Synthesis and biological activities of 5′-ethylenic and acetylenic modified L-nucleosides and isonucleosides

Tetrahedron 60 (2004) 8535–8546

Synthesis and biological activities of 5⁰-ethylenic and acetylenic

modiﬁed ^L-nucleosides and isonucleosides

*

Jun-Feng Wang, Xiao-Da Yang, Liang-Ren Zhang, Zhen-Jun Yang and Li-He Zhang

National Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, People’s Republic of China

Received 30 March 2004; revised 22 June 2004; accepted 24 June 2004

Available online 3 August 2004

Abstract—Two series of 6⁰-halovinyl-adenosine stereoisomers including 5⁰-ethylenic and acetylenic substituted L-adenosine, 5⁰-ethylenic

and acetylenic substituted isonucleosides were synthesized. In the ^L-nucleoside series, compounds 6b, 8b, 10b and 13b showed modest

inhibition of SAH hydrolase (21, 44, 50 and 26% respectively) at 100 mM. The ^L-isomers of 5⁰-ethylenic and acetylenic modiﬁed

isonucleoside 23, 24 exhibited no activity for the inhibition of SAH hydrolase, however, the ^D-isomers 30 and 31 showed some activities in

the same test (35 and 21%). It indicated clearly the strict stereochemical requirement for the substrate of SAH hydrolase. Compounds 6b, 8b,

8c, 11b exhibited modest to good inhibition effects on the growth of HeLa cells or Bel-7420 cells at 1 mM (64, 44, 53 and 82% respectively).

1. Introduction

S-Adenosyl-^L-methionine is involved in the methylation of

many biomolecules and SAH is a potent feedback inhibitor

of crucial transmethylation enzymes.^3–5De Clercq and

Cools found that some adenosine analogues showed the

inhibition of vaccinia virus replication and a good

correlation between the antiviral effectiveness and their

ability to inhibit SAH hydrolase.⁶According to the

mechanism of the hydrolysis of SAH by SAH hydrolase,

many adenosine analogues, including some acyclic sugar

mimics, have been designed and displayed interesting

broad-spectrum antiviral properties. These ﬁrst-generation

inhibitors act as substrates for the ﬁrst step in the hydrolysis

reaction, where, the enzyme oxidizes the 3⁰-hydroxyl group

of SAH to ketone and converts the NAD^Cto NADH in the

process.^7,8However, some dihalohomovinyl adenosine

analogues were reported as the inhibitors for the ‘type II’

inactivation of SAH hydrolase involving its ‘5⁰/6⁰-hydro-

lytic activity’. The type II inhibitors contain an electrophilic

entity at 5⁰-position of adenosine which could bin₀d

covalently to the enzyme without prior oxidation at 3 -

position.⁹This discovery may open a way to the rational

design of antiviral and antitumour drugs. Many 5⁰-halovinyl

adenosine analogues or its conjugated diene analogues

and acetylenic analogues were reported.^10–15It was sug-

gested that enzyme-mediated addition of water across the

5⁰,6⁰-double bond could generate electrophilic acyl halids or

a-halo ketone species that could undergo nucleophilic attack

by proximal groups on the enzyme and addition of water

across the 5⁰,6⁰-triple bond followed by tautomerization of

the hydroxyvinyl intermediates could also generate similar

electrophiles at the enzyme active site. ^L-Nucleosides are

the enantiomers of the natural nucleosides. Among these

Methylated 5⁰-cap structure plays an essential role for the

stability of mRNA against phosphatases and ribonucleases,

for proper binding to ribosomes, and for the promotion of

splicing. Hence, an uncapped mRNA is much less likely to

be translated into its respective protein. Since many types of

viruses also require methylated 5⁰-capped mRNA for proper

translation into proteins, interference with the formation of

these 5⁰-caps could lead to the inhibition of replication.^1,2

The various methyltransferases which catalyze these

reactions have been targeted for drug design. The normal

cellular role of SAH hydrolase is regulating S-adenosyl-^L-

methionine dependent biological methylation reactions.

Keywords: Stereoisomers; Biological activity; L-Nucleoside series.

Abbreviations: Ado, adenosine; AIBN, 2,2⁰-azobisisobutyronitrile; BSA,

N,O-bis(trimethylsilyl)acetamide; ACN, acetonitrile; DCC, N,N⁰-dicyclo-

hexylcarbodiimide; DCE, 1,2-dichloroethane; DCM, dichloromethane;

DEAD, diethyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride;

DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; Hcy, homocysteine;

HBV, heptitis B virus; HIV, human immunodeﬁciency virus; HPLC, high-

performance liquid chromatography; HRMS (TOF), high resolution mass

spectrometry; NBS, N-bromosuccinimide; NIS, N-iodosuccinimide;

NAD^C, the oxidized form of NAD; NADH, the reduced form of NAD;

NAD, nicotinamide–adenine dinucleotide; NMR, nuclear magnetic reson-

ance; PBE, 20 mM phosphate buffer and 5 mM EDTA; Py, pyridine; RP-

HPLC, reverse phase high-performance liquid chromatography; SAH, S-

adenosyl-^L-homocysteine; 3TC, (K)-b-L-1,3-oxathiolanyl-cytosine; TFA,

triﬂuoroacetic acid; TMSOTf, trimethylsilyl triﬂuromethanesulfonate; Ts,

toluenesulfonyl; TSA, p-toluenesulfonic acid; THF, tetrahydrofuran.

* Corresponding author. Tel.: C86-10-82801700; fax: C86-10-828-

02724; e-mail: zdszlh@bjmu.edu.cn

doi:10.1016/j.tet.2004.06.131

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J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

compounds, the separated enantiomer of L-2⁰-deoxy-3⁰-

thiacytidine (3TC) demonstrated more potent activities against

both HIV and HBV than their corresponding ^D-conﬁguration

counterparts with much less host toxicity.¹⁶Since then, a

number of ^L-nucleoside analogues have been synthesized

and biologically evaluated.^17–21Isonucleosides represent a

novel class of carbohydrate modiﬁed nucleosides in which

the nucle₀obase is linked to various positions of ribose other

than C-1 . We have reported a series of syntheses of iso-

nucleosides.^22–25The conformations of such L-nucleosides

and isonucleosides exhibit profound changes compared to

natural nucleosides. These alternations in conformation

might be interesting to understand the recognition of

L-nucleosides and isonucleosides as substrates by SAH

hydrolase and explain the speciﬁc substrates requirement of

SAH hydrolase. Herein, we report the synthesis of such

L-nucleoside and isonucleoside analogues and their inacti-

vation of SAH hydrolase.

to synthesize the 5⁰-ethylenic adenosine, we tried ﬁrst to

isolate 5⁰-aldehyde adenosine after Moffatt oxidation of

compound 3. But many efforts failed due to the instability of

5⁰-aldehyde intermediate. However, the 5⁰-aldehyde inter-

mediate in solution (without separation) could react with a

stable Wittig reagent [(p-tolylsulfonyl)methylene]triphenyl

phosphorane^27,28to give compound 4 in 97% yield (Scheme

1

1). H NMR data showed that compound 4 had E-form

0

double bond signals at 5 and 6 position (dZ6.30, H₆; dZ

0

6.99, H₅; J_{5 ,6}Z15.0 Hz). Isomerization of compound 4

was observed in basic condition. When compound 4 was

treated with 1 M NaOH, a pure 6⁰-tosylallylic adenosine 5a

was obtained in 70% yield. 5a was deprotected to give 5b

which can b₀e conﬁrmed by ¹³C NMR, the double bond

0

shifted to 4 ,5 position (dZ161.0, C₄; dZ86.4, C₅).

According to the computer simulation, the Z-conﬁguration

of the double bond in compound 5b is thermodynamicly

more stable than the E-conﬁguration. Treatment of 4 with

sodium borohydride in aqueous methanol resulted in

reduction of the tosylvinyl to give 6a in 93% yield.

Deprotection of 6a gave 6b in 54% yield.

2. Chemistry

2.1. Synthesis of 5⁰-ethylenic and acetylenic substituted

L-adenosine analogues

Stannyldesulfonylation (Bu₃SnH/AIBN/toluene)²⁹of 4 pro-

vided the 6-N-benzoyl-9-[6⁰-(E)-(tributylstannyl)-5⁰,6⁰-

dideoxy-2⁰,3⁰-O-isopropylidene-b-L-ribo-hex-5⁰(E)-enofur-

anosyl] adenine 7. Compound 7 was reacted with N-bromo-

succinimide (NBS), N-iodosuccinimide (NIS) and chlorine

respectively to provide the 6-N-benzoyl-9-[6⁰-halo-5⁰,6⁰-

dideoxy-2⁰,3⁰-O-isopropylidene-b-L-ribo-hex-5⁰(E)-eno-

furanosyl] adenine 8a, 9a, 10a (89, 98 and 77% respec-

tively) and only E-form isomers were formed (Scheme 2).

According to a known procedure, condensation of 1,2,3,5-

tetra-O-acetyl-^L-ribose 1 with benzoyl adenine provided the

fully protected nucleoside₀2 ₀in 86% yield.²⁶After removal

of the acetyl groups, the 2 ,3 -hydroxy groups of compound

2 were protected in the presence of HC(OEt)₃and a catalytic

amount of TSA at room temperature to give 6-N-benzoyl-

2⁰,3⁰-O-isopropylidene-L-adenosine 3 in 81% yield. In order

Scheme 1. Reagents and conditions: (i) BSA, 80 8C, DCE; TMSOTf, 80 8C, toluene. (ii) K₂CO₃/MeOH, rt; TSA, HC(OEt)₃, rt. (iii) DCC, Cl₂CHCOOH,

DMSO; TsCH]PPh₃, rt. (iv) 1 N NaOH/H₂O, ACN, rt. (v) NH₃/MeOH, rt; CF₃COOH/H₂O, 0 8C. (vi) NaBH₄, MeOH/H₂O, rt. (vii) CF₃COOH/H₂O, 0 8C.

(viii) Bu₃SnH, AIBN, toluene, reﬂux.

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

8537

Scheme 2. Reagents and conditions: (i) Cl₂, DCM/CCl₄, K50 8C. (ii) NBS, DCM/CCl₄, K30 8C. (iii) NIS, DCM/CCl₄, K20 8C. (iv) NH₄F, EtOH, reﬂux. (v)

Pb(AcO)₄, MeCN, rt. (vi) CF₃COOH/H₂O, 0 8C; NH₃/MeOH. (vii) NH₃/MeOH.

After deprotection, 8b, 9b, 10b were obtained and in the

case of compound 8a, the deprotected product was a mixture

of E- and Z-form isomers, Z-form isomer 8c was separated

in 16% yield. Destannnylation of 7 with ammonium ﬂuoride

in ethanol at reﬂux gave compound 11a and treatment of 7

with lead tetraacetate in acetonitrile resulted in oxidative

destannylation to give the 6-N-benzoyl-9-(5⁰,6⁰-dideoxy-

2⁰,3⁰-O-isopropylidene-b-L-ribo-hex-5⁰-ynofuranosyl)

adenine 12a. Deprotection of 11a and 12a provided 11b and

12b respectively (Scheme 2). To extend the structure types

of 5⁰-modiﬁed L-nucleosides, 5⁰-iodo-L-adenosine 13b was

synthesized from compound 3 by the substitution of iodine

Scheme 3. Reagents and conditions: (i) Ph₃P, I₂, 1,4-dioxane, py, rt. (ii) NH₃/MeOH, rt; CF₃COOH/H₂O, 0 8C. (iii) DCC, DMSO, Cl₂CHCOOH,

EtO₂CCH]PPh₃. (iv) DIBAL-H, DCM, K78 8C. (v) Phthalimide, Ph₃P, DEAD, THF, rt. (vi) CH₃NH₂/EtOH, rt. (vii) CF₃COOH/H₂O, rt.

8538

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

Table 1. ¹HNMR Spectral data^a,bof 5⁰-modiﬁed L-nucleosides

f

c

d

c

H1⁰

H2⁰

H3⁰

H4⁰

H5⁰

H6⁰

0

Compound

3^g

H2^e

H8^e

NH₂

Others^e

0

(J_{1 –2}

0

(J_{2 –3}

0

(J_{3 –4}

0

(J_{4 –5}

0

(J_{5 –6}

0

(J_{6 –4})

)

5.94 (4.8)

6.17 (1.5)

5.23^h(5.7)

5.13 (2.1)

5.22 (3.6)

5.78^d(1.8)

4.86ⁱ(4.2)

3.81ⁱ, 4.03^j

8.07

8.08

8.79

8.60

9.04^k

9.23^k

1.28, 1.66 (CH₃),

7.54–7.63ⁱ(Bz)

1.38, 1.60, 2.39^l

(CH₃),

4^g

5.50 (6.0)

6.99 (15.0)

6.30 (1.8)

3.84^d

7.21–7.64ⁱ(Bz)

1.52, 1.61, 2.39^l

(CH₃),

5a^g

5b

6.24^e

5.61^c(6.0)

4.56 (7.5)

5.24^c

4.52^c

4.85^h(8.1)

4.85^h(5.5)

8.02

8.07

8.75

8.14

8.91^k

7.35

7.18–8.00ⁱ(Bz)

2.31^l(CH₃), 5.62

(OH3⁰) 5.71

6.00 (4.0)

3.90ⁱ

(OH2⁰),

7.21–7.64ⁱ(Bz)

1.35, 1.57, 2.44^l

(CH₃) 7.27–7.68ⁱ

(Bz)

6a^g

6b

5.95 (2.4)

5.79 (4.5)

5.43 (6.3)

4.63 (5.0)

4.90 (3.9)

4.10 (5.5)

4.21ⁱ(7.2)

3.90ⁱ(6.0)

2.16ⁱ(8.4)

1.95ⁱ(8.0)

3.12ⁱ

3.34ⁱ

7.87

8.02

8.19

8.27

7.27

7.30

2.38^l(CH₃), 5.23

(OH3⁰), 5.45

(OH2⁰),

7.44–7.75ⁱ(Bz)

1.37, 1.63 (CH₃),

0.79–1.52ⁱ

7^g

6.19 (1.8)

6.15 (1.5)

5.57 (6.3)

5.04 (3.0)

5.12 (3.3)

4.72 (6.0)

4.69 (6.0)

6.03 (18.9)

6.27^d(1.2)

8.10

8.35

8.83

8.96^k

9.03^k

(Bu₃Sn),

7.57–8.05ⁱ(Bz)

1.41, 1.63 (CH₃),

7.58–8.03ⁱ(Bz)

8a^g

6.34–6.36ⁱ

8b

8c

5.90 (5.0)

5.91 (6.0)

6.15 (1.5)

4.70 (10.0)

4.85 (5.0)

5.59 (6.3)

4.19 (5.0)

4.14 (3.5)

5.13 (3.0)

4.36 (8.0)

4.73 (8.0)

4.69 (6.9)

6.56 (13.5)

6.72^h(7.0)

6.66 (14.7)

6.71^c

6.75^c

6.38^d

8.15

8.08

8.37

8.84

7.31

8.97^k

9a^g

1.43, 1.63 (CH₃),

7.58–8.03ⁱ(Bz)

9b

10a^g

5.89 (5.1)

6.14 (2.1)

4.70 (5.4)

5.56 (6.3)

4.18 (4.5)

5.10 (3.3)

4.32 (7.5)

4.71 (7.8)

6.85 (14.4)

6.08 (13.2)

6.67^d

6.23^d

8.15

8.24

8.35

8.81

7.31

9.14^k

1.40, 1.64 (CH₃),

7.54–8.03ⁱ(Bz)

10b

11a^g

5.89 (4.5)

6.19 (1.5)

4.38 (8.3)

5.59 (6.3)

3.36–3.41

6.31 (13.0)

5.89^m

(17.1, 10.8)

6.08^m

(17.0, 10.5)

6.60^d

5.16, 5.27

8.15

8.11

8.36

8.84

7.31

8.92^k

5.05 (3.6)

4.12^h(4.5)

5.13 (1.2)

4.72 (6.9)

4.32^h(6.5)

5.07^c

1.42, 1.63 (CH₃),

7.57–8.03ⁱ(Bz)

11b

6.25 (5.0)

6.28^e

4.68^h(5.0)

5.75^c(5.7)

5.19, 5.30

2.44

8.16

8.30

8.31

8.82

7.29

12a^g

9.07^k

1.41, 1.58 (CH₃),

7.47–8.00ⁱ(Bz)

12b

13a^g

5.94 (5.0)

6.21 (2.4)

4.78^h(5.0)

5.52 (5.7)

4.35^h(4.0)

5.11 (3.3)

4.55

4.45^j(10.2)

3.75^c(2.0)

8.21

8.19

8.32

8.83

7.59

9.08^k

3.29, 3.46

3.45, 3.60

6.96 (15.6)

1.42, 1.64 (CH₃),

7.58–8.03ⁱ(Bz)

5.50 (OH3⁰),

5.79 (OH2⁰)

1.40, 1.63 (CH₃),

1.22^h(H9⁰),

4.11ⁿ(H8⁰)

13b

14^g

5.90 (6.0)

6.13 (1.8)

4.80 (6.0)

5.56 (6.3)

4.16 (5.0)

5.14 (3.6)

3.98^j(10.5)

4.81ⁱ

8.14

7.86

8.36

8.33

7.30

5.59

5.81^d(1.5)

15^g

16^g

17a

6.10 (2.1)

6.07 (2.0)

6.09 (1.5)

5.85–5.86

4.72ⁱ

5.02 (6.3)

5.54^d(2.1^o)

5.73^j(5.5)

7.87

7.85

7.89

8.36

8.25

8.35

5.60

5.47

5.80

1.40, 1.63 (CH₃),

3.97^c,p(H7⁰,2.4)

1.37, 1.60 (CH₃),

7.72–7.85ⁱ(Bz)

1.39, 1.62 (CH₃),

3.28^e(H7⁰), 1.99^f

(CH₂NH₂)

5.49 (6.0)

5.52 (5.7)

4.98 (3.5)

5.00 (3.3)

4.68 (7.0)

4.70 (6.3)

5.84 (15.5)

5.76–5.86^j

3.46^c(H7⁰)

17b

5.94 (5.1)

4.66^h(5.1)

4.14^h(4.5)

4.37^h(5.7)

6.07 (15.3)

5.77^j,p(6.9)

8.19

8.35

5.80

^aChemical shifts(d) in DMSO-d₆at 300 MHz (unless otherwise noted). ^b‘Apparent’ ﬁrst-order coupling constants (Hz, in parentheses). ^cDoublet (unless

otherwise noted). ^dDoublet of doublets (unless otherwise noted). ^eSinglet (unless otherwise noted). ^fBroad singlet. ^gIn CDCl₃. ^hTriplet (unless otherwise noted).

ⁱMultiplet. ^jDoublet of triplets. ^kNH. ^lPhCH₃. ^mDoublet of doublets of doublets. ⁿQuartet. ^o3J_{H6 –H7}

0

.

1

(Scheme 3). For the synthesis of compound 17b, the 2⁰,3⁰-O-

isopropylidene-^L-adenosine was oxidized by Moffat reac-

tion and followed by the treatment of Wittig reagent to give

the a,b-unsaturated ester 14 in 50% yield. Reduction of

compound 14 with DIBAL-H gave the allylic alcohol 15,

which was converted into phthalimide 16 by a Mitsunobu

reaction in 81% yield. After the cleavage with methylamine,

and deprotection with TFA, compound 17b was obtained

in 81% yield (Scheme 3). The H and ¹³C NMR data of

L-adenosine derivatives were listed in Tables 1 and 2.

2.2. Synthesis of 5⁰-ethylenic and acetylenic substituted

isonucleosides

The [5-(R)-dimethoxy-4-(R)-hydroxy-3(S)-adenin-9-yl]tetra-

hydrofuran 18 was synthesized starting from D-xylose²²

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

Table 2. ¹³C NMR Spectral data^a,bof 5⁰-modiﬁed L-nucleosides

8539

c

C1⁰

C2⁰

C3⁰

C4⁰

C5⁰

C6⁰

Compound

C2

C4

C5

C6

C8

5b^d

6b^e

7^f,g

8b

8c

152.8

152.5

152.8

152.6

152.7

152.8

152.0

152.7

153.2

153.1

153.2

152.8

149.1

149.6

149.3

149.4

149.3

149.5

152.6

151.3

149.4

149.5

119.1

119.3

123.4

119.2

119.4

119.2

119.0

119.1

122.6

122.4

123.4

120.2

119.2

156.1

152.8

156.1

155.4

156.1

155.4

155.6

155.5

156.1

139.3

144.4

142.3

140.0

140.3

143.7

140.0

139.9

139.6

139.9

140.0

139.9

139.8

88.2

88.0

90.8

87.6

87.7

87.6

87.4

90.6

90.5

90.4

87.5

71.9

72.7

84.6

73.4

74.2

73.2

73.7

73.9

73.5

73.2

84.3

84.6

84.5

84.6

73.9

69.4

72.6

84.2

72.6

72.7

72.6

72.7

72.8

73.1

72.7

83.9

84.2

84.1

84.2

72.8

161.0

81.6

90.5

83.6

82.2

85.7

82.3

84.8

81.1

83.9

86.3

87.5

87.2

87.7

83.5

86.4

21.1

52.6

51.6

133.6

109.9

111.1

82.1

121.5

117.1

75.2

143.8

136.0

133.6

140.0

132.2

136.7

78.7

9b

10b

11b

12b

13b

14^g,h

15^g,i

16^g,j

17a^k

17b^l

7.8

143.4

133.7

132.0

136.0

133.6

131.9

127.4

120.2

126.4

124.7

a

Chemical shifts (d) in DMSO-d₆at 75 MHz.

Proton-decoupled singlets.

Assignment might be reversed.

Peaks also at d 21.5, 127.86, 129.3, 135.9, 144.0 (PhCH₃).

Peaks also at d 26.3, 127.7, 129.9, 135.9, 144.4 (PhCH₃).

b

c

d

e

f

Peaks also at d 9.4, 13.6, 27.2, 28.9 (Bu₃Sn); 25.4, 27.1, 114.5 (CMe₂); 127.8, 128.9, 132.8, 133.8, 164.3 (PhCO).

In CDCl₃.

Peaks also at d 14.1, 60.6, 165.6 (CO₂CH₂CH₃); 25.3, 27.1, 114.7 (CMe₂).

Peaks also at d 25.4, 27.1, 114.5 (CMe₂); 62.4 (CH₂OH).

Peaks also at d 25.3, 27.0, 114.4 (CMe₂); 38.6 (CH₂NR); 123.4, 127.4, 130.4, 134.0 (Ph); 155.9, 167.0 (CO).

Peaks also at d 25.4, 27.1, 114.5 (CMe₂); 43.2 (CH₂NR).

Peaks also at d 63.1 (CH₂NR).

g

h

i

j

k

l

with a published procedure in our laboratory. Protection of

18 with benzoyl chloride gave compound 19. One-pot

reaction of 19 by treatment with 1% HCl/THF at 70–90 8C

and followed by reduction with NaBH₄provided the

isonucleoside 20 in 52% yield. As the procedure described

above, Moffatt oxidation was applied to 20 and followed by

Using the similar procedure as described above, the

syntheses of 30 and 31 were shown in Scheme 5. The H

1

and ¹³C NMR data of iso-adenosine derivatives were listed

in Tables 3 and 4.

2.3. Biological results and discussion

treatment

with

wittig

reagent

[(p-tolylsul-

fonyl)methylene]triphenylphosphorane to give the 6⁰-(E)-

tosylvinyl derivative 21 in 85% yield. Stannyldesulfonyla-

tion (Bu₃SnH/AIBN/toluene) of 21 provided compound 22.

Compound 22 was treated in the presence of lead

tetraacetate at ambient temperature to provide the 5⁰-vinyl

isonucleoside 23. Treatment of 22 with ammonium ﬂuoride

in ethanol at reﬂux gave the 5⁰-ethynyl isonucleoside 24

(Scheme 4). The compound 25, enantiomer of isonucleoside

18, were synthesized using our published procedure.²⁵

Compounds 5b, 6b, 8b, 8c, 9b, 10b, 11b, 12b, 13b, 17b and

23, 24, 30, 31 were evaluated for the inactivation of SAH

hydrolase and known active compound, 6⁰(E)-bromovinyl

D-adenosine (D–B),³⁶was used as control (Table 5). In the

L-nucleoside series, compounds 6b, 8b, 10b and 13b

showed modest inhibition of SAH hydrolase (21, 44, 50

and 26% respectively) at 100 mM. Compound 8c, the Z-form

isomer of 8b, and compound 9b exhibited no inhibition in

this assay. Wnuk et al. reported the synthesis of 6⁰-(E) and

Scheme 4. Reagents and conditions: (i) BzCl, Py, 0–40 8C. (ii) 1% HCl, THF, 70–90 8C. (iii) DCC, DMSO, Cl₂CHCOOH; TsCH]PPh₃. (iv) Bu₃SnH, AIBN,

toluene, reﬂux. (v) NH₄F, EtOH, reﬂux. (vi) Pb(OAc)₄, ACN, rt. (vii) NaOCH₃/MeOH, rt.

8540

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

Scheme 5. Reagents and conditions: (i) BzCl, Py, 0–40 8C. (ii) 1% HCl, THF, 70–90 8C. (iii) DCC, DMSO, Cl₂CHCOOH; TsCH]PPh₃. (iv) Bu₃SnH, AIBN,

toluene, reﬂux. (v) NH₄F, EtOH, reﬂux. (vi) Pb(OAc)₄, ACN, rt. (vii) NaOCH₃/MeOH, rt.

(Z)-halohomovinyl derivatives of adenosine and indicated

that the order of inhibitory potency of these derivatives for

the inactivation of S-adenosyl-^L-homocysteine hydrolase

was IOBrOClOF and E form OZ form.¹⁴However, in our

L-nucleoside series, the inhibitory potency of SAH hydro-

lase was weaker than that of their ^D-isomers and the order of

inhibitory potency seemed confusion. The activities of

chloro-substituted derivative 10b and the bromo partner 8b

were almost the same but iodo-substituted compound 9b

showed no activity. According to the mechanism studies on

the inhibition of SAH hydrolase,³³the acetylenic derivative

of adenosine showed marked inhibition at 100 mM¹⁴but the

L-isomer of 9-(5⁰,6⁰-dideoxy-b-D-ribo-hex-5⁰-ynofuranosy-

l)adenine 12b was inactive at the same condition. The 5⁰-

ethylenic and acetylenic modiﬁed isonucleoside analogues

23, 24, 30, 31 were also evaluated in this assay. The ^L-

isomers of 5⁰-ethylenic and acetylenic modiﬁed isonucleo-

side 23, 24 exhibited no activity for the inhibition of SAH

hydrolase, however, the ^D-isomers 30 and 31 showed some

activities in the same test (35 and 21%). Computer modeling

study showed that each pair of ^D- and L-enantiomer of 6⁰-

halohomovinyl derivative of adenosine was ﬁtted together

by pairing base moiety and 5⁰-moiety (Fig. 1), but sugar ring

was out from the model. ^L-5⁰-Ethylenic and acetylenic

isonucleoside analogues could not ﬁt to the 5⁰-acetylenic

modiﬁed adenosine derivative but the ^D-isomer was more

similar to the ^D-nucleoside partner (Fig. 2). In the cases of L-

2⁰-deoxy-3⁰-thiacytidine (3TC) and other L-nucleoside

antiviral drugs, the monophosphorylation of a nucleoside

analogues was the crucial step for the biological activity and

the certain kinase could recognize both of the ^D- and L-

isomers without limitation of conﬁguration,³⁰however, this

study indicated clearly the strict stereochemical requirement

for the substrate of SAH hydrolase.³¹

Wnuk et al. reported the observation of a direct

correlation of cytostatic activity with inhibition of SAH

hydrolase and found that the most potent inhibitors of

Figure 1. Overlap of structure of D- and L-6⁰(E)-bromovinyl adenosine. The

modeling was performed based on the energy optimized structure.

Enatiomers were ﬁt together by pairing N3, N6, N7, N9, C5⁰and C6⁰

(the modeling was performed on SGI Indy workstation and MSI Insight II

was used).

Figure 2. Overlap of structure of 6⁰-ethynyl D-adenosine. The modeling

was performed based on the energy optimized structure. Enantiomers were

ﬁt together by pairing N3, N6, N7, N9, C5⁰and C6⁰(the modeling was

performed on SGI Indy workstation and MSI Insight II was used).

1

Table 3. HNMR Spectral data^a,bof 5⁰-modiﬁed isonucleosides

Others^g

f

e

H2⁰a^c,d(J_{2 –3}

)

H2⁰b (J_{2 –3}

)

H3⁰^e(J_{3 –4}

)

H4⁰(J_{4 –5}

)

H5⁰^e(J_{5 –6}

)

0

H6⁰

H7⁰

0

Compound

H2

H8

NH₂

19

20

21

22

23^l

24^l

26

28

29

30^l

31^l

4.39 (6.0, 10.5)

4.14 (5.7, 10.5)

4.44–4.46 (5.1, 10.5)

4.44 (5.7, 10.5)

4.29 (3.0)

4.04 (3.3)

5.72 (2.4)

5.75 (4.2)

5.36 (7.5)

5.34ⁱ(7.8)

4.88 (6.0)

4.59 (4.2)

5.73 (2.4)

5.36 (7.8)

5.34 (7.8)

4.88 (6.0)

4.59 (5.0)

5.32 (5.4)

5.34 (6.0)

4.77^j(4.5)

4.60ⁱ(4.5)

4.36 (6.5)

4.85 (6.0)

5.33 (5.4)

4.76 (4.5)

4.60ⁱ(4.5)

4.36 (6.5)

4.85 (5.0)

4.28 (3.6)

4.08 (4.2)

5.56 (3.0)

5.49 (4.5)

4.11^j(6.5)

4.40 (4.2)

4.28^c(3.6)

5.56 (3.0)

5.48 (4.8)

4.11^j(6.5)

4.40

4.77^c

4.36^c

8.32

8.25

7.84

8.01

8.13

8.14

8.32

7.84

8.01

8.14

8.33

8.32

8.29

8.39

8.14

8.15

8.33

8.29

8.36

8.15

6.35^h

6.59^h

5.83^h

5.69^h

7.24^h

7.25^h

6.35^h

5.88^h

5.57^h

7.24^h

7.25^h

3.51, 3.55^g(OCH₃), 7.47–8.03ⁱ(Bz)

7.42–8.01ⁱ(Bz)

7.28^k(15.0)

6.25^k

6.80^d

6.53^d

5.17

2.63^g(PhCH₃), 7.27–8.03ⁱ(Bz)

0.94–1.48ⁱ(Bu₃Sn), 7.55–8.06ⁱ(Bz)

4.33 (2.7)

4.19–4.20 (6.0, 10.5)

4.30 (5.7, 10.5)

4.38 (6.0, 10.5)

5.92^i,k(17.5, 10.5)

4.20 (3.3)

4.29 (3.0)

2.49^g

6.20^g(OH4⁰)

4.79^c

7.47–8.03ⁱ(Bz)

4.44–4.46^c(5.1, 10.5)

4.44 (5.7, 10.5)

7.30^k(15.0)

6.25^k(19.2)

5.92^i,k(17.5, 10.5)

6.83^e

6.53^e

5.17^e

2.49^g

2.44^g(PhCH₃), 7.27–8.03ⁱ(Bz)

0.85–1.46ⁱ(Bu₃Sn), 7.27–8.05ⁱ(Bz)

6.21^g(OH4⁰)

4.32 (2.7)

4.19^e(6.0, 10.5)

4.30 (5.0, 10.5)

4.20 (2.0)

^aChemical shifts (d) in CDCl₃at 300 MHz (unless otherwise noted). ^b‘Apparent’ ﬁrst-order coupling constants (Hz, in parentheses). ^cDoublet (unless otherwise noted). ^d2J_{H2 –H2}. Doublet of doublets (unless otherwise

e

0

j

k3

J

l

n2

J

o2

J

noted). ^fBroad singlet. ^gSinglet (unless otherwise noted). ^hNH₂. ⁱMultiplet. Triplet (unless otherwise noted).

. In Me₂SO-d₆. ^mDoublet of triplets.

.

0

H6 –H7

H6 –H6

H7 –H7

Table 4. ¹³CNMR Spectral data^a,bof 5⁰-modiﬁed isonucleosides

C2⁰

C3⁰

C4⁰

C5⁰

C6⁰

C7⁰

Compound

C2

C4

C5

C6

C8

CH₃

20^c,d

21^c,e

22^c,g

23

152.5

153.3

153.1

152.4

153.3

153.1

152.4

149.3

149.8

149.3

149.5

149.8

148.9

149.5

149.4

118.6

119.6

118.9

119.0

119.3

119.6

118.9

119.0

119.3

156.0

155.5

156.0

155.6

156.0

139.3

140.2

140.3

139.4

139.2

140.2

141.9

139.4

139.2

70.4

70.9

71.4

68.7

69.0

70.9

71.3

68.7

69.0

60.5

59.9

60.3

61.7

61.2

59.9

60.8

61.7

61.2

79.1

84.4

59.9

128.3

142.2

136.5

77.9

128.3

142.3

136.5

77.9

81.6^f

82.5

78.6

79.9

81.5

82.6

78.6

79.9

82.6^f

86.3

84.7

81.4

82.6

86.1

84.6

81.4

144.7

133.9

116.9

73.7

144.7

134.0

116.8

73.7

24

28^c,h

29^c,i

30

31

^aChemical shifts (d) in DMSO-d₆at 75 MHz. ^bProton-decoupled singlets. ^cPeaks also at d 128.5, 129.7, 133.8, 135.4, 166.1 (PhCO). ^dPeaks also at d 128.8, 128.9, 129.5, 133.8, 165.2 (PhCO). ^ePeaks also at d 12.6, 127.9,

128.6, 129.9, 130.0, 132.4, 134.0, 136.8, 138.3, 165.6 (PhCO). ^fAssignment might be reversed. ^gPeaks also at d 9.5, 13.6, 17.5, 19.1, 26.8, 27.2 27.7, 27.8, 29.0, 29.7, (Bu₃Sn), 128.6, 129.9, 130.9, 133.8, 165.6 (PhCO).

i

^hPeaks also at d 127.8, 128.6, 129.9, 130.0, 132.4, 134.0, 136.8, 138.3, 165.6 (PhCO). Peaks also at d 9.5, 13.6, 17.5, 19.2, 26.8, 27.2, 27.7, 29.0, 29.7 (Bu₃Sn), 128.8, 129.9, 130.9, 134.0, 165.6 (PhCO).

8542

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

Table 5. Inhibition of S-adenosyl-L-homocysteine hydrolase by synthetic adenosine analogues^a

Conc.\compound

D–B

6b

8b

10b

11b

100 mM

10 mM

1 mM

94%

92%

68%

21%

—

44%

10%

—

50%

16%

—

30%

—

b

Conc\compound

100 mM

10 mM

13b

26%

24%

—

23

12%

—

24

20%

11%

—

30

35%

16%

—

31

21%

12%

—

1 mM

—

a

The data shown are the average results of three experiments.

Dash means no activity in this condition.

b

SAH hydrolase also showed the most potent cytostatic

activities.¹⁴In our case, compounds 6b, 8b, 8c, 9b, 10b,

11b, 12b, 13b, 17b and 23, 24, 30, 31 were screened by

culture of tumor cells (Table 6), only HeLa cells and

Bel-7420 cells were sensitive to these compounds.

Compounds 6b, 8b, 8c, 11b exhibited modest to good

inhibition effects on the growth of HeLa cells or Bel-

7420 cells at 1 mM (64, 44, 53 and 82% respectively),

compounds 9b, 12b and 13b were active at 10 mM (55,

64 and 73%). Therefore, the cytostatic activites of ^L-nucleo-

side and isonucleoside derivatives reported may correlate

with inhibition of SAH hydrolase and other mechanisms.

active at 10 mM (55, 64 and 73%). Therefore, the cyto-

static activites of ^L-nucleoside and isonucleoside deriva-

tives reported may correlate with inhibition of SAH

hydrolase and other mechanisms.

4. Experimental

4.1. General

Uncorrected melting points were determined on a XT-4A

melting point apparatus. NMR spectra were recorded on a

Varian 300 MHz spectrometer. Mass spectra were obtained

on PE SCLEX QSTAR mass spectrometer. Elemental

analyses were performed on Varian ELIII analyzer.

Solvents were dried by reﬂux over CaH₂and distilled

before use, chromatographic puriﬁcations were carried out

using silica gel (200–300 mesh, Qingdao chemicals).

Preparative RP-HPLC was performed with spectra physics

SP 8800 ternary pump system and Pynamax C₁₈columns.

3. Conclusions

The synthesis of stereoisomers of 6⁰-halovinyl modiﬁed

adenosine analogues could explain the speciﬁc substrates

requirement of SAH hydrolase. We designed and

synthesized two series of 6⁰-halovinyl D-adenosine

stereoisomers including 5⁰-ethylenic and acetylenic sub-

stituted ^L-adenosine and 5⁰-ethylenic substituted iso-

nucleosides. In the ^L-nucleoside series, compounds 6b,

8b, 10b and 13b showed modest inhibition of SAH

hydrolase (21, 44, 50 and 26% respectively) at 100 mM.

The isonucleoside analogues 31, 32 showed some weaker

activities in the same test (35 and 21%). It indicated

clearly the strict stereochemical requirement for the

substrate of SAH hydrolase. Compounds 6b, 8b, 8c, 11b

exhibited modest to good inhibition effects on the growth

of HeLa cells or Bel-7420 cells at 1 mM (64, 44, 53 and

82% respectively), compounds 9b, 12b and 13b were

4.1.1. 6-N-Benzoyl-2⁰,3⁰-O-isopropylidene-L-adenosine

(3). Compound 2 (240 mg, 0.48 mmol) was dissolved in

K₂CO₃/CH₃OH (20 mL), the resulting solution was stirred at

ambient temperature for 1.2 h. The solution was neutralized to

pHZ7 with HCl/H₂O and then was evaporated, the residue

was dissolved into acetone (20 mL) and TSA (400 mg,

2.1 mmol), triethyl orthoformate (0.5 mL, 2.9 mmol) were

added. The mixture was stirred at ambient temperature

overnight. The solution was neutralized with NaHCO₃/H₂O

and evaporated. The residue was partitioned (CHCl₃/H₂O),

dried (MgSO₄) and evaporated. Puriﬁed on column chro-

matograph (EtOAc/acetoneZ4/1), 3 (161 mg, 81%) was

obtained as a white foam. TOF-MS (M^CCH): 412.

Table 6. The inhibitory effect of 5⁰-modiﬁed L-nucleosides on Hela cells

and Bel-7420 cells in vitro^a

4.1.2. 6-N-Benzoyl-9-[5⁰,6⁰-dideoxy-2⁰,3⁰-O-isopropyli-

dene-6⁰-(p-toluenesulfonyl)-b-L-ribo-hex-5⁰(E)-enofura-

nosyl] adenosine (4). A solution of 3 (375 mg, 0.91 mmol)

and N,N⁰-dicyclohexylcarbodiimide (DCC, 564 mg,

2.7 mmol) in dried DMSO (2 mL) was cooled (0 8C) under

argon, Cl₂CHCOOH (38 mL, 0.47 mmol) was added, and

stirred for 2 h at ambient temperature, then [(p-tolylsul-

fonyl)methylene]triphenyl phosphorane was added to the

solution and stirred overnight. Oxalic acid dihydrate (453 mg,

3.6 mmol) in MeOH (4 mL) was added. After 30 min the

dicyclohexylurea was ﬁltered and washed with cold MeOH,

and the combined ﬁltrates were evaporated (in vacuo). The

residue was partitioned (EtOAc/H₂O), the organic layer was

washed with H₂O (3!10 mL), NaHCO₃/H₂O, and NaCl/

H₂O, dried (MgSO₄) and evaporated. Puriﬁed on column

chromatography, 4 (500 mg, 97%) was obtained as a white

Compound

Tumor cell line

Bel-7420

Conc. (mM)

Inhibition rate (%)

6b

1.0

10.0

1.0

10.0

0.1

1.0

10.0

1.0

10.0

1.0

10.0

0.1

1.0

10.0

64

81

44

67

23

53

67

22

55

10

64

27

82

35

73

8b

8c

Hela

9b

Hela

13b

11b

12b

Hela

Bel-7420

a

The data shown are the average results of three experiments.

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

8543

solid. Mp 108–109 8C. HRMS (TOF) calcd for C₂₈H₂₈N₅O₆S

(M^CCH): 562.1760; found: 562.1770.

CH₂Cl₂/CCl₄(1:1, 10 mL) was added dropwise to a stirred

solution of 7 (169 mg, 0.243 mmol) in CH₂Cl₂/CCl₄(1:1,

6 mL) at K30 8C. After 30 min, the mixture was poured into

saturated NaHCO₃/H₂O and extracted by CHCl₃. The

combined organic phase was washed (brine), dried

(MgSO₄), and evaporated, and the residue was puriﬁed on

column chromotography (EtOAc/petroleum ether, 1:1) to

provide 8a (106 mg, 89%). TOF-MS (M^CCH): 486.

4.1.3. 6-N-Benzoyl-9-[5⁰,6⁰-dideoxy-6⁰-(p-toluenesul-

fonyl)-b-^L-ribo-hex-5⁰(Z)-enofuranosyl] adenosine (5a).

To a stirred solution of 4 (181 mg, 0.32 mmol) in CH₃CN/

H₂O (4:1, 10 mL) was added 1 M NaOH/H₂O(1 mL) and

stirring was continued at ambient temperature for 4 h, the

solution was concentrated to half volume and EtOAc

(10 mL) and 0.05 M HCl/H₂O (2 mL) were added. The

organic layer was washed with saturated NaHCO₃/H₂O,

brine, dried (Na₂SO₄) and puriﬁed on column chromato-

graph to give 5a (127 mg, 70%) as a white foam. Mp 103–

104 8C. TOF-MS (M^CCH): 562.

4.1.9. 9-[6⁰-Bromo-5⁰,6⁰-dideoxy-b-L-ribo-hex-5⁰(E)-eno-

furanosyl] adenosine (8b). 9-[6⁰-Bromo-5⁰,6⁰-dideoxy-b-

L-ribo-hex-5⁰(Z)-enofuranosyl] adenosine (8c). Deprotec-

tion of 8a (35 mg, 0.072 mmol) (as described for 5b) gave a

residue that was puriﬁed by RP-HPLC (preparative column;

program: 17% CH₃CN/H₂O for 1 h at 3 mL/min) to give 8b

(E) (15 mg, 61%, t_RZ47 min. Mp 141–142 8C) and 8c (Z)

(4 mg, 16%, t_RZ31 min. Mp 135–136 8C). 8b. HRMS

(TOF) calcd for C₁₁H₁₃BrN₅O₃(M^CCH): 342.0202;

found: 342.0218; 8c. TOF-MS (M^CCH): 342.

4.1.4. 9-[4⁰,5⁰-Dideoxy-6⁰-(p-toluenesulfonyl)-b-L-ribo-

hex-5⁰(Z)-enofuranosyl] adenosine (5b). Compound 5a

(25 mg, 0.044 mmol) was dissolved in 15 mL saturated

NH₃/CH₃OH solution, the resulting solution was stirred

overnight at ambient temperature and evaporated, CF_3-

COOH/H₂O (9:1, 3 mL) was added to the residue and

stirring was continued for 1 h at 0 8C (ice bath). After co-

evaporation with EtOH (2!3 mL), the residue was

separated with a short silica gel column (EtOAc/EtOAc/

i-PrOH/H₂O, 20:1:2, upper layer), compound 5b (12 mg,

64%) was collected as a white solid. Mp 115–117 8C.

HRMS (TOF) calcd for C₁₈H₂₀N₅O₅S (M^CCH): 418.1185;

found: 418.1152

4.1.10. 6-N-Benzoyl-9-[6⁰-iodo-5⁰,6⁰-dideoxy-2⁰,3⁰-O-iso-

propylidene-b-^L-ribo-hex-5⁰(E)-enofuranosyl] adenosine

(9a). A solution of NIS (70 mg, 0.312 mmol) in CH₂Cl₂/

CCl₄(1:1, 10 mL) was added dropwise to a stirred solution

of 7 (150 mg, 0.216 mmol) in CH₂Cl₂/CCl₄(1:1, 10 mL) at

wK20 8C. After 1.5 h the slightly pink mixture was poured

into saturated NaHCO₃/H₂O and extracted by CHCl₃, the

combined organic phase was washed with 2% NaHSO₃/H₂O

and brine, dried (MgSO₄), and evaporated. Puriﬁed on

column chromatography (EtOAc/petroleum ether, 1:1) gave

9a (113 mg, 98%). TOF-MS (M^CCH): 534.

4.1.5. 9-[2⁰,3⁰-O-Isopropylidene-6⁰-(p-toluenesulfonyl)-b-

L-ribo-hex-5⁰(E)-enofuranosyl] adenosine (6a). To a

stirred solution of 4 (12 mg, 0.021 mmol) in MeOH/H₂O

(1:1, 3 mL) was added sodium borohydride (3 mg,

0.08 mmol). After 18 h at ambient temperature, the solution

was concentrated to half volume and the residue was

partitioned (CHCl₃/H₂O). The organic layer was washed

with brine, H₂O, dried (MgSO₄), and evaporated. Puriﬁed

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

compond 6a (9 mg, 93%) was obtained as a white foam.

TOF-MS (M^CCH): 460.

4.1.11. 9-[6⁰-Iodo-5⁰,6⁰-dideoxy-b-L-ribo-hex-5⁰(E)-eno-

furanosyl] adenosine (9b). Deprotection of 9a (20 mg,

0.0375 mmol) (as described for 5b) gave a residue, which

was puriﬁed byRP-HPLC (preparative column; program: 17%

CH₃CN/H₂O for 100 min at 3 mL/min) to give a white solid 9b

(8 mg, 54%, t_RZ93 min. Mp 113–114 8C). HRMS (TOF)

calcd for C₁₁H₁₃N₅O₃I (M^CCH): 390.0063; found: 390.0076.

4.1.12. 6-N-Benzoyl-9-[6⁰-chloro-5⁰,6⁰-dideoxy-2⁰,3⁰-O-

isopropylidene-b-^L-ribo-hex-5⁰(E)-enofuranosyl] adeno-

sine (10a). Cl₂was gently bubbled through a solution

of 7 (35 mg, 0.05 mmol) in CH₂Cl₂/CCl₄(1:1, 2 mL) at

w–50 8C, and stirring was continued for 5 min. The

solution was carefully washed (NaHCO₃/H₂O, 2%

NaHSO₃/H₂O, and brine), dried (MgSO₄), and evaporated.

Puriﬁed on column chromatography [EtOAc/petroleum

ether, 1:1] gave 10a (17 mg, 77%) as a white foam. Mp

80–83 8C. TOF-MS (M^CCH): 512.

4.1.6. 9-[6⁰-(p-Toluenesulfonyl)-b-L-ribo-hex-5⁰(E)-eno-

furanosyl] adenosine (6b). The deprotection of 6a was

performed (as described for 5b) gave a white solid 6b

(11 mg, 55%). Mp 124–126 8C. HRMS (TOF) calcd for

C₁₈H₂₂N₅O₅S (M^CCH): 420.1342; found: 420.1329.

4.1.7. 6-N-Benzoyl-9-[6⁰-(E)-(tributylstannyl)-5⁰,6⁰-

dideoxy-2⁰,3⁰-O-isopropylidene-b-L-ribo-hex-5⁰(E)-eno-

furanosyl] adenosine (7). The oxygen in the solution of 4

(93 mg, 0.165 mmol) in toluene (10 mL) was removed by

argon for 30 min, and Bu₃SnH (0.2 mL, 0.66 mmol) was

added. Argon was passed through the solution continually

for 15 min, and AIBN (70 mg, 0.33 mmol) was added. The

solution was reﬂuxed for 5 h and evaporated, and the residue

was puriﬁed on column chromatography (EtOAc/petroleum

ether, 1:3). Compound 7 (70 mg, 61%) was obtained as a

colorless oil. Analysis calcd for C₃₃H₄₇N₅O₄Sn: C, 56.91;

H, 6.80; N, 10.06; found: C, 57.07; H, 7.01; N, 9.66.

4.1.13. 9-[6⁰-Chloro-5⁰,6⁰-dideoxy-b-L-ribo-hex-5⁰(E)-

enofuranosyl] adenosine (10b). Deprotection of 10a

(17 mg, 0.0385 mmol) (as described for 5b) gave a residue

which was puriﬁed by RP-HPLC (preparative column;

program: 17% CH₃CN/H₂O for 60 min at 3 mL/min) to give

a white solid 10b (5 mg, 43%, t_RZ54 min. Mp 140–

141 8C). HRMS (TOF) calcd for C₁₁H₁₃N₅O₃Cl (M^CCH):

298.0707; found: 298.0690

4.1.8. 6-N-Benzoyl-9-[6⁰-bromo-5⁰,6⁰-dideoxy-2⁰,3⁰-O-

isopropylidene-b-^L-ribo-hex-5⁰(E)-enofuranosyl] adeno-

sine (8a). A solution of NBS (59 mg, 0.329 mmol) in

4.1.14. 6-N-Benzoyl-9-[5⁰,6⁰-dideoxy-2⁰,3⁰-O-isopropyl-

idene-b-^L-ribo-hex-5⁰-enofuranosyl] adenosine (11a). A

solution of 7 (200 mg, 0.287 mmol) and NH₄F (190 mg,

8544

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

5.75 mmol) in anhydrous EtOH (15 mL) was reﬂuxed for

19 h and evaporated. The residue was partitioned (NaHCO₃/

H₂O/CHCl₃), and the organic layer was washed (brine),

dried (MgSO₄), and concentrated. Puriﬁed on column

chromatography (EtOAc/petroleum ether, 1:1) gave 11a

(116 mg, 99%) as a foam. TOF-MS (M^CCH): 408.

(14). 2⁰,3⁰-O-Isopropylidene-L-adenosine (500 mg, 1.63 mmol),

DCC (1 g, 4.89 mmol) were added to a stirred solution of

DMSO (4.5 mL) under argon, Cl₂CHCOOH (78 ml,

0.96 mmol) was added at w0 8C, and stirring was continued

for 2 h at ambient temperature. Ph₃PZCHCO₂Et (680 mg,

1.95 mmol) was added and the resulting mixture was stirred

overnight. Oxalic acid dihydrate (1.1 g, 8.7 mmol) in MeOH

(10 mL) was added, after 30 min the dicyclohexylurea was

ﬁltered and the ﬁltrate was evaporated (in vacuo). The

residue was partitioned (EtOAc/H₂O), the organic layer was

washed with H₂O (3!40 mL), NaHCO₃/H₂O, and brine,

dried (Na₂SO₄) and the solution was evaporated. Puriﬁed on

column chromatography (EtOAc/petroleum ether, 2:1) gave

14 (306 mg, 50%) as a white solid. Mp 105–107 8C. HRMS

(TOF) calcd for C₁₇H₂₂N₅O₅(M^CCH): 376.1621; found:

376.1608.

4.1.15. 9-[5⁰,6⁰-Dideoxy-b-L-ribo-hex-5⁰-enofuranosyl]

adenosine (11b). Deprotection of 11a (20 mg,

0.049 mmol) (as described for 5b) gave a residue which

puriﬁed by a short silica gel column (EtOAc/EtOAc/

i-PrOH/H₂O, 20:1:2) to give 11b (7 mg, 54%) as a white

solid. Mp 160–161 8C. HRMS (TOF) calcd for C₁₁H₁₄N₅O₃

(M^CCH): 264.1097; found: 264.1111

4.1.16. 9-[2⁰,3⁰-O-Isopropylidene-5⁰,6⁰-dideoxy-b-L-ribo-

hex-5⁰-ynofuranosyl] adenosine (12a). A deoxygenated

solution of 7 (341 mg, 0.49 mmol) in anhydrous CH₃CN

(25 mL) under argon was treated with Pb(OAc)₄(272 mg,

0.61 mmol), and stirred at w0 8C (ice bath) for 5 h and then

for 2 h at ambient temperature, the mixture was evaporated,

the residue was partitioned (NaHCO₃/H₂O/CHCl₃) and the

organic phase was washed (NaHCO₃/H₂O and brine), dried

(MgSO₄), and evaporated. Puriﬁed on column chromato-

graphy (EtOAc/petroleum ether, 2:1) gave 12a (105 mg,

71%) as a foam. TOF-MS (M^CCH): 406.

4.1.21. 9-[5⁰,6⁰-Dideoxy-2⁰,3⁰-O-isopropylidene-6⁰-hydr-

oxymethyl-b-^L-ribo-hex-5⁰(E)-enofuranosyl] adenosine

(15). To 14 (306 mg, 0.816 mmol) in CH₂Cl₂(10 mL), a

20% solution of DIBAL-H in toluene (5.25 mL, 8.16 mmol)

was added drop wise. The mixture was stirred at K78 8C for

2 h, and then quashed with MeOH (10 mL). A saturated

aqueous solution of potassium sodium tartrate monohydrate

(80 mL) was added, and the resulting suspension was stirred

vigorously for 16 h at ambient temperature, then extracted

with EtOAc (3!100 mL). The combined organic phase

were dried (Na₂SO₄) and evaporated. The residue was

puriﬁed by column chromatography (CH₂Cl₂/MeOH, 25:1) to

give 15 (180 mg, 66%) as a white foam. HRMS (TOF) calcd

for C₁₅H₂₀N₅O₄(M^CCH): 334.1515; found: 334.1538.

4.1.17. 9-[5⁰,6⁰-Dideoxy-b-L-ribo-hex-5⁰-ynofuranosyl]

adenosine (12b). Compound 12a (20 mg, 0.066 mmol)

was added to a solution of CF₃COOH/H₂O (9:1, 3 mL), the

mixture was stirred for 1 h at w0 8C and evaporated, co-

evaporated with EtOH (2!3 mL), the residue was separated

with a short silica gel column (EtOAc/EtOAc/i-PrOH/

H₂O, 20:1:2). 12b (8 mg, 46%) was collected as a solid. Mp

114–116 8C. HRMS (TOF) calcd for C₁₁H₁₂N₅O₃(M^CC

H): 262.0940; found: 262.0922

4.1.22. 9-[5⁰,6⁰-Dideoxy-2⁰,3⁰-O-isopropylidene-6⁰-phtha-

limido-b-^L-ribo-hex-5⁰(E)-enofuranosyl] adenosine (16).

DEAD (40% wt in toluene, 0.23 mL, 0.51 mmol) was added

dropwise to a stirred suspension of 15 (170 mg, 0.51 mmol),

phthalimide (75 mg, 0.51 mmol) and Ph₃P (133 mg,

0.51 mmol) in THF (3 mL). After stirring for 3 h at ambient

temperature, the solvents were evaporated. Puriﬁed on

column chromatography gave 16 (160 mg, 67%) as a white

solid. Mp 198–200 8C. HRMS (TOF) calcd for C₂₃H₂₃N₆O₅

(M^CCH): 463.1730; found: 463.1714.

4.1.18. 6-N-Benzoyl-9-[5⁰-deoxy-5⁰-iodo-2⁰,3⁰-O-isopro-

pylidene-b-^L-ribo-hex-5⁰(E)-enofuranosyl] adenosine

(13a). To a stirred solution of 3 (233 mg, 0.56 mmol) in

anhydrous dioxane (15 mL) containing anhydrous pyri-

dine (0.1 mL, 1.12 mmol), triphenylphosphine (222 mg,

0.84 mmol), I₂(220 mg, 0.84 mmol) were added and

stirring was continued for 20 h at ambient temperature,

methanol (1.0 mL) was added, and then the solvents were

removed by evaporation. The residue was partitioned

(EtOAc/H₂O), and the organic phase was washed (10%

Na₂S₂O₃and brine), dried (Na₂SO₄), and evaporated.

Puriﬁed on column chromatography (EtOAc/petroleum

ether, 1:1) gave 13a (263 mg, 89%) as a foam. TOF-MS

(M^CCH): 522.

4.1.23. 9-[5⁰,6⁰-Dideoxy-2⁰,3⁰-O-isopropylidene-6⁰-amino-

methyl-b-^L-ribo-hex-5⁰(E)-enofuranosyl] adenosine

(17a). To phthalimide 16 (150 mg, 0.32 mmol), a solution

of 30% MeNH₂in EtOH (20 mL) was added and the

mixture was stirred at 20 8C for 24 h. After evaporation in

vacuo, the residue was separated with a silica gel column

(CH₂Cl₂/MeOH, 35:1), compound 17 (101 mg, 93%) was

collected as a colorless oil. HRMS (TOF) calcd for

C₁₅H₂₁N₆O₃(M^CCH): 333.1675; found: 333.1693.

4.1.19. 9-[5⁰-Deoxy-5⁰-iodo-b-L-ribo-hex-5⁰(E)-enofura-

nosyl] adenosine (13b). Deprotection of 13a (25 mg,

0.046 mmol) (as described for 5b) gave a residue that puri-

ﬁed by RP-HPLC (preparative column; program: 0–10%

CH₃CN/H₂O for 3 min followed by a gradient of 10–40%

for 50 min) to give 13b as a white foam (8 mg, 45%, t_RZ

34 min). HRMS (TOF) calcd for C₁₀H₁₃N₅O₃I (M^CCH):

378.0063; found: 378.0071.

4.1.24. 9-[5⁰,6⁰-Dideoxy-6⁰-aminomethyl-b-L-ribo-hex-

5⁰(E)-enofuranosyl] adenosine (17b). A solution of 17a

(28 mg, 0.084 mmol) in TFA-H₂O (5:2, 1.5 mL), was

stirred at 20 8C for 2 h and then evaporated to dryness.

The residue was dissolved with H₂O and puriﬁed by

RP-HPLC (preparative column; program: a gradient of

0–10% CH₃CN/H₂O for 30 min at 3 mL/min) to give 17b

(20 mg, 81%, t_RZ22 min) as a colorless foam. HRMS

(TOF) calcd for C₁₂H₁₇N₆O₃(M^CCH): 293.1362; found:

293.1381.

4.1.20. 9-[5⁰,6⁰-Dideoxy-2⁰,3⁰-O-isopropylidene-6⁰-ethoxy-

carbonyl-b-L-ribo-hex-5⁰(E)-enofuranosyl] adenosine

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

8545

4.1.25. [5-(R)-Dimethoxymethyl-4-(R)-benzoyloxy-3(S)-

(adenine-9⁰-yl)]-tetrahydrofuran (19). A stirred solution

of compound 18 (2.3 g, 8.0 mmol) in anhydrous pyridine

(50 mL), benzoyl chloride (1.0 mL, 8.8 mmol) was added

dropwise at 0 8C and stirring was continued for 3 h at 0 8C.

The solvents were evaporated and the residue was

partitioned (saturated NaHCO₃/H₂O/EtOAc), the organic

layer was washed with brine, dried (Na₂SO₄) and evapor-

ated. Puriﬁed on column chromatography (EtOAc/petro-

leum ether, 2:1) gave 19 (2.1 g, 66%) as a colorless foam,

and recovered 18 (0.56 g, 23%). HRMS (TOF) calcd for

C₁₉H₂₂N₅O₅(M^CCH): 400.1621; found: 400.1618.

protected 23. The product was dissolved in CH₂Cl₂(10 mL)

and catalytic amount of 30% NaOMe/MeOH was added

under stirring. After stirred at room temperature for 1 h, the

mixture was evaporated and puriﬁed by a silica gel column,

to give 23 (9 mg, 68%) as a white solid. Mp 179–181 8C.

HRMS (TOF) calcd for C₁₁H₁₄N₅O₂(M^CCH): 248.1147;

found: 248.1152.

4.1.30. [5-(S)-(2-Ethynyl)-4-(R)-hydroxy-3(S)-(adenine-

9⁰-yl)]-tetrahydrofuran (24). The procedure of reaction is

the same as the synthesis for 12a, but Pb(OAc)₄(4 equiv to

compound 22) was added and stirring was continued for 3

days at ambient temperature. The procedure gave the

residue no further puriﬁcation for the removal of the

benzoyl group. The procedure of deprotection was

described as compound 23 that gave 24 (18 mg, 67%) as a

colorless foam. HRMS (TOF) calcd for C₁₁H₁₂N₅O₂

(M^CCH): 246.0991; found: 246.1005.

4.1.26. [5-(S)-Hydroxymethyl-4-(R)-benzoyloxy-3(S)-

(adenine-9⁰-yl)]-tetrahydrofuran (20). Compound 19

(1.2 g, 3.0 mmol) was added to a mixed solution of THF

(12 mL) and 1% HCl (12 mL). The resulting solution was

stirred for 8 h at 90 8C, 2 N NaOH/H₂O was used to

neutralize the solution to pHZ7, NaBH₄(370 mg,

9.2 mmol) was added and stirred at ambient temperature

for 1 h. After neutralization with 1 N HCl/H₂O, the solution

was evaporated to dryness and the residue was puriﬁed on

silica gel column to give compound 20 (851 mg, 80%) as a

colorless foam. HRMS (TOF) calcd for C₁₇H₁₈N₅O₄

(M^CCH): 356.1359; found: 356.1349.

4.1.31. [5-(S)-Dimethoxymethyl-4-(S)-benzoyloxy-3(R)-

(adenine-9⁰-yl)]-tetrahydrofuran (26). Similar to the

synthesis of 19, compound 26 was obtained in a yield of

85% as a white solid. Mp 183–185 8C.

4.1.32. [5-(R)-Hydroxymethyl-4-(S)-benzoyloxy-3(R)-

(adenine-9⁰-yl)]-tetrahydrofuran (27). Similar to the

synthesis of 20, compound 27 was obtained in a yield of

70% as a colorless oil.

4.1.27. {5-(S)-[2(E)-p-Toluenesulfonylethylene]-4-(R)-

benzoyloxy-3(S)-(adenine-9⁰-yl)}-tetrahydrofuran (21).

A stirred solution of compound 20 (476 mg, 1.3 mmol),

DCC (1.08 g, 5.9 mmol) in DMSO (3.5 mL), Cl₂CHCOOH

(66 ml, 0.7 mmol) was added dropwise at 0 8C (ice bath),

stirring was continued for 2 h at ambient temperature,

[(p-tolylsulfonyl)methylene]triphenyl phosphorane (900 mg,

7.2 mmol) was added and stirred overnight. MeOH (8 mL)

was added and stirred for 30 min at room temperature, the

resulted dicyclohexylurea was ﬁltered and washed with cold

MeOH, and the combined ﬁltrates were evaporated (in

vacuo). The residue was partitioned (EtOAc/H₂O), the

organic layer was washed with H₂O (3!30 mL), NaHCO₃/

H₂O, and brine, dried (MgSO₄) and evaporated. Puriﬁed on

column chromatography gave 21 (350 mg, 51%) as a white

solid. Mp 136–138 8C. HRMS (TOF) calcd for

C₂₅H₂₄N₅O₅S (M^CCH): 506.1498; found: 506.1484.

4.1.33. {5-(R)-[2(E)-p-Toluenesulfonylethylene]-4-(S)-

benzoyloxy-3(R)-(adenine-9⁰-yl)}-tetrahydrofuran (28).

Similar to the synthesis of 21, compound 28 was obtained

in a yield of 54% as a white solid. Mp 141–143 8C. HRMS

(TOF) calcd for C₂₅H₂₄N₅O₅S (M^CCH): 506.1498; found:

506.1485.

4.1.34. {5-(R)-[2(E)-tributylstannylvinyl]-4-(S)-benzoyl-

oxy-3(R)-(adenine-9⁰-yl)}-tetrahydrofuran (29). Similar

to the synthesis of 22, compound 29 was obtained in a yield

of 63% as a white foam, with recovered 28 (29%). HRMS

(TOF) calcd for C₃₀H₄₄N₅O₃Sn (M^CCH): 642.2466;

found: 642.2427.

4.1.35. [5-(R)-(2-Vinyl)-4-(S)-hydroxy-3(R)-(adenine-9⁰-

yl)]-tetrahydrofuran (30). Similar to the synthesis of 23,

compound 30 was obtained in a yield of 41% as a white

solid. Mp 172–174 8C. HRMS (TOF) calcd for C₁₁H₁₄N₅O₂

(M^CCH): 248.1147; found: 248.1143.

4.1.28. {5-(S)-[2(E)-Tributylstannylvinyl]-4-(R)-benzoyl-

oxy-3(S)-(adenine-9⁰-yl)}-tetrahydrofuran (22). A solu-

tion of 21 (320 mg, 0.6 mmol) in toluene (50 mL) was

deoxygenated (argon, 30 min), and Bu₃SnH (0.67 mL,

2.2 mmol) was added. Deoxygenation was continued for

15 min, and AIBN (40 mg, 0.2 mmol) was added. The

solution was reﬂuxed for 6 h and evaporated, and the residue

was puriﬁed by column chromatograph (EtOAc/petroleum

ether, 2:1). The elution gave 22 (219 mg, 54%) as a

colorless oil, and recovered 21 (85 mg, 26%). HRMS (TOF)

calcd for C₃₀H₄₄N₅O₃Sn (M^CCH): 642.2466; found:

642.2449.

4.1.36. [5-(R)-(2-Ethynyl)-4-(S)-hydroxy-3(R)-(adenine-

9⁰-yl)]-tetrahydrofuran (31). Similar to the synthesis of

24, compound 31 was obtained in a yield of 64% as a white

foam. HRMS (TOF) calcd for C₁₁H₁₂N₅O₂(M^CCH):

246.0991; found: 246.1002.

4.2. Puriﬁcation of AdoHcy hydrolase and evaluation of

the effectiveness of synthetic compounds

4.1.29. [5-(S)-(2-Vinyl)-4-(R)-hydroxy-3(S)-(adenine-9⁰-

yl)]-tetrahydrofuran (23). A solution of compound 22

(76 mg, 0.12 mmol) in dried EtOH (10 mL), NH₄F (200 mg,

6.0 mmol), was added, the resulting solution was reﬂuxed

for 3 days, and then evaporated. The residue was puriﬁed by

silica gel column (EtOAc/petroleum ether, 3:1) to give

Recombination human placental AdoHcy hydrolase was

puriﬁed from cell free extracts of Escherichia coli

transformed with plasmid pPROK-19.³²To evaluate the

inhibitory potential of the compounds, different concentra-

tions (0.01–100 mM) were preincubated with 10 mL (10 mg)

8546

J. -F. Wang et al. / Tetrahedron 60 (2004) 8535–8546

10. Yuan, C.-S.; Liu, S.; Wnuk, S. F.; Robin, M. J.; Borchardt,

R. T. Biochemistry 1994, 33, 3758–3765.

enzyme at 37 8C for 10 min at pH 7.4 in 240 mL PBE buffer.

The mixture was then incubated with 50 mL Ado (10 mM)

and 200 mL Hcy (10 mM) at 37 8C for 10 min. This reaction

was terminated by addition of 20 mL sulfuric acid, and the

mixture was cooled with ice bath. After centrifugalization

(10,000 rpm for 5 min), the AdoHcy formed was analyzed

by HPLC on a C-18 reversed-phase column (Diamonsile;

250!4.6 mm). Elution was performed with a gradient:

(94%A: 6% B, 1 mL/min) over 10 min. (mobile phase A

was 0.1% CF₃COOH and mobile phase B was acetonitrile).

Quantitative analysis of AdoHcy was monitored (UV) at

258 nm.

11. Yuan, C.-S.; Wnuk, S. F.; Liu, S.; Robin, M. J.; Borchardt,

R. T. Biochemistry 1994, 33, 12305–12311.

12. Liu, S.; Wnuk, S. F.; Yuan, C.-S.; Robins, M. J.; Borchardt,

R. T. J. Med. Chem. 1993, 36, 883–887.

13. Wnuk, S. F.; Dalley, N. K.; Robins, M. J. J. Org. Chem. 1993,

58, 111–117.

14. Wnuk, S. F.; Yuan, C.-S.; Borchardt, R. T.; Balzarini, J.; De

Clercq, E.; Robins, M. J. J. Med. Chem. 1994, 37, 3579–3587.

15. Wnuk, S. F.; Ro, B.-O.; Valdez, C. A.; Lewandowska, E.;

Valdez, N. X.; Sacasa, P. R.; Yin, D.; Zhang, J.; Borchardt,

R. T.; De Clercq, E. J. Med. Chem. 2002, 45, 2651–2658.

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4.3. Assays for the inhibition on the growth of various

tumor cells

Growth inhibition of the synthetic compounds to various

tumor cells were determined by MTT and SRB assays.^34,35

Brieﬂy, tumor cells (1–2.5!10⁴cells mL^K1) were inocu-

lated in 96-well culture plates (180 mL/well). After 24 h

culture, 20 mL of culture medium containing synthetic

compound of various concentrations were added to the

wells, and RPMI-1640 medium in control cells, then the

cells were incubated for 48 h. The growth inhibition of

HL-60 cells were determined by MTT method, and the other

cell lines were assayed by SRB method. The absorbance of

each well was measured using a microculture plate reader at

570 nm (MTT) and 540 nm (SRB).

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Acknowledgements

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We thank National Natural Science Foundation of China for

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Article Doi

Synthesis and biological activities of 5′-ethylenic and acetylenic modified L-nucleosides and isonucleosides

DOI: 10.1016/j.tet.2004.06.131

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