6-(Het)aryl-7-deazapurine ribonucleosides as novel potent cytostatic agents

Article abstract of DOI:10.1021/jm901428k

A series of novel 7-deazapurine ribonucleosides bearing an alkyl, aryl, or hetaryl group in position 6 and H, F, or Cl atom in position 7 has been prepared either by Pd-catalyzed cross-coupling reactions of the corresponding protected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with alkyl- or (het)-arylorganometallics followed by deprotection, or by single-step aqueous phase cross-coupling reactions of unprotected 6-chloro-(7-halogenated-)7- deazapurine ribonucleosides with (het)arylboronic acids. Significant cytostatic effect was detected with a substantial proportion of the prepared compounds. The most potent were 7-H or 7-F derivatives of 6-furyl- or 6-thienyl-7-deazapurines displaying cytostatic activity in multiple cancer cell lines with a geometric mean of 50% growth inhibition concentration ranging from 16 to 96 nM, a potency comparable to or better than that of the nucleoside analogue clofarabine. Intracellular phosphorylation to mono- and triphosphates and the inhibition of total RNA synthesis was demonstrated in preliminary study of metabolism and mechanism of action studies. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901428k

460 J. Med. Chem. 2010, 53, 460–470
DOI: 10.1021/jm901428k
6-(Het)aryl-7-Deazapurine Ribonucleosides as Novel Potent Cytostatic Agents
†
Petr Naus, Radek Pohl, Ivan Votruba, Petr Dzubak, Marian Hajduch, Ria Ameral, Gabriel Birkus, Ting Wang,
†
‡
‡
§
§
ꢀ †
ꢀ
ꢁ
ꢁ
ꢁ
ꢀ §
Adrian S. Ray,§ Richard Mackman,§ Tomas Cihlar,§ and Michal Hocek*,†
†Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences & IOCB Research Center,
Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic, ‡Laboratory of Experimental Medicine, Faculty of Medicine, Palacky University,
Puꢀskinova 6, Olomouc 77520, Czech Republic, and §Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404, U.S.A.
Received September 25, 2009
A series of novel 7-deazapurine ribonucleosides bearing an alkyl, aryl, or hetaryl group in position 6 and
H, F, or Cl atom in position 7 has been prepared either by Pd-catalyzed cross-coupling reactions of the
corresponding protected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with alkyl- or (het)-
arylorganometallics followed by deprotection, or by single-step aqueous phase cross-coupling reactions
of unprotected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with (het)arylboronic acids.
Significant cytostatic effect was detected with a substantial proportion of the prepared compounds. The
most potent were 7-H or 7-F derivatives of 6-furyl- or 6-thienyl-7-deazapurines displaying cytostatic
activity in multiple cancer cell lines with a geometric mean of 50% growth inhibition concentration
ranging from 16 to 96 nM, a potency comparable to or better than that of the nucleoside analogue
clofarabine. Intracellular phosphorylation to mono- and triphosphates and the inhibition of total RNA
synthesis was demonstrated in preliminary study of metabolism and mechanism of action studies.
Introduction
inhibition of both viral and host RNA synthesis6 was ob-
served, suggesting that these nucleosides are phosphorylated
to triphosphates that may subsequently interfere with the
activity of viral and/or host RNA polymerases. However,
the lack of selectivity toward HCV prevented further devel-
opment of these compounds as antivirals. Therefore, novel
modifications of 6-hetaryl nucleosides were pursued with a
goal to achieve either selective antiviral activity through the
specific inhibition of HCV RNA polymerase or more potent
cytostatic activity that could potentially lead to novel antic-
ancer therapeutics. Sugar-modified derivatives (20- and 50-
deoxyribonucleosides,7 30-deoxyribonucleosides,8 as well as
20-C-methyl-ribonucleosides9) of the 6-aryl- or 6-hetarylpur-
ine series are all inactive, while some carbocyclic homonucleo-
sides were reported10 to still exert cytostatic effects. Some
L-ribonucleosides were found11 to exhibit weak anti-HCV
effect in replicon assay, but their triphoshates did not inhibit
HCVRNA polymerase. Inaddition, nucleoside withmodified
purine ring such as 2-substituted12 and 8-substituted13 6-
arylpurine ribonucleosides as well as purine ribonucleosides
bearing partially or fully saturated hetaryl groups14 do not
exhibit any notable biological activities. Replacing purine-
ring N-atoms by carbon to form deazapurine analogues has
also been explored. While some 6-aryl-1-deazapurine nucleo-
sides15 possessed moderate cytostatic activities, the corres-
ponding 6-aryl-3-deazapurine nucleosides 2 were devoid of
cytostatic and antiviral activities.16 This shows that the N-3
nitrogen is crucial for the interaction of these compounds with
the target biological system (presumably a kinase or RNA
polymerase), while the N-1 nitrogen is not. Therefore, the next
logical step was to assess the role of N-7 nitrogen, which is
engaged neither in H-bonds with the complementary pyrimi-
dine nucleobase during biosynthesis of RNA nor in minor
groove interactions in the active site of RNA polymerase. We
Purine nucleosides and their analogues represent an impor-
tant class of biologically active compounds that have been
extensively explored in the last five decades for their antiviral
and antitumor properties. A number of active compounds in
this class are being used as clinical drugs for the treatment of
viral infections such as HIVa (abacavir, didanosine) and
chronic hepatitis B (entecavir). Many purine (fludarabine,
cladribine, and clofarabine)1 and other (gemcitabin, ara-C,
5-F-dU, 2-deoxycoformycin, capecitabine, nelarabine, and
decitabine)2 nucleosides are clinical therapeutics for variety
of hematological malignancies. Despite systematic studies of
modified purine nucleosides, there still remains a space for the
design of new analogues and development of novel nucleo-
side-based therapeutics. In particular, there is a need for new
cytostatics3 for the treatment of drug-resistant tumors and
antivirals for therapeutic interventions against infections by
various RNA viruses, particularly hepatitis C virus (HCV).4
In our prior studies, we have discovered a new type of biolo-
gically active nucleosides 1: purine ribonucleosides bearing
aryl or hetaryl substituents in position 6.5 This group of com-
pounds exhibits5 low micromolar cytostatic activities toward
leukemia cell-lines. Moreover, some 6-hetarylpurine ribonu-
cleosides also exert6 strong anti-HCV activities. Although the
mechanism of action remains to be fully identified, potent
*To whom correspondence should be addressed. Phone: þ420
220183324. Fax: þ420 220183559. E-mail: hocek@uochb.cas.cz.
a Abbreviations: BrdU, 5-bromo-20-deoxyuridine; BrU, bromouri-
dine; HCV, hepatitis C virus; HIV, human immunodeficiency virus;
HPFC, high performance flash chromatography; NMP, nucleoside
50-O-monophosphate; NTP, nucleoside 50-O-triphosphate; SRB, sulfor-
hodamine; TCA, trichloroacetic acid; TDA-1, tris(2-(2-methoxyethoxy)-
ethyl)amine; TIPS, triisopropylsilyl, TTPTS, tris(3-sulfonatophenyl)pho-
sphine.
r
pubs.acs.org/jmc
Published on Web 11/24/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 461
Scheme 1a
Chart 1
a Conditions: (a) CCl4, P(NMe2)3, THF or toluene; (b) KOH, TDA-1,
MeCN, or toluene (40% from 9).
were interested whether the replacement of the N-7 nitrogen
by C-H, C-F, or C-Cl would result in improved selectivity
toward viral RNA polymerase or enhanced cytostatic effect,
presumably through the inhibition of cellular RNA poly-
merases. Here we report on the synthesis and evaluation of
cytostatic and anti-HCV activity of novel 7-unsubstituted or
7-halogenated 6-aryl-7-deazapurine ribonucleosides 3-5
(Chart 1). Because 6-ethyl-17 and 6-(hydroxymethyl)purine18
ribonucleosides are known to be potent cytostatics, we have
also included 6-ethyl and 6-hydroxymethyl substituents into
the series.
7-Deazapurine (correct IUPAC name is pyrrolo[2,3-d]-
pyrimidine but purine numbering will be used throughout
the paper) nucleosides are important compounds showing
broad spectrum of activities.19 Tubercidin (7-deazaadeno-
sine)20 is an antibiotic exerting potent cytotoxicity and anti-
viral activity. Tubercidin and some other 7-deazapurine nu-
cleosides are known to be inhibitors of adenosine kinases21
and display antiparasitic activity.22 20-C-Me-ribonucleo-
sides derived from 7-deazaadenine (including 7-fluoro- and
7-chloro derivatives) were found to be highly potent inhibitors
of HCV and are being evaluated as potential antiviral thera-
peutics.23
using toluene as a solvent in both steps (chlorination and
glycosylation). Our application of the same conditions for
glycosylation of 6-chloro-7-deazapurine 6 with chlorose 7
gave the desired 6-chloro-7-deazapurine riboside 8 as a single
β-anomer in an overall yield of 63% from 9.
Palladium catalyzed cross-coupling reactions27 of com-
pound 8 (Scheme 2) with the corresponding alkyl- or arylbo-
ronic acids, -zinc, -tin, and -aluminum reagents were per-
formed to provide protected 6-substituted 7-deazapurines
ribonucleosides 10a-l in good yields. The reaction conditions
were derived from previously reported procedures for the
modification of purine derivatives.5,18,28 The subsequent
deprotection step was performed by treatment with 90%
aqueous trifluoroacetic acid to afford the desired ribonucleo-
sides 3a-3l. N-Protecting Boc (10k) and trityl (10l) groups
were also simultaneously removed under the acidic deprotec-
tion conditions. In the case of 6-hydroxymethyl derivative
(10b), the benzoyl group was quantitatively deprotected
with sodium methoxide in methanol before final acidic
deprotection.
Alternative 6-hetaryl-7-deazapurine ribosides 3m-3s
(Scheme 3) were prepared directly from unprotected 6-
chloro-7-deazapurine riboside 11, using aqueous-phase Suzu-
ki cross-coupling reaction29 (3m-3r) or Stille coupling (3s). In
the case of 3-pyrrolyl derivative, the N-protecting triisopro-
pylsilyl moiety was simultaneously deprotected under the
strongly basic conditions of the aqueous coupling (3o). It
should be noted that in the case of NH containing boronic
acids, we also observed the formation of the N-arylated
product formed by coupling of the 5-membered heterocycle
with chloro derivative 11. Thus in the case of 4-pyrazolyl
derivative 3r, the concomitant N-arylation gave 1,4-bis{7-(β-
D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl}-1H-pyra-
zole (3rX) as side-product in 18% yield.
Chemistry
Our synthesis of novel 6-substituted 7-deazapurine ribonu-
cleosides 3a-s relied on palladium catalyzed cross-coupling
reactions of either protected or unprotected 6-chloro-7-dea-
zapurine ribonucleosides. The glycosylation (Scheme 1) of
sodium24 or (more preferably) potassium salt25 of 6-chloro-
7-deazapurine 6 with “nonparticipating” R-D-halogenose 726
affording protected riboside 8 represents hitherto the only
practically usable access to 6-chloro-7-deazapurine ribonu-
cleosides unsubstituted in position 7. Unstable halogenose 7 is
generated from lactol 9 by Appel’s chlorination and is imme-
diately used in the displacement reaction. In accordance with
literature reports,22 the protocol25 utilizing (KOH/TDA-1/
MeCN-THF) provided the β-anomer 8 contaminated with
unseparable amounts of R-anomer in an overall yield of up to
40% from 9.
The synthesis of analogous 7-fluoro-6-hetaryl(aryl)-7-dea-
zapurine ribosides started with cross-coupling reactions of
per-O-benzoylated 6-chloro-7-fluoro-7-deazapurine riboside
1230 (Scheme 4), under analogous conditions described for the
coupling reactions on 8. The resulting 6-substituted 7-fluoro-
7-deazapurine nucleosides 13d,h-n were then deprotected
ꢁ
During extensive glycosylation studies utilizing pyrrolopyr-
imidines base anions with a series of analogous R-D-ribofura-
nosyl chlorides, Ugarkar21 developed modified conditions
under Zemplen conditions to afford the unprotected 7-fluoro-
6-substituted-7-deazapurine ribonucleosides 4d,h-n in good
yields.

462 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Nauꢀs et al.
Scheme 2a
Scheme 3a
a Conditions: (A) M = B(OH)2:Pd(OAc)2, TPPTS, Na2CO3, water/
MeCN (2:1), 100°C; (B) M = SnBu3:PdCl2(PPh3)2, DMF, 100 °C.
5d,h,i,m,n (again, no nucleophilic substitution of the 7-Cl was
observed) in excellent yields.
Biological Profiling
a Conditions: (A) M = ZnX or AlEt2:Pd(PPh3)4, THF, 70°C (rt for
10b); (B) M = B(OH)2:Pd(PPh3)4, K2CO3, toluene, or dimethoxyetha-
ne-water [3:1 for 10g and 4:1 for 10k], 100°C; (C) M = SnBu3:PdCl2-
(PPh3)2, DMF, 100°C.
Cytostatic Activity. Cytostatic activity of all prepared
compounds was initially evaluated against four different cell
lines derived from various human solid tumors including
lung (A-549 cells), prostate (Du-145 cells), colon (HCT-116
cells), and breast (HS-578 cells) carcinomas. Concentrations
inhibiting the cell growth by 50% (GIC50) were determined
using a quantitative cellular staining with sulforhodamine B
(SRB)32 following a 5-day treatment. The SRB method
allowed for determining a net effect on cell growth by
subtracting background signal generated by the cell culture
inoculum at the beginning of treatment.
In general, 6-substituted 7-deaza-nucleosides modified
with a 6-member (het)aryl ring either with or without sub-
stitution (compounds 3c-f,3g, and 3s) showed minimal to no
cytostatic activity against the tested cell lines (Table 1). In
contrast, a number of nucleosides containing 5-member
heterocyclic aryl in 6-position exhibited potent cytostatic
effects, with those containing furan or thiophene being
among the most active. In particular, compounds substituted
with furan-2-yl (3h) and thiophen-2-yl (3i) showed the most
potent cytostatic activity across all four tested cell lines with
GIG50 values ranging from 7 to 100 nM. In comparison,
A different approach was used for the synthesis of 3-pyrro-
lyl derivative 4o (Scheme 5). Glycosylation of R-chlorose 7
using the potassium salt of 4-chloro-5-fluoropyrrolo[2,3-d]-
pyrimidine 15 provided protected nucleoside 16 in 43% yield
(in two steps from 9). Acidic deprotection afforded nucleo-
side 14 in 85% yield. Aqueous Suzuki reaction of 6-chloro-
7-fluoro-7-deazapurine riboside 14 with TIPS-protected pyr-
role-3-boronic acids gave the desired nucleoside 4o in 73%
yield.
Synthesis of compounds in the 7-chloro-7-deazapurine
series consisted of palladium-catalyzed regioselective cross-
coupling reactions on per-O-benzoyl-6,7-dichloro-7-deaza-
purine riboside 1731 (Scheme 6) to provide benzoylated 6-het-
aryl(aryl) products 18d,h,i,m,n. The cross-coupling reactions
proceeded selectively at position 6 leaving the chlorine in
position 7 intact. Deprotection of intermediates 18 with
methanolic sodium methoxide afforded free ribonucleosides

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 463
Scheme 6a
Scheme 4a
a Conditions: (A) M = B(OH)2:Pd(PPh3)4, K2CO3, toluene, 100°C;
(B) M = SnBu3:PdCl2(PPh3)2, DMF, 100 °C.
5-member N-heterocycles (pyrrole, pyrazole, or imidazole;
3k, 3l, 3q, and 3r) with the exception of 3o (pyrrol-3-yl)
showed further reduced potency with some of them being
inactive even at 10 μM concentration. A number of potent
compounds were also identified among analogues further
modified with 7-halogen atom; active compounds were iden-
tified both among 7-fluoro and 7-chloro derivatives. For
example, compounds 4h and 4i exhibited similar cytostatic
potency as the corresponding compounds without 7-substi-
tution (3h and 3i). In general, analogues with 7-fluoro substi-
tution tended to be more active than corresponding com-
pounds bearing 7-chloro modifications. Notably, examples
of compounds were identified that demonstrated a pro-
foundly negative effect of 7-chloro on the cytostatic potency
(e.g., 5h).
a Conditions: (A) M = B(OH)2:Pd(PPh3)4, K2CO3, toluene, 100°C;
(B) M = SnBu3:PdCl2(PPh3)2, DMF, 100°C; (C) M = ZnX or AlEt2:
Pd(PPh3)4, THF, 70 °C.
Scheme 5a
The synthesized nucleosides were also tested for their
cytotoxic activity in human T-lymphoid (CCRF-CEM),
promyelocytic leukemia (HL60), and cervical carcinoma
(HeLa) cell lines using and XTT-based cell viability assay33
following a shorter 72 h incubation. The potency of most
tested compounds in the XTT-based assays was lower com-
pared to the SRB-based cytostatic screening assays. In
general, the compound potency ranking followed similar
trends as those observed in the SRB assays, but the differ-
ences among individual nucleosides were substantially less
pronounced compared to what has been observed in the SRB
assay. Shorter duration of incubation during the XTT-based
assay is likely the main reason for the lower activity and
narrower potency differentiation observed in this assay,
suggesting a delayed cytostatic and/or cytotoxic effect of
these compounds.
Five nucleoside analogues (3h, 3i, 3m, 4h, and 4i) that were
among the most potent compounds in the initial screening
were further characterized against additional solid tumor cell
lines and compared with the activity of two established
anticancer nucleosides gemcitabine and clofarabine. Table
2 summarizes results from an independent set of tests con-
ducted for all compounds in parallel against total of eight
a Conditions: (a) KOH, TDA-1, toluene (43% from 9); (b) 90% TFA
(aq) (85%); (c) Pd(OAc)2, TPPTS, Na2CO3, H2O-MeCN (73%).
analogues substituted with furan-3-yl (3m) and thiophen-
3-yl (3n) were slightly less active, exhibiting GIC50 values of
approximately 30-700 nM. Most nucleosides containing

464 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Nauꢀs et al.
Table 1. Cytostatic Activity (GIC50) and Cytotoxic Activity (IC50) of 6-(Het)aryl-7-deaza-purine Nucleosides
GIC50 (μM)a
IC50 (μM)a
compd
A549
Du145
HCT116
Hs578
HL-60
CCRF-CEM
HeLa S3
3a
3b
3c
3d
3e
3f
4.4
0.25
1.2
0.030
>10
1.1
2.6
0.45
>10
>10
>10
>10
>10
0.078
0.049
0.48
>10
3.7
4.7
0.053
5.6
2.2
1.25
0.45
3.8
3.6
0.35
>7.5
>40
>10
2.9
>10
>10
>10
ndb
>10
>10
>10
>10
>10
>10
4.0
1.3
0.73
>10
52.7
>10
0.81
1.2
10.9
23.2
>10
0.31
0.29
0.39
10.0
0.71
1.9
2.5
>10
0.049
3g
3h
3i
>10
0.007
0.009
0.019
0.63
>10
0.77
8.8
0.088
0.045
0.41
0.10
0.29
1.3
3j
1.2
0.13
nd
3.3
3k
3l
>10
n.d.
n.d.
1.5
2.6
0.094
0.036
0.032
0.030
0.028
0.41
1.9
3m
3n
3o
3p
3q
3r
3s
0.073
0.63
0.36
0.23
3.3
0.092
0.22
0.50
0.34
7.3
0.26
0.71
0.44
0.29
3.9
4.6
>40
1.2
2.8
4.0
n.d.
0.26
7.4
0.28
1.5
10.1
nd
0.91
1.0
1.1
4.1
6.2
4.9
0.075
0.39
5.7
3.1
>10
>10
>40
1.5
n.d.
4d
4h
4i
>10
0.58
>10
0.039
0.009
1.3
>10
0.18
>40
1.2
4.3
1.0
3.3
n.d.
2.0
4.9
1.9
3.1
1.4
2.6
8.0
0.11
0.061
0.90
0.19
0.005
0.009
0.024
0.009
0.89
0.13
0.57
0.31
2.1
0.27
0.55
0.41
1.8
4.3
3.1
4j
4k
4l
0.45
>10
0.018
0.14
0.25
0.23
12.2
0.17
0.21
8.9
>10
4m
4n
4o
0.060
0.38
0.13
0.005
0.066
0.010
0.18
0.81
0.36
1.0
0.56
0.93
5d
5h
5i
2.5
0.14
>10
1.4
>10
0.27
2.0
>10
>10
1.6
1.2
21.6
5.5
0.91
3.8
2.7
1.1
1.0
0.42
>40
28.2
>10
0.38
1.1
0.013
0.054
0.018
5m
5n
1.6
0.11
2.5
1.6
2.7
0.34
0.38
0.10
a Cytostatic and cytotoxic activities were determined by SRB (GIC50) and XTT (IC50) assay following a 5-day and 3-day incubation with tested
compounds, respectively. Values represent means from 2 to 4 independent experiments. b nd, not determined.
Table 2. Cytostatic Activity of Selected 6-(Het)aryl-7-deaza-purine Nucleosides in Comparison with Clofarabine and Gemcitabine
GIC50 (μM)a
lung
NCI H23
prostate
Du145
colon
breast
compd
A549
PC3
HCT116
HCT15
HS578
BT549
geometric mean
3h
3i
0.033
0.11
0.13
0.43
0.28
4.3
0.007
0.009
0.036
0.005
0.009
0.003
0.125
0.014
0.033
0.096
0.075
0.090
0.006
0.063
0.006
0.007
0.026
0.039
0.020
0.002
0.106
0.020
0.078
0.086
0.057
0.018
0.003
0.180
0.009
0.024
0.051
0.18
0.012
0.023
0.035
0.11
0.016
0.038
0.062
0.096
0.081
0.003
0.12
3m
4h
0.055
0.11
0.38
4i
1.5
0.002
0.040
0.14
0.001
1.241
0.081
0.004
0.065
gemcitabine
clofarabine
0.007
0.086
a Cytostatic activity was determined by SRB assay following a 5-day incubation with tested compounds in parallel. Values represent means from three
independent experiments.
human solid tumor cell lines. Results of these assays indicate
that particularly the compounds 3h and 3i retain their potent
cytostatic effect against all the tested cell lines. Although
being less potent than gemcitabine, all five explored com-
pounds showed comparable or better cytostatic effects than
clofarabine either in the individual assays or as a composite
geometric mean of all GIC50 values (Table 2).
Intracellular Metabolism. Similar to other nucleoside ana-
logues, the 6-hetaryl-7-deaza purines are likely to undergo
intracellular phosphorylation. Compounds 3h and 5h were
selected to explore their intracellular metabolism; although
Table 3. Intracellular Metabolism of Compounds 3h and 5h in Du145
Prostate Cancer Cells
intracellular concentrationa [pmol/million cells]
6 h
24 h
compd nucleoside NMP
NTP
nucleoside NMP
NTP
3h
5h
950
20.1
4124
164
88.4
19.2
207
45.0
6842
332
109
21.2
a Intracellular levels of parent compound (nucleoside), monopho-
sphate (NMP), and triphosphate (NTP) were determined following 6
and 24 h incubation of cells with 10 μM compounds.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 465
Table 4. Summary of Cell Cycle, Apoptosis, Mitosis (pH3þ), RNA (BrUþ), and DNA (BrdUþ) Synthesis Analysis in CCRF-CEM Cells Treated with
Selected Nucleosides at GIC50 for 24 ha
% of total cell populations
compd
sub-G1
G1
S
G2/M
pH3Ser10
þ
BrdUþ
BrUþ
control
3h 1 ꢀ GIC50
10.6 ( 1.9
16.5 ( 7.2
60.2 ( 8.3
13.7 ( 3.8
55 ( 9.3
17.1 ( 7.2
43.4 ( 10.8
39.8 ( 8.9
61.2 ( 10.3
24.1 ( 1.2
54.0 ( 2.4
48.0 ( 4.0
61.5 ( 3.4
51.9 ( 4.1
44.9 ( 5.7
46.7 ( 6.7
45.5 ( 7.5
47.0 ( 7.7
43.9 ( 7.2
40.4 ( 4.9
55.6 ( 4.9
54.4 ( 8.4
27.1 ( 3.8
67.1 ( 5.2
73.2 ( 3.6
61.6 ( 6.6
36.7 ( 1.6
42.2 ( 7.1
44.6 ( 4.1
42.2 ( 7.7
47.0 ( 4.4
46.7 ( 9.8
50.5 ( 4.3
37.3 ( 3.9
39.7 ( 6.9
72.3 ( 4.2
30.5 ( 5.6
24.9 ( 3.5
30.8 ( 5.2
9.8 ( 2.5
12.9 ( 4.8
6.8 ( 2.6
12.2 ( 7.5
6.0 ( 3.5
9.3 ( 6.4
9.1 ( 6.2
7.1 ( 1.6
5.9 ( 2.1
0.6 ( 0.6
2.3 ( 0.7
1.9 ( 1.2
7.6 ( 3.6
1.96 ( 0.32
1.21 ( 0.07
0.48 ( 0.44
0.87 ( 0.33
0.44 ( 0.25
0.85 ( 0.48
0.47 ( 0.43
0.84 ( 0.29
0.39 ( 0.17
0.78 ( 0.59
0.53 ( 0.43
0.85 ( 0.71
0.44 ( 0.39
63.9 ( 4.8
54.3 ( 9.2
31.3 ( 2.2
55.5 ( 6.8
38.3 ( 9.4
56.2 ( 10.9
47 ( 12.12
44.6 ( 10.5
28.9 ( 7.5
56.7 ( 10.7
9.5 ( 6.6
51.8 ( 1.1
28.7 ( 4.3
4.7 ( 1.5
28.5 ( 5.2
3.5 ( 2.4
28.4 ( 6.8
5.7 ( 1.8
14.0 ( 7.3
7.2 ( 1.0
54.7 ( 7.2
44.8 ( 2.2
46.2 ( 1.8
41.3 ( 4.1
3h 5 ꢀ GIC50
3i 1 ꢀ GIC50
3i 5 ꢀ GIC50
4i 1 ꢀ GIC50
4i 5 ꢀ GIC50
3m 1 ꢀ GIC50
3m 5 ꢀ GIC50
gemcitabine 1 ꢀ GIC50
gemcitabine 5 ꢀ GIC50
cladribine 1 ꢀ GIC50
cladribine 5 ꢀ GIC50
32.1 ( 8.4
13.0 ( 3.6
a Data are expressed as a percentage of total cellular population plus/minus standard deviation.
Figure 1. Specificity of RNA synthesis inhibition (BrUþ cells) in CCRF-CEM cells treated with compound 3h in comparison with cladribine
at 5ꢀ GIC50
.
they differ only in 7-substitution (7-H and 7-Cl, respectively),
there is a striking difference in their cytostatic activity
(Table 1). DU145 cells are among the most sensitive to 3h
but show no response to 5h. Du145 cells were treated with the
two compounds for 6 and 24 h and cellular extracts were
analyzed using high-performance liquid chromatography

466 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Nauꢀs et al.
coupled to tandem mass spectrometery (LC/MS/MS). Re-
sults demonstrated the intracellular phosphorylation of both
compounds as evidenced by the presence of increasing con-
centrations of monophosphates (MP) and triphophates (TP)
in cell extracts over time (Table 3). Formation of dipho-
sphate was not followed due to the lack of chemical standard
required for the quantitative LC/MS/MS analysis. Intracel-
lular levels of MP and TP of 3h were approximately 20-fold
and 5-fold higher, respectively, compared to those of 5h.
Although we have not formally established the role of
intracellular metabolites in the cytostatic activity of these
novel nucleosides, our data support a notion that, similar to
many other cytostatic nucleosides, these compounds or one
of their phosphorylated metabolites may act directly through
incorporation of their TP by RNA polymerases, inhibiting
the elongation of cellular RNAs and/or indirectly through
the modulation of intracellular nucleotide pools.
Inhibition of RNA/DNA Synthesis and Cell Cycle Analysis.
To better understand molecular mechanism of the cytostatic
effect of the prepared nucleosides, we have performed the
analysis of cell cycle and DNA and RNA synthesis in CCRF-
CEM T-lymphoblastic leukemia cells treated for 24 h with
selected active compounds at their 1ꢀ and 5ꢀ GIC50. Con-
sistently with data from intracellular metabolism showing
formation of intracellular TP metabolites (Table 3), the
6-hetaryl-7-deaza purines exhibited a dose-dependent inhi-
bition of RNA synthesis measured via the incorporation of
bromouridine into the total pool of cellular RNA (Table 4,
Figure 1). Specifically, the treatment of cells with concentra-
tions corresponding to 1ꢀ and 5ꢀ GIC50 resulted in approxi-
mately 50 and 90% inhibition of total RNA synthesis,
respectively. In contrast, the effect of nucleosides on total
DNA synthesis and the DNA content in specific cell cycle
phases was much less pronounced and likely nonspecific.
Notably, profound inhibition of cellular RNA synthesis was
accompanied by a decrease of mitotic phospho-histone H3
positive cells and rapid onset of apoptosis as evidenced by the
high proportion of sub-G1 cells (Table 4, Figure 1).
cellular RNA polymerases) have been detected in treated cells.
Additional studies of the mechanism as well as in vivo cyto-
static activity and pharmacokinetic testing are under way and
will be reported in due course. The title 6-hetaryl-7-deaza-
purine ribonucleosides represent a new promising lead class
for further development as potential cytostatics.
Experimental Section
General. NMR spectra were recorded on a 400 MHz (1H at
400 MHz, 13C at 100.6 MHz), a 500 MHz (500 MHz for 1H and
1
125.7 MHz for 13C), or a 600 MHz (600 MHz for H and 151
MHz for 13C) spectrometer. Melting points were determined on
a Kofler block and are uncorrected. Optical rotations were
measured at 25 °C, [R]D values are given in 10-1 deg cm2 g-1
.
The high resolution mass spectra were measured using electro-
spray ionization. High performance flash chromatography
(HPFC) purifications were performed on C-18 columns using
water-methanol gradient. The purity of final compounds
(>95%) was confirmed by elemental analyses and clean
NMR spectra. Only selected most important procedures and
compound characterizations are given here, for complete ex-
perimental part including full characterization of all compounds
including assigment of NMR signals; see Supporting Informa-
tion.
4-Chloro-7-(2,3-O-isopropylidene-5-O-tert-butyldimethylsilyl-
β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (8). Tris(dime-
thylamino)phosphine (9.7 mL, 53.5 mmol) was added dropwise
to a stirred solution of 2,3-O-isopropylidene-5-O-tert-butyldi-
methylsilyl-β-D-ribofuranose 9 (12.5 g, 41 mmol) and carbon
tetrachloride (6.4 mL, 61.5 mmol) in toluene (70 mL) over
35 min at -30 °C. The temperature of the reaction mixture
was raised to 0 °C within 1 h. The cloudy toluene solution of
halogenose 7 was then quickly washed with ice-cold brine
(70 mL), dried over MgSO4, and added to a vigorously stirred
suspension of 4-chloropyrrolo[2,3-d]pyrimidine 6 (4.16 g, 27.1
mmol), powdered KOH (3.4 g, 60.7 mmol), and TDA-1 (4.3 mL,
13.4 mmol) in toluene (70 mL). The mixture was stirred for 24 h
and then aqueous NH4Cl (sat, 250 mL) was added and the
mixture extracted with chloroform (400 mL, then 2 ꢀ 75 mL).
The combined organic extracts were dried over MgSO4, evapo-
rated, and the residue chromatographed on silica (hexanes-
AcOEt, 22:1) to afford 8 (7.57 g, 63%) as a colorless oil. It
should be noted that the yield of 8 was variable and ranged from
39% to 63%. Handling of preprepared solution of unstable
halogenose 7 should be as quick as possible. Analytical data of
compound 8 are in agreement with published data.25
4-Ethyl-7-(2,3-O-isopropylidene-5-O-tert-butyldimethylsilyl-
β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (10a). An argon
purged mixture of compound 8 (200 mg, 0.454 mmol), triethy-
laluminum (1 M in hexanes, 910 μL 0.91 mmol), and Pd(PPh3)4
(26 mg, 0.022 mmol) in THF (5 mL) was stirred at 70 °C for 20 h.
The mixture was diluted with hexane (30 mL) and washed with
aqueous NH4Cl (sat, 10 mL). The aqueous phase was re-
extracted with hexane (2 ꢀ 10 mL). The combined organic
extracts were dried over MgSO4, concentrated in vacuo, and
the residue chromatographed on silica (hexanes-AcOEt, 10:1
f 6:1) to afford 10a (162 mg, 82%) as a colorless oil. 1H NMR
(600 MHz, CDCl3): 0.046 and 0.053 (2 ꢀ s, 2 ꢀ 3H, CH3Si), 0.90
(s, 9H, (CH3)3C), 1.39 (q, 3H, J=0.6, (CH3)2C), 1.393 (t, 3H,
Antiviral Activity. All synthesized compounds were tested
in Huh-7 cells harboring subgenomic reporter replicon de-
rived from HCV subtype 1B. Although a number of tested
compounds inhibited the replicon reporter signal, the activ-
ity largely correlated with cytostatic effect in several different
cell lines, suggesting that the activity detected in HCV
replicon are likely a consequence of interference with host
target(s). This conclusion is supported by observation that
TP metabolites of the nucleosides active in HCV replicon do
not inhibit the enzymatic activity of HCV RNA polymerase
(data not shown).
Conclusions
6-Hetaryl-7-deazapurine and -7-fluoro-7-deazapurine ribo-
nucleosides have been found to possess cytostatic effects at
low nanomolar concentrations with the potency comparable
to clofarabine. On the other hand, the corresponding 6-alkyl-
and 6-phenyl-7-deazapurine and 6-hetaryl-7-chloro-7-deaza-
purine ribonucleosides were much less active. The highest
activities were observed with 6-furyl and 6-thienyl derivatives,
while the derivatives bearing N-heterocycles were less potent.
The mechanism by which these compounds exert their cyto-
static effect has not been fully elucidated yet, but intracellular
triphosphate metabolites together with a potent inhibition of
RNA synthesis (potentially through the direct inhibition of
J
J
vic=7.7, CH3CH2), 1.65 (q, 3H, J=0.6, (CH3)2C), 3.04 (q, 2H,
0
0
vic=7.7, CH2CH3), 3.79 (dd, 1H, Jgem=11.2, J5 b,4 =4.0, H-
0
50b), 3.87 (dd, 1H, Jgem=11.2, J5 a,4 =3.8, H-5 a), 4.33 (m, 1H,
0
0
0
0
0
0
0
0
0
0
0
4 ,5 =4.0, 3.8, J4 ,3 =3.1, J4 ,2 =0.4, H-4 ), 4.98 (ddd, 1H, J3 ,2
0
J
6.3, J3 ,4 =3.1, J3 ,1 =0.5, H-3 ), 5.13 (ddd, 1H, J2 ,3 =6.3, J2 ,1
=
=
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3.1, J2 ,4 =0.4, H-2 ), 6.41 (d, 1H, J1 ,2 =3.1, H-1 ), 6.58 (d, 1H,
C
J
5,6=3.7, H-5), 7.43 (d, 1H, J6,5=3.7, H-6), 8.81 (s, 1H, H-2). 13
NMR (151 MHz, CDCl3): -5.50, -5.40, 12.87, 18.37, 25.47,
25.90, 27.34, 28.61, 63.37, 80.94, 84.80, 85.96, 90.17, 100.09,
114.11, 117.70, 125.60, 150.39, 151.64, 164.25. MS ESI, m/z:

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 467
434[M þ H]. HR MS (ESI): calcd for C22H36N3O4Si [M þ H]
434.2475, found 434.2468.
(d, 1H, J6,5=3.7, H-6), 7.72 (dd, 1H, J5,4=1.7, J5,3=0.8, H-5-
furyl), 8.87 (s, 1H, H-2). 13C NMR (151 MHz, CDCl3): -5.50,
-5.38, 18.38, 25.45, 25.90, 27.33, 63.36, 80.85, 84.92, 85.94,
90.04, 102.11, 112.36, 112.97, 113.55, 114.13, 126.80, 145.11,
147.12, 151.41, 151.82, 152.95. MS FAB, m/z (rel %): 73 (100),
186 (20), 472 (45) [M þ H]. HR MS (FAB): calcd for
C24H34N3O5Si [M þ H] 472.2268, found 472.2274.
4-Benzyl-7-(2,3-O-isopropylidene-5-O-tert-butyldimethylsilyl-
β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (10c). An argon
purged mixture of compound 8 (191 mg, 0.43 mmol), benzylzinc
bromide (0.5 M in THF, 1.75 mL, 0.875 mmol), and Pd(PPh3)4
(25 mg, 0.022 mmol) in THF (5 mL) was stirred at 70 °C for 24 h.
The mixture was diluted with hexane (25 mL) and washed with
aqueous NH4Cl (satd, 10 mL). The aqueous phase was re-
extracted with hexane (2 ꢀ 10 mL) and the combined organic
extracts dried over MgSO4, concentrated in vacuo, and the
residue chromatographed on silica (hexanes-AcOEt, 6:1) to
afford 10c (201 mg, 93%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): 0.02 and 0.04 (2 ꢀ s, 2 ꢀ 3H, CH3Si), 0.88 (s, 9H,
(CH3)3C), 1.38 (q, 3H, J=0.6, (CH3)2C), 1.64 (q, 03H, J=0.6,
4-Ethyl-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (3a).
Compound 10a (149 mg, 0.34 mmol) was treated with aqueous
TFA (90% v/v, 0.5 mL) for 1 h at RT. The volatiles were
removed in vacuo, and the residue was several times coevapo-
rated with MeOH. Chromatography on silica (3.5% MeOH in
CHCl3) afforded compound 3a (100 mg, 99%) as a colorless
glassy solid. After reverse phase chromatography, compound 3a
crystallized from water/MeOH as colorless needles; mp 120-
121 °C; [R]D -58.8 (c 0.473, DMSO). UV (MeOH): λmax (ε) 271
0
0
(CH3)2C), 3.77 (dd, 1H, Jgem=11.2, J5 b,4 =4.0, H-5 b), 3.86 (dd,
0
1
0
0
0
0
1H, Jgem=11.2, J5 a,4 =3.8, H-5 a), 4.31 (q, 1H, J4 ,5 =4.0, 3.8,
(4407), 223 (25061). H NMR (600 MHz, DMSO-d6): 1.30 (t,
0
0
0
0
0
J
4 ,3 =3.1, H-4 ), 4.35 (s, 20H, CH2Ph), 4.96 (ddd, 1H, J3 ,2 =6.3,
3H, Jvic=7.6, CH3CH2), 2.99 (q, 2H, Jvic=7.6, CH2CH3), 3.54
0
0
0
0
0
0
0
0
0
J
3 ,4 =3.1, J3 ,1 =0.4, H-3 ), 5.10 (dd, 1H, J2 ,3 =6.3, J2 ,1 =3.1,
0
0
(ddd, 1H, Jgem=11.9, J5 b,OH=5.8, J5 b,4 =4.0, H-5 b),; 3.63
0
H-20), 6.39 (d, 1H, J1 ,2 =3.1, H-1 ), 6.43 (d, 1H, J5,6=3.7, H-5),
7.21 (m, 1H, H-p-Ph), 7.25-7.33 (m, 4H, H-o,m-Ph),; 7.39 (d,
1H, J6,5=3.7, H-6), 8.83 (s, 1H, H-2). 13C NMR (100.6 MHz,
CDCl3): -5.50, -5.40, 18.37, 25.47, 25.90, 27.34, 42.27, 63.38,
80.96, 84.79, 85.99, 90.21, 100.37, 114.15, 118.28, 126.00, 126.60,
128.57, 129.07, 138.11, 150.81, 151.65,161.14. MS FAB, m/z (rel
%): 73 (100), 210 (30), 292 (10), 496 (95) [M þ H]. HR MS
(FAB): calcd for C27H38N3O4Si [M þ H] 496.2632, found
496.2636.
0
0
0
0
0
(ddd, 1H, Jgem=11.9, J5 a,OH=5.3, J5 a,4 =4.0, H-5 a), 3.91 (q,
0
1H, J4 ,5 =4.0, J4 ,3 =3.3, H-4 ), 4.11 (td, 1H, J3 ,2 =5.1, J3,OH
0
0
0
0
0
0
0
0
0
=
=
0
0
0
0
0
0
0
0
4.8, J3 ,4 =3.3, H-3 ), 4.43 (td, 1H, J2 ,OH=6.5, J2 ,1 =6.3, J2 ,3
0
5.1, H-20), 5.13 (t, 1H, JOH,5 =5.8, 5.3, OH-5 ), 5.19 (d, 1H, JOH,3
0
=4.8, OH-30), 5.35 (d, 1H, JOH,2 =6.5, OH-2 ), 6.18 (d, 1H, J1 ,2
=6.3, H-10), 6.77 (dd, 1H, J5,6=3.7, J5,2=0.4, H-5), 7.78 (d, 1H,
J6,5=3.7, H-6), 8.69 (s, 1H, H-2). 13C NMR (151 MHz, DMSO-
d6): 12.93, 27.97, 61.87, 70.87, 74.18, 85.38, 87.02, 100.09,
117.38, 126.78, 150.73, 151.15, 163.77. MS FAB, m/z (rel %):
149 (45), 280 (100) [M þ H]. HR MS (FAB): calcd for
C13H18N3O4 [M þ H] 280.1297, found 280.1293. Anal.
0
0
0
0
7-(2,3-O-Isopropylidene-5-O-tert-butyldimethylsilyl-β-D-ribo-
furanosyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (10d). An argon
purged mixture of compound 8 (309 mg, 0.7 mmol), phenyl-
boronic acid (128 mg, 1.05 mmol), K2CO3 (193 mg, 1.4 mmol),
and Pd(PPh3)4 (41 mg, 0.035 mmol) in toluene (5 mL) was stirred
at 100 °C for 5 h. The mixture was diluted with chloroform
(20 mL) and washed with aqueous NH4Cl (sat., 20 mL). The
aqueous phase was re-extracted with chloroform (2 ꢀ 5 mL) and
the combined organic extracts dried over MgSO4, concentrated
in vacuo, and the residue chromatographed on silica (hexanes-
AcOEt, 10:1f 7:1) to afford 10d (320 mg, 95%) as a colorless oil.
1H NMR (600 MHz, CDCl3): 0.06 and 0.07 (2 ꢀ s, 2 ꢀ 3H,
(C13H17N3O4 1/2H2O): C, H, N.
3
4-(Furan-2-yl)-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine (3h). Deprotection of 10h as described for 3a gave com-
pound 3h as colorless needles after crystallization from 2-pro-
panol (no silica column); yield 63%; mp 162-164 °C; [R]D -72.0
(c 0.261, DMSO). UV (MeOH): λmax (ε) 326 (15338), 234
1
(19556). H NMR (400 MHz, DMSO-d6): 3.57 and 3.66 (2 ꢀ
0
0
0
0
dd, 2H, Jgem=11.9, J5 ,4 =4.0, H-5 ), 3.94 (q, 1H, J4 ,5 =4.0,
0
0
0
0
4 ,3 =3.3, H-4 ), 4.13 (dd, 1H, J3 ,2 =5.1, J3 ,4 =3.3, H-3 ), 4.45
0
0
0
0
0
0
J
(dd, 1H, J2 ,1 =6.2, J2 ,3 =5.1, H-2 ), 6.25 (d, 1H, J1 ,2 =6.2, H-
0
0
0
0
0
0
10), 6.80 (dd, 1H, J4,3=3.5, J4,5=1.7, H-4-furyl), 7.08 (d, 1H,
CH3Si), 0.90 (s, 9H, (CH3)3C), 1.40 and 1.67 (2 ꢀ q, 2 ꢀ 3H, J=
0
0
0.6, (CH3)2C), 3.82 (dd, 1H, Jgem=11.2, J5 b,4 =3.8, H-5 b), 3.91
0
J
5,6=3.7, H-5), 7.50 (dd, 1H, J3,4=3.5, J3,5=0.7, H-3-furyl), 7.95
0
0
(dd, 1H, Jgem=11.2, J5 a,4 =3.7, H-5 a), 4.36(ddd, 1H, J4 ,5 =3.8,
0
0
0
(d, 1H, J6,5=3.7, H-6), 8.07 (dd, 1H, J5,4=1.7, J5,3=0.7, H-5-
furyl), 8.78 (s, 1H, H-2). 13C NMR (100.6 MHz, DMSO-d6):
61.74, 70.76, 74.24, 85.40, 86.88, 101.41, 112.79, 112.89, 113.62,
0
0
0
0
0
0
0
0
0
3.7, J4 ,3 =3.5, H-4 ), 5.00 (ddd, 1H, J3 ,2 =6.3, J3 ,4 =3.5, J3 ,1
0
=
0.4, H-30), 5.150(dd, 1H, J2 ,3 =6.3, J2 ,1 =3.1, H-2 ), 6.49 (d, 1H,
0
0
0
0
0 0
1 ,2 =3.1, H-1 ), 6.84 (d, 1H, J5,6=3.8, H-5), 7.51 (m, 1H, H-p-
J
128.32, 146.36, 146.60, 151.00, 152.24, 152.43. IR (KBr): ν =
1675, 1601, 1564, 1462, 1353, 1237, 1207, 1188, 1099, 1051, 1016
cm-1. MS FAB, m/z (rel %): 318 (100) [M þ H]. HR MS (FAB):
calcd for C15H16N3O5 [M þ H] 318.1090, found 318.1089. Anal.
Ph), 7.55 (m, 2H, H-m-Ph), 7.56 (d, 1H, J6,5=3.8, H-6), 8.09 (m,
2H, H-o-Ph), 8.98 (s, 1H, H-2). 13C NMR (151 MHz, CDCl3):
-5.49, -5.37, 18.39, 25.49,; 25.91, 27.37, 63.40, 80.92, 84.95,
86.03, 90.23, 101.44, 114.16, 116.43, 126.75, 128.77, 128.82,
130.02, 138.06, 151.65, 151.71, 157.63. MS FAB, m/z (rel %):
73 (100), 196 (40), 482 (90) [M þ H]. HR MS (FAB): calcd for
C26H36N3O4Si [M þ H] 482.2475, found 482.2479.
(C15H15N3O5 1/2H2O): C, H, N.
3
4-(Furan-3-yl)-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine (3m). An argon purged mixture of compound 11 (226 mg,
0.79 mmol), furane-3-boronic acid (111 mg, 0.99 mmol),
Na2CO3 (251 mg, 2.37 mmol), Pd(OAc)2 (9 mg, 0.04 mmol),
and TPPTS (57 mg, 0.1 mmol) in water/MeCN (2:1, 3 mL) was
stirred at 100 °C for 3 h. After cooling, the mixture was
neutralized by the addition of aqueous HCl (3M), volatiles were
removed in vacuo, and the residue chromatographed on silica
(3.5% MeOH in CHCl3) to afford 3m (172 mg, 69%) as a
yellowish solid. Compound 3m was recrystallized from 2-pro-
panol to provide a white microcrystalline solid; mp 142-143 °C;
[R]D -64.3 (c 0.524, DMSO). UV (MeOH): λmax (ε) 305 (8343),
4-(Furan-2-yl)-7-(2,3-O-isopropylidene-5-O-tert-butyldimethyl-
silyl-β-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (10h). An
argon purged mixture of compound 8 (294 mg, 0.67 mmol),
2-(tributylstannyl)furane (252 μL, 0.80 mmol), and PdCl2-
(PPh3)2 (24 mg, 0.03 mmol) in DMF (3 mL) was stirred at 100
°C for 2 h. Volatiles were removed in vacuo and the residue
coevaporated with toluene several times. Silica gel chromatog-
raphy of the residue (hexanes-AcOEt, 20:1 f 10:1) afforded
product 10h (293 mg, 93%) as a colorless foam. 1H NMR (600
MHz, CDCl3): 0.069 and 0.074 (2 ꢀ s, 2 ꢀ 3H, CH3Si), 0.91 (s,
1
214 (25297). H NMR (500 MHz, DMSO-d6): 3.57 (ddd, 1H,
0
0
gem =11.9, J5 b,OH =5.8, J5 b,4 =4.0, H-5 b), 3.66 (ddd, 1H,
0
0
9H, (CH3)3C), 1.40 and 1.67 (2 ꢀ q, 2 ꢀ 3H, J=0.6, (CH3)2C),
J
J
0
0
0
3.81 (dd, 1H, Jgem=11.2, J5 b,4 =3.7, H-5 b), 3.90 (dd, 1H, Jgem
0
0
gem=11.9, J5 a,OH=5.3, J5 b,4 =4.0, H-5 a), 3.94 (td, 1H, J4 ,5
0
4.0, J4 ,3 =3.4, H-4 ), 4.14 (ddd, 1H, J3 ,2 =5.1, J3 ,OH =4.9,
0
0
0
0
=
=
=
0
0
0
0
0
0
0
0
0
0
0
0
11.2, J5 a,4 =3.5, H-5 a), 4.36 (ddd, 1H, J4 ,5 =3.7, 3.5, J4 ,3
0
3.1, H-40), 4.99 (ddd, 1H, J3 ,2 =6.3, J3 ,4 =3.1, J3 ,1 =0.4, H-3 ),
J
3 ,4 =3.4, H-3 ), 4.45 (ddd, 1H, J2 ,OH=6.3, J2 ,1 =6.1, J2 ,3
0
=
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5.12 (dd, 1H, J2 ,3 =6.3, J2 ,1 =3.1, H-2 ), 6.47 (d, 1H, J1 ,2 =3.1,
H-10), 6.64 (dd, 1H, J4,3=3.5, J4,5=1.7, H-4-furyl), 7.05 (d, 1H,
5.1, H-20), 5.09 (dd, 1H, JOH,5 =5.8, 5.3, OH-5 ), 5.018 (d, 1H,
0
0
0
0
0
0
0
0
0
0
J
1H, J1 ,2 =6.1, H-1 ), 7.10 (d, 1H, J5,6=3.8, H-5), 7.26 (dd, 1H,
OH,3 =4.9, OH-3 0), 5.37 (d, 1H, JOH,2 =6.3, OH-2 ), 6.24 (d,
0
0
J5,6=3.7, H-5), 7.41 (dd, 1H, J3,4=3.5, J3,5=0.8, H-3-furyl), 7.56

468 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Nauꢀs et al.
J4,5=1.9, J4,2=0.8, H-4-furyl), 7.90 (dd, 1H, J5,4=1.9, J5,2=1.5,
H-5-furyl), 7.92 (d, 1H, J6,5=3.8, H-6),; 8.74 (dd, 1H, J2,5=1.5,
J
(d, JC,F=1), 150.54 (d, JC,F=4), 151.66. 19F NMR (470.3 MHz,
CDCl3, ref (C6F6)=-163 ppm): -168.82. MS ESI, m/z: 458
[M þ H]. HR MS (ESI): calcd for C20H30ClFN3O4Si [M þ H]
458.1678, found 458.1669.
2,4=0.8, H-2-furyl),; 8.78 (s, 1H, H-2). 13C NMR (125.7 MHz,
DMSO-d6): 61.73, 70.73, 74.20, 85.32, 86.92, 100.86, 109.55,
114.65, 125.19, 127.77, 144.74, 145.01, 150.15, 151.12, 151.73.
MS FAB, m/z (rel %): 73 (100), 217 (45), 318 (55) [M þ H]. HR
MS (FAB): calcd for C15H16N3O5 [M þ H] 318.1090, found
318.1086. Anal. (C15H15N3O5 0.1C3H8O 0.2H2O): C, H, N.
4-Chloro-5-fluoro-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyri-
midine (14). Compound 16 was deprotected as described for 3a
to afford 14 as a white foam after chromatography on silica (4%
MeOH in CHCl3); yield 85%. 1H NMR (600 MHz, DMSO-d6):
3
3
0
0
3.56 (ddd, 1H, Jgem=12.0, J5 b,OH=5.4, J5 b,4 =3.9, H-5 b), 3.64
0
0
5-Fluoro-4-(furan-2-yl)-7-(2,3,5-tri-O-benzoyl-β-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidine (13h). Compound 13h was pre-
pared as described for 10h by coupling compound 12 and
2-(tributylstannyl)furane. A yellowish foam was obtained after
chromatography on silica (hexanes-AcOEt, 20:1 f 6:1); yield
99%. 1H NMR0(600 MHz, CDCl3): 4.69 (dd, 1H, Jgem=12.2,
0
0
0
0
(ddd, 1H, Jgem=12.0, J5 a,OH=5.4,0J5 a,4 =4.0, H-5 a), 3.93 (ddd,
0
1H, J4 ,5 =4.0, 3.9, J4 ,3 =3.3, H-4 ), 4.10 (td, 1H, J3 ,2 =J3,OH
0
0
0
0
0
=
5.0, J3 ,4 =3.3, H-3 ), 4.33 (ddd, 1H, J2 ,OH =6.2, J2 ,1 =5.9,
0
0
0
0
0
0
0
0
0
0
2 ,3 =5.0, H-2 ), 5.09 (t, 1H, JOH,5 =5.4, OH-5 ), 5.22 (d, 1H,
OH,3 =5.0, OH-3 ), 5.44 (d, 1H, JOH,2 =6.2, OH-2 ), 6.25 (dd,
0
J
J
0
0
0
0
J
5 b,4 =3.7, H-5 b), 4.80 (ddd, 1H, J4 ,3 =4.1, J4 ,5 =3.7, 3.1, H-
1H, J1 ,2 =5.9, JH,F=1.9, H-10), 8.02 (d, 1H, JH,F=2.0, H-6),;
8.70 (s, 1H, H-2). 13C NMR (151 MHz, DMSO-d6): 61.48, 70.55,
74.53, 85.66, 86.98, 106.55 (d, JC,F=14), 111.42 (d, JC,F=27),
140.45 (d, JC,F=249), 146.97 (d, JC,F=1), 149.09 (d, JC,F=4),
151.65. 19F NMR (470.3 MHz, DMSO-d6, ref (C6F6)=-163
ppm): -169.72. MS ESI, m/z: 304 [M þ H]. HR MS (ESI): calcd
for C11H12ClFN3O4 [M þ H] 304.0500, found 304.0506.
Biology. Cytostatic Activity Assays. All cell lines were obtai-
ned from ATCC (Manassas, VA). Colon (HCT116, HCT 15),
breast (BT549, HS 578), and lung (A549, NCI-H23) cell lines
were maintained in the RPMI cultivation medium (Invitrogen;
Carlsbad, CA) supplemented with 10% fetal bovine serum
(FBS). Prostate cell lines (Du145, PC3) were cultivated in
MEM and F12K medium containing 10% FBS, respectively.
Doxorubicin, clofarabine, trichloroacetic acid (TCA), and sul-
forhodamine B (SRB) were from Sigma-Aldrich (St. Louis,
MO). Gemcitabine was obtained from Moravek Biochemicals
(Brea, CA)
0
0
0
0
0
0
0
0
40), 4.88 (dd, 1H, Jgem=12.2, J5 a,4 =3.1, H-5 a), 6.09 (dd, 1H,
0
3 ,2 =5.9, J3 ,4 =4.1, H-3 ), 6.14 (t, 1H, J2 ,3 =J2 ,1 =5.9, H-2 ),
0
0
0
0
0
0
0
0
0
0
0
0
J
0
0
=
6.63 (dd, 1H, J4,3=3.5, J4,5=1.7, H-4-furyl), 6.84 (dd, 1H, J1 ,2
5.9, JH,F=1.3, H-10), 7.199 (d, 1H, JH,F=2.4 H-6), 7.36 and 7.42
(2 ꢀ m, 2 ꢀ 2H, H-m-Bz), 7.50 (dd, 1H, J3,4=3.5, J3,5=0.7, H-3-
furyl), 7.51 (m, 2H, H-m-Bz), 7.54, 7.60, and 7.62 (3 ꢀ m, 3 ꢀ 1H,
H-p-Bz), 7.71 (dd, 1H, J5,4=1.7, J5,3=0.7, H-5-furyl), 7.93, 8.02,
and 8.15 (3 ꢀ m, 3 ꢀ 2H, H-o-Bz), 8.85 (s, 1H, H-2). 13C NMR
(151 MHz, CDCl3): 63.73, 71.40, 73.69, 80.26, 85.41, 103.47 (d,
JC,F=16), 108.46 (d, JC,F=31), 112.66, 115.68 (d, JC,F=11),
128.30, 128.48, 128.54, 128.60, 128.72, 129.23, 129.66, 129.81,
129.82, 133.56, 133.76, 142.79 (d, JC,F=253), 145.84, 147.07 (d,
J
C,F =4), 148.16 (d, JC,F =3), 150.45, 152.25, 165.11, 165.42,
166.15. 19F NMR (470.3 MHz, CDCl3): -159.30. MS FAB, m/z
(rel %): 648 (100) [M þ H]. HR MS (FAB): calcd for C36H27-
FN3O8 [M þ H] 648.1782, found 648.1775.
5-Fluoro-4-(furan-2-yl)-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (4h). Deprotection of 13h as described for 4d gave
compound 4h as a beige solid; yield 78%. Compound 4h crystal-
lized from MeOH as a beige microcrystalline solid; mp 177-
179 °C; [R]D -66.5 (c 0.409, DMSO). UV (MeOH): λmax (ε) 333
A modified protocol of sulforhodamine B colorimetric assay
was used for the cytostatic activity screening.32 Cells were
distributed into the 96-well plates in 150 μL of media (HCT-
116 and Du145 5300 cell/mL; HCT15 and A549 10600cells/mL;
Hs578 and BT549 26600 cells/mL; PC3 16600 cells/mL; NCI-
H23 40000 cells/mL) and incubated overnight in humidified
CO2 incubator at 37 °C. Next day, one plate of each cell line was
fixed with TCA by removing media and adding 100 μL cold 10%
(v/v) TCA to each well. After 1 h incubation at 4 °C, TCA was
discarded and plates were washed four times with tap water.
These plates represented cell counts at day zero. The tested
compounds were 5-fold serially diluted and distributed to cells in
50 μL of media. After five days of incubation, the plates were
fixed with TCA as mentioned above and 100 μL of 0.057% SRB
solution in 1% (v/v) acetic acid was added to each well. After 30
min incubation at room temperature, SRB was removed and the
plates were rinsed four times 1% (v/v) acetic acid. Next, 200 μL
of 10 mM Tris base solution (pH 10.5) was added to each well of
completely dried plates and absorbance of cell associated SRB
was read at 500 nm. The percentage of cell-growth inhibition
was calculated using the following formula: % of control cell
(12452), 297 (12588), 235 (19787). 1H NMR (600 MHz, DMSO-
0
0
0
0
d6): 3.56 (ddd, 1H, Jgem=11.9, J5 b,OH=5.5, J5 b,4 =3.9,0H-5 b),
0
3.64 (ddd, 1H, Jgem=11.9, J5 a,OH=5.5, J5 a,4 =4.1, H-5 a), 3.92
0
0
0
0
(ddd, 1H, J4 ,5 =4.1, 3.9, J4 ,3 =3.3, H-4 ), 4.11 (ddd, 1H, J3 ,2 =
5.1, J3,OH=4.9, J3 ,4 =3.3, H-3 ), 4.36 (ddd, 1H, J2 ,OH=6.3,
0
J2 ,1 =6.1, J2 ,3 =5.1, H-2 ), 5.10 (t, 1H, JOH,5 =5.5, OH-5 ), 5.22
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
(d, 1H, JOH,3 =4.9, OH-3 ), 5.43 (d, 1H, JOH,2 =6.3, OH-2 ),
0
6.31 (dd, 1H, J1 ,2 =6.1, JH,F=1.8, H-10), 6.80 (dd, 1H, J4,3=3.5,
J4,5 =1.7, H-4-furyl), 7.48 (dd, 1H, J3,4=3.5, J3,5=0.8, H-3-
0
furyl), 7.96 (d, 1H, JH,F=1.9, H-6), 8.08 (dd, 1H, J5,4=1.7, J5,3
=
0.8, H-5-furyl), 8.81 (s, 1H, H-2). 13C NMR (151 MHz, DMSO-
d6): 61.64, 70.68, 74.34, 85.47, 86.36, 102.12 (d, JC,F=16), 110.75
(d, JC,F=30), 113.15, 114.93 (d, JC,F=6), 141.46 (d, JC,F=249),
146.04 (d, JC,F=4), 147.02, 147.80 (d, JC,F=3), 151.12, 151.81.
19F NMR (470.3 MHz, DMSO-d6, ref (C6F6) = -163 ppm):
-161.79. IR (KBr): ν=1586, 1485, 1461, 1395, 1249, 1209, 1101,
1046, 1021 cm-1. MS FAB, m/z (rel %): 204 (90), 336 (100) [M þ
H]. HR MS (FAB): calcd for C15H15FN3O5 [M þ H] 336.0996,
growth = 100 ꢀ (ODsample - mean ODday0)/(ODnegcontrol
-
mean ODday0). For GIC50 determination, plot a dose-response
curves between the compound concentration and percent of
growth inhibition. GIC50 values can be derived by fitting
dose-response curves using a sigmoidal dose-response equa-
tion.
found 336.1003. Anal. (C15H14FN3O5 0.25H2O): C, H, N.
3
4-Chloro-5-fluoro-7-(2,3-O-isopropylidene-5-O-tert-butyldi-
methylsilyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (16).
Compound 16 was prepared from 4-chloro-5-fluoropyrrolo[2,3-
d]pyrimidine 15 following the same procedure used for the
1
synthesis of compound 8; yield 43%; colorless oil. H NMR
(600 MHz, CDCl3): 0.10 and 0.11 (2 ꢀ s, 2 ꢀ 3H, CH3Si), 0.92 (s,
Intracellular Metabolism. Du145 cells were seeded into T25
flasks at 60% confluence in the MEM medium supplemented
with 10% FBS. The next day, the medium was replaced with
fresh media containing the tested compounds (3h or 5h at
10uM). After 6 and 24 h of incubation, cells were washed with
phosphate buffer and detached with trypsin. Trypsin was neu-
tralized by adding cultivation medium and the cells were spun
for 5 min at 500g. The supernatants were removed and cell
pellets were resuspended in 0.5 mL of media. Cell suspension
was layered onto 0.25 mL of Nyosil M25 oil and centrifuged for
3 min. The media was aspirated off and the top of the oil layer
9H, (CH3)3C), 1.38 (q, 3H, J=0.5, (CH3)2C), 1.65 (q, 3H, J=0.5,
0
0
(CH3)2C), 3.81 (dd, 1H, Jgem=11.4, J5 b,4 =3.2, H-5 b), 3.91 (dd,
0
0
0
0
0
1H, Jgem=11.4, J5 a,4 =2.9, H-5 a), 4.38 (ddd, 1H, J4 ,5 =3.2,
0
0
0
0
2.9, J4 ,3 =2.4, H-4 ), 4.91 (dd, 1H, J3 ,2 =6.2, J3 ,4 =2.4, H-3 ),
0
0
0
0
0
0
0
0
0
0
0
0
4.93 (dd, 1H, J2 ,3 =6.2, J2 ,1 =2.6, H-2 ), 6.47 (dd, 1H, J1 ,2
=
2.6, JH,F=1.5, H-10), 7.44 (d, 1H, JH,F=2.5, H-6), 8.65 (s, 1H, H-
2). 13C NMR (151 MHz, CDCl3): -5.33, -5.44, 18.38, 25.41,
25.87, 27.33, 63.53, 80.73, 85.32, 86.19, 90.16, 107.56 (d, JC,F=
14), 107.62 (d, JC,F=27), 114.24, 141.49 (d, JC,F=253), 146.50

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 469
was washed with water. Both water and oil were aspirated off
without disturbing the cell pellet. The cells were extracted with
500 μL of 70% MetOH and cell lysates were centrifuged,
supernatants was collected, dried by vacuum, and resuspended
in 10 μL of tetrabutyl ammonium acetate containing an internal
standard. Transient ion-pairing high-performance liquid chro-
matography coupled to positive ion electrospray tandem mass
spectrometery (LC/MS/MS) was used to quantitate intracellu-
lar nucleotides. Standard curves and quality control samples
were generated for all analytes using extracts from untreated
cells. Methods were adapted from those described for the acyclic
phosphonate nucleotide analogue adefovir, its phosphorylated
metabolites, and natural nucleotides.34
Cell Cycle and Apoptosis Analysis. Subconfluent CCRF-
CEM cells (ATCC), seeded at the density of 5 ꢀ 105 cells/mL
in 6-well panels, were cultivated with the 1ꢀ or 5ꢀ GIC50 of
tested compounds in a humidified CO2 incubator at 37 °C in
RMPI 1640 cell culture medium containing 10% fetal calf
serum, 10 mM glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin. Controls containing vehicle were harvested at the
same time point (24 h). Cells were washed with cold PBS and
fixed in 70% ethanol overnight at -20 °C. The next day the cells
were washed in hypotonic citrate buffer, treated with RNase (50
μg/mL), stained with propidium iodide, and analyzed by flow
cytometry using a 488 nm single beam laser (Becton Dickinson).
Cell cycle was analyzed in the program ModFitLT (Verity), and
apoptosis was measured in logarithmic mode as percentage of
the particles with propidium content lower than cells in G0/G1
phase (sub-G1) of the cell cycle. Half of the sample was used for
phospho-histon H3Ser10 antibody (Sigma) labeling and subse-
quent flow cytometry analysis of mitotic cells.35
Supporting Information Available: Complete experimental
section with characterization data and elemental analyses. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Acknowledgment. This work is a part of the research
project Z4 055 0506. It was supported by the Ministry of
Education, Youth and Sports (grant 1M0508), by the Pro-
gramme for Targeted Research (1QS400550501) and by
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