Synthesis and biological evaluation of some 4′-C-(hydroxymethyl)- α- and -β-D-arabinofuranosyl pyrimidine and adenine nucleosides

Article abstract of DOI:10.1135/cccc20061063

A series of 4′-C-(hydroxymethyl) analogs of pyrimidine and purine nucleosides have been prepared utilizing standard methodologies, and the α and β anomers were separated. These analogs are part of our continuing efforts to identify new anticancer drugs as well as to explore the substrate specificities of these analogs with the initial activating enzymes in the metabolic pathway leading to nucleoside triphosphates. Although not cytotoxic to CCRF-CEM cells (an acute lymphoblastic leukemia of T-cell origin), many of these compounds were utilized as substrates for the various human nucleoside kinases, including deoxycytidine kinase, thymidine kinase 1, and thymidine kinase 2. Because the 4′-C-(hydroxymethyl) analog of arabinofuranosyl cytosine was identified as a good substrate with deoxycytidine kinase, its metabolism in CEM cells was evaluated. These results indicated that nucleosides with this modification could be activated in human cells without cytotoxicity, which suggested that they should be examined for antiviral activity.

Full text of DOI:10.1135/cccc20061063

4′-C-(Hydroxymethyl)nucleosides
1063
SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME
4′-C-(HYDROXYMETHYL)-α- AND -β-D-ARABINOFURANOSYL
PYRIMIDINE AND ADENINE NUCLEOSIDES
a2
Jean-François GRIFFONa,+, Sue C. SHADDIXa1, William B. PARKER
,
b
c1
a
Ashraf S. AL-MADHOUN , Staffan ERIKSSON , John A. MONTGOMERY and
John A. SECRIST IIIa3,
*
a
Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, U.S.A.;
1
2
3
e-mail: shaddix@sri.org, parker@sri.org, secrist@sri.org
Department of Biochemistry, Microbiology and Immunology, University of Ottawa,
451 Smyth Street, Ottawa, Ontario, Canada
b
c
Department of Molecular Biosciences, Swedish University of Agricultural Sciences, The Biomedical
1
Centre, SE 753 24 Uppsala, Sweden; e-mail: staffan.eriksson@vmk.slu.se
Received March 23, 2006
Accepted Jun e 6, 2006
Dedicated to Professor Antonín Holý on the occasion of his 70th birthday.
A series of 4′-C-(h ydroxym eth yl) an alogs of pyrim idin e an d purin e n ucleosides h ave been
prepared utilizin g stan dard m eth odologies, an d th e α an d β an om ers were separated. Th ese
an alogs are part of our con tin uin g efforts to iden tify n ew an tican cer drugs as well as to ex-
plore th e substrate specificities of th ese an alogs with th e in itial activatin g en zym es in th e
m etabolic path way leadin g to n ucleoside triph osph ates. Alth ough n ot cytotoxic to
CCRF-CEM cells (an acute lym ph oblastic leukem ia of T-cell origin ), m an y of th ese com -
poun ds were utilized as substrates for th e various h um an n ucleoside kin ases, in cludin g
deoxycytidin e kin ase, th ym idin e kin ase 1, an d th ym idin e kin ase 2. Because th e
4′-C-(h ydroxym eth yl) an alog of arabin ofuran osyl cytosin e was iden tified as a good substrate
with deoxycytidin e kin ase, its m etabolism in CEM cells was evaluated. Th ese results in di-
cated th at n ucleosides with th is m odification could be activated in h um an cells with out
cytotoxicity, wh ich suggested th at th ey sh ould be exam in ed for an tiviral activity.
Keyw ord s: 4′-C-(Hydroxym eth yl)n ucleosides; 4′-C-Bran ch ed n ucleosides; Deoxycytidin e
kin ase; Cytotoxicity; Ph osph orylation ; Glycosidation ; Pyrim idin es; Purin es.
+ Curren t address: Laboratoire Cooperatif Iden ix – CNRS – Un iversité Mon tpellier, Case
Courrier 008, Place Eugen e Bataillon , 34095 Mon tpellier Cedex 5, Fran ce.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20061063

1064
Griffon et al.:
In terest in 4′-substituted n ucleosides dates back to th e early work of
Moffatt, Verh eyden an d oth ers1. Sin ce th at tim e a large n um ber of 4′-substi-
tuted n ucleosides h ave been prepared an d som e of th em h ave been foun d
to h ave in terestin g biological activity2–21. Com poun ds th at h ave been
foun d to h ave som e cytotoxicity in clude 2′-deoxyn ucleosides with
α-4′-C-m eth yl3,11, cyan o3,13, eth yn yl2,4,13, eth en yl2 an d fluorom eth yl19
groups. In our laboratory we h ave focused for m an y years on th e develop-
m en t of n ew n ucleosides with an tican cer activity, in cludin g recen tly sev-
eral series of com poun ds th at h ave 4′-C-substitution . In addition , we h ave
focused on oth er substitution pattern s an d oth er carboh ydrate con figura-
tion s as we explore wh at ch an ges are allowed by th e in itial activatin g en -
zym es, typically deoxycytidin e kin ase, th ym idin e kin ase an d guan osin e
kin ase. On e of th e first groups th at was of in terest was th e
α-4′-C-(h ydroxym eth yl) group because it h ad th e poten tial of offerin g a
secon d site for in itial ph osph orylation in th e m etabolism of a n ucleoside
an alog up to th e triph osph ate level. Man y 4′-C-(h ydroxym eth yl)n ucleo-
sides h ave been prepared, in cludin g an alogs with β-D-xylo, ribo an d
2′-deoxyribo con figuration s1,23–28, as well as an alogs of AZT, d4T, ddT pre-
pared as poten tial an tiviral agen ts29,30. We report h erein th e syn th esis of a
series of 4′-C-(h ydroxym eth yl)-α- an d -β-D-threo-pen tofuran osyl pyrim idin e
an d purin e n ucleosides31. Th ese target com poun ds are addition al exam ples
of 4′-derivatives of arabin on ucleosides. Th e n ewly syn th esized com poun ds
h ave been exam in ed for th eir cytotoxicity as well as for th eir ability to
serve as substrates for th e appropriate in itial activatin g kin ases.
Chemistry
Our syn th etic approach to th e target h ydroxym eth yl-substituted n ucleo-
sides follows gen erally alon g stan dard lin es. Th e suitably protected carbo-
h ydrate 10 was prepared in 13 steps from L-xylose (Sch em e 1).
1,2-O-Isopropyliden e-α-L-xylo-furan ose in term ediate 1 was prepared from
L-xylose followin g th e procedure described by Gosselin et al.32 in 94% yield.
Selective ben zoylation of th e 5-h ydroxyl group of 1 with ben zoyl ch loride
in dry pyridin e at 0 °C was perform ed in 77% yield an d was followed by th e
protection of th e 3-h ydroxy group by reaction with tert-butyldim eth ylsilyl
ch loride (TBDMSCl) in dry pyridin e at room tem perature to give com poun d
3 in quan titative yield. Th e 5-position of 3 was deben zoylated with sodium
m eth oxide in a m eth an ol/toluen e m ixture in 88% yield an d subjected to an
oxidation . Oxidation of th e 5-position of 4 was perform ed un der
Pfitzn er–Moffatt con dition s33,34, with dim eth yl sulfoxide (DMSO), dicyclo-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

4′-C-(Hydroxymethyl)nucleosides
1065
h exylcarbodiim ide (DCC) an d dich loroacetic acid an d led to th e pen to-
dialdofuran ose in term ediate 5 wh ich was subsequen tly con verted to th e
4-C-(h ydroxym eth yl) derivative 6.
H3C
O
H3C
O
CH3
CH3
O
O
O
O
(vi)
(i), (ii)
L-xylose
R1O
OHC
OR2
OR2
5
1: R1 = R2 = H
(vii)
(iii)
H3C
O
2: R1 = Bz, R2 = H
CH3
(iv)
(v)
HO
HO
3: R1 = Bz, R2 = TBDMS
4: R1 = H, R2 = TBDMS
O
O
HO
6
(viii)
H3C
O
CH3
BnO
BnO
BnO
O
BnO
BnO
O
BnO
O
O
(ix), (x)
(xi), (xii)
R3O
OAc
OMe
BnO
BnO
BnO
BnO
10
7
8: R3 = H
(viii)
9: R3 = Bn
(i) Me2CO, H2SO4, CuSO4; (ii) aqueous 0.2% HCl; (iii) C6H5COCl, pyridine, 0 oC; (iv) TBDMSCl,
imidazole, pyridine, r.t.; (v) MeONa/MeOH/toluene; (vi) DMSO, DCC, Cl2CHCO2H, benzene/pyridine,
r.t.; (vii) CH2O, NaOH, H2O/dioxane, r.t.; (viii) BnBr, NaH, Bu4N+I-, THF, r.t.; (ix) CH3CO2H, H2SO4,
r.t.; (x) MeOH, H2SO4, r.t.; (xi) 90% CF3COOH, 5-15 oC; (xii) Ac2O, pyridine, r.t.
SCHEME 1
Our approach of th e syn th esis of 6 was based upon th e preceden t estab-
lish ed by Sch affer35,36 an d Moffatt an d co-workers24,25, utilizin g an aldol
con den sation -crossed Can n izzaro reaction sequen ce between a suitably
p rotected pen todialdofuran ose an d form aldeh yde in aqu eou s sodiu m
h ydroxide. Wh en th e 3-O-(tert-butyldim eth ylsilyl)-1,2-O-isopropyliden e-
α-L-xylo-pen todialdo-1,4-furan ose in term ediate 5 was subjected to th e aldol
con den sation -crossed Can n izzaro reaction sequen ce, in troduction of th e
h ydroxym eth yl group was accom pan ied by loss of th e 3-O-tert-butyl-
17
dim eth ylsilyl group2 but with out an y epim erisation at C-3
.
4-C-(Hydroxym eth yl)-1,2-O-isopropyliden e-β-D-arabino-fu ran ose 6 was
obtain ed in 66% yield from 4.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

1066
Griffon et al.:
Th e th ree h ydroxy groups of 6 were th en protected by reaction with ben -
zyl ch loride in th e presen ce of sodium h ydride an d tetrabutylam m on ium
iodide in THF to afford com poun d 7 in 91% yield. Cleavage of th e
isopropyliden e group of 7 with 85% acetic acid an d con cen trated sulfuric
acid followed by m eth ylation of th e an om eric h ydroxyl group with m eth a-
n ol an d sulfuric acid afforded 8 in 95% yield. Th e last OH group of 8 was
ben zylated un der th e sam e con dition described above to give th e
tetra-O-ben zylated derivative 9 in 92% yield. Hydrolysis of th e m eth yl
glycoside 9 with 90% trifluoroacetic acid followed by acetylation of th e
an om eric OH with acetic an h ydride in dry pyridin e afforded th e desired in -
term ediate 10.
As represen tative n ucleoside targets, we ch ose 12, 14, 16, 18, 20, an d 22,
both th e α an d β an om ers. Again , stan dard routes were utilized to prepare
th e twelve target com poun ds, th ough sligh tly differen t con dition s were uti-
lized for pyrim idin es versus purin es.
To prepare th e 4-oxopyrim idin e n ucleosides (Sch em e 2), blocked precur-
sor 10 was respectively con den sed with th e com m ercially available uracil,
th ym in e or 5-fluorouracil, un der Vörbruggen 37 con dition s usin g trim eth yl-
silyltrifluorom eth an esulfon ate (TMSOTf) as a catalyst, to give th e fully cor-
respon din g ben zylated n ucleosides as an α:β m ixture, separable by silica gel
ch rom atograph y, in 90% yield (α/β 1.8:1) for th e uracil n ucleosides 11␣,
11␤, 87% yield (α/β 3.3:1) for th e th ym in e n ucleosides 13␣, 13␤, an d 88%
yield (α/β 1.4:1) for 5-fluorouracil n ucleosides 15␣, 15␤. Fin ally, ben zyl
eth er protective groups of 11␣, 13␣, 15␣ an d 11␤, 13␤, 15␤ were cleaved
by catalytic h ydrogen ation in good yields to give target com poun ds 12␣,
14␣, 16␣ an d 12␤, 14␤, 16␤.
In order to gen erate cytosin e n ucleoside an alogs (Sch em e 2), con version
of ben zylated uracil an d 5-fluorouracil derivatives 11␣,␤ an d 15␣,␤ in to
th e correspon din g cytosin e an d 5-fluorocytosin e n ucleosides 17␣,␤ an d
19␣,␤ was carried out via a treatm en t with Lawesson ’s reagen t38,39 wh ich
led to th e 4-th ioam ide derivatives, followed by
a treatm en t with
m eth an olic am m on ia at 100 °C in a stain less-steel bom b. Th is tran sform a-
tion was perform ed in quan titative yield for 17␣, 82% yield for 17␤ an d
90% yield for 19␣, 19␤. Fin ally, ben zyl eth er protective groups of 17␣, 19␣
an d 17␤, 19␤ were cleaved with a boron trich loride solution in an h ydrous
dich lorom eth an e in good yields to give target com poun ds 18␣, 20␣ an d
18␤, 20␤.
In order to prepare th e two aden in e n ucleoside an om ers 24␣ an d 24␤
(Sch em e 3), con den sation of 10 with silylated 6-ch loropurin e was per-
form ed at room tem perature in th e presen ce of tin (IV) ch loride in an h yd-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

4′-C-(Hydroxymethyl)nucleosides
1067
O
N
X
O
(CH3)3SiO
N
HN
X
X
(ii)
HN
(i)
O
RO
RO
O
O
N
H
(CH3)3SiO
N
10
RO
RO
X = H: uracil
X = CH3: thymine
X = F: 5-fluorouracil
11α,β: R = Bn, X = H
13α,β: R = Bn, X = CH3
15α,β: R = Bn, X = F
O
N
NH2
X
X
HN
O
N
O
N
RO
RO
RO
(iii), (iv)
O
O
RO
RO
RO
RO
RO
11β: R = Bn, X = H
13β: R = Bn, X = CH3
15β: R = Bn, X = F
17β: R = Bn, X = H
19β: R = Bn, X = F
(vi)
(v)
12β: R = H, X = H
14β: R = H, X = CH3
16β: R = H, X = F
18β: R = H, X = H
20β: R = H, X = F
RO
RO
RO
RO
O
RO
O
RO
N
O
N
O
O
(iii), (iv)
RO
RO
N
NH
X
X
NH2
11α: R = Bn, X = H
13α: R = Bn, X = CH3
15α: R = Bn, X = F
17α: R = Bn, X = H
19α: R = Bn, X = F
(vi)
(v)
12α: R = H, X = H
14α: R = H, X = CH3
16α: R = H, X = F
18α: R = H, X = H
20α: R = H, X = F
(i) HMDS, (NH4)2SO4, reflux; (ii) TMSOTf, (CH2Cl)2, r.t.; (iii) Lawesson's reagent, (CH2Cl)2, reflux;
(iv) MeOH/NH3, 100 oC; (v) atm H2, Pd/C, methanol/AcOH, r.t.; (vi) BCl3, CH2Cl2, -78 oC, then -25 oC
SCHEME 2
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

1068
Griffon et al.:
rous aceton itrile an d afforded in 74% yield a 1.8:1 m ixture of N-7 an d N-9
isom ers (21␣␤ an d 22␣␤). After separation by silica gel ch rom atograph y N-7
isom er (21␣␤) was con verted to th e N-9 isom er (22␣␤) in 54% yield by
treatm en t with TMSOTf in aceton itrile at room tem perature. Separation of
22␣ an d 22␤ (α/β 0.8:1) was perform ed by silica gel ch rom atograph y. Dis-
placem en t of th e ch lorin e atom at th e 6-position of com poun ds 22␣ an d
22␤ was perform ed with eth an olic am m on ia at 75 °C in a stain less-steel
Cl
N
N
N
N
N
N
Cl
N
Cl
N
BnO
BnO
N
N
N
O
BnO
BnO
BnO
N
(ii)
(i)
N
N
O
BnO
Cl
N
H
N
10
+
Si(CH3)3
BnO
BnO
22α,β
6-chloropurine
21α,β
Cl
N
N
N
N
N
N
N
BnO
BnO
BnO
BnO
N
O
BnO
O
BnO
Cl
(iii)
BnO
BnO
21α,β
22α,β
Cl
NH2
N
NH2
N
N
N
N
N
N
N
N
BnO
O
BnO
HO
N
N
N
O
BnO
O
HO
(iv)
(v)
BnO
BnO
BnO
HO
BnO
BnO
HO
22β
23β
24β
BnO
BnO
BnO
BnO
HO
HO
O
BnO
O
BnO
O
HO
(iv)
(v)
N
N
N
N
N
N
N
N
N
BnO
BnO
23α
HO
N
N
NH2
N
22α
24α
Cl
NH2
Synthesis of compounds 24α and 24β. (i) BSA, reflux; (ii) SnCl4, CH3CN, r.t.; (iii) TMSTf,
CH3CN, r.t.; (iv) EtOH-NH3, 75 oC; (v) 50 Pa H2, Pd(OH)2/C, methanol/AcOH, r.t.
SCHEME 3
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

4′-C-(Hydroxymethyl)nucleosides
1069
bom b in 94 an d 82% yields, respectively. Fin ally, ben zyl eth er protective
groups of 23␣ an d 23␤ were cleaved by h ydrogen ation usin g Pd(OH)2 as
catalyst un der 50 Pa of H2 to give target com poun ds 24␣ an d 24␤ in 66 an d
49% yields, respectively.
Biological Results
Th e target com poun ds were prepared as 4′-substituted arabin on ucleoside
an alogs, an d th ey are referred to in th at m an n er. Th us, th e cytosin e-
con tain in g target 18␤ can be con sidered to be 4′-C-(h ydroxym eth yl)-ara-C,
abbreviated HM-araC below.
Non e of th e 4′-C-(h ydroxym eth yl)-substituted target n ucleosides was
cytotoxic to CEM cells at a con cen tration of 40 µg/m l. Th e 4′-C-h ydroxy-
m eth yl com poun ds were evaluated as appropriate as substrates with
th ym idin e kin ase 1, th ym idin e kin ase 2, deoxycytidin e kin ase an d
deoxyguan osin e kin ase to determ in e wh eth er or n ot th ey could be
ph osph orylated in h um an cells (Table I). Th e uracil, th ym in e an d aden in e
an alogs were poor substrates for th eir respective kin ases, wh ich suggested
th at th eir lack of cytotoxicity was due to th eir lack of con version to
cytotoxic n ucleotides. However, th e β isom ers of th e two cytosin e an alogs
were ph osph orylated by dCyd kin ase. Th e Km values for 2′-deoxycytidin e
(dCyd), HM-araC (18␤) an d 5-F-HM-araC (20␤) with th e recom bin an t en -
zym e were 1.0 ± 0.3, 18 ± 6, an d 32 ± 19 µM, respectively, an d Kcat values
for th ese agen ts were 0.11 ± 0.02, 0.07 ± 0.01, an d 0.11 ± 0.02 s–1, respec-
tively. Th e catalytic efficien cy of HM-araC with th is en zym e was 14% of
th at seen with dCyd.
Because of th e activity of th e cytosin e an alogs with th e recom bin an t
dCyd kin ase, th e m etabolism [3H]HM-araC was studied in CEM cells. In our
in itial studies usin g our stan dard SAX HPLC system 40, th ere was a lin ear in -
crease in a m etabolite in CEM cells th at eluted at 20 m in (data n ot sh own ).
Th is m etabolite eluted with a reten tion tim e th at was sim ilar to th at of
dCTP an d is th erefore likely to be th e 5′-triph osph ate of HM-araC
(HM-araCTP). Because th ere was con siderable in crease in radioactivity in
th e void of th is SAX HPLC colum n , th e sam ples were an alyzed usin g
BioBasic HPLC, wh ich retain s m on oph osph ates better th an our stan dard
SAX HPLC system . Usin g th is system it was clear th at HM-araC was con -
verted to 2 m etabolites (Fig. 1). Th e addition of dCyd (100 µM) to th e in cu-
bation s with HM-araC com pletely in h ibited th e form ation of both
m etabolites, an d n eith er m etabolite was form ed in CEM cells th at were de-
ficien t in dCyd kin ase (data n ot sh own ). Th e peak elutin g at 8 m in was de-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

1070
Griffon et al.:
term in ed to be HM-araC by spikin g th e sam ple with [3H]HM-araC. Th e
5′-ph osph ate of HM-araC (HM-araCMP) was created by in cubatin g
HM-araC with dCyd kin ase th at h ad been purified from CEM cells an d ATP,
an d th e peak of radioactivity th at eluted at 24 m in in Fig. 1 eluted with th is
HM-araCMP stan dard. Th e peak elutin g at 36 m in is HM-araCTP, based on
its elution with dCTP in th e first SAX HPLC colum n . Th e rate of con version
of 10 µM dCyd to dCTP in CEM cells was 0.91 pm ol/106 cells/m in , wh ereas
th e rate of con version of 10 µM HM-araC to HM-araCMP an d HM-araCTP in
CEM cells was 0.01 pm ol/106 cells/m in , wh ich in dicated th at th e rate of ac-
tivation of HM-araC in CEM cells was approxim ately 100-fold less th an th at
of dCyd.
TABLE I
Ph osph orylation of 4′-C-(h ydroxym eth yl)arabin ofuran osyl an alogs by recom bin an t dTh d
kin ase 1, dTh d kin ase 2, or dCyd kin ase
dTh d kin ase 1 dTh d kin ase 2
dCyd kin ase
dGuo kin ase
Base
% of con trol activity
Uracil
Uracil
(α)
(β)
3.5 ± 0.4
3.0 ± 0.1
1.0 ± 0.5
1.0 ± 0.5
n d
n d
n d
n d
Th ym in e
Th ym in e
(α)
(β)
<1
<1
3.1 ± 0.8
3.7 ± 0.9
n d
n d
n d
n d
5-Fluorouracil
5-Fluorouracil
(α)
(β)
<1
<1
1.1 ± 0.1
1.7 ± 0.4
2.2 ± 1.8
5.8 ± 0.8
n d
n d
5-Fluorocytosin e
5-Fluorocytosin e
(α)
(β)
n d
n d
<1
<1
2.1 ± 0.7
103 ± 5
n d
n d
Cytosin e
Cytosin e
(α)
(β)
n d
n d
<1
<1
<1
n d
n d
72 ± 0.9
Aden in e
Aden in e
(α)
(β)
n d
n d
n d
n d
<1
<1
<1
<1
Th e ph osph oryl tran sfer assays were perform ed as described in th e Biological Materials an d
Meth ods. Th e fin al con cen tration of each n ucleoside was 100 µM an d th e values (m ean an d
stan dard deviation s of th ree determ in ation s) represen t percen t activity com pared to eith er
dTh d or dCyd. n d, n ot don e.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

4′-C-(Hydroxymethyl)nucleosides
1071
FIG. 1
Metabolism of HM-araC (18␤) in CEM cells. CEM cells (1 × 106 cells/m l) were in cubated with
10 µM [3H]HM-araC (1 µCi/m l) for 4 h . Th e cells from 15 m l cultures were collected by
cen trifugation , an d an acid-soluble extract was prepared. Th e m etabolites of HM-araC were
separated usin g BioBasic AX ion exch an ge HPLC. Fraction s were collected as th ey eluted from
th e colum n an d coun ted for radioactivity
FIG. 2
Rate of production of HM-araCMP an d HM-araCTP in CEM cells. CEM cells (1 × 106 cells/m l)
were in cubated with 10 µM [3H]HM-araC (1 µCi/m l). Cells were collected from 15 m l of culture
an d th e am oun t of HM-araCMP an d HM-araCTP were determ in ed as described in th e legen d to
Fig. 1
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1072
Griffon et al.:
Th ere was a con siderable accum ulation of HM-araCMP in CEM cells,
wh ich in dicated th at th e rate of its syn th esis was m uch greater th an th e
rate of its use as a substrate by th e m on oph osph ate kin ase (Fig. 2). At early
tim e poin ts th e predom in an t in tracellular m etabolite was HM-araCMP.
However, by 6 h th e in tracellular con cen tration of HM-araCTP was equal to
th at of th e m on oph osph ate (Fig. 2). Th is result in dicated th at HM-araCMP
was a relatively poor substrate for CMP kin ase.
With a partially pure preparation of dCyd kin ase from CEM cells we com -
pared th e rate of ph osph orylation of dCyd an d HM-araC usin g both ATP
an d UTP as th e ph osph ate don or (Table II). With ATP as th e ph osph ate do-
n or th e HM-araC was ph osph orylated at a rate th at was 0.7% of th at seen
with dCyd, an d with UTP th e rate of HM-araC ph osph orylation was 6% of
th at of dCyd. Th ese results are in gen eral agreem en t with th e in tact cells
studies an d in dicate th at HM-araC was on ly a m odest substrate for dCyd
kin ase isolated from CEM cells. Th e kin etic data with th e recom bin an t en -
zym e in dicates th at th e ph osph orylation with HM-araC at 10 µM would be
15% of th at of dCyd. Previous studies in our laboratory41 in dicate th at th e
ph osph orylation of 10 µM araC by dCyd kin ase isolated from CEM cells
would be sim ilar to th at of dCyd usin g both UTP an d ATP as th e ph osph ate
don or. Th erefore, th ese results in dicate th at HM-araC is approxim ately
100-fold less efficien t as a substrate for dCyd kin ase th an araC.
HM-araC was n ot toxic to CEM cells. Th e con cen tration of HM-araC re-
quired to in h ibit growth by 50% durin g a 96-h in cubation was greater th an
150 µM. Alth ough th e m etabolism is n ot as great as th at seen with araC, a
con siderable am oun t of HM-araCTP was form ed in CEM cells, wh ich sug-
gested th at HM-araCTP was poorly recogn ized by th e h um an DNA
polym erases in volved in DNA replication . Sin ce HM-araCTP was form ed in
TABLE II
HM-araC (18␤) an d dCyd as substrates for dCyd kin ase purified from CEM cells
Specific activity
n m ol/m g/m in
Percen t of activity
with dCyd
Substrate, 10 µM
dCyd
Ph osph ate don or
ATP
UTP
5.80
0.66
HM-araC
ATP
UTP
0.04
0.04
0.7
6.0
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4′-C-(Hydroxymethyl)nucleosides
1073
cells with out toxicity, th is result suggests th at th is com poun d sh ould be
exam in ed for an tiviral activity.
EXPERIMENTAL
Biological Materials an d Meth ods
Determination of cytotoxicity. CCRF-CEM cells (obtain ed from th e Am erican Type Culture
Collection ) were grown in RPMI 1640 m edium (Gibco-BRL) con tain in g 10% of fetal bovin e
serum (Atlan ta Biologicals), 10 U/m l of pen icillin , 10 µg/m l of streptom ycin , an d 50 µg/m l
of gen tam ycin . Th e cells were exposed con tin uously to various con cen tration s of th e com -
poun ds at 37 °C for 72 h , an d cell n um bers were determ in ed with a Coulter Coun ter.
Measurement of nucleoside kinase activity with recombinant nucleoside kinases. Th e recom bi-
n an t h um an en zym es dTh d kin ase 1, dTh d kin ase 2, an d dCyd kin ase were expressed an d
purified from th e bacterial expression system accordin g to th e procedures described previ-
ously42 with out rem oval of th e h istidin e tag sequen ces. Th e en zym es were at least 95% pure
as judged by SDS-PAGE. dTh d kin ase 1 an d dTh d kin ase 2 h ad specific activities of 17 an d
70 n m ol/m in /m g protein , respectively, usin g dTh d as substrate. Th e specific activity of dCyd
kin ase was 18 n m ol/m in /m g usin g dCyd as substrate. Th e assays were perform ed as de-
scribed previously42 with m in or m odification s. Th e reaction m ixtures con tain ed fin al con -
cen tration s of 100 µM of n ucleosides, 100 µM [32P]ATP (with 0.0325 µM γ-[32P]ATP from
Am ersh am ), 50 m M Tris-HCl (pH 7.6), 5 m M MgCl2, 125 m M KCl, 10 m M dith ioth reitol,
0.5 m g/m l bovin e serum album in an d 2 µg/m l en zym e. After in cubation at 37 °C for 20 m in
th e sam ples were h eated at 95 °C for 2 m in followed by cen trifugation . Five µl of each sam -
ple was applied on to PEI-cellulose TLC plates (Merck), wh ich were developed overn igh t with
a solven t system con tain in g isobutyric acid/am m on ium h ydroxide/water (66:1:33). Th e
radiolabeled spots were visualized by a ph osph or-im ager (Fuji Film , Scien ce Lab., Im age
Gauge V3.3).
Measurement of [3H]HM-araC intracellular metabolites. CEM cells were in cubated with
[3H]HM-araC (obtain ed from Moravek Bioch em icals, Brea, CA, U.S.A.). At th e en d of th e
in cubation periods, th e cells were collected by cen trifugation , wash ed twice with sterile
ph osph ate buffered salin e, an d th en treated with 0.5 M ice-cold perch loric acid as de-
scribed43. Th e perch loric extracts were cen trifuged at 12 000 g for 10 m in , an d th e
supern atan t fluid was n eutralized with th e appropriate am oun t of 4 M KOH an d th en buf-
fered with 1 M tripotassium ph osph ate (pH 7.4). Th e KClO4 precipitate was rem oved by
cen trifugation , an d th e extract was in jected on to a stron g an ion exch an ge (SAX) HPLC sys-
tem . A BioBasic ion exch an ge HPLC colum n (Th erm o Electron Corp., Bellefon te, PA, U.S.A.)
was used to an alyze th e ph osph orylated m etabolites of HM-araC, because of its superior re-
ten tion of m on on ucleotides. Elution of n ucleotides with th is colum n was accom plish ed with
30-m in lin ear salt an d pH gradien t from 6 m M am m on ium ph osph ate (pH 2.8) to 900 m M
am m on ium ph osph ate (pH 6) with a flow rate of 2 m l/m in . Radioactive m etabolites of
HM-araC were detected by coun tin g 1-m in fraction s th at eluted from th e colum n . Natural
n ucleotides were detected by th eir absorban ce at 260 n m .
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1074
Griffon et al.:
Ch em ical Syn th esis
Meltin g poin ts were determ in ed in open capillary tubes on a Mel-Tem p apparatus an d are
un corrected. Th e UV absorption spectra (λ, n m ; ε) were recorded on Perkin –Elm er Lam bda 9
spectroph otom eter in m eth an ol. 1H NMR spectra were run in CDCl3 or DMSO-d6 on
a
Nicolet NT-300 spectrom eter operatin g at 300.625 MHz. Ch em ical sh ifts (δ) are given in
ppm down field from tetram eth ylsilan e. Wh en n ecessary, deuterium exch an ge, selective de-
couplin g experim en ts an d NOE experim en ts were perform ed in order to con firm proton as-
sign m en ts. All J values are in Hz. Mass spectra were recorded on a Varian /MAT 311A
dou ble-focu sin g m ass spectrom eter in th e positive fast atom bom bardm en t ion m ode
(FAB > 0) usin g 3-n itroben zyl alcoh ol/lith ium ch loride m atrix (NBA, LiCl) or on a PE Sciex
API3 m ass spectrom eter in an electro-spray (ES) tech n ique. HPLC an alyses were carried out
on a Hewlett–Packard 1100 series liquid ch rom atograph with a Ph en om en ex sph ereclon e 5 µ
ODS(1) colum n (4.6 × 250 m m ) an d UV m on itorin g (254 n m ). Elem en tal an alyses were car-
ried out by th e Spectroscopic an d An alytical Laboratory of South ern Research In stitute or by
Atlan tic Microlabs, Atlan ta, GA, U.S.A. Th in layer ch rom atograph y (TLC) was perform ed on
precoated alum in um sh eets of Silica Gel 60 F254 (Merck, Art. 5554), visualization of products
bein g accom plish ed by UV absorption followed by ch arrin g with a saturated am m on ium sul-
fate solution . Colum n ch rom atograph y was carried out on Merck Silica Gel 60 (230–400
m esh ) at atm osph eric pressure.
1,2-O-Isopropyliden e-α-L-xylofuran ose (1)
To an h ydrous aceton e (1 l) were successively added 97% sulfuric acid (5 m l), an h ydrous cop-
per sulfate (100 g) an d L-xylose (50 g, 0.33 m ol). Th e m ixture was stirred at room tem pera-
ture for 24 h with exclusion of m oisture an d filtered. Th e filtrate was n eutralized with 20%
am m on ium h ydroxide (35 m l), filtered an d evaporated to dryn ess un der reduced pressure.
Th e residue was th en co-evaporated several tim es with absolute eth an ol to afford
1,2:3,5-di-O-isopropyliden e-α-L-xylofuran ose as a syrup, on ly con tam in ated with a trace of 1.
To th is syrup, aqueous 0.2% h ydroch loric acid (440 m l) was added an d th e m ixture was
stirred at room tem perature for 3 h an d n eutralized with solid sodium h ydrogen carbon ate.
Th e solution was evaporated to dryn ess un der reduced pressure an d th e crude m aterial was
dissolved in ch loroform (200 m l), dried over an h ydrous sodium sulfate an d evaporated
un der reduced pressure to give pure 1 as a yellow syrup (59.1 g, 94%). 1H NMR (CDCl3):
5.99 (d, 1 H, H-1, J1-2 = 3.6); 4.53 (d, 1 H, H-2, J2-1 = 3.6); 4.34 (d, 1 H, H-3, J3-4 = 2.4);
4.2–4.0 (m , 3 H, H-4, H-5 an d H-5′); 3.7–2.9 (br s, 2 H, OH-5 an d OH-3); 1.49 (s, 3 H, CH3);
1.33 (s, 3 H, CH3). MS (m atrix NBA, LiCl): FAB > 0 m/z 429 (2 M + Li + LiCl)+, 387 (2 M +
Li)+, 239 (M + Li + LiCl)+, 197 (M + Li)+.
5-O-Ben zoyl-1,2-O-isopropyliden e-α-L-xylofuran ose (2)
A solution of 1 (59.1 g, 0.31 m ol) in an h ydrous pyridin e (400 m l) was stirred un der argon
an d cooled to 0 °C. A solution of ben zoyl ch loride (37.8 m l, 0.327 m ol) in an h ydrous pyri-
din e (100 m l) was added dropwise an d th e reaction m ixture was stirred at 0 °C for 45 m in .
Th en th e reaction was quen ch ed with water (5 m l) an d th e m ixture was partially con cen -
trated un der reduced pressure an d diluted in m eth ylen e ch loride (500 m l). Th e organ ic layer
was wash ed with a saturated aqueous sodium h ydrogen carbon ate solution (2 × 300 m l),
with water (2 × 300 m l) an d th en dried over sodium sulfate an d evaporated un der reduced
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

4′-C-(Hydroxymethyl)nucleosides
1075
pressure. Th e residue was purified by silica gel colum n ch rom atograph y (eluen t: stepwise
gradien t of m eth an ol, 0–8% in m eth ylen e ch loride) to give pure 2 (70.3 g, 77%) as a syrup.
1H NMR (CDCl3): 8.1–7.4 (m , 5 H, C6H5CO); 5.96 (d, 1 H, H-1, J1-2 = 3.6); 4.81 (dd, 1 H,
H-5, J4-5 = 9.5, J5-5′ = 12.6); 4.60 (d, 1 H, H-2, J2-1 = 3.6); 4.4–4.3 (m , 2 H, H-4 an d H-5′); 4.18
(br t, 1 H, H-3, J3-4 = 2.2, J3-OH = 3.9); 3.29 (d, 1 H, OH-3, JOH-3 = 3.9); 1.51 (s, 3 H, CH3);
1.33 (s, 3 H, CH3). MS (m atrix NBA, LiCl): FAB > 0 m/z 301 (M + Li)+, 295 (M + H)+, 105
(C6H5CO)+.
5-O-Ben zoyl-3-O-(tert-butyldim eth ylsilyl)-1,2-O-isopropyliden e-α-L-xylofuran ose (3)
To a solution of 2 (69.0 g, 0.23 m ol) in dry pyridin e (800 m l) un der argon atm osph ere were
added tert-butyldim eth ylsilyl ch loride (53.0 g, 0.35 m ol) an d im idazole (40.0 g, 0.59 m ol),
an d th e m ixture was stirred at 30 °C for 24 h . Th en th e reaction was quen ch ed with 10%
aqueous sodium h ydrogen carbon ate solution (10 m l), partially con cen trated un der reduced
pressure an d diluted in ch loroform (500 m l). Th e organ ic layer was wash ed with a 10%
aqueous sodium h ydrogen carbon ate solution (2 × 300 m l), with water (2 × 300 m l) an d th en
dried over sodium sulfate an d evaporated un der reduced pressure. Th e residue was purified
by silica gel colum n ch rom atograph y (eluen t: stepwise gradien t of m eth an ol, 0–8% in ch loro-
form ) to give pure 3 (96.6 g, quan titative) as a colorless syrup. 1H NMR (CDCl3): 8.1–7.4 (m ,
5 H, C6H5CO); 5.97 (d, 1 H, H-1, J1-2 = 3.6); 4.6–4.4 (m , 3 H, H-4, H-5 an d H-5′); 4.40 (d, 1 H,
H-2, J2-1 = 3.6); 4.30 (br d, 1 H, H-3, J3-4 = 1.5); 1.51 (s, 3 H, CH3); 1.34 (s, 3 H, CH3); 0.90
(s, 9 H, (CH3)3C-Si); 0.14 (s, 3 H, (CH3)Si); 0.09 (s, 3 H, (CH3)Si). MS (m atrix NBA, LiCl):
FAB > 0 m/z 425 (M + Li)+, 409 (M + H)+, 351 (M + H – [(CH3)3CH]), 115 (TBDMS)+, 105 (C6H5CO)+.
3-O-(tert-Butyldim eth ylsilyl)-1,2-O-isopropyliden e-α-L-xylofuran ose (4)
Sodium (5.37 g, 0.23 m ol) was added at 0 °C to a solution of 3 (90.9 g, 0.22 m ol) in a m ix-
ture of an h ydrous toluen e (225 m l) an d m eth an ol (450 m l). Th e resultin g solution was
stirred at 0 °C durin g 1 h an d th en n eutralized at 0 °C by addition of Dowex 50WX2 (H+
form ). Th e resin was filtered an d wash ed with warm m eth an ol, an d th e com bin ed filtrates
were evaporated to dryn ess. Colum n ch rom atograph y of th e residue on silica gel (eluen t:
stepwise gradien t of m eth an ol, 0–6% in ch loroform ) afforded 4 (59.4 g, 88%), wh ich was
recrystallized from cycloh exan e, m .p. 67–68 °C. 1H NMR (CDCl3): 5.96 (d, 1 H, H-1, J1-2
=
3.7); 4.37 (d, 1 H, H-2, J2-1 = 3.7); 4.3–4.2 (m , 2 H, H-3 an d H-4); 4.0–3.7 (m , 2 H, H-5 an d
H-5′); 2.01 (dd, OH-5, JOH-5 = 8.8, JOH-5′ = 3.3); 1.50 (s, 3 H, CH3); 1.33 (s, 3 H, CH3); 0.90 (s,
9 H, (CH3)3C-Si); 0.15 (s, 3 H, (CH3)Si); 0.12 (s, 3 H, (CH3)Si). MS (m atrix NBA, LiCl): FAB > 0
m/z 657 (2 M + Li + LiCl)+, 615 (2 M + Li)+, 353 (M + Li + LiCl)+, 311 (M + Li)+.
4-C-(Hydroxym eth yl)-1,2-O-isopropyliden e-β-D-threo-pen tofuran ose (6)
Dicycloh exylcarbodiim ide (DCC; 50.3 g, 0.24 m ol) an d dich loroacetic acid (4 m l, 48.7
m m ol) were added to a solution of 4 (49.5 g, 0.16 m ol) in an h ydrous ben zen e (490 m l),
DMSO (325 m l) an d pyridin e (13 m l). Th e resultin g solution was stirred at room tem pera-
ture un der argon atm osph ere for 23 h an d diluted with eth yl acetate (650 m l). Oxalic acid
(2.3 g, 25.5 m m ol) dissolved in DMSO (30 m l) was added an d th e reaction m ixture was
stirred at room tem perature durin g 1 h an d th en filtered to elim in ate precipitated dicyclo-
h exylurea (DCU). Th e filtrate was wash ed with brin e (2 × 500 m l), with a saturated aqueous
sodium h ydrogen carbon ate solution (2 × 500 m l) an d fin ally with water (3 × 500 m l) before
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

1076
Griffon et al.:
bein g dried over sodium sulfate an d evaporated un der reduced pressure. Th e presen ce of th e
aldeh yde group was sh own on TLC by a positive test after sprayin g with a solution of
2,4-din itroph en ylh ydrazin e in 1% eth an olic h ydroch loric acid an d h eatin g. Th e crude resi-
due was quickly ch rom atograph ed th rough a pad of silica gel (eluen t: stepwise gradien t of
ch loroform , 70–100% in h exan es). Th e collected m aterial (46.7 g, 0.15 m ol) was dissolved in
a m ixture of 37% aqueous form aldeh yde (30.9 m l), water (155 m l) an d dioxan e (310 m l),
an d aqueous sodium h ydroxide (154.5 m l of 1 M solution ) was added. Th e m ixture was
stirred at room tem perature durin g 23 h an d th en n eutralized by addition of Dowex 50WX2
(H+ form ). Th e resin was filtered an d wash ed with warm m eth an ol, an d th e com bin ed
filtrates were evaporated to dryn ess. Th e residue was purified by silica gel colum n ch rom ato-
graph y (eluen t: stepwise gradien t of m eth an ol, 4–10% in ch loroform ) to give pure 6 (27.2 g,
76%), wh ich was recrystallized from m eth ylen e ch loride, m .p. 90–92 °C. 1H NMR (DMSO-d6):
6.01 (d, 1 H, H-1, J1-2 = 4.2); 4.65 (d, 1 H, H-2, J2-1 = 4.2); 4.24 (d, 1 H, H-3, J3-OH = 5.3);
3.89 (d, 2 H, H-5 an d H-5′, J5-OH = J5′-OH = 6.2); 3.82 (dd, 1 H, H-6, J6-OH = 3.7, J6-6′ = 11.6);
3.71 (d, OH-3, JOH-3 = 5.5); 3.59 (dd, 1 H, H-6′, J6′-OH = 8.8, J6-6′ = 11.6); 2.52 (t, 1 H, OH-5,
JOH-5 = JOH-5′ = 6.2); 2.26 (dd, 1 H, OH-6, JOH-6 = 4.0, JOH-6′ = 8.8); 1.54 (s, 3 H, CH3); 1.32 (s,
3 H, CH3). MS (m atrix NBA, LiCl): FAB > 0 m/z 227 (M + Li)+.
3,5-Di-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-1,2-O-isopropyliden e-β-D-threo-pen tofuran ose (7)
To an ice-cold solution of 6 (26.8 g, 0.12 m ol) in dry THF (730 m l) was added portion wise
un der argon sodium h ydride (60% dispersion in m in eral oil, 29.2 g, 0.73 m ol), an d th e
reaction m ixture was stirred for 15 m in un der argon atm osph ere. Solid tetrabutylam m on ium
iodide (2.7 g, 7.3 m m ol) was added to th e reaction m ixture, followed by a dropwise addition
of ben zyl brom ide (72.4 m l, 0.61 m m ol). Th e reaction m ixture was stirred at room tem pera-
ture for 24 h un der argon atm osph ere. After addition of m eth an ol (25 m l), th e solution was
partially con cen trated un der reduced pressure an d diluted with m eth ylen e ch loride (500 m l).
Th e organ ic layer was wash ed with brin e (2 × 400 m l), with water (400 m l) an d th en dried
over sodium sulfate an d evaporated un der reduced pressure. Th e residue was purified by silica
gel colum n ch rom atograph y (eluen t: ch loroform /h exan es, 8:2) to give pure 7 (53.3 g, 91%)
as a syrup. 1H NMR (CDCl3): 7.3–7.2 (m , 15 H, 3 C6H5); 6.00 (d, 1 H, H-1, J1-2 = 4.5); 4.76
(dd, 1 H, H-2, J2-1 = 4.6, J2-3 = 2.3); 4.8–4.4 (m , 6 H, 3 Ph CH2); 4.12 (d, 1 H, H-3, J3-2 = 2.3);
3.7–3.5 (m , 4 H, H-5, H-5′, H-6 and H-6′); 1.46 (s, 3 H, CH3); 1.34 (s, 3 H, CH3). MS (m atrix NBA,
LiCl): FAB > 0 m/z 497 (M + Li)+, 91 (C6H5CH2)+.
Meth yl 3,5-Di-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α,β-D-threo-pen tofuran oside (8)
To an ice-cold solution of 7 (53.2 g, 92 m m ol) in 85% acetic acid (185 m l) was added con -
cen trated sulfuric acid (0.75 m l, 13.8 m m ol). Th e tem perature was raised to 50 °C an d th e
reaction was stirred at th is tem perature durin g 3.5 h . Th en th e reaction m ixture was cooled
in an ice-cold bath , diluted with water (100 m l) an d n eutralized with solid sodium
h ydrogen carbon ate. Th e organ ic layer was extracted with ch loroform (2 × 300 m l) an d th e
com bin ated extracts were successively wash ed with
a saturated aqueous sodium
h ydrogen carbon ate solution (2 × 400 m l) an d fin ally with water (2 × 400 m l) before bein g
dried over sodium sulfate an d evaporated to dryn ess. Th e resultin g crude residue was dis-
solved in dry m eth an ol (550 m l) an d cooled to 0 °C. To th is ice-cold solution was added
con cen trated sulfuric acid (1.5 m l, 27.6 m m ol) an d th e reaction was strirred at room tem pe-
rature durin g 23 h , th en n eutralized with pyridin e, con cen trated un der reduced pressure an d
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4′-C-(Hydroxymethyl)nucleosides
1077
diluted with m ethylene chloride (300 m l). The organic layer was washed with water (2 × 300 m l)
before bein g dried over sodium sulfate an d evaporated to dryn ess. Th e resultin g residue was
purified by silica gel colum n ch rom atograph y (eluen t: ch loroform /m eth an ol, 0.5%) to give
pure 8 as a pale yellow syrup (40.6 g, 95%) as a ~1:2 α:β m ixture. 1H NMR (CDCl3): 7.3–7.2
(m , 15 H, 3 C6H5); 4.88 (br s, 1 H, H-1 α an om er); 4.85 (d, 1 H, H-1 β an om er, J1-2 = 4.8);
4.8–4.4 (m , 6 H, 3 Ph CH2); 4.4–4.3 (m , 1 H, H-2); 3.94 (m , 1 H, H-3); 3.7–3.5 (m , 4 H, H-5,
H-5′, H-6 an d H-6′); 3.41 (s, 3 H, OCH3); 2.43 (d, 1 H, OH-2, JOH-2 = 9.9). MS (m atrix NBA,
LiCl): FAB > 0 m/z 513 (M + Li + LiCl)+, 471 (M + Li)+, 91 (C6H5CH2)+.
Meth yl 2,3,5-Tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α,β-D-threo-pen tofuran oside (9)
To an ice-cold solution of 8 (10.2 g, 22.0 m m ol) in dry THF (180 m l) was added portion wise
un der argon sodium h ydride (60% dispersion in m in eral oil, 2.63 g, 65.9 m ol), an d th e
reaction m ixture was stirred for 15 m in un der argon atm osph ere. Solid tetrabutylam m on ium
iodide (162 m g, 0.44 m m ol) was added to th e reaction m ixture followed by a dropwise addi-
tion of ben zyl brom ide (6.5 m l, 54.9 m m ol). Th e reaction m ixture was stirred at room tem -
perature for 22 h un der argon atm osph ere. After th e addition of m eth an ol (9 m l), th e
solution was partially con cen trated un der reduced pressure an d diluted with m eth ylen e
ch loride (400 m l). Th e organ ic layer was wash ed with brin e (2 × 300 m l), with water (1 ×
400 m l) an d th en dried over sodium sulfate an d evaporated un der reduced pressure. Th e resi-
due was purified by silica gel colum n ch rom atograph y (eluen t: stepwise gradien t of ch loro-
form , 70–100% in h exan es) an d th en a stepwise gradien t of m eth an ol (0–1%) in ch loroform
to give pure 9 as a pale yellow syrup (11.3 g, 92%) as a ~1:2 α:β m ixture. 1H NMR (CDCl3):
7.4–7.1 (m , 20 H, 4 C6H5); 4.94 (d, 1 H, H-1 α an om er, J1-2 = 1.8); 4.70 (d, 1 H, H-1 β
an om er, J1-2 = 4.4); 4.7–4.4 (m , 8 H, 3 Ph CH2); 4.24 (d, 1 H, H-3 β an om er, J3-2 = 8.1); 4.12
(m , 2 H, H-2 β an om er an d H-3 α an om er); 4.02 (dd, 1 H, H-2 α an om er, J2-1 = 1.8, J2-3
=
3.5); 3.8–3.4 (m , 4 H, H-5, H-5′, H-6 an d H-6′); 3.39 (s, 3 H, OCH3 α an om er); 3.33 (s, 3 H,
OCH3 β an om er). MS (m atrix NBA, LiCl): FAB > 0 m/z 561 (M + Li)+, 91 (C6H5CH2)+.
1-O-Acetyl-2,3,5-tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α,β-D-threo-pen tofuran ose (10)
An ice-cold 90% aqueous trifluoroacetic acid solution (51 m l) was added to 9 (9.5 g, 17.1
m m ol) an d th e reaction m ixture was stirred between 3 an d 10 °C for 4.5 h an d at room tem -
perature for 1.5 h . Th en th e reaction m ixture was cooled in an ice-cold bath , diluted with
water (50 m l) an d n eutralized with solid sodium h ydrogen carbon ate. Th e organ ic layer was
extracted with ch loroform (2 × 150 m l) an d th e com bin ed extracts were successively wash ed
with a saturated aqueous sodium h ydrogen carbon ate solution (2 × 200 m l) an d fin ally with
water (2 × 200 m l) before bein g dried over sodium sulfate an d evaporated to dryn ess. Th e re-
sultin g residue was co-evaporated twice with an h ydrous pyridin e an d diluted in th is solven t
(120 m l). To th e solution was added acetic an h ydride (16.2 m l, 171.3 m m ol) an d th e reac-
tion was stirred at room tem perature durin g 5 h , th en partially con cen trated un der reduced
pressure an d diluted with m eth ylen e ch oride (300 m l). Th e organ ic layer was successively
wash ed with a saturated aqueous sodium h ydrogen carbon ate solution (2 × 200 m l) an d fi-
n ally with water (2 × 200 m l) before bein g dried over sodium sulfate an d evaporated to dry-
n ess an d co-evaporated with toluen e. Th e crude m aterial was purified by silica gel colum n
ch rom atograph y (eluen t: stepwise gradien t of ch loroform , 70–100% in h exan es) to give pure
10 as a pale yellow syrup (8.1 g, 80%) as a 3:1 α:β m ixture. 1H NMR (CDCl3): 7.4–7.2 (m , 20 H,
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1078
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4 C6H5); 6.29 (d, 1 H, H-1 β an om er, J1-2= 4.4); 6.21 (d, 1 H, H-1 α an om er, J1-2 = 1.1);
4.7–4.4 (m , 8 H, 3 Ph CH2); 4.36 (dd, 1 H, H-2 β an om er, J2-1 4.4, J2-3 = 8.3); 4.22 (d, 1 H,
H-3 β an om er, J3-2 = 8.3); 4.16 (d, 1 H, H-3 α an om er, J3-2 = 2.5); 4.12 (dd, 1 H, H-2 α
an om er, J2-1 = 1.4, J2-3 = 2.9); 3.8–3.3 (m , 4 H, H-5, H-5′, H-6 an d H-6′); 1.99 (s, 3 H,
CH3CO2 α an om er); 1.99 (s, 3 H, CH3CO2 β an om er). MS (m atrix NBA, LiCl): FAB > 0 m/z
589 (M + Li)+, 91 (C6H5CH2)+.
1-(2,3,5-Tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α- an d -β-D-threo-pen tofuran osyl)uracil
(11␣) an d (11␤)
A m ixture of uracil (276 m g, 2.46 m m ol), h exam eth yldisilazan e (HMDS; 16 m l) an d a cata-
lytic am oun t of am m on ium sulfate was h eated durin g 18 h un der reflux. After coolin g to
room tem perature, th e m ixture was evaporated un der reduced pressure, an d th e residue ob-
tain ed as a colorless oil was diluted with an h ydrous 1,2-dich loroeth an e (17 m l). To th e re-
sultin g solution was added 10 (0.99 g, 1.70 m m ol) in an h ydrous 1,2-dich loroeth an e (10 m l),
followed by addition of trim eth ylsilyl trifluorom eth an esulfon ate (TMSTf, 0.62 m l, 3.40
m m ol). Th e solution was stirred at room tem perature for 2.5 h un der argon atm osph ere,
th en diluted with ch loroform (150 m l), wash ed with th e sam e volum e of a saturated aque-
ous sodium h ydrogen carbon ate solution an d fin ally with water (2 × 100 m l). Th e organ ic
ph ase was dried over sodium sulfate, th en evaporated un der reduced pressure. Th e resultin g
crude m aterial, as a 1.8:1 α:β m ixture, was purified by silica gel colum n ch rom atograph y
(eluen t: ch loroform /m eth an ol, 0.5%) to afford as pale yellow foam s pure 11␤ (295 m g,
27%), followed by a m ixture of 11␣ an d 11␤ (134 m g, 12%) an d pure 11␣ (550 m g, 51%).
11␤: 1H NMR (CDCl3): 7.99 (br s, 1 H, NH); 7.69 (d, 1 H, H-6, J6-5 = 8.2); 7.4–7.1 (m , 20 H,
4 C6H5); 6.49 (d, 1 H, H-1′, J1′-2′ = 6.1); 5.33 (dd, 1 H, H-5, J5-6 = 8.2, J5-NH = 2.3); 4.7–4.3 (m ,
9 H, H-2′ an d 4 Ph CH2); 4.32 (d, 1 H, H-3′, J3′-2′ = 6.3); 3.64 (d, 1 H, H-6′, J6′-6′′ = 10.0); 3.55
(d, 1 H, H-5′, J5′-5′′ = 10.2); 3.41 (d, 1 H, H-5′′, J5′′-5′ = 10.2); 3.40 (d, 1 H, H-6′′, J6′′-6′ = 10.0).
MS (m atrix NBA, LiCl): FAB > 0 m/z 641 (M + Li)+, 91 (C6H5CH2)+.
11␣: 1H NMR (CDCl3): 8.05 (br s, 1 H, NH); 7.62 (d, 1 H, H-6, J6-5 = 8.2); 7.4–7.1 (m , 20 H,
4 C6H5); 6.08 (d, 1 H, H-1′, J1′-2′ = 5.4); 5.22 (dd, 1 H, H-5 J5-6 = 8.2, J5-NH = 2.3); 4.7–4.3 (m ,
8 H, 4 Ph CH2); 4.22 (d, 1 H, H-3′, J3′-2′ = 5.6); 4.17 (t, 1 H, H-2′, J2′-1′ = J2′-3′ 5.5); 3.75 (d, 1 H,
H-6′, J6′-6′′ = 10.0); 3.69 (d, 1 H, H-6′′, J6′′-6′ = 10.0); 3.49 (d, 1 H, H-5′, J5′-5′′ = 9.8); 3.44 (d, 1 H,
H-5′′, J5′′-5′ = 9.8). MS (m atrix NBA, LiCl): FAB > 0 m/z 641 (M + Li)+, 91 (C6H5CH2)+.
1-[4-C-(Hydroxym eth yl)-β-D-threo-pen tofuran osyl]uracil (12␤)
A m ixture of 11␤ (280 m g, 0.441 m m ol) an d 67 m g of 10% Pd/C in a 1:1 solution of m eth a-
n ol/acetic acid (13 m l) was h ydrogen ated at room tem perature an d atm osph eric pressure
durin g 2 days. Th e m ixture was filtered th rough a pad of Celite an d th e filtrate was con cen -
trated un der reduced pressure. Th e crude m aterial was purified by silica gel colum n ch rom a-
tograph y (eluen t: stepwise gradien t of m eth an ol, 10–20% in ch loroform ) to give pure 12␤
(90 m g, 74%), wh ich was recrystallized from eth an ol, m .p. 182–184 °C. UV (m eth an ol): λm ax
263 (11300), λm in 242 (6200). HPLC 99.8%, 99:1 H2O/MeCN. 1H NMR (DMSO-d6): 11.21 (s,
1 H, NH); 7.73 (d, 1 H, H-6, J6-5 = 8.1); 6.12 (d, 1 H, H-1′, J1′-2′ = 5.8); 5.55 (d, 1 H, H-5, J5-6
=
8.1); 5.44 (d, 1 H, OH-2′, JOH-2′ = 5.2); 5.35 (d, 1 H, OH-3′, JOH-3′ = 5.1); 5.03 (t, 1 H, OH-5′,
JOH-5′ = JOH-5′′ = 5.3); 4.57 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′ = 5.8); 4.28 (dd, 1 H, H-2′, J = 5.4, J =
10.9); 4.03 (t, 1 H, H-3′, J3′-2′ = J3′-OH′ = 5.3); 3.56 (dd, 1 H, H-6′, J6′-6′′ = 11.5, JOH-6′ = 5.6);
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4′-C-(Hydroxymethyl)nucleosides
1079
3.48 (br d, 2 H, H-5′ an d H-5′′, J = 4.2); 3.34 (dd, 1 H, H-6′′, J6′′-6′ = 11.5, JOH-6′ = 5.9). MS
(m atrix NBA, LiCl): FAB > 0 m/z 287 (M – H + 2 Li)+, 281 (M + Li)+. For C10H14N2O7 (274.2)
calculated: 43.80% C, 5.15% H, 10.22% N; foun d: 43.83% C, 5.03% H, 10.09% N.
1-[4-C-(Hydroxym eth yl)-α-D-threo-pen tofuran osyl]uracil (12␣)
Hydrogen ation of 11␣ (534 m g, 0.841 m m ol) was perform ed followin g th e sam e procedure
as described for th e deprotection of com poun d 11␤, an d provided, after purification by
silica gel ch rom atograph y, pure 12␣ (218 m g, 94%), wh ich was precipitated from a m eth a-
n ol/eth yl eth er m ixture, m .p. 79–85 °C (h ygroscopic). UV (m eth an ol): λm ax 262 (8000), λm in
231 (1900). HPLC 99.0%, 99:1 H2O/MeCN. 1H NMR (DMSO-d6): 11.26 (s, 1 H, NH); 8.07 (d,
1 H, H-6, J6-5 = 8.1); 5.76 (d, 1 H, H-1′, J1′-2′′ = 6.4); 5.69 (d, 1 H, H-5, J5-6 = 8.1); 5.57 (d, 1 H,
OH-2′, JOH-2′ = 5.0); 5.37 (d, 1 H, OH-3′, JOH-3′ = 4.9); 4.89 (t, 1 H, OH-5′, JOH-5′ = JOH-5′′
=
5.7); 4.69 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′ = 5.4); 4.1–4.0 (m , 2 H, H-2′ an d H-3′); 3.60 (dd, 1 H,
H-6′, J6′-6′′ = 11.7, JOH-6′ = 5.0); 3.4–3.3 (m , 3 H, H-5′, H-5′′ an d H-6′′). MS (m atrix NBA, LiCl):
FAB > 0 m/z 287 (M – H + 2 Li)+, 281 (M + Li)+. For C10H14N2O7·H2O (292.2) calculated:
41.10% C, 5.52% H, 9.59% N; foun d: 41.35% C, 5.11% H, 9.17% N.
1-{2,3,5-Tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α- an d -β-D-threo-pen tofuran osyl}th ym in e
(13␣) an d (13␤)
Con den sation of th ym in e (311 m g, 2.46 m m ol) with 10 (0.99 g, 1.70 m m ol) was perform ed
followin g th e sam e procedure as described for th e preparation of com poun ds 11␣ an d 11␤
an d provided a 3.3:1 α:β m ixture of 13␣ an d 13␤, wh ich was separable by silica gel ch rom a-
tograph y (eluen t: stepwise gradien t of m eth an ol, 0–0.5% in ch loroform ): 13␤ (213 m g,
19%), m ixture of 13␣ an d 13␤ (170 m g, 15%), an d 13␣ (590 m g, 53%). Both com poun ds
were pale yellow foam s.
13␤: 1H NMR (CDCl3): 8.06 (br s, 1 H, NH); 7.47 (d, 1 H, H-6, J6-Me = 1.1); 7.3–7.1 (m , 20 H,
4 C6H5); 6.51 (d, 1 H, H-1′, J1′-2′ = 6.2); 4.7–4.3 (m , 9 H, 4 Ph CH2); 4.58 (t, 1 H, H-2′, J2′-1′
=
J2′-3′ = 6.2); 4.37 (d, 1 H, H-3′, J3′-2′ = 6.2); 3.64 (d, 1 H, H-6′, J6′-6′′ = 9.9); 3.57 (d, 1 H, H-5′,
J5′-5′′ = 9.9); 3.44 (d, 1 H, H-5′′, J5′′-5′ = 10.1); 3.40 (d, 1 H, H-6′′, J6′′-6′ = 10.0); 1.58 (d, 3 H,
5-CH3, JMe-6 = 1.1). MS (m atrix NBA, LiCl): FAB > 0 m/z 655 (M + Li)+, 91 (C6H5CH2)+.
13␣: 1H NMR (CDCl3): 7.95 (br s, 1 H, NH); 7.39 (d, 1 H, H-6, J6-Me = 1.3); 7.3–7.1 (m , 20 H,
4 C6H5); 6.11 (m , 1 H, H-1′); 4.7–4.4 (m , 8 H, 4 Ph CH2); 4.24 (m , 2 H, H-2′ an d H-3′); 3.73
(d, 1 H, H-6′, J6′-6′′ = 10.1); 3.69 (d, 1 H, H-6′′, J6′′-6′ = 10.2); 3.47 (d, 1 H, H-5′, J5′-5′′ = 9.8);
3.43 (d, 1 H, H-5′′, J5′′-5′ = 9.8); 1.42 (d, 3 H, 5-CH3, JMe-6 = 1.2). MS (m atrix NBA, LiCl): FAB > 0
m/z 655 (M + Li)+, 91 (C6H5CH2)+.
1-[4-C-(Hydroxym eth yl)-β-D-threo-pen tofuran osyl]th ym in e (14␤)
Hydrogen ation of 13␤ (213 m g, 0.328 m m ol) was perform ed followin g th e sam e procedure as
described for th e deprotection of com poun d 11␤, an d provided, after purification by silica
gel ch rom atograph y, pure 14␤ (90 m g, 95%), wh ich was lyoph ilized, m .p. 92–98 °C. UV
(m eth an ol): λm ax 268 (9900), λm in 234 (2900). HPLC 98.9%, 99:1 H2O/MeCN. 1H NMR
(DMSO-d6): 11.18 (s, 1 H, NH); 7.65 (d, 1 H, H-6, J6-Me = 1.2); 6.11 (d, 1 H, H-1′, J1′-2′ = 5.9);
5.49 (d, 1 H, OH-2′, JOH-2′ = 5.5); 5.32 (d, 1 H, OH-3′, JOH-3′ = 5.3); 5.10 (t, 1 H, OH-5′, JOH-5′
= JOH-5′′ = 5.3); 4.59 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′ = 5.8); 4.29 (dd, 1 H, H-2′, J = 5.8, J = 11.4);
4.03 (t, 1 H, H-3′, J3′-2′ = J3′-OH′ = 5.4); 3.56 (dd, 1 H, H-6′, J6′-6′′ = 11.5, JOH-6′ = 5.8); 3.5–3.4
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1080
Griffon et al.:
(m , 2 H, H-5′ an d H-5′′); 3.4–3.3 (m , 1 H, H-6′′); 1.75 (d, 3 H, 5-CH3, JMe-6 = 1.1). MS (ES):
m/z 599 (2 M + Na)+, 577 (2 M + H)+, 311 (M + Na)+, 289 (M + H)+. For C11H16N2O7·2/3H2O
(300.3) calculated: 43.99% C, 5.82% H, 9.33% N; foun d: 44.03% C, 5.78% H, 9.09% N.
1-[4-C-(Hydroxym eth yl)-α-D-threo-pen tofuran osyl]th ym in e (14␣)
Hydrogen ation of 13␣ (309 m g, 0.476 m m ol) was perform ed followin g th e sam e procedure
as described for th e deprotection of com poun d 11␤, an d provided, after purification by
silica gel ch rom atograph y, pure 14␣ (125 m g, 91%), wh ich was lyoph ilized from water, m .p.
92–98 °C. UV (m eth an ol): λm ax 266 (9300), λm in 233 (2300). HPLC 99.9%, 99:1 H2O/MeCN.
1H NMR (DMSO-d6): 11.26 (s, 1 H, NH); 7.93 (d, 1 H, H-6, J6-Me = 1.2); 5.75 (d, 1 H, H-1′,
J1′-2′ = 6.8); 5.52 (d, 1 H, OH-2′, JOH-2′ = 5.1); 5.32 (d, 1 H, OH-3′, JOH-3′ = 5.4); 4.88 (t, 1 H,
OH-5′, JOH-5′ = JOH-5′′ = 5.7); 4.7 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′ = 5.4); 4.2–4.1 (m , 2 H, H-2′
an d H-3′); 3.60 (dd, 1 H, H-6′, J6′-6′′ = 11.7, JOH-6′ = 5.0); 3.41 (dd, 1 H, H-6′′, J6′′-6′ = 11.7,
JOH-6′ = 6.0); 3.4–3.3 (m , 1 H, H-5′ an d H-5′′); 1.78 (d, 3 H, 5-CH3, JMe-6 = 1.0). MS (ES): m/z
599 (2 M + Na)+, 577 (2 M + H)+, 311 (M + Na)+, 289 (M + H)+. For C11H16N2O7·2/3H2O
(300.3) calculated: 43.99% C, 5.82% H, 9.33% N; foun d: 43.99% C, 5.53% H, 9.16% N.
5-Fluoro-1-{2,3,5-tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl-α- an d
-β-D-threo-pen tofuran osyl}uracil (15␣) an d (15␤)
Con den sation of 5-fluorouracil (324 m g, 2.49 m m ol) with 10 (1.00 g, 1.72 m m ol) was per-
form ed followin g th e sam e procedure as described for th e preparation of com poun ds 11␣
an d 11␤ an d provided a 1.4:1 α:β m ixture, wh ich was separable by silica gel ch rom atogra-
ph y (eluen t: stepwise gradien t of m eth an ol, 0–0.2% in ch loroform ): 15␤ (258 m g, 23%),
m ixture of 15␣ an d 15␤ (146 m g, 13%), an d 15␣ (584 m g, 52%). Both com poun ds were
pale yellow foam s.
15␤: 1H NMR (CDCl3): 8.10 (br d, 1 H, NH, JNH-F = 5.0); 7.95 (d, 1 H, H-6, J6-F = 6.5);
7.4–7.1 (m , 20 H, 4 C6H5); 6.43 (d d , 1 H, H-1′, J1′-2′ = 6.2, J1′-F = 1.7); 4.7–4.3 (m , 9 H,
4 Ph CH2); 4.59 (t, 1 H, H-2′, J2′-1′ = 6.2, J2′-3′ = 6.4); 4.34 (d, 1 H, H-3′, J3′-2′ = 6.4); 3.61 (d, 1 H,
H-6′, J6′-6′′ = 9.9); 3.54 (d, 1 H, H-5′, J5′-5′′ = 10.2); 3.36 (d, 1 H, H-5′′, J5′′-5′ = 10.2); 3.35 (d, 1 H,
H-6′′, J6′′-6′ = 9.9). MS (m atrix NBA, LiCl): FAB > 0 m/z 659 (M + Li)+, 91 (C6H5CH2)+.
15␣: 1H NMR (CDCl3): 8.13 (br d, 1 H, NH, JNH-F = 4.1); 7.80 (d, 1 H, H-6, J6-F = 6.4);
7.4–7.1 (m , 20 H, 4 C6H5); 6.00 (d d , 1 H, H-1′, J1′-2′ = 5.9, J1′-F = 1.8); 4.7–4.4 (m , 8 H,
4 Ph CH2); 4.23 (d, 1 H, H-3′, J3′-2′ = 6.3); 4.18 (t, 1 H, H-2′, J2′-1′ = 5.9, J2′-3′ = 6.3); 3.49 (d, 1 H,
H-6′, J6′-6′′ = 10.1); 3.89 (d, 1 H, H-6′′, J6′′-6′ = 10.1); 3.42 (d, 1 H, H-5′, J5′-5′′ = 9.8). MS (m atrix
NBA, LiCl): FAB > 0 m/z 659 (M + Li)+, 91 (C6H5CH2)+.
5-Fluoro-1-[4-C-(h ydroxyl)m eth yl-β-D-threo-pen tofuran osyl]uracil (16␤)
Hydrogen ation of 15␤ (215 m g, 0.329 m m ol) was perform ed followin g th e sam e procedure
as described for th e deprotection of com poun d 11␤, an d provided, after purification by silica
gel ch rom atograph y, 16␤ (92 m g, 96%), wh ich was lyoph ilized from water, m .p. 86–90 °C.
UV (m eth an ol): λm ax 271 (8100), λm in 235 (1700). HPLC 99.3%, 98:2 0.01 M NH4H2PO4
(pH 5.1)/MeOH. 1H NMR (DMSO-d6): 11.76 (br s, 1 H, NH); 8.14 (d, 1 H, H-6, J6-F = 7.4);
6.09 (dd, 1 H, H-1′, J1′-2′ = 5.9, J1′-F = 1.9); 5.58 (d, 1 H, OH-2′, JOH-2′ = 5.5); 5.38 (d, 1 H,
OH-3′, JOH-3′ = 5.3); 5.23 (t, 1 H, OH-5′, JOH-5′ = JOH-5′′ = 5.0); 4.63 (t, 1 H, OH-6′, JOH-6′
=
JOH-6′′ = 5.7); 4.33 (dd, 1 H, H-2′, J = 5.8, J = 11.5); 4.05 (t, 1 H, H-3′, J3′-2′ = J3′-OH′ = 5.7);
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4′-C-(Hydroxymethyl)nucleosides
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3.55 (dd, 1 H, H-6′, J6′-6′′ = 11.7, JOH-6′ = 5.8); 3.5–3.4 (m , 2 H, H-5′ an d H-5′′); 3.28 (dd, 1 H,
H-6′′, J6′′-6′ = 11.7, JOH-6′ = 5.9). MS (m atrix NBA, LiCl): FAB > 0 m/z 293 (M + H)+. For
C
10H13FN2O7·1/2H2O (301.2) calculated: 39.87% C, 4.69% H, 9.30% N; foun d: 39.65% C,
4.71% H, 8.92% N.
5-Fluoro-1-[4-C-(h ydroxym eth yl)-α-D-threo-pen tofuran osyl]uracil (16␣)
Hydrogen ation of 15␣ (226 m g, 0.346 m m ol) was perform ed followin g th e sam e procedure
as described for th e deprotection of com poun d 11␤, an d provided, after purification by silica
gel ch rom atograph y, 16␣ (100 m g, 99%), wh ich was lyoph ilized from water, m .p. 80–84 °C.
UV (m eth an ol): λm ax 268 (8400), λm in 234 (2100). HPLC 98.1%, 98:2 0.01 M NH4H2PO4
(pH 5.1)/MeOH. 1H NMR (DMSO-d6): 11.80 (br s, 1 H, NH); 8.43 (d, 1 H, H-6, J6-F = 7.3);
5.75 (dd, 1 H, H-1′, J1′-2′ = 6.5, J1′-F = 1.8); 5.55 (d, 1 H, OH-2′, JOH-2′ = 4.9); 5.40 (d, 1 H,
OH-3′, JOH-3′ = 5.1); 5.0–4.9 (m , 2 H, OH-5′ an d OH-6′); 4.2–4.0 (m , 2 H, H-2′ an d H-3′); 3.62
(dd, 1 H, H-6′, J6′-6′′ = 11.7, JOH-6′ = 4.9); 3.4–3.3 (m , 3 H, H-5′, H-5′′an d H-6′′). MS (m atrix
NBA, LiCl): FAB > 0 m/z 293 (M + H)+. For C10H13FN2O7·5/4H2O (315.3) calculated: 38.16%
C, 4.96% H, 8.90% N; foun d: 38.10% C, 4.64% H, 8.82% N.
1-{2,3,5-Tri-O-benzyl-4-C-[(benzyloxy)methyl]-β-D-threo-pentofuranosyl}cytosine (17␤)
Lawesson ’s reagen t (140 m g, 0.345 m m ol) was added un der argon to a solution of 11␤ (313 mg,
0.493 m m ol) in an h ydrous 1,2-dich loroeth an e (12 m l) an d th e reaction m ixture was stirred
un der reflux overn igh t. Th e solven t was th en evaporated un der reduced pressure an d th e
residue was purified by silica gel colum n ch rom atograph y (eluen t: stepwise gradien t of
m eth an ol, 0–1% in ch loroform ) to give th e correspon din g 4-th io in term ediate wh ich was
treated by m eth an olic am m on ia (12 m l previously saturated at –10 °C an d tigh tly stoppered)
at 100 °C in a stain less-steel bom b for 3 h , th en cooled to room tem perature. Th e solution
was evaporated to dryn ess un der reduced pressure an d th e residue coevaporated several
tim es with m eth an ol. Th e crude residue was purified by silica gel colum n ch rom atograph y
(eluen t: stepwise gradien t of m eth an ol, 0–4% in ch loroform ) to give pure 17␤ as a brigh t
yellow foam (256 m g, 82%). 1H NMR (CDCl3): 7.98 (d, 1 H, H-6, J6-5 = 7.4); 7.3–7.1 (m , 20 H,
4 C6H5); 6.65 (d, 1 H, H-1′, J1′-2′ = 5.6); 5.37 (d, 1 H, H-5, J5-6 = 7.5); 5.3–5.1 (br s, 2 H, NH2);
4.6–4.3 (m , 9 H, H-2′ an d 4 Ph CH2); 4.25 (d, 1 H, H-3′, J3′-2′ = 5.1); 3.66 (d, 1 H, H-6′, J6′-6′′
=
10.1); 3.58 (d, 1 H, H-5′, J5′-5′′ = 10.1); 3.53 (d, 1 H, H-5′′, J5′′-5′ = 10.1); 3.49 (d, 1 H, H-6′′,
J6′′-6′ = 10.1). MS (m atrix NBA, LiCl): FAB > 0 m/z 640 (M + Li)+, 91 (C6H5CH2)+.
1-[4-C-(Hydroxym eth yl)-β-D-threo-pen tofuran osyl]cytosin e (18␤)
A solution of 17␤ (250 m g, 0.394 m m ol) in an h ydrous dich lorom eth an e (4 m l) was treated
at –78 °C un der argon with a 1 M boron trich loride solution in dich lorom eth an e (6.3 m l).
After stirrin g at –78 °C for 3 h an d at –25 °C overn igh t, th e reaction m ixture was cooled at
–78 °C, a 1:1 m ixture of an h ydrous m eth an ol an d pyridin e was added dropwise an d th e so-
lution was evaporated to dryn ess. Th e residue was purified by silica gel colum n ch rom ato-
graph y (eluen t: ch loroform /m eth an ol/am m on ium h ydroxide, 65:30:5) to give pure 18␤
(81 m g, 75%), wh ich was recrystallized from m eth an ol, m .p. 226–227 °C. UV (m eth an ol):
λm ax 274 (6500), λm in 253 (4500), λ 218 sh (5900). HPLC 99.7%, 99:1 H2O/MeCN. 1H NMR
(DMSO-d6): 7.65 (d, 1 H, H-6, J6-5 = 7.4); 7.1–6.9 (br d, 2 H, NH2); 6.15 (d, 1 H, H-1′, J1′-2′
=
5.0); 5.64 (d, 1 H, H-5, J5-6 = 7.3); 5.37 (d, 1 H, OH-2′, JOH-2′ = 5.6); 5.29 (d, 1 H, OH-3′,
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1082
Griffon et al.:
JOH-3′ = 5.0); 4.97 (t, 1 H, OH-5′, JOH-5′ = JOH-5′′ = 5.3); 4.49 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′
=
5.8); 4.15 (dd, 1 H, H-2′, J = 5.3, J = 9.6); 4.00 (t, 1 H, H-3′, J3′-2′ = J3′-OH′ = 4.5); 3.57 (dd,
1 H, H-6′, J6′-6′′ = 11.7, JOH-6′ = 5.6); 3.52 (d, 2 H, H-5′ an d H-5′′, J = 5.4); 3.41 (dd, 1 H, H-6′′,
J6′′-6′ = 11.5, JOH-6′ = 6.1). MS (ES): m/z 547 (2 M + H)+, 274 (M + H)+, 112 (BH2)+. For
C
10H15N3O6 (273.3) calculated: 43.96% C, 5.53% H, 15.38% N; foun d: 43.81% C, 5.33% H,
15.26% N.
1-{2,3,5-Tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α-D-threo-pen tofuran osyl}cytosin e (17␣)
Th ion ation of 11␣ (760 m g, 1.20 m m ol) with Lawesson ’s reagen t (339 m g, 0.83 m m ol) in
an h ydrous 1,2-dich loroeth an e (12 m l), was perform ed followin g th e sam e procedure as de-
scribed for th e th ion ation of com poun d 11␤, an d provided th e correspon din g 4-th io in ter-
m ediate wh ich was treated with m eth an olic am m on ia followin g th e sam e procedure as
descibed above to give, after purification by silica gel ch rom atograph y (eluen t: stepwise gra-
dien t of m eth an ol, 0–4% in toluen e), 17␣ (300 m g, 100%). 1H NMR (CDCl3): 7.69 (d, 1 H,
H-6, J6-5 = 7.5); 7.3–7.1 (m , 20 H, 4 C6H5); 6.26 (d, 1 H, H-1′, J1′-2′ = 3.4); 5.38 (d, 1 H, H-5,
J5-6 = 7.4); 6.0–5.0 (br s, 2 H, NH2); 4.8–4.3 (m , 8 H, 4 Ph CH2); 4.13 (t, 1 H, H-2′, J2′-1′ = J2′-3′
3.3); 4.09 (d, 1 H, H-3′, J3′-2′ = 3.2); 3.83 (d, 1 H, H-6′, J6′-6′′ = 9.9); 3.78 (d, 1 H, H-6′′, J6′′-6′
=
=
10.2); 3.69 (d, 1 H, 1 H, H-5′, J5′-5′′= 9.5); 3.64 (d, 1 H, H-5′′, J5′′-5′ = 9.6). MS (m atrix NBA,
LiCl): FAB > 0 m/z 640 (M + Li)+, 91 (C6H5CH2)+.
1-[4-C-(Hydroxym eth yl)-α-D-threo-pen tofuran osyl]cytosin e (18␣)
Deprotection of 17␣ (695 m g, 1.10 m m ol) with a 1 M boron trich loride solution in dich loro-
m eth an e (17.5 m l), was perform ed followin g th e sam e procedure as described for th e
deprotection of 17␤, an d provided, after purification by silica gel ch rom atograph y (eluen t:
ch loroform /m eth an ol/am m on ium h ydroxide, 65:30:5), pure 18␣ wh ich was recrystallized
from m eth an ol, m .p. 205–206 °C. UV (m eth an ol): λm ax 235 (7200), 272 (7600); λm in 228
(7100), 255 (6400). HPLC 100%, 99:1 H2O/MeCN. 1H NMR (DMSO-d6): 7.91 (d, 1 H, H-6,
J6-5 = 7.4); 7.2–7.0 (br d, 2 H, NH2); 5.78 (d, 1 H, H-1′, J1′-2′ = 6.0); 5.73 (d, 1 H, H-5, J5-6
=
7.4); 5.48 (d, 1 H, OH-2′, JOH-2′ = 5.1); 5.27 (d, 1 H, OH-3′, JOH-3′ = 5.3); 4.83 (t, 1 H, OH-5′,
JOH-5′ = JOH-5′′ = 5.7); 4.59 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′ = 5.5); 4.1–4.0 (m , 2 H, H-2′ an d
H-3′); 3.58 (dd, 1 H, H-6′, J6′-6′′ = 11.6, JOH-6′ = 5.4); 3.43 (dd, 1 H, H-6′′, J6′′-6′ = 11.8, JOH-6′
=
5.8); 3.37 (d, 2 H, H-5′ an d H-5′′, J = 5.6). MS (ES): m/z 547 (2 M + H)+, 274 (M + H)+, 112
(BH2)+. For C10H15N3O6·CH3OH (305.3) calculated: 43.28% C, 6.27% H, 13.76% N; foun d:
43.29% C, 5.98% H, 13.53% N.
5-Fluoro-1-{2,3,5-tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α- an d
-β-D-threo-pen tofuran osyl}cytosin e (19␣) an d (19␤)
A m ixture of 15␣ an d 15␤ (273 m g, 0.418 m m ol) was treated with Lawesson ’s reagen t (118 m g,
0.293 m m ol) in an h ydrous 1,2-dich loroeth an e (10.5 m l), followin g th e sam e procedure as
described for th e th ion ation of com poun d 11␤, an d provided th e correspon din g 4-th io in ter-
m ediates, wh ich were treated with m eth an olic am m on ia followin g th e sam e procedure as
described above to give, after several purification s by silica gel ch rom atograph y (eluen t:
stepwise gradien t of m eth an ol, 2–3% in ch loroform ), pure 19␤ (100 m g, 42%), a m ixture of
19␣ an d 19␤ (7 m g, 3%), an d pure 19␣ (130 m g, 55%). Both com poun ds were pale yellow
foam s.
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4′-C-(Hydroxymethyl)nucleosides
1083
19␤: 1H NMR (CDCl3): 7.92 (d, 1 H, H-6, J6-F = 6.6); 7.4–7.1 (m , 20 H, 4 C6H5); 6.57 (dd, 1 H,
H-1′, J1′-2′ = 5.7, J1′-F = 1.9); 6.6–6.3 (br s, 1 H, NH2); 6.4–6.1 (br s, 1 H, NH2); 4.6–4.2 (m , 9 H,
H-2′ an d 4 Ph CH2); 4.26 (d, 1 H, H-3′, J3′-2′ = 5.5); 3.63 (d, 1 H, H-6′, J6′-6′′ = 10.1); 3.58 (d, 1 H,
H-5′, J5′-5′′ = 10.0); 3.47 (d, 1 H, H-5′′, J5′′-5′ = 10.1); 3.44 (d, 1 H, H-6′′, J6′′-6′ = 10.0). MS (m atrix
NBA, LiCl): FAB > 0 m/z 658 (M + Li)+, 91 (C6H5CH2)+.
19␣: 1H NMR (CDCl3): 7.80 (d, 1 H, H-6, J6-F = 6.5); 7.4–7.1 (m , 20 H, 4 C6H5); 6.19 (br s,
1 H, H-1′); 5.5–5.1 (br s, 2 H, NH2); 4.8–4.4 (m , 8 H, 4 Ph CH2); 4.13 (m , 2 H, H-2′ an d H-3′);
3.81 (d, 1 H, H-6′, J6′-6′′ = 10.3); 3.77 (d, 1 H, H-6′′, J6′′-6′ = 10.3); 3.63 (d, 1 H, H-5′, J5′-5′′
=
9.7); 3.54 (d, 1 H, H-5′′, J5′′-5′ = 9.6). MS (m atrix NBA, LiCl): FAB > 0 m/z 658 (M + Li)+, 91
(C6H5CH2)+.
5-Fluoro-1-[4-C-(h ydroxym eth yl)-β-D-threo-pen tofuran osyl]cytosin e (20␤)
Deprotection of 19␤ (162 m g, 0.248 m m ol) with a 1 M boron trich loride solution in dich loro-
m eth an e (4.0 m l) was perform ed followin g th e sam e procedure as described for th e
deprotection of 17␤, an d provided, after purification by silica gel ch rom atograph y (eluen t:
ch loroform /m eth an ol/am m on ium h ydroxide, 65:30:5), pure 20␤ (62 m g, 86%), wh ich was
precipitated from an eth an ol/dieth yl eth er m ixture, m .p. 83–88 °C (h ygroscopic). UV (m eth -
an ol): λm ax 239 (7000), 284 (6800); λm in 226 (6300), 263 (4600). HPLC 98.3%, 99:1
H2O/MeCN. 1H NMR (DMSO-d6): 7.93 (d, 1 H, H-6, J6-F = 7.3); 7.7–7.3 (br d, 2 H, NH2); 6.10
(dd, 1 H, H-1′, J1′-2′ = 5.4, J1′-F = 2.0); 5.45 (d, 1 H, OH-2′, JOH-2′ = 5.6); 5.33 (d, 1 H, OH-3′,
JOH-3′ = 5.1); 5.14 (t, 1 H, OH-5′, JOH-5′ = JOH-5′′ = 5.2); 4.56 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′
=
5.9); 4.22 (dd, 1 H, H-2′, J = 5.4, J = 10.8); 4.00 (t, 1 H, H-3′, J3′-2′ = J3′-OH′ = 4.5); 3.6–3.5 (m ,
3 H, H-5′, H-5" an d H-6); 3.34 (dd, 1 H, H-6′′, J6′′-6′ = 10.5, JOH-6′ = 6.1). MS (ES): m/z 605
(2 M + Na)+, 583 (2 M + H)+, 314 (M + Na)+, 292 (M + H)+. For C10H14FN3O6·1.5H2O (318.2)
calculated: 37.74% C, 5.38% H, 13.20% N; foun d: 37.83% C, 4.90% H, 12.50% N.
5-Fluoro-1-[4-C-h ydroxym eth yl)-α-D-threo-pen tofuran osyl]cytosin e (20␣)
Deprotection of 19␣ (212 m g, 0.325 m m ol) with a 1 M boron trich loride solution in dich loro-
m eth an e (5.2 m l) was perform ed followin g th e sam e procedure as described for th e
deprotection of 17␤, an d provided, after purification by silica gel ch rom atograph y (eluen t:
ch loroform /m eth an ol/am m on ium h ydroxide, 65:30:5), pure 20␣ (94 m g, 99%), wh ich was
recrystallized from eth an ol, m .p. 132–135 °C (h ygroscopic). UV (m eth an ol): λm ax 242 (8500),
283 (7200); λm in 225 (6800), 265 (5900). HPLC 100%, 99:1 H2O/MeCN. 1H NMR (DMSO-d6):
8.22 (d, 1 H, H-6, J6-F = 7.2); 7.5–7.8 (br d, 2 H, NH2); 5.78 (br d, 1 H, H-1′, J1′-2′ = 5.1, J1′-F
=
2.0); 5.49 (d, 1 H, OH-2′, JOH-2′ = 4.4); 5.33 (d, 1 H, OH-3′, JOH-3′ = 4.6); 4.86 (t, 1 H, OH-5′,
JOH-5′ = JOH-5′′ = 5.3); 4.77 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′ = 5.1); 4.1–4.0 (m , 2 H, H-2′ an d
H-3′); 3.61 (dd, 1 H, H-6′, J6′-6′′ = 11.6, JOH-6′ = 4.9); 3.41 (dd, 1 H, H-6′′, J6′′-6′ = 11.7, JOH-6′
=
5.6); 3.34 (m , 2 H, H-5′ an d H-5′′). MS (ES): m/z 605 (2 M + Na)+, 583 (2 M + H)+, 314 (M +
Na)+, 292 (M + H)+. For C10H14FN3O6·H2O (309.2) calculated: 38.84% C, 5.22% H, 13.59% N;
foun d: 39.04% C, 4.91% H, 13.83% N.
6-Ch loro-9-{2,3,5-tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α- an d
-β-D-threo-pen tofuran osyl}purin e (22␣) an d (22␤)
A suspen sion of 6-ch loropurin e (3.60 g, 6.18 m m ol) in an h ydrous aceton itrile (40 m l) was
treated with N,O-bis(trim eth ylsilyl)acetam ide (BSA; 9.2 m l, 37.07 m m ol) durin g 18 h un der
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1084
Griffon et al.:
reflux. To th e resultin g solution was added at 0 °C 10 (0.99 g, 1.70 m m ol) in an h ydrous ace-
ton itrile (18.5 m l), followed by addition of tin (IV) ch loride (0.795 m l, 6.80 m m ol). Th e solu-
tion was stirred at room tem perature for 7 h un der argon atm osph ere. To th e reaction
m ixture was added carefully a saturated aqueous sodium h ydrogen carbon ate solution (7 m l)
an d th e resultin g m ixture was filtered th rough a sin tered fun n el covered with Celite. Th e
Celite was th en triturated an d wash ed several tim es with boilin g ch loroform (100 m l). Th e
com bin ed filtrates were con cen trated un der reduced pressure an d th e resultin g residue was
diluted with ch loroform (150 m l), wash ed with water (3 × 100 m l). Th e organ ic layer was
dried over sodium sulfate an d evaporated un der reduced pressure. Colum n ch rom atograph y
of th e residue usin g a stepwise gradien t of eth yl acetate (20–40%) in h exan es afforded
6-ch loro-7-{2,3,5-tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α- an d -β-D-threo-pen tofuran osyl}-
purin es 21␣ an d 21␤ (2.02 g, 48%), an d 22␣ an d 22␤ (1.07 g, 26%). To a solution of 21␣
an d 21␤ (2.02 g, 2.98 m m ol) in an h ydrous aceton itrile (20 m l) was added at
0 °C
trim eth ylsilyl trifluorom eth an esulfon ate (TMSOTf; 0.864 m l, 4.77 m m ol). Th e solution was
stirred at room tem perature for 1.5 h un der argon atm osph ere, th en diluted with ch loro-
form (150 m l), wash ed with th e sam e volum e of
a saturated aqueous sodium
h ydrogen carbon ate solution , with brin e (2 × 100 m l) an d fin ally with water (1 × 100 m l).
Th e organ ic ph ase was dried over sodium sulfate, th en evaporated un der reduced pressure.
Colum n ch rom atograph y of th e residue usin g a stepwise gradien t of eth yl acetate (20–40%)
in h exan es afforded 22␣ an d 22␤ (1.09 g, 54%). An om eric purification of th e com bin ed m a-
terial obtain ed above as a 0.8:1 α:β m ixture was perform ed by silica gel ch rom atograph y
(eluen t: h exan es/eth yl acetate, 8:2) an d afforded as pale yellow foam s pure 22␤ (1.00 g,
48%), a m ixture of 22␣ an d 22␤ (0.29 g, 14%), an d pure 22␣ (0.81 g, 39%).
22␤: 1H NMR (CDCl3): 8.64 (s, 1 H, H-2); 8.46 (s, 1 H, H-8); 7.4–6.7 (m , 20 H, 4 C6H5);
6.60 (d, 1 H, H-1′, J1′-2′ = 6.1); 4.72 (t, 1 H, H-2′, J2′-1′ = J2′-3′ = 6.3); 4.7–4.0 (m , 9 H, H-3′ an d
4 Ph CH2); 3.72 (d, 1 H, H-6′, J6′-6′′ = 10.0); 3.53 (d, 1 H, H-5′, J5′-5′′ = 9.7); 3.47 (m , 2 H, H-5′′
an d H-6′′). 13C NMR (CDCl3): 151.6 (2-C); 151.5 (4-C); 150.5 (6-C); 145.7 (8-C); 137.9–136.3
(C6H5); 131.3 (9-C); 128.5–127.4 (C6H5); 84.9 (4′-C); 83.0 (2′-C); 82.4 (1′-C); 81.7 (3′-C);
73.8–73.1 (Ph CH2); 70.0 (5′-C or 6′-C); 69.3 (5′-C or 6′-C). MS (m atrix NBA, LiCl): FAB > 0
m/z 683 (M + Li)+, 91 (C6H5CH2)+.
22␣: 1H NMR (CDCl3): 8.66 (s, 1 H, H-2); 8.37 (s, 1 H, H-8); 7.4–6.9 (m , 20 H, 4 C6H5);
6.20 (d, 1 H, H-1′, J1′-2′ = 5.4); 4.73 (t, 1 H, H-2′, J2′-1′ = J2′-3′ = 5.6); 4.7–4.3 (m , 9 H, H-3′ an d
4 Ph CH2); 3.81 (d, 1 H, H-6′, J6′-6′′ = 10.2); 3.72 (d, 1 H, H-6′′, J6′′-6′ = 10.2); 3.55 (br s, 2 H,
H-5′ an d H-5). 13C NMR (CDCl3): 151.7 (2-C); 151.3 (4-C); 150.6 (6-C); 144.0 (8-C);
137.7–136.7 (C6H5); 131.7 (9-C); 128.4–127.6 (C6H5); 87.0 (4′-C); 86.0 (1′-C an d 2′-C); 83.3
(3′-C); 73.8–73.0 (Ph CH2); 70.7 (5′-C or 6′-C); 69.9 (5′-C or 6′-C). MS (m atrix NBA, LiCl):
FAB > 0 m/z 683 (M + Li)+, 91 (C6H5CH2)+.
9-{2,3,5-Tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-β-D-threo-pen tofuran osyl}aden in e (23␤)
Com poun d 22␤ (0.98 g, 1.45 m m ol) was treated with eth an olic am m on ia (29 m l previously
saturated at –10 °C an d tigh tly stoppered) at 75 °C in a stain less-steel bom b for 5.5 h , th en
cooled to room tem perature. Th e solution was evaporated to dryn ess un der reduced pres-
sure. Th e resultin g residue was diluted with ch loroform (150 m l) an d th e organ ic layer was
wash ed with brin e (3 × 150 m l), dried over sodium sulfate an d evaporated to dryn ess. Th e
crude residue was purified by silica gel colum n ch rom atograph y (eluen t: stepwise gradien t of
m eth an ol, 0–4% in ch loroform ) to give pure 23␤ as a pale yellow foam (782 m g, 82%).
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4′-C-(Hydroxymethyl)nucleosides
1085
1H NMR (CDCl3): 8.32 (s, 1 H, H-2); 8.17 (s, 1 H, H-8); 7.4–6.8 (m , 20 H, 4 C6H5); 6.64 (d, 1 H,
H-1′, J1′-2′ = 5.9); 5.60 (br s, 2 H, NH2); 4.7–4.0 (m , 10 H, H-2′, H-3′ an d 4 Ph CH2); 3.72 (d, 1 H,
H-6′, J6′-6′′ = 10.1); 3.6–3.5 (m , 3 H, H-5′, H-5′′ an d H-6′′). MS (ES): m/z 658 (M + H)+.
9-[4-C-(Hydroxym eth yl)-β-D-threo-pen tofuran osyl]aden in e (24␤)
To a solution of 23␤ (617 m g, 0.938 m m ol) in a 1:1 solution of m eth an ol/acetic acid (20 m l)
was added 375 m g of 20% Pd(OH)2/C an d th e suspen sion was sh aken on a Parr apparatus
un der 50 Pa of H2. After 1, 2 an d 3 days 94 m g portion s of 20% Pd(OH)2/C were added.
After 5 days th e m ixture was filtered th rough a pad of Celite an d filtrate was con cen trated
un der reduced pressure. Th e crude m aterial was purified by silica gel colum n ch rom ato-
graph y (eluen t: ch loroform /m eth an ol/am m on ium h ydroxide, 80:18:2) to give pure 24␤ (136
m g, 49%), wh ich was recrystallized from m eth an ol, m .p. 240–242 °C. UV (m eth an ol): λm ax
259 (13700), λm in 237 (2600). HPLC 100%, 90:10 0.01 M NH4H2PO4 (pH 5.1)/MeOH. 1H NMR
(DMSO-d6): 8.20 (s, 1 H, H-8); 8.11 (s, 1 H, H-2); 7.20 (s, 2 H, NH2); 6.32 (d, 1 H, H-1′, J1′-2′
=
6.0); 5.48 (d, 1 H, OH-2′, JOH-2′ = 5.6); 5.41 (d, 1 H, OH-3′, JOH-3′ = 5.3); 5.12 (t, 1 H, OH-5′,
JOH-5′ = JOH-5′′ = 5.4); 4.62 (t, 1 H, OH-6′, JOH-6′ = JOH-6′′ = 5.8); 4.47 (dd, 1 H, H-2′, J = 6.1, J =
12.0); 4.32 (t, 1 H, H-3′, J3′-OH = J3′-2′ = 5.6); 3.61 (dd, 1 H, H-6′, J6′-6′′ = 11.6, JOH-6′′ = 5.8);
3.51 (d, 2 H, H-5′ an d H-5′′, J5′-OH = J5′′-OH = 5.3); 3.39 (dd, 1 H, H-6′′, J6′′-6′ = 11.7, JOH-6′
=
5.8). MS (ES): m/z 320 (M + Na)+, 298 (M + H)+, 136 (BH2)+. For C11H15N5O5 (297.3) calcu-
lated: 44.45% C, 5.09% H, 23.56% N; foun d: 44.16% C, 5.01% H, 23.38% N.
9-{2,3,5-Tri-O-ben zyl-4-C-[(ben zyloxy)m eth yl]-α-D-threo-pen tofuran osyl}aden in e (23␣)
Com poun d 22␣ (0.720 g, 1.06 m m ol) was treated with eth an olic am m on ia (21 m l), follow-
in g th e sam e procedure as described for am in ation of com poun d 22␤, an d provided, after
purification by silica gel colum n ch rom atograph y (eluen t: stepwise gradien t of m eth an ol,
0–4% in ch loroform ), pure 23␣ (657 m g, 94%). 1H NMR (CDCl3): 8.33 (s, 1 H, H-2); 8.05 (s,
1 H, H-8); 7.4–7.0 (m , 20 H, 4 C6H5); 6.21 (d, 1 H, H-1′, J1′-2′ = 5.3); 5.56 (br s, 2 H, NH2);
4.72 (t, 1 H, H-2′, J2′-1′ = J2′-3′ = 5.4); 4.7–4.4 (m , 8 H, 4 Ph CH2); 3.84 (d, 1 H, H-6′, J6′-6′′
=
10.2); 3.73 (d, 1 H, H-6′′, J6′′-6′ = 10.1); 3.60 (d, 1 H, H-5′, J5′-5′′ = 10.1); 3.56 (d, 1 H, H-5′′,
J5′′-5′ = 9.9). MS (ES): m/z 658 (M + H)+.
9-[4-C-(Hydroxym eth yl)-α-D-threo-pen tofuran osyl]aden in e (24␣)
To a solution of 23␣ (650 m g, 0.988 m m ol) in a 1:1 solution of m eth an ol/acetic acid (20 m l)
was added 395 m g of 20% Pd(OH)2/C an d th e suspen sion was sh aken on a Parr apparatus
un der 50 Pa of H2. After 1, 2 an d 3 days 99 m g portion s of 20% Pd(OH)2/C were added. After
5 days th e m ixture was filtered th rough a pad of Celite an d filtrate was con cen trated un der
reduced pressure. Th e crude m aterial was purified by silica gel colum n ch rom atograph y
(eluen t: ch loroform /m eth an ol/am m on ium h ydroxide, 80:18:2) to give pure 24␣ (194 m g,
66%), wh ich was recrystallized from m eth an ol, m .p. 246–248 °C. UV (m eth an ol): λm ax 259
(15400), λm in 228 (2600). HPLC 100%, 90:10 0.01 M NH4H2PO4 (pH 5.1)/MeOH. 1H NMR
(DMSO-d6): 8.37 (s, 1 H, H-8); 8.14 (s, 1 H, H-2); 7.34 (s, 2 H, NH2); 5.77 (d, 1 H, H-1′, J1′-2′
=
6.9); 5.67 (d, 1 H, OH-2′, JOH-2′ = 5.6); 5.38 (d, 1 H, OH-3′, JOH-3′ = 6.3); 5.15 (dd, 1 H, OH-6′,
JOH-6′ = 7.1, JOH-6′′ = 4.8); 4.91 (t, 1 H, OH-5′, JOH-5′ = JOH-5′′ = 5.8); 4.67 (m , 1 H, H-2′); 4.21
(t, 1 H, H-3′, J3′-OH = J3′-2′ = 5.6); 3.59 (dd, 1 H, H-6′, J6′-6′′ = 11.9, JOH-6′ = 7.2); 3.47 (dd, 1 H,
H-6′′, J6′′-6′ = 11.7, JOH-6′′ = 4.8); 3.40 (d, 2 H, H-5′ an d H-5′′, J5′-OH = J5′′-OH = 6.0). MS (ES):
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1063–1087

1086
Griffon et al.:
m/z 320 (M + Na)+, 298 (M + H)+, 136 (BH2)+. For C11H15N5O5 (297.3) calculated: 44.45% C,
5.09% H, 23.56% N; foun d: 44.30% C, 5.09% H, 23.23% N.
The investigation was supported by the National Cancer Institute, National Institutes of Health
(PO1-CA34200). The authors are indebted to Dr J. M. Riordan for recording and interpreting NMR
spectra, to Mr M. Richardson for mass-spectrum data, to Ms J. Bearden for UV and elemental
analyses, and to Ms S. Campbell for HPLC analyses.
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