °C, 3 h, 36% over the two steps for 52; (f) K₂CO₃(0.30 equiv), MeOH, 25 °C,

30 min, 83% over one step or 38% over the three steps from compound 86; (g) n-

BuOH (1.2 equiv), DCC (1.2 equiv), DMAP (0.15 equiv), CH₂Cl₂, 25 °C, 24 h; then DBU

N

HO

O

N

N(Bz)₂

HO

N

H

O

OBn

15: anticancer

OBn

(1.2 equiv), 25 °C, 24 h, 67%; (h) 1,2-cis-dichloroethylene (16.9 equiv), MeCN, hm,

25 °C, 6.5 d, 33% based on 12% recovered starting material. TMSCHN₂= trimethyl-

silyl diazomethane; DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene; Ac₂O = acetic anhy-

dride; AcOH = acetic acid; DCC = N,N⁰-dicyclohexylcarbodiimide; DMAP = 4-

dimethylaminopyridine.

19: antiviral

(IC₅₀SF162 = 60.0 µM)

(IC₅₀CCRF-CEM = 0.36 µM)

(IC₅₀Ra ji = 0 .25 µM)

NHBn

NH₂

N

compound 85 (step b) and the cycloaddition product 86. To cir-

cumvent the problem, a larger ester group was employed. Thus,

substrate 84 was esteriﬁed with n-BuOH, DCC, and DMAP, and

the resulting product was treated with DBU, leading to unsaturated

n-butyl ester 87 in 67% yield (see Scheme 8). Light-induced [2+2]

cycloaddition of compound 87 with 1,2-cis-dichloroethylene gave

product 88 in an improved 33% yield (based on 12% recovered

starting material), along with the other two diastereomers (27%

combined yield). Intermediate 88 was then exposed to the same

one-pot acetonide cleavage–bis-acetylation procedure and Vor-

brüggen coupling to afford, upon potassium carbonate-induced

deacetylation, desired product 53 in 38% overall yield for the three

steps. This approach provided a more efﬁcient and reliable access

to these new 3⁰,4⁰-carbocyclic bridged nucleoside analogues.

N

HO

N

Cl

N

O

HO

N

H

O

OBn

21: antiviral

(IC₅₀SF162 = 68.0 µM)

(IC₅₀HxB2 = 28.0 µM)

33: antiviral

(IC₅₀SF162 = 7.0 µM)

(IC₅₀HxB2 = 2.4 µM)

Me

Bn

N

HO

Cl

N

O

OH

36: antibacterial + antiviral

(MIC = 16 µM)

OBn

40: anticancer + antiviral

(IC₅₀CCRF-CEM = 7.6 µM)

(IC₅₀Raji = 5.8 µM)

2.3. Biological evaluation

E. coli

(MIC S. aureus = 8 µM)

(IC₅₀SF162 = 27.9 µM)

(IC₅₀HxB2 = 24.9 µM)

(IC₅₀HxB2 = 2.4 µM)

The library of bridged nucleoside analogues 15–53 (Fig. 3) were

tested for antibacterial, antitumor, and antiviral properties. Of

these thirty-nine compounds, six demonstrated signiﬁcant biolog-

ical activities (see Table 1 and Fig. 4). Compound 36 was the only

compound to demonstrate antibacterial activity, showing moder-

O

NH₂

Me

N

NH

N

O

N

Cl

HO

O

ate potency against both Escherichia coli (MIC = 16

lM) and Staph-

ylococcus aureus (MIC = 8 M). It is interesting to note that several

l

OH

cladribine

N₃

other compounds with very similar structures displayed no signif-

icant biological activity. The N-methylbenzylamine structural mo-

tif speciﬁcally appears to be essential, as compound 36 differs from

compound 34 (Fig. 3) only by a methyl group, and compound 29

(Fig. 3) differs from nucleoside 36 by having no substituents on

the C6 amino group. Neither of these two compounds (i.e., 34

azidothy midine (AZT)

(IC₅₀SF162 = 0.078 µM)

(IC₅₀HxB2 = 0.037 µM)

(IC₅₀CCRF-CEM = 0.0005 µM)

(IC₅₀Raji = 0.009 µM)

Figure 4. Structures of biologically active, bridged nucleoside analogues 15, 19, 21,

33, 36, and 40 as compared to those of cladribine and azidothymidine (AZT).

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5655

and 29) displayed signiﬁcant antibacterial activity. Additionally,

properties (e.g., compound 33, SF162 IC₅₀= 7.0

lM; HxB2

benzylation of the 3⁰-hydroxyl group of compound 36 also results

in complete loss of its biological activity since the resulting nucle-

oside (i.e., 35, Fig. 3) is inactive against E. coli and S. aureus.

Two bridged nucleosides were found to exhibit signiﬁcant anti-

tumor activity: compound 15 and compound 40 (see Table 1 and

Fig. 4). Nucleoside 15 was found to possess the most potent activ-

IC₅₀= 2.4 lM). While the mechanism of action of these nucleosides

remains unknown, it has been shown that the biological activity is

dependent on not only the bridged system on the sugar, but the

speciﬁc modiﬁcations of the purine residue. These compounds

are, to the best of our knowledge, the ﬁrst examples of 2⁰,4⁰-

bridged nucleosides exhibiting antibacterial, anticancer, or antivi-

ral activity.

ity of the two against CEM (IC₅₀= 0.36

lM) and Raji

(IC₅₀= 0.25 M) cancer cell lines. However, these activities are con-

l

siderably lower than those of the anticancer nucleoside agent

cladribine (CEM: IC₅₀= 0.5 nM; Raji: IC₅₀= 9 nM). Compound 40

(Fig. 4) showed less potent activities than compound 15, with an

4. Experimental methods

4.1. Chemical synthesis

IC₅₀of 7.6 lM against the T cell line CEM and an IC₅₀of 5.8 lM

against the B cell line Raji. Important to note is the fact that the

2⁰,4⁰-bridged nucleoside analogue corresponding to the structure

of cladribine, compound 29 (Fig. 3), was also synthesized, but did

All reactions were carried out under an argon atmosphere with

dry solvents under anhydrous conditions, unless otherwise noted.

Dry tetrahydrofuran (THF), toluene, benzene, diethyl ether (Et₂O),

N,N⁰-dimethylformamide (DMF), and methylene chloride (CH₂Cl₂)

were obtained by passing commercially available pre-dried, oxy-

gen-free formulations through activated alumina columns. Yields

refer to chromatographically and spectroscopically (¹H NMR)

homogeneous materials, unless otherwise stated. Reagents were

purchased at the highest commercial quality and used without fur-

ther puriﬁcation, unless otherwise stated. Reactions were moni-

tored by thin-layer chromatography (TLC) carried out on

0.25 mm E. Merck silica gel plates (60F-254) using UV light as visu-

alizing agent and an ethanolic solution of anisaldehyde and heat as

developing agents. E. Merck silica gel (60, particle size 0.040–

0.063 mm) was used for ﬂash column chromatography. Preparative

thin-layer chromatography (PTLC) separations were carried out on

0.25 or 0.50 mm E. Merck silica gel plates (60F-254 for normal sil-

ica, RF-18 F-254 for C₁₈-silica).

not exhibit signiﬁcant cytotoxicity below 10 lM. It appears that

the act of locking the conformation of cladribine, or the addition

of an extra CH₂O moiety, changes the structure of the molecule en-

ough to deplete its antitumor properties.

The most promising biological activities of the synthesized

bridged nucleosides were discovered from screening the library

against HIV-1 (see Table 1). Thus, compound 36 (Fig. 4), which

had also demonstrated antibacterial activity, showed moderate

activity against both the SF162 (IC₅₀= 27.9

(IC₅₀= 24.9 M) pseudoviruses. Compound 19 (Fig. 4), with an eth-

ylamine functionality, also showed moderate activity, but only

against SF162 (IC₅₀= 60.0 M). Similarly, n-butylamine nucleoside

21 (Fig. 4) displayed moderate activity against both pseudoviruses

(SF162 IC₅₀= 68.0 M; HxB2 IC₅₀= 28.0 M). However, octyl ana-

lM) and HxB2

l

logue 20 (Fig. 3) and isobutyl nucleoside 23 (Fig. 3) did not exhibit

any signiﬁcant antiviral activities against the viruses. This suggests

that an alkyl amine containing approximately two to four carbon

atoms with no branching is an essential structural motif for antivi-

ral properties.

The highest antiviral activities were demonstrated by com-

pounds 33 and 40 (see Table 1). Nucleoside 40 (Fig. 4), which

had also displayed moderate antitumor activity, was found to be

toxic to the TZM-bl cells used in the antiviral assay at concentra-

NMR spectra were recorded on Bruker AV-400, DRX-500, or

DRX-600 instruments and calibrated using residual undeuterated

solvent (CDCl₃: d_H= 7.26 ppm, d_C= 77.16 ppm; acetone-d₆: d_H

=

2.05 ppm, d_C= 29.84 ppm; CD₃CN: d_H= 1.94 ppm, d_C= 1.32 ppm;

CD₃OD: d_H= 3.31 ppm, d_C= 49.00 ppm; D₂O: d_H= 4.79 ppm)³¹as

an internal reference. The following abbreviations were used to

designate the multiplicities: s = singlet, d = doublet, t = triplet,

q = quartet, quin = quintet, m = multiplet, br = broad. Infrared (IR)

spectra were recorded on a Perkin-Elmer 100 FT-IR spectrometer.

High-resolution mass spectra (HRMS) were recorded on an Agilent

ESI-TOF (time of ﬂight) mass spectrometer using MALDI (matrix-

assisted laser desorption ionization) or ESI (electrospray ioniza-

tion). Optical rotations were recorded on a Perkin-Elmer Model

343 polarimeter at 589 nm, and are reported in units of 10^ꢁ1

(deg cm²g^ꢁ1).

tions at or above 33 lM. However, compound 40 (Fig. 4) did exhi-

bit some actual antiviral activity against pseudovirus HxB2 below

the concentration at which it was causing cell death. Therefore,

an IC₅₀was approximated by ignoring the higher concentrations

at which it was toxic; this approximation led to an IC₅₀against

HxB2 of 2.4

ited the most potent antiviral activity without concomitant cellular

toxicity (SF162: IC₅₀= 7.0 M; HxB2: IC₅₀= 2.4 M). Analogue 35

lM for this analogue (40). Nucleoside 33 (Fig. 4) exhib-

l

(Fig. 3), which contains a N-methylbenzylamine moiety instead

of a benzylamine substituent, did not display any antiviral activity.

While compounds 33 (Fig. 4) and 40 (Fig. 4) still remain less potent

4.1.1. (2R,3S,5S)-4-(Benzyloxy)-5-((tert-butyldiphenylsilyloxy)-

methyl)-2-(2,6-diamino-9H-purin-9-yl)-5-(tosyloxymethyl)-

tetrahydrofuran-3-yl acetate (57)

than the antiviral nucleoside AZT (SF162: IC₅₀= 0.078

lM; HxB2:

Compound 56 (1.35 g, 1.81 mmol) and 2,6-diaminopurine

(407 mg, 2.71 mmol) were suspended in MeCN (17.4 mL), and

BSA (1.06 mL, 4.14 mmol) was added. The mixture was heated at

65 °C for 1.5 h, after which the mixture was cooled to 0 °C and

TMSOTf (0.72 mL, 3.62 mmol) was added dropwise. The solution

was then stirred at 65 °C for 3 h. The reaction mixture was

quenched with cold satd aq NaHCO₃(10 mL) and extracted with

CH₂Cl₂. The organics were washed with satd aq NaHCO₃

(2 ꢂ 10 mL) and brine (2 ꢂ 10 mL), dried over MgSO₄, ﬁltered, con-

centrated, and puriﬁed by ﬂash column chromatography (silica,

EtOAc) to give compound 57 (1.51 g, 1.81 mmol, 100%). Compound

IC₅₀= 0.037 M), these activities suggest considerable potential

l

for these bridged nucleosides as possible lead compounds for fur-

ther optimization.

3. Conclusion

In conclusion, we have synthesized a focused compound library

of both 2⁰,4⁰- and 3⁰,4⁰-bridged nucleoside analogues with various

modiﬁcations on the purine base. This study included the develop-

ment of a [2+2] cycloaddition strategy to synthesize novel 3⁰,4⁰-

carbocyclic bridged systems expediently and with the ﬂexibility

to target considerable molecular diversity. Some of the compounds

synthesized were found to exhibit diverse but selective biological

activities, with the best compounds displaying potent antiviral

57: white foam; R_f= 0.50 (silica, EtOAc); ½a _D²⁵

= ꢁ7.0 (MeCN, c

ꢃ

1.57); FT-IR (ﬁlm) m_max3371, 2932, 1735, 1599, 1472, 1427,

1409, 1371, 1238, 1189, 1176, 1095, 1042, 975, 915, 813, 790,

741, 601, 665 cm^ꢁ1

;

¹H NMR (CDCl₃, 500 MHz) d = 7.70

5656

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

(d, J = 8.3 Hz, 2H), 7.64–7.59 (m, 4H), 7.44 (s, 1H), 7.44–7.31 (m, 7H),

7.28–7.25 (m, 4H), 7.18 (d, J = 8.0 Hz, 2H), 6.04 (t, J = 5.7 Hz, 1H), 5.83

(d, J = 5.8 Hz, 1H), 5.53 (s, 2H), 4.78 (d, J = 5.5 Hz, 1H), 4.58 (d,

J = 11.4 Hz, 1H), 4.52 (d, J = 11.4 Hz, 1H), 4.42 (d, J = 10.5 Hz, 1H),

4.35 (d, J = 10.6 Hz, 1H), 4.27 (s, 2H), 3.88 (d, J = 10.8 Hz, 1H), 3.74

(d, J = 10.9 Hz, 1H), 2.37 (s, 3H), 2.03 (s, 3H), 0.99 (s, 9H) ppm; ¹³C

NMR (CDCl₃, 126 MHz) d = 170.05, 159.58, 155.88, 151.61, 144.72,

137.39, 137.39, 137.19, 135.71, 135.65, 133.01, 132.85, 132.27,

130.08, 130.06, 129.74, 128.68, 128.31, 128.14, 128.09, 128.05,

127.99, 115.00, 86.21, 86.14, 78.26, 74.72, 73.57, 68.75, 63.86,

26.88, 21.71, 20.76, 19.22 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd

for C₄₅H₄₉N₆O₈SSi⁺837.3096, found 837.3100.

4.1.4. N,N⁰-(9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyl-

diphenylsilyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-

9H-purine-2,6-diyl)bis(N-benzoylbenzamide) (60)

Compound 59 (30 mg, 0.0483 mmol) was co-evaporated twice

with anhydrous pyridine (2 ꢂ 0.6 mL) and then dissolved in anhy-

drous pyridine (0.6 mL). The solution was cooled to 0 °C, benzoyl

chloride (0.023 mL, 0.193 mmol) was added, and the mixture was

stirred at rt for 18 h. The mixture was diluted with EtOAc (4 mL)

and washed with H₂O (2 mL). The organics were dried over MgSO₄,

ﬁltered, concentrated, and puriﬁed by preparative-plate chroma-

tography (silica, hexanes:EtOAc 2:1) to give compound 60

(26 mg, 0.0251 mmol, 52%). Compound 60: yellow oil; R_f= 0.49

(silica, hexanes:EtOAc 2:1); ½a _D²⁵

ꢃ

= +27.3 (CHCl₃, c 1.20); FT-IR

(ﬁlm) m_max3068, 2932, 2858, 1704, 1599, 1576, 1491, 1449,

4.1.2. (2R,3R,4S,5S)-4-(Benzyloxy)-5-((tert-butyldiphenylsilyloxy)-

methyl)-2-(2,6-diamino-7H-purin-7-yl)-5-(tosyloxymethyl)-

tetrahydrofuran-3-yl acetate (58)

1428, 1365, 1244, 1177, 1112, 1048, 1027, 1001, 976, 932, 909,

864, 823, 794, 733, 701 cm^ꢁ^{1 1}H NMR (CDCl₃, 500 MHz) d = 8.13

;

Compound 56 (200 mg, 0.268 mmol) and 2,6-diaminopurine

(61.8 mg, 0.412 mmol) were suspended in MeCN (2.6 mL), and BSA

(0.34 mL, 1.33 mmol) was added. The reaction mixture was heated

at 65 °C for 1 h, after which the mixture was cooled to 0 °C and

TMSOTf (0.15 mL, 0.753 mmol) was added dropwise. The solution

was then stirred at 65 °C for 3.5 h. The reaction was quenched with

cold satd aq NaHCO₃(2 mL) and extracted with CH₂Cl₂. The organics

were washed with satd aq NaHCO₃(2 ꢂ 2 mL) and brine (2 ꢂ 2 mL),

dried over MgSO₄, ﬁltered, concentrated, and puriﬁed by ﬂash col-

umn chromatography (silica, EtOAc) to give compound 58

(81.1 mg, 0.0964 mmol, 36%) along with isomer 57 (133 mg,

0.158 mmol, 59%). Compound 58: white foam; R_f= 0.21 (silica,

(s, 1H), 7.79 (dd, J = 8.4, 1.2 Hz, 4H), 7.67–7.62 (m, 8H), 7.51 (dd,

J = 10.6, 4.3 Hz, 2H), 7.43–7.30 (m, 19H), 7.20 (dd, J = 6.8, 2.7 Hz,

2H), 5.79 (s, 1H), 4.47 (d, J = 11.3 Hz, 1H), 4.38 (d, J = 11.3 Hz,

1H), 4.16 (s, 1H), 4.03 (d, J = 8.2 Hz, 2H), 3.97 (d, J = 12.1 Hz, 1H),

3.91 (d, J = 12.1 Hz, 1H), 3.87 (d, J = 7.9 Hz, 1H), 1.04 (s, 9H) ppm;

¹³C NMR (CDCl₃, 126 MHz) d = 172.44, 172.30, 152.97, 152.58,

143.01, 137.31, 135.97, 135.86, 134.80, 134.44, 133.39, 133.01,

132.95, 132.90, 130.39, 130.36, 129.83, 129.58, 129.12, 129.04,

128.92, 128.50, 128.32, 128.26, 127.98, 126.17, 88.95, 87.19,

77.62, 77.00, 73.06, 72.68, 59.87, 27.23, 19.63 ppm; HRMS (ESI-

TOF) (m/z): [M+H]⁺calcd for C₆₂H₅₅N₆O₈Si⁺1039.3845, found

1039.3826.

EtOAc); ½a ²_D⁵

= ꢁ40.1 (CHCl₃, c 1.85); FT-IR (ﬁlm) m_max3342, 3191,

ꢃ

2932, 1735, 1668, 1625, 1576, 1470, 1428, 1359, 1234, 1189, 1176,

4.1.5. N,N⁰-(9-((1S,3R,4R,7S)-7-(Benzyloxy)-1-(hydroxymethyl)-

2,5-dioxabicyclo[2.2.1]heptan-3-yl)-9H-purine-2,6-diyl)bis(N-

benzoylbenzamide) (15)

1105, 1045, 972, 812, 792, 741, 701, 665 cm^ꢁ^{1 1}H NMR (CDCl₃,

;

500 MHz) d = 7.67 (s, 1H), 7.67–7.64 (m, 2H), 7.53–7.51 (m, 2H),

7.50–7.48 (m, 2H), 7.45–7.41 (m, 2H), 7.35–7.31 (m, 7H), 7.22 (d,

J = 8.6 Hz, 2H), 7.19 (dd, J = 3.8, 1.8 Hz, 2H), 5.87 (d, J = 7.1 Hz, 1H),

5.50 (s, 2H), 5.45 (dd, J = 7.0, 5.8 Hz, 1H), 4.92 (s, 2H), 4.54 (d,

J = 5.7 Hz, 1H), 4.46 (dd, J = 11.1, 2.7 Hz, 2H), 4.41 (d, J = 11.3 Hz,

1H), 4.09 (d, J = 11.1 Hz, 1H), 3.62 (s, 2H), 2.39 (s, 3H), 2.04 (s, 3H),

0.99 (s, 9H) ppm; ¹³C NMR (CDCl₃, 126 MHz) d = 159.95, 151.75,

145.11, 141.00, 136.26, 135.43, 135.25, 132.39, 131.73, 131.66,

130.13, 129.75, 128.60, 128.43, 127.93, 127.88, 127.69, 86.82,

85.84, 77.53, 75.21, 73.27, 67.64, 64.15, 26.72, 21.51, 20.30,

18.97 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₄₅H₄₉N₆O₈SSi⁺

837.3096, found 837.3096.

Compound 60 (91 mg, 0.0880 mmol) was dissolved in THF

(0.55 mL) and cooled to 0 °C. HFꢀpyridine (0.011 mL, 0.440 mmol)

was added and the reaction solution stirred at rt for 12 h. The reac-

tion solution was poured into cold satd aq NaHCO₃(1 mL) and stir-

red for 1 h. The resulting mixture was ﬁltered through Celite and

washed with CH₂Cl₂. The aqueous was extracted with CH₂Cl₂,

and then the organics were dried over MgSO₄, ﬁltered, and concen-

trated. The residue was puriﬁed by preparative-plate chromatogra-

phy (silica, EtOAc:hexanes 3:1) to give compound 15 (35.2 mg,

0.0440 mmol, 50%). Compound 15: white foam; R_f= 0.46 (silica,

EtOAc:hexanes 3:1); ½a _D²⁵

ꢃ

= +37.1 (CHCl₃, c 1.24); FT-IR (ﬁlm) m_max

3506, 2947, 1702, 1598, 1577, 1491, 1449, 1407, 1365, 1244,

4.1.3. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-9H-

purine-2,6-diamine (59)

1177, 1143, 1052, 1002, 977, 932, 905, 864, 830, 795, 774, 732,

694 cm^ꢁ^{1 1}H NMR (CDCl₃, 500 MHz) d = 8.12 (s, 1H), 7.81–7.75

;

(m, 4H), 7.66–7.60 (m, 4H), 7.51 (dd, J = 10.6, 4.3 Hz, 2H), 7.43 (t,

J = 7.5 Hz, 2H), 7.34 (dt, J = 22.9, 7.7 Hz, 11H), 7.25–7.22 (m, 2H),

5.77 (s, 1H), 4.49 (d, J = 11.5 Hz, 1H), 4.42 (d, J = 11.4 Hz, 1H),

4.20 (s, 1H), 4.12 (s, 1H), 4.06 (d, J = 8.0 Hz, 1H), 3.90 (d,

J = 12.6 Hz, 1H), 3.84 (dd, J = 10.3, 7.0 Hz, 2H) ppm; ¹³C NMR

(CDCl₃, 126 MHz) d = 172.18, 172.01, 154.18, 152.75, 143.07,

137.10, 134.39, 134.02, 133.23, 132.75, 129.52, 129.30, 128.90,

128.77, 128.67, 128.29, 127.74, 125.61, 88.17, 86.92, 77.30, 76.95,

Compound 57 (1.12 g, 1.34 mmol) was dissolved in THF (56 mL)

at 0 °C, and 2 M NaOH (56 mL) was added. The reaction solution was

warmed to rt and stirred for 2 h. The solution was extracted with

CH₂Cl₂and the organic layer was dried over MgSO₄, ﬁltered, and

concentrated to give compound 59 (780 mg, 1.26 mmol, 94%). Com-

pound 59: white foam; R_f= 0.50 (silica, EtOAc); ½a _D²⁵

ꢃ

= +13.0 (CHCl₃,

c 0.5); FT-IR (ﬁlm) m_max3330, 3187, 2931, 2857, 1591, 1471, 1427,

1408, 1363, 1279, 1198, 1111, 1038, 939, 791, 743, 701 cm^ꢁ1

;

¹H

72.49, 72.47, 57.96 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

NMR (CDCl₃, 600 MHz) d = 7.75 (s, 1H), 7.71–7.67 (m, 3H), 7.45–

7.37 (m, 4H), 7.34 (t, J = 7.4 Hz, 3H), 7.29 (dd, J = 5.4, 1.7 Hz, 2H),

7.26–7.24 (m, 3H), 5.92 (s, 1H), 5.48 (s, 2H), 4.75 (s, 2H), 4.72 (s,

1H), 4.64 (d, J = 11.6 Hz, 1H), 4.56 (d, J = 11.6 Hz, 1H), 4.28 (s, 1H),

4.06 (d, J = 7.7 Hz, 1H), 4.03 (d, J = 11.9 Hz, 1H), 3.97 (d, J = 12.0 Hz,

1H), 3.91 (d, J = 7.7 Hz, 1H), 1.08 (s, 9H) ppm; ¹³C NMR (CDCl₃,

151 MHz) d = 159.70, 155.67, 151.05, 137.20, 135.77, 135.68,

135.48, 132.74, 132.72, 130.08, 128.63, 128.17, 128.03, 127.98,

127.80, 114.85, 88.27, 86.44, 77.27, 77.03, 72.73, 72.49, 59.41,

26.91, 19.37 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

C

₄₆H₃₇N₆O₈801.2667, found 801.2687.

4.1.6. ((1S,3R,4R,7S)-7-(Benzyloxy)-3-(2,6-diamino-9H-purin-9-

yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol (16)

From compound 59, using the same procedure as for compound

15, compound 16 was obtained and puriﬁed by ﬂash column chro-

matography to give nucleoside 16 (409 mg, 1.06 mmol, 85%). Com-

pound 16: white powder; R_f= 0.18 (C₁₈silica, 5% MeOH/DCM);

½

a ²_D⁵

ꢃ

= +31.0 (MeOH, c 0.62); FT-IR (ﬁlm) m_max3332, 3195, 2927,

1595, 1455, 1407, 1280, 1201, 1036, 932, 908, 881, 809,

789, 740, 698 cm^ꢁ^{1 1}H NMR (CD₃OD, 500 MHz) d = 7.86 (s, 1H),

C

₃₄H₃₉N₆O₄Si⁺623.2796, found 623.2795.

;

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5657

7.31–7.22 (m, 5H), 5.85 (s, 1H), 4.63 (s, 2H), 4.58 (s, 1H), 4.22 (s,

1H), 4.05 (d, J = 7.8 Hz, 1H), 3.93 (d, J = 1.8 Hz, 2H), 3.87 (d,

J = 7.8 Hz, 1H) ppm; ¹³C NMR (CD₃OD, 151 MHz) d = 161.60,

157.34, 151.72, 138.80, 136.59, 129.39, 129.14, 128.99, 114.44,

89.49, 87.48, 78.44, 77.96, 73.49, 73.27, 58.15 ppm; HRMS (ESI-

preparative-plate chromatography (silica, hexanes:EtOAc:MeOH

1:1:0.1) to give compound 62 (29 mg, 0.0325 mmol, 29%) along with

recovered starting material 59 (21 mg, 0.0336 mmol, 30%). Com-

pound 62: yellow oil; R_f= 0.70 (silica, hexanes:EtOAc:MeOH

1:1:0.1); ½a ²_D⁵

ꢃ

= +50.4 (CHCl₃, c 0.81); FT-IR (ﬁlm) m_max3325, 2927,

2855, 1635, 1599, 1537, 1470, 1427, 1408, 1362, 1324, 1263, 1198,

TOF) (m/z): [M+H]⁺calcd for C₁₈H₂₁N₆O₄385.1619, found

þ

385.1614.

1112, 1036, 939, 911, 885, 862, 805, 789, 740, 701 cm^ꢁ^{1 1}H NMR

;

(CDCl₃, 600 MHz) d = 7.75 (s, 1H), 7.69 (dd, J = 8.0, 6.7 Hz, 4H),

7.45–7.38 (m, 3H), 7.35–7.27 (m, 6H), 7.23 (dd, J = 6.8, 2.8 Hz, 2H),

5.93 (s, 1H), 5.69 (s, 2H), 5.12 (s, 1H), 4.77 (s, 1H), 4.62 (d,

J = 11.6 Hz, 1H), 4.54 (d, J = 11.6 Hz, 1H), 4.25 (s, 1H), 4.06 (d,

J = 7.7 Hz, 1H), 4.03 (d, J = 12.0 Hz, 1H), 3.97 (d, J = 12.0 Hz, 1H),

3.91 (d, J = 7.7 Hz, 1H), 3.37 (td, J = 7.0, 1.7 Hz, 2H), 1.59–1.57 (m,

2H), 1.29–1.25 (m, 10H), 1.08 (s, 9H), 0.88 (d, J = 5.1 Hz, 3H) ppm;

¹³C NMR (CDCl₃, 151 MHz) d = 151.90, 138.37, 137.83, 136.50,

136.40, 133.46, 133.40, 130.82, 129.39, 129.32, 128.90, 128.76,

128.71, 128.65, 128.53, 88.98, 87.27, 73.47, 73.28, 63.98, 60.14,

42.66, 33.68, 32.69, 30.52, 30.27, 30.14, 27.89, 27.63, 23.53, 20.08,

14.98 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₄₂H₅₅N₆O₄Si⁺

735.4048, found 735.4053.

4.1.7. (1S,3R,4R,7S) and (1S,3S,4R,7S)-3-(2,6-Diamino-9H-purin-

9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol

(17and 18)

Compound 16 (640 mg, 1.67 mmol) was partially dissolved in

CH₂Cl₂(3.3 mL) at 0 °C. Boron trichloride (3.33 mL, 1.0 M in DCM,

3.33 mmol) was slowly added dropwise and the solution was stir-

red at rt for 1 h. The reaction was quenched with MeOH (4 mL) and

allowed to stir for 1.5 h, after which the mixture was evaporated

and azeotroped with MeOH. Puriﬁcation by ﬂash column chroma-

tography (C₁₈silica, 4% H₂O/MeCN) was completed to give com-

pound 17 (416 mg, 1.42 mmol, 85%) and epimer 18 (50 mg,

0.167 mmol, 10%). Compound 17: characterization previously re-

ported.³²Compound 18: white powder; R_f= 0.33 (C₁₈silica, 4%

H₂O/MeCN); ½a ²_D⁵

ꢃ

= ꢁ1.7 (DMSO, c 0.2); FT-IR (ﬁlm) m_max3318,

4.1.10. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-N2-butyl-

9H-purine-2,6-diamine (63)

3142, 2922, 1632, 1594, 1481, 1460, 1410, 1386, 1286, 1226,

1178, 1140, 1119, 1035, 1009, 965, 941, 916, 872, 832, 808,

785 cm^ꢁ1

;

¹H NMR (D₂O, 600 MHz) d = 7.94 (s, 1H), 5.92 (s, 1H),

Compound 59 (145 mg, 0.234 mmol) and NaBH₃CN (87 mg,

1.38 mmol) were suspended in MeOH (2.9 mL), and butyraldehyde

(0.17 mL, 1.82 mmol) was added. The mixture was stirred at rt for

48 h. The solvent was evaporated and the residue was puriﬁed by

ﬂash column chromatography (silica, hexanes:EtOAc 1:1 then hex-

anes:EtOAc:MeOH 1:1:0.1) to give compound 63 (90 mg,

0.133 mmol, 57%) along with recovered starting material 59

(35 mg, 0.056 mmol, 24%). Compound 63: yellow oil; R_f= 0.22 (sil-

4.63 (s, 1H), 4.47 (s, 1H), 4.07 (d, J = 8.5 Hz, 1H), 4.01 (d,

J = 10.2 Hz, 3H) ppm; ¹³C NMR (D₂O, 151 MHz) d = 151.00,

137.32, 113.80, 89.21, 85.78, 80.29, 72.30, 71.07, 57.62 ppm; HRMS

(ESI-TOF) (m/z): [M+H]⁺calcd for C₁₁H₁₅N₆O₄295.1149, found

þ

295.1154.

4.1.8. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-N2-ethyl-

9H-purine-2,6-diamine (61)

ica, hexanes:EtOAc 1:1); ½a _D²⁵

ꢃ

= +61.2 (CHCl₃, c 0.20); FT-IR (ﬁlm)

m_max3322, 2930, 2858, 1633, 1600, 1537, 1471, 1427, 1408,

Compound 59 (70 mg, 0.112 mmol) and NaBH₃CN (42 mg,

0.668 mmol) were suspended in MeOH (1.4 mL), and acetaldehyde

(0.05 mL, 0.899 mmol) was added. The mixture was stirred at rt for

48 h. The solvent was evaporated and the residue was puriﬁed by

preparative-plate chromatography (silica, hexanes:EtOAc:MeOH

1:1:0.1) to give compound 61 (35 mg, 0.0538 mmol, 48%) along

with recovered starting material 59 (6.4 mg, 0.0101 mmol, 9%).

Compound 61: white foam; R_f= 0.42 (silica, hexanes:EtOAc:MeOH

1362, 1324, 1267, 1199, 1112, 1036, 938, 911, 823, 789, 740,

701 cm^ꢁ^{1 1}H NMR (CDCl₃, 400 MHz) d = 7.73–7.66 (m, 5H), 7.46–

;

7.36 (m, 4H), 7.33 (dd, J = 9.0, 5.9 Hz, 2H), 7.27 (dd, J = 5.2, 1.9 Hz,

3H), 7.25–7.21 (m, 2H), 5.93 (s, 1H), 5.32 (s, 2H), 4.82–4.75 (m,

2H), 4.62 (d, J = 11.5 Hz, 1H), 4.53 (d, J = 11.5 Hz, 1H), 4.26 (s,

1H), 4.06 (d, J = 7.7 Hz, 1H), 4.02 (d, J = 11.9 Hz, 1H), 3.97 (d,

J = 11.9 Hz, 1H), 3.91 (d, J = 7.7 Hz, 1H), 3.38 (d, J = 6.9 Hz, 1H),

3.36 (d, J = 7.0 Hz, 1H), 1.60–1.52 (m, 2H), 1.40 (dq, J = 14.2,

7.2 Hz, 2H), 1.07 (s, 9H), 0.94 (t, J = 7.4 Hz, 3H) ppm; ¹³C NMR

(CDCl₃, 151 MHz) d = 159.78, 155.52, 151.19, 137.17, 135.78,

135.69, 134.83, 132.77, 132.73, 130.07, 128.59, 128.14, 128.03,

127.97, 127.80, 114.39, 88.16, 86.55, 77.31, 77.05, 72.77, 72.52,

59.49, 41.60, 32.05, 26.91, 20.29, 19.36, 14.07 ppm; HRMS (ESI-

TOF) (m/z): [M+H]⁺calcd for C₃₈H₄₆N₆O₄Si⁺679.3422, found

679.3417.

1:1:0.1); ½a ²_D⁵

ꢃ

= +54.5 (CHCl₃, c 1.03); FT-IR (ﬁlm) m_max3328, 3178,

3071, 2931, 2858, 1733, 1630, 1595, 1536, 1485, 1472, 1427, 1409,

1373, 1345, 1324, 1255, 1199, 1109, 1036, 938, 910, 885, 865, 823,

805, 789, 737, 701 cm^ꢁ1; ¹H NMR (CDCl₃, 500 MHz) d = 7.73 (s, 1H),

7.70 (t, J = 6.6 Hz, 4H), 7.47–7.37 (m, 4H), 7.34 (t, J = 7.5 Hz, 2H),

7.28 (dd, J = 5.3, 1.9 Hz, 3H), 7.23 (dd, J = 6.9, 2.5 Hz, 2H), 5.95 (s,

1H), 5.47 (d, J = 0.7 Hz, 2H), 4.83 (t, J = 5.5 Hz, 1H), 4.80 (s, 1H),

4.63 (d, J = 11.5 Hz, 1H), 4.54 (d, J = 11.5 Hz, 1H), 4.27 (s, 1H),

4.07 (d, J = 7.7 Hz, 1H), 4.04 (d, J = 12.0 Hz, 1H), 3.98 (d,

J = 11.9 Hz, 1H), 3.92 (d, J = 7.7 Hz, 1H), 3.44–3.38 (m, 2H), 1.21

(t, J = 7.2 Hz, 3H), 1.08 (s, 9H) ppm; ¹³C NMR (CDCl₃, 126 MHz)

d = 159.66, 155.61, 151.27, 137.28, 135.87, 135.77, 134.98,

132.89, 132.84, 130.16, 128.68, 128.23, 128.12, 128.06, 127.88,

114.43, 88.28, 86.66, 77.46, 77.15, 72.87, 72.62, 59.62, 36.79,

27.01, 19.46, 15.31 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

4.1.11. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-N2-

isobutyl-9H-purine-2,6-diamine (64)

Compound 59 (140 mg, 0.225 mmol) and NaBH₃CN (84 mg,

1.34 mmol) were suspended in MeOH (2.8 mL), and isobutyralde-

hyde (0.17 mL, 1.82 mmol) was added. The mixture was stirred

at rt for 48 h. The solvent was evaporated and the residue was puri-

ﬁed by ﬂash column chromatography (silica, hexanes:EtOAc 1:1

then hexanes:EtOAc:MeOH 1:1:0.1) to give compound 64 (80 mg,

0.117 mmol, 52%) along with recovered starting material 59

(8 mg, 0.0135 mmol, 6%). Compound 64: yellow oil; R_f= 0.38 (sil-

C

₃₆H₄₃N₆O₄Si⁺651.3109, found 651.3103.

4.1.9. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-N2-octyl-

9H-purine-2,6-diamine (62)

Compound 59 (70 mg, 0.112 mmol) and NaBH₃CN (42 mg,

0.668 mmol) were suspended in MeOH (1.4 mL), and octyl aldehyde

(0.14 mL, 0.899 mmol) was added. The mixture was stirred at rt for

48 h. The solvent was evaporated and the residue was puriﬁed by

ica, 1:1 hexanes:EtOAc); ½a _D²⁵

ꢃ

= +40.6 (CHCl₃, c 2.0); FT-IR (ﬁlm)

m_max3323, 2956, 1602, 1536, 1471, 1427, 1364, 1253, 1199,

1112, 1037, 939, 910, 789, 739, 702 cm^ꢁ1

;

¹H NMR (CDCl₃,

600 MHz) d = 7.73 (s, 1H), 7.72–7.68 (m, 4H), 7.45–7.37 (m, 5H),

7.33 (t, J = 7.5 Hz, 2H), 7.28 (dt, J = 4.6, 2.4 Hz, 2H), 7.25–7.22

5658

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

(m, 2H), 5.94 (s, 1H), 5.49 (s, 2H), 4.96 (s, 1H), 4.79 (s, 1H), 4.62 (d,

J = 11.5 Hz, 1H), 4.54 (d, J = 11.5 Hz, 1H), 4.25 (s, 1H), 4.06 (d,

J = 7.7 Hz, 1H), 4.03 (d, J = 11.9 Hz, 1H), 3.97 (d, J = 12.0 Hz, 1H),

3.92 (d, J = 7.7 Hz, 1H), 3.21 (t, J = 6.3 Hz, 2H), 1.85 (m, 1H), 1.08

(s, 9H), 0.96 (d, J = 6.7 Hz, 6H) ppm; ¹³C NMR (CDCl₃, 151 MHz)

d = 155.45, 151.12, 137.14, 135.78, 135.69, 134.84, 132.76,

132.71, 130.07, 128.60, 128.16, 128.03, 127.98, 127.80, 114.22,

88.18, 86.54, 77.27, 77.03, 72.77, 72.55, 59.47, 49.45, 28.70,

26.91, 20.50, 19.36 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

chromatography (silica, hexanes:EtOAc:MeOH 1:1:0.1) was com-

pleted to give nucleoside 21 (5 mg, 0.0114 mmol, 77%). Compound

21: yellow oil; R_f= 0.30 (silica, hexanes:EtOAc:MeOH 1:1:0.1);

½

a ²_D⁵

ꢃ

= +19.0 (CHCl₃, c 0.45); FT-IR (ﬁlm) m_max3337, 2928, 2871,

1634, 1600, 1542, 1466, 1409, 1365, 1326, 1273, 1207, 1144,

1036, 935, 909, 882, 789, 736, 698 cm^ꢁ^{1 1}H NMR (CDCl₃,

;

500 MHz) d = 7.56 (s, 1H), 7.34–7.27 (m, 5H), 5.89 (s, 1H), 5.82–

5.66 (s, 1H), 4.99 (dd, J = 3.5, 1.7 Hz, 1H), 4.72 (s, 1H), 4.66 (d,

J = 11.7 Hz, 1H), 4.64–4.60 (m, 1H), 4.43 (s, 1H), 4.15–4.10 (m,

1H), 4.03 (d, J = 12.8 Hz, 1H), 3.95 (d, J = 12.7 Hz, 1H), 3.91 (d,

J = 7.8 Hz, 1H), 3.36 (dt, J = 7.2, 4.7 Hz, 2H), 2.08 (s, 1H), 1.56 (dd,

J = 8.5, 6.3 Hz, 2H), 1.44–1.35 (m, 2H), 0.94 (d, J = 7.3 Hz, 3H)

ppm; ¹³C NMR (CDCl₃, 151 MHz) d = 159.43, 155.53, 151.13,

137.25, 135.00, 128.63, 128.23, 127.85, 113.76, 88.26, 86.95,

77.73, 77.30, 72.61, 72.50, 57.84, 41.60, 31.96, 20.28, 14.05 ppm;

C

₃₈H₄₇N₆O₄Si⁺679.3422, found 679.3424.

4.1.12. ((1S,3R,4R,7S)-3-(6-Amino-2-(ethylamino)-9H-purin-9-

yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol

(19)

Compound 61 (30 mg, 0.0461 mmol) was dissolved in THF

(0.46 mL) at 0 °C, and tetra-n-butylammonium ﬂuoride

(0.092 mL, 0.0922 mmol, 1.0 M in THF) was slowly added drop-

wise. The reaction was allowed to warm to rt and stirred for

16 h. The solution was quenched with H₂O (0.5 mL) and stirred

for 10 min. The aqueous layer was extracted with 5% MeOH/DCM

and the organics dried over MgSO₄, ﬁltered, and concentrated.

Puriﬁcation by preparative-plate chromatography (silica, hex-

anes:EtOAc:MeOH 1:1:0.1) was completed to give compound 19

(15 mg, 0.0364 mmol, 79%). Compound 19: white foam; R_f= 0.21

HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₂₂H₂₉N₆O₄441.2245,

þ

found 441.2249.

4.1.15. (1S,3R,4R,7S)-3-(6-Amino-2-(butylamino)-9H-purin-9-

yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (22)

From compound 21, using the same procedure as for com-

pound 64, compound 22 was obtained and puriﬁcation by pre-

parative-plate chromatography (C₁₈silica, 5% MeOH/DCM) was

completed to give nucleoside 22 (18 mg, 0.0514 mmol, 71%).

Compound 22: yellow oil; R_f= 0.13 (C₁₈silica, 5% MeOH/DCM);

(silica, hexanes:EtOAc:MeOH 1:1:0.1);

0.50); FT-IR (ﬁlm) m_max3344, 2928, 1631, 1598, 1540, 1473,

½

a ²_D⁵

= +13.7 (CHCl₃, c

ꢃ

½

a ²_D⁵

ꢃ

= +11.1 (MeOH, c 0.93); FT-IR (ﬁlm) m_max3258, 2957, 1683,

1624, 1512, 1463, 1416, 1367, 1327, 1222, 1130, 1031, 924,

902, 875, 833, 809, 762, 722, 675 cm^ꢁ^{1 1}H NMR (CD₃OD,

1409, 1345, 1260, 1212, 1143, 1035, 935, 909, 789, 752,

698 cm^ꢁ1

;

¹H NMR (CDCl₃, 600 MHz) d = 7.55 (s, 1H), 7.34–7.27

;

(m, 5H), 5.89 (s, 1H), 5.66 (s, 2H), 4.93 (s, 1H), 4.67 (d,

J = 11.7 Hz, 2H), 4.63 (d, J = 11.7 Hz, 1H), 4.49 (s, 1H), 4.12 (d,

J = 7.7 Hz, 1H), 4.04 (d, J = 12.6 Hz, 1H), 3.96 (d, J = 12.6 Hz, 1H),

3.91 (d, J = 7.7 Hz, 1H), 3.43–3.36 (m, 2H), 1.20 (t, J = 7.2 Hz, 3H)

ppm; ¹³C NMR (CDCl₃, 151 MHz) d = 159.37, 155.53, 151.17,

137.31, 135.11, 128.62, 128.20, 127.82, 114.02, 88.31, 86.99,

77.80, 72.58, 72.50, 57.76, 53.57, 36.70, 15.20 ppm; HRMS (ESI-

600 MHz) d = 8.07 (s, 1H), 5.86 (s, 1H), 4.51 (s, 1H), 4.32 (s, 1H),

4.05 (d, J = 7.9 Hz, 1H), 3.95 (s, 2H), 3.87 (d, J = 8.0 Hz, 1H), 3.45

(td, J = 6.9, 4.9 Hz, 2H), 1.65 (dd, J = 10.0, 4.9 Hz, 2H), 1.45 (dd,

J = 15.0, 7.5 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H) ppm; ¹³C NMR (CD₃OD,

151 MHz) d = 152.90, 152.84, 139.61, 139.59, 112.88, 90.21, 87.51,

80.92, 72.85, 71.40, 58.08, 42.29, 32.09, 21.04, 14.12 ppm; HRMS

(ESI-TOF) (m/z): [M+H]⁺calcd for C₁₅H₂₃N₆O₄351.1775, found

þ

TOF) (m/z): [M+H]⁺calcd for C₂₀H₂₅N₆O₄413.1932, found

351.1781.

þ

413.1938.

4.1.16. ((1S,3R,4R,7S)-3-(6-Amino-2-(isobutylamino)-9H-purin-

9-yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)-

methanol (23)

4.1.13. ((1S,3R,4R,7S)-3-(6-Amino-2-(octylamino)-9H-purin-9-

yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol

(20)

From compound 64, using the same procedure as for compound

19, compound 23 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc:MeOH 1:1:0.1) was com-

pleted to give nucleoside 23 (38 mg, 0.0863 mmol, 98%). Com-

pound 23: white foam; R_f= 0.22 (silica, hexanes:EtOAc:MeOH

From compound 62, using the same procedure as for compound

19, compound 20 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc:MeOH 1:1:0.1) was com-

pleted to give nucleoside 20 (15 mg, 0.0302 mmol, 77%). Com-

pound 20: white foam; R_f= 0.33 (silica, hexanes:EtOAc:MeOH

1:1:0.1); ½a ²_D⁵

ꢃ

= +20.7 (CHCl₃, c 1.83); FT-IR (ﬁlm) m_max3339,

2956, 1633, 1601, 1542, 1485, 1467, 1409, 1384, 1354, 1277,

1:1:0.1); ½a ²_D⁵

ꢃ

= +27.0 (CHCl₃, c 1.30); FT-IR (ﬁlm) m_max3335,

2924, 2854, 1634, 1598, 1539, 1467, 1408, 1366, 1326, 1264,

1207, 1143, 1036, 935, 909, 863, 807, 789, 734, 698 cm^ꢁ^{1 1}H

;

1206, 1143, 1034, 935, 909, 881, 809, 788, 738, 697 cm^ꢁ1

;

¹H

NMR (CDCl₃, 500 MHz) d = 7.61 (s, 1H), 7.32–7.26 (m, 5H), 5.88

(s, 1H), 5.84 (s, 2H), 5.10 (s, 1H), 4.77 (s, 1H), 4.65 (d, J = 11.6 Hz,

1H), 4.61 (d, J = 11.6 Hz, 1H), 4.38 (s, 1H), 4.12 (d, J = 7.7 Hz, 1H),

4.03 (d, J = 12.9 Hz, 1H), 3.94 (d, J = 12.9 Hz, 1H), 3.91 (d,

J = 7.8 Hz, 1H), 3.19 (dd, J = 9.2, 3.3 Hz, 2H), 2.07 (s, 1H), 1.84 (dp,

J = 13.4, 6.8 Hz, 1H), 0.94 (d, J = 6.7 Hz, 6H) ppm; ¹³C NMR (CDCl₃,

126 MHz) d = 159.73, 155.58, 151.09, 137.31, 134.99, 128.60,

128.17, 127.79, 113.77, 99.72, 88.38, 86.82, 77.38, 72.59, 72.54,

57.63, 49.44, 28.63, 20.46 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺

NMR (CDCl₃, 500 MHz) d = 7.56 (s, 1H), 7.32–7.27 (m, 5H), 5.89

(s, 1H), 5.54 (s, 2H), 4.82 (t, J = 5.3 Hz, 1H), 4.75 (s, 1H), 4.66 (d,

J = 11.6 Hz, 1H), 4.62 (d, J = 11.7 Hz, 1H), 4.45 (s, 1H), 4.12 (d,

J = 7.8 Hz, 1H), 4.03 (d, J = 12.7 Hz, 1H), 3.94 (d, J = 12.8 Hz, 1H),

3.91 (d, J = 7.7 Hz, 1H), 3.38–3.32 (m, 2H), 1.59–1.52 (m, 2H),

1.37–1.25 (m, 10H), 0.87 (t, J = 6.9 Hz, 3H) ppm; ¹³C NMR (CDCl₃,

126 MHz) d = 160.03, 155.94, 151.51, 137.62, 135.30, 128.89,

128.47, 128.10, 114.38, 100.01, 88.58, 87.28, 78.06, 72.87, 72.80,

58.08, 42.28, 32.25, 30.22, 29.83, 29.69, 27.47, 23.08, 14.52 ppm;

þ

calcd for C₂₂H₂₉N₆O₄441.2245, found 441.2242.

HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₂₆H₃₇N₆O₄497.2871,

þ

found 497.2872.

4.1.17. (1S,3R,4R,7S)-3-(6-Amino-2-(isobutylamino)-9H-purin-

9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol

(24)

Compound 23 (19.8 mg, 0.0449 mmol) was dissolved in CH₂Cl₂

(0.45 mL) at 0 °C, and boron trichloride (0.09 mL, 1.0 M in DCM,

0.0899 mmol) was added slowly dropwise. The solution was

allowed to stir at rt for 16 h. The reaction was quenched with

4.1.14. ((1S,3R,4R,7S)-3-(6-Amino-2-(butylamino)-9H-purin-9-

yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol

(21)

From compound 63, using the same procedure as for compound

19, compound 21 was obtained and puriﬁcation by ﬂash column

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5659

MeOH (1 mL) and allowed to stir for 1.5 h, after which the solution

was evaporated and azeotroped with MeOH. Puriﬁcation by ﬂash

column chromatography (C₁₈silica, 5% MeOH/DCM) was com-

pleted to give compound 24 (10.2 mg, 0.0287 mmol, 64%). Com-

pound 24: white powder; R_f= 0.29 (C₁₈silica, 5% MeOH/DCM);

½

a ²_D⁵

ꢃ

= ꢁ131.8 (CHCl₃, c 0.98); FT-IR (ﬁlm) m_max3351, 2956, 1630,

1579, 1530, 1488, 1467, 1411, 1362, 1229, 1147, 1095, 1039,

911, 856, 790, 733, 699 cm^ꢁ^{1 1}H NMR (CDCl₃, 500 MHz) d = 7.39

;

(ddd, J = 27.1, 17.2, 7.2 Hz, 5H), 7.07 (s, 1H), 5.63 (s, 1H), 5.48 (s,

2H), 4.97–4.90 (m, 1H), 4.87 (d, J = 12.1 Hz, 1H), 4.77 (d,

J = 12.1 Hz, 1H), 4.65 (d, J = 11.9 Hz, 1H), 4.57 (s, 1H), 4.15 (d,

J = 7.9 Hz, 1H), 4.05 (d, J = 13.3 Hz, 1H), 3.97 (d, J = 13.3 Hz, 1H),

3.91 (d, J = 7.9 Hz, 1H), 3.21 (dt, J = 12.8, 6.2 Hz, 1H), 3.14–3.07

(m, 1H), 2.15 (s, 1H), 1.82 (dt, J = 13.5, 6.7 Hz, 1H), 0.93 (d,

J = 6.7 Hz, 6H) ppm; ¹³C NMR (CDCl₃, 126 MHz) d = 159.67,

151.56, 138.80, 137.58, 128.87, 128.54, 128.17, 105.58, 99.74,

90.76, 85.89, 77.91, 77.04, 73.08, 57.57, 49.37, 28.49, 20.45,

½

a ²_D⁵

ꢃ

= +3.9 (MeOH, c 0.17); FT-IR (ﬁlm) m_max3274, 2958, 1684,

1627, 1575, 1512, 1465, 1420, 1385, 1283, 1223, 1175, 1132,

1032, 928, 904, 808 cm^ꢁ^{1 1}H NMR (CD₃OD, 600 MHz) d = 7.90 (s,

;

1H), 5.85 (s, 1H), 4.52 (s, 1H), 4.36 (s, 1H), 4.04 (d, J = 7.8 Hz, 1H),

3.93 (s, 2H), 3.88 (d, J = 7.8 Hz, 1H), 3.19 (ddd, J = 26.4, 13.1,

6.9 Hz, 2H), 1.94–1.88 (m, 1H), 0.96 (d, J = 6.7 Hz, 6H) ppm; ¹³C

NMR (CD₃OD, 151 MHz) d = 152.05, 136.47, 114.05, 89.82, 87.30,

81.06, 72.88, 71.56, 58.35, 50.21, 29.63, 20.66 ppm; HRMS (ESI-

20.41 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₂₂H₂₉N₆O₄

441.2245, found 441.2245.

þ

TOF) (m/z): [M+H]⁺calcd for C₁₅H₂₃N₆O₄351.1775, found

þ

351.1781.

4.1.21. ((1S,3R,4R,7S)-7-(Benzyloxy)-3-(2,6-diamino-7H-purin-

7-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol (26)

4.1.18. 7-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-7H-

purine-2,6-diamine (65)

From compound 65, using the same procedure as for compound

15, compound 26 was obtained and puriﬁcation for characterization

was completed by preparative-plate chromatography (C₁₈silica,

MeCN/H₂O 10:1). Compound 26: white semi-solid; R_f= 0.12 (C₁₈

From compound 58, using the same procedure as for compound

59, compound 65 (636 mg, 1.02 mmol, 85%) was obtained. Com-

pound 65: white foam; R_f= 0.21 (silica, EtOAc); ½a _D²⁵

ꢃ

= ꢁ119.1

silica, 5% MeOH/DCM); ½a _D²⁵

= ꢁ62.8 (MeOH, c 1.11); FT-IR (ﬁlm)

ꢃ

(CHCl₃, c 1.15); FT-IR (ﬁlm) m_max3330, 3182, 2931, 2857, 1574,

m_max3115, 1648, 1545, 1455, 1389, 1316, 1219, 1143, 1047, 937,

1470, 1427, 1401, 1361, 1217, 1105, 1036, 923, 883, 856, 823,

884, 858, 733, 696, 676 cm^ꢁ^{1 1}H NMR (D₂O, 600 MHz) d = 7.83 (s,

;

792, 744, 699, 666 cm^ꢁ1

;

¹H NMR (CDCl₃, 500 MHz) d = 7.56 (dd,

1H), 7.37 (s, 5H), 6.03 (s, 1H), 4.75 (s, 1H), 4.70 (s, 1H), 4.67 (d,

J = 11.7 Hz, 1H), 4.11 (s, 1H), 4.09 (d, J = 8.5 Hz, 1H), 4.04 (d,

J = 8.5 Hz, 1H), 4.01 (d, J = 13.4 Hz, 1H), 3.96 (d, J = 13.4 Hz, 1H)

ppm; ¹³C NMR (D₂O, 151 MHz) d = 153.65, 153.35, 150.03, 140.93,

136.10, 128.77, 128.75, 104.92, 89.66, 85.90, 77.11, 76.27, 72.70,

J = 10.4, 3.8 Hz, 4H), 7.44–7.30 (m, 13H), 5.75 (s, 1H), 5.45 (s, 2H),

4.81 (d, J = 0.9 Hz, 2H), 4.72 (d, J = 12.0 Hz, 1H), 4.57 (d,

J = 12.0 Hz, 1H), 4.54 (s, 1H), 4.14 (d, J = 7.5 Hz, 2H), 3.98 (t,

J = 6.9 Hz, 3H), 0.95 (s, 9H) ppm; ¹³C NMR (CDCl₃, 126 MHz)

d = 160.34, 151.92, 139.19, 136.80, 135.57, 135.52, 132.86,

132.54, 130.13, 130.09, 128.95, 128.76, 128.13, 127.96, 106.10,

89.93, 86.19, 78.07, 76.64, 73.10, 72.95, 59.59, 26.70, 19.34 ppm;

HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₃₄H₃₉N₆O₄Si⁺623.2796,

found 623.2778.

72.16, 56.43 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C

₁₈H₂₁N₆O₄385.1619, found 385.1624.

4.1.22. (1S,3R,4R,7S)-3-(2,6-Diamino-7H-purin-7-yl)-1-

(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (27)

From compound 26, using the same procedure as for compound

17, compound 27 was obtained and puriﬁcation by ﬂash column

chromatography (C₁₈silica, 4% H₂O/MeCN) was completed to give

nucleoside 27 (100 mg, 0.469 mmol, 46% over two steps). Com-

pound 27: white semi-solid; R_f= 0.39 (C₁₈silica, 20% H₂O/MeOH);

4.1.19. 7-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-N2-

isobutyl-7H-purine-2,6-diamine (66)

From compound 65, using the same procedure as for compound

17, compound 66 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc 1:1 then hex-

anes:EtOAc:MeOH 1:1:0.1) was completed to give nucleoside 66

(43 mg, 0.0634 mmol, 44%). Compound 66: white foam; R_f= 0.22

½

a ²_D⁵

ꢃ

= ꢁ95.1 (MeOH, c 0.21); FT-IR (ﬁlm) m_max3129, 1648, 1466,

1392, 1224, 1172, 1091, 1039, 1012, 975, 929, 874, 832,

764 cm^ꢁ^{1 1}H NMR (D₂O, 400 MHz) d = 8.16 (s, 1H), 6.02 (s, 1H),

;

4.97 (s, 1H), 4.42 (s, 1H), 4.07 (d, J = 10.6 Hz, 2H), 3.97 (s, 2H)

ppm; ¹³C NMR (D₂O, 126 MHz) d = 154.29, 153.86, 150.48,

142.01, 141.78, 90.69, 86.27, 79.34, 72.06, 70.81, 57.03 ppm; HRMS

(silica, hexanes:EtOAc:MeOH 1:1:0.1);

2.13); FT-IR (ﬁlm) m_max3331, 2956, 1629, 1588, 1470, 1427,

1385, 1361, 1233, 1112, 1039, 910, 856, 823, 791, 736, 701 cm^ꢁ1

½

a ²_D⁵

ꢃ

= ꢁ67.6 (CHCl₃,

c

þ

;

(ESI-TOF) (m/z): [M+H]⁺calcd for C₁₁H₁₅N₆O₄295.1149, found

¹H NMR (CDCl₃, 500 MHz) d = 7.58 (td, J = 8.4, 1.3 Hz, 4H), 7.45–

7.31 (m, 12H), 5.77 (s, 1H), 5.68 (d, J = 0.4 Hz, 2H), 4.72 (d,

J = 11.9 Hz, 1H), 4.63–5.59 (m, 2H), 4.14 (dd, J = 7.0, 2.2 Hz, 2H),

4.00 (d, J = 1.8 Hz, 2H), 3.98 (d, J = 8.0 Hz, 1H), 3.31–3.24 (m, 2H),

2.04 (s, 1H), 1.91 (dt, J = 13.4, 6.7 Hz, 1H), 0.99–0.95 (m, 15H)

ppm; ¹³C NMR (CDCl₃, 126 MHz) d = 151.86, 139.01, 136.86,

135.60, 132.91, 132.64, 130.16, 130.14, 129.00, 128.81, 128.25,

128.01, 128.00, 105.38, 99.74, 90.08, 86.29, 78.03, 76.70, 73.17,

73.00, 59.61, 49.37, 28.58, 26.78, 20.42, 19.40 ppm; HRMS (ESI-

TOF) (m/z): [M+H]⁺calcd for C₃₈H₄₇N₆O₄Si⁺679.3422, found

679.3421.

295.1157.

4.1.23. (2R,3R,4S,5S)-2-(6-Amino-2-chloro-9H-purin-9-yl)-4-

(benzyloxy)-5-((tert-butyldiphenylsilyloxy)methyl)-5-

(tosyloxymethyl)tetrahydrofuran-3-yl acetate (67)

Compound 56 (400 mg, 0.526 mmol) and 2-chloroadenine

(136 mg, 0.803 mmol) were dissolved in MeCN (5.2 mL), and BSA

(0.30 mL, 1.23 mmol) was added. The mixture was heated to

65 °C for 1.5 h. The reaction mixture was then cooled to 0 °C and

TMSOTf (0.24 mL, 1.07 mmol) was added, after which the reaction

was heated at 65 °C for 3 h. The reaction solution was quenched

with cold satd aq NaHCO₃(2 mL) and extracted with CH₂Cl₂. The

organics were washed with satd aq NaHCO₃(2 ꢂ 5 mL) and brine

(2 ꢂ 5 mL), dried over MgSO₄, ﬁltered, concentrated, and puriﬁed

by ﬂash column chromatography (silica, hexanes:EtOAc 1:1) to

give compound 67 (390 mg, 0.447 mmol, 85%). Compound 67:

white foam; R_f= 0.44 (silica, hexanes:EtOAc:MeOH 1:1:0.1);

4.1.20. ((1S,3R,4R,7S)-3-(6-Amino-2-(isobutylamino)-7H-purin-

7-yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)-

methanol (25)

From compound 66, using the same procedure as for compound

19, compound 25 was obtained and puriﬁcation by preparative-

plate chromatography (silica, 5% MeOH/DCM) was completed to

give nucleoside 25 (20 mg, 0.0454 mmol, 77%). Compound 25:

white foam; R_f= 0.11 (silica, hexanes:EtOAc:MeOH 1:1:0.1);

½

a ²_D⁵

ꢃ

= ꢁ5.6 (CHCl₃, c 1.64); FT-IR (ﬁlm) m_max3320, 3174, 2931,

2858, 1745, 1643, 1593, 1497, 1461, 1428, 1360, 1308, 1227,

1189, 1175, 1105, 978, 936, 909, 813, 790, 731, 700, 666 cm^ꢁ1

;

5660

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

¹H NMR (CDCl₃, 400 MHz) d = 7.75 (s, 1H), 7.71–7.67 (m, 2H), 7.58–

7.52 (m, 4H), 7.45–7.27 (m, 11H), 7.20 (d, J = 8.0 Hz, 2H), 6.06 (s,

2H), 5.94 (d, J = 4.9 Hz, 1H), 5.71 (dd, J = 5.7, 5.0 Hz, 1H), 4.71 (d,

J = 5.8 Hz, 1H), 4.54 (s, 1H), 4.51 (s, 1H), 4.36 (d, J = 10.7 Hz, 1H),

4.28 (d, J = 10.7 Hz, 1H), 3.76 (d, J = 11.0 Hz, 1H), 3.73 (d,

J = 11.1 Hz, 1H), 2.38 (s, 3H), 2.04 (s, 3H), 1.00 (s, 9H) ppm; ¹³C

NMR (CDCl₃, 101 MHz) d = 169.99, 156.22, 154.35, 147.46,

144.85, 139.78, 137.03, 135.72, 135.58, 132.92, 132.63, 132.32,

130.09, 130.07, 129.83, 128.67, 128.38, 128.27, 128.13, 128.00,

127.90, 86.79, 86.54, 78.15, 74.86, 74.67, 68.68, 64.75, 27.01,

21.73, 20.74, 19.31 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

ﬁltered through Celite to remove most salts. Puriﬁcation by pre-

parative-plate chromatography (C₁₈silica, EtOAc) was completed

to give compound 29 (1.8 mg, 0.00570 mmol, 46%). Compound

29: white powder; R_f= 0.61 (C₁₈silica, EtOAc); ½a _D²⁵

ꢃ

= +1.8 (MeOH,

c 0.15); FT-IR (ﬁlm) m_max3179, 2925, 1667, 1597, 1571, 1498, 1460,

1445, 1354, 1311, 1265, 1204, 1180, 1131, 1034, 1009, 934, 900,

877, 848, 821, 786, 749, 725, 678 cm^ꢁ^{1 1}H NMR (CD₃OD,

;

500 MHz) d = 8.20 (s, 1H), 5.92 (s, 1H), 4.48 (s, 1H), 4.30 (s, 1H),

4.03 (d, J = 7.9 Hz, 1H), 3.92 (s, 2H), 3.86 (d, J = 7.9 Hz, 1H) ppm;

¹³C NMR (CD₃OD, 151 MHz) d = 158.08, 155.48, 139.99, 90.18,

87.55, 81.01, 72.82, 71.36, 58.16 ppm; HRMS (ESI-TOF) (m/z):

C

₄₃H₄₇ClN₅O₈SSi⁺856.2598, found 856.2587.

[M+H]⁺calcd for C₁₁H₁₃ClN₅O₄314.0651, found 314.0657.

þ

4.1.24. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-2-chloro-

9H-purin-6-amine (68)

4.1.27. (2R,3R,4S,5S)-2-(6-Amino-2-ﬂuoro-9H-purin-9-yl)-4-

(benzyloxy)-5-((tert-butyldiphenylsilyloxy)methyl)-5-

(tosyloxymethyl)tetrahydrofuran-3-yl acetate (69)

Compound 67 (29 mg, 0.0339 mmol) was dissolved in THF

(3.42 mL) at 0 °C, and 2 M NaOH (0.28 mL) was added. The mixture

was allowed to warm to rt with stirring over 15 h. The reaction

solution was worked up by extraction with CH₂Cl₂, drying over

MgSO₄, ﬁltration, and concentration. Puriﬁcation by ﬂash column

chromatography was completed to produce compound 68

(19 mg, 0.0285 mmol, 87%). Compound 68: white foam; R_f= 0.53

Acetylated compound 56 (200 mg, 0.268 mmol) was dissolved

in dry MeCN (2.0 mL) along with 2-ﬂuoro-9H-purine-6-amine

(81 mg,

0.530 mmol)

and

N,O-bis(trimethylsilyl)acetamide

(0.17 mL, 0.670 mmol). The reaction mixture was heated to

80 °C for 1.5 h. The reaction mixture was cooled to 0 °C and

TMSOTf (0.132 mL, 0.530 mmol) was slowly added. Heating was

resumed for an additional 3 h at 80 °C. The reaction mixture

was quenched with satd aq NaHCO₃(2 mL) and extracted with

EtOAc. The combined organic extracts were washed with brine

(2 mL), dried over MgSO₄, ﬁltered, and concentrated under re-

duced pressure. Puriﬁcation by ﬂash column chromatography (sil-

ica, hexanes:EtOAc 4:1) was completed to give compound 69

(176 mg, 0.210 mmol, 78%). Compound 69: light yellow oil;

(silica, hexanes:EtOAc:MeOH 1:1:0.1);

1.55); FT-IR (ﬁlm) m_max3314, 3170, 2931, 2858, 1645, 1592,

½

a ²_D⁵

= +33.0 (CHCl₃, c

ꢃ

1571, 1498, 1456, 1427, 1345, 1310, 1245, 1202, 1112, 1038,

;

937, 909, 857, 823, 804, 735, 701 cm^ꢁ^{1 1}H NMR (CDCl₃,

500 MHz) d = 8.01 (s, 1H), 7.69 (ddd, J = 8.0, 3.7, 1.4 Hz, 4H),

7.48–7.32 (m, 7H), 7.29–7.27 (m, 2H), 7.26–7.22 (m, 2H), 6.09 (s,

2H), 6.02 (s, 1H), 4.79 (s, 1H), 4.66 (d, J = 11.6 Hz, 1H), 4.57 (d,

J = 11.6 Hz, 1H), 4.24 (s, 1H), 4.07 (d, J = 7.8 Hz, 1H), 4.04 (d,

J = 11.9 Hz, 1H), 3.98 (d, J = 12.0 Hz, 1H), 3.90 (d, J = 7.8 Hz, 1H),

1.08 (s, 9H) ppm; ¹³C NMR (CDCl₃, 126 MHz) d = 156.18, 154.34,

138.69, 137.03, 135.75, 135.64, 132.74, 132.66, 130.12, 128.59,

128.23, 128.06, 128.00, 127.88, 88.62, 86.72, 77.36, 76.88, 72.71,

72.56, 59.31, 26.92, 19.38 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺

calcd for C₃₄H₃₆ClN₅O₄Si⁺642.2298, found 642.2280.

R_f= 0.49 (silica, hexanes:EtOAc 2:3); ½a _D²⁵

= ꢁ6.9 (MeCN, c 0.30);

ꢃ

FT-IR (ﬁlm) m_max3459, 2931, 1652, 1496, 1438, 1410, 1387,

;

1255, 1224, 1094, 1062, 865, 659 cm^ꢁ^{1 1}H NMR (CDCl₃,

600 MHz) d = 7.74 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.55 (dd,

J = 11.5, 6.8 Hz, 4H), 7.42–7.30 (m, 9H), 7.26–7.23 (m, 2H), 7.19

(d, J = 8.0 Hz, 2H), 5.89 (d, J = 4.8 Hz, 1H), 5.74–5.71 (m, 1H),

4.78 (s, 2H), 4.73 (d, J = 5.8 Hz, 1H), 4.54 (d, J = 11.3 Hz, 1H),

4.49 (d, J = 11.2 Hz, 1H), 4.36 (d, J = 10.7 Hz, 1H), 4.26 (d,

J = 10.7 Hz, 1H), 3.77 (d, J = 11.0 Hz, 1H), 3.71 (d, J = 11.0 Hz,

1H), 2.37 (s, 3H), 2.02 (s, 3H), 0.99 (s, 9H) ppm; ¹³C NMR (CDCl₃,

151 MHz) d = 176.75, 169.98, 144.91, 139.65, 139.63, 136.96,

135.70, 135.54, 132.77, 132.57, 132.27, 130.09, 130.08, 129.83,

128.65, 128.35, 128.24, 128.20, 128.13, 128.10, 127.99, 127.90,

86.64, 86.48, 77.95, 74.78, 74.46, 68.64, 64.58, 26.95, 21.73,

20.74, 19.29 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

4.1.25. ((1S,3R,4R,7S)-3-(6-Amino-2-chloro-9H-purin-9-yl)-7-

(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol (28)

From compound 68, using the same procedure as for compound

15, compound 28 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc:MeOH 1:1:0.1) was com-

pleted to give nucleoside 28 (28 mg, 0.0695 mmol, 61%). Com-

pound

28:

white

semi-solid;

R_f= 0.18

(silica,

C

₄₃H₄₇N₅O₈SSi⁺840.2893, found 840.2892.

hexanes:EtOAc:MeOH 1:1:0.1); ½a _D²⁵

ꢃ

= +18.0 (CHCl₃, c 0.56); FT-IR

(ﬁlm) m_max3322, 3176, 2926, 2248, 1644, 1593, 1572, 1497,

4.1.28. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-2-ﬂuoro-

9H-purin-6-amine (70)

1455, 1347, 1309, 1249, 1203, 1182, 1143, 1097, 1035, 987, 933,

907, 883, 821, 789, 729, 697, 681 cm^ꢁ1

;

¹H NMR (CDCl₃,

500 MHz) d = 7.84 (s, 1H), 7.34–7.27 (m, 5H), 6.49 (s, 2H), 5.96 (s,

1H), 4.78 (s, 1H), 4.72 (d, J = 11.8 Hz, 1H), 4.68 (d, J = 11.9 Hz,

1H), 4.43 (s, 1H), 4.13 (d, J = 7.9 Hz, 1H), 4.04 (d, J = 12.2 Hz, 2H),

3.96 (d, J = 7.8 Hz, 1H), 3.91 (d, J = 7.8 Hz, 1H) ppm; ¹³C NMR

(CDCl₃, 126 MHz) d = 156.19, 154.45, 138.83, 137.25, 128.63,

128.26, 127.88, 99.71, 88.57, 87.08, 77.56, 77.27, 72.52,

From compound 69, using the same procedure as for com-

pound 30, compound 70 was obtained and puriﬁcation by ﬂash

column chromatography (silica, hexanes:EtOAc 4:1) was

completed to give nucleoside 70 (6 mg, 0.00964 mmol, 81%).

Compound 70: colorless foam; R_f= 0.60 (silica, hexanes:EtOAc

2:3); ½a ²_D⁵

ꢃ

= +12.9 (MeCN, c 0.30); FT-IR (ﬁlm) m_max3490, 2293,

57.69 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₁₈H₁₉ClN₅O₄

2253, 1443, 1375, 1039, 918, 749 cm^ꢁ^{1 1}H NMR (CD₃CN,

;

+

404.1120, found 404.1120.

600 MHz) d = 7.95 (s, 1H), 7.71–7.65 (m, 4H), 7.49–7.43 (m, 2H),

7.42–7.35 (m, 4H), 7.32–7.25 (m, 5H), 6.32 (s, 2H), 5.92 (s, 1H),

4.70 (s, 1H), 4.65 (d, J = 11.8 Hz, 1H), 4.59 (d, J = 11.8 Hz, 1H),

4.38 (s, 1H), 4.08–4.01 (m, 2H), 3.98 (d, J = 7.9 Hz, 1H), 3.87 (d,

J = 7.9 Hz, 1H), 1.02 (s, 9H) ppm; ¹³C NMR (CD₃CN, 151 MHz)

d = 160.56, 159.19, 158.48, 158.34, 139.07, 139.05, 138.59,

136.35, 136.29, 133.67, 133.60, 130.89, 129.23, 128.76, 128.68,

128.58, 88.97, 87.00, 78.11, 77.69, 73.09, 72.70, 60.22, 27.01,

19.66 ppm; HRMS (ESI-TOF) (m/z): [M+Na]⁺calcd for

4.1.26. (1S,3R,4R,7S)-3-(6-Amino-2-chloro-9H-purin-9-yl)-1-

(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (29)

Compound 28 (5 mg, 0.0124 mmol) was dissolved in CH₂Cl₂

(0.13 mL) at 0 °C, and methanesulfonic acid (0.06 mL, 0.963 mmol)

was added. The reaction was stirred at this temperature for

1.5 h. The reaction was neutralized with concentrated aq

NaOH (0.13 mL, 0.963 mmol) and the mixture concentrated. The

residue was diluted with CH₂Cl₂(2 mL) and MeOH (0.5 mL) then

C

₃₄H₃₆FN₅O₄SiNa⁺648.2413, found 648.2411.

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5661

4.1.29. ((1S,3R,4R,7S)-3-(6-Amino-2-ﬂuoro-9H-purin-9-yl)-7-

(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol (30)

From compound 70, using the same procedure as for compound

15, compound 30 was obtained and puriﬁcation by ﬂash column

chromatography (silica, EtOAc:hexanes 8:1) was completed to give

nucleoside 30 (21 mg, 0.0543 mmol, 85%). Compound 30: colorless

with satd aq NaHCO₃(5 mL) and extracted with EtOAc. The com-

bined organic extracts were washed with brine (2 ꢂ 5 mL), dried

over MgSO₄, ﬁltered, and concentrated. Puriﬁcation by ﬂash col-

umn chromatography (silica, hexanes:EtOAc:acetone 8:1:1) was

completed to give compound 71 (374 mg, 0.428 mmol, 64%). Com-

pound 71: yellow foam; R_f= 0.51 (silica, 10% MeOH/ DCM);

oil; R_f= 0.25 (silica, 10% MeOH/DCM); ½a _D²⁵

ꢃ

= ꢁ8.0 (MeCN, c 0.10);

½

a ²_D⁵

ꢃ

= ꢁ2.8 (CHCl₃, c 0.10). FT-IR (ﬁlm) m_max2932, 1750, 1595,

1557, 1428, 1360, 1227, 1189, 1177, 1112, 980, 883, 814, 744,

702, 667 cm^ꢁ^{1 1}H NMR (CDCl₃, 600 MHz) d = 8.09 (s, 1H), 7.69

FT-IR (ﬁlm) m_max3311, 2949, 2837, 1646, 1408, 1113, 1014 cm^ꢁ1

;

¹H NMR (CD₃OD, 400 MHz) d = 8.13 (s, 1H), 7.29–7.24 (m, 5H),

5.92 (s, 1H), 4.97 (s, 1H), 4.63 (s, 2H), 4.22 (s, 1H), 4.06 (d,

J = 7.8 Hz, 1H), 3.94 (d, J = 1.8 Hz, 2H), 3.89 (d, J = 7.8 Hz, 1H)

ppm; ¹³C NMR (CD₃OD, 151 MHz) d = 161.19, 159.80, 159.10,

139.81, 138.73, 129.35, 129.16, 129.00, 89.70, 87.69, 78.38, 78.03,

;

(d, J = 8.2 Hz, 2H), 7.55–7.49 (m, 4H), 7.40–7.32 (m, 8H), 7.28 (d,

J = 5.8 Hz, 2H), 7.22–7.19 (m, 3H), 6.02 (d, J = 5.2 Hz, 1H), 5.67 (t,

J = 6.0 Hz, 1H), 4.60 (d, J = 5.9 Hz, 1H), 4.55 (d, J = 11.4 Hz, 1H),

4.48 (d, J = 11.4 Hz, 1H), 4.36 (d, J = 10.7 Hz, 1H), 4.26 (d,

J = 10.7 Hz, 1H), 3.76 (d, J = 10.8 Hz, 1H), 3.71 (d, J = 10.8 Hz, 1H),

2.38 (s, 3H), 2.05 (s, 3H), 0.99 (s, 9H) ppm; ¹³C NMR (CDCl₃,

151 MHz) d = 176.98, 170.23, 153.42, 152.57, 152.49, 145.20,

144.80, 135.92, 135.77, 133.04, 132.60, 132.35, 131.71, 130.39,

130.08, 128.97, 128.77, 128.55, 128.32, 128.23, 128.09, 87.60,

87.28, 77.99, 77.58, 74.77, 68.68, 64.86, 27.22, 21.99, 20.92,

19.49 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

73.52, 73.30, 58.08 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C

₁₈H₁₉FN₅O₄388.1416, found 388.1411.

4.1.30. (1S,3R,4R,7S)-3-(6-Amino-2-ﬂuoro-9H-purin-9-yl)-1-

(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (31)

From compound 30, using the same procedure as for compound

12, compound 31 was obtained and puriﬁcation by ﬂash column

chromatography (silica, 5% MeOH/DCM) was completed to give

nucleoside 31 (5.3 mg, 0.0178 mmol, 69%). Compound 31: white

C

₄₃H₄₅Cl₂N₄O₈SSi⁺875.2099, found 875.2097.

foam; R_f= 0.12 (silica, 10% MeOH/DCM); ½a _D²⁵

ꢃ

= ꢁ3.5 (MeCN, c

4.1.33. ((3S,4R,5R)-5-(6-(Benzylamino)-2-chloro-9H-purin-9-yl)-

3-(benzyloxy)-2-((tert-butyldiphenylsilyloxy)methyl)-4-hydr-

oxytetrahydrofuran-2-yl)methyl 4-methylbenzenesulfonate

(72)

0.10); FT-IR (ﬁlm) m_max3343, 2948, 2836, 2502, 2238, 2073,

1655, 1449, 1119, 1021, 977 cm^ꢁ^{1 1}H NMR (CD₃OD, 600 MHz)

;

d = 8.21 (s, 1H), 5.92 (s, 1H), 4.50 (s, 1H), 4.34 (s, 1H), 4.05 (d,

J = 7.9 Hz, 1H), 3.94 (s, 2H), 3.88 (d, J = 7.9 Hz, 1H) ppm; ¹³C NMR

(CD₃OD, 151 MHz) d = 161.30, 159.91, 139.83, 90.14, 87.50, 80.97,

72.84, 71.42, 58.18 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

Compound 71 (104 mg, 0.119 mmol) was dissolved in MeOH

(7.0 mL) and treated with benzylamine (0.065 mL, 0.594 mmol).

The reaction mixture was heated at 55 °C for 12 h. The solvent

was evaporated and puriﬁcation of the residue by ﬂash column

chromatography (silica, hexanes:EtOAc 1:1) was completed to give

compound 72 (100 mg, 0.111 mmol, 93%). Compound 72: white

C₁₁H₁₃FN₅O₄298.0946, found 298.0945.

4.1.31. ((1S,3R,4R,7S)-3-(6-Amino-2-morpholino-9H-purin-9-

yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol

(32)

foam; R_f= 0.48 (silica, hexanes:EtOAc 1:1); ½a _D²⁵

= ꢁ7.7 (CHCl₃, c

ꢃ

1.07); FT-IR (ﬁlm) m_max3325, 3069, 2931, 2858, 1619, 1581,

Compound 70 (18 mg, 0.0288 mmol) was dissolved in dry

1533, 1496, 1472, 1454, 1428, 1354, 1310, 1189, 1176, 1103,

DMSO

(0.30 mL)

along

with

morpholine

(0.0050 mL,

1029, 1019, 977, 910, 813, 788, 736, 701, 667 cm^ꢁ^{1 1}H NMR

;

0.0570 mmol). The reaction mixture was heated to 95 °C for 24 h.

The mixture was placed under vacuum to remove solvent, and

the residue was dissolved in dry THF (0.2 mL). The solution was

cooled to 0 °C and treated with HFꢀpyridine (0.03 mL, 0.280 mmol).

After 1 h at 0 °C, the reaction mixture was quenched with satd aq

NaHCO₃(1 mL) and extracted with CH₂Cl₂. The combined organic

extracts were washed brine (2 ꢂ 1 mL), dried over MgSO₄, ﬁltered,

and concentrated. Puriﬁcation by ﬂash column chromatography

(silica, 5% MeOH/DCM) was completed to give compound 32

(10 mg, 0.0220 mmol, 76% over two steps). Compound 32: light

(CDCl₃, 500 MHz) d = 7.75–7.71 (m, 2H), 7.54 (ddd, J = 17.0, 8.0,

1.4 Hz, 5H), 7.42–7.28 (m, 16H), 7.23 (d, J = 7.9 Hz, 2H), 6.36 (s,

1H), 5.65 (d, J = 4.9 Hz, 1H), 4.77 (s, 2H), 4.70 (d, J = 11.4 Hz, 1H),

4.64 (d, J = 11.4 Hz, 1H), 4.59 (dd, J = 11.3, 6.0 Hz, 1H), 4.48 (d,

J = 5.9 Hz, 1H), 4.38 (q, J = 10.5 Hz, 2H), 3.77 (d, J = 10.8 Hz, 1H),

3.71 (d, J = 6.7 Hz, 1H), 3.66 (d, J = 10.8 Hz, 1H), 2.39 (s, 3H), 1.00

(s, 9H) ppm; ¹³C NMR (CDCl₃, 126 MHz) d = 144.93, 139.28,

136.88, 135.68, 135.58, 132.86, 132.69, 132.31, 130.07, 129.88,

128.89, 128.84, 128.58, 128.44, 128.17, 128.14, 127.97, 127.89,

127.86, 89.73, 86.91, 79.47, 74.82, 74.55, 69.08, 64.92, 26.99,

21.75, 19.30 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

yellow oil; R_f= 0.54 (silica, 5% MeOH/DCM); ½a _D²⁵

ꢃ

= +50.0 (MeCN,

c 0.10); FT-IR (ﬁlm) m_max3399, 2962, 1605, 1474, 1408, 1293,

C

₄₈H₅₁ClN₅O₇SSi⁺904.2961, found 904.2961.

1056, 1017 cm^ꢁ^{1 1}H NMR (CD₃CN, 500 MHz) d = 7.69 (s, 1H),

;

7.34–7.25 (m, 5H), 5.83 (s, 1H), 5.73 (s, 2H), 4.67 (s, 1H), 4.61 (s,

2H), 4.29 (s, 1H), 3.99 (d, J = 7.9 Hz, 1H), 3.88 (dd, J = 7.7, 3.2 Hz,

2H), 3.84 (d, J = 7.9 Hz, 1H), 3.67 (s, 8H), 3.19–3.15 (m, 1H) ppm;

¹³C NMR (CD₃CN, 151 MHz) d = 160.12, 156.51, 152.08, 138.84,

136.40, 129.26, 128.70, 128.60, 88.78, 86.85, 78.39, 78.12, 73.06,

72.73, 67.34, 58.23, 45.72 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺

4.1.34. N-Benzyl-9-((1R,3R,4R,7S)-7-(benzyloxy)-1-((tert-butyl-

diphenylsilyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-

2-chloro-9H-purin-6-amine (73)

From compound 72, using the same procedure as for compound

68, compound 73 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc 7:3) was completed to af-

ford nucleoside 73 (59 mg, 0.0806 mmol, 68%). Compound 73:

þ

calcd for C₂₂H₂₇N₆O₅455.2037, found 455.2050.

white foam; R_f= 0.87 (silica, hexanes:EtOAc 1:1); ½a _D²⁵

ꢃ

= +18.0

4.1.32. (2R,3R,4S,5S)-4-(Benzyloxy)-5-((tert-butyldiphenyl-

silyloxy)methyl)-2-(2,6-dichloro-9H-purin-9-yl)-5-(tosyl-

oxymethyl)tetrahydrofuran-3-yl acetate (71)

Peracetylated compound 56 (500 mg, 0.669 mmol) was dis-

solved in dry MeCN (5.0 mL) along with 2,6-dichloro-9H-purine

(240 mg, 1.33 mmol) and BSA (0.41 mL 1.67 mmol). The reaction

mixture was heated to 95 °C for 1.5 h. After cooling to 0 °C, TMSOTf

(0.33 mL, 1.33 mmol) was slowly added and heating was resumed

for 3 h at 80 °C. The reaction mixture was brought to rt, quenched

(MeCN, c 0.10); FT-IR (ﬁlm) m_max2930, 1619, 1577, 1455, 1428,

1346, 1311, 1208, 1112, 1046, 937, 858, 824, 741, 701 cm^ꢁ^{1 1}H

;

NMR (CDCl₃, 500 MHz) d = 7.94 (s, 1H), 7.68 (ddd, J = 8.0, 3.6,

1.4 Hz, 4H), 7.46–7.26 (m, 15H), 7.25–7.23 (m, 1H), 6.32 (s, 1H),

6.02 (s, 1H), 4.81 (d, J = 9.3 Hz, 3H), 4.66 (d, J = 11.6 Hz, 1H), 4.57

(d, J = 11.6 Hz, 1H), 4.23 (s, 1H), 4.07 (d, J = 7.8 Hz, 1H), 4.03 (d,

J = 12.0 Hz, 1H), 3.97 (d, J = 12.0 Hz, 1H), 3.90 (d, J = 7.8 Hz, 1H),

1.07 (s, 9H) ppm; ¹³C NMR (CDCl₃, 126 MHz) d = 137.99, 137.10,

135.75, 135.65, 132.75, 132.68, 130.10, 128.91, 128.59, 128.25,

5662

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

128.19, 128.04, 127.99, 127.92, 127.87, 99.72, 88.54, 86.67, 77.41,

76.91, 72.71, 72.53, 59.36, 26.90, 19.35 ppm; HRMS (ESI-TOF) (m/

z): [M+H]⁺calcd for C₄₁H₄₃ClN₅O₄Si⁺732.2767, found 732.2765.

4.1.38. N-Benzyl-9-((1R,3R,4R,7S)-7-(benzyloxy)-1-((tert-butyl-

diphenylsilyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-

2-chloro-N-methyl-9H-purin-6-amine (75)

From 74, using the same procedure as for 68, compound 75 was

obtained (73 mg, 0.0979 mmol, 98%). Compound 75: white foam;

4.1.35. ((1S,3R,4R,7S)-3-(6-(Benzylamino)-2-chloro-9H-purin-9-

yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol

(33)

R_f= 0.77 (silica, hexanes:EtOAc 2:1); ½a _D²⁵

ꢃ

= +13.1 (CHCl₃, c 1.18);

FT-IR (ﬁlm) m_max3030, 2930, 2857, 1590, 1582, 1524, 1495, 1472,

From compound 73, using the same procedure as for compound

15, compound 33 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc 7:3) was completed to give

nucleoside 33 (39 mg, 0.0790 mmol, 96%). Compound 33: light yel-

1454, 1427, 1402, 1354, 1314, 1241, 1207, 1110, 1037, 939, 910,

885, 856, 824, 806, 784, 736, 701, 677 cm^ꢁ^{1 1}H NMR (CD₃CN,

;

500 MHz) d = 7.92 (s, 1H), 7.66 (dd, J = 9.3, 8.1 Hz, 4H), 7.43 (dd,

J = 10.6, 4.2 Hz, 2H), 7.40–7.24 (m, 14H), 5.94 (s, 1H), 4.73 (s, 1H),

4.66 (d, J = 11.8 Hz, 1H), 4.59 (d, J = 11.8 Hz, 1H), 4.35 (s, 1H),

4.06 (d, J = 12.1 Hz, 1H), 4.03 (d, J = 12.1 Hz, 1H), 3.98 (d,

J = 8.0 Hz, 1H), 3.88 (d, J = 7.9 Hz, 1H), 3.64 (s, 1H), 3.12 (s, 1H),

1.00 (s, 9H) ppm; ¹³C NMR (CD₃CN, 126 MHz) d = 138.66, 137.49,

136.43, 136.38, 133.81, 133.77, 130.98, 130.96, 129.61, 129.33,

128.86, 128.80, 128.69, 128.37, 120.10, 89.12, 87.12, 78.31, 77.80,

73.23, 72.87, 60.36, 27.16, 19.80 ppm; HRMS (ESI-TOF) (m/z):

[M+H]⁺calcd for C₄₂H₄₅ClN₅O₄Si⁺746.2929, found 746.2922.

low oil; R_f= 0.17 (silica, hexanes:EtOAc 1:1); ½a _D²⁵

ꢃ

= +10.0 (MeCN, c

0.50); FT-IR (ﬁlm) m_max3316, 2930, 1703, 1618, 1578, 1496, 1454,

1348, 1310, 1212, 1145, 1097, 1050, 931, 883, 857, 787, 739, 698,

679 cm^ꢁ1; ¹H NMR (CDCl₃, 600 MHz) d = 7.73 (s, 1H), 7.38–7.28 (m,

10H), 6.56 (s, 1H), 5.95 (s, 1H), 4.80 (s, 2H), 4.70 (d, J = 13.3 Hz, 2H),

4.66 (d, J = 11.8 Hz, 1H), 4.40 (s, 1H), 4.11 (d, J = 7.8 Hz, 1H), 3.98 (d,

J = 12.5 Hz, 1H), 3.88 (d, J = 7.7 Hz, 2H), 3.38 (d, J = 0.5 Hz, 1H) ppm;

¹³C NMR (CDCl₃, 151 MHz) d = 155.08, 154.97, 149.05, 138.05,

137.69, 137.21, 128.92, 128.63, 128.28, 128.22, 127.99, 127.95,

119.09, 88.47, 87.19, 77.83, 77.16, 72.59, 72.47, 57.84,

44.95 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

4.1.39. ((1S,3R,4R,7S)-3-(6-(Benzyl(methyl)amino)-2-chloro-9H-

purin-9-yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)-

methanol (35)

þ

C₂₅H₂₅ClN₅O₄495.1589, found 495.1591.

From compound 75, using the same procedure as for compound

15, compound 35 was obtained and preparative-plate chromatog-

raphy (silica, EtOAc) was completed to give nucleoside 35 (36 mg,

0.0710 mmol, 75%). Compound 35: white foam; R_f= 0.67 (silica,

4.1.36. (1S,3R,4R,7S)-3-(6-(Benzylamino)-2-chloro-9H-purin-9-

yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (34)

From compound 33, using the same procedure as for compound

39, compound 34 was obtained and puriﬁcation by preparative-

plate chromatography (C₁₈silica, EtOAc) was completed to give

nucleoside 34 (10 mg, 0.0248 mmol, 61%). Compound 34: yellow

EtOAc); ½a ²_D⁵

= ꢁ13.3 (CHCl₃, c 1.13); FT-IR (ﬁlm) m_max3378,

ꢃ

2942, 1592, 1454, 1403, 1354, 1313, 1211, 1145, 1039, 938, 910,

856, 783, 751, 700, 677 cm^ꢁ^{1 1}H NMR (CDCl₃, 500 MHz) d = 7.75

;

oil; R_f= 0.71 (C₁₈silica, EtOAc); ½a _D²⁵

ꢃ

= ꢁ8.3 (MeOH, c 0.86); FT-IR

(s, 1H), 7.35–7.26 (m, 10H), 5.99 (s, 1H), 5.53 (s, 1H), 5.00 (s, 1H),

4.72–4.63 (m, 3H), 4.42 (s, 1H), 4.13 (d, J = 7.8 Hz, 1H), 4.05–3.99

(m, 1H), 3.98–3.93 (m, 1H), 3.92 (t, J = 5.5 Hz, 1H), 3.65 (s, 1H),

3.21 (s, 1H), 2.78 (s, 1H) ppm; ¹³C NMR (CDCl₃, 126 MHz)

d = 155.12, 154.13, 137.24, 136.52, 128.82, 128.62, 128.25,

127.97, 127.72, 119.39, 88.21, 87.12, 77.89, 77.36, 77.29, 72.58,

(ﬁlm) m_max3269, 2926, 1619, 1577, 1533, 1453, 1351, 1300,

1205, 1138, 1094, 1040, 930, 876, 847, 821, 786, 694, 678 cm^ꢁ1

;

¹H NMR (CD₃OD, 500 MHz) d = 8.25 (s, 1H), 7.46 (d, J = 7.3 Hz,

2H), 7.39 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 6.02 (s, 1H),

4.82 (s, 2H), 4.57 (s, 1H), 4.40 (s, 1H), 4.12 (d, J = 7.9 Hz, 1H), 4.02

(s, 2H), 3.95 (d, J = 7.9 Hz, 1H) ppm; ¹³C NMR (CD₃OD, 126 MHz)

d = 149.14, 138.90, 138.55, 128.55, 127.84, 127.80, 127.34, 89.15,

86.53, 80.06, 71.83, 70.41, 57.19 ppm; HRMS (ESI-TOF) (m/z):

72.47, 58.12 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C

₂₆H₂₇ClN₅O₄508.1746, found 508.1750.

[M+H]⁺calcd for C₁₈H₁₉ClN₅O₄404.1120, found 404.1122.

4.1.40. (1S,3R,4R,7S)-3-(6-(Benzyl(methyl)amino)-2-chloro-9H-

purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-

7-ol (36)

þ

4.1.37. ((2S,3S,4R,5R)-5-(6-(Benzyl(methyl)amino)-2-chloro-9H-

purin-9-yl)-3-(benzyloxy)-2-((tert-butyldiphenylsilyloxy)-

methyl)-4-hydroxytetrahydrofuran-2-yl)methyl 4-methyl-

benzenesulfonate (74)

From compound 35, using the same procedure as for compound

29, compound 36 was obtained and puriﬁcation by preparative-

plate chromatography (silica, EtOAc) was completed to give

nucleoside 36 (36 mg, 0.0863 mmol, 75%). Compound 36: white

Compound 71 (107 mg, 0.122 mmol) was dissolved in MeOH

(2.14 mL), and N-benzylmethylamine (0.08 mL, 0.610 mmol) was

added. The solution was heated to 55 °C for 3 h. Evaporation and

puriﬁcation by ﬂash column chromatography (silica, hex-

anes:EtOAc 3:1) were completed to give compound 74 (95 mg,

0.100 mmol, 82%). Compound 74: white foam; R_f= 0.3 (silica, hex-

semi-solid; R_f= 0.34 (silica, EtOAc); ½a _D²⁵

= ꢁ7.8 (MeOH, c 0.80);

ꢃ

FT-IR (ﬁlm) m_max3354, 2928, 1592, 1453, 1403, 1356, 1313, 1215,

1131, 1038, 936, 905, 874, 845, 820, 783, 738, 701, 676 cm^ꢁ^{1 1}H

;

NMR (CD₃OD, 600 MHz) d = 8.15 (s, 1H), 7.35–7.22 (m, 5H), 5.96

(s, 1H), 5.56 (s, 2H), 4.49 (s, 1H), 4.32 (s, 1H), 4.05 (d, J = 7.9 Hz,

1H), 3.94 (s, 2H), 3.88 (d, J = 7.9 Hz, 1H), 3.65 (s, 3H) ppm; ¹³C

NMR (CD₃OD, 151 MHz) d = 156.23, 155.04, 138.25, 129.68,

129.35, 129.17, 128.81, 128.55, 120.20, 90.10, 87.48, 81.03, 72.81,

anes:EtOAc 2:1); ½a _D²⁵

ꢃ

= ꢁ6.3 (CHCl₃, c 0.70); FT-IR (ﬁlm)

m_max2930,

2858, 1592, 1454, 1427, 1402, 1359, 1313, 1237, 1210, 1189, 1176,

1095, 974, 941, 908, 812, 730, 699, 666 cm^ꢁ^{1 1}H NMR (CDCl₃,

;

500 MHz) d = 7.73 (d, J = 8.3 Hz, 2H), 7.60 (s, 1H), 7.57–7.51 (m,

4H), 7.43–7.28 (m, 15H), 7.23 (d, J = 8.0 Hz, 3H), 5.65 (d,

J = 4.8 Hz, 1H), 4.71 (d, J = 11.4 Hz, 1H), 4.64 (d, J = 11.4 Hz, 1H),

4.58 (dd, J = 11.5, 6.0 Hz, 1H), 4.49 (d, J = 5.9 Hz, 1H), 4.38 (d,

J = 10.5 Hz, 1H), 4.36 (d, J = 10.5 Hz, 1H), 3.77 (d, J = 10.8 Hz, 1H),

3.65 (dd, J = 8.7, 4.7 Hz, 3H), 3.17 (s, 1H), 2.39 (s, 3H), 0.99 (s, 9H)

71.37, 58.16 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C

₁₉H₂₀ClN₅O₄418.1277, found 418.1280.

4.1.41. ((1S,3R,4R,7S)-7-(Benzyloxy)-3-(2,6-dimethoxy-9H-

purin-9-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol (37)

Compound 71 (150 mg, 0.171 mmol) was dissolved in MeOH

(2.0 mL) and THF (2.0 mL) and solid NaOH (100 mg, 2.50 mmol)

was added. The reaction mixture was stirred at rt for 12 h. The

reaction mixture was diluted with water and extracted with EtOAc.

The combined organic extracts were washed with brine (2 ꢂ 4 mL),

dried over MgSO₄, ﬁltered, and concentrated to give the cyclic

compound (10 mg, 0.153 mmol, 90%), which was taken to the next

ppm; ¹³C NMR (CDCl₃, 151 MHz)

d 144.94, 136.94, 135.71,

135.60, 132.83, 132.70, 132.34, 130.07, 129.89, 128.85, 128.80,

128.56, 128.46, 128.21, 127.99, 127.88, 127.70, 119.42, 89.85,

86.93, 79.58, 74.83, 74.68, 69.16, 64.93, 27.00, 21.79, 19.31 ppm;

HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₄₉H₅₃ClN₅O₇SSi⁺

918.3124, found 918.3110.

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5663

step without further puriﬁcation. The oil (10 mg, 0.153 mmol) was

dissolved in dry THF (2.0 mL) along with HFꢀpyridine (0.20 mL,

2.00 mmol) at 0 °C. The reaction mixture was stirred at rt for

12 h. The reaction was quenched with satd aq NaHCO₃(1 mL)

and extracted with CH₂Cl₂. The combined organic extracts were

washed with brine (2 ꢂ 4 mL), dried over MgSO₄, ﬁltered, and con-

centrated. Puriﬁcation by ﬂash column chromatography (silica, 7:3

hexanes:acetone) was completed to afford compound 37 (49 mg,

77%). Compound 37: colorless semi-solid; R_f= 0.41 (silica, 10%

28.45, 27.58 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C₂₂H₂₃N₄O₆439.1612, found 439.1612.

4.1.44. 2-Allyl-9-((1R,3R,4R,7S)-7-(benzyloxy)-1-((tert-butyl-

diphenylsilyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-

6-((E)-prop-1-enyl)-9H-purine (76)

Compound 71 (150 mg, 0.171 mmol) was dissolved in dry, de-

gassed DMF (0.50 mL) along with allyl(tri-n-butyl)tin (0.16 mL,

0.513 mmol) and Pd(PPh₃)₂Cl₂(12 mg, 0.0171 mmol). The reaction

mixture was stirred at 95 °C for 6 h. It was allowed to cool down

and directly passed through a silica gel plug, affording a mixture

of inseparable compounds that were taken to the next step without

further puriﬁcation. The yellow oil residue was dissolved in THF

(0.60 mL) along with 2 M NaOH (0.10 mL) and stirred at rt for

12 h. The reaction mixture was diluted with H₂O (1 mL) and ex-

tracted with EtOAc. The combined organic extracts were washed

with brine (2 ꢂ 2 mL), dried over MgSO₄, ﬁltered, and concen-

trated. Puriﬁcation by ﬂash column chromatography (silica, 4:1

hexanes:EtOAc) was completed to give compound 76 (48 mg,

0.0714 mmol, 42% over two steps). Compound 76: light yellow

MeOH/DCM); ½a ²_D⁵

ꢃ

= +28.0 (CHCl₃, c 0.50); FT-IR (ﬁlm) m_max3300,

2951, 1594, 1480, 1397, 1363, 1255, 1144, 1040, 791, 742,

700 cm^ꢁ^{1 1}H NMR (CDCl₃, 600 MHz) d = 7.85 (d, J = 8.5 Hz, 1H),

;

7.35–7.27 (m, 5H), 6.01 (d, J = 3.2 Hz, 1H), 4.68–4.57 (m, 3H),

4.33 (s, 1H), 4.19–4.12 (m, 4H), 4.06–3.97 (m, 5H), 3.93 (dd,

J = 7.8, 3.5 Hz, 1H) ppm; ¹³C NMR (CDCl₃, 151 MHz) d = 162.42,

162.19, 152.48, 138.35, 137.27, 128.89, 128.57, 128.19, 118.07,

88.48, 87.02, 77.90, 72.93, 72.70, 58.15, 55.65, 54.76 ppm; HRMS

(ESI-TOF) (m/z): [M+H]⁺calcd for C₂₀H₂₃N₄O₆415.1612, found

þ

415.1629.

4.1.42. (1S,3R,4R,7S)-3-(2,6-Dimethoxy-9H-purin-9-yl)-1-

(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (38)

From compound 37, using the same procedure as for compound

17, compound 38 was obtained and puriﬁcation by ﬂash column

chromatography (silica, DCM:MeOH 99:1) was completed to give

nucleoside 38 (18 mg, 0.0555 mmol, 77%). Compound 38: colorless

oil; R_f= 0.90 (silica, 5% MeOH/DCM); ½a _D²⁵

ꢃ

= +35.5 (MeOH, c 0.20);

FT-IR (ﬁlm) m_max2931, 1579, 1428, 1365, 1211, 1112, 1038, 911,

823, 741, 702 cm^{ꢁ1 1}H NMR (CD₃CN, 600 MHz) d = 8.20 (s, 1H),

;

7.68 (dd, J = 6.7, 4.9 Hz, 4H), 7.66–7.60 (m, 1H), 7.47–7.42 (m,

2H), 7.37 (dt, J = 12.8, 7.4 Hz, 4H), 7.29–7.23 (m, 5H), 6.90 (dd,

J = 15.7, 1.7 Hz, 1H), 6.22 (ddt, J = 17.0, 10.2, 6.8 Hz, 1H), 6.04 (s,

1H), 5.16 (dd, J = 17.2, 1.9 Hz, 1H), 5.09 (dd, J = 10.1, 1.9 Hz, 1H),

4.82 (s, 1H), 4.65 (d, J = 11.8 Hz, 1H), 4.59 (d, J = 11.8 Hz, 1H),

4.44 (s, 1H), 4.08 (d, J = 12.1 Hz, 1H), 4.04 (d, J = 12.1 Hz, 1H),

4.00 (d, J = 7.9 Hz, 1H), 3.90 (d, J = 7.9 Hz, 1H), 3.71 (dt, J = 6.8,

1.5 Hz, 2H), 2.04 (dd, J = 6.9, 1.7 Hz, 3H), 1.01 (s, 9H) ppm; ¹³C

NMR (CD₃CN, 151 MHz) d = 163.74, 154.46, 152.40, 142.59,

140.85, 138.66, 136.71, 136.40, 136.35, 133.74, 133.69, 130.94,

130.00, 129.27, 128.82, 128.81, 128.72, 128.57, 127.63, 116.70,

89.02, 87.15, 78.46, 77.79, 73.22, 72.80, 60.35, 44.52, 27.08,

19.73, 19.18 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

oil; R_f= 0.18 (silica, 10% MeOH/DCM); ½a _D²⁵

ꢃ

= +25.6 (MeCN, c 0.20);

FT-IR (ﬁlm) m_max3341, 2951, 1594, 1507, 1480, 1397, 1362, 1255,

1240, 1138, 1082, 1034, 1012, 959, 937, 905, 875, 829, 806, 791,

770, 742, 722, 677 cm^ꢁ^{1 1}H NMR (CD₃CN, 600 MHz) d = 7.96 (s,

;

1H), 5.90 (s, 1H), 4.48 (s, 1H), 4.40 (s, 1H), 4.08 (s, 3H), 4.01–3.95

(m, 4H), 3.84 (dt, J = 17.7, 6.1 Hz, 3H) ppm; ¹³C NMR (CD₃CN,

151 MHz) d = 162.32, 162.17, 153.15, 139.10, 89.39, 86.95, 80.46,

72.43, 71.60, 58.11, 55.56, 54.77 ppm; HRMS (ESI-TOF) (m/z):

[M+H]⁺calcd for C₁₃H₁₇N₄O₆325.1143, found 325.1151.

þ

4.1.43. 1,1⁰-(9-((1S,3R,4R,7S)-7-(Benzyloxy)-1-(hydroxymethyl)-

2,5-dioxabicyclo[2.2.1]heptan-3-yl)-9H-purine-2,6-diyl)-

diethanone (39)

C

₄₀H₄₅N₄O₄Si⁺673.3204, found 673.3211.

4.1.45. ((1S,3R,4R,7S)-3-(2-Allyl-6-((E)-prop-1-enyl)-9H-purin-9-

yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol

(40)

Compound 71 (65 mg, 0.0742 mmol) was dissolved in dry, de-

gassed DMF (2.0 mL) along with tributyl(1-ethoxyvinyl)tin

(0.050 mL, 0.148 mmol) and Pd(PPh₃)₂Cl₂(5 mg, 0.00713 mmol).

The reaction mixture was stirred at 95 °C for 18 h. It was then al-

lowed to cool down and directly passed through a silica gel plug,

affording a mixture of inseparable compounds that was taken to

the next step without further puriﬁcation. The oil was dissolved

in THF (3.3 mL) along with 2 M NaOH (0.30 mL) and stirred at rt

for 12 h. The reaction mixture was diluted with H₂O (5 mL) and ex-

tracted with EtOAc. The combined organic extracts were washed

with brine (2 ꢂ 5 mL), dried over MgSO₄, ﬁltered, and concentrated

to afford a yellow foam, which was dissolved in dry THF (0.50 mL)

along with HFꢀpyridine (0.10 mL, 0.860 mmol) at 0 °C. The reaction

mixture was stirred at rt for 6 h. It was then quenched with satd aq

NaHCO₃(3 mL) and extracted with CH₂Cl₂. The combined organic

extracts were washed with brine (2 ꢂ 3 mL), dried over MgSO₄, ﬁl-

tered, and concentrated. Puriﬁcation by ﬂash column chromatogra-

phy (silica, hexanes:EtOAc:acetone 5:4:1) was completed to give

compound 39 (10 mg, 0.0228 mmol, 31% over three steps). Com-

pound 39: colorless oil; R_f= 0.44 (silica, 10% MeOH/DCM);

From compound 76, using the same procedure as for compound

40, compound 40 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc 1:1) was completed to give

nucleoside 40 (28 mg, 0.0645 mmol, 87%). Compound 40: colorless

oil; R_f= 0.31 (silica, 5% MeOH/chloroform); ½a _D²⁵

ꢃ

= +22.0 (MeOH, c

0.70); FT-IR (ﬁlm) m_max3347, 2941, 1653, 1578, 1492, 1454,

1368, 1288, 1213, 1142, 1096, 1054, 1033, 976, 909, 882, 860,

811, 738, 698 cm^ꢁ^{1 1}H NMR (CD₃CN, 600 MHz) d = 8.19 (s, 1H),

;

7.68–7.58 (m, 1H), 7.32–7.22 (m, 5H), 6.92–6.85 (m, 1H), 6.21

(ddt, J = 17.0, 10.2, 6.8 Hz, 1H), 6.02 (s, 1H), 5.16 (ddd, J = 17.2,

3.4, 1.6 Hz, 1H), 5.10 (ddd, J = 10.1, 1.9, 1.4 Hz, 1H), 4.71 (s, 1H),

4.64–4.56 (m, 2H), 4.34 (s, 1H), 4.02 (d, J = 7.8 Hz, 1H), 3.93–3.87

(m, 3H), 3.70 (dd, J = 6.8, 1.5 Hz, 2H), 3.46 (s, 1H), 2.04 (dd,

J = 6.9, 1.7 Hz, 3H) ppm; ¹³C NMR (CD₃CN, 151 MHz) d = 163.64,

154.47, 152.52, 142.83, 140.88, 138.81, 136.70, 130.09, 129.24,

128.68, 128.59, 127.62, 116.76, 89.17, 87.41, 78.31, 78.29, 73.13,

72.73, 58.13, 44.47, 19.17 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺

þ

calcd for C₂₄H₂₇N₄O₄435.2027, found 435.2015.

½

a ²_D⁵

ꢃ

= +24.0 (MeOH, c 0.10); FT-IR (ﬁlm) m_max3408, 2925, 1708,

1580, 1363, 1210, 1138, 1054, 1033, 700 cm^ꢁ^{1 1}H NMR (CD₃CN,

;

4.1.46. (1S,3R,4R,7S)-3-(2,6-Dipropyl-9H-purin-9-yl)-1-(hydr-

oxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (41)

Protected compound 40 (10 mg, 0.0230 mmol) was dissolved in

wet ethanol (1.2 mL) along with Pd(OH)₂(1 mg, 0.00712 mmol).

The mixture was degassed and put under hydrogen atmosphere.

The reaction mixture was heated to 50 °C for 12 h. The mixture

600 MHz) d = 8.66 (s, 1H), 7.32–7.23 (m, 5H), 6.16 (s, 1H), 4.75 (s,

1H), 4.60 (s, 2H), 4.30 (s, 1H), 4.07–4.02 (m, 1H), 3.93 (m, 3H),

2.86 (s, 3H), 2.81 (s, 3H) ppm; ¹³C NMR (CD₃CN, 151 MHz)

d = 199.20, 197.38, 177.55, 149.42, 148.99, 138.77, 129.23,

128.69, 128.62, 89.53, 87.59, 78.22, 78.09, 73.21, 72.78, 57.99,

5664

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

þ

was ﬁltered and the solvent was concentrated under reduced pres-

sure. Puriﬁcation by ﬂash column chromatography (silica,

DCM:MeOH 95:5) was completed to give compound 41 (4 mg,

0.0115 mmol, 50%). Compound 41: colorless oil; R_f= 0.19 (silica,

TOF) (m/z): [M+H]⁺calcd for C₂₆H₂₃N₄O₆487.1612, found

487.1623.

4.1.49. (1S,3R,4R,7S)-3-(2,6-Di(furan-2-yl)-9H-purin-9-yl)-1-

(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (43)

From compound 42, using the same procedure as for compound

12, compound 43 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc 7:3) was completed to af-

ford nucleoside 43 (11 mg, 0.0278 mmol, 90%). Compound 43:

hexanes:acetone 7:3); ½a _D²⁵

ꢃ

= +13.0 (MeCN, c 0.10); FT-IR (ﬁlm)

m_max3300, 2927, 1592, 1490, 1368, 1050, 810, 738, 698 cm^ꢁ1

;

¹H

NMR (CD₃CN, 600 MHz) d = 8.20 (s, 1H), 5.99 (s, 1H), 4.47 (s, 1H),

4.43 (s, 1H), 4.00 (d, J = 7.9 Hz, 1H), 3.88 (d, J = 4.6 Hz, 2H), 3.84

(d, J = 8.0 Hz, 1H), 3.06–3.03 (m, 2H), 2.92–2.88 (m, 2H), 1.88–

1.82 (m, 4H), 0.97 (t, J = 7.4 Hz, 6H) ppm; ¹³C NMR (CD₃CN,

151 MHz) d = 165.96, 162.85, 151.35, 142.25, 131.84, 89.23,

86.70, 80.49, 72.49, 71.74, 58.27, 41.80, 35.63, 22.95, 22.44,

14.28, 13.95 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

off-white foam; R_f= 0.15 (silica, hexanes:EtOAc 7:3); ½a _D²⁵

ꢃ

= +26.0

(MeOH, c 0.25); FT-IR (ﬁlm) m_max3340, 2949, 2838, 1647, 1407,

1113, 1014, 550, 530 cm^ꢁ^{1 1}H NMR (CD₃OD, 400 MHz) d = 8.57

;

(s, 1H), 7.91 (dd, J = 1.7, 0.7 Hz, 1H), 7.83 (dd, J = 3.5, 0.7 Hz, 1H),

7.75 (dd, J = 1.7, 0.8 Hz, 1H), 7.40 (dd, J = 3.4, 0.8 Hz, 1H), 6.76

(dd, J = 3.5, 1.7 Hz, 1H), 6.65 (dd, J = 3.4, 1.8 Hz, 1H), 6.18 (s, 1H),

4.70 (s, 1H), 4.45 (s, 1H), 4.10 (d, J = 7.9 Hz, 1H), 3.99 (s, 2H), 3.94

(d, J = 7.9 Hz, 1H) ppm; ¹³C NMR (CD₃OD, 151 MHz) d = 153.68,

153.57, 151.15, 147.59, 147.23, 146.10, 144.57, 118.24, 114.21,

113.68, 113.26, 90.27, 87.64, 81.06, 72.91, 71.58, 58.18 ppm; HRMS

þ

C₁₇H₂₅N₄O₄349.187, found 349.1884.

4.1.47. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-2,6-

di(furan-2-yl)-9H-purine (77)

Compound 71 (83 mg, 0.0948 mmol) was dissolved in dry, de-

gassed DMF (2.0 mL) along with 2-(tributylstannyl)furan

(0.060 mL, 0.189 mmol) and Pd(PPh₃)₂Cl₂(5 mg, 0.00712 mmol).

The reaction mixture was stirred at 95 °C for 13 h. It was then al-

lowed to cool to rt and directly passed through a silica gel plug,

affording the compound as a yellow oil (88 mg, 0.0937 mmol,

99%), which was taken to the next step without further puriﬁca-

tion. The yellow oil (85 mg, 0.0905 mmol) was dissolved in THF

(5.0 mL) along with 2 M NaOH (0.40 mL) and stirred at rt for

12 h. The reaction mixture was diluted with H₂O (5 mL) and ex-

tracted with EtOAc. The combined organic extracts were washed

with brine (2 ꢂ 5 mL), dried over MgSO₄, ﬁltered, and concen-

trated. Puriﬁcation by ﬂash column chromatography (silica, hex-

anes:EtOAc 4:1) was completed to give compound 77 (50 mg,

0.0690 mmol, 76%). Compound 77: yellow foam; R_f= 0.94 (silica,

(ESI-TOF) (m/z): [M+H]⁺calcd for C₁₉H₁₇N₄O₆397.1143, found

þ

397.1148.

4.1.50. 9-((1R,3R,4R,7S)-7-(Benzyloxy)-1-((tert-butyldiphenyl-

silyloxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-2,6-

di(thiophen-2-yl)-9H-purine (78)

Compound 71 (40 mg, 0.0457 mmol) was dissolved in dry, de-

gassed DMF (1.0 mL) along with 2-(tributylstannyl)thiophene

(0.029 mL, 0.091 mmol) and Pd(PPh₃)₂Cl₂(4 mg, 0.00570 mmol).

The reaction mixture was stirred at 80 °C for 7 h. It was then al-

lowed to cool to rt and directly passed through a silica gel plug,

affording the compound as yellow oil (32 mg, 0.0330 mmol,

72%), which was advanced to the next chemical transformation

without further puriﬁcation. The yellow oil (28 mg, 0.0289 mmol)

was dissolved in THF (2.4 mL) along with 2 M NaOH (0.20 mL)

and stirred at rt for 13.5 h. The reaction mixture was diluted with

H₂O (4 mL) and extracted with EtOAc. The combined organic

extracts were washed with brine (2 ꢂ 5 mL), dried over MgSO₄,

ﬁltered, and concentrated. Puriﬁcation by ﬂash column chroma-

tography (silica, hexanes:EtOAc 7:3) was completed to give com-

pound 78 (21.3 mg, 0.0282 mmol, 98%). Compound 78: white

hexanes:EtOAc 7:3); ½a _D²⁵

ꢃ

= +52.0 (MeOH, c 1.00); FT-IR (ﬁlm) m_max

3602, 2937, 1584, 1053, 1033, 1012, 702 cm^ꢁ1

;

¹H NMR (CD₃CN,

500 MHz) d = 8.31 (s, 1H), 7.92–7.86 (m, 2H), 7.72 (dd, J = 1.7,

0.9 Hz, 1H), 7.71–7.66 (m, 4H), 7.46–7.42 (m, 2H), 7.41–7.34 (m,

6H), 7.27–7.23 (m, 4H), 6.75 (dt, J = 2.9, 1.4 Hz, 1H), 6.65 (dt,

J = 2.9, 1.4 Hz, 1H), 6.13 (s, 1H), 4.91 (s, 1H), 4.68 (d, J = 11.8 Hz,

1H), 4.64 (d, J = 11.8 Hz, 1H), 4.48 (s, 1H), 4.09 (q, J = 12.1 Hz,

2H), 4.03 (d, J = 7.9 Hz, 1H), 3.94 (d, J = 7.9 Hz, 1H), 1.03 (s, 9H)

ppm; ¹³C NMR (CD₃CN, 126 MHz) d = 153.68, 152.84, 152.61,

150.49, 147.13, 146.62, 145.89, 143.67, 138.74, 136.45, 136.40,

133.83, 133.76, 130.97, 129.30, 128.86, 128.74, 128.61, 128.46,

118.75, 113.72, 113.69, 113.19, 89.23, 87.34, 78.51, 77.90, 73.30,

72.88, 60.45, 27.14, 19.79 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺

calcd for C₄₂H₄₁N₄O₆Si⁺725.2790, found 725.2798.

foam; R_f= 0.97 (silica, hexanes:EtOAc 7:3); ½a _D²⁵

ꢃ

= +38.0 (MeOH,

c 1.00); FT-IR (ﬁlm) m_max3477, 2979, 1709, 1422, 1360, 1221,

1066, 903 cm^{ꢁ1 1}H NMR (CD₃CN, 600 MHz) d = 8.70–8.68 (m,

;

1H), 8.34 (s, 1H), 8.08–8.06 (m, 1H), 7.80–7.78 (m, 1H), 7.74–

7.70 (m, 4H), 7.61–7.59 (m, 1H), 7.49–7.44 (m, 3H), 7.41 (ddd,

J = 21.6, 11.2, 3.8 Hz, 5H), 7.35–7.33 (m, 1H), 7.27–7.25 (m, 3H),

7.23–7.20 (m, 1H), 6.16 (s, 1H), 4.97 (s, 1H), 4.68 (d, J = 11.8 Hz,

1H), 4.61 (d, J = 11.8 Hz, 1H), 4.46 (s, 1H), 4.15 (d, J = 12.0 Hz,

1H), 4.11 (d, J = 12.1 Hz, 1H), 4.06 (d, J = 7.8 Hz, 1H), 3.97 (d,

J = 7.8 Hz, 1H), 1.06 (s, 9H) ppm; ¹³C NMR (CD₃CN, 151 MHz)

d = 177.49, 156.17, 152.74, 150.31, 144.42, 143.53, 140.94,

138.61, 136.42, 136.36, 133.79, 133.68, 132.33, 130.95,

130.58, 129.91, 129.43, 129.28, 129.25, 128.85, 128.83, 128.70,

128.58, 89.20, 87.40, 78.38, 77.87, 73.27, 72.85, 60.46, 27.12,

19.76 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

4.1.48. ((1S,3R,4R,7S)-7-(Benzyloxy)-3-(2,6-di(furan-2-yl)-9H-

purin-9-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol (42)

From compound 77, using the same procedure as for compound

15, compound 42 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc 7:3) was completed to give

nucleoside 42 (27 mg, 0.0552 mmol, 100%). Compound 42: light

yellow foam; R_f= 0.38 (silica, hexanes:EtOAc 7:3); ½a _D²⁵

ꢃ

= +64.0

(MeOH, c 0.50); FT-IR (ﬁlm) m_max3375, 2943, 1584, 1487, 1367,

C₄₂H₄₁N₄O₄S₂Si 757.2333, found 757.2332.

1208, 1144, 1102, 1050, 1012, 938, 895, 885, 827, 801, 747,

698 cm^ꢁ1

;

¹H NMR (1% D₂O/CD₃CN, 400 MHz) d = 8.34 (s, 1H),

4.1.51. ((1S,3R,4R,7S)-7-(Benzyloxy)-3-(2,6-di(thiophen-2-yl)-

9H-purin-9-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methanol

(44)

7.90 (dd, J = 3.5, 0.8 Hz, 1H), 7.87 (dd, J = 1.8, 0.8 Hz, 1H), 7.73

(dd, J = 1.8, 0.9 Hz, 1H), 7.35 (dd, J = 3.4, 0.9 Hz, 1H), 7.29–7.22

(m, 5H), 6.75 (dd, J = 3.5, 1.8 Hz, 1H), 6.65 (dd, J = 3.4, 1.8 Hz, 1H),

6.10 (s, 1H), 4.80 (s, 1H), 4.62 (s, 2H), 4.34 (s, 1H), 4.06–4.04 (m,

1H), 3.93–3.91 (m, 3H) ppm; ¹³C NMR (CD₃CN, 151 MHz)

d = 153.54, 152.67, 152.48, 150.32, 147.00, 146.44, 145.78,

143.71, 138.74, 129.17, 128.60, 128.52, 118.68, 113.59, 113.09,

89.21, 87.23, 78.26, 78.08, 73.13, 72.70, 58.05 ppm; HRMS (ESI-

From compound 78, using the same procedure as for compound

15, compound 44 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:EtOAc 7:3) was completed to give

nucleoside 44 (27 mg, 0.0521 mmol, 99%). Compound 44: white

foam; R_f= 0.43 (silica, hexanes:EtOAc 7:3); ½a _D²⁵

ꢃ

= +64.0 (MeOH, c

0.50); FT-IR (ﬁlm) m_max3338, 2951, 1642, 1572, 1428, 1391,

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5665

1327, 1206, 1032, 1015, 820, 591, 541, 528 cm^ꢁ1; ¹H NMR (CD₃CN,

400 MHz) d = 8.68 (dd, J = 3.8, 1.2 Hz, 1H), 8.34 (s, 1H), 8.05 (dd,

J = 3.7, 1.3 Hz, 1H), 7.76 (dd, J = 5.0, 1.2 Hz, 1H), 7.58 (dd, J = 5.0,

1.3 Hz, 1H), 7.31 (dt, J = 6.1, 3.0 Hz, 1H), 7.29–7.25 (m, 4H), 7.25–

7.21 (m, 1H), 7.20 (dd, J = 5.0, 3.7 Hz, 1H), 6.10 (s, 1H), 4.85 (s,

1H), 4.61 (m, 2H), 4.34 (s, 1H), 4.05 (d, J = 8.0 Hz, 1H), 3.98–3.90

(m, 3H), 3.27 (t, J = 6.0 Hz, 1H) ppm; ¹³C NMR (CD₃CN, 151 MHz)

d = 156.12, 152.74, 150.25, 144.42, 143.67, 140.93, 138.76,

133.64, 132.29, 130.55, 129.89, 129.40, 129.28, 129.22, 128.67,

128.59, 128.58, 89.28, 87.35, 78.32, 78.12, 73.19, 72.79,

58.15 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for C₂₆H₂₂N₄O₄S₂

519.1155, found 519.1151.

7.86 (dt, J = 5.0, 1.2 Hz, 1H), 7.66 (dt, J = 5.0, 1.2 Hz, 1H), 7.36–

7.30 (m, 3H), 7.25 (ddd, J = 7.1, 4.3, 1.1 Hz, 2H), 7.21 (ddd, J = 6.1,

3.9, 1.2 Hz, 2H), 7.01 (s, 2H), 6.25 (s, 1H), 5.09 (s, 1H), 4.73 (ddd,

J = 28.3, 14.7, 6.4 Hz, 3H), 4.65 (d, J = 11.3 Hz, 2H), 4.22 (d,

J = 7.8 Hz, 1H), 4.05 (d, J = 7.8 Hz, 1H) ppm; ¹³C NMR (acetone-d₆,

151 MHz) d = 156.11, 152.60, 150.22, 144.37, 143.70, 140.96,

138.60, 133.70, 132.25, 130.47, 129.67, 129.33, 129.09, 129.02,

128.58, 128.54, 128.46, 87.55, 86.32, 78.78, 78.03, 73.06, 72.74,

65.93 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C

₂₆H₂₄N₅O₆S₃598.0883, found 598.0885.

4.1.54. (2R,3R,4S)-4-(Benzyloxy)-2-(2,6-dichloro-9H-purin-9-yl)-

5,5-bis((methylsulfonyloxy)methyl)tetrahydrofuran-3-yl

acetate (80)

4.1.52. (1S,3R,4R,7S)-3-(2,6-Di(thiophen-2-yl)-9H-purin-9-yl)-1-

(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (45) and

(1S,3S,4R,7S)-3-(2,6-di(thiophen-2-yl)-9H-purin-9-yl)-1-

Literature-known compound 79 (290 mg, 0.569 mmol) was dis-

solved in dry MeCN (1.5 mL) along with 2,6-dichloropurine

(214 mg, 1.13 mmol) and BSA (0.51 mL, 1.99 mmol). After 5 min,

the reaction mixture was cooled to ꢁ40 °C followed by slow addi-

tion of TMSOTf (0.21 mL, 0.850 mmol), and then heated to 80 °C in

the microwave (CEM Discover, 300 W). The reaction mixture was

brought to rt, quenched with satd aq NaHCO₃(2 mL), and extracted

with EtOAc. The combined organic extracts were washed with

brine (3 mL), dried over MgSO₄, ﬁltered, and concentrated. Puriﬁ-

cation by ﬂash column chromatography (silica, hexanes:EtOAc

4:1) was completed to give compound 80 (30 mg, 0.478 mmol,

84%). Compound 80: yellow foam; R_f= 0.56 (silica, 5% MeOH/

(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol (46)

From compound 44, using the same procedure as for compound

17, compounds 45 and 46 were obtained. Puriﬁcation by ﬂash col-

umn chromatography (silica, hexanes:EtOAc 7:3) was completed

to give compounds 45 (4.0 mg, 0.00934 mmol, 40%) and 46

(3.3 mg, 0.00770 mol, 33%). Compound 45: white foam; R_f= 0.14

(silica, hexanes:EtOAc 7:3); ½a _D²⁵

ꢃ

= +51.2 (MeOH, c 0.20); FT-IR

(ﬁlm) m_max3361, 1572, 1535, 1443, 1426, 1376, 1222, 1038, 907,

823, 799, 711 cm^ꢁ1

;

¹H NMR (CD₃OD, 500 MHz) d = 8.61 (d,

J = 3.7 Hz, 1H), 8.50 (s, 1H), 8.04 (d, J = 3.5 Hz, 1H), 7.75 (d,

J = 5.0 Hz, 1H), 7.56 (d, J = 4.9 Hz, 1H), 7.32–7.25 (m, 1H), 7.18–

7.13 (m, 1H), 6.13 (s, 1H), 4.73 (s, 2H), 4.48 (s, 1H), 4.12 (d,

J = 7.8 Hz, 1H), 3.99 (s, 2H), 3.96 (d, J = 7.8 Hz, 1H) ppm; ¹³C NMR

(CD₃OD, 151 MHz) d = 156.93, 152.85, 150.98, 144.73, 144.09,

141.36, 133.66, 132.29, 130.54, 129.61, 129.03, 128.54, 90.19,

87.63, 81.10, 72.97, 71.76, 58.30 ppm; HRMS (ESI-TOF) (m/z):

[M+H]⁺calcd for C₁₉H₁₇N₄S₂O₄429.0686, found 429.0691. Com-

pound 46: colorless oil; R_f= 0.54 (silica, hexanes:EtOAc 7:3);

DCM); ½a ²_D⁵

ꢃ

= +13.0 (CHCl₃, c 0.10); FT-IR (ﬁlm) m_max3019, 1749,

1595, 1560, 1496, 1357, 1226, 1175, 1030, 963, 883, 826, 755,

702 cm^ꢁ^{1 1}H NMR (CDCl₃, 400 MHz) d = 8.18 (s, 1H), 7.43–7.32

;

(m, 5H), 6.17 (d, J = 3.6 Hz, 1H), 5.87 (dd, J = 5.9, 3.7 Hz, 1H), 4.97

(d, J = 6.0 Hz, 1H), 4.71–4.59 (m, 3H), 4.42 (d, J = 10.9 Hz, 1H),

4.35 (d, J = 11.7 Hz, 1H), 4.30 (d, J = 10.9 Hz, 1H), 3.04 (s, 3H),

2.93 (s, 3H), 2.14 (s, 3H) ppm; ¹³C NMR (CDCl₃, 151 MHz)

d = 207.26, 169.83, 153.77, 152.49, 151.89, 145.32, 136.39,

128.94, 128.82, 88.40, 84.96, 77.26, 75.08, 73.66, 67.48, 67.38,

½

a ²_D⁵

ꢃ

= ꢁ26.4 (MeOH, c 0.40); FT-IR (ﬁlm) m_max3330, 2982, 1574,

1444, 1426, 1378, 1212, 1057, 1033, 800, 723 cm^ꢁ^{1 1}H NMR

;

38.00, 37.71, 31.10 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

(CD₃OD, 400 MHz) d = 8.68 (s, 1H), 8.64 (dd, J = 3.7, 1.2 Hz, 1H),

8.08 (dd, J = 3.7, 1.2 Hz, 1H), 7.79 (dd, J = 5.0, 1.1 Hz, 1H), 7.60

(dd, J = 5.0, 1.2 Hz, 1H), 7.30 (dd, J = 5.0, 3.8 Hz, 1H), 7.19 (ddd,

J = 5.3, 3.7, 1.6 Hz, 1H), 6.82 (d, J = 7.4 Hz, 1H), 4.76 (dd, J = 7.4,

3.5 Hz, 1H), 4.29 (d, J = 9.6 Hz, 1H), 4.12 (d, J = 9.5 Hz, 1H), 4.04

(d, J = 9.5 Hz, 1H), 3.90–3.86 (m, 2H) ppm; ¹³C NMR (CD₃OD,

151 MHz) d = 157.51, 153.16, 151.41, 144.91, 144.41, 141.01,

133.95, 132.67, 130.94, 129.96, 129.77, 129.13, 111.74, 89.41,

88.39, 77.81, 76.31, 67.80, 66.35 ppm; HRMS (ESI-TOF) (m/z):

[M+H]⁺calcd for C₁₉H₁₇N₄O₄S₂429.0686, found 429.0688.

C

₂₂H₂₅Cl₂N₄O₁₀S₂639.0389, found 639.0388.

4.1.55. ((3S,4R,5R)-5-(2,6-Dichloro-9H-purin-9-yl)-3,4-dihydr-

oxytetrahydrofuran-2,2-diyl)bis(methylene) dimethane-

sulfonate (81)

From compound 80, using the same procedure as for compound

17, compound 81 was obtained and puriﬁcation by ﬂash column

chromatography (silica, hexanes:acetone 7:3) was completed to

give nucleoside 81 (270 mg, 0.534 mmol, 90%). Compound 81: col-

orless semi-solid; R_f= 0.40 (silica, 5% MeOH/DCM); ½a _D²⁵

ꢃ

= +2.5

(MeCN, c 0.10); FT-IR (ﬁlm) m_max3368, 1726, 1660, 1596, 1561,

1355, 1252, 1174, 1031, 999, 965, 884, 830 cm^ꢁ1; ¹H NMR (CD₃CN,

600 MHz) d = 8.47 (d, J = 2.2 Hz, 1H), 6.08 (dd, J = 6.3, 2.1 Hz, 1H),

4.97–4.93 (m, 1H), 4.57–4.54 (m, 2H), 4.45 (ddd, J = 15.6, 13.0,

6.5 Hz, 3H), 4.21–4.09 (m, 2H), 3.09 (t, J = 2.6 Hz, 6H) ppm;

¹³C NMR (CD₃CN, 151 MHz) d = 153.97, 152.71, 151.45,

146.37, 132.10, 99.58, 89.05, 85.08, 73.50, 72.25, 69.17, 37.53,

4.1.53. ((1R,3R,4R,7S)-7-(Benzyloxy)-3-(2,6-di(thiophen-2-yl)-

9H-purin-9-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methyl

sulfamate (47)

Formic acid (0.055 mL, 1.45 mmol) was added to chlorosulfonyl

isocyanate (0.13 mL, 1.45 mmol) at 0 °C with stirring. The resulting

white solid was dissolved in CH₂Cl₂(1.4 mL) and the solution

warmed to rt and stirred for 14 h. The solution was cooled to

0 °C and a portion of this reagent (0.04 mL) was added dropwise

to a solution of compound 44 (5 mg, 0.00964 mmol) in CH₂Cl₂

(0.08 mL) at 0 °C. Pyridine (0.005 mL, 0.0556) was added dropwise

and the reaction mixture was warmed to rt and stirred 24 h. The

solvent was evaporated and puriﬁcation by preparative-plate chro-

matography (silica, hexanes:EtOAc 1:1) was completed to give

compound 47 (2 mg, 0.00337 mmol, 35%). Compound 47: white

29.24 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C

₁₃H₁₇Cl₂N₄O₉S₂506.9814, found 506.9810.

4.1.56. ((1R,3R,4R,5S)-3-(2,6-Dichloro-9H-purin-9-yl)-4-

hydroxy-2,6-dioxabicyclo[3.2.0]heptan-1-yl)methyl

methanesulfonate (48)

Compound 81 (200 mg, 0.364 mmol) was dissolved in wet THF

(15 mL) along with K₂CO₃(200 mg, 1.45 mmol). The reaction mix-

ture was stirred at rt for 18 h. The reaction was quenched with satd

aq NH₄Cl (10 mL) and extracted with CH₂Cl₂. The combined

organic extracts were washed with brine (2 ꢂ 10 mL), dried over

MgSO₄, ﬁltered, and concentrated. Puriﬁcation by ﬂash column

chromatography (silica, hexanes:acetone 1:1) was completed to

semi-solid; R_f= 0.37 (silica, hexanes:EtOAc 1:1); ½a _D²⁵

ꢃ

= +33.6 (ace-

tone, c 0.15); FT-IR (ﬁlm) m_max2925, 1572, 1535, 1487, 1443, 1426,

1376, 1245, 1183, 1148, 1094, 1039, 994, 911, 855, 818, 800, 782,

724 cm^ꢁ^{1 1}H NMR (acetone-d₆, 600 MHz) d = 8.73 (dt, J = 3.7,

1.1 Hz, 1H), 8.49 (d, J = 1.1 Hz, 1H), 8.07 (dt, J = 3.6, 1.2 Hz, 1H),

;

5666

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

afford compound 48 (118 mg, 0.288 mmol, 79%). Compound 48:

colorless semi-solid; R_f= 0.48 (silica, 5% MeOH/DCM);

chromatography (silica, DCM:MeOH 95:5) was completed to give

nucleoside 83 (20 mg, 0.0351 mmol, 82%). Compound 83: white

½

a ²_D⁵

ꢃ

= ꢁ42.0 (MeCN, c 0.30); FT-IR (ﬁlm) m_max3615, 2293, 2253,

foam; R_f= 0.36 (silica, 10% MeOH/DCM); ½a _D²⁵

= ꢁ17.0 (MeOH, c

ꢃ

1443, 1375, 1039, 918, 750 cm^ꢁ^{1 1}H NMR (CD₃CN, 600 MHz)

;

0.10); FT-IR (ﬁlm) m_max3300, 2922, 1586, 1489, 1354, 1174,

d = 8.51 (s, 1H), 6.43 (d, J = 7.3 Hz, 1H), 5.21 (dd, J = 16.9, 4.6 Hz,

1H), 4.94–4.86 (m, 1H), 4.67 (ddd, J = 21.2, 12.0, 6.5 Hz, 2H), 4.54

(tt, J = 7.2, 3.5 Hz, 2H), 3.75 (dd, J = 7.7, 1.4 Hz, 1H), 3.07 (s, 3H)

ppm; ¹³C NMR (CD₃CN, 151 MHz) d = 154.49, 153.31, 151.76,

146.28, 132.77, 89.19, 85.10, 84.51, 78.45, 75.12, 69.06,

37.78 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

1054, 1033, 1001, 832, 754 cm^ꢁ^{1 1}H NMR (CD₃CN, 600 MHz)

;

d = 8.37 (s, 1H), 7.94 (dd, J = 11.6, 3.5 Hz, 1H), 7.88 (m, 1H), 7.75

(dd, J = 1.7, 0.8 Hz, 1H), 7.58 (s, 1H), 7.38 (s, 1H), 6.76 (ddd,

J = 3.5, 1.7, 0.7 Hz, 1H), 6.66 (ddd, J = 3.4, 1.7, 0.7 Hz, 1H), 6.15 (d,

J = 6.4 Hz, 1H), 5.21 (d, J = 11.6 Hz, 1H), 4.68 (s, 1H), 4.60 (dd,

J = 24.0, 10.8 Hz, 2H), 4.50 (t, J = 11.1 Hz, 2H), 4.12 (d, J = 12.5 Hz,

1H), 3.10 (s, 3H), 3.03 (s, 3H) ppm; ¹³C NMR (CD₃CN, 151 MHz)

d = 153.56, 153.28, 152.86, 150.39, 147.21, 146.95, 146.11,

145.69, 128.69, 118.98, 113.80, 113.76, 113.22, 90.16, 89.38,

85.09, 73.69, 72.78, 69.34, 69.23, 37.69, 37.64 ppm; HRMS (ESI-

C

₁₂H₁₃Cl₂N₄O₆S⁺410.9933, found 410.9937.

4.1.57. ((1R,3R,4R,5S)-3-(6-(Benzoyloxy)-2-chloro-9H-purin-9-

yl)-4-hydroxy-2,6-dioxabicyclo[3.2.0]heptan-1-yl)methyl

benzoate (49)

TOF) (m/z): [M+H]⁺calcd for C₁₂H₂₃N₄O₁₁S₂571.0805, found

þ

Compound 48 (100 mg, 0.244 mmol) was dissolved in dry DMF

(0.50 mL) along with sodium benzoate (70 mg, 0.486 mmol). The reac-

tion mixture was heated to 90 °C for 4.5 h. Direct puriﬁcation of the

mixture by ﬂash column chromatography (silica, 7:3 hexanes:EtOAc)

was completed to give compound 49 (105 mg, 0.201 mmol, 82%). Com-

571.0801.

4.1.60. ((1R,3R,4R,5S)-3-(2,6-Di(furan-2-yl)-9H-purin-9-yl)-4-

hydroxy-2,6-dioxabicyclo[3.2.0]heptan-1-yl)methyl

methanesulfonate (50)

pound 49: white foam; R_f= 0.45 (silica, 5% MeOH/DCM); ½a _D²⁵

ꢃ

= ꢁ65.7

From compound 83, using the same procedure as for com-

pound 64, compound 50 was obtained and puriﬁcation by ﬂash

column chromatography (silica, EtOAc:hexanes 8:1) was com-

pleted to give nucleoside 50 (7 mg, 0.0148 mmol, 91%). Com-

pound 50: slightly yellow foam; R_f= 0.31 (silica, 10% MeOH/

(MeCN, c 0.30); FT-IR (ﬁlm) m_max3618, 2293, 2253, 1443, 1375, 1038,

918, 749 cm^{ꢁ1 1}H NMR (CD₃CN, 600 MHz) d = 8.08–7.99 (m, 5H),

;

7.68–7.62 (m, 2H), 7.52 (dt, J = 19.1, 7.7 Hz, 4H), 6.74 (d, J = 6.8 Hz,

1H), 5.68 (dt, J = 9.9, 4.6 Hz, 2H), 5.01 (d, J = 8.3 Hz, 1H), 4.78 (d,

J = 8.3 Hz, 1H), 4.73 (d, J = 12.2 Hz, 1H), 4.69 (d, J = 12.2 Hz, 1H) ppm;

¹³C NMR (CD₃CN, 151 MHz) d = 166.75, 166.03, 157.89, 149.13,

145.52, 139.43, 134.70, 134.42, 130.53, 130.51, 130.39, 129.75,

129.69, 129.64, 124.93, 87.16, 85.69, 84.48, 79.14, 77.35,

DCM); ½a ²_D⁵

ꢃ

= ꢁ18.0 (MeOH,

c

0.10); FT-IR (ﬁlm) m_max3300,

2922, 1586, 1354, 1175, 1054, 1033, 1001, 832 cm^ꢁ1

;

¹H NMR

(CD₃CN, 600 MHz) d = 8.45 (s, 1H), 7.94 (d, J = 3.5 Hz, 1H), 7.89

(s, 1H), 7.75 (s, 1H), 7.40 (d, J = 3.3 Hz, 1H), 6.77 (dd, J = 3.4,

1.7 Hz, 1H), 6.66 (dd, J = 3.4, 1.7 Hz, 1H), 6.53 (d, J = 7.2 Hz, 1H),

5.30 (d, J = 4.6 Hz, 1H), 4.94 (d, J = 8.4 Hz, 1H), 4.84 (td, J = 7.1,

4.6 Hz, 1H), 4.67 (d, J = 8.4 Hz, 1H), 4.59 (q, J = 11.5 Hz, 1H), 3.92

(d, J = 7.1 Hz, 1H), 3.62–3.54 (m, 1H), 3.10–2.94 (m, 3H) ppm;

¹³C NMR (CD₃CN, 151 MHz) d = 153.72, 153.59, 153.05, 150.42,

147.21, 146.94, 146.06, 145.10, 126.37, 118.98, 113.84, 113.76,

113.24, 88.96, 85.60, 84.54, 78.60, 75.33, 69.40, 37.73 ppm; HRMS

(ESI-TOF) (m/z): [M+H]⁺calcd for C₂₀H₁₉N₄O₈S⁺475.0918, found

475.0927.

64.33 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C

₂₅H₂₀ClN₄O₇523.1015, found 523.1015.

4.1.58. (2R,3R,4S)-4-(Benzyloxy)-2-(2,6-di(furan-2-yl)-9H-purin-

9-yl)-5,5-bis((methylsulfonyloxy)methyl)tetrahydrofuran-3-yl

acetate (82)

Compound 80 (50 mg, 0.0781 mmol) was dissolved in dry, de-

gassed DMF (0.20 mL) along with 2-(tributylstannyl)furan

(0.098 mL, 0.310 mmol) and Pd(PPh₃)₂Cl₂(5 mg, 0.0071 mmol).

The reaction mixture was stirred at 95 °C for 3 h, quenched with

a solution of potassium ﬂuoride (4 mL), and extracted with EtOAc.

The combined organic extracts were washed with brine

(2 ꢂ 2 mL), dried over MgSO₄, ﬁltered, and concentrated. Puriﬁca-

tion by ﬂash column chromatography (silica, EtOAc:hexanes 4:1)

was completed to give compound 82 (46 mg, 0.0655 mol, 84%).

Compound 82: yellow foam; R_f= 0.58 (silica, 10% MeOH/DCM);

4.1.61. (1S,3R,4R,5S)-3-(2,6-Di(furan-2-yl)-9H-purin-9-yl)-1-

(hydroxymethyl)-2,6-dioxabicyclo[3.2.0]heptan-4-ol (51)

Compound 50 (15 mg, 0.0316 mmol) was dissolved in dry DMF

(0.20 mL) along with sodium benzoate (9.1 mg, 0.0630 mmol). The

reaction mixture was heated to 90 °C for 4.5 h. The resulting white

mixture was passed through a silica plug to give a white solid com-

pound (15.3 mg, 0.0306 mmol, 97%). The benzoyl derivative (5 mg,

0.00999 mmol) was dissolved in MeOH (0.40 mL), and 0.1 M

NaOMe (0.20 mL) was added. The mixture was stirred at rt for

30 min and quenched with a solution of 0.1 M HCl (1 mL). The

reaction mixture was extracted with 10% MeOH:DCM, dried over

MgSO₄, ﬁltered, and concentrated. Puriﬁcation by ﬂash column

chromatography (silica, DCM:MeOH 9:1) was completed to give

compound 51 (2.8 mg, 0.00707 mmol, 71%). Compound 51: white

semi-solid; R_f= 0.15 (silica, hexanes:acetone:MeOH 5:4:1);

½

a ²_D⁵

ꢃ

= ꢁ6.5 (MeOH, c 0.20); FT-IR (ﬁlm) m_max1748, 1585, 1488,

1357, 1226, 1176, 966, 825, 753 cm^ꢁ^{1 1}H NMR (CD₃CN,

;

600 MHz) d = 8.31 (s, 1H), 7.92 (d, J = 3.5 Hz, 1H), 7.88 (d,

J = 1.7 Hz, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.45–7.32 (m, 6H), 6.76

(dd, J = 3.5, 1.7 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 6.34 (d,

J = 3.4 Hz, 1H), 6.16 (dd, J = 5.9, 3.4 Hz, 1H), 5.28 (d, J = 5.9 Hz,

1H), 4.75 (s, 2H), 4.63 (d, J = 11.3 Hz, 1H), 4.55 (d, J = 10.6 Hz,

1H), 4.49 (d, J = 10.7 Hz, 1H), 4.44 (d, J = 11.3 Hz, 1H), 3.07 (s,

3H), 2.91 (s, 3H), 2.08 (s, 3H) ppm; ¹³C NMR (CD₃CN, 151 MHz)

d = 170.51, 153.48, 152.78, 152.67, 150.19, 147.25, 146.83,

146.06, 145.76, 137.85, 129.28, 129.16, 129.09, 118.98, 113.74,

113.22, 88.77, 85.17, 79.59, 75.66, 74.33, 69.20, 69.14, 37.77,

½

a ²_D⁵

ꢃ

= ꢁ28.0 (MeOH, c 0.10); FT-IR (ﬁlm) m_max3300, 2926, 1680,

1354, 1175, 1054, 1033, 1001, 830 cm^ꢁ^{1 1}H NMR (CD₃CN,

;

400 MHz) d = 8.45 (s, 1H), 7.97 (dd, J = 3.5, 0.8 Hz, 1H), 7.91 (dd,

J = 1.8, 0.8 Hz, 1H), 7.76 (dd, J = 1.8, 0.9 Hz, 1H), 7.42 (dd, J = 3.4,

0.9 Hz, 1H), 6.79 (dd, J = 3.5, 1.8 Hz, 1H), 6.68 (dd, J = 3.4, 1.8 Hz,

1H), 6.47 (d, J = 7.3 Hz, 1H), 5.25 (d, J = 4.5 Hz, 1H), 4.89 (d,

J = 8.1 Hz, 1H), 4.80 (dd, J = 7.3, 4.5 Hz, 1H), 4.60 (d, J = 7.6 Hz,

1H), 3.90 (d, J = 12.3 Hz, 1H), 3.81 (d, J = 12.3 Hz, 1H) ppm; ¹³C

NMR (CD₃CN, 151 MHz) d = 153.44, 153.41, 152.90, 150.37,

147.26, 147.00, 145.98, 145.68, 129.43, 119.10, 113.82, 113.78,

113.26, 90.33, 88.09, 86.33, 78.78, 75.55, 62.57 ppm; HRMS

37.34, 20.71 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

þ

C

₃₀H₃₁N₄O₁₂S₂703.1374, found 703.1374.

4.1.59. ((3S,4R,5R)-5-(2,6-Di(furan-2-yl)-9H-purin-9-yl)-3,4-

dihydroxytetrahydrofuran-2,2-diyl)bis(methylene)

dimethanesulfonate (83)

From compound 82, using the same procedure as for compound

17, compound 83 was obtained and puriﬁcation by ﬂash column

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

5667

(ESI-TOF) (m/z): [M+H]⁺calcd for C₁₉H₁₇N₄O₆397.1143, found

397.1145.

crude oil. Puriﬁcation by preparative-plate chromatography (C₁₈

silica, 5% MeOH/DCM) was completed to give the title compound

52 (10.5 mg, 0.0243 mmol, 36% over two steps). Compound 52:

þ

4.1.62. (3aR,5S,6R,6aR)-Methyl 2,2-dimethyl-6-(tosyloxy)tetra-

hydrofuro[2,3-d][1,3]dioxole-5-carboxylate (85)

yellow oil; R_f= 0.71 (C₁₈silica, 5% MeOH/DCM); ½a _D²⁵

= ꢁ30.4

ꢃ

(MeCN, c 0.90); FT-IR (ﬁlm) m_max3335, 3193, 2958, 1747, 1686,

Literature-known compound 84 (770 mg, 2.15 mmol) was dis-

solved in dry Et₂O (10 mL) and MeOH (10 mL) at 0 °C, and trimeth-

ylsilyldiazomethane (1.29 mL, 2.58 mmol, 2.0 M in hexanes) was

slowly added. The reaction solution was stirred at this temperature

for 30 min, then the solvent was evaporated. Puriﬁcation by ﬂash

column chromatography (silica, hexanes:EtOAc 3:1) was com-

pleted to give compound 85 (700 mg, 1.88 mmol, 88%). Compound

1644, 1528, 1420, 1333, 1282, 1208, 1180, 1159, 1110, 1044,

1031, 884, 794, 760, 711 cm^ꢁ1

;

¹H NMR (CD₃CN, 600 MHz)

d = 7.84 (s, 1H), 7.40 (s, 2H), 6.58–6.55 (m, 1H), 6.25 (s, 2H), 6.09

(d, J = 7.1 Hz, 1H), 5.45 (d, J = 8.1 Hz, 1H), 4.72 (t, J = 8.4 Hz, 1H),

3.95–3.91 (m, 1H), 3.82 (s, 3H) ppm; ¹³C NMR (CD₃CN, 600 MHz)

d = 170.47, 167.75, 155.10, 152.99, 152.74, 92.08, 87.27, 74.26,

66.18, 54.79, 53.42, 51.22, 20.73 ppm; HRMS (ESI-TOF) (m/z):

85: white foam; R_f= 0.67 (silica, hexanes:EtOAc 1:1); ½a _D²⁵

ꢃ

= ꢁ28.8

[M+H]⁺calcd for C₁₅H₁₆Cl₂N₆O₅431.0632, found 431.0630.

þ

(CHCl₃, c 0.82); FT-IR (ﬁlm) m_max2988, 1769, 1741, 1597, 1495,

1440, 1372, 1294, 1215, 1190, 1176, 1163, 1094, 1063, 1034,

4.1.65. (3aR,6aR)-Butyl 2,2-dimethyl-3a,6a-dihydrofuro[2,3-

d][1,3]dioxole-5-carboxylate (87)

964, 901, 866, 846, 816, 772, 740, 704, 665 cm^ꢁ^{1 1}H NMR (CDCl₃,

;

500 MHz) d = 7.76 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 6.07 (d,

J = 3.5 Hz, 1H), 5.13 (d, J = 3.3 Hz, 1H), 4.83 (d, J = 3.4 Hz, 1H), 4.77

(d, J = 3.5 Hz, 1H), 3.58 (s, 3H), 2.46 (s, 3H), 1.48 (s, 3H), 1.31 (s, 3H)

ppm; ¹³C NMR (CDCl₃, 126 MHz) d = 166.73, 145.95, 133.22,

130.35, 128.42, 113.57, 105.78, 83.11, 82.45, 78.46, 52.81, 27.16,

26.72, 22.13 ppm; HRMS (ESI-TOF) (m/z): [M+H]⁺calcd for

C₁₆H₂₁O₈S⁺373.0952, found 373.0957.

Literature-known compound 84 (470 mg, 1.31 mmol) and

anhydrous n-butanol (0.14 mL, 1.57 mmol) were dissolved in

CH₂Cl₂(11.8 mL), and DCC (325 mg, 1.57 mmol) and DMAP

(24 mg, 0.196 mmol) were added. The reaction solution was stir-

red at rt for 24 h. DBU (0.22 mL, 1.57 mmol) was added and the

solution stirred at rt for 24 h. The reaction solution was evapo-

rated and puriﬁcation by ﬂash column chromatography (silica,

hexanes:EtOAc 5:1) was completed to give compound 87

(200 mg, 0.878 mmol, 67%). Compound 87: white semi-solid;

4.1.63. (1R,3S,4S,5S,6S,7S)-Methyl 6,7-dichloro-3,4-dimethyl-

tetrahydrofuro[2,3-d][1,3]dioxole-2-oxabicyclo[3.2.0]heptane-

1-carboxylate (86)

R_f= 0.70 (silica, hexanes:EtOAc 2:1);

½

a ²_D⁵

ꢃ

= ꢁ17.7 (MeOH,

c

0.26); FT-IR (ﬁlm) m_max2993, 2957, 2935, 2873, 1722, 1639,

1627, 1575, 1458, 1397, 1381, 1371, 1332, 1320, 1305, 1256,

1248, 1234, 1213, 1156, 1127, 1082, 1039, 1009, 970, 897, 886,

Compound 85 (254 mg, 0.682 mmol) was dissolved in benzene

(6.8 mL), and DBU (0.12 mL, 0.818 mmol) was added. The reaction

solution was stirred at rt for 5 h. The mixture was worked up by

evaporation to half volume, then elution through a silica gel plug

with EtOAc. This residue was dissolved in MeCN (8 mL) under ar-

gon in a Quartz test tube tightly capped with a septum. 1,2-Cis-

dichloroethylene (0.51 mL, 6.82 mmol) was added and the reaction

degassed with argon for 20 min. The reaction solution was exposed

to UV light (Hanovia 400 W high-pressure Hg lamp) for 36 h. Addi-

tional 1,2-cis-dichloroethylene (0.3 mL, 4.0 mmol) was added and

the solution degassed for 20 min. The reaction solution was ex-

posed to UV light for an additional 48 h, then worked up by evap-

oration. Puriﬁcation by ﬂash column chromatography

(hexanes:EtOAc 5:1) was completed to give compound 86

(36 mg, 0.123 mmol, 18% over two steps) along with a mix of

two other diastereomers (27 mg, 0.0887 mmol, 13% over two

steps). Compound 86: yellow oil; R_f= 0.50 (silica, hexanes:EtOAc

852, 835, 805, 757, 668 cm^ꢁ1

;

¹H NMR (CDCl₃, 500 MHz)

d = 6.17 (d, J = 5.4 Hz, 1H), 6.06 (d, J = 2.5 Hz, 1H), 5.36 (dd,

J = 5.4, 2.5 Hz, 1H), 4.23 (t, J = 6.7 Hz, 2H), 1.68 (dd, J = 9.9,

5.2 Hz, 2H), 1.39 (dt, J = 14.8, 7.5 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H)

ppm; ¹³C NMR (CDCl₃, 126 MHz) d = 159.88, 150.48, 113.22,

110.31, 106.84, 82.93, 65.74, 30.60, 28.10, 27.84, 19.18,

13.79 ppm; HRMS (ESI-TOF) (m/z): [M+Na]⁺calcd for C₁₂H₁₈O₅Na⁺

265.1046, found 265.1034.

4.1.66. (1R,3S,4S,5S,6S,7S)-Methyl 6,7-dichloro-3,4-dimethyl-

tetrahydrofuro[2,3-d][1,3]dioxole-2-oxabicyclo[3.2.0]heptane-

1-carboxylate (88)

Compound 87 (200 mg, 0.826 mmol) was dissolved in MeCN

(16 mL) under argon in a Quartz test tube tightly capped with a

septum. 1,2-Cis-dichloroethylene (0.86 mL, 8.86 mmol) was added

and the reaction solution degassed with argon for 30 min. The

mixture was exposed to UV light (Hanovia 400 W high-pressure

Hg lamp) for 108 h. Additional 1,2-cis-dichloroethylene (0.50 mL,

5.14 mmol) was added and the solution degassed for 30 min.

The reaction mixture was exposed to UV light for an additional

48 h and then worked up by evaporation. Puriﬁcation by ﬂash

column chromatography (silica, hexanes:EtOAc 20:1) was com-

pleted to give compound 76 (59 mg, 0.174 mmol, 21%) along with

two other diastereomers (76 mg, 0.224 mmol, 27%) and recovered

starting material 87 (23 mg, 0.0949 mmol, 12%). Compound 88:

4:1); ½a ²_D⁵

ꢃ

= +3.0 (CHCl₃, c 2.70); FT-IR (ﬁlm) m_max2992, 1738,

1439, 1375, 1337, 1211, 1161, 1124, 1053, 990, 934, 899, 783,

708 cm^ꢁ^{1 1}H NMR (CDCl₃, 500 MHz) d = 6.08 (d, J = 3.8 Hz, 1H),

;

5.08 (d, J = 3.8 Hz, 1H), 4.50 (dd, J = 9.5, 6.4 Hz, 1H), 4.17 (d,

J = 6.6 Hz, 1H), 3.87 (d, J = 0.7 Hz, 3H), 3.79 (d, J = 9.7 Hz, 1H),

1.41 (d, J = 15.5 Hz, 6H) ppm; ¹³C NMR (CDCl₃, 151 MHz)

d = 167.03, 114.98, 109.11, 88.90, 81.99, 65.55, 54.11, 53.04,

50.48, 27.93, 27.34 ppm; HRMS (ESI-TOF) (m/z): [M+Na]⁺calcd

for C₁₁H₁₄Cl₂O₅Na⁺319.0110, found 319.0103.

4.1.64. (1R,3R,4R,5R,6S,7S)-Methyl 4-acetoxy-6,7-dichloro-3-

(2,6-diamino-9H-purin-9-yl)-2-oxabicyclo[3.2.0]heptane-1-

carboxylate (52)

yellow oil; R_f= 0.75 (silica, hexanes:EtOAc 4:1);

(CHCl₃, c 1.00); FT-IR (ﬁlm) m_max2961, 2937, 2875, 1757, 1731,

½

a ²_D⁵

= +16.0

ꢃ

1459, 1384, 1374, 1325, 1270, 1244, 1190, 1160, 1122, 1052,

Compound 86 (20 mg, 0.0675 mmol) was dissolved in acetic

acid (0.72 mL) and acetic anhydride (0.08 mL, 0.810 mmol). Two

drops of sulfuric acid were added and the reaction solution stirred

at rt for 16 h. The reaction solution was poured into ice water

(4 mL) and stirred for 30 min. Satd aq NH₄Cl (4 mL) was added

and the aqueous phase extracted with 5% MeOH/DCM. The com-

bined organics were dried over MgSO₄, ﬁltered, and concentrated

to give a residue. This crude material was exposed to the same pro-

cedure as for compound 57, and compound 52 was obtained as a

987, 933, 899, 852, 782, 746, 707 cm^ꢁ^{1 1}H NMR (CDCl₃,

;

600 MHz) d = 6.08 (d, J = 3.8 Hz, 1H), 5.08 (d, J = 3.8 Hz, 1H), 4.49

(dd, J = 9.7, 6.8 Hz, 1H), 4.31–4.24 (m, 2H), 4.16 (dd, J = 6.8,

1.2 Hz, 1H), 3.77 (d, J = 9.7 Hz, 1H), 1.69 (dq, J = 13.6, 6.7 Hz,

2H), 1.43–1.38 (m, 8H), 0.93 (t, J = 7.4 Hz, 3H) ppm; ¹³C NMR

(CDCl₃, 151 MHz) d = 166.61, 114.90, 109.03, 88.68, 81.95, 66.21,

65.58, 54.09, 50.56, 30.62, 27.90, 27.37, 19.14, 13.77 ppm; HRMS

(ESI-TOF) (m/z): [M+H]⁺calcd for C₁₄H₂₁Cl₂O₅339.0760, found

þ

339.0768.

5668

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

4.1.67. (1R,3R,4R,5R,6S,7S)-Methyl 6,7-dichloro-3-(2,6-diamino-

9H-purin-9-yl)-4-hydroxy-2-oxabicyclo[3.2.0]heptane-1-

carboxylate (53)

samples for an additional 48 h. The degree of virus neutralization by

each sample was achieved by measuring luciferase activity. The wells

were aspirated and washed once with PBS, and 60

culture lysis reagent (Promega, Madison, WI) was added. The lysate is

mixed by pipetting, and 50 L was transferred to a round-bottom

plate (Corning), and the plate is centrifuged at 1800g for 10 min at

4 °C. Then 20 L was transferred to an opaque assay plate (Corning,

lL of luciferase cell

Method 1: Compound 88 (4.3 mg, 0.00986 mmol) was dissolved

in MeOH (0.47 mL), and K₂CO₃(0.40 mg, 0.00296 mmol) was

added. The reaction was stirred at rt for 30 min. The reaction mix-

ture was quenched with one drop AcOH and puriﬁcation by pre-

parative-plate chromatography (C₁₈silica, 5% MeOH/DCM) was

completed to give compound 53 (3.2 mg, 0.00818 mmol, 83%).

Method 2: From compound 88, using the same procedures as for

compound 52, a crude residue was obtained. This residue was then

dissolved in MeOH (0.3 mL), and K₂CO₃(0.26 mg, 0.00190 mmol)

was added. The reaction mixture was stirred at rt for 1 h. The mix-

ture was quenched with one drop AcOH and puriﬁcation by pre-

parative-plate chromatography (C₁₈silica, 5% MeOH/DCM) was

completed to give compound 53 (2.2 mg, 0.00564 mmol, 38% over

three steps). Compound 53: white powder; R_f= 0.10 (silica, 5%

l

Corning, NY), and the luciferase activity was measured on a lumino-

meter (EG&G Berthold LB 96 V; Perkin Elmer, Gaithersburg, MD) by

using luciferase assay reagent (Promega, Madison, WI).

Acknowledgments

We thank Dr. D. H. Huang and Dr. L. Pasternack for NMR spec-

troscopic assistance and Dr. G. Siuzdak for mass spectrometric

assistance. Financial support for this work was provided by the

National Institutes of Health (USA) and the Skaggs Institute for

research, along with fellowships from Novartis (to S.P.E.) and

UCSD/SDSU IRACDA (to F.R.).

MeOH/DCM);

3333, 2925, 1742, 1688, 1643, 1528, 1414, 1331, 1281, 1208,

1157, 1100, 1031, 959, 888, 791, 707 cm^ꢁ^{1 1}H NMR (CD₃CN,

½

a ²_D⁵

ꢃ

= ꢁ17.7 (MeOH, c 0.26); FT-IR (ﬁlm) m_max

;

600 MHz) d = 7.93 (s, 1H), 7.30 (s, 2H), 6.32 (s, 2H), 5.89 (d,

J = 7.8 Hz, 1H), 5.43 (t, J = 7.3 Hz, 1H), 5.20 (dd, J = 8.0, 1.0 Hz,

1H), 4.72–4.68 (m, 1H), 4.21 (s, 1H), 3.81 (s, 3H), 3.66 (ddd,

J = 6.8, 5.0, 1.1 Hz, 1H) ppm; ¹³C NMR (CD₃CN, 151 MHz)

d = 168.07, 154.25, 153.11, 152.15, 141.45, 125.82, 93.25, 86.46,

74.31, 66.88, 55.22, 53.30, 52.41 ppm; HRMS (ESI-TOF) (m/z):

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.bmc.2011.07.022.

[M+H]⁺calcd for C₁₃H₁₅Cl₂N₆O₄389.0526, found 389.0534.

þ

References and notes

1. Zamecnik, P. C.; Stephenson, M. L. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 280.

2. Reviews: (a) Petersen, M.; Wengel, J. Trends Biotechnol. 2003, 21, 74; (b)

Kurreck, J. Eur. J. Biochem. 2003, 270, 1628; (c) Kaur, H.; Babu, B. R.; Maiti, S.

Chem. Rev. 2007, 107, 4672; (d) Rahman, S. M. A.; Imanishi, T.; Obika, S. Chem.

Lett. 2009, 38, 512; (e) D’Alonzo, D.; Guaragna, A.; Palumbo, G. Chem. Biodivers.

2011, 8, 373.

4.2. Biological assays

4.2.1. Bacterial minimum inhibitory concentration (MIC) assays

Minimum inhibitory concentrations (MIC) were determined by

CLSI micro-broth dilution guidelines.³³Brieﬂy, compounds were

3. (a) Altona, C.; Sunderalingam, M. J. Am. Chem. Soc. 1972, 94, 8205; (b) Altona, C.;

Sunderalingam, M. J. Am. Chem. Soc. 1973, 95, 2333.

serially diluted in 96-well plates containing 100 lL of cation-ad-

4. (a) Koshkin, A. A.; Singk, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.;

Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607; (b) Koshkin,

A. A.; Rajwanshi, V. K.; Wengel, J. Tetrahedron Lett. 1998, 39, 4381; (c)

Rajwanshi, V. K.; Håkansson, A. E.; Sørensen, M. D.; Pitsch, S.; Singh, S. K.;

Kumar, R.; Nielsen, P.; Wengel, J. Angew. Chem., Int. Ed. 2000, 39, 1656; (d)

Obika, S.; Uneda, T.; Sugimoto, T.; Nanbu, D.; Minami, T.; Doi, T.; Imanishi, T.

Bioorg. Med. Chem. 2001, 9, 1001; (e) Koshkin, A. A.; Fensholdt, J.; Pfundheller,

H. M.; Lomholt, C. J. Org. Chem. 2001, 66, 8504.

5. (a) Obika, S.; Morio, K.-i.; Nanbu, D.; Imanishi, T. Chem. Commun. 1997, 1643;

(b) Obika, S.; Morio, K.-i.; Hari, Y.; Imanishi, T. Chem. Commun. 1999, 2423; (c)

Obika, S.; Morio, K.-i.; Nanbu, D.; Hari, Y.; Itoh, H.; Imanishi, T. Tetrahedron

2002, 58, 3039.

justed Mueller-Hinton broth (MHB) per well. E. coli MG1655 and

S. aureus 8325 were inoculated into MHB from a fresh overnight

plate scrape on Mueller-Hinton agar (MHA) plates to a ﬁnal con-

centration of 1 ꢂ 10⁷colony forming units ml^ꢁ1, and 5

lL of this

suspension was added to each well. The plates were incubated at

37 °C and the MIC determined after 24 h of growth. MICs were de-

ﬁned as the lowest concentration of the compound that yielded no

observable growth, and reported values were the median value of

at least three independent replicates with an error of no greater

than two-fold.

6. Rahman, S. M. A.; Seki, S.; Obika, S.; Yoshikawa, H.; Miyashita, K.; Imanishi, T. J.

Am. Chem. Soc. 2008, 130, 4886.

7. (a) Umemoto, T.; Wengel, J.; Madsen, A. S. Org. Biomol. Chem. 2009, 7, 1793; (b)

Astakhova, I. V.; Lindegaard, D.; Korshun, V. A.; Wengel, J. Chem. Commun. 2010,

46, 8362.

8. Liu, Y.; Xu, J.; Karimiahmadabadi, M.; Zhou, C.; Chattopadhyaya, J. J. Org. Chem.

2010, 75, 7112.

9. Seth, P. P.; Allerson, C. R.; Berdeja, A.; Siwkowski, A.; Pallan, P. S.; Gaus, H.;

Prakash, T. P.; Watt, A. T.; Egli, M.; Swayze, E. E. J. Am. Chem. Soc. 2010, 132,

14942.

10. Review: Mathé, C.; Périgaud, C. Eur. J. Org. Chem. 2008, 1489.

11. (a) Webb, T. R.; Mitsuya, H.; Broder, S. J. Med. Chem. 1988, 31, 1475; (b) Okabe,

M.; Sun, R.-C. Tetrahedron Lett. 1989, 30, 2203; (c) Beard, A. R.; Butler, P. I.;

Mann, J.; Partlett, N. K. Carbohydr. Res. 1990, 205, 87; (d) Rodriguez, J. B.;

Marquez, V. E.; Nicklaus, M. C.; Mitsuya, H.; Barchi, J. J., Jr. J. Med. Chem. 1994,

37, 3389; (e) Park, A.-Y.; Moon, H. R.; Kim, K. R.; Chun, M. W.; Jeong, L. S. Org.

Biomol. Chem. 2006, 4, 4065; (f) Moon, H. R.; Park, A.-Y.; Kim, K. R.; Chun, M.

W.; Jeong, L. S. Nucleosides, Nucleotides Nucleic Acids 2007, 26, 1653.

12. (a) Tchilibon, S.; Joshi, B. V.; Kim, S.-K.; Duong, H. T.; Gao, Z.-G.; Jacobson, K. A. J.

Med. Chem. 2005, 48, 1745; (b) Jacobson, K. A.; Joshi, B. V.; Tchilibon, S.

International Patent WO 031505 A1, 2006; (c) Tosh, D. K.; Chinn, M.; Ivanov, A.

A.; Klutz, A. M.; Gao, Z.-G.; Jacobson, K. A. J. Med. Chem. 2009, 52, 7580.

13. Eoff, R. L.; McGrath, C. E.; Maddukuri, L.; Salamanca-Pinzón, S. G.; Marquez, V.

E.; Marnett, L. J.; Guengerich, F. P.; Egli, M. Angew. Chem., Int. Ed. 2010, 49, 1.

14. O-Yang, C.; Kurz, W.; Eugui, E. M.; McRoberts, M. J.; Verheyden, J. P. H.; Kurz, L.

J.; Walker, K. A. M. Tetrahedron Lett. 1992, 33, 41.

4.2.2. Cytotoxicity assays

Raji and CEM cells were incubated for 72 h in 96-well plates

with the test compounds. Cell proliferation by reduction of the yel-

low dye MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-

zolium bromide] to a blue formazan product was used to test

viability. The amount of formazan dye formed is a direct indication

of the number of metabolically active cells in the culture. The opti-

cal density of the blue formazan product was measured at 570 nm

with an Inﬁnite M200 (Tecan, Switzerland) and analyzed using

one-way ANOVA using GraphPad Prism version 5.0b for OSX

(GraphPad Software, San Diego, CA).

4.2.3. Pseudovirus neutralization assays

Pseudoviruses were generated in 293T cells and neutralization

with single-round infectious pseudovirus was performed using

TZM-bl cells as targets for infection as described previously.³⁴

Brieﬂy, pseudovirus was titrated on TZM-bl cells and a predeter-

mined amount of virus that produces ꢄ1 ꢂ 10⁶RLUs was incubated

for 1 h at 37 °C with serially diluted samples and controls. TZM-bl

cells were resuspended in media, washed, counted and plated at

1 ꢂ 10⁵cells per well over the incubated solution of virus and

15. Kim, M. J.; Kim, H. O.; Kim, H.-D.; Kim, J. H.; Jeong, L. S.; Chun, M. W. Bioorg.

Med. Chem. Lett. 2003, 13, 3499.

16. Díaz-Rodríguez, A.; Sanghvi, Y. S.; Fernández, S.; Schinazi, R. F.; Theodorakis, E.

A.; Ferrero, M.; Gotor, V. Org. Biomol. Chem. 2009, 7, 1415.

K. C. Nicolaou et al. / Bioorg. Med. Chem. 19 (2011) 5648–5669

17. (a) Ravn, J.; Qvortrup, K.; Rosenbohm, C.; Koch, T. Bioorg. Med. Chem. 2007, 15, 26. Langli, G.; Gundersen, L.-L.; Rise, F. Tetrahedron 1996, 52, 5625.

5669

5440; (b) Ravn, J.; Rosenbohm, C.; Qvortrup, K.; Koch, T. International Patent

WO 006369 A1, 2008.

18. (a) Olsen, A. G.; Nielsen, C.; Wengel, J. J. Chem. Soc., Perkin Trans. 1 2001, 900;

(b) Bryld, T.; Sørensen, M. H.; Nielsen, P.; Koch, T.; Nielsen, C.; Wengel, J. J.

Chem. Soc., Perkin Trans. 1 2002, 1655; (c) Nair, V.; Jeon, G.-S. ARKIVOC 2004,

133.

19. (a) Chun, M. W.; Kim, M. J.; Kim, H. O.; Kim, H.-D.; Kim, J. H.; Moon, H. R.; Jeong,

L. S. Nucleosides, Nucleotides Nucleic Acids 2003, 22, 915; (b) Hrdlicka, P. J.;

Jepsen, J. S.; Wengel, J. Nucleosides, Nucleotides Nucleic Acids 2005, 24, 397.

20. (a) Fleet, G. W. J.; Ramsden, N. G.; Dwek, R. A.; Rademacher, T. W.; Fellows, L. E.;

Nash, R. J.; Green, St. C. D.; Winchester, B. Chem. Commun. 1988, 483; (b)

Yoshikawa, M.; Okaichi, Y.; Cha, B. C.; Kitagawa, I. Tetrahedron 1990, 46, 7459.

21. Youssefyeh, R. D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem. 1979, 44, 1301.

22. Niedballa, U.; Vorbrüggen, H. Angew. Chem., Int. Ed. Engl. 1970, 9, 461.

23. Vorbrüggen, H.; Höﬂe, G. Chem. Ber. 1981, 114, 1256.

27. Thornton, A. R.; Martin, V. I.; Blakey, S. B. J. Am. Chem. Soc. 2009, 131, 2434.

28. Bookser, B. C.; Raffaele, N. B. J. Org. Chem. 2007, 72, 173.

29. (a) Timoshchuk, V. A. Carbohydr. Res. 1985, 145, 131; (b) Mezzina, E.; Savoia, D.;

Tagliavini, E.; Trombini, C.; Umani-Ronchi, A. J. Chem. Soc., Perkin Trans. 1 1989,

845.

30. Wilson, P. K.; Szabados, E.; Mulligan, S. P.; Christopherson, R. I. Int. J. Biochem.

Cell Biol. 1998, 30, 833.

31. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J.Org. Chem. 1997, 62, 7512.

32. (a) Koshkin, A. A. J. Org. Chem. 2004, 69, 3711; Ramsing, N. B.; Nielsen, A. T.;

Koshkin, E. A.; Tolstrup, N.; Pfundheller, H. M.; Lomholdt, C. U.S. Patent

0,147,924, A1, 2006.; (c) Pasternack, A.; Kierzek, E.; Pasternack, K.; Turner, D.

H.; Kierzek, R. Nucleic Acids Res. 2007, 35, 4055.

33. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial

Susceptibility Tests for Bacteria that Grow Aerobically. CLSI, Wayne, PA, USA,

2006.

24. Hirota, K.; Kazaoka, K.; Sajiki, H. Bioorg. Med. Chem. 2003, 11, 2715.

25. Geng, B.; Breault, G.; Comita-Prevoir, J.; Petrichko, R.; Eyerman, C.; Lundqvist,

T.; Doig, P.; Gorseth, E.; Noonan, B. Bioorg. Med. Chem. Lett. 2008, 18, 4368.

34. Zwick, M. B.; Wang, M.; Poignard, P.; Stiegler, G.; Katinger, H.; Burton, D. R.;

Parren, P. W. J. Virol. 2001, 75, 12198.

Article Doi

Synthesis and biological evaluation of 2′,4′- and 3′,4′-bridged nucleoside analogues

DOI: 10.1016/j.bmc.2011.07.022

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2011.07.022

Full text of DOI:10.1016/j.bmc.2011.07.022

Products guided by the article

R&D Labs maybe for 1341075-14-6

Relevant to this article

Hot Product