Synthesis and cytotoxicity of 9-(2-deoxy-2-alkyldithio-β-D-arabinofuranosyl)purine nucleosides which are stable precursors to potential mechanistic probes of ribonucleotide reductases

Article abstract of DOI:10.1039/b311504f

A series of 2′-thionucleosides, as potential inhibitors of ribonucleotide reductases, has been synthesized. Treatment of the 3′,5′-O-TPDS-2′-O-(trifluoromethanesulfonyl)adenosine with potassium thioacetate gave the arabino epimer of 2′-S-acetyl-2′-thioade

Full text of DOI:10.1039/b311504f

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Synthesis and cytotoxicity of 9-(2-deoxy-2-alkyldithio-ꢀ-D-arabino-
furanosyl)purine nucleosides which are stable precursors to
potential mechanistic probes of ribonucleotide reductases
Stanislaw F. Wnuk,*a Elzbieta Lewandowska,†a Dania R. Companioni,a Pedro I. Garcia Jra
and John A. Secrist IIIb
a Department of Chemistry and Biochemistry, Florida International University, Miami,
FL 33199, USA. E-mail: wnuk@fiu.edu; Fax: (305) 348-3772
b Southern Research Institute, Birmingham, AL 35205, USA. E-mail: secrist@sri.org
Received 19th September 2003, Accepted 28th October 2003
First published as an Advance Article on the web 20th November 2003
A series of 2Ј-thionucleosides, as potential inhibitors of ribonucleotide reductases, has been synthesized. Treatment
of the 3Ј,5Ј-O-TPDS-2Ј-O-(trifluoromethanesulfonyl)adenosine with potassium thioacetate gave the arabino epimer
of 2Ј-S-acetyl-2Ј-thioadenosine which was deacetylated to give 9-(3,5-O-TPDS-2-thio-β--arabinofuranosyl)adenine
in high yield. Treatment of the latter with diethyl azodicarboxylate–C3H7SH–THF gave 2Ј-propyl disulfide which
was desilylated to give 9-(2-deoxy-2-propyldithio-β--arabinofuranosyl)adenine. Subsequent tosylation (O5Ј) and
displacement of the tosylate with pyrophosphate afforded the 5Ј-O-diphosphate in a stable form as propyl mixed-
disulfide, which upon treatment with dithiothreitol releases 9-(2-thio-β--arabinofuranosyl)adenine 5Ј-diphosphate.
The arabino 2Ј-mercapto group might interact with the crucial thiyl radical at cysteine 439 leading to the inhibition
of ribonucleotide reductases via formation of a Cys439–2Ј-mercapto disulfide bridge. The 2,6-diamino-, 2-amino-
6-chloro- and 2-amino-6-methoxypurine ribosides were also converted to the corresponding 2Ј-deoxy-2Ј-propyl-
dithio-β--arabinofuranosyl nucleosides, which might serve as convenient precursors to the arabino epimer of
2Ј-thioguanosine. Analogously, 2Ј-deoxy-2Ј-propyldithioadenosine was prepared from 9-(β--arabinofuranosyl)-
adenine. The nucleoside disulfides show modest cytotoxicity in a panel of human tumor cell lines.
Fontecave and Decout and their coworkers demonstrated
Introduction
that 2Ј-thiouridine 5Ј-diphosphate A is a potent inactivator of
Ribonucleotide reductases (RNRs) are ubiquitous enzymes
RDPR (Fig. 1).10 They postulated that the mode of inactivation
that execute conversion of 5Ј-(di- or tri)phosphate esters of
involved substrate-protein disulfide B which after generation of
ribonucleosides into the requisite 2Ј-deoxy building blocks.1
a radical at C3Ј C underwent fragmentation to the protein-
Inhibition of RNRs disrupts the primary source of DNA com-
based perthiyl radical R1–SSؒD.10a The perthiyl radical D can
ponents and is an appealing target for the rational drug design.2
then add to the proximal 2Ј-deoxy-3Ј-ketouridine intermediate
generated at the active site. Such an addition may produce new
radicals which would resemble structures that were proposed to
The ribonucleoside diphosphate reductase (RDPR) from
Escherichia coli consists of two subunits (R1 and R2) whose
X-ray structure have been determined.3 The R1 subunit con-
be formed during inactivation of RDPR by 2Ј-azido-2Ј-deoxy-
tains allosteric control sites and cysteine residues that partici-
uridine 5Ј-diphosphate.11
pate in catalytic turnover and/or as redox dithiol/disulfide pairs.
The R2 subunit contains an essential tyrosine (Tyr122) free
radical that is responsible for generation of a proximate thiyl
radical (Cys439) on R1. A thiyl radical initiates nucleotide
reduction by abstraction of H3Ј from the substrate ribonucleo-
tide and water (O2Ј) is then lost from C2Ј of the resulting
hydrogen-bonded C3Ј radical.1a,4
In spite of numerous attempts using modified substrates as
radical traps for both enzyme and substrate radicals, direct
observation of nucleotide radical intermediates has remained
elusive until very recently.5–7 Elegant EPR studies with (E)-2Ј-
deoxy-2Ј-(fluoromethylene)cytidine 5Ј-diphosphates and its [2H]
and [13C] isotopomers has allowed detection and characteriz-
ation of a substrate-derived allyl radical during inactivation of
RDPR.2,5 Spectroscopic evidence has been provided for new
radical intermediates in the reduction of cytidine 5Ј-diphos-
phate (CDP) by Glu441Gln R1 mutant of RDPR.6,7 Further-
more, radicals at C3Ј of adenosine,8 and 2Ј-substituted homo-
nucleosides9a and homoribofuranose9b have been selectively
generated to simulate the initiation–elimination cascades that
occur during enzymatic 2Ј-deoxygenation of ribonucleotides.
† On faculty leave from the Department of Chemistry, University of
Agriculture, Poznan, Poland.
Fig. 1 The proposed mechanism10a for the inhibition of RDPR by
2Ј-deoxy-2Ј-thiouridine 5Ј-diphosphate A.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 0 – 1 2 6
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
120

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Fig. 2 Possible interaction of RDPR with 9-(2-thio-β--arabinofuranosyl)adenine 5Ј-diphosphate.
Scheme 1 Reagents and conditions: (a) CH3COSK–DMF; (b) NH3–MeOH; (c) TBAF–THF; (d) DEAD–PrSH or MeSH–THF; (e) NH4F–MeOH;
(f ) TsCl–pyridine; (g) (Bu4N)3ؒHP2O7–CH3CN; (h) DTT–H2O; (i) Me3SiCH2CH2SH–K2CO3–DMF.
We now report the syntheses of stable mixed-disulfide pre-
cursors to 9-(2-thio-β--arabinofuranosyl)adenine 5Ј-diphos-
phate (E, Fig. 2). The arabino 2Ј-mercapto group in E may
interact with Cys439 leading to the inhibition of RDPR via
formation of R1 Cys439–2Ј-SH disulfide bridge F. Altern-
atively, competitive abstraction of hydrogen from the 2Ј-thiol
group (path a) rather than the H3Ј (path b) by Cys439 radical G
may also be considered as a possible interaction pathway. The
homolytic bond dissociation energies of 381 kJ molϪ1 for S–H
and 393 kJ molϪ1 for H–C–O make hydrogen atom abstraction
from the 2Ј-mercapto group (path a) thermodynamically more
feasible.4 It is noteworthy that arabinonucleosides have some
preferences12 for C2Ј endo conformation which is conducive for
the formation of disulfide bridge F or abstraction of a proton
from the 2Ј-thiol (G, path a).
The thiols protected as mixed-disulfides were prepared using
diethyl azodicarboxylate (DEAD) as an oxidizing agent.10b,17
Thus, treatment of 4 with DEAD and 1-propanethiol in THF
gave disulfide 6a (18%) plus unchanged 4 (25%). In order
to overcome the limited solubility of deprotected thiol 4 in
THF, 9-(3,5-O-TBDS-2-thio-β--arabinofuranosyl)adenine 3
was prepared by deacetylation of 2 with NH3–MeOH (97%).
Treatment of 3 with DEAD–C3H7SH–THF gave protected
disulfide 5a (45%) but separation from the diethyl hydrazine-
dicarboxylate byproduct was tedious. Desilylation (NH4F–
MeOH18) gave 6a (60%) which was readily purified. Diagnostic
1
peaks for the propyl group were observed in the H and 13C
NMR spectra of 5a and 6a.
In the second approach, chemistry utilizing 2-(trimethyl-
silyl)ethyl sulfides19 was employed for the attempted prepar-
ation of mixed-methyl disulfide 6b. Thus, displacement of
triflate from 1 with 2-(trimethylsilyl)ethanethiolate in DMF
proceeded smoothly at 60 ЊC to give 2Ј-S-[(2-trimethylsilyl)-
ethyl]-2Ј-thionucleoside 10 in good yield. Deprotection of 10
with TBAF gave 11, which also demonstrated stability of
the 2-(trimethylsilyl)ethyl group towards fluoride.20 However,
reaction of 10 or 11 with dimethyl(methylthio)sulfonium
tetrafluoroborate19,20 in the presence of large excess of dimethyl
sulfide failed to produce methyl mixed-disulfides 5b or 6b.
Instead unchanged 10 or 11 and fluorescent-type byproduct(s)
were isolated from the reaction mixtures indicating instability
of the purine ring (as opposed to pyrimidine19) under the
reaction conditions. However, treatment of thiol 3 with MeSH–
DEAD–THF gave 5b that was deprotected to yield less
sterically hindered 6b.
From the methods available for the 5Ј-phosphorylation of
nucleosides,21 we chose the methodology of Poulter and co-
workers22 that is based on the displacement of the 5Ј-O-tosylate
ester with the corresponding pyrophosphate ion. Thus, disulfide
6a was converted to the 5Ј-O-tosylate 7 using standard chem-
istry with the bulky propyl group allowing selective 5Ј-O-tosyl-
ation. Treatment of 7 with tris(tetrabutylammonium) hydrogen
Results and discussion
From the available arabino thionucleosides,13 we chose an
adenosine 2Ј-mercapto analog (e.g., 414) because attempts to
synthesize 1-(2-thio-β--arabinofuranosyl)uracil failed due to
the rapid Michael addition of the arabino 2Ј-mercapto function
across the double bond of the uracil moiety to give 2Ј-deoxy-
2Ј,6-epithio-5,6-dihydro derivative.15 The synthesis of arabino
2Ј-thio analogs was attempted using two different approaches.
In our first approach, treatment of the 3Ј,5Ј-O-[1,1,3,3-tetra-
isopropyldisiloxane(TPDS)-1,3-diyl]-2Ј-O-(trifluoromethane-
sulfonyl)adenosine16 (1) with potassium thioacetate gave the
arabino epimer of the protected 2Ј-S-acetyl-2Ј-thioadenosine
(2, Scheme 1). Sequential removal of the 3Ј,5Ј-O-TPDS [tetra-
butylammonium fluoride (TBAF)] and 2Ј-S-acetyl (NH3–
MeOH) groups afforded 9-(2-thio-β--arabinofuranosyl)-
adenine14 (4; 41% from 1) after purification on Dowex 1 × 2
(OHϪ) column. Compound 4 had spectroscopic data as
reported14b but slow formation of the corresponding disulfide
was apparent since treatment of the mixture with dithiothreitol
(DTT) led to a sharper spot on TLC.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 0 – 1 2 6
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pyrophosphate22 effected displacement to give 5Ј-diphosphate
8. The nucleotide 8 was purified by ion-exchange chromato-
graphy on a DEAE Sephadex A-25 (HCO3Ϫ) column using
Cytotoxic activities
The disulfide nucleosides 6a, 6b, 16, and 23a–c were evaluated
for their cytotoxicities in a panel of human tumor cell lines.
This panel was comprised of SNB-7 (CNS), DLD-1 (colon),
CCRF-CEM (leukemia), NCI-H23 (lung), ZR-75-1 (mam-
mary), LOX IMVI (melanoma), PC-3 (prostate), and CAKI-1
(renal) cells. The two mixed arabino disulfides with adenine as
the base, 6a and 6b, were not cytotoxic up to the highest doses
evaluated (>150 µM) in any of the eight cell lines. The mixed
ribo disulfide 16 had a weak IC50 of 110 µM in CCRF-CEM
cells, and exhibited no cytotoxicity up to the highest doses in
any other cell lines. The three mixed arabino disulfides contain-
ing 2,6-disubstituted purines were similarly non-toxic to most
of the cell lines. In CCRF-CEM cells, which are known to be
sensitive to many nucleosides, 23a had an IC50 of 14 µM and
23b had an IC50 of 19 µM. The only other cytotoxicities seen
were for 23a (100 µM, NCI-H23), 23b (160 µM, DLD-1, and
140 µM, NCI-H23), and 23c (130 µM, CCRF-CEM). The lack
of or very modest cytotoxicity seen with these analogs may be
the result of their inability to be activated by the appropriate
enzymes to the monophosphate level and then up to the di-
and/or triphosphate levels.
triethyl ammonium bicarbonate (TEAB, 0.05
0.50 M)
as an elutant. The 31P NMR spectrum showed two doublets
(J = 23.5 Hz) at δ Ϫ9.75 and Ϫ5.26 and the 1H NMR spectrum
confirmed the structure. This material was converted to its
sodium salt by passage through a Dowex 50 (Naϩ) column.
Reduction of diphosphate 8 with a slight excess of DTT
immediately generated10a the mercaptonucleotide 9. Dithio-
threitol was chosen as a reducing agent since it is one of the
electron sources used in the assays for ribonucleotide reductase
activity in vitro.
In addition to the propyl 6a and methyl 6b mixed-disulfides,
the ribo analogue 16 was synthesized in order to test differ-
ences in the biological activities between ribo and arabino
epimers. Subjection of 9-(3,5-O-TPDS-2-O-triflate-β--
arabinofuranosyl)adenine16 12 to the similar dithiolation
sequence (12
16, Scheme 2) gave ribo disulfide 16 in 11%
overall yield.
We have synthesized 9-(2-deoxy-2-propyldithio-β--arabino-
furanosyl)adenine and 2-amino-6-substituted(amino/chloro/
methoxy)purine analogues which are stable precursors of
arabino 2Ј-deoxy-2Ј-mercaptonucleotides. The mixed propyl-
disulfides were chosen as in general they could be deprotected
under mild reductive conditions compatible with biological
assays to form the corresponding mercaptonucleosides. These
mercaptonucleosides can serve as valuable probes for under-
standing the role of the Cys439 radical during enzymatic
deoxygenation reaction catalyzed by ribonucleotide reductases.
Studies on inhibition of RDPR with 9 are in progress.24
Scheme 2 Reagents and conditions: (a) CH3COSK–DMF; (b) NH3–
MeOH; (c) DEAD–PrSH–THF; (d) NH4–MeOH.
The 2-amino-6-chloro-, 2-amino-6-methoxy-, and 2,6-di-
aminopurine ribosides are known to be substrates of adenosine
deaminase.23 The deaminase-mediated hydrolysis at C6 of
2-amino-6-(substituted) mixed-disulfides 23a–c (Scheme 3)
would generate the arabino epimer of 2Ј-thioguanosine. Subjec-
tion of 2-amino-6-chloro- (17a), 2-amino-6-methoxy- (17b) and
2,6-diaminopurine riboside (17c) to our triflation–dithiolation
Experimental
1H (Me4Si) NMR spectra were determined with solution in
CDCl3 at 400 MHz, 13C (Me4Si) at 100.6 MHz and 31P (H3PO4)
at 161.9 MHz unless otherwise noted. Mass spectra (MS) were
obtained by atmospheric pressure chemical ionization (APCI)
technique. Reagent grade chemicals were used and solvents
were dried by reflux over and distillation from CaH2 under
an argon atmosphere except THF (K–benzophenone). TLC
was performed on Merck kieselgel 60-F254 with MeOH–CHCl3
(1 : 19) and EtOAc–hexane (4 : 1) as developing systems,
and products were detected with 254 nm light. Merck kieselgel
60 (230–400 mesh) was used for column chromatography.
Elemental analyses were determined by Galbraith Laborator-
ies, Knoxville, TN. The 2,6-(disubstituted)purine ribosides
17a–c were purchased from Berry & Associates, Inc, Ann
Arbor, MI.
sequence (17
23), produced arabino mixed-disufides 23a
(14%), 23b (10%), and 23c (24%; overall yield).
9-[2-S-Acetyl-3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-2-
thio-ꢀ-D-arabinofuranosyl]adenine (2)
Procedure A. CH3COSK (434 mg, 3.8 mmol) was added to a
solution of 116 (1.53 g, 2.38 mmol) in dried DMF (10 mL) and
the resulting mixture was stirred overnight at ambient temper-
ature. Volatiles were evaporated and the residue was partitioned
(EtOAc–NaHCO3–H2O). The organic layer was washed (NaCl–
H2O), dried (MgSO4), evaporated and the residue was column
chromatographed (EtOAc–hexanes; 70 : 30) to give 225 (1.01 g,
75%): mp 68–76 ЊC; UV max 260 nm (ε 15 100), min 233 nm
1
(ε 6200); H NMR δ 0.8–1.3 (m, 28, 4 × i-Pr), 2.19 (s, 3, Ac),
3.99–4.08 (m, 2, H4Ј,5Ј), 4.28 (dd, J = 4.5, 12.5 Hz, 1, H5Љ), 4.59
(dd, J = 7.3, 10.3 Hz, 1, H2Ј), 5.06 (dd, J = 8.2, 10.0 Hz, 1, H3Ј),
6.45 (d, J = 7.3 Hz, 1, H1Ј), 6.30 (br s, 2, NH2), 7.95 (s, 1, H2),
8.29 (s, 1, H8); MS m/z 568 (100, MHϩ). Anal. calcd for
C24H41N5O5Si2S (567.86): C, 50.76; H, 7.28; N, 12.33. Found: C,
50.39; H, 7.49; N, 11.94%.
Scheme
3
Reagents and conditions: (a) TPDS-Cl2–pyridine;
(b) Tf2NPh–DMAP–CHCl2; (c) CH3COSK–DMF; (d) NH3–MeOH;
(e) DEAD–PrSH–THF; (f ) NH4F–MeOH.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 0 – 1 2 6
122

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9-[3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanyl)-2-thio-ꢀ-D-
arabinofuranosyl]adenine (3)
CH3), 1.53 (sextet, J = 7.3 Hz, 2, CH2), 2.57 (t, J = 7.2 Hz, 2,
CH2S), 3.86–3.95 (m, 4, H2Ј,4Ј,5Ј,5Љ), 4.53 (t, J = 7.6 Hz, 1,
H3Ј), 6.58 (d, J = 7.1 Hz, 1, H1Ј), 8.21 (s, 1, H2), 8.44 (s, 1, H8);
13C NMR (MeOH-d4) δ 12.13 (CH3), 22.03 (CH2), 40.96
(SCH2), 60.22 (C2Ј), 62.54 (C5Ј), 71.94 (C3Ј), 84.92 & 85.32
(C4Ј & C1Ј), 119.0 (C5), 140.79 (C8), 149.50 (C4), 152.68 (C2),
156.30 (C6); MS m/z 358 (100, MHϩ). Anal. calcd. for C13H19-
N5O3S2ؒH2O (375.47): C, 41.59; H, 5.64; N, 18.65. Found: C,
41.95; H, 5.65; N, 18.27%.
Procedure B. A solution of 2 (241 mg, 0.43 mmol) in satur-
ated (0 ЊC) NH3–MeOH (50 mL) was stirred for 30 min at
∼0 ЊC. Volatiles were evaporated, and the residue was column
chromatographed (CHCl3–MeOH; 95 : 5) to give 3 [254 mg,
97%; contaminated (1H NMR) with 0.5 equiv. of CH3CONH2]:
mp 78–82 ЊC; UV max 261 nm (ε 14 400), min 230 nm (ε 3100);
1H NMR δ 1.02–1.19 (m, 28, 4 × i-Pr), 1.47 (d, J = 8.1 Hz, 1,
SH), 3.87 (dd, J = 7.6, 9.7 Hz, 1, H2Ј), 3.91 (dt, J = 2.8, 8.2 Hz,
1, H4Ј), 4.07 (dd, J = 2.7, 12.9 Hz, 1, H5Ј), 4.24 (dd, J = 2.8, 12.9
Hz, 1, H5Љ), 4.62 (t, J = 8.9 Hz, 1, H3Ј), 6.12 (br s, 2, NH2), 6.42
(d, J = 7.1 Hz, 1, H1Ј), 8.14 (s, 1, H2), 8.35 (s, 1, H8); 13C NMR
δ 12.8–17.9 (12, 4 × i-Pr), 48.7 (C2Ј), 61.4 (C5Ј), 75.7 (C3Ј), 84.2
(C4Ј), 85.3 (C1Ј), 120.1 (C5), 139.7 (C8), 150.2 (C4), 153.2 (C2),
155.9 (C6); MS m/z 526 (100, MHϩ). For elemental analysis,
this material was repurified [column chromatography (CHCl3–
MeOH; 97 : 3)]. Anal. calcd for C22H39N5O4Si2S (525.54): C,
50.28; H, 7.43; N, 13.33. Found: C, 49.91; H, 7.68; N, 12.91%.
Treatment of 4 (159 mg, 0.56 mmol) with DEAD (98 mg,
0.57 mmol) and propane-1-thiol (2.9 mL, 2.4 g, 32 mmol) in
THF (15 mL), as described for 5a, also gave 6a (36 mg, 18%).
9-(2-Deoxy-2-methyldithio-ꢀ-D-arabinofuranosyl)adenine (6b)
Treatment of 5b (19 mg, 0.033 mmol) by procedure D (chrom-
atography: EtOAc
4% MeOH–EtOAc) gave 6b (7.3 mg, 67%)
as a white powder: UV max 261 nm (ε 12 700), min 230 nm
(ε 2800); 1H NMR (MeOH-d4) δ 2.33 (s, 3, CH3S), 3.87–4.00 (m,
4, H2Ј,4Ј,5Ј,5Љ), 4.57 (t, J = 7.7 Hz, 1, H3Ј), 6.57 (d, J = 7.0 Hz,
1, H1Ј), 8.20 (s, 1, H2), 8.39 (s, 1, H8); MS m/z 330 (100, MHϩ).
1
Anal. calcd. for C11H15N5O3S2ؒ0.5 C4H8O2 (373.45; H NMR
9-(2-Thio-ꢀ-D-arabinofuranosyl)adenine (4)
integration of residual EtOAc): C, 41.81; H, 5.13; N, 18.75.
Found: C, 41.47; H, 5.38; N, 18.39%.
Treatment of 2 (283 mg, 0.50 mmol) with TBAF (1 M solution
in THF; 1 mL) in THF (15 mL) at 0 ЊC for 30 min followed by
NH3–MeOH (10 mL; 2 h, ambient temperature) gave crude 4.
This material was partitioned (EtOAc–H2O) and the water
layer was chromatographed [Dowex 1 × 2 (OHϪ); H2O
25% MeOH–H2O) to give 414b [89 mg, 55%; contaminated
(1H NMR) by the corresponding dimer-disulfide].
9-[2-Deoxy-2-propyldithio-5-O-(p-tolylsulfonyl)-ꢀ-D-arabino-
furanosyl]adenine (7)
p-Tolylsulfonyl chloride (106 mg, 0.55 mmol) was added to a
solution of 6a (40 mg, 0.11 mmol) in anhydrous pyridine (2 mL)
and the reaction mixture was stirred for 3 h at 0 ЊC. Volatiles
were evaporated, and the residue was partitioned (NaHCO3–
H2O–EtOAc). The organic layer was washed (AcOH–H2O,
NaHCO3–H2O), dried (MgSO4), evaporated and column
9-[3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanyl)-2-deoxy-2-
propyldithio-ꢀ-D-arabinofuranosyl]adenine (5a)
Procedure C. A solution of 3 (370 mg, 0.70 mmol) and
DEAD (113 µL, 0.72 mmol) in THF (7 mL) was stirred over-
night at ambient temperature and then propane-1-thiol
(3.68 mL, 3.09 g, 41 mmol) was added. Reaction mixture was
refluxed for 8 h and volatiles were evaporated. The residue was
partitionated (Na2CO3–H2O–EtOAc) and the organic layer was
washed (NaCl), dried (MgSO4), evaporated and column
chromatographed (EtOAc–hexane; 1 : 1) to give 5a (190 mg,
45%): mp 110–118 ЊC (soften); UV max 261 nm (ε 7600), min
233 nm (ε 1400); 1H NMR δ 0.94 (t, J = 7.4 Hz, 3, CH3), 1.07–
1.17 (m, 28, 4 × i-Pr), 1.62 (sextet, J = 7.4 Hz, 2, CH2), 2.61 (t,
J = 7.4 Hz, 2, SCH2), 3.91–3.97 (m, 2, H2Ј,4Ј), 4.05–4.10 (m, 2,
H5Ј,5Љ), 4.60 (t, J = 8.5 Hz, 1, H3Ј), 6.54 (d, J = 6.8 Hz, 1, H1Ј),
8.15 (s, 1, H2), 8.33 (s, 1, H8); MS m/z 600 (100, MHϩ).
chromatographed (EtOAc
2.5% MeOH–EtOAc) to give 7
1
(28 mg, 52%): H NMR δ 0.90 (t, J = 7.2 Hz, 3, CH3), 1.56
(sextet, J = 7.2 Hz, 2, CH2), 2.42 (s, 3, PhCH3), 2.59 (m, 2,
CH2S), 3.90 (t, J = 8.2 Hz, 1, H2Ј), 4.16 (m, 1, H4Ј), 4.42 (dd,
J = 3.1, 11.2 Hz, 1, H5Ј), 4.45 (dd, J = 4.5, 11.3 Hz, 1, H5Љ),
4.81 (t, J = 8.2 Hz, 1, H3Ј), 6.48 (d, J = 7.2 Hz, 1, H1Ј), 7.27 (d,
J = 8.0 Hz, 2, HAr), 7.87 (d, J = 8.1 Hz, 2, HAr), 7.99 (s, 1, H2),
8.29 (s, 1, H8); MS m/z 512 (100, MHϩ).
9-(2-Deoxy-2-propyldithio-ꢀ-D-arabinofuranosyl)adenine
5Ј-diphosphate (8)
Tris(tetra-n-butylammonium) hydrogenpyrophosphate22 (31
mg, 0.035 mmol) was added to a stirred solution of 7 (12 mg,
0.023 mmol) in CH3CN (0.5 mL) and stirring was continued at
ambient temperature for 72 h. Volatiles were removed in vacuo
and the residue was dissolved in water and was purified on
an ion exchange resin [Sephadex-DEAE A-25, 40–125 µm;
gradient elution with triethylammonium bicarbonate (0.05
0.5 M)]. Appropriated fractions were evaporated, and the resi-
due was dissolved in H2O and was converted to a sodium salt by
passing the solution through a Dowex (Naϩ) column to yield 8
(4 mg, 29%) as a trisodium salt: 1H NMR (MeOH-d4) δ 0.64 (t,
J = 6.3 Hz, 3, CH3), 1.27 (sextet, J = 6.5 Hz, 2, CH2), 2.32 (t,
J = 6.7 Hz, 2, CH2S), 3.88 (dd, J = 7.4, 8.8 Hz, 1, H2Ј), 4.03–
4.07 (m, 1, H4Ј), 4.19–4.23 (m, 2, H5Ј,5Љ), 4.49 (t, J = 8.3 Hz, 1,
H3Ј), 6.84 (d, J = 7.1 Hz, 1, H1Ј), 8.11 (s, 1, H2), 8.33 (s, 1, H8);
31P NMR δ Ϫ9.75 (d, J = 23.5 Hz, 1, Pα), Ϫ5.26 (d, J = 23.5 Hz,
1, Pβ).
9-[3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanyl)-2-deoxy-2-
methyldithio-ꢀ-D-arabinofuranosyl]adenine (5b)
A solution of 3 (100 mg, 0.19 mmol) and DEAD (29 µL,
0.19 mmol) in THF (5 mL) was stirred overnight at ambient
temperature and then methanethiol (183 mg, 3.8 mmol) in THF
(3 mL) was added. Stirring was continued at ambient temper-
ature for 96 h, and then volatiles were evaporated and the resi-
due was column chromatographed (EtOAc–hexane; 1 : 1) to
1
give 5b (41 mg, 38%): H NMR δ 1.04–1.14 (m, 28, 4 × i-Pr),
2.32 (s, 3, SCH3), 3.92–3.99 (m, 2, H2Ј,4Ј), 4.05–4.21 (m, 2,
H5Ј,5Љ), 4.76 (t, J = 7.8 Hz, 1, H3Ј), 6.07 (br s, 2, NH2), 6.53 (d,
J = 7.1 Hz, 1, H1Ј), 8.01 (s, 1, H2), 8.33 (s, 1, H8); MS m/z 572
(100, MHϩ).
Treatment of 8 (1 mg) with dithiothreitol (1 mg) in H2O
(1 mL) produced 9 as judged by the appearance of a new spot
on TLC and immediate releasing of the PrSH odor.
9-(2-Deoxy-2-propyldithio-ꢀ-D-arabinofuranosyl)adenine (6a)
Procedure D. NH4F (222 mg, 6.0 mmol) was added to a
stirred solution of 5a (180 mg, 0.3 mmol) in MeOH (25 ml).
After 18 h, volatiles were removed in vacuo and the residue was
chromatographed (CHCl3–MeOH; 95 : 5) to give 6a (64 mg,
60%): mp 120–124 ЊC (dec.); UV max 261 nm (ε 14 800), min
231 nm (ε 5100); 1H NMR (MeOH-d4) δ 0.87 (t, J = 7.3 Hz, 3,
9-[2-S-(2-Trimethylsilylethyl)-3,5-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanyl)-2-thio-ꢀ-D-arabinofuranosyl]adenine (10)
2-(Trimethylsilyl)ethanethiol (0.1 mL, 90 mg, 0.7 mmol) was
added to a stirred suspension of 1 (371 mg, 0.60 mmol) and
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 0 – 1 2 6
123

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K2CO3 (276 mg, 2.0 mmol) in DMF (3 mL) at ambient temper-
ature, and the reaction mixture was heated at 60 ЊC overnight.
Volatiles were evaporated and the residue was partitioned
[CHCl3–NaHCO3–H2O (ice-cold)]. The organic layer was
washed (NaCl–H2O), dried (MgSO4), evaporated and column
chromatographed (EtOAc–hexanes; 1 : 1) to give 2 (108 mg,
30%): 1H NMR δ Ϫ0.07 (s, 9, SiMe3), 0.57 (dt, J = 5.2, 14.0 Hz,
1, CH2Si), 0.61 (dt, J = 5.2, 14.0 Hz, 1, CH2Si), 0.80–1.31 (m,
28, 4 × i-Pr), 2.49 (dt, J = 5.6, 12.7 Hz, 1, SCH2), 2.53 (dt,
J = 5.6, 12.7 Hz, 1, SCH2), 3.72 (dd, J = 7.0, 9.7 Hz, 1, H2Ј),
3.89 (dt, J = 3.0, 7.9 Hz, 1, H4Ј), 4.09 (dd, J = 2.8, 12.9 Hz, 1,
H5Ј), 4.16 (dd, J = 3.2, 12.9 Hz, 1 H5Љ), 4.55 (dd, J = 8.3, 9.3 Hz,
1, H3Ј), 6.50 (d, J = 7.0 Hz, 1, H1Ј), 8.08 (s, 1, H2), 8.32 (s, 1,
H8); MS m/z 626 (100, MHϩ). Anal. calcd for C27H51N5O4SSi3ؒ
H2O (644.07): C, 50.35; H, 8.29; N, 10.87. Found: C, 50.39; H,
8.11; N, 10.49%.
δ 0.74 (t, J = 7.3 Hz, 3, CH3), 1.36 (sextet, J = 7.1 Hz, 2, CH2),
2.18–2.24 (m, 2, CH2S), 3.78 (dd, J = 2.4, 12.6 Hz, 1, H5Ј), 3.92
(dd, J = 2.6, 12.6 Hz, 1, H5Љ), 4.19–4.25 (m, 1, H4Ј), 4.30 (dd,
J = 5.0, 9.2 Hz, 1, H2Ј), 4.57 (d, J = 5.0 Hz, 1, H3Ј), 6.20 (d,
J = 9.2 Hz, 1, H1Ј), 8.22 (s, 1, H2), 8.35 (s, 1, H8); MS m/z
358 (100, MHϩ). Anal. calcd. for C13H19N5O3S2ؒ0.5 C4H8O2
(401.50; 1H NMR integration of residual EtOAc): C, 44.87; H,
5.77; N, 17.44. Found: C, 44.48; H, 5.71; N, 17.21%.
2-Amino-6-methoxy-9-[3,5-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanyl)-ꢀ-D-ribofuranosyl]purine (18b)
TPDS-Cl2 (317 mg, 0.32 mL, 1.01 mmol) was added to a
stirred solution of 2-amino-6-methoxy-(β--ribofuranosyl)-
purine (17b; 300 mg, 1.01 mmol) in dry pyridine (8 mL), and the
mixture was stirred overnight at ambient temperature. Volatiles
were evaporated and the residue was partitioned (EtOAc–H2O).
The organic phase was washed [cold 0.1 M HCl–H2O (2 ×
30 mL), H2O, saturated NaHCO3–H2O, and brine], dried
(Na2SO4), and evaporated. Column chromatography (CHCl3
4% MeOH–CHCl3) gave 18b (375 mg, 69%): 1H NMR
δ 0.93–1.12 (m, 28, 4 × i-Pr), 4.03–4.15 (m, 6, H4Ј,5Ј,5Љ, OCH3),
4.49 (d, J = 4.7 Hz, 1, H2Ј), 4.82 (m, 1, H3Ј), 5.32 (br s, 2, NH2),
6.04 (s, 1, H1Ј), 7.82 (s, 1, H8); MS m/z 540 (100, MHϩ). Anal.
calcd for C23H41N5O6Si2 (539.77): C, 51.18; H, 7.66; N, 12.97.
Found: C, 51.38; H, 7.88; N, 12.88%.
Treatment (ambient temperature to ∼45 ЊC, 2 h to overnight)
of 10 (97 mg, 0.16 mmol) [or 11 (27 mg, 0.07 mmol)] with
dimethyl(methylthio)sulfonium tetrafluoroborate (219 mg, 1.12
mmol) and dimethyl sulfide (0.72 mL, 496 mg, 8 mmol) in
anhydrous THF (5 mL) gave recovered 10 or 11 (∼20% to 80%
depends on reaction conditions) plus other byproduct(s).
9-[2-S-(2-Trimethylsilylethyl)-2-thio-ꢀ-D-arabinofuranosyl]-
adenine (11)
TBAF–THF (1M; 0.3 mL, 0.3 mmol) was added to a solution
of 10 (50 mg, 0.08 mmol) in THF (2 mL) and was stirred for 2h
at ambient temperature. Volatiles were evaporated and the resi-
2-Amino-6-chloro-9-[2-O-trifluoromethanesulfonyl-3,5-O-
(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-ꢀ-D-ribofuranosyl]purine
(19a)
due was column chromatographed (EtOAc
8% MeOH–
EtOAc) to give 11 (27 mg, 90%): 1H NMR δ Ϫ0.08 (s, 9, SiMe3),
0.59–0.63 (m, 2, SiCH2), 2.40–2.45 (m, 2, SCH2), 3.74 (dd,
J = 6.9, 8.8 Hz 1, H2Ј), 3.80–4.01 (m, 3, H4Ј,5Ј,5Љ), 4.32–4.38
(m, 1, H3Ј), 6.57 (d, J = 6.9 Hz, 1, H1Ј), 8.20 (s, 1, H2), 8.41
(s, 1, H8); MS m/z 384 (100, MHϩ).
Procedure E. N-Phenyltrifluoromethanesulfonimide (237 mg,
0.66 mmol) was added to a cold (0 ЊC) stirred solution of 18a26
(300 mg, 0.55 mmol) and DMAP (201 mg, 1.65 mmol) in
anhydrous CH2Cl2 (5 mL). The reaction mixture was stirred for
2 h and partitioned between cold 0.1 M AcOH–H2O (50 mL)
and CH2Cl2 (2 × 50 mL). The combined organic phase was
washed with cold NaHCO3–H2O (50 mL), brine (50 mL) and
dried MgSO4, filtered and evaporated. The residue was column
chromatographed (EtOAc–hexane; 30 : 70) to give 19a (310 mg,
83%): 1H NMR δ 1.04–1.12 (m, 28, 4 × i-Pr), 4.07 (dd, J = 2.7,
13.5 Hz, 1, H5Ј), 4.13 (dt, J = 2.4, 7.0 Hz, 1, H4Ј), 4.50 (d,
J = 13.5 Hz, 1, H5Љ), 4.86 (dd, J = 4.5, 9.3 Hz, 1, H3Ј), 5.64 (d,
J = 4.5 Hz, 1, H2Ј), 6.10 (s, 1, H1Ј), 8.02 (s, 1, H8); MS m/z 676
(100, MHϩ[35Cl]), 678 (30, MHϩ[37Cl]).
2Ј-S-Acetyl-3Ј,5Ј-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-2Ј-
thioadenosine (13)
Treatment of 1216 (290 mg, 0.45 mmol) by procedure A gave
1325 (200 mg, 78%): 1H NMR δ 1.05–1.12 (m, 28, 4 × i-Pr), 2.31
(s, 3, Ac), 4.02–4.09 (m, 3, H4Ј,5Ј,5Љ), 4.70 (dd, J = 4.8, 7.4 Hz,
1, H2Ј), 5.26 (dd, J = 5.4, 7.2 Hz, 1, H3Ј), 6.07 (d, J = 4.8 Hz, 1,
H1Ј), 7.97 (s, 1, H2), 8.29 (s, 1, H8); MS m/z 568 (100, MHϩ).
3Ј,5Ј-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanyl)-2Ј-thio-
adenosine (14)
2-Amino-6-methoxy-9-[2-O-trifluoromethanesulfonyl-3,5-O-
(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-ꢀ-D-ribofuranosyl]purine
(19b)
Treatment of 13 (180 mg, 0.31 mmol) by procedure B gave 14
(160 mg, 96%): 1H NMR δ 1.03–1.15 (m, 28, 4 × i-Pr), 2.19 (d,
J = 6.4 Hz, 1, SH), 4.08 (d, J = 4.7 Hz, 1, H2Ј), 4.17–4.25 (m, 3,
H4Ј,5Ј,5Љ), 4.88 (t, J = 6.4 Hz, 1, H3Ј), 5.98 (d, J = 7.1 Hz, 1,
H1Ј), 6.22 (br s, 2, NH2), 8.01 (s, 1, H2), 8.34 (s, 1, H8); MS m/z
526 (100, MHϩ).
Treatment of 18b (200 mg, 0.37 mmol) by procedure E gave 19b
(156 mg, 64%): 1H NMR δ 1.04–1.12 (m, 28, 4 × i-Pr), 4.03–4.23
(m, 6, H4Ј,5Ј,5Љ,OCH3), 4.85 (br s, 2, NH2), 4.94 (dd, J = 4.7,
9.0 Hz, 1, H3Ј), 5.70 (d, J = 4.7 Hz, 1, H2Ј), 6.04 (s, 1, H1Ј), 7.82
(s, 1, H8); MS m/z 672 (100, MHϩ).
3Ј,5Ј-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanyl)-2Ј-deoxy-2Ј-
propyldithioadenosine (15)
2,6-Diamino-9-[2-O-trifluoromethanesulfonyl-3,5-O-(1,1,3,3-
tetraisopropyl-1,3-disiloxanyl)-ꢀ-D-ribofuranosyl]purine (19c)
Treatment of 18c27 (370 mg, 0.706 mmol) by procedure E
Treatment of 14 (140 mg, 0.27 mmol) by procedure C gave 15
1
(67 mg, 42%): H NMR δ 0.89 (t, J = 7.3 Hz, 3, CH3), 1.07–
(column chromatography: EtOAc–hexane, 75
: 25) gave
1.12 (m, 28, 4 × i-Pr), 1.59 (sextet, J = 7.2 Hz, 2, CH2), 2.56 (t,
J = 7.4 Hz, 2, SCH2), 4.05–4.07 (m, 2, H2Ј,4Ј), 4.19–4.26 (m, 2,
H5Ј,5Љ), 5.20 (t, J = 7.3 Hz, 1, H3Ј), 6.07 (br s, 2, NH2), 6.34 (d,
J = 3.5 Hz, 1, H1Ј), 8.03 (s, 1, H2), 8.29 (s, 1, H8); MS m/z 600
(100, MHϩ).
amorphous 19c (360 mg, 78%), which failed to crystallize
(MeOH): 1H NMR δ 1.01–1.12 (m, 28, 4 × i-Pr), 4.04–4.20 (m,
3, H4Ј,5Ј,5Љ), 4.66 (br s, 2, NH2), 4.99 (dd, J = 4.5, 8.9 Hz, 1,
H3Ј), 5.41 (br s, 2, NH2), 5.81 (d, J = 4.5 Hz, 1, H2Ј), 6.05 (s, 1,
H1Ј), 7.71 (s, 1, H8); 13C NMR δ 13.2–17.4 (12, 4 × i-Pr), 60.1
(C2Ј), 68.6 (C5Ј), 81.6 (C3Ј), 86.9 (C4Ј), 88.4 (C1Ј), 115.0 (C5),
136.7 (C8), 151.7 (C4), 155.8 (C2), 159.7 (C6); MS m/z 657 (100,
2Ј-Deoxy-2Ј-propyldithioadenosine (16)
1
Treatment of 15 (60 mg, 0.1 mmol) by procedure D gave 16
(12.4 mg, 35%): mp 145–147 ЊC (dec; EtOAc–MeOH); UV max
260 nm (ε 15 400), min 229 nm (ε 4100); 1H NMR (MeOH-d4)
MHϩ). Anal. calcd for C23H39F3N6O7SSi2ؒCH4O (688.86, H
NMR integration of MeOH): C, 41.84; H, 6.29; N, 12.20.
Found: C, 41.56; H, 5.99; N, 12.25%.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 0 – 1 2 6
124

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2-Amino-6-chloro-9-[2-S-acetyl-3,5-O-(1,1,3,3-tetraisopropyl-
1,3-disiloxanyl)-2-thio-ꢀ-D-arabinofuranosyl]purine (20a)
J = 2.6, 13.0 Hz, 1, H5Ј), 4.19 (dd, J = 2.6, 13.0 Hz, 1, H5Љ), 4.51
(t, J = 8.7, Hz, 1, H3Ј), 4.75 (br s, 2, NH2), 5.46 (br s, 2, NH2),
6.27 (d, J = 7.1 Hz, 1, H1Ј), 7.84 (s, 1, H8); 13C NMR δ 12.8–
17.9 (12, 4 × i-Pr), 48.5 (C2Ј), 61.2 (C5Ј), 75.7 (C3Ј), 83.9 (C4Ј),
84.4 (C1Ј), 114.5 (C5), 137.0 (C8), 152.3 (C4), 155.8 (C2), 159.6
(C6); MS m/z 541 (100, MHϩ). Anal. calcd for C22H40N6O4SSi2ؒ
Treatment of 19a (250 mg, 0.37 mmol) by procedure A (column
chromatography: EtOAc–hexanes, 20 : 80) gave 20a (150 mg,
67%): mp 78–86 ЊC; 1H NMR δ 0.8–1.3 (m, 28, 4 × i-Pr), 2.20 (s,
3, Ac), 3.94–3.97 (m, 1, H4Ј), 4.09 (dd, J = 3.2, 12.0 Hz, 1, H5Ј),
4.16 (dd, J = 3.2, 12.0 Hz, 1, H5Љ), 4.56 (dd, J = 7.0, 10.5 Hz, 1,
H2Ј), 4.68 (t, J = 10.5 Hz, 1, H3Ј), 5.24 (br s, 2, NH2), 6.39 (d,
J = 7.0 Hz, 1, H1Ј), 7.96 (s, 1, H8); 13C NMR δ 13.3–17.6 (12,
4 × i-Pr), 30.7 (CH3) 52.2 (C2Ј), 61.4 (C5Ј), 71.66 (C3Ј), 83.7
(C4Ј), 83.9 (C1Ј), 125.7 (C5), 141.3 (C8), 151.5 (C4), 153.8 (C2),
159.1 (C6), 194.0 (CO); MS m/z 602 (100, MHϩ[35Cl]), 604 (40,
MHϩ[37Cl]). Anal. calcd for C24H40ClN5O5SSi2 (602.30): C,
47.86; H, 6.69; N, 11.63. Found: C, 47.87; H, 6.67; N, 11.24.
1
CH4O (572.87; H NMR integration of MeOH): C, 48.22; H,
7.74; N, 14.67. Found: C, 48.65; H, 7.56; N, 14.92%.
2-Amino-6-chloro-9-[3,5-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanyl)-2-deoxy-2-propyldithio-ꢀ-D-arabinofuranosyl]purine
(22a)
Treatment of 21a (122 mg, 0.212 mmol) by procedure C (after
thiol was added, the reaction mixture was stirred overnight at
ambient temperature) and column chromatography (EtOAc–
1
hexane, 30 : 70) gave 22a (66 mg, 48%): H NMR δ 0.92 (t,
2-Amino-6-methoxy-9-[2-S-acetyl-3,5-O-(1,1,3,3-tetraisopropyl-
1,3-disiloxanyl)-2-thio-ꢀ-D-arabinofuranosyl]purine (20b)
J = 7.2 Hz, 3, CH3), 1.05–1.27 (m, 28, 4 × i-Pr), 1.60 (sextet,
J = 7.2 Hz, 2, CH2), 2.56 (t, J = 7.2 Hz, 2, SCH2), 3.86–3.89 (m,
2, H2Ј,4Ј), 4.08 (dd, J = 3.0, 12.7 Hz, 1, H5Ј), 4.10 (dd, J = 3.0,
12.7 Hz, 1, H5Љ), 4.51 (dd, J = 8.0, 9.6 Hz, 1, H3Ј), 5.20 (br s, 2,
NH2), 6.42 ( d, J = 7.0 Hz, 1, H1Ј), 7.98 (s, 1, H8); MS m/z 634
(100, MHϩ[35Cl]), 636 (55, MHϩ[37Cl]).
Treatment of 19b (100 mg, 0.15 mmol) by procedure A (column
chromatography: EtOAc–hexanes, 20 : 80) gave 20b (65 mg,
1
73%): H NMR δ 1.04–1.20 (m, 28, 4 × i-Pr), 2.19 (s, 3, Ac),
3.93–3.96 (m, 1, H4Ј), 4.05–4.09 (m, 4, H5Ј, OCH3), 4.16 (dd,
J = 3.8, 12.8 Hz, 1, H5Љ), 4.55 (dd, J = 7.2, 10.4 Hz, 1, H2Ј),
4.77 (dd, J = 8.2, 10.4 Hz, 1, H3Ј), 4.87 (br s, 2, NH2), 6.35 (d,
J = 7.2 Hz, 1, H1Ј), 7.79 (s, 1, H8); MS m/z 598 (100, MHϩ).
2-Amino-6-methoxy-9-[3,5-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanyl)-2-deoxy-2-propyldithio-ꢀ-D-arabinofuranosyl]purine
(22b)
2,6-Diamino-9-[2-S-acetyl-3,5-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanyl)-2-thio-ꢀ-D-arabinofuranosyl]purine (20c)
Treatment of 21b (110 mg, 0.2 mmol) by procedure C (after
thiol was added, the reaction mixture was stirred overnight at
ambient temperature) and column chromatography (EtOAc–
Treatment of 19c (340 mg, 0.52 mmol) by procedure A (column
chromatography: EtOAc–hexanes, 20 : 80) gave 20c (220 mg,
1
hexane, 30 : 70) gave 22b (52 mg, 42%): H NMR δ 0.90 (t,
1
73%): H NMR δ 1.04–1.20 (m, 28, 4 × i-Pr), 2.22 (s, 3, Ac),
J = 7.2 Hz, 3, CH3), 1.05–1.27 (m, 28, 4 × i-Pr), 1.58 (sextet,
J = 7.2 Hz, 2, CH2), 2.56 (t, J = 7.2 Hz, 2, SCH2), 3.85–3.90 (m,
2, H2Ј,4Ј), 4.07–4.09 (m, 5, H5Ј,5Љ,OCH3), 4.59 (dd, J = 8.4, 9.5
Hz, 1, H3Ј), 4.82 (br s, 2, NH2), 6.41 (d, J = 7.0 Hz, 1, H1Ј), 7.80
(s, 1, H8); MS m/z 630 (100, MHϩ).
3.95 (dt, J = 3.2, 7.9 Hz, 1, H4Ј), 4.06 (dd, J = 3.1, 12.8 Hz, 1,
H5Ј), 4.09 (dd, J = 4.2, 12.8 Hz, 1, H5Љ), 4.57 (dd, J = 7.2, 10.4
Hz, 1, H2Ј), 4.65 (br s, 2, NH2), 4.84 (t, J = 10.2 Hz, 1, H3Ј),
5.35 (br s, 2, NH2), 6.30 (d, J = 7.2 Hz, 1, H1Ј), 7.72 (s, 1, H8);
13C NMR δ 12.8–17.8 (12, 4 × i-Pr), 30.6 (CH3), 52.4 (C2Ј), 61.9
(C5Ј), 72.6 (C3Ј), 83.4 (C4Ј), 83.8 (C1Ј), 114.9 (C5), 137.3 (C8),
152.2 (C4), 156.0 (C2), 159.9 (C6), 194.0 (CO); MS m/z 583
(100, MHϩ).
2,6-Diamino-9-[3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-2-
deoxy-2-propyldithio-ꢀ-D-arabinofuranosyl]purine (22c)
Treatment of 21c (145 mg, 0.268 mmol) by procedure C (after
thiol was added, the reaction mixture was stirred overnight at
ambient temperature) and column chromatography (EtOAc–
2-Amino-6-chloro-9-[3,5-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanyl)-2-thio-ꢀ-D-arabinofuranosyl]purine (21a)
1
hexane, 90 : 10) gave 22c (110 mg, 67%): H NMR δ 0.87 (t,
Treatment of 20a (110 mg, 0.18 mmol) by procedure B (column
chromatography: EtOAc–hexanes, 35 : 65) gave 21a (95 mg,
93%): mp 270 ЊC (dec.); 1H NMR δ 1.02–1.19 (m, 28, 4 × i-Pr),
1.42 (d, J = 8.0 Hz, 1, SH), 3.83–3.89 (m, 2, H2Ј,4Ј), 4.12 (dd,
J = 2.8, 13.2 Hz, 1, H5Ј), 4.15 (dd, J = 2.8, 13.2 Hz, 1, H5Љ), 4.44
(t, J = 8.5 Hz, 1, H3Ј), 5.25 (br s, 2, NH2), 6.32 (d, J = 7.0 Hz, 1,
H1Ј), 8.10 (s, 1, H8); MS m/z 560 (100, MHϩ[35Cl]), 562 (40,
MHϩ[37Cl]).
J = 7.2 Hz, 3, CH3), 1.04–1.27 (m, 28, 4 × i-Pr), 1.55 (sextet,
J = 7.2 Hz, 2, CH2), 2.53 (t, J = 7.2 Hz, 2, SCH2), 3.84–3.87 (m,
2, H2Ј,4Ј), 4.06–4.12 (m, 2, H5Ј,5Љ), 4.56 (t, J = 8.2 Hz, 1, H3Ј),
4.92 (br s, 2, NH2), 5.86 (br s, 2, NH2), 6.39 (d, J = 7.0 Hz, 1,
H1Ј), 7.70 (s, 1, H8); MS m/z 615 (100, MHϩ).
2-Amino-6-chloro-9-(2-deoxy-2-propyldithio-ꢀ-D-arabino-
furanosyl)purine (23a)
Treatment of 22a (30 mg, 0.047 mmol) by procedure D (column
chromatography: CHCl3–MeOH, 96 : 4) gave 23a (17 mg, 91%):
mp 190–192 ЊC (dec.); UV max 309 nm (ε 8800), max 248 nm
2-Amino-6-methoxy-9-[3,5-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanyl)-2-thio-ꢀ-D-arabinofuranosyl]purine (21b)
1
Treatment of 20b (120 mg, 0.2 mmol) by procedure B (column
chromatography: EtOAc–hexane, 35 : 65) gave 21b (102 mg,
91%): 1H NMR δ 1.03–1.18 (m, 28, 4 × i-Pr), 1.39 (d, J = 8.2 Hz,
1, SH), 3.80–3.86 (m, 2, H2Ј,4Ј), 4.04–4.07 (m, 4, H5Ј,OCH3),
4.15 (dd, J = 2.6, 13.0 Hz, 1, H5Љ), 4.46 (t, J = 8.5 Hz, 1, H3Ј),
5.02 (br s, 2, NH2), 6.31 (d, J = 7.1 Hz, 1, H1Ј), 7.95 (s, 1, H8);
MS m/z 556 (100, MHϩ).
(ε 8200), min 272 nm (ε 1900), min 236 nm (ε 6800); H NMR
(MeOH-d4) δ 0.89 (t, J = 7.2 Hz, 3, CH3), 1.53 (sextet, J = 7.2
Hz, 2, CH2), 2.58 (t, J = 7.2 Hz, 2, CH2S), 3.84–3.92 (m, 4,
H2Ј,4Ј,5Ј,5Љ), 4.49 (t, J = 8.0 Hz, 1, H3Ј), 6.52 (d, J = 7.0 Hz, 1,
H1Ј), 8.35 (s, 1, H8); 13C NMR (MeOH-d4) δ 12.1 (CH3), 22.0
(CH2), 41.0 (SCH2), 60.3 (C2Ј), 62.7 (C5Ј), 72.7 (C3Ј), 84.8 &
84.9 (C4Ј & C1Ј), 123.7 (C5), 142.4 (C8), 150.6 (C4), 154.0 (C2),
160.6 (C6); MS m/z 392 (100, MHϩ[35Cl]), 394 (55, MHϩ[37Cl]).
Anal. calcd. for C13H18ClN5O3S2 (391.90): C, 39.84; H, 4.63; N,
17.87. Found: C, 39.63; H, 4.79; N, 17.49%.
2,6-Diamino-9-[3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-2-
thio-ꢀ-D-arabinofuranosyl]purine (21c)
Treatment of 20c (210 mg, 0.36 mmol) by procedure B (column
chromatography: EtOAc–hexane, 75 : 15) gave amorphous 21c
(160 mg, 82%), which failed to crystallize (MeOH): H NMR
2-Amino-6-methoxy-9-(2-deoxy-2-propyldithio-ꢀ-D-arabino-
furanosyl)purine (23b)
1
δ 1.03–1.19 (m, 28, 4 × i-Pr), 1.51 (d, J = 8.0 Hz, 1, SH), 3.81
(dd, J = 7.5, 13.2 Hz, 1, H2Ј), 3.87–3.89 (m, 1, H,4Ј), 4.07 (dd,
Treatment of 22b (40 mg, 0.064 mmol) by procedure D (column
chromatography: CHCl3–MeOH, 96 : 4) gave 23b (20 mg,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 0 – 1 2 6
125

View Article Online
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81%): mp 81–85 ЊC; UV max 282 nm (ε 9700), max 249 nm
(ε 9600), min 262 nm (ε 5300), min 227 nm (ε 4600); 1H
NMR (MeOH-d4) δ 0.89 (t, J = 7.3 Hz, 3, CH3), 1.53 (sextet,
J = 7.3 Hz, 2, CH2), 2.58 (t, J = 7.3 Hz, 2, CH2S), 3.82–3.96
(m, 4, H2Ј,4Ј,5Ј,5Љ), 4.07 (s, 3, OCH3), 4.52 (t, J = 8.2 Hz, 1,
H3Ј), 6.47 (d, J = 7.1 Hz, 1, H1Ј), 8.08 (s, 1, H8); 13C NMR
(MeOH-d4) δ 12.1 (CH3), 22.0 (CH2), 41.0 (SCH2), 53.3
(OCH3), 60.4 (C2Ј), 63.1 (C5Ј), 72.4 (C3Ј), 84.7 & 84.9 (C4Ј &
C1Ј), 114.1 (C5), 139.1 (C8), 153.7 (C4), 160.7 (C2), 161.7 (C6);
MS m/z 388 (100, MHϩ). Anal. calcd. for C14H21N5O4S2ؒCH4O
(419.52; 1H NMR integration of MeOH): C, 42.94; H, 6.01; N,
16.69. Found: C, 43.01; H, 5.76; N, 16.66%.
2,6-Diamino-9-(2-deoxy-2-propyldithio-ꢀ-D-arabinofuranosyl)-
purine (23c)
Treatment of 22c (110 mg, 0.18 mmol) by procedure D (column
chromatography: CHCl3–MeOH, 96 : 4) gave 23c (57 mg, 85%):
mp 82–90 ЊC (soften), 110 ЊC; UV max 283 nm (ε 11 200), max
257 nm (ε 9600), min 267 nm (ε 7900), min 238 nm (ε 6300);
1H NMR (MeOH-d4) δ 0.89 (t, J = 7.2 Hz, 3, CH3), 1.53 (sextet,
J = 7.2 Hz, 2, CH2), 2.58 (t, J = 7.2 Hz, 2, CH2S), 3.81–3.96 (m,
4, H2Ј,4Ј,5Ј,5Љ), 4.51 (t, J = 8.8 Hz, 1, H3Ј), 6.41 (d, J = 7.2 Hz,
1, H1Ј), 7.98 (s, 1, H8); 13C NMR (MeOH-d4) δ 12.1 (CH3), 22.0
(CH2), 41.0 (SCH2), 60.4 (C2Ј), 63.2 (C5Ј), 72.4 (C3Ј), 84.7 &
84.9 (C4Ј & C1Ј), 113.1 (C5), 137.8 (C8), 151.6 (C4), 156.6 (C2),
160.7 (C6); MS m/z 373 (100, MHϩ). Anal. calcd. for C13H20-
N6O3S2ؒH2O (390.49): C, 39.99; H, 5.68; N, 21.52. Found: C,
40.35; H, 5.78; N, 21.33%.
Cell culture
The eight human cell lines used were obtained from the Devel-
opmental Therapeutics Program Tumor Repository, National
Cancer Institute (Frederick, MD). The cell lines were grown in
RPMI 1640 medium containing 9% fetal bovine serum, 1%
iron-supplemented calf serum, and 2 mmol -glutamine. For
the in vitro evaluation of the sensitivity of the human cell lines
to the compounds, cells were plated in 96-well microtiter plates
(5.0 × 103 cells per well) and then were exposed continuously to
various concentrations of the compounds for 72 h at 37 ЊC. Cell
viability was based on the reduction of MTS (in conjunction
with phenazine ethosulfate) to a water soluble formazan prod-
uct, which was measured with a microplate reader at 490 nm.
The background absorbance mean was subtracted from the
data followed by conversion to percent of control. The drug
concentrations producing survival just above and below the
50% level were used in a linear regression analysis to calculate
the IC50.
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Acknowledgements
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This work was partially supported by an award from the Amer-
ican Heart Association, Florida/Puerto Rico Affiliate. D.R.C
and P.I.G were sponsored by MBRS RISE program (NIH/
NIGMS; R25 GM61347). We thank Professor J. Stubbe for
helpful suggestions on the manuscript. We thank Dr William R.
Waud and his staff at the Southern Research Institute for
supplying the cytotoxicity data.
24 In collaboration with Professor J. Stubbe at Massachusetts Institute
of Technology.
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 0 – 1 2 6
126