Stereoselective synthesis of D - And L -carbocyclic nucleosides by enzymatically catalyzed kinetic resolution

FULL PAPER

DOI: 10.1002/chem.201200733

Stereoselective Synthesis of d- and l-Carbocyclic Nucleosides by

Enzymatically Catalyzed Kinetic Resolution

Miriam Mahler,^[a]Bastian Reichardt,^[a]Philip Hartjen,^[b]Jan van Lunzen,^[b]and

Chris Meier*^[a]

Abstract: An efficient synthesis of (S)-

or (R)-3-(benzyloxy-methyl)-cyclopent-

3-enol was developed by appling an

syntheses of carbocyclic d- and l-nu-

cleoside analogues were readily acces-

sible. 3’,4’-Unsaturated d- or l-carbocy-

clic nucleosides were obtained from

the condensation of various nucleo-

bases with (S)- or (R)-cyclopentenol.

Functionalization of the double bond

in 3’-deoxy-3’,4’-didehydro-carba-d-thy-

midine led to a variety of new nucleo-

side analogues. By using the cycloSal

approach, their corresponding phos-

phorylated metabolites were readily ac-

cessable. Moreover, a new synthetic

route to carbocyclic 2’-deoxy-nucleo-

sides was developed, thereby leading to

d- and l-carba-dT. d-Carba-dT was

tested for antiviral activity against

multidrug-resistance HIV-1 strain E2-2

and compared to the known antiviral

agent d4T, as well as l-carba-dT.

Whilst l-carba-dT was found to be in-

active, its d-analogue showed remarka-

bly high activity against the resistant

virus and significantly better than that

of d4T. However, against the wild-type

virus strain NL4/3, d4T was found to be

more-active than d-carba-dT.

enzyme-catalyzed

kinetic-resolution

approach. This procedure allowed the

syntheses of the enantiomeric building

blocks (S)- and (R)-cyclopentenol with

high optical purity (>98% ee). In con-

trast to previous approaches, the key

advantage of this procedure is that the

resolution is done on the level of enan-

tiomers that only contain one stereo-

genic center. Owing to this feature, it

was possible to chemically convert the

enantiomers into each other. By using

this route, the starting materials for the

Keywords: antiviral agents · carbo-

cycles · enzyme catalysis · kinetic

resolution · nucleotides

Introduction

Over the last few decades, carbocyclic d- and l-nucleosides

have attracted much interest owing to their antiviral activity

against a large number of viruses.^[1]Carbocyclic nucleoside

analogues, such as entecavir (1), d-[(E)-5-(2-bromovinyl)]-

2’-deoxy-carba-uridine (carba-BVdU, 2), and carba-d-thymi-

dine (carba-dT, 3), exhibit interesting biological activity

(Figure 1). Purine analogue entecavir has been shown to be

an anti-HBV agent, but it also possesses activity against

HIV, HSV-1, VZV, and influenza.^[2]Morover, it was ap-

proved for the therapy of chronic HBV infections by the

FDA in 2005. Carba-BVdU is antivirally active against HSV

and VZV.^[3]Carba-dT (3) has shown high in vitro activity

against several different viruses, such as HIV-1, HIV-2,

HSV-1, and VV.^[4]Furthermore, it blocks DNA synthesis by

a unique mechanism that is termed kinetic- or delayed chain

Figure 1. Examples of antivirally active carbocyclic nucleosides.

termination, which makes carba-dT a very promising lead

compound for the development of antiviral agents.^[5]

The advantages of carbocyclic compounds compared to

their natural counterparts include their higher metabolic

and chemical stability owing to the replaced oxygen atom.^[6]

In addition, the replacement of the oxygen atom by a meth-

ylene group often leads to lower cytotoxicity and increased

bioavailability.^[7]In many cases, carbocyclic nucleosides

show revised biological properties owing to the conforma-

tionally changed cyclopentane ring compared to their

normal nucleoside analogues.^[1a,c,7,8]

[a] Dipl. Chem. M. Mahler, Dr. B. Reichardt, Prof. Dr. C. Meier

Organic Chemistry, Department of Chemistry

The most challenging obstacle to the development of new

antivirally active carbocyclic agents is the synthetic ap-

proach. Usually, it is necessary to perform a multistep syn-

thesis to introduce the required stereoinformation. There

are many synthetic approaches to carbocyclic nucleosides

and they can be divided into two groups:^[1]The first group is

the “linear synthetic strategy”, in which the nucleoside is

Faculty of Sciences, University of Hamburg

Martin-Luther-King-Platz 6, 20146 Hamburg (Germany)

E-mail: chris.meier@chemie.uni-hamburg.de

[b] Dr. P. Hartjen, Prof. Dr. J. van Lunzen

Infectious Diseases Unit, Department of Medicine

University Medical Center Hamburg-Eppendorf

and Heinrich Pette Institute

Leibniz Institute for experimental Virology, Hamburg (Germany)

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prepared by the initial functionalization of the cyclopentane

moiety followed by stepwise construction of the nucleobase.

The second approach is the “convergent synthesis”, which

offers the formation of a variety of nucleoside analogues by

the direct coupling of a cyclopentanol derivative and differ-

ent heterocyclic bases.

Herein, we report a stereoselective, chemoenzymatic ki-

netic resolution of racemic cyclopentenol 8. Based on this

reaction, enantiomerically pure key intermediates (R)-8 or

(S)-8 were obtained and used for the preparation of carbo-

cyclic d- or l-3’-deoxy-3’,4’-didehydronucleosides and -nu-

cleotides. Furthermore, a new route to carbocyclic 2’-deoxy-

nucleosides, such as carba-dT, is presented; the antiviral ac-

tivities of these compounds were studied against a multi-

drug-resistant HIV strain.

Biggadike et al. reported the synthesis of (1S,2R)-2-(ben-

zyloxymethyl)-cyclopent-3-enol (4) starting from cyclopenta-

diene (5), which was first deprotonated with NaH and then

alkylated with benzylchloromethylether (BOMCl).^[9]The cy-

clopentadiene derivative was stereoselectively hydroborated

by using (ꢀ)-diisopinocampheylborane to give (1S,2R)-2-

(benzyloxymethyl)-cyclopent-3-enol. In this reaction, two

new stereocenters were created with excellent stereoselec-

tivity. However, the carbocyclic precursor was isolated in

low chemical yield and the introduction of two new stereo-

centers in this molecule at the same time did not allow a gen-

eral access to carbocyclic d- and l-nucleosides. We devel-

oped a convergent synthetic strategy towards carbocyclic 2’-

deoxynucleoside analogues with improved chemical yields

by using compound 4; in addition, we showed that symmet-

ric cyclopentadiene intermediate 6 isomerized into two ther-

modynamically stable cyclopentadienes (7a and 7b) above

08C.^[10]Subsequently, hydroboration of these two unsymmet-

rical dienes was achieved by using BH₃·THF. After the hy-

droboration and alkaline-oxidation steps, only one product,

racemic 3-(benzyloxymethyl)-cyclopent-3-enol (8), was

formed in an overall yield of 59% starting from cyclopenta-

diene (Scheme 1).

Results and Discussion

Kinetic resolution: Our previously reported method, which

used rac-3-(benzyloxymethyl)-cyclopent-3-enol (8), only led

to racemic carbocyclic nucleosides.^[11]Different approaches

were tested for preparing cyclopentenol 8 and their corre-

sponding nucleoside analogues in their optically pure form:

The first approach was to convert cyclopentenol 8 into

a b,g-ketone by Swern oxidation and then preparing the

enantiomerically pure product by stereoselective reduction.

Several reducing reagents, such as the CBS-catalyst, (R)-

BINAL-H, and diisopinocampheylchloroborane, were

tested, but none of them led to the desired enantiomeric cy-

clopentenol (8). Another possibility to obtain chiral alcohol

8 was the stereoselective hydroboration of the two cyclopen-

tadienes (7a and 7b). Diisopinocampheylborane and dilon-

gifolylborane, which were prepared from a-pinene and long-

ifolene, respectively, were used as stereoselective hydro-

borating reagents.^[13]Unfortunately, hydroboration only gave

the required product with an enantiomeric excess of ꢁ81%

and 33% chemical yield. Next, cyclopentenol 8 was convert-

ed into diastereomers by reacting with different chiral acids.

We tried to separate the diastereomers by column chroma-

tography on silica gel. Although esterification reactions with

(S)-mandelic acid, (R)-(ꢀ)-methoxyphenylacetic acid, and

(S)-(ꢀ)-camphanic acid chloride were successful, the separa-

tion of the diastereomers on the preparative scale failed in

all three cases.

One further option to achieve the separation of the enan-

tiomers of compound 8 was to perform a kinetic resolution.

In this reaction, the two enantiomers show different rates of

reaction in the presence of a chiral entity, such as a chiral

catalyst or a biocatalyst.^[14]In contrast to normal asymmetric

reactions, the theoretical maximum yield for kinetic resolu-

tions is 50%. The parameter that describes the efficiency of

an enzyme-catalyzed kinetic resolution is the E value; for

a good kinetic resolution, the value should exceed 30.^[15]

Finally, we found that an enzyme-catalyzed acylation reac-

tion by using vinyl acetate and triethylamine in the presence

of porcine pancreatin was successful. This approach was mo-

tivated by a previously reported desymmetrization reaction

that led to a different cyclopentene building block for the

syntheses of spinosyn A analogues.^[16]However, with our

substrate, rac-3-(benzyloxymethyl)-cyclopent-3-enol (8), the

rate of the esterification reaction was very low, although the

enantiomeric excess of the formed product was high (95%).

Scheme 1. a) NaH, BOMCl, THF; b) (ꢀ)-diisopinocampheylborane ((ꢀ)-

(ipc)₂BH), THF, ꢀ608C; c) H₂O₂/NaOH 1:1; d)>08C; e) BH₃·THF, THF.

Furthermore, we demonstrated that racemic cyclopente-

nol 8 is a good building block for the convergent synthesis

of many different racemic carbocyclic d-nucleoside ana-

logues by using a modified Mitsunobu reaction.^[11]However,

again, this synthetic route is not generally applicable for pre-

paring either carbocyclic d- or l-nucleosides.

The preparation of enantiomerically pure compounds is

still challenging. There are various routes for the enantiose-

lective synthesis of new carbocyclic nucleosides, such as the

use of natural chiral molecules as starting material or an

asymmetric synthetic strategy for the syntheses of optically

pure building blocks. In particular, chemoenzymatic meth-

ods have become quite popular in organic synthesis over the

past few decades.^[12]

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Synthesis of d- and l-Carbocyclic Nucleosides

FULL PAPER

The enantiomeric excesses of the acetylated compound

((R)-9) and the residual alcoholic enantiomer ((S)-8) were

determined by HPLC by using a CHIRALPAK OD column.

The conversion was calculated from the enantiomeric ex-

cesses of the substrate and the product.^[17]

This promising stereoselectivity led us to optimize the

procedure to improve the enantiomeric excess, reaction rate,

conversion, and the E value.

ture when 1 g of cyclopentenol rac-8 was used. Then, 1, 2, 3,

and 5 equivalents of pancreatin were tested. We found that

higher amounts of pancreatin not only led to higher reaction

rates, but also to a higher selectivity of the reaction.

For studying the temperature dependence of pancreatin,

the kinetic resolution was performed at different tempera-

tures, starting at room temperature and rising in 108C steps

up to 608C (Table 2).

First, eight different solvents, that is, acetone, Et₂O, THF,

CH₂Cl₂, 1,4-dioxane, toluene, n-hexane, and cyclohexane,

were used for the kinetic resolution with pancreatin. Ace-

tone, THF, n-hexane, and toluene were the best solvents for

the esterification reaction with pancreatin. This result corre-

lated well with the study by Secundo et al. regarding the

effect on the enantioselectivity of pancreatin in organic sol-

vents.^[18]In acetone, the acetylated compound, (R)-9, was

obtained with the highest enantiomeric excess of 95%, but

it took 3 days to achieve 38% conversion with 3 mass equiv-

alents of pancreatin annualized to the substrate (8). The

enantioselectivity (E value) of the kinetic resolution in ace-

tone was quite high (E=69, Table 1). The fastest conver-

Table 2. Temperature dependence of pancreatin in the kinetic resolution

with rac-8.^[a]

T

[8C]

t

Conversion

[%]

ee (R)-9

[%]

ee (S)-8

[%]

E

[h]

RT

30

40

50

60

28

20

24

28

31

26

27

96

97

96

95

94

43

44

33

34

75

101

76

54

45

[a] Reagents and conditions: rac-8 (100 mg), pancreatin (300 mg), Et₃N

(2 equiv), vinyl acetate (2 equiv), solvent (4 mL).

The optimum temperature of pancreatin as described in

the literature is between 308C and 408C.^[18,19]In our experi-

ments, we observed that the conversion increased at higher

temperature. However, after a couple of hours, the enzyme

activity and the enantioselectivity of the reactions decreased

at 508C and 608C, as shown by smaller enantiomeric excess-

es and slower reaction progress. As expected, the optimal

temperature for pancreatin in this reaction was between

308C and 408C.

Table 1. Kinetic resolution of rac-8 with pancreatin in different sol-

vents.^[a]

Solvent

t

Conversion

[%]

ee (R)-9

[%]

ee (S)-8

[%]

E

[h]

acetone

THF

1,4-dioxane

n-hexane

toluene

67

48

47

3

38

36

39

40

95

92

94

93

91

57

56

52

59

61

69

42

54

59

51

40

cyclohexane

Furthermore, different enzymes, especially lipases, were

investigated in the esterification reaction. As mentioned

above, porcine pancreatin is a mixture of different enzymes.

To determine which enzyme was responsible for the cataly-

sis and to examine if the pure enzyme was a better biocata-

lyst than porcine pancreatin, different isolated lipases and

esterases were used for the kinetic resolution of racemic 3-

(benzyloxymethyl)-cyclopent-3-enol (8). All of the chemo-

enzymatic reactions were tested by using 2 equivalents of

vinyl acetate and triethylamine in acetone. For comparing

the influence of the isolated enzymes on the reaction, the

amount of enzyme was calculated from the specific activity

of the respective enzyme, so that, in each reaction,

50 enzyme units were used. The results for the kinetic reso-

lution with the isolated esterases were found to be really

poor. Even after a couple of days, almost no conversion was

observed; thus, no further investigation was done for the es-

terases. However, the results for the lipases looked to be

quite promising. Most of the lipases did work in the kinetic

resolution in acetone, but, in general, the results were not as

good as for pancreatin. The best results were achieved for

the reactions with the enzymes PPL (porcine pancreas

lipase), HP (hog pancreas lipase), PFL (pseudomonas fluo-

rescens lipase), CAL (candida antarctica lipase), PCC (candi-

da cylindraca lipase), and PCL (pseudomonas cepacia lipase;

Table 3).

[a] Reagents and conditions: rac-8 (50 mg), pancreatin (150 mg), Et₃N

(2 equiv), vinyl acetate (2 equiv), solvent (2 mL), RT.

sions were observed in highly nonpolar solvents: Almost

40% of the substrate was converted into the product within

3 h and the decrease in enantiomeric excess was insignifi-

cant. After 3 h, the ee value was 94% for the reaction in n-

hexane with 39% conversion (Table 1). The enantioselectivi-

ty of the kinetic resolution was still good (E=59). The re-

sults of the enzymatic syntheses with pancreatin in different

solvents are summarized in Table 1.

Besides the solvent, the number of equivalents of the re-

agents (vinyl acetate and triethylamine) was varied. In these

experiments, up to 5 equivalents of these components were

tested. The best reaction conditions were achieved with

2 equivalents of both reagents. Less then 2 equivalents of

the acyl donor and triethylamine gave lower conversions

and reaction rates, whilst using more than 2 equivalents led

to a decrease in the enantioselectivity of the acetylated com-

pound ((R)-9).

In addition to these studies, the effect of the amount of

porcine pancreatin on the reaction was examined. Owing to

the fact that pancreatin is a mixture of different enzymes,

such as lipases, amylases, and proteases, we defined that

1 equivalent of pancreatin correlated to 1 g of enzyme mix-

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Table 3. Kinetic resolution of rac-8 with different lipases.^[a]

Enzyme Amount of Conversion ee (R)-9 ee (S)-8

enzyme [mg] [h] [%]

t

E

[%]

pancreatin 150

92 31

92 20

92 13

94

80

72

37

70

42

23

14

4

10

56

33

3

49

41

37

34

36

16

8

PPL

HP

PFL

CAL

PCC

PCL

150

100

0.5

3.3

1.3

33.3

25

92

4

73 10

73 41

73 31

73

8

2

8

1

73 37

41

[a] Reagents and conditions: rac-8 (15 mg), Et₃N (2 equiv), vinyl acetate

(2 equiv), acetone (0.6 mL), RT.

In the reactions with the last four lipases, the enantiomer-

ic excesses of the product were not as high as for the resolu-

tions with pancreatin, PPL, and HP. Because of this result,

no further optimization was done for these enzymes. Be-

cause PPL was the cheapest enzyme and the selectivity of

the chemoenzymatic reaction looked promising, further op-

timization was performed. By increasing the amount of

PPL, it was possible to increase the conversion and reaction

rate of the kinetic resolution.

Scheme 2. a) Pancreatin, Et₃N, vinyl acetate, acetone, 23 h; b) NaOH,

MeOH; c) pancreatin, Et₃N, vinyl acetate, acetone, 6 h; d) DIAD, PPh₃,

benzoic acid, Et₂O.

and the remaining (S)-alcohol (8) could be easily separated

by silica gel column chromatography.

In addition, the effect of the solvent on the kinetic resolu-

tion was investigated (Table 4). The highest E value was

Product (R)-9 was deacetylated in quantitative yield by

using a solution of sodium hydroxide in MeOH, thereby

leading to the R-configured cyclopentenol (8) in an overall

yield of 39% starting from rac-8. The S-configured alcohol

(8), which had an enantiomeric excess of 77%, was generat-

ed in its optically pure form by a second kinetic resolution/

transesterification reaction with pancreatin. (S)-Cyclopente-

nol (8) was isolated in 37% overall yield from rac-cyclopen-

tenol. Both enantiomers could be transformed into each

other by Mitsunobu inversion in very good yields.^[21]Finally,

total chemical yields of up to 80% were obtained for either

enantiomer. The significant advantage of this procedure is

that the resolution is done on the level of a precursor with

only one stereogenic center. In the literature, several other

enzymatic approaches have been reported that led to the

formation of compounds with at least two stereogenic cen-

ters such that only diastereomers were obtained that could

not be interconverted.^[12c]Thus, our approach led directly to

compounds that could be used as starting materials for the

synthesis of d- and l-carbocyclic nucleoside analogues

(Scheme 3). Other advantages of this kinetic resolution by

using pancreatin and 3-(benzyloxymethyl)-cyclopent-3-enol

(rac-8) include the low cost of the enzyme and the reagents.

Table 4. Kinetic resolution of rac-8 with PPL in different solvents.^[a]

Solvent

t

Conversion

[%]

ee (R)-9

[%]

ee (S)-8

[%]

E

[h]

acetone

toluene

1,4-dioxane

n-hexane

cyclohexane

49

47

15

46

16

31

16

95

91

95

85

87

17

78

18

38

18

46

49

47

18

20

[a] Reagents and conditions: rac-8 (50 mg), PPL (150 mg), Et₃N

(2 equiv), vinyl acetate (2 equiv), solvent (2 mL), RT.

found with toluene after 47 h (ee value of (R)-9: 91% and

46% conversion).

In addition, two other acyl donors were tested. Isopropen-

yl acetate and p-ClPhOAc are two literature-known re-

agents that have been used in kinetic resolutions and dy-

namic kinetic resolutions.^[20]However, their application

showed no advantage over our established procedures with

pancreatin and PPL.

In the end, the best results for the kinetic resolution were

found when 5 g of the cyclopentenol rac-8, 5 equivalents of

pancreatin, and 2 equivalents of vinyl acetate and triethyl-

amine were used in 170 mL acetone (Scheme 2). After 23 h

and 44% conversion, an excellent enantiomeric excess of

>98% was obtained for the acetylated compound ((R)-9).

The enantiomeric excess of the remaining (S)-cyclopentenol

(8) was 77%. A further scale-up of reaction to 10 g of rac-8

was easily accomplished and it seems there is no obvious

limit to the tolerable scale of the reaction. Compound (R)-9

3’-Deoxy-3’,4’-didehydronucleosides and -nucleotides: For

the syntheses of carbocyclic 3’-deoxy-3’,4’-didehydro-d-nu-

cleosides, R-configured alcohol 8 was used as the starting

material and, for the corresponding l-nucleoside analogues,

S-cyclopentenol 8 was used (Scheme 3).

The introduction of the pyrimidine heterocycles was ach-

ieved by using modified Mitsunobu reaction conditions.^[22]In

addition to the N1-alkylated product, the O2-regioisomer

was formed in minor amounts. These mixtures of the N1-

and O2-alkylated products were easily separated by column

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Synthesis of d- and l-Carbocyclic Nucleosides

FULL PAPER

Mitsunobu coupling of precursor (R)-8 with 6-chloropur-

ine in THF led to the formation of the benzyl-protected

purine nucleoside (d-17) in 80%. Next, nucleoside d-17 was

converted into its adenine-nucleoside analogue (d-18) in

75% yield by treatment with ammonia in MeOH; however,

in contrast to previous syntheses,^[25]the reaction was carried

out in a microwave reactor. The fully deprotected d-nucleo-

side (19) was obtained after treatment with ZnCl₂under al-

kaline conditions. Starting from (R)-8 and 2-amino-6-chloro-

purine, benzyl-protected d-20 was formed. Guanine ana-

logue d-21 was isolated in 34% yield by using formic acid

and subsequent treatment with ammonia in MeOH.

Scheme 3. Stereoselective access to carbocyclic l- and d-nucleosides from

(S)-8 and (R)-8.

chromatography on silica gel before alkaline cleavage of the

benzoyl group. Condensation of N3-benzoylthymine and

N3-benzoyluracil with (R)-cyclopentenol (8) and subsequent

alkaline deprotection led to carbocyclic 5’-O-benzylated d-

thymidine derivative 10 in 75% yield and its d-uridine ana-

logue (13) in 67% yield (Scheme 4). Cleavage of the benzyl

Therefore, starting from cyclopentadiene and R-config-

ured building block 8, we were able to prepare various 3’-

deoxy-3’,4’-didehydro-carba-d-nucleosides by a short chemo-

enzymatic route in good overall yields. Furthermore, the op-

posite enantiomer of cyclopentenol, (S)-8, was used for the

preparation of l-nucleoside analogues 10–21 with thymine,

uracil, cytosine, adenine, and guanine as nucleobases under

identical reaction conditions (Scheme 5). Thus, by using this

Scheme 4. a) DIAD, PPh₃, N3-benzoylthymine, CH₃CN; b) ZnCl₂, Ac₂O,

AcOH; c) NaOH, MeOH; d) DIAD, PPh₃, N3-benzoyluracil, CH₃CN;

e) triazole, POCl₃, Et₃N, CH₃CN; f) NH₃, CH₃CN; g) DIAD, PPh₃, 6-

chloropurine, THF; h) NH₃, MeOH; i) DIAD, PPh₃, 2-amino-6-chloro-

purine, THF; j) HCOOH k) NH₃, MeOH.

Scheme 5. a) DIAD, PPh₃, N3-benzoylthymine, CH₃CN; b) ZnCl₂, Ac₂O,

AcOH; c) NaOH, MeOH; d) DIAD, PPh₃, N3-benzoyluracil, CH₃CN;

e) triazole, POCl₃, Et₃N, CH₃CN; f) NH₃, CH₃CN; g) DIAD, PPh₃, 6-

chloropurine, THF; h) NH₃, MeOH; i) DIAD, PPh₃, 2-amino-6-chloro-

purine, THF; j) HCOOH; k) NH₃, MeOH.

ethers was achieved by ZnCl₂in Ac₂O/AcOH (5:1) and

yielded the acetylated d-thymidine analogue (11) in 82%

yield.^[23]Alkaline treatment led to the fully deprotected thy-

midine (12) in 73% yield. Deprotection to the d-uridine de-

rivative (14) was achieved in 53% yield without isolating

the acetylated intermediate. Transformation into the corre-

sponding d-cytidine analogue (15) was achieved by using

a method reported by Divakar and Reese.^[24]The uridine de-

rivative (13) was treated with triazole, phosphoryl chloride,

and triethylamine in CH₃CN to afford the triazolide inter-

mediate. This material was then stirred in a mixture of aque-

ous ammonia in CH₃CN to give compound 15 in 74% yield.

After cleavage of the benzyl group, 3’-deoxy-3’,4’-didehydro-

carba-d-cytidine (16) was obtained in 52% yield.

route, the preparation of numerous new enantiomerically

pure carbocyclic nucleosides was successfully achieved,

except for the corresponding d-thymidine analogue (12),

which has been isolated previously as a byproduct by Bꢁres

et al.^[4b]

All of the chiral precursors were isolated as oils. There-

fore, it was not possible to assign the absolute configuration

of these molecules. Because both enantiomers of 3-(benzyl-

oxymethyl)-cyclopent-3-enol (8) had not been prepared

before, the determination of their optical rotations alone did

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C. Meier et al.

not allow for their assignment. To assign the configurations

of (S)- and (R)-cyclopentenol (8) and, hence, of all of the

other synthesized compounds, 6-chloropurine nucleosides d-

17 and l-17 were used, which were obtained as crystalline

compounds. The assignment of the absolute configuration of

these purine derivatives was achieved by using X-ray crys-

tallography (Figure 2 and Figure 3).^[26]This result also al-

Scheme 6. a) (R)-(ꢀ)-methoxyphenylacetic acid, DCC, DMAP, CH₂Cl₂.

using CH₃CN, water, and triethylamine, d-nucleoside mono-

phosphates 27 and 28 were obtained by hydrolysis of cyclo-

Sal compounds 23 and 25. Their corresponding di- and tri-

phosphates (29–32) were synthesized from cycloSal-phos-

phate triesters 24 and 26 by reaction with either phosphate-

or pyrophosphate salts as nucleophiles (Scheme 7). In the

Figure 2. ORTEP of 5’-O-benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-d-6-

chloro-6-deamino-adenosine (d-17).

Scheme 7. a) Saligenyl chlorophosphite, DIPEA, CH₃CN; b) oxone in

H₂O; c) CH₃CN, H₂O, Et₃N; d) bis(tetra-n-butylammonium)hydrogen

phosphate, DMF; e) tris(tetra-n-butylammonium)hydrogen pyrophos-

phate, DMF.

Figure 3. ORTEP of 5’-O-benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-l-6-

chloro-6-deamino-adenosine (l-17).

future, these nucleotides will be used in biological assays

with kinases and polymerases, as well as viral enzymes, to

evaluate the intracellular metabolic pathway of the carbocy-

clic nucleosides.

lowed an assignment of the chirality of acetylated com-

pound (R)-9 and of cyclopentenol (S)-8 that was obtained

by kinetic resolution.

In addition, the optical purity of the prepared carbocyclic

nucleoside d-14 was examined by transformation into its

corresponding diastereomers with (R)-(ꢀ)-methoxyphenyl-

acetic acid (Scheme 6). The diastereomeric ratio of 5’-((2R)-

methoxy-phenylacetate)-2’,3’-dideoxy-3’,4’-didehydro-carba-

d-uridine (22) and, hence, the enantiomeric excess of d-14

were determined by ¹H NMR spectroscopy. The excellent

optical purity of the nucleoside correlates to that of its car-

bocyclic building block, which showed that all of the syn-

thetic steps in this convergent route were highly stereospe-

cific.

Modification of the 3’-deoxy-3’,4’-didehydronucleosides:

Furthermore, we established an enantioselective route to

various modified carbocyclic nucleoside analogues starting

from (S)-cyclopentenol (8, Scheme 8). In this process, it is

possible to functionalize the double bond before or after the

introduction of the nucleobase.^[11]Cyclopropanation for the

synthesis of carbobicyclic thymidine analogue d-36 was car-

ried out starting from (S)-cyclopentenol 8.

The reaction, using the conditions reported by Furukawa

et al., gave the trans product (33) in 60% yield.^[28]The ste-

reochemistry was confirmed by NOE experiments. In the

NMR spectra, a strong NOE effect between the H1 atom

The carbocyclic d-nucleotides were synthesized from

cycloSal nucleotides 23–26 as the starting materials.^[27]By

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Synthesis of d- and l-Carbocyclic Nucleosides

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Scheme 9. a) Imidazole, TBDMSCl, DMF; b) disiamylborane, THF;

c) NaOH/H₂O₂; d) NaH, BnBr, TBAI, THF; e) TBAF, THF; f) DIAD,

PPh₃, benzoic acid, Et₂O; g) DIAD, PPh₃, N3-benzoylthymine, CH₃CN;

h) Pd/C, H₂, MeOH.

Scheme 9) that was obtained by chemoenzymatic kinetic

resolution. First, alcohol (S)-8 was protected by using tert-

butyldimethylsilylchloride (TBDMS-Cl) in 98% yield. Then,

the introduction of the additional hydroxy group with di-

siamylborane afforded diastereomeric cyclopentenols 42 and

43 in a ratio of 11:1 and chemical yields of 74% and 7%, re-

spectively. The correct stereochemistry at the new stereo-

centers was confirmed by NOE spectroscopy. The two dia-

stereomers were separated by column chromatography on

silica gel and trans-cyclopentanol was benzylated in 91%

yield. Deprotection of the TBDMS group was achieved with

tetrabutylammonium fluoride (TBAF), thus yielding com-

pound 45 in 95% yield. The inversion of the configuration

at the secondary alcohol was easily carried out by a Mitsuno-

bu inversion in 92% yield. Subsequent coupling of carbocy-

clic building block 46 and the nucleobase was again accom-

plished under Mitsunobu conditions in 78% yield by using

N3-benzoylthymine as the nucleophile. The removal of the

benzyl groups for the formation of compound d-3 was ach-

ieved by Pd/C-catalyzed hydrogenation in 83% yield. The

overall yield of carba-d-dT (3) averages 17% over 10 steps

starting from cyclopentadiene. With this approach, a few

grams of compound d-3 were synthesized. Furthermore, this

synthetic route also allows for the preparation of their corre-

sponding l-nucleosides starting from compound (R)-8. Con-

sequently, the chemoenzymatic approach reported herein is

far more convenient then the previously published synthetic

routes to carbocyclic 2’-deoxynucleosides.

Scheme 8. a) PPh₃, DIAD, benzoic acid, Et₂O; b) CH₂I₂, Et₂Zn, CH₂Cl₂;

c) DIAD, PPh₃, N3-benzoylthymine, CH₃CN; d) Pd/C, HCOOH; e) Pd/

C, H₂, MeOH; f) disiamylborane, THF; g) NaOH/H₂O₂; h) NMO,

K₂OsO₄, DMF.

and one proton of the cyclopropane group was observed,

which confirmed the formation of the trans product (33).

Conversion of the hydroxy group with PPh₃, DIAD, and

benzoic acid led to cis-cyclopropanated alcohol 34 in 89%

yield. In addition, the condensation of N3-benzoylthymine

and cis-34 was also achieved (Scheme 8). Finally, the benzyl

protecting group in nucleoside d-35 was cleaved by using

Pd/C and formic acid to give bicyclic d-thymidine derivative

36 in 43% yield.

(R)-Configured alcohol 8 was used as a precursor to 5’-O-

benzylated d-thymidine analogue 10. This compound was

transferred into the diastereomeric carbocylic compound 3’-

deoxy-2’,3’-didehydro-d-thymidine (37 and iso-37, 3:1) in

95% yield by Pd/C-catalyzed hydrogenation and simultane-

ous debenzylation. The relative stereochemistry was deter-

mined by NOE spectroscopy. cis-Dihydroxylation was per-

formed with d-10, K₂OsO₄·2H₂O, and N-methylmorpholine-

N-oxide (NMO) in DMF. The electrophilic addition at the

cyclopentene moiety led to cis-diol d-38 in 62% yield.

Again, debenzylation to afford nucleoside d-39 was ach-

ieved by hydrogenation.

Unfortunately, the hydroboration of thymidine analogue

d-10 with 9-borabicycloACTHNUGRTENUNG[3.3.1]nonane (9-BBN) failed. There-

fore, the nucleoside was treated with disiamylborane. How-

ever, from this reaction with anti-Markownikow orientation,

carbocyclic carba-d-dT (3) was generated in a poor yield

(12%).

Antiviral actvity of carba-thymidine (3): carba-dT has been

studied previously concerning its antiviral potency against

HIV. In contrast to a previous report,^[4b]we found that

carba-dT showed promising antiviral activity without in-

creased toxicity.^[4a,5]Moreover, studies with the triphosphate

of carba-dT (carba-dTTP) in primer-extension assays by

using DNA- or RNA-templates and the viral enzyme re-

verse transcriptase revealed an interesting mechanism of

action. We observed that carba-dTTP was incorporated op-

carba-dT (3): Thus, a new synthetic route to carba-thymi-

dine d-3 was elaborated by using (S)-cyclopentenol (8,

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posite the canonical 2’-deoxyadenosine and was further

elongated with the following nucleotides but, after several

carba-dT incorporations, the elongation of the growing

DNA strand stopped. This mechanism was entitled a “de-

layed chain-termination”.^[5]A growing problem in antiviral

chemotherapy is the development of drug-resistant virus

strains. This problem has primarily been shown for different

antiretroviral-active drugs that are currently being used in

combination therapy against HIV/AIDS. Today, multicom-

ponent highly active antiretroviral therapies (HAART) are

very effective in decreasing the viral load in HIV-infected

patients. However, if resistance develops in such a scenario,

multidrug-resistant virus strains may easily emerge. Thus,

there is an urgent need for new, effective drugs that target

multidrug-resistant viruses. The efficacy of carba-dT (3) as

an RT inhibitor was tested against wildtype HIV-1 (strain

NL4/3) and two multidrug-resistant (MDR) HIV-1 clones.

The MDR clones, E2-2 and 8ka3, contain a number of well-

defined drug-resistant mutations that confer low susceptibili-

ty against a number of clinically approved anti-HIV drugs,

such as nucleoside analogues and protease inhibitors. As

given in the Experimental Section, both MDR viruses exhib-

it a number of thymidine-analogue mutations (TAMS),

thereby conferring resistance to most of the currently li-

censed nucleo(t)side analogues that are used in clinical prac-

tice. Interestingly, d-carba-dT (3) showed a marked inhibito-

ry effect against the virus replication of both MDR HIV-

1 clones. d-carba-dT (3) inhibited the replication of MDR

clone E2-2 in a clearly dose-dependent manner with an IC₅₀

value of 0.03 mm (Figure 4). 8ka3 was also potently inhibited,

albeit in a less-pronounced manner, with an IC₅₀value of

0.14 mm (not shown). The IC₅₀value for wildtype HIV-

1 (NL4/3) was 0.18 mm, which is of the same order of magni-

tude as the IC₅₀values that were reported previously for dif-

ferent cell-lines and wildtype HIV-1 strains.^[5]As expected,

the IC₅₀values for MDR HIV-1 clones were lower than the

IC₅₀values for wildtype HIV-1 because drug-resistant muta-

tions are commonly associated with a viral-fitness cost (re-

viewed for RT-inhibitor-resistance mutations).^[29]For com-

parison, the l-configured counterpart and the clinically used

antiretroviral drug d4T (Stavudine) were also tested. l-

carba-dT was entirely inactive in the tested concentration

range for all of the tested viruses (E2-2, 8ka3, and NL4/3).

d4T inhibited the replication of wildtype HIV-1 (NL4/3)

with an IC₅₀value of <0.03 in our experimental setup and

MDR clone E2-2, as expected, with a considerably higher

IC₅₀value of 0.38 mm, owing to resistance mutations. Nota-

bly, none of the tested compounds exerted cellular toxicity

in the tested concentration range (Figure 4, bottom). The

IC₅₀value for the inhibition of NL4/3 by d4T is considerably

lower than the values reported by Rosenblum et al. for dif-

ferent cell-lines and wildtype HIV-1 strains,^[30]possibly

owing to the relatively long duration of the virus culture of

6 days until readout in our assay. In general, assays with

multiple rounds of viral replication give low IC₅₀values.

Although the underlying mechanism that is responsible

for overcoming the resistance is still unclear, the strong anti-

viral activity against multidrug-resistant virus strains makes

d-carba-dT a promising new compound to combat drug re-

sistance in HIV infection.

Conclusion

Our chemoenzymatic approach offers a convenient route for

the preparation of enantiomeric precursors (S)-8 and (R)-8

in high optical purity. Owing to the fact that, by using this

procedure, only one stereocenter in this building block is

created, it is an excellent key intermediate that allows direct

access to either carbocyclic d- or l-2’,3’-dideoxy-3’,4’-didehy-

dronucleosides. Moreover, various functionalization reac-

tions lead to a series of new enantiomerically pure carbocy-

clic nucleoside and -nucleotide analogues. Furthermore, the

general applicability of this carbocyclic precursor was also

demonstrated by its use in a new synthetic route to carbocy-

clic 2’-deoxynucleosides. Finally, we demonstrated that d-

carba-dT (3) exerts strong antiviral activity against multi-

drug-resistant virus strains, including multiple classical thy-

midine-analogue mutations, which makes it an attractive

compound for thereapeutic approaches that aim to combat

drug resistance in HIV.

Experimental Section

General: All experiments that involved water-sensitive compounds were

conducted under rigorously dry conditions and under a nitrogen atmos-

phere. CH₂Cl₂, pyridine, and CH₃CN were distilled from CaH₂and

stored over molecular sieves. Et₂O and THF were distilled from sodium

or potassium with benzophenone and stored over molecular sieves. Pe-

troleum ether, EtOAc, CH₂Cl₂, and MeOH that were employed in

column chromatography were distilled before use. Column chromatogra-

phy on silica gel was performed by using silica gel 60, (230–400 mesh,

Merck). Reversed-phase column chromatography was performed on re-

verse-phase silica gel with distilled water as the eluent at RT in a glass

column. Ion-exchange chromatography was performed on Dowex

(50WX8, 100–200 mesh, ion-exchange resin) in a glass column. Some sep-

arations were performed on a chromatotron by using glass plates that

were coated with silica gel (60 PF₂₅₄, layer thickness 1 mm) that con-

tained a fluorescent indicator. Analytical TLC was performed on pre-

coated aluminium plates 60F254 (Merck) with a 0.2 mm layer of silica gel

that contained a fluorescence indicator; the compounds were visualized

with a spray reagent (4-methoxybenzaldehyde (0.5 mL), EtOH (9 mL),

conc. H₂SO₄(0.5 mL), and glacial AcOH (0.1 mL)) by heating with

a heat gun. UV/Vis detection was performed at l_max=254 nm. Pancreatin

from porcine pancreas (CAS 8049–47–6, EC 232–468–9) and all of the

isolated enzymes (CAS 9001–62–1, EC 232–619–9) were purchased from

Sigma–Aldrich. ¹H NMR spectroscopy was either recorded on a Bruker

AMX 400 at 400 MHz, on a Bruker DMX 500 at 500 MHz, or on

a Bruker AV 400 at 400 MHz with residual solvent peaks used as the in-

ternal standard. ¹³C NMR spectra were either recorded on a Bruker

AMX 400 at 101 MHz, a Bruker AV 400 at 101 MHz, or on a DMX 500

at 126 MHz with residual solvent peaks used as the internal standard. All

¹H and ¹³C NMR chemical shifts (d) are quoted in parts per million

(ppm). The spectra were recorded at RT and all ¹³C NMR spectra were

recorded in proton-decoupled mode. MS was either performed on a VG

Analytical VG/70–250 F spectrometer (FAB, matrix: m-nitrobenzyl alco-

hol), a VG Analytical VG/70–250S spectrometer (double focused), or on

a Finnigan ThermoQuest MAT 95XL (HRMS ESI). Optical rotations

were measured on a P8000 polarimeter (A. Kruss Optonic GmbH) at

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FULL PAPER

Figure 4. Antiretroviral activity and toxicity of d-carba-dT, l-carba-dT, and d4T. Top: Percentage of virus replication compared with replication under

solvent-control (DMSO) cultures, which was arbitrarily set to 100% and shown as a dotted line. Bottom: AlamarBlue assays, which indicate cell viability

from the same cultures. The dotted line indicates the absorbance in DMSO cultures. Mean values of two independent infections are shown. Error bars

represent the standard deviation.

589 nm. IR spectra were recorded with a ThermoNicolet Avatar 370

FTIR spectrometer. For the X-ray crystal-structure analysis, data collec-

tion was performed on a Bruker Smart APEX CCD area-detector dif-

fractometer. Analytical HPLC was carried out on a Merck-Hitachi D-

7000 HPLC system that consisted of a Merck-Hitachi L-7100 pump, an

autosampler, and a diode-array detector l-7455. The column used was

a CHIRALPAK OD (CS-Chromatographie Service GmbH, Langerwehe,

Germany; 250 mmꢂ4.6 mm) with cellulose tris(3,5-dimethylphenylcarba-

mate) coated on 100 mm silica gel (method 1: n-hexane/2-propanol (95:5,

0–30 min), n-hexane/2-propanol (60:40, 30–35 min), n-hexane/2-propanol

(95:5, 30–40 min), flow rate: 1 mLmin^ꢀ1, UV/Vis detection was per-

formed at l_max=210 nm; method 2: n-hexane/2-propanol (98:2, 0–

15 min), n-hexane/2-propanol (60:40, 15–20 min), n-hexane/2-propanol

(98:2, 20–25 min), flow rate: 1 mLmin^ꢀ1, UV/Vis detection was per-

formed at l_max=210 nm).

Compounds rac-8, d-36, and d-39 were synthesized according to a litera-

ture procedure.^[11]Hydroboration with disiamylborane (d-10) for the

preparation of compound d-3 was carried out in a similar manner to

a previously reported synthesis with 9-BBN.^[11]

Kinetic resolution of rac-3-(benzyloxymethyl)-cyclopent-3-enol (rac-8):

Optimal conditions: A suspension of racemic 3-(benzyloxymethyl)-cyclo-

pent-3-enol (rac-8, 5.00 g, 24.5 mmol), pancreatin (25 g), vinyl acetate

(4.54 mL, 49.0 mmol), and Et₃N (6.79 mL, 49.0 mmol) in acetone

(170 mL) was stirred at room temperature. After 23 h, the mixture was

filtered through celite and the filtrate was concentrated under reduced

pressure. The crude product was analyzed by HPLC and then purified by

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C. Meier et al.

column chromatography on silica gel (petroleum ether/EtOAc, 4:1!2:1)

in MeOH) and the mixture was stirred overnight at room temperature.

After neutralization with aqueous HCl (1m), the solvent was evaporated

and the crude product was purified by column chromatography on silica

gel (petroleum ether/EtOAc, 2:1).

to afford acetylated compound (R)-9 (2.01 g, 8.16 mmol, 33%, >98% ee)

25

589

as a yellow oil after 44% conversion. [a] =ꢀ15.48 (c=0.5, CHCl₃); R_f

(petroleum ether/EtOAc, 2:1)=0.86; ¹H NMR (400 MHz, CDCl₃): d=

7.38–7.27 (m, 5H; aromatic CH), 5.66–5.61 (m, 1H; H4), 5.42–5.36 (m,

1H; H1), 4.52–4.49 (m, 2H; benzyl CH₂), 4.07 (s, 2H; OCH₂), 2.84–2.73

(m, 2H; H2a, H5a), 2.46–2.37 (m, 2H; H2b, H5b), 2.03 ppm (s, 3H; Ac

CH₃); ¹³C NMR (101 MHz, CDCl₃): d=171.2 (C_qAc), 139.2 (C3), 138.3

(aromatic C_q), 128.5 (aromatic CH), 127.9 (aromatic CH), 127.8 (aromat-

ic CH), 125.0 (C4), 74.6 (C1), 72.3 (benzyl CH₂), 68.6 (OCH₂), 40.2 (C2),

39.7 (C5), 21.5 ppm (Ac CH₃); IR (film): n˜ =2856, 1732, 1454, 1362, 1241,

2072, 1026, 736, 697, 606 cm^ꢀ1; HRMS (FAB): m/z calcd for C₁₅H₁₈O₃:

247.1334 [M+H]⁺; found: 247.1333.

5’-O-Benzyl-3’-deoxy-3’,4’-didehydro-carba-d-thymidine (d-10): The reac-

tion was carried out according to the general coupling procedure (see

above) with a solution of (R)-3-(benzyloxymethyl)-cyclopent-3-enol ((R)-

8, 500 mg, 2.45 mmol) and N3-benzoyl-protected thymine (1.13 g,

4.90 mmol) in CH₃CN (45 mL) and a preformed complex of PPh₃(1.95 g,

7.45 mmol) and DIAD (1.35 mL, 6.86 mmol) in CH₃CN (30 mL). The re-

action mixture was stirred for 36 h and, after purification, benzylated nu-

cleoside d-17 (573 mg, 1.84 mmol, 75%) was obtained as a yellow oil.

25

589

[a] =+28.38 (c=1.0, CHCl₃); R_f(petroleum ether/EtOAc, 1:2)=0.37;

¹H NMR (500 MHz, C₆D₆): d=9.84 (br s, 1H; NH), 7.28–7.03 (m, 5H; ar-

omatic CH), 6.44 (d, ⁴J=1.1 Hz, 1H; H6), 5.28–5.25 (m, 1H; H3’), 5.12–

5.11 (m, 1H; H1’), 4.26 (s, 2H; benzyl CH₂), 3.73–3.63 (m, 2H; H5’),

2.41–2.30 (m, 2H; H6’a, H2’a), 1.96–1.92 (m, 1H; H2’b), 1.91–1.88 (m,

1H; H6’b), 1.65 ppm (d, ⁴J=1.0 Hz, 3H; thymine CH₃); ¹³C NMR

(101 MHz, C₆D₆): d=162.3 (C4), 151.5 (C2), 138.5 (aromatic C_q), 136.3

(C6), 128.9 (aromatic CH), 128.7 (aromatic CH), 128.3 (aromatic CH),

125.3 (C3’), 124.8 (C4’) 111.5 (C5), 72.8 (benzyl CH₂), 68.6 (C5’), 53.6

(C1’), 39.4 (C2’), 39.3 (C6’), 12.9 ppm (thymine CH₃); IR (film): n˜ =3854,

3450, 2081, 1666, 1459, 1383, 1203, 1093, 1028, 699 cm^ꢀ1; HRMS (FAB):

m/z calcd for C₁₈H₂₀N₂O₃: 313.1552 [M+H]⁺; found: 313.1541.

Alcohol (S)-8 (2.81 g, 13.8 mmol, 56%, 77% ee) was used in a second ki-

netic resolution to increase the enantiomeric excess of compound (S)-8.

The reaction was done under the same conditions as described above,

but with only 1.5 equiv of pancreatin. In this reaction, alcohol (S)-8

(1.86 g, 9.11 mmol, 37%, 93% ee) was obtained after 6 h. [a]²_D⁵=+20.08

(c=0.25, CH₂Cl₂/MeOH 1:1). The spectroscopic data for alcohol (S)-8

were identical to those described for rac-8.

Conditions for different enzymes: A suspension of racemic 3-(benzyloxy-

methyl)-cyclopent-3-enol rac-8 (15 mg, 0.073 mmol), vinyl acetate (14 mL,

0.15 mmol), Et₃N (20 mL, 0.15 mmol), and the respective enzyme in ace-

tone or toluene (2 mL) was stirred at room temperature. After different

time intervals, samples were taken, filtered through celite, and the filtrate

was concentrated under reduced pressure. The crude product was ana-

lyzed by HPLC.

5’-O-Benzyl-3’-deoxy-3’,4’-didehydro-carba-l-thymidine (l-17): The reac-

tion was carried out according to the general coupling procedure (see

above) with a solution of (S)-3-(benzyloxymethyl)-cyclopent-3-enol (S)-8

(214 mg, 0.685 mmol) and N3-benzoyl-protected thymine (539 mg,

(R)-3-(Benzyloxymethyl)-cyclopent-3-en-1-ol ((R)-8): For the deacetyla-

tion reaction, (R)-9 (0.878 g, 4.30 mmol) was stirred in NaOH (2.5m in

MeOH, 25 mL) for 2 h at room temperature. After neutralization with

aqueous HCl (1m), MeOH was evaporated and the aqueous layer was ex-

tracted with Et₂O (3ꢂ25 mL). The combined organic fractions were

dried with Na₂SO₄and the solvent was removed under reduced pressure.

2.06 mmol) in CH₃CN (15 mL) and

a preformed complex of PPh₃

(539 mg, 2.06 mmol) and DIAD (0.380 mL, 1.92 mmol) in CH₃CN

(20 mL). The reaction mixture was stirred for 36 h and, after purification,

benzylated nucleoside l-17 (154 mg, 0.493 mmol, 72%) was obtained as

25

589

a yellow syrup. [a] =ꢀ25.98 (c=1.0, CHCl₃). The spectroscopic data

were identical to those described for d-17.

Alcohol (R)-8 (4.26 mmol, 99%, 96% ee) was obtained as a yellow oil.

25

589

[a] =ꢀ19.48 (c=1.0, CHCl₃). The spectroscopic data were identical to

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-d-uridine (d-13): The re-

action was carried out according to the general coupling procedure (see

above) with a solution of (R)-3-(benzyloxymethyl)-cyclopent-3-enol (R)-

those described for rac-8.

General procedure for the inversion of the stereochemistry of the hy-

droxy group: Diisopropylazodicarboxylate (DIAD, 2 equiv) was slowly

added to a suspension of PPh₃(2 equiv) in Et₂O at 08C under a nitrogen

atmosphere and stirred for 0.5 h. This yellow suspension was added to

a solution of the alcohol (1 equiv) and benzoic acid (2 equiv) in Et₂O at

08C. The reaction mixture was stirred at room temperature until com-

plete conversion was observed by TLC analysis; the mixture was then fil-

tered and concentrated. The residue was treated with a solution of

NaOH (1% in MeOH) and stirred overnight. After neutralization with

HCl (1m), the solvent was evaporated and the crude product was puri-

fied.

8

(482 mg, 2.36 mmol) and N3-benzoyl-protected uracil (1.02 g,

4.72 mmol) in CH₃CN (50 mL) and a preformed complex of PPh₃(1.86 g,

7.08 mmol) and DIAD (1.30 mL, 6.61 mmol) in CH₃CN (35 mL). The re-

action mixture was stirred for 24 h and, after purification, benzylated nu-

cleoside d-13 (473 mg, 1.59 mmol, 67%) was obtained as a yellow oil.

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589

[a] =+21.58 (c=1.0, CHCl₃); R_f(petroleum ether/EtOAc, 1:2)=0.20;

¹H NMR (500 MHz, CDCl₃): d=7.95 (br s, 1H; NH), 7.30–7.18 (m, 5H;

3

aromatic CH), 7.09 (d, J=8.2 Hz, 1H; H6), 5.65–5.63 (m, 1H; H3’), 5.59

(d, ³J=7.9 Hz, 1H; H5), 5.29–5.24 (m, 1H; H1’), 4.44 (s, 2H; benzyl

CH₂), 4.06–3.95 (m, 2H; H5’) 2.91–2.81 (m, 2H; H2’a, H6’a), 2.34–

2.28 ppm (m, 2H; H2’b, H6’b); ¹³C NMR (101 MHz, CDCl₃): d=163.1

(C4), 150.8 (C2), 140.9 (C5), 140.6 (C4’), 138.1 (aromatic C_q), 128.6 (aro-

matic CH), 128.0 (aromatic CH), 127.8 (aromatic CH), 125.4 (C3’), 103.2

(C6), 72.9 (benzyl CH₂), 68.4 (C5’), 53.4 (C1’), 40.0 (C2’), 39.8 ppm (C6’);

IR (film): n˜ =3032, 2853, 1672, 1463, 1370, 1263, 1074, 820, 696, 552,

417 cm^ꢀ1; HRMS (FAB): m/z calcd for C₁₇H₁₈N₂O₃: 299.1396 [M+H]⁺;

found: 299.1384.

(R)-3-(Benzyloxymethyl)-cyclopent-3-en-1-ol ((R)-8): The Mitsunobu-in-

version reaction was carried out according to the general procedure (see

above) with (S)-3-(benzyloxymethyl)-cyclopent-3-enol ((S)-8, 1.00 g,

4.90 mmol), benzoic acid (1.20 g, 9.80 mmol) in Et₂O (30 mL), and

DIAD (1.92 mL, 9.80 mmol), PPh₃(2.57 g, 9.80 mmol) in Et₂O (30 mL).

The reaction mixture was stirred for 16 h and was treated with a solution

of NaOH (1% in MeOH, 25 mL). The crude product was purified by

column chromatography on silica gel (petroleum ether/EtOAc, 2:1) to

yield compound (R)-8 (995 mg, 4.87 mmol, 99%) as a yellow syrup. The

spectroscopic data were identical to those described for rac-8.

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-l-uridine (l-13): The re-

action was carried out according to the general coupling procedure (see

above) with a solution of (S)-3-(benzyloxymethyl)-cyclopent-3-enol (S)-

13 (425 mg, 2.08 mmol) and N3-benzoyl-protected uracil (740 mg,

4.16 mmol) in CH₃CN (50 mL) and a preformed complex of PPh₃(1.64 g,

6.24 mmol) and DIAD (1.03 mL, 5.83 mmol) in CH₃CN (35 mL). The re-

action mixture was stirred for 24 h and, after purification, benzylated nu-

General procedure for the coupling of N3-protected pyrimidines to cyclo-

pentenol: DIAD (2.8 equiv) was slowly added to a suspension of PPh₃

(3 equiv) in CH₃CN at 08C under a nitrogen atmosphere and the mixture

was stirred for 0.5 h. This preformed complex was slowly added to a solu-

tion of N3-protected pyrimidine (2 equiv) and cyclopentenol (1 equiv) in

CH₃CN at ꢀ508C. The reaction mixture was stirred at ꢀ408C for 2 h

before being slowly warmed to room temperature and stirred until com-

plete conversion was observed by TLC. The solvent was removed and

the residue was purified by column chromatography on silica gel (petro-

leum ether/EtOAc, 1:2) to remove the triphenylphosphine oxide. The

benzoyl-protected nucleoside was treated with a solution of NaOH (1%

cleoside l-13 (376 mg, 1.26 mmol, 61%) was obtained as a yellow solid.

25

589

[a] =ꢀ18.98 (c=1.0, CHCl₃). The spectroscopic data were identical to

those described for d-13.

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-d-cyctidine (d-15): Phos-

phoryl chloride (0.078 mL, 0.86 mmol) and Et₃N (0.53 mL, 3.8 mmol)

were added dropwise to a solution of 1,2,4-triazole (0.266 g, 3.85 mmol)

in CH₃CN (5 mL) under a nitrogen atmosphere at 08C and the mixture

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Synthesis of d- and l-Carbocyclic Nucleosides

FULL PAPER

was stirred for 0.5 h at the same temperature. To the resulting product

was slowly added a solution of benzyl-protected uridine derivative d-13

(85.0 mg, 0.285 mmol) in CH₃CN (3 mL) and the reaction mixture was

stirred for 2 days at room temperature. The solvent was evaporated

under reduced pressure and the residue was partitioned between EtOAc

(5 mL) and a saturated aqueous solution of sodium hydrogen carbonate

(3ꢂ5 mL). The organic layer was washed with a saturated aqueous solu-

tion of sodium chloride (3ꢂ5 mL) and the combined aqueous layer was

extracted with EtOAc (5 mL). The combined organic fractions were

dried with Na₂SO₄, filtered, and the solvent was removed under reduced

pressure. The residue was dissolved in CH₃CN (5 mL) and treated with

a solution of aqueous ammonia (25%, 3 mL). The reaction mixture was

stirred for 7 days. The solvent was evaporated and the residue was parti-

tioned between CH₂Cl₂(5 mL) and water (5 mL). The aqueous layer was

extracted with CH₂Cl₂(4ꢂ5 mL) and the organic layer was washed with

a saturated aqueous solution of sodium chloride (2ꢂ5 mL). The com-

bined organic fractions were dried with Na₂SO₄and the solvent was

evaporated. The crude product was purified by column chromatography

(101 MHz, CDCl₃): d=170.8 (Ac C_q), 163.9 (C4), 151.0 (C2), 138.2 (C4’),

136.4 (C6), 126.7 (C3’), 111.9 (C5), 62.5 (C5’), 52.9 (C1’), 39.8 (C2’/C6’),

39.6 (C2’/C6’), 20.9 (Ac CH₃), 12.7 ppm (thymine CH₃); IR (film): n˜ =

3171, 3044, 2925, 2854, 1739, 1681, 1469, 1387, 1270, 1228, 1118, 1027,

870, 667 cm^ꢀ1

; HRMS (FAB): m/z calcd for C₁₃H₁₆N₂O₄: 265.1188

[M+H]⁺; found: 265.1186.

5’-O-Acetyl-3’-deoxy-3’,4’-didehydro-carba-l-thymidine (l-11):

A solu-

tion of benzylated nucleoside l-10 (147 mg, 0.470 mmol) in Ac₂O/AcOH

(5:1, 1.5 mL) was deprotected according to general debenzylation proce-

dure 1 (see above) with a solution of ZnCl₂(320 mg, 2.35 mmol) in

Ac₂O/AcOH (5:1, 3 mL). The mixture was stirred for 23 h purified by

column chromatography on silica gel (CH₂Cl₂/MeOH, 20:1) to obtain nu-

25

589

cleoside l-11 (83.7 mg, 0.371 mmol, 79%) as a colorless solid. [a]

=

ꢀ16.88 (c=1.0, CHCl₃). The spectroscopic data were identical to those

described for d-11.

3’-Deoxy-3’,4’-didehydro-carba-d-thymidine (d-12): Acetylated nucleo-

side d-11 (75.7 mg, 0.286 mmol) was treated with a solution of NaOH

(1% in MeOH, 10 mL) at room temperature until complete conversion

was observed by TLC analysis. The reaction mixture was neutralized with

HCl (1m), MeOH was evaporated, and, after lyophilization, the crude

product was purified by column chromatography on silica gel (CH₂Cl₂/

on silica gel (CH₂Cl₂/MeOH, 9:1) to yield compound d-15 (62.8 mg,

25

589

0.211 mmol, 74%) as a yellow solid. [a] =ꢀ15.68 (c=1.0, CHCl₃); m.p.

1378C; R_f(CH₂Cl₂/MeOH, 9:1)=0.64; ¹H NMR (400 MHz, CDCl₃): d=

7.38–7.28 (m, 5H; aromatic CH), 7.23 (d, ³J=7.3 Hz, 1H; H6), 5.77 (d,

³J=7.3 Hz, 1H; H5), 5.73–5.68 (m, 1H; H3’), 5.49–5.41 (m, 1H; H1’),

4.52 (s, 2H; benzyl CH₂), 4.10–4.03 (m, 2H; H5’), 2.99–2.85 (m, 2H;

H2’a, H6’a), 2.42–2.34 ppm (m, 2H; H2’b, H6’b); ¹³C NMR (126 MHz,

CDCl₃): d=165.3 (C2), 156.4 (C4), 141.9 (C6), 140.4 (C4’), 138.2 (aromat-

ic C_q), 128.6 (aromatic CH), 127.8 (aromatic CH), 127.8 (aromatic CH),

125.6 (C3’), 95.3 (C5), 72.7 (benzyl CH₂), 68.5 (C5’), 54.0 (C1’), 40.3 (C2’/

C6’), 40.1 ppm (C2’/C6’); IR (film): n˜ =3344, 1920, 2852, 1618, 1524, 1483,

1398, 1279, 1070, 906, 786, 726, 598 cm^ꢀ1; HRMS (FAB): m/z calcd for

C₁₇H₁₉N₃O₂: 298.1556 [M+H]⁺; found: 298.1554.

MeOH, 15:1) to yield carbocyclic nucleoside d-12 (46.5 mg, 0.209 mmol,

25

589

73%) as a colorless solid. [a] =+24.18 (c=0.5, MeOH); m.p. 1378C; R_f

(CH₂Cl₂/MeOH, 9:1)=0.42; ¹H NMR (400 MHz, [D₆]DMSO): d=11.20

(br s, 1H; NH), 7.14 (d, ⁴J=1.3 Hz, 1H; H6), 5.46–5.42 (m, 1H; H3’),

3

4.98–4.88 (m, 1H; H1’), 4.06 (s, 2H; H5’), 3.17 (d, J=4.1 Hz, 1H; OH’),

2.48–2.42 (m, 1H; H2’a), 2.42–2.31 (m, 1H; H6’a), 2.30–2.20 (m, 1H;

H2’b), 1.76 (d, ⁴J=1.3 Hz, 3H; thymine CH₃), 1.71–1.59 ppm (m, 1H;

H6’b); ¹³C NMR (101 MHz, [D₆]DMSO): d=163.9 (C4), 153.1 (C4’),

150.9 (C2), 137.1 (C6), 121.2 (C6’), 109.1 (C5), 60.4 (C1’), 59.9 (C5’), 30.9

(C3’), 30.3 (C2’), 12.2 ppm (thymine CH₃); IR (film): n˜ =3448, 2252,

2126, 1676, 1054, 1027, 1008, 824, 762, 627 cm^ꢀ1; UV/Vis (CH₃CN): l_max

=

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-l-cyctidine (l-15): The

reaction was carried out as described for compound d-15 with a solution

of benzyl-protected uridine derivative l-13 (150 mg, 0.503 mmol) in

CH₃CN (5 mL) and a mixture of 1,2,4-triazole (0.931 g, 6.78 mmol), phos-

phoryl chloride (0.140 mL, 1.51 mmol) and Et₃N (0.940 mL, 6.80 mmol)

in CH₃CN (12 mL). The reaction mixture was stirred for 2 days. The resi-

due was dissolved in CH₃CN (5 mL) and treated with aqueous ammonia

(25%, 4 mL). The mixture was stirred for 7 days at room temperature.

After column chromatography on silica gel (CH₂Cl₂/MeOH, 9:1), com-

271 nm; HRMS (FAB): m/z calcd for C₁₁H₁₄N₂O₃: 223.1083 [M+H]⁺;

found: 223.1100.

3’-Dideoxy-3’,4’-didehydro-carba-l-thymidine (l-12): Acetylated nucleo-

side l-11 (51.1 mg, 0.286 mmol) was treated with a solution of NaOH

(1% in MeOH, 10 mL) at room temperature until complete conversion

was observed. The reaction mixture was neutralized with HCl (1m), the

MeOH was evaporated off and after lyophilization the crude product was

purified by column chromatography on silica gel (CH₂Cl₂/MeOH, 15:1)

to yield the carbocyclic nucleoside l-12 (32.3 mg, 0.145 mmol, 75%) as

a colorless solid. [a]²₅₈⁵₉=ꢀ22.48 (c=0.7, MeOH). The spectroscopic data

were identical to those described for d-12.

pound l-15 (118 mg, 0.397 mmol, 79%) was isolated as a yellow solid.

25

589

[a] =+14.48(c=1.0, CHCl₃). The spectroscopic data were identical to

those described for d-15.

General procedures for the debenzylation reaction: Procedure 1: Benzyl-

protected nucleoside (1 equiv), was dissolved in Ac₂O/AcOH (5:1) and

the solution was added to a solution of freshly melted ZnCl₂(5 equiv) in

Ac₂O/AcOH (5:1). The mixture was stirred at room temperature until

complete conversion was observed by TLC. The reaction was stopped by

the addition of water and the mixture was extracted with CH₂Cl₂. The

combined organic layers were neutralized with a saturated aqueous solu-

tion of sodium hydrogen carbonate, dried with Na₂SO₄, and the solvent

was evaporated. Procedure 2: The residue was treated with a solution of

NaOH (1% in MeOH) at room temperature until complete conversion

was observed by TLC. The reaction mixture was neutralized with HCl

(1m), MeOH was evaporated, and, after lyophilization, the crude product

was purified by column chromatography on silica gel.

2’,3’-Dideoxy-3’,4’-didehydro-carba-d-uridine (d-14): A solution of ben-

zylated nucleoside d-13 (157 mg, 0.526 mmol) in Ac₂O/AcOH (5:1,

4 mL) was deprotected according to general debenzylation procedure 2

(see above) with a solution of ZnCl₂(541 mg, 3.97 mmol) in Ac₂O/AcOH

(5:1, 4 mL). The mixture was stirred for 18 h at room temperature and

the obtained intermediate was treated with a solution of NaOH (1% in

MeOH). After column chromatography on silica gel (CH₂Cl₂/MeOH,

15:1), debenzylated nucleoside d-14 (58.1 mg, 0.279 mmol, 53%) was ob-

25

589

tained as a colorless solid. [a] =+20.28 (c=0.58, MeOH); R_f(CH₂Cl₂/

MeOH, 15:1)=0.48; ¹H NMR (400 MHz, [D₆]DMSO): d=11.25 (s, 1H;

NH), 7.41 (d, ³J=8.0 Hz, 1H; H6), 5.85–5.83 (m, 1H; H3’), 5.58 (d, ³J=

8.0 Hz, 1H; H5), 5.16–5.08 (m, 1H; H1’), 4.34 (s, 2H; H5’), 2.87–2.76 (m,

2H; H2’a, H6’a), 2.46–2.42 ppm (m, 2H; H2’b, H6’b); ¹³C NMR

(101 MHz, [D₆]DMSO): d=163.3 (C4), 150.8 (C2), 145.7 (C4’), 142.0

(C6), 128.7 (C3’), 109.3 (C5), 53.6 (C1’), 43.0 (C5’), 38.4 (C6’) 38.4 ppm

(C2’); IR (film): n˜ =3433, 3006, 2819, 1686, 1460, 1379, 1272, 1055, 1008,

760 cm^ꢀ1; UV/Vis (CH₃CN): l_max=263, 213 nm; HRMS (FAB): m/z calcd

for C₁₀H₁₂N₂O₃: 209.0926 [M+H]⁺; found: 209.0925.

5’-O-Acetyl-3’-deoxy-3’,4’-didehydro-carba-d-thymidine (d-11): A solu-

tion of benzylated nucleoside d-10 (307 mg, 0.983 mmol) in Ac₂O/AcOH

(5:1, 5 mL) was deprotected according to general debenzylation proce-

dure 1 (see above) with a solution of ZnCl₂(670 mg, 4.92 mmol) in

Ac₂O/AcOH (5:1, 5 mL). The mixture was stirred for 12 h and purified

by column chromatography on silica gel (CH₂Cl₂/MeOH, 20:1) to obtain

d-(212 mg, 0.802 mmol, 82%) as a colorless solid. [a]²₅₈⁵₉=+17.28 (c=1.0,

CHCl₃); m.p. 1378C; R_f(CH₂Cl₂/MeOH, 20:1)=0.37; ¹H NMR

2’,3’-Dideoxy-3’,4’-didehydro-carba-l-uridine (l-14): A solution of ben-

zylated nucleoside l-13 (131 mg, 0.439 mmol) in Ac₂O/AcOH (5:1, 3 mL)

was deprotected according to general debenzylation procedure 2 (see

above) with a solution of ZnCl₂(500 mg, 3.70 mmol) in Ac₂O/AcOH

(5:1, 8 mL). The mixture was stirred for 13 h at room temperature and

the obtained intermediate was treated with a solution of NaOH (1% in

MeOH). After column chromatography on silica gel (CH₂Cl₂/MeOH,

4

(500 MHz, CDCl₃): d=9.17 (br s, 1H; NH), 6.97 (d, J=1.0 Hz, 1H; H6),

5.78–5.73 (m, 1H; H3’), 5.44–5.37 (m, 1H; H1’), 4.71–4.62 (m, 2H; H5’),

3.01–2.87 (m, 2H; H2a’, H6a’), 2.46–2.34 (m, 2H; H2b’, H6b’), 2.09 (s,

3H; Ac CH₃), 1.91 ppm (d, ⁴J=1.0 Hz, 3H; thymine CH₃); ¹³C NMR

Chem. Eur. J. 2012, 00, 0 – 0

ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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C. Meier et al.

15:1), debenzylated nucleoside l-14 (60.6 mg, 0.291 mmol, 62%) was ob-

tained as a colorless solid. [a]²₅₈⁵₉=ꢀ19.58 (c=0.14, MeOH). The spectro-

scopic data were identical to those described for d-14.

ꢀ18.38 (c=1.0, CHCl₃). The spectroscopic data were identical to those

described for d-17.

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-d-adenosine

(d-18):

Chloropurine derivative d-17 (180 mg, 0.528 mmol) was dissolved in a so-

lution of ammonia (7 m in MeOH, 6.5 mL) and the mixture was stirred

for 55 min at 1008C in a microwave reactor. The solvent was removed

under reduced pressure and the crude product was purified by column

2’,3’-Dideoxy-3’,4’-didehydro-carba-d-cytidine (d-16): A solution of ben-

zylated nucleoside d-15 (53.1 mg, 0.179 mmol) in Ac₂O/AcOH (5:1,

1 mL) was deprotected according to general debenzylation procedure 2

(see above) with a solution of ZnCl₂(122 mg, 0.893 mmol) in Ac₂O/

AcOH (5:1, 4 mL). The mixture was stirred for 24 h at room temperature

and the obtained intermediate was treated with a solution of NaOH (1%

in MeOH). After column chromatography on silica gel (CH₂Cl₂/MeOH,

chromatography on silica gel (CH₂Cl₂/MeOH, 20:1!15:1) to yield com-

25

589

pound d-18 (127 mg, 0.396 mmol, 75%) as a colorless solid. [a]

=

+29.88 (c=1.0, CHCl₃); m.p. 1298C; R_f(CH₂Cl₂/MeOH, 9:1)=0.25;

¹H NMR (400 MHz, CDCl₃): d=8.35 (s, 1H; H2), 7.86 (s, 1H; H8), 7.39–

7.28 (m, 5H; aromatic CH), 6.25 (br s, 2H; NH₂), 5.84–5.77 (m, 1H;

H3’), 5.40–5.30 (m, 1H; H1’), 4.54 (s, 2H; benzyl CH₂), 4.15–4.10 (m,

2H; H5’), 3.12–2.99 (m, 2H; H2’a, H6’a), 2.77–2.65 ppm (m, 2H; H2’b,

H6’b); ¹³C NMR (126 MHz, CDCl₃): d=155.8 (C6), 152.8 (C2), 149.8

(C4), 140.3 (C4’), 138.5 (C8), 138.1 (aromatic C_q), 128.5 (aromatic CH),

127.8 (aromatic CH), 127.7 (aromatic CH), 125.1 (C3’), 119.7 (C5), 72.7

(benzyl CH₂), 68.4 (C5’), 53.3 (C1’), 40.6 (C6’), 40.3 ppm (C2’); IR (film):

n˜ =2904, 1673, 1597, 1569, 1417, 1303, 1121, 796, 730, 656 cm^ꢀ1; HRMS

(FAB): m/z calcd for C₁₈H₁₉N₅O: 322.1668 [M+H]⁺; found: 322.1671.

5:1), debenzylated nucleoside d-16 (27.6 mg, 0.093 mmol, 52%) was ob-

25

589

tained as a colorless solid. [a] =+8.48 (c=0.28, MeOH); R_f(CH₂Cl₂/

MeOH, 5:1)=0.14; ¹H NMR (400 MHz, CD₃OD): d=7.55 (d, ³J=

7.3 Hz, 1H; H6), 5.95 (d, ³J=7.1 Hz, 1H; H5), 5.70–5.64 (m, 1H; H3’),

5.40–5.31 (m, 1H; H1’), 4.20–4.10 (m, 2H; H5’), 2.97–2.83 (m, 2H; H2’a,

H6’a), 2.51–2.41 ppm (m, 2H; H2’b, H6’b); ¹³C NMR (101 MHz,

CD₃OD): d=166.9 (C2), 159.1 (C4), 144.5 (C6), 143.9 (C4’), 96.9 (C5),

61.4 (C5’), 56.4 (C1’), 40.2 (C2’/C6’), 40.1 ppm (C2’/C6’); IR (film): n˜ =

3342, 1635, 1568, 1498, 1450, 1013, 787 cm^ꢀ1; UV/Vis (CH₃CN): l_max

=

271 nm; HRMS (FAB): m/z calcd for C₁₀H₁₃N₃O₂: 208.1086 [M+H]⁺;

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-l-adenosine (l-18): The

reaction was carried out as described for compound d-18 with chloropur-

ine-derivative l-17 (201 mg, 0.590 mmol). The residue was purified by

column chromatography on silica gel (CH₂Cl₂/MeOH, 20:1!15:1) to

found: 208.1091.

2’,3’-Dideoxy-3’,4’-didehydro-carba-l-cytidine (l-16): A solution of ben-

zylated nucleoside l-15 (82.1 mg, 0.276 mmol) in Ac₂O/AcOH (5:1,

1.5 mL) was deprotected according to general debenzylation procedure 2

(see above) with a solution of ZnCl₂(282 mg, 2.07 mmol) in Ac₂O/AcOH

(5:1, 3 mL). The mixture was stirred for 35 h at room temperature and

the obtained intermediate was treated with a solution of NaOH (1% in

MeOH). After column chromatography on silica gel (CH₂Cl₂/MeOH,

yield compound l-18 (129 mg, 0.401 mmol, 68%) as a colorless solid.

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589

[a] =ꢀ26.68 (c=1.0, CHCl₃). The spectroscopic data were identical to

those described for d-18.

2’,3’-Dideoxy-3’,4’-didehydro-carba-d-adenosine (d-19):

A solution of

benzylated nucleoside d-18 (103 mg, 0.320 mmol) in Ac₂O/AcOH (5:1,

1 mL) was deprotected according to general debenzylation procedure62

(see above) with a solution of ZnCl₂(136 mg, 1.60 mmol) in Ac₂O/AcOH

(5:1, 2 mL). The mixture was stirred for 30 h at room temperature and

the obtained intermediate was treated with a solution of NaOH (1% in

MeOH). After column chromatography on silica gel (CH₂Cl₂/MeOH,

5:1), debenzylated nucleoside l-16 (22.1 mg, 0.107 mmol, 39%) was ob-

25

tained as a colorless solid. [a] =ꢀ9.58(c=0.21, CHCl₃). The spectro-

589

scopic data were identical to those described for d-16.

General procedure for the coupling of chloropurine to cyclopentenol: To

a

suspension of PPh₃(3 equiv) in THF was slowly added DIAD

(2.8 equiv) at 08C under a nitrogen atmosphere and the mixture was

stirred for 0.5 h. The preformed complex was slowly added to a solution

of 6-chloropurine or 2-amino-6-chloropurine (1.5 equiv) and cyclopente-

nol (1 equiv) in THF at ꢀ508C. The mixture was stirred at ꢀ408C for

2 h, slowly warmed to room temperature, and stirred until complete con-

version was observed by TLC. The solvent was evaporated and the crude

product was purified by column chromatography on silica gel (petroleum

ether/EtOAc, 2:1!1:2).

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-d-6-chloro-6-deamino-

adenosine (d-17): The reaction was carried out according to the general

coupling procedure (see above) with a solution of compound (R)-8

(400 mg, 1.96 mmol) and 6-chloropurine (454 mg, 2.94 mmol) in THF

(35 mL) and a preformed complex of PPh₃(1.54 g, 5.87 mmol) and

DIAD (1.08 mL, 5.48 mmol) in THF (30 mL). The mixture was stirred

9:1), nucleoside d-19 (73.9 mg, 0.269 mmol, 66%) was obtained as a color-

25

589

less solid. [a] =+28.78 (c=0.23, DMSO); m.p. 2128C; R_f(CH₂Cl₂/

MeOH, 9:1)=0.18; ¹H NMR (400 MHz, [D₆]DMSO): d=8.13 (s, 1H;

H2), 8.09 (s, 1H; H8), 5.65–5.60 (m, 1H; H3’), 5.26–5.16 (m, 1H; H1’),

4.07–4.01 (m, 2H; H5’), 2.94–2.79 (m, 2H; H2’a, H6’a), 2.73–2.63 ppm

(m, 2H; H2’b, H6’b); ¹³C NMR (126 MHz, [D₆]DMSO): d=156.1 (C6),

152.4 (C2), 149.5 (C4), 144.0 (C4’), 139.1 (C8), 121.7 (C3’), 59.9 (C5’),

53.7 (C1’), 39.3 (C6’), 39.1 ppm (C2’); IR (film): n˜ =3093, 2923, 2850,

1675, 1600, 1569, 1307, 1074, 795, 731, 696, 545 cm^ꢀ1; UV/Vis (CH₃CN):

l_max=261, 210 nm; HRMS (FAB): m/z calcd for C₁₁H₁₃N₅O: 232.1198

[M+H]⁺; found: 232.1208.

2’,3’-Dideoxy-3’,4’-didehydro-carba-l-adenosine (l-19):

A solution of

benzylated nucleoside l-18 (86.2 mg, 0.268 mmol) in Ac₂O/AcOH (5:1,

1 mL) was deprotected according to general debenzylation procedure 2

(see above) with a solution of ZnCl₂(184 mg, 1.34 mmol) in Ac₂O/AcOH

(5:1, 3 mL). The mixture was stirred for 30 h at room temperature and

the obtained intermediate was treated with a solution of NaOH (1% in

MeOH). After column chromatography on silica gel (CH₂Cl₂/MeOH,

9:1), debenzylated nucleoside l-19 (48.3 mg, 0.209 mmol, 78%) was ob-

tained as a colorless solid. [a]²₅₈⁵₉=ꢀ25.88 (c=0.19, DMSO). The spectro-

scopic data were identical to those described for d-19.

for 3 days and, after purification, benzylated nucleoside d-17 (536 mg,

25

589

1.57 mmol, 80%) was obtained as a crystalline, colorless solid. [a]

=

+19.58 (c=1.0, CHCl₃); m.p. 678C; R_f(petroleum ether/EtOAc, 1:2)=

1

0.41; H NMR (400 MHz, CDCl₃): d=8.53 (s, 1H; H2), 7.97 (s, 1H; H8),

7.37–7.27 (m, 5H; aromatic CH), 5.84–5.80 (m, 1H; H3’), 5.43–5.36 (m,

1H; H1’), 4.55 (s, 2H; benzyl CH₂), 4.18 (s, 2H; H5’), 3.14–3.02 (m, 2H;

H2’a, H6’a), 2.77–2.67 ppm (m, 2H; H2’b, H6’b); ¹³C NMR (126 MHz,

CDCl₃): d=161.2 (C6), 152.0 (C2), 151.8 (C4), 140.4 (C8), 138.1 (aromat-

ic C_q), 128.6 (aromatic CH), 127.8 (aromatic CH), 125.1 (C3’), 72.8

(benzyl CH₂), 68.4 (C5’), 53.7 (C1’), 40.7 (C6’), 40.3 ppm (C2’); IR (film):

n˜ =2953, 2854, 1471, 1252, 1067, 892, 832, 773, 669 cm^ꢀ1; HRMS (FAB):

m/z calcd for C₁₈H₁₇ClN₄O: 341.1169 [M+H]⁺; found: 341.1182.

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-d-2-amino-6-chloro-6-

deamino-adenosine (d-20): The reaction was carried out according to the

general coupling procedure (see above) with a solution of (R)-8 (400 mg,

1.96 mmol) and 2-amino-6-chloropurine (498 mg, 2.94 mmol) in THF

(25 mL) and a preformed complex of PPh₃(1.54 g, 5.87 mmol) and

DIAD (1.08 mL, 5.48 mmol) in THF (30 mL). The mixture was stirred

for 5 days and, after purification, benzylated nucleoside d-20 (474 mg,

1.33 mmol, 68%) was obtained as a yellow syrup. [a]²₅₈⁵₉=+9.68 (c=1.0,

CHCl₃); R_f(petroleum ether/EtOAc, 1:2)=0.17; ¹H NMR (400 MHz,

CDCl₃): d=7.81 (s, 1H; H8), 7.38–7.28 (m, 5H; aromatic CH), 5.84–5.77

(m, 1H; H3’), 5.21 (br s, 3H; H1’, NH₂), 4.54 (s, 2H; benzyl CH₂), 4.16–

4.10 (m, 2H; H5’), 3.08–2.96 (m, 2H; H2’a, H6’a), 2.74–2.63 ppm (m, 2H;

H2’b, H6’b); ¹³C NMR (101 MHz, CDCl₃): d=159.0 (C2), 153.6 (C6),

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-l-6-chloro-6-deamino-

adenosine (l-17): The reaction was carried out according to the general

coupling procedure (see above) with a solution of compound (S)-8

(200 mg, 1.47 mmol) and 6-chloropurine (277 mg, 1.47 mmol) in THF

(12 mL) and a preformed complex of PPh₃(700 mg, 2.34 mmol) and

DIAD (0.540 mL, 2.74 mmol) in THF (10 mL). The mixture was stirred

for 3 days and, after purification, benzylated nucleoside l-17 (246 mg,

25

589

0.722 mmol, 74%) was obtained as a crystalline, colorless solid. [a]

=

&

12

&

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Chem. Eur. J. 0000, 00, 0 – 0

ÝÝ

These are not the final page numbers!

Synthesis of d- and l-Carbocyclic Nucleosides

FULL PAPER

151.3 (C4), 140.6 (C8), 140.3 (C4’), 138.1 (aromatic C_q), 128.6 (aromatic

CH), 127.9 (aromatic CH), 127.8 (aromatic CH), 125.1 (C3’), 124.4 (C5),

72.7 (benzyl CH₂), 68.4 (C5’), 53.4 (C1’), 40.4 (C6’), 40.0 ppm (C2’); IR

(film): n˜ =3318, 3199, 2854, 1607, 1559, 1455, 1403, 1229, 1070, 905, 731,

697 cm^ꢀ1; HRMS (FAB): m/z calcd for C₁₈H₁₈ClN₅O: 356.1278 [M+H]⁺;

found: 356.1276.

1267, 1170, 1101, 807, 732, 679 cm^ꢀ1

; HRMS (FAB): m/z calcd for

C₁₉H₂₀N₂O₅: 357.1451 [M+H]⁺; found: 357.1456.

General procedure for the synthesis of 3-methyl- and 5-chloro-substituted

cycloSal nucleotides: The carbocyclic nucleosides (1 equiv) were dis-

solved in CH₃CN, and the solution was cooled to ꢀ208C. Then, diisopro-

pylethylamine (DIPEA, 2 equiv) and a solution of either 3-methyl-sali-

genyl chlorophosphite or 5-chloro-saligenyl chlorophosphite (2 equiv) in

anhydrous CH₃CN were added. The mixture was warmed to room tem-

perature and stirred for 2 h at this temperature. Subsequently, a solution

of oxone (4 equiv) in cold water was added at ꢀ108C. Cooling was re-

moved and stirring was continued for an additional 15 min. The mixture

was directly extracted with EtOAc (2ꢂ5 mL) and cold water (2ꢂ5 mL).

The combined organic layers were dried over Na₂SO₄and the solvent

was removed under reduced pressure. The resulting solid was purified on

a chromatotron.

5’-O-Benzyl-2’,3’-dideoxy-3’,4’-didehydro-carba-l-2-amino-6-chloro-6-

deamino-adenosine (l-20): The reaction was carried out according to the

general coupling procedure (see above) with a solution of (S)-8 (400 mg,

1.96 mmol) and 2-amino-6-chloropurine (498 mg, 2.94 mmol) in THF

(25 mL) and a preformed complex of PPh₃(1.54 g, 5.87 mmol) and

DIAD (1.08 mL, 5.48 mmol) in THF (30 mL). The mixture was stirred

for 5 days and, after purification, benzylated nucleoside l-20 (460 mg,

1.29 mmol, 66%) was obtained as a yellow syrup. [a]²₅₈⁵₉=ꢀ10.48 (c=1.0,

CHCl₃). The spectroscopic data were identical to those described for d-

20.

3-Methyl-cycloSal-3’-deoxy-3’,4’-didehydro-carba-d-thymidine

mono-

phosphate (d-23): Following the general procedure for the synthesis of

cycloSal-nucleotides (see above), compound d-12 (70 mg, 0.31 mmol), 3-

methyl-saligenyl chlorophosphite (0.13 g, 0.63 mmol), DIPEA (0.11 mL,

0.63 mmol), oxone (0.78 g, 1.3 mmol), and CH₃CN (7 mL) were used.

The crude product was purified on a chromatotron (EtOAc) to yield

2’,3’-Dideoxy-3’,4’-didehydro-carba-d-guanosine (d-21): To compound d-

20 (179 mg, 0.502 mmol) was added formic acid (10 mL) and the mixture

was stirred at 808C for 5 h. The solvent was evaporated and the residue

was dissolved in ammonia (25% in MeOH, 10 mL) and the mixture was

stirred for 12 h at room temperature. The solvent was removed under re-

duced pressure and the crude product was purified by column chroma-

tography on silica gel (CH₂Cl₂/MeOH, 9:1!1:1) to yield compound d-21

(42.3 mg, 0.171 mmol, 34%) as a colorless solid. [a]²₅₈⁵₉=+23.88 (c=0.6,

DMSO); R_f(CH₂Cl₂/MeOH, 1:1)=0.67; ¹H NMR (400 MHz,

[D₆]DMSO): d=11.10 (br s, 1H; NH), 7.61 (s, 1H; H8), 6.63 (br s, 2H;

NH₂), 5.62–5.58 (m, 1H; H3’), 5.01–4.93 (m, 1H; H1’), 4.04–4.00 (m, 2H;

H5’), 2.87–2.75 (m, 2H; H2a’, H6a’), 2.59–2.53 ppm (m, 2H; H2b’, H6b’);

¹³C NMR (101 MHz, [D₆]DMSO): d=154.3 (C6), 151.0 (C4), 143.9 (C4’),

134.8 (C8), 121.4 (C3’), 116.7 (C5), 59.7 (C5’), 52.7 (C1’), 39.8 (C6’),

39.6 ppm (C2’); IR (film): n˜ =3115, 1689, 1605, 1534, 1371, 1183, 1023,

779, 696, 641 cm^ꢀ1; UV/Vis (MeOH): l_max=270, 252 nm; HRMS (FAB):

m/z calcd for C₁₈H₁₈ClN₅O: 248.1148 [M+H]⁺; found: 248.1156.

compound d-23 (28 mg, 0.070 mmol, 22%) as

a colorless foam. R_f

(CH₂Cl₂/MeOH, 9:1)=0.62; ¹H NMR (400 MHz, CDCl₃): d=8.95 (br s,

2ꢂ1H; 2ꢂNH), 7.19–7.17 (m, 2ꢂ1H; 2ꢂH4’’), 7.08–6.96 (m, 2ꢂ2H; 2ꢂ

H6, 2ꢂH5’’), 6.94–6.90 (m, 2ꢂ1H; 2ꢂH6’’), 5.88–5.85 (m, 1H; H3’),

5.85–5.81 (m, 1H; H3’), 5.43–5.24 (m, 2ꢂ3H; 2ꢂH1’, 2ꢂH7’’), 4.84–4.70

(m, 2ꢂ2H; 2ꢂH5’), 2.99–2.86 (m, 2ꢂ2H; 2ꢂH2a’, 2ꢂH6a’), 2.48–2.41

ꢀ

(m, 2ꢂ2H; 2ꢂH2b’, 2ꢂH6b’), 2.28 (s, 3H; Ar CH₃) 2.27 (s, 3H; Ar

CH₃), 1.88 ppm (d, ⁴J=1.4 Hz, 2ꢂ3H; 2ꢂthymine CH₃); ¹³C NMR

(101 MHz, CDCl₃): d=163.6 (2ꢂC4), 150.7 (2ꢂC2), 148.6 (2ꢂC2’’),

137.7 (d, ³J(C,P)=2.5 Hz, C4’), 137.7 (d, ³J

ACTHNUGRTNENUG AHCTUNGTRNEN(UGN C,P)=2.5 Hz, C4’), 136.4

(C4’’), 136.4 (C4’’), 131.2 (2ꢂC6), 128.4 (C3’), 128.1 (C3’), 127.9 (C3’’),

127.8 (C3’’), 123.9 (C5’’), 123.9 (C5’’), 122.8 (C6’’), 122.8 (C6’’), 120.5

2

(C1’’), 120.5 (C1’’), 111.9 (C5), 111.8 (C5), 68.7 (d, J

(C,P)=7.4 Hz, C7’’),

68.6 (d, ²J

(C,P)=7.4 Hz, C7’’), 66.3 (d, ²J(C,P)=4.8 Hz, C5’), 66.2 (d, ²J-

ACHUTGTNRENNUG CAHTUNGTRENNUGN

2’,3’-Dideoxy-3’,4’-didehydro-carba-l-guanosine (l-21): The reaction was

carried out as described for compound d-21 with compound l-20

(57.4 mg, 0.161 mmol) and formic acid (3 mL). After column chromatog-

raphy on silica gel, compound l-21 (16.7 mg, 67.8 mmol, 42%) was afford-

ed as a colorless solid. [a]²₅₈⁵₉=ꢀ16.98 (c=0.3, DMSO). The spectroscopic

data were identical to those described for d-21.

AHCTUNGERTG(NNUN C,P)=4.8 Hz, C5’), 52.8 (C1’), 52.6 (C1’), 39.5 (C2’), 39.4 (C2’), 39.2

ꢀ

(C6’), 39.1 (C6’), 15.3 (2ꢂAr CH₃), 12.5 (thymine CH₃), 12.4 ppm (thy-

mine CH₃); ³¹P NMR (162 MHz, CDCl₃): d=ꢀ8.43 (s), ꢀ8.65 ppm (s) (2

diastereomers in a ratio of 1.0:0.9); IR (film): n˜ =3043, 2925, 1672, 1469,

1294, 1267, 1188, 988, 935, 771, 727, 647 cm^ꢀ1; HRMS (FAB): m/z calcd

for C₁₉H₂₁N₂O₆P: 405.1216 [M+H]⁺; found: 405.1209.

5’-((2R)-Methoxyphenylacetate)-2’,3’-dideoxy-3’,4’-didehydro-carba-d-ur-

idine (d-22): Carbocyclic nucleoside d-14 (0.10 g, 0.48 mmol), (R)-(ꢀ)-

methoxyphenylacetic acid (0.80 g, 0.53 mmol), and N,N’-dicyclohexylcar-

bodiimide (DCC, 0.11 g, 0.53 mmol) were dissolved in CH₂Cl₂(12 mL).

4-Dimethylaminopyridine (DMAP, 6 mg, 0.05 mmol) was added and the

reaction mixture was stirred for 48 h at room temperature. After filtra-

tion, the solution was washed with saturated aqueous solutions of sodium

hydrogen carbonate (2ꢂ10 mL) and ammonium chloride (2ꢂ10 mL).

The organic fraction was dried with Na₂SO₄and the solvent was removed

under reduced pressure. The crude product was purified on a chromato-

tron (EtOAc) to yield compound d-22 (0.12 g, 0.35 mmol, 73%) as a col-

orless oil and as a mixture of two diastereomers in a ratio of 1.00:0.03. R_f

(CH₂Cl₂/MeOH, 9:1)=0.67; ¹H NMR (400 MHz, CDCl₃): d=8.85 (br s,

1H; NH), 8.80 (br s, 1H; NH), 7.37–7.30 (m, 2ꢂ5H; 2ꢂaromatic CH),

5-Chloro-cycloSal-3’-deoxy-3’,4’-didehydro-carba-d-thymidine

mono-

phosphate (d-24): Following the general procedure for the synthesis of

cycloSal-nucleotides (see above), compound d-12 (0.19 g, 0.85 mmol), 5-

chloro-saligenyl chlorophosphite (0.38 g, 1.7 mmol), DIPEA (0.29 mL,

1.7 mmol), oxone (2.1 g, 3.4 mmol), and CH₃CN (7 mL) were used. The

crude product was purified on a chromatotron (EtOAc) to yield com-

pound d-24 (0.13 g, 0.30 mmol, 35%) as a colorless foam. R_f(CH₂Cl₂/

MeOH, 9:1)=0.44; ¹H NMR (400 MHz, CDCl₃): d=8.22 (br s, 2ꢂ1H;

2ꢂNH), 7.32–7.30 (m, 1H; H4’’), 7.30–7.28 (m, 1H; H4’’), 7.11–7.09 (m,

2ꢂ1H; 2ꢂH6’’), 7.04–6.98 (m, 2ꢂ2H; 2ꢂH3’’, 2ꢂH6), 5.89–5.87 (m, 2ꢂ

1H; 2ꢂH3’), 5.41–5.31 (m, 2ꢂ3H; 2ꢂH1’, 2ꢂH7’’), 4.87–4.70 (m, 2ꢂ2H;

2ꢂH5’), 3.01–2.89 (m, 2ꢂ2H; 2ꢂH2a’, 2ꢂH6a’), 2.50–2.45 (m, 2ꢂ2H;

2ꢂH2b’, 2ꢂH6b’), 1.90 ppm (d, ⁴J=3.0 Hz, 2ꢂ3H; 2ꢂthymine CH₃);

¹³C NMR (101 MHz, CDCl₃): d=163.5 (C4), 163.5 (C4), 150.9 (C2), 150.8

(C2), 148.7 (C2’’), 148.6 (C2’’), 137.5 (C4’), 137.4 (C4’), 136.5 (2ꢂC6),

3

7.01 (d, J=7.5 Hz, 1H; H6), 6.90 (d, ³J=7.5 Hz, 1H; H6), 5.64–5.62 (m,

1H; H5), 5.60–5.57 (m, 1H; H5), 5.56–5.53 (m, 1H; H3’), 5.53–5.49 (m,

1H; H3’), 5.35–5.31 (m, 1H; H1’), 5.25–5.19 (m, 1H; H1’), 4.73–4.60 (m,

2ꢂ3H; 2ꢂCH, 2ꢂH5’), 3.34 (s, 3H; CH₃), 3.34 (s, 3H; CH₃), 2.98–2.92

(m, 2H; H2a’, H6a’), 2.87–2.79 (m, 1H; H2a’/H6a’), 2.71–2.64 (m, 1H;

H2a’/H6a’), 2.41–2.33 (m, 2H; H2b’, H6b’), 2.27–2.22 (m, 1H; H2b’/

H6b’), 2.04–2.00 ppm (m, 1H; H2b’/H6b’); ¹³C NMR (101 MHz, CDCl₃):

d=170.5 (C=O), 170.2 (C=O), 163.2 (C4), 163.2 (C4), 150.8 (C2), 150.7

(C2), 140.7 (C6), 140.5 (C6), 137.8 (C4’), 137.7 (C4’), 137.8 (aromatic C_q),

129.2 (aromatic CH), 129.1 (aromatic CH), 128.9 (aromatic CH), 128.9

(aromatic CH), 127.4 (aromatic CH), 127.3 (aromatic CH), 127.1 (C3’),

127.1 (C3’), 103.4 (C5), 103.3 (C5), 82.7 (CH), 82.6 (CH), 62.8 (C5’), 62.8

(C5’), 57.5 (CH₃), 57.4 (CH₃), 52.9 (C1’), 52.9 (C1’), 39.7 (C2’), 39.7 (C2’),

39.6 (C6’), 39.6 ppm (C6’); IR (film): n˜ =3055, 2928, 2852, 1456, 1374,

130.0 (2ꢂC4’’), 128.8 (C3’), 128.5 (C3’), 125.4 (2ꢂC6’’), 129.6 (C1’’), 129.5

3

(C1’’), 122.8 (C5’’), 122.7 (C5’’), 120.1 (d, J

A

³J

(d, ²J

²J

C

ACHTUNGTRENNUNG

A

ACHTUNGTRENNUNG

(C6’), 39.2 (C6’), 12.5 (thymine CH₃), 12.5 ppm (thymine CH₃); ³¹P NMR

(162 MHz, CDCl₃): d=ꢀ9.63 (s), ꢀ9.86 ppm (s) (2 diastereomers in

a ratio of 1.0:0.9); IR (film): n˜ =3055, 1670, 1479, 1265, 1186, 988, 934,

861, 815, 718, 613 cm^ꢀ1; HRMS (FAB): m/z calcd for C₁₈H₁₈ClN₂O₆P:

425.0659 [M+H]⁺; found: 425.0659.

3-Methyl-cycloSal-2’,3’-dideoxy-3’,4’-didehydrocarba-d-uridine

mono-

phosphate (d-25): Following the general procedure for the synthesis of

Chem. Eur. J. 2012, 00, 0 – 0

ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

&

13

&

ÞÞ

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C. Meier et al.

cycloSal-nucleotides (see above), compound d-14 (0.10 g, 0.48 mmol), 3-

methyl-saligenyl chlorophosphite (0.20 g, 0.96 mmol), DIPEA (0.16 mL,

0.96 mmol), oxone (1.2 g, 1.9 mmol), and CH₃CN (12 mL) were used.

The crude product was purified on a chromatotron (EtOAc) to yield

(162 MHz, D₂O): d=1.34 ppm; MS (ESI^ꢀ): m/z calcd for C₁₁H₁₄N₂O₆P^ꢀ:

301.0 [M]^ꢀ; found: 301.0.

2’,3’-Dideoxy-3’,4’-didehydro-carba-d-uridine monophosphate (d-28):

cycloSal-nucleotide d-25 (60 mg, 0.12 mmol) was dissolved in CH₃CN

(6 mL) and water (1 mL). To this solution was slowly added Et₃N (35

drops) and the mixture was stirred at room temperature until complete

hydrolysis of the triester was observed by TLC. The reaction mixture was

diluted with water (10 mL) and CH₃CN (10 mL) and the solution was

lyophilized. The crude product was purified by column chromatography

on RP-C18-silica gel (water). The obtained triethylammonium salt was

transferred into the sodium salt by using ion-exchange resin (Dowex

50X8). After lyophilization, compound d-28 (49 mg, 0.12 mmol 84%)

was obtained as a colorless solid. R_f(2-propanol/1m aqueous ammonium

acetate, 2:1)=0.45; ¹H NMR (400 MHz, D₂O): d=7.58 (d, ³J=8.0 Hz,

1H; H6), 5.85–5.73 (m, 2H; H3’, H5), 5.24–5.18 (m, 1H; H1’), 4.47 (d,

compound d-25 (87 mg, 0.22 mmol, 46%) as

a colorless foam. R_f

(CH₂Cl₂/MeOH, 9:1)=0.34; ¹H NMR (400 MHz, CDCl₃): d=8.11 (br s,

2ꢂ1H; 2ꢂNH), 7.23–7.18 (m, 2ꢂ1H; 2ꢂH4’’), 7.20 (d, ³J=8.1 Hz, 1H;

H6), 7.14 (d, ³J=8.1 Hz, 1H; H6), 7.06–7.03 (m, 2ꢂ1H; 2ꢂH5’’), 6.96–

6.90 (m, 2ꢂ1H; 2ꢂH6’’), 5.89–5.86 (m, 1H; H3’), 5.86–5.83 (m, 1H;

H3’), 5.70–5.66 (m, 2ꢂ1H; 2ꢂH5), 5.41–5.30 (m, 2ꢂ3H; 2ꢂH1’, 2ꢂ

H7’’), 4.85–4.71 (m, 2ꢂ2H; 2ꢂH5’), 3.03–2.86 (m, 2ꢂ2H; 2ꢂH2a’, 2ꢂ

H6a’), 2.48–2.42 (m, 2ꢂ2H; 2ꢂH2b’, 2ꢂH6b’), 2.29 ppm (s, 2ꢂ3H; 2ꢂ

Ar CH₃); ¹³C NMR (101 MHz, CDCl₃): d=162.7 (C4), 162.6 (C4), 151.8

ꢀ

(C2), 151.8 (C2), 148.8 (2ꢂC2’’), 140.7 (C6), 140.6 (C6), 137.8 (C4’),

137.8 (C4’), 131.3 (2ꢂC4’’), 129.6 (C3’’), 129.4 (C3’’), 128.3 (C3’), 128.0

(C3’), 124.0 (C5’’), 124.0 (C5’’), 122.9 (C6’’), 122.9 (C6’’), 121.6 (C1’’),

²J=7.5 Hz, 2H; H5’), 3.25–3.09 (m, 6H; HN

2H; H2a’, H6a’), 2.49–2.45 (m, 2H; H2b’, H6b’), 1.32–1.18 ppm (m, 9H;

HN

(CH₂CH₃)₃); ¹³C NMR (126 MHz, D₂O): d=166.4 (C4), 152.1 (C2),

144.1 (C6), 139.0 (C4’), 125.6 (C3’), 102.1 (C5), 63.6 (C5’), 54.7 (C1’), 46.6

ACHTNUGTRENN(UGN CH₂CH₃)₃), 2.97–2.91 (m,

121.5 (C1’’), 103.2 (C5), 103.2 (C5), 68.7 (d, ²J

(d, ²J(C,P)=6.7 Hz, C7’’), 66.1 (d, ²J

²J

ACHTUNGTRENNUNG(C,P)=4.3 Hz, C5’), 53.2 (C1’), 53.1 (C1’), 39.7 (C2’), 39.6 (C2’), 39.3

ACHTUNGTRENNUNG

A

ACHTUNGTRENNUNG

AHCTUNGTRENNUNG

(C6’), 39.2 (C6’), 15.0 ppm (2ꢂAr CH₃); ³¹P NMR (162 MHz, CDCl₃):

d=ꢀ8.47 (s), ꢀ8.60 ppm (s) (2 diastereomers in a ratio of 1.0:0.8); IR

ꢀ

(HNACHTUNGTRNNEU(G CH₂CH₃)₃), 38.6 (C2’), 38.5 (C6’) 8.2 ppm (HNCAHTUNGTREN(NNGU CH₂CH₃)₃);

³¹P NMR (162 MHz, D₂O): d=0.39 ppm; MS (ESI^ꢀ): m/z calcd for

C₁₀H₁₂N₂O₆P^ꢀ: 287.0 [M]^ꢀ; found: 287.0.

(film): n˜ =3052, 2925, 1673, 1459, 1266, 1188, 987, 933, 809, 767, 647 cm^ꢀ1

;

HRMS (FAB): m/z calcd for C₁₈H₁₉N₂O₆P: 391.1059 [M+H]⁺; found:

General procedure for the synthesis of nucleoside di- and triphosphates:

Freshly prepared bis(tetra-n-butylammonium)hydrogen phosphate

(1.5 equiv) or tris(tetra-n-butylammonium)hydrogen pyrophosphate

(1.5 equiv) was dried in vacuo for 3 h and further dried for 2 h in anhy-

drous DMF over activated molecular sieves (4 ꢃ). This solution was

added dropwise to a solution of the cycloSal nucleotide (1 equiv) in

DMF. After stirring at room temperature, the solvent was removed

under reduced pressure and the residue was extracted twice each with

EtOAc and water. The combined aqueous layer was dried by lyophiliza-

tion. Then, the crude product was converted into its corresponding

sodium salt by using ion-exchange resin (Dowex). After lyophilization,

the crude product was purified by column chromatography on RP-18

silica gel in a glass column (water).

391.1071.

5-Chloro-cycloSal-2’,3’-dideoxy-3’,4’-didehydro-carba-d-uridine

mono-

phosphate (d-26): Following the general procedure for the synthesis of

cycloSal-nucleotides (see above), compound d-14 (0.30 g, 1.4 mmol), 5-

chloro-saligenyl chlorophosphite (0.64 g, 2.9 mmol), DIPEA (0.49 mL,

2.9 mmol), oxone (3.5 g, 5.8 mmol), and CH₃CN (14 mL) were used. The

crude product was purified on a chromatotron (CH₂Cl₂, MeOH gradient

0–5%) to yield compound d-26 (0.20 g, 0.49 mmol, 34%) as a colorless

foam. R_f(CH₂Cl₂/MeOH, 9:1)=0.36; ¹H NMR (400 MHz, CDCl₃): d=

8.44 (br s, 2ꢂ1H; 2ꢂNH), 7.32–7.31 (m, 1H; H4’’), 7.30–7.28 (m, 1H;

H4’’), 7.21 (d, ³J=8.1 Hz, 1H; H6), 7.18 (d, ³J=8.1 Hz, 1H; H6), 7.11–

7.09 (m, 2ꢂ1H; 2ꢂH6’’), 7.01 (d, ³J=3.8 Hz, 1H; H3’’), 6.99 (d, ³J=

3.8 Hz, 1H; H3’’), 5.88–5.86 (m, 2ꢂ1H; 2ꢂH3’), 5.73–5.72 (m, 1H; H5),

5.71–5.69 (m, 1H; H5), 5.42–5.30 (m, 2ꢂ3H; 2ꢂH1’, 2ꢂH7’’), 4.86–4.70

(m, 2ꢂ2H; 2ꢂH5’), 3.03–2.91 (m, 2ꢂ2H; 2ꢂH2a’, 2ꢂH6a’), 2.49–

2.44 ppm (m, 2ꢂ2H; 2ꢂH2b’, 2ꢂH6b’); ¹³C NMR (101 MHz, CDCl₃):

d=162.7 (C4), 162.6 (C4), 150.4 (C2), 150.4 (C2), 148.6 (C2’’), 148.5

(C2’’), 140.8 (C6), 140.7 (C6), 137.6 (C4’), 137.5 (C4’), 130.0 (2ꢂC4’’),

128.6 (C3’), 128.4 (C3’), 127.1 (C1’’), 127.1 (C1’’), 125.4 (2ꢂC6’’), 122.8

3’-Deoxy-3’,4’-didehydro-carba-d-thymidine diphosphate (d-29): Bis(-

tetra-n-butylammonium)hydrogen phosphate (48 mg, 0.14 mmol) and

compound d-24 (40 mg, 0.094 mmol) were stirred in DMF (5 mL) for

16 h at room temperature. Nucleoside diphosphate d-29 (12 mg,

0.028 mmol, 30%) was obtained as a colorless solid. R_f(2-propanol/1m

aqueous ammonium acetate, 2:1)=0.15; ¹H NMR (400 MHz, D₂O): d=

7.43 (d, ⁴J=1.0 Hz, 1H; H6), 5.82–5.79 (m, 1H; H3’), 5.30–5.24 (m, 1H;

H1’), 4.55 (d, ²J=7.0 Hz, 2H; H5’), 3.00–2.95 (m, 2H; H2a’, H6a’), 2.53–

2.45 (m, 2H; H2b’, H6b’), 1.87 ppm (d, ⁴J=0.8 Hz, 3H; thymine CH₃);

¹³C NMR (101 MHz, D₂O): d=166.7 (C4), 152.2 (C2), 139.5 (C6), 139.4

(C4’), 125.3 (C3’), 111.6 (C5), 64.1 (C5’), 54.3 (C1’), 38.8 (C2’), 38.7 (C6’),

11.5 ppm (thymine CH₃); ³¹P NMR (162 MHz, D₂O): d=ꢀ10.3 (d, ²J=

22.0 Hz, 1P; P-b), ꢀ6.65 ppm (d, ²J=21.8 Hz, 1P; P-a); HRMS (ESI^ꢀ):

m/z calcd for C₁₁H₁₅N₂O₉P₂^ꢀ: 381.0258 [M]^ꢀ; found: 381.0257.

(C5’’), 122.7 (C5’’), 120.1 (d, ³J

2.4 Hz, C3’’), 103.3 (C5), 103.2 (C5), 68.0 (d, ²J

(d, ²J(C,P)=7.2 Hz, C7’’), 66.5 (d, ²J

(C,P)=4.3 Hz, C5’), 66.4 (d,

²J

ACHTUNGTRENNUNG(C,P)=4.3 Hz, C5’), 53.3 (C1’), 53.2 (C1’), 39.5 (C2’), 39.5 (C2’), 39.3

G

ACHTUNGTRNE(NUNG C,P)=

AHCTUNGTRENNUNG

G

ACHTUNGTRENNUNG

(C6’), 39.3 ppm (C6’); ³¹P NMR (162 MHz, CDCl₃): d=ꢀ9.61 (s),

ꢀ9.77 ppm (s) (2 diastereomers in a ratio of 1.0:0.9); IR (film): n˜ =3055,

1672, 1480, 1265, 1185, 987, 934, 860, 807, 716, 613 cm^ꢀ1; HRMS (FAB):

m/z calcd for C₁₇H₁₆ClN₂O₆P: 411.0513 [M+H]⁺; found: 411.0500.

2’,3’-Dideoxy-3’,4’-didehydro-carba-d-uridine diphosphate (d-30): Bis(-

tetra-n-butylammonium)hydrogen phosphate (0.11 g, 0.33 mmol) and

compound d-26 (90 mg, 0.22 mmol) were stirred in DMF (5 mL) for 16 h

at room temperature. Nucleoside diphosphate d-30 (15 mg, 0.041 mmol,

19%) was obtained as a colorless solid. R_f(2-propanol/1m aqueous am-

monium acetate, 2:1)=0.11; ¹H NMR (400 MHz, D₂O): d=7.60 (d, ³J=

8.0 Hz, 1H; H6), 5.82–5.79 (m, 2H; H3’, H5), 5.26–5.20 (m, 1H; H1’),

4.55 (d, ³J=3.9 Hz, 2H; H5’), 2.99–2.93 (m, 2H; H2a’, H6a’), 2.53–

2.46 ppm (m, 2H; H2b’, H6b’); ¹³C NMR (101 MHz, D₂O): d=166.5

(C4), 152.2 (C2), 144.2 (C6), 138.6 (C4’), 126.3 (C3’), 102.2 (C5), 64.8

(C5’), 54.8 (C1’), 38.8 (C2’), 38.6 ppm (C6’); ³¹P NMR (162 MHz, D₂O):

3’-Deoxy-3’,4’-didehydro-carba-d-thymidine

monophosphate

(d-27):

cycloSal-nucleotide d-23 (18 mg, 0.045 mmol) was dissolved in CH₃CN

(1 mL) and water (330 mL). To this solution was slowly added Et₃N

(20 drops) and the mixture was stirred at room temperature until com-

plete hydrolysis of the triester was observed by TLC. The reaction mix-

ture was diluted with water (5 mL) and CH₃CN (5 mL) and the solution

was lyophilized. The crude product was purified by column chromatogra-

phy on RP-C18-silica gel (water). The obtained triethylammonium salt

was transferred into the sodium salt by using ion-exchange resin (Dowex

50X8). After lyophilization, compound d-27 (7 mg, 0.2 mmol 39%) was

obtained as a colorless solid. R_f(2-propanol/1m aqueous ammonium ace-

tate, 2:1)=0.37; ¹H NMR (400 MHz, D₂O): d=7.40 (d, ⁴J=0.8 Hz, 1H;

d=ꢀ10.7 ppm (br s,

2

P; P-a, P-b); HRMS (ESI^ꢀ): m/z calcd for

C₁₀H₁₃N₂O₉P₂^ꢀ: 367.0113 [M]^ꢀ; found: 367.0093.

2

H6), 5.80–5.76 (m, 1H; H3’), 5.27–5.21 (m, 1H; H1’), 4.42 (d, J=8.0 Hz,

3’-Deoxy-3’,4’-didehydro-carba-d-thymidine triphosphate (d-31): Tris-

(tetra-n-butylammonium)hydrogen pyrophosphate (0.18 g, 0.12 mmol)

and compound d-24 (50 mg, 0.12 mmol) were stirred in DMF (5 mL) for

16 h at room temperature. Nucleoside diphosphate d-31 (12 mg,

0.023 mmol, 19%) was obtained as a colorless solid. R_f(2-propanol/1m

aqueous ammonium acetate, 2:1)=0.18; ¹H NMR (400 MHz, D₂O): d=

2H; H5’), 3.21–3.16 (m, 6H; HN

H6a’), 2.50–2.45 (m, 2H; H2b’, H6b’), 1.85 (d, ⁴J=0.8 Hz, 3H; thymine

CH₃), 1.28–1.24 ppm (m, 9H; HN

(CH₂CH₃)₃); ¹³C NMR (126 MHz,

D₂O): d=166.6 (C4), 152.1 (C2), 139.4 (C6), 139.3 (C4’), 125.3 (C3’),

111.5 (C5), 63.4 (C5’), 54.2 (C1’), 46.7 (HN(CH₂CH₃)₃), 38.6 (C2’), 38.6

(C6’), 11.4 (thymine CH₃), 8.2 ppm (HN

(CH₂CH₃)₃); ³¹P NMR

ACHTUGNTREN(NUNG CH₂CH₃)₃), 2.97–2.88 (m, 2H; H2a’,

AHCTUNGTRENNUNG

&

14

&

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Chem. Eur. J. 0000, 00, 0 – 0

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Synthesis of d- and l-Carbocyclic Nucleosides

FULL PAPER

7.41 (d, ⁴J=1.1 Hz, 1H; H6), 5.84–5.80 (m, 1H; H3’), 5.29–5.23 (m, 1H;

H1’), 4.58 (d, ²J=7.8 Hz, 2H; H5’), 2.99–2.92 (m, 2H; H2a’, H6a’), 2.52–

2.45 (m, 2H; H2b’, H6b’), 1.85 ppm (d, ⁴J=0.9 Hz, 3H; thymine CH₃);

¹³C NMR (101 MHz, D₂O): d=166.5 (C4), 152.2 (C2), 139.7 (C6), 138.2

(C4’), 125.8 (C3’), 111.6 (C5), 64.6 (C5’), 54.2 (C1’), 38.6 (C2’), 38.6 (C6’),

11.4 ppm (thymine CH₃); ³¹P NMR (162 MHz, D₂O): d=ꢀ22.9 (dd, ²J=

19.8 Hz, ²J=19.8 Hz, 1P; P-b), ꢀ10.9 (d, ²J=19.7 Hz, 1P; P-a),

ꢀ9.8 ppm (d, ²J=19.7 Hz, 1P; P-g); HRMS (ESI^ꢀ): m/z calcd for

C₁₁H₁₆N₂O₁₂P₃^ꢀ: 460.9933 [M]^ꢀ; found: 460.9909.

0.10 mmol, 48%) as a colorless solid. [a]²₅₈⁵₉=+7.88(c=1.0, MeOH). The

spectroscopic data were identical to that previously reported for the race-

mich nucleoside.^[11]

3’-Deoxy-carba-d-thymidine (d-37 and iso-d-37): Benzylated nucleoside

d-10 (109 mg, 0.671 mmol) was dissolved in MeOH (4 mL) and Pd/C

(5 mg) was added. The mixture was stirred for 2 days under a hydrogen

atmosphere at room temperature. The reaction mixture was filtered

through celite and washed with MeOH. The filtrate was concentrated

and the crude product was purified by column chromatography on silica

gel (CH₂Cl₂/MeOH, 9:1) to yield nucleosides d-37 and iso-d-37 (74 mg,

0.33 mmol, 95%) as a colorless solid and as a mixture of two dia-

stereomers in a ratio of 3:1. R_f(CH₂Cl₂/MeOH, 15:1)=0.12; ¹H NMR

(400 MHz, CD₃OH): d=7.51 (d, ⁴J=0.8 Hz, 1H; H6-A), 7.47 (d, ⁴J=

0.8 Hz, 1H; H6-B), 4.91–4.80 (m, 2H; H1’-A, H1’-B), 3.59–3.55 (m, 2H;

H5’-A), 3.48 (dd, ²J=6.7 Hz, ³J=3.8 Hz, 2H; H5’-B), 2.47–2.35 (m, 1H;

H4’-B), 2.26–1.96 (m, 5H; H4’-A, H6’a-A, H2’a-A, H3’a-B, H2’a-B),

1.92–1.88 (m, 6H; thymine CH3A, thymine CH3-B), 1.87–1.74 (m, 5H;

H3’a-A, H2’b-A, H3’b-B, H2’b-B, H6’a-B), 1.70–1.61 (m, 1H; H3’b-A),

1.55–1.46 (m, 1H; H6’b-A), 1.44–1.34 ppm (m, 1H; H6’b-B); ¹³C NMR

(101 MHz, CD₃OH): d=166.4 (C4-A/B), 153.1 (C2-A/B), 139.7 (C6-B),

139.6 (C6-A), 111.5 (C5-A/B), 66.7 (C5’-B), 66.6 (C5’-A), 58.0 (C1’-A),

57.7 (C1’-B), 41.5 (C4’-B), 41.2 (C4’-A), 35.2 (C6’-A), 34.4 (C6’-B), 32.2

(C2’-B), 30.6 (C2’-A), 28.6 (C3’-A), 27.5 (C3’-B), 12.3 ppm (thymine

CH₃-A/B); IR (film): n˜ =3174, 2949, 1656, 1472, 1395, 1269, 1222, 1124,

1056, 589, 420 cm^ꢀ1; UV/Vis (MeOH): l_max=269, 218 nm; HRMS (FAB):

m/z calcd for C₁₁H₁₆N₂O₃: 225.1239 [M+H]⁺; found: 225.1237.

2’,3’-Dideoxy-3’,4’-didehydro-carba-d-uridine triphosphate (d-32): Tris-

(tetra-n-butylammonium)hydrogen pyrophosphate (0.22 g, 0.33 mmol)

and compound d-26 (90 mg, 0.22 mmol) were stirred in DMF (5 mL) for

16 h at room temperature. Nucleoside diphosphate d-32 (20 mg,

0.039 mmol, 18%) was obtained as a colorless solid. R_f(2-propanol/1m

aqueous ammonium acetate, 2:1)=0.00; ¹H NMR (400 MHz, D₂O): d=

7.63 (d, ³J=8.0 Hz, 1H; H6), 5.85–5.83 (m, 2H; H3’, H5), 5.29–5.23 (m,

1H; H1’), 4.59 (d, ²J=7.9 Hz, 2H; H5’), 3.02–2.96 (m, 2H; H2a’, H6a’),

2.55–2.48 ppm (m, 2H; H2b’, H6b’); ¹³C NMR (101 MHz, D₂O): d=166.5

(C4), 152.2 (C2), 144.2 (C6), 138.8 (C4’), 125.8 (C3’), 102.2 (C5), 64.6

(C5’), 54.7 (C1’), 38.8 (C2’), 38.6 ppm (C6’); ³¹P NMR (162 MHz, D₂O):

d=ꢀ22.9 (dd, ²J=19.5 Hz, ²J=19.5 Hz, 1P; P-b), ꢀ10.9 (d, ²J=19.8 Hz,

1P; P-a), ꢀ9.9 ppm (d, ²J=19.7 Hz, 1P; P-g); HRMS (ESI^ꢀ): m/z calcd

for C₁₀H₁₄N₂O₁₂P₃^ꢀ: 446.9765 [M]^ꢀ; found: 446.9772.

(1S,3R,4R)-trans-3-(Benzyloxymethyl)-bicycloACTHNUTRGNEUG[N 3.1.0]hexanol (33): A so-

lution of Et₂Zn (1m in toluene, 1.63 mL, 1.80 mmol) was added dropwise

to a solution of cyclopentenol (S)-8 (330 mg, 1.62 mmol) in CH₂Cl₂

(10 mL) at ꢀ108C and the mixture was stirred for 15 min. To this mixture

was rapidly added a solution of diiodomethane (145 mL, 1.80 mmol) in

CH₂Cl₂(5 mL). After 15 min, Et₂Zn (1m in toluene, 1.63 mL, 1.80 mmol)

was added dropwise. A solution of diiodmethane (145 mL, 1.80 mmol) in

CH₂Cl₂(5 mL) was added rapidly and the mixture was stirred at 08C

overnight. After quenching with a saturated aqueous solution of NH₄Cl

(15 mL), the mixture was stirred at room temperature overnight. The

aqueous layer was washed with EtOAc (3ꢂ10 mL) and the organic phase

was washed with a saturated aqueous solution of NH₄Cl (10 mL), dried

with Na₂SO₄, and the solvent was removed under reduced pressure. Pu-

rification of the residue by column chromatography on silica gel (petrole-

(S)-3-(Benzyloxymethyl)-1-tert-butyldimethylsilyloxycyclopent-3-enol

(41): Alcohol (S)-8 (2.44 g, 11.9 mmol) was dissolved in DMF (35 mL)

under a nitrogen atmosphere. Afterwards, imidazole (2.11 g, 31.0 mmol)

and tert-butyldimethylsilylchloride (2.52 g, 16.7 mmol) were added at

room temperature and the reaction mixture was stirred for 24 h. The sol-

vent was evaporated and the residue was partitioned between CH₂Cl₂

(50 mL) and water (40 mL). The organic layer was washed with water

(2ꢂ30 mL) and dried with Na₂SO₄. The crude product was purified by

column chromatography on silica gel (petroleum ether/EtOAc, 4:1) to

25

589

yield compound 41 (3.72 g, 11.7 mmol, 98%) as a colorless oil. [a]

=

+1.68 (c=1.0, CHCl₃); R_f(petroleum ether/EtOAc, 4:1)=0.93; ¹H NMR

(500 MHz, CDCl₃): d=7.38–7.28 (m, 5H; aromatic CH), 5.60–5.57 (m,

1H; H4), 4.60–4.54 (m, 1H; H1), 4.49 (d, ³J=4.4 Hz, 2H; benzyl CH₂),

4.09–4.01 (m, 2H; OCH₂), 2.67–2.58 (m, 2H; H2a, H5a), 2.35–2.26 (m,

2H; H2b, H5b), 0.89 (s, 9H; TBDMS tBu), 0.06 ppm (s, 6H; 2ꢂTBDMS

CH₃); ¹³C NMR (101 MHz, CDCl₃): d=139.3 (aromatic C_q), 128.6 (aro-

matic CH), 128.0 (aromatic CH), 127.8 (aromatic CH), 125.4 (C4), 73.0

(C3), 72.1 (benzyl CH₂), 69.2 (OCH₂), 43.3 (C2/C5), 42.9 (C2/C5), 26.2

um ether/EtOAc, 2:1) afforded compound 33 (213 mg, 0.976 mmol, 60%)

25

589

as a colorless oil. [a] =ꢀ14.58 (c=1.0, CHCl₃). The spectroscopic data

were identical to those previously reported for the racemic hexanol.^[11]

(1R,3R,4R)-cis-3-(Benzyloxymethyl)-bicyclo

Mitsunobu-inversion reaction was carried out according to the general

procedure (see above) with solution of compound 33 (213 mg,

ACHTUNGERTN[NUNG 3.1.0]hexanol (34): The

a

0.976 mmol) and benzoic acid (328 mg, 3.13 mmol) in Et₂O (20 mL) and

a preformed complex of DIAD (575 mL, 2.92 mmol) and PPh₃(820 mg,

3.13 mmol) in Et₂O (30 mL). The reaction mixture was stirred for 16 h

and was treated with NaOH (1% in MeOH, 25 mL). The crude product

was purified by column chromatography on silica gel (petroleum ether/

(TBDMS C

(CH₃)₃), ꢀ4.5 (TBDMS CH₃), ꢀ4.5 ppm (TBDMS CH₃); IR

(film): n˜ =2928, 2854, 1361, 1252, 1194, 1067, 892, 832, 773, 733, 696 cm^ꢀ1

;

MS (FAB): m/z calcd for C₁₉H₃₀O₂Si: 319.2 [M+H]⁺; found: 319.2.

(1S,2R,4R)-2-(Benzyloxymethyl)-4-tert-butyldimethylsilyloxycyclopenta-

nol (42): Under a nitrogen atmosphere, BH₃·SACHTNUGTRNEG(UN CH₃)₂(450 mL, 4.73 mmol)

EtOAc, 2:1) to yield compound 34 (190 mg, 0.870 mmol, 89%) as a color-

25

589

less oil. [a] =+15.98 (c=1.0, CHCl₃). The spectroscopic data were

was added to 2-methyl-2-butene (985 mL, 9.30 mmol) at 08C and the solu-

tion was stirred 12 h at room temperature to form disiamylborane. Cyclo-

pentene 41 (603 mg, 1.89 mmol) was dissolved in THF (15 mL) and the

previously prepared disiamylborane was added dropwise at 08C. The so-

lution was stirred at this temperature for 2 days. The reaction mixture

was quenched with NaOH (1.7 mL, 3m) followed by H₂O₂(1.7 mL, 30%)

at 08C and the mixture was stirred overnight. After filtration, the phases

were separated and the aqueous layer was washed with EtOAc (4ꢂ

20 mL). The combined organic fractions were dried with Na₂SO₄and the

solvent was removed under reduced pressure. The residue was purified

by column chromatography on silica gel (petroleum ether/EtOAc, 4:1!

identical to those previously reported for the racemic hexanol.^[11]

(1S,3R,4R)-5’-O-Benzyl-3’-deoxy-3’,4’-methano-carba-d-thymidine (35):

The reaction was carried out according to the general coupling procedure

(see above) with a solution of compound 34 (200 mg, 0.916 mmol) and

N3-benzoyl-protected thymine (412 mg, 1.83 mmol) in CH₃CN (20 mL)

and

a preformed complex of PPh₃(0.721 g, 2.75 mmol) and DIAD

(0.505 mL, 2.57 mmol) in CH₃CN (30 mL. The mixture was stirred for

48 h and, after purification, benzylated nucleoside 35 (162 mg,

0.495 mmol, 54%) was obtained as a yellow oil. [a]²₅₈⁵₉=+17.28 (c=1.0,

CHCl₃). The spectroscopic data were identical to those previously report-

ed for the racemich nucleoside.^[11]

3:1) to yield compound 42 (467 mg, 1.39 mmol, 74%) as a pale yellow

25

589

oil. [a] =+5.08 (c=1.0, CHCl₃); R_f(petroleum ether/EtOAc, 4:1)=

(1S,3R,4R)-3’-Deoxy-3’,4’-methano-carba-d-thymidine (36): Benzylated

nucleoside 35 (106 mg, 0.335 mmol) was dissolved in MeOH (4 mL) and

formic acid (3 mL) and Pd/C (5 mg) were added. The mixture was stirred

for 24 h under a hydrogen atmosphere (balloon) at 508C. The reaction

mixture was filtered through celite and washed with MeOH. The filtrate

was concentrated and the crude product was purified by column chroma-

tography on silica gel (CH₂Cl₂/MeOH, 9:1) to yield nucleoside 36 (26 mg,

0.27; ¹H NMR (400 MHz, CDCl₃): d=7.38–7.27 (m, 5H; aromatic CH),

4.53 (s, 2H; benzyl CH₂), 4.38–4.31 (m, 1H; H1), 4.25–4.17 (dd, ³J=

13.6 Hz, ³J=7.0 Hz, 1H; H4) 3.58 (dd, ²J=8.8 Hz, ³J=5.3 Hz, 1H;

OCH₂a), 3.45 (d, ²J=9.0 Hz, ³J=8.8 Hz, 1H; OCH₂b), 2.28 (br s, 1H;

OH), 2.15–2.08 (m, 1H; H3a), 2.07–2.00 (m, 1H; H2), 1.95–1.88 (m, 1H;

H5a), 1.80–1.73 (m, 1H; H5b), 1.28–1.18 (m, 1H; H3b), 0.86 (s, 9H;

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TBDMS C

N

aromatic CH), 4.58–4.45 (m, 4H; 2ꢂbenzyl CH₂), 4.31–4.26 (m, 1H; H1),

4.00–3.95 (m, 1H; H3), 3.41 (dd, ²J=9.4 Hz, ³J=5.6 Hz, 1H; OCH₂a),

3.26 (dd, ²J=9.1 Hz, ³J=7.6 Hz, 1H; OCH₂b), 2.71–2.61 (m, 1H; H4),

2.10–1.97 (m, 2H; H2a, H5a), 1.88–1.80 (m, 1H; H2b), 1.55 ppm (ddd,

²J=13.6 Hz, ³J=7.9 Hz, ³J=5.6 Hz, 1H; H5b); ¹³C NMR (101 MHz,

CDCl₃): d=139.1 (aromatic C_q), 138.2 (aromatic C_q), 128.9 (aromatic

CH), 128.8 (aromatic CH), 128.2 (aromatic CH), 128.1 (aromatic CH),

82.4 (C1), 74.1 (benzyl CH₂), 73.70 (benzyl CH₂), 72.8 (C3), 71.5 (OCH₂),

44.9 (C4), 40.8 (C2), 37.8 ppm (C5); IR (film): n˜ =3410, 2857, 1497, 1453,

1358, 1205, 1065, 1027, 733, 696, 607, 456 cm^ꢀ1; HRMS (FAB): m/z calcd

for C₂₀H₂₄O₃: 313.1804 [M+H]⁺; found: 313.1799.

26.0 (TBDMS C

3406, 2954, 2928, 2855, 1471, 1361, 1252, 1096, 1044, 844, 774, 735,

697 cm^ꢀ1; HRMS (FAB): m/z calcd for C₁₉H₃₂O₃Si: 337.2199 [M+H]⁺;

found: 337.2200.

(1R,3S,4R)-3-Benzyloxy-4-(benzyloxymethyl)-1-tert-butyldimethylsilyl-

oxycyclopentanol (44): Alcohol 42 (100 mg, 0.297 mmol) was added drop-

wise to a suspension of NaH (8.6 mg, 0.39 mmol) in THF (8 mL) at 08C

under

a

nitrogen atmosphere. After 1 h, benzyl bromide (46.0 mL,

3’,5’-Di-O-benzyl-carba-d-thymidine (d-47): The reaction was carried out

according to the general coupling procedure (see above) with a solution

of compound 46 (321 mg, 1.03 mmol) and N3-benzoyl-protected thymine

(621 mg, 2.70 mmol) in CH₃CN (35 mL) and a preformed complex of

PPh₃(1.06 g, 4.05 mmol) and DIAD (744 mL, 3.78 mmol) in CH₃CN

(30 mL). The mixture was stirred for 2 days and, after purification, ben-

0.386 mmol) and tetrabutylammonium iodide (TBAI, 0.5 mg,

0.001 mmol) were added at room temperature and the mixture was

stirred overnight. The reaction was stopped by the addition of water and

the mixture was stirred for a further 1 hour. The aqueous layer was ex-

tracted with EtOAc (15 mL) and the combined organic fractions were

dried with Na₂SO₄. The solvent was removed under reduced pressure and

the residue was purified by column chromatography on silica gel (petro-

zylated nucleoside d-47 (422 mg, 0.804 mmol, 78%) was obtained as a col-

25

589

orless syrup. [a] =+13.68 (c=1.0, CHCl₃); R_f(petroleum ether/EtOAc,

leum ether/EtOAc, 4:1) to obtain compound 44 (116 mg, 0.271 mmol,

91%) as a colorless oil. [a] =+3.78 (c=1.0, CHCl₃); R_f(petroleum

1:2)=0.48; ¹H NMR (400 MHz, CD₃OD): d=8.53 (br s, 1H; NH), 7.40–

25

589

4

7.27 (m, 10H; aromatic CH), 7.09 (d, J=1.3 Hz, 1H; H6), 5.19–5.08 (m,

ether/EtOAc, 4:1)=0.80; ¹H NMR (400 MHz, CDCl₃): d=7.41–7.27 (m,

10H; aromatic CH), 4.53–4.47 (m, 4H; 2ꢂbenzyl CH₂), 4.41–4.33 (m,

1H; H1), 3.93–3.86 (m, 1H; H3), 3.52 (dd, ²J=9.1 Hz, ³J=7.6 Hz, 1H;

OCH₂a), 3.42 (dd, ²J=9.1 Hz, ³J=6.6 Hz, 1H; OCH₂b), 2.36–2.26 (m,

1H; H4), 2.14–2.10 (m, 1H; H5a), 1.97–1.89 (m, 1H; H2a), 1.88–1.79 (m,

1H; H2b), 1.39–1.30 (m, 1H; H5b), 0.86 (s, 9H; TBDMS tBu), 0.03 ppm

(s, 6H; 2ꢂTBDMS CH₃); ¹³C NMR (101 MHz, CDCl₃): d=139.0 (aro-

matic C_q), 138.8 (aromatic C_q), 128.6 (aromatic CH),128.5 (aromatic

CH),128.4 (aromatic CH),127.9 (aromatic CH), 127.8 (aromatic CH),

127.7 (aromatic CH), 127.6 (aromatic CH), 127.5 (aromatic CH), 81.7

(C3), 73.4 (OCH₂), 73.2 (benzyl CH₂), 72.6 (C1), 71.3 (benzyl CH₂), 44.6

1H; H1’), 4.56–4.43 (m, 4H; 2ꢂbenzyl CH₂), 4.02–3.97 (m, 1H; H3’),

3.59 (dd, ²J=9.3 Hz, ³J=4.5 Hz, 1H; H5’a), 3.54 (dd, ²J=9.4 Hz, ³J=

4.6 Hz, 1H; H5’b), 2.44–2.29 (m, 2H; H4’, H6’a), 2.23–2.15 (m, 1H;

H2’a), 2.01–1.92 (m, 1H; H2’b), 1.78 (d, ⁴J=1.3 Hz, 1H; thymine CH₃),

1.67–1.58 ppm (m, 1H; H6’b); ¹³C NMR (101 MHz, CD₃OD): d=164.1

(C4), 151.24 (C2), 138.3 (aromatic C_q), 138.2 (aromatic C_q), 137.2 (C6),

128.5 (aromatic CH), 128.4 (aromatic CH), 127.8 (aromatic CH), 127.6

(aromatic CH), 127.6 (aromatic CH), 110.8 (C5), 80.5 (C3’), 73.3 (C5’),

71.6 (benzyl CH₂), 71.0 (benzyl CH₂), 54.9 (C1’), 44.6 (C4’), 36.7 (C2’),

32.1 (C6’), 12.4 ppm (thymine CH₃); IR (film): n˜ =2858, 1672, 1553, 1362,

1270, 1066, 1027, 908, 729, 696, 646, 595, 421 cm^ꢀ1; HRMS (FAB): m/z

calcd for C₂₅H₂₈N₂O₄: 421.2127 [M+H]⁺; found: 421.2121.

(C4), 42.1 (C5), 37.9 (C2), 26.0 (TBDMS C

G

ꢀ4.7 ppm (TBDMS CH₃); IR (film): n˜ =2927, 2856, 1454, 1251, 1090,

1068, 775, 692, 603, 547 cm^ꢀ1; MS (EI): m/z calcd for C₂₆H₃₈O₃Si: 426

[M]; found: 426.

carba-d-Thymidine (d-3): Benzylated nucleoside d-47 (282 mg,

0.671 mmol) was dissolved in MeOH (7 mL) and Pd/C (5 mg) was added.

The mixture was stirred for 2 days under a hydrogen atmosphere at room

temperature. The mixture was filtered through celite and washed with

MeOH. The filtrate was concentrated and the crude product was purified

by column chromatography on silica gel (CH₂Cl₂/MeOH, 9:1!6:1) to

(1R,3S,4R)-3-Benzyloxy-4-(benzyloxymethyl)-cylopentanol (45): A solu-

tion of compound 44 (0.952 g, 2.23 mmol) in THF (10 mL) was slowly

treated with TBAF (5.6 mL, 5.6 mmol, 1m in THF) under a nitrogen at-

mosphere at room temperature and the mixture was stirred for 8 h. The

solvent was evaporated and the crude product was purified by column

chromatography on silica gel (petroleum ether/EtOAc, 1:2) to yield com-

pound 45 (633 mg, 2.12 mmol, 95%) as a colorless oil. [a]²₅₈⁵₉=+36.98 (c=

1.0, CHCl₃); R_f(petroleum ether/EtOAc, 2:1)=0.18; ¹H NMR

(400 MHz, CDCl₃): d=7.37–7.27 (m, 10H; aromatic CH), 4.53 (s, 2H;

yield nucleoside d-3 (134 mg, 0.556 mmol, 83%) as a colorless solid.

25

[a] =+5.98(c=1.0, MeOH); R_f(CH₂Cl₂/MeOH, 9:1)=0.21; ¹H NMR

589

(400 MHz, CDCl₃): d=7.52 (d, ⁴J=1.0 Hz, 1H; H6), 5.12–5.01 (m, 1H;

H1’), 4.22–4.14 (m, 1H; H3’), 3.69 (dd, ²J=10.9 Hz, ³J=5.3 Hz, 1H;

H5’a), 3.63 (dd, ²J=10.9 Hz, ³J=6.1 Hz, 1H; H5’b), 2.24 (ddd, ²J=

3

12.7 Hz, J=7.5 Hz, J=7.5 Hz, 1H; H6’a), 2.15–2.02 (m, 2H; H4’, H2’a),

2.02–1.93 (m, 1H; H2’b), 1.89 (d, ⁴J=1.0 Hz, 1H; thymine CH₃), 1.64–

1.54 ppm (m, 1H; H6’b); ¹³C NMR (101 MHz, CDCl₃): d=166.4 (C2),

152.9 (C4), 111.5 (C5), 73.7 (C3’), 64.3 (C5’), 55.9 (C1’), 50.3 (C4’), 40.0

(C2’), 33.4 (C6’), 12.3 ppm (thymine CH₃); IR (film): n˜ =3383, 2910,

2496, 1669, 1625, 1470, 1264, 1029, 761, 589, 426 cm^ꢀ1; UV/Vis (MeOH):

l_max=272, 211 nm; HRMS (FAB): m/z calcd for C₁₁H₁₆N₂O₄: 241.1188

[M+H]⁺; found: 241.1192.

2

benzyl CH₂), 4.50 (d, J=11.7 Hz, 1H; benzyl CH₂), 4.45 (d, J=11.7 Hz,

1H; benzyl CH₂), 4.35–4.29 (m, 1H; H1), 4.12–4.06 (m, 1H; H3), 3.55

(dd, ²J=9.0 Hz, ³J=3.9 Hz, 1H; OCH₂a), 3.50 (dd, ²J=9.0 Hz, ³J=

4.3 Hz, 1H; OCH₂b), 2.40 (br s, 1H; OH), 2.37–2.25 (m, 2H; H4, H5a),

2.11–2.04 (m, 1H; H2a), 1.90–1.83 (m, 1H; H2b), 1.55–1.48 ppm (m, 1H;

H5b); ¹³C NMR (101 MHz, CDCl₃): d=138.8 (aromatic C_q), 138.0 (aro-

matic C_q), 128.6 (aromatic CH), 128.5 (aromatic CH), 127.9 (aromatic

CH), 127.9 (aromatic CH), 127.8 (aromatic CH), 127.7 (aromatic CH),

82.1 (C1), 73.5 (benzyl CH₂), 72.5 (OCH₂), 72.4 (C3), 71.6 (benzyl CH₂),

44.7 (C4), 42.8 (C2), 37.5 ppm (C5); IR (film): n˜ =3395, 2857, 1453, 1348,

1204, 1062, 1027, 733, 695, 607 cm^ꢀ1; MS (FAB): m/z calcd for C₂₀H₂₄O₃:

313.1 [M+H]⁺; found: 313.1.

Antiviral testing: For the in vitro infection assays, roughly 1ꢂ10⁷LuSIV

cells^[31](a CEMx174-derived cell-line) were resuspended in the culture

medium (500 mL) without the presence of any additional drugs and the

medium was incubated at 378C for 3 h in the presence of X4 tropic HIV-

1 viral stocks that corresponded to p24 NL4/3 (14 ng). After infection,

the cells were washed twice with PBS, seeded into 24 well plates at a den-

sity of 1ꢂ10⁶cellsml^ꢀ1, and cultivated in the presence of specific antiviral

compounds or DMSO as a solvent control. At day 3 post-infection, the

culture medium was replaced and the cells cultures were divided. At

day 6 post-infection, the p24-levels in the supernatant were determined

by an enzyme-linked immunosorbent assay (Innogenetics NV). Cellular

viability was tested in parallel by using AlamarBlue (AbD Serotec).

LuSIV cells were maintained in an RPMI solution (Invitrogen) that con-

tained 10% FCS (Biochrom AG), penicillin (100 unitsmL^ꢀ1), and strep-

tomycin (100 mgmL^ꢀ1, Invitrogen). The HIV-1 isolate NL4/3 was ob-

tained from the NIH AIDS Research and Reference Reagent Program.

(1S,3S,4R)-3-Benzyloxy-4-(benzyloxymethyl)-cyclopentanol (46): The

Mitsunobu-inversion reaction was carried out according to the general

procedure (see above) with

a solution of compound 45 (587 mg,

1.88 mmol) and benzoic acid (459 mg, 3.76 mmol) in Et₂O (35 mL) and

a preformed complex of DIAD (728 mL, 3.76 mmol) and PPh₃(1.10 g,

4.18 mmol) in Et₂O (60 mL). The reaction mixture was stirred for 16 h

and treated with NaOH (1% in MeOH, 25 mL). The crude product was

purified by column chromatography on silica gel (petroleum ether/

EtOAc, 2:1!1:2) to yield compound 46 (601 mg, 1.92 mmol, 92%) as

25

589

a colorless oil. [a] =+19.98 (c=1.0, CHCl₃); R_f(petroleum ether/

EtOAc, 1:2)=0.47; ¹H NMR (400 MHz, CDCl₃): d=7.37–7.27 (m, 10H;

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Synthesis of d- and l-Carbocyclic Nucleosides

FULL PAPER

MDR HIV-1 strains E2–2 and 8ka3 were a kind gift from Dr. Hauke

Walter (University Erlangen-Nꢄrnberg). IC₅₀values were calculated by

fitting the data to a dose-response curve with the software Graphpad

Prism 5. MDR HIV-1 strains were constructed as described by Walter

and Schmidt);^[32]they contained the following known drug-resistant mu-

tations: E2–2: L10I, K20I, I54 V, A71V, T74S, V82F, L90M, K65R,

D67N, T69N, V75I, K103N, V108I, Q151M, M184V, K219E; 8ka3: L10F,

V11I, I13V, K20I, M36I, K43T, I54 V, A71T, G73S, V82C, I84 V, L89V,

L90M, M41L, D67N, T69N, K70R, V75M, K101E, V108I, Y181C,

M184V, G190A, L210W, T215Y, K219E. Classical thymidine analogue

mutations (TAMs) are italicized.

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The authors (C.M., M.M., and B.R.) are grateful to the German Federal

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to the University of Hamburg for financial support. J.v.L. and P.H. were

funded by the German Federal Ministry of Education and Research

(Grant No. 01GU0716). We thank Dr. Hauke Walter (University Erlan-

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Received: March 5, 2012

Published online: && &&, 0000

Chem. Eur. J. 2012, 00, 0 – 0

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www.chemeurj.org

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C. Meier et al.

Drug Resistance

M. Mahler, B. Reichardt, P. Hartjen,

J. van Lunzen, C. Meier* . . . &&&& — &&&&

Stereoselective Synthesis of d- and l-

Carbocyclic Nucleosides by Enzymati-

cally Catalyzed Kinetic Resolution

Fighting chance: Various carbocyclic

d- and l-nucleosides were synthesized

from cyclopentadiene by enzyme-cata-

lyzed kinetic resolution with high opti-

cal purity. In contrast to its l-analogue,

d-carba-dT was antivirally active

against multidrug-resistant HI-viruses.

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Chem. Eur. J. 0000, 00, 0 – 0

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Stereoselective synthesis of D - And L -carbocyclic nucleosides by enzymatically catalyzed kinetic resolution

DOI: 10.1002/chem.201200733

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Article abstract of DOI:10.1002/chem.201200733

Full text of DOI:10.1002/chem.201200733

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