Synthetic strategies toward carbocyclic purine-pyrimidine hybrid nucleosides

Article abstract of DOI:10.1016/j.bmc.2009.06.039

The blending of key structural features from the purine and pyrimidine nucleobase scaffolds gives rise to a new class of hybrid nucleosides. The purine-pyrimidine hybrid nucleosides can be viewed as either N-3 ribosylated purines or 5,6-disubstituted pyri

Full text of DOI:10.1016/j.bmc.2009.06.039

Bioorganic & Medicinal Chemistry 17 (2009) 5520–5525
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthetic strategies toward carbocyclic purine–pyrimidine hybrid nucleosides
Joshua M. Sadler a, Sylvester L. Mosley a, , Kathleen M. Dorgan b, Zhaohui Sunny Zhou b,à
,
Katherine L. Seley-Radtke a,
*
a Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
b Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The blending of key structural features from the purine and pyrimidine nucleobase scaffolds gives rise to
a new class of hybrid nucleosides. The purine–pyrimidine hybrid nucleosides can be viewed as either N-3
ribosylated purines or 5,6-disubstituted pyrimidines, thus recognition by both purine- and pyrimidine-
metabolizing enzymes is possible. Given the increasing reports of the development of resistance in many
enzymatic systems, a drug that could be recognized by more than one enzyme could prove highly advan-
tageous in overcoming resistance mechanisms related to binding site mutations. In that regard, the
design, synthesis and results of preliminary biological activity for a series of carbocyclic uracil derivatives
with either a fused imidazole or thiazole ring are presented herein.
Received 15 April 2009
Revised 13 June 2009
Accepted 16 June 2009
Available online 23 June 2009
Keywords:
Carbocyclic
Purine
Pyrimidine
Nucleosides
Hybrid
Ó 2009 Elsevier Ltd. All rights reserved.
Dual inhibitor
1. Introduction
highly effective approach for circumventing some resistance
mechanisms.
Efforts in our laboratories have focused on the strategic manip-
ulation of key aspects of the nucleoside scaffold in an effort to
probe structure and function for biologically significant enzymes.
Increasing reports of viral and antibacterial resistance has rendered
many current chemotherapeutics less effective, however recent
examples of the success of dual inhibitors have opened new
avenues for investigation. The most common example of a dual
inhibitor involves two known drugs covalently linked together.
The drugs bind to two different sites in the same enzyme, but
can be delivered at the same time and in the same levels, thereby
ensuring more effective inhibition. Another type is a drug that can
be recognized and inhibit two different enzymes in the same rep-
lication pathway, or in two different mechanistic pathways that
are involved in the same disease.1–4 This should also increase the
chances for effective inhibition, especially if mutations have devel-
oped, thus a nucleoside analogue that could be recognized by both
purine- and pyrimidine-metabolizing enzymes might prove to be a
In that regard, exploitation of the nonstandard connectivity of
the purine analogue isoadenosine (1, IsoA, Fig. 1) was envisioned
as a strategic starting point for constructing hybrid nucleosides
that resemble both purines and pyrimidines. IsoA is a structural
isomer of adenosine where the purine ring is connected to the
sugar moiety at N-3 (purine numbering), rather than the tradi-
tional N-9.5,6 As depicted in Figure 2, N-3 glycosolated purines
can be viewed as 5,6-disubstituted pyrimidines, and conversely,
5,6-disubstituted pyrimidines are often considered as purine mim-
ics, thus providing impetus for the design goals.7,8
Although IsoA was initially considered an interesting analogue
since it was recognized by a number of biologically significant
enzymes, the proclivity to undergo 1,3-migration in both basic and
acidic conditions to result in adenosine, made synthesis tedious and
low-yielding.6 As an answer to this, employing the carbocyclic nucle-
* Corresponding author. Tel.: +1 410 455 8684; fax: +1 410 455 2608.
E-mail address: kseley@umbc.edu (K.L. Seley-Radtke).
Present address: U.S. FDA, Office of Food Additive Safety Division of Biotechnology
and GRAS Notice Review, College Park, MD 20740, USA.
à
Present address: Department of Chemistry and Chemical Biology and the Barnett
Institute of Chemical and Biochemical Analysis, Northeastern University, Boston, MA
02115-5000, USA.
Figure 1. Isoadenosine (IsoA) and DHCe-isoA.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.039

J. M. Sadler et al. / Bioorg. Med. Chem. 17 (2009) 5520–5525
5521
Figure 2. Purine–pyrimidine hybrid nucleosides.
oside scaffold was viewed ashighly advantageousfor several reasons;
first, carbocyclic nucleosides are stable to glycosidic bond cleavage,
since replacement of the furanose oxygen with a methylene group
converts the bond between the sugar and the base from an unstable
hemiaminal to a morerobust tertiaryamine. Thisstructural modifica-
tion would provide an answer to IsoA’s instability towards acids and
bases, as well as the propensity to undergo glycosidic migration, since
it would require cleavage of a tertiary amine bond.9 Second, carbocy-
clic purine nucleosides as a class, are known potent inhibitors of S-
adenosylhomocysteine hydrolase (SAHase), and indirectly, DNA
methyltransferase (DNA MeTase).10–12 Both are critical enzymes in
the replication pathway of many viruses, parasites and cancers, thus
an analogue that could inhibit both steps in a single pathway would
increase the likelihood of achieving complete inhibition.10,13 Drawing
upon these leads, it appeared that a hybrid of the pyrimidine and pur-
ineringscaffoldsmergedwiththecarbocyclic‘sugar’mightofferforth
a successful strategy for the design of more effective inhibitors.
Scheme 1. Reagents and conditions: (a) LDA, THF, I2, ꢀ78 °C, 5 h; (b) NaN3, DMF, rt,
1 h; (c) 10% PdC, MeOH, H2, rt, 1 h; (d) NaHCO3, Br2, rt, 1 h; (e) HC(OEt)3, reflux,
1.5 h; (f) TFA/H2O (2:1), rt, 2 h.
the substituents, this intermediate could provide a facile starting
point to realize both of the desired bicyclic analogues. Treatment
of 5 with lithium diisopropyl amide (LDA) and iodine provided
the 6-iodo intermediate 6.19 Subsequent displacement with
sodium azide, followed by reduction afforded amine 8. Bromina-
tion of C-5 gave the difunctionalized 9, which was then subjected
to the same set of reactions as was used to functionalize the C-6
position, resulting in diamino 11. It should be noted that these
reactions were not particularly high-yielding, since several of the
intermediates were unstable, however the reaction times were
short, thus the entire series of reactions, including ring closure,
could be accomplished in a matter of a few days. Finally, ring clo-
sure with triethylorthoformate and deprotection gave 3 in a 16.5%
overall yield from 5.
Attention then turned to the thiazole analogue 4. Using a two-
step procedure reported in the literature20,21, the synthesis of 4 ini-
tially appeared to be much more facile than the low-yielding and
tedious route employed for 3. As shown below in Scheme 2, start-
ing with 6, treatment with methylamine gave 13 in a 94% yield.
Treatment with thionylchloride should have resulted in the bicy-
clic intermediate 14 similarly to the reports of Mizuno et al.,21
however no reaction was observed, despite repeated manipulation
of the reaction times and temperatures.
2. Chemistry
Although initial efforts for this project had focused on the syn-
thesis of the parent purine isoA ring system (2, Fig. 1)9, the ultimate
goal was to construct a more versatile hybrid of the pyrimidine and
purine ring systems. In considering the design of possible targets, it
was necessary to retain two key structural features; the aromatic-
ity and hydrogen bonding capabilities of the heterocyclic ring sys-
tem. In addition to the parent carbocyclic isoxanthosine (3, Fig. 3),
the isoelectronic exchange of a sulfur atom for the nitrogen was
also considered (4, Fig. 3). This would allow for retention of the aro-
maticity desired for the heterocyclic base, as well as to retain uri-
dine’s acceptor–donor–acceptor pattern.
Although Townsend and co-workers had previously reported7,8
several thiazolopyrimidine ribose-based nucleosides, there were
no reports of any carbocyclic analogues. Since the routes to the car-
bocyclic analogues differ significantly from those used to obtain
the ribose analogues, these were of interest synthetically. Addi-
tionally, because the 40-deoxy derivatives of Aristeromycin and
Neplanocin A exhibited less toxicity as compared to the parent
compounds14–17, the truncated analogues were initially chosen
for the design of the carbocyclic portion of the nucleoside. As a re-
sult, the first targets considered were the fused uridine–imidazole
and uridine–thiazole analogues shown in Figure 3.
Since the original paper contained a thiazole substituted with
methyl groups on N-3 and N-1, speculation that the electronics
of the pyrimidine nitrogen might be hindering the ring closure
reaction dictated investigating the possibility that a protecting
group for N-3 might solve the problem. Several traditional protect-
ing groups were considered, since a facile method for selective N-3
protection was available in the literature.22 Surprisingly, the out-
come of these trials were not as straightforward as anticipated,
and are summarized in Table 1.
The synthesis of 3 was envisioned from functionalizing the C-5
and C-6 positions of a carbocyclic uridine analogue recently
reported18 from our laboratories (Scheme 1). Depending upon
Scheme 2. Reagents and conditions: (a) 33%, NH2Me, EtOH, rt, 1.5 h, 94%; (b) SOCl2,
Figure 3. Hybrid targets.
pyridine, reflux, 3 h.

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J. M. Sadler et al. / Bioorg. Med. Chem. 17 (2009) 5520–5525
Table 1
Entry
R=
Results
5a
5b
5c
5d
Bz
Bn
BOM
PMB
Iodination at multiple sites, including the Bz group
Removal of Bn problematic; NR to very low-yielding, to complete loss of the thiazole ring
Removal of BOM problematic; results ranged from NR, conversion of the BOM group to Me, which proved intractable to removal
Removal of the PMB resulted in the loss of the isopropylidine protecting group, and a complex, inseparable product mixture
Starting with the most obvious choice, benzoylation was tried
first, however upon treatment of 5a with LDA and iodine as before,
iodination occurred at multiple sites, including on the benzoyl
group. Turning next to the Bn group, iodination proceeded with
no problems, and the ring closure to give the desired fused pyrim-
idine–thiazole ring system was accomplished in good yield,
removal of the Bn group proved difficult. The presence of the sulfur
precluded the use of the traditional deblocking systems that em-
ploy hydrogenation catalysts such as Pd/C, thus other methods
such as the use of ethanethiol, which had been successful in
deblocking our thieno-expanded purine nucleosides23–25, were
attempted. These proved to be very low-yielding, as did the Lewis
acid deblocking methods. Additionally, the use of dissolving metal
catalysis with sodium and ammonia led to the complete loss of the
thiazole portion of the bicyclic base, reverting to the starting mate-
rial, Bn-protected uridine 5b, which was similar to other reports.
The benzyloxymethyl (BOM) group appeared to be a reasonable
option, however again, all attempts to remove the BOM group re-
sulted in either no reaction under mild conditions, or with more
stringent conditions, conversion to a methyl group, which then
proved to be completely recalcitrant to removal.
both groups using excess CAN gave rise to an inseparable mixture
of highly polar products. Finally, reprotection of the unmasked
hydroxyls of 16 with the acetate group gave 17. Deprotection of
the PMB group with CAN afforded 18, followed by removal of the
acetates, provided 4 in a 75% yield.
Once in hand, the targets were screened for their ability to inhi-
bit SAHase and DNA MeTase. Weak to no activity was observed
against DNA MeTase (data not shown) for either compound, how-
ever 3 exhibited good activity against SAHase while 4 showed no
appreciable activity. For SAHase, in the first experiment the con-
centration of inhibitor was varied while the concentration of sub-
strate (SAH) was held constant at 10
lM. Conversion of SAH to
adenosine and then to inosine was monitored at 265 nm (Fig. 4).
In the second experiment the concentration of inhibitor was held
constant at 45 lM while the concentration of substrate was varied.
Initial rates in the presence and absence of inhibitors were mea-
sured (Fig. 5). There was no change in absorbance at 265 nm in
the absence of SAH. These experimental data best fit a competitive
inhibition mechanism. The Ki value for compound 3 was found to
be 5.0 0.9
on a Km value of 7.9
l
M against SAHase in the hydrolysis direction, based
l
M for the SAH substrate.26 Interestingly, in
Finally, the p-methoxybenzyl (PMB) group was considered. As
detailed in Scheme 3, the iodination, followed by displacement
with methylamine, and subsequent ring closure gave PMB-pro-
tected 16 in very good yield. At this point, deblocking of the PMB
group was undertaken, but initial attempts with the traditional
method employing ceric ammonium nitrate (CAN), surprisingly
resulted in loss of the isopropylidine group instead, leaving the
PMB group untouched. To our knowledge this observation has
not previously been noted in the literature. All attempts to remove
the presence of the inhibitor, biphasic enzyme kinetics were
observed; albeit, the initial phase was much shorter than the later
one. These data suggested that the mechanism may be more com-
plicated than a simple competitive inhibition mechanism. Detailed
mechanistic studies will be reported in the future.
O
N
O
N
O
N
PMB
O
R
O
PMB
O
S
N
S
N
N
N
N
X
c
d
O
O
O
O
R'O
OR'
17, R=PMB; R'=Ac
18, R=H; R'=Ac
4, R=R'=H
e
f
5d, X=H
6d, X=I
15, X=NHCH3
16
a
b
Scheme 3. Reagents and conditions: (a) LDA, THF, I2, ꢀ78 °C, 3 h, 58%; (b) 33%
NH2Me, EtOH, rt, 1.5 h, 94%; (c) SOCl2, pyridine, reflux, 3 h, 80%; (d) (i) TFA/H2O
(2:1), rt, 18 h; (ii) Ac2O, pyridine, DMAP, CH2Cl2, 12 h, 82% for two steps; (e) 10:1
CH3CN/H2O, CAN, 55 °C, 3 h, 86%; (f) NH3, MeOH, rt 15 h, 65%.
Figure 4. Inhibition of SAHase by 3. Assay solutions contained 50 mM potassium
phosphate at pH 7.4, 0.39 units of adenosine deaminase, 132 nM of SAHase, 10 lM
of SAH (substrate), and various concentrations of 10. Consumption of substrate was
monitored at 265 nm.

J. M. Sadler et al. / Bioorg. Med. Chem. 17 (2009) 5520–5525
5523
and n-BuLi was added dropwise (2.5 M in hexanes, 2.27 mmol,
0.91 mL). After stirring for 15 min, solution of (0.26 g,
a
5
1.03 mmol) in anhydrous THF (10 mL) was added. The mixture
was stirred at ꢀ78 °C for an additional 30 min, at which point a
solution of I2 (0.58 g, 2.27 mmol) in anhydrous THF (5 mL) was
added and the mixture was allowed to continue stirring at this
temperature for 2 h. Following quenching with acetic acid (10
drops) and NaHCO3 (1 mL), the solvent was removed by evapora-
tion and the residue dissolved in EtOAc (50 mL), washed with so-
dium thiosulfate (25 mL) and brine (25 mL), dried (MgSO4),
filtered, and concentrated under vacuum. The residue was purified
by column chromatography eluting with n-hexanes/EtOAc (1:4) to
afford 0.23 g of 6 as a yellow solid (60%). mp 180–182 °C. 1H NMR
(DMSO-d6): d 1.38 (s, 3H), 1.41 (s, 3H), 1.51–1.61 (m, 2H), 1.76–
1.86 (m, 2H), 3.88 (m, 1H), 3.93 (m, 1H), 4.50 (t, 1H), 6.57 (s,
1H), 11.00 (br s, 1H). 13C NMR (DMSO-d6): d 19.3, 24.3, 25.9,
26.6, 56.1, 81.9, 88.4, 102.1, 107.3, 110.7, 150.9, 163.6. HRMS:
calcd for C12H15IN2O4 (M+H)+, 379.0077; found, 379.0176.
Figure 5. The concentration of inhibitor 3 was held constant at 45 lM while the
concentration of substrate was varied. Initial rates in the presence (data points at
the bottom of the plot) and absence (data points at the top of the plot) of inhibitors
were measured. There was no change in absorbance at 265 nm in the absence of
SAH.
4.2.2. Preparation of 6-azido-1-(20,30-O-isopropylidenedioxycy-
clopent-1-yl)uracil (7)
A
solution of 6 (0.10 g, 0.26 mmol) and NaN3 (0.094 g,
3. Summary
1.45 mmol) in anhydrous DMF (3 mL) was stirred at rt for
30 min, at which point H2O (20 mL) was added and the mixture ex-
tracted with EtOAc (3 ꢁ 50 mL), washed with brine (50 mL), dried
(MgSO4), filtered, and concentrated. The crude product was recrys-
tallized from ethanol to afford 7 as a white solid (0.066 g,
0.23 mmol, 85%), mp 159–160 °C. 1H NMR (DMSO-d6): d 1.36 (s,
3H), 1.41 (s, 3H), 1.51–1.61 (m, 2H), 1.76–1.86 (m, 2H), 3.88 (m,
1H), 3.93 (m, 1H), 4.50 (t, 1H), 5.80 (s, 1H), 11.00 (br s, 1H). 13C
NMR (DMSO-d6): d 20.2, 24.3, 25.9, 26.6, 52.5, 81.9, 89.3, 100.0,
110.7, 142.0, 150.9, 163.6. Anal. Calcd for C12H15N5O4 (0.25 EtOH):
C, 49.28; H, 5.46; N, 23.00. Found: C, 49.41; H, 5.31; N, 22.88.
In summary, given the unique electronic nature of many het-
erocyclic ring systems, it is clear that the choice of protecting
groups is not always straightforward, since reactivity for these
systems varies greatly. Additional analogues are currently being
pursued, the results of which should provide potentially interest-
ing information for further investigation of the hybrid purine–
pyrimidine scaffold.
4. Experimental
4.1. General
4.2.3. Preparation of 6-amino-1-(20,30-O-isopropylidenedioxy-
cyclopent-1-yl)uracil (8)
All chemicals were obtained from commercial sources and
used without further purification unless otherwise noted. Anhy-
drous DMF, MeOH, DMSO and toluene were purchased from Fish-
er Scientific. Anhydrous THF, acetone, CH2Cl2, CH3CN and ether
were obtained using a solvent purification system (mBraun Lab-
master 130). Melting points are uncorrected. NMR solvents were
purchased from Cambridge Isotope Laboratories (Andover, MA).
All 1H and 13C NMR spectra were obtained on a JEOL ECX
400 MHz NMR, operated at 400 and 100 MHz, respectively, and
referenced to internal tetramethylsilane (TMS) at 0.0 ppm. The
spin multiplicities are indicated by the symbols s (singlet), d (dou-
blet), dd (doublet of doublets), t (triplet), q (quartet), m (multi-
plet), and br (broad). Reactions were monitored by thin-layer
chromatography (TLC) using 0.25 mm Whatman Diamond Silica
Gel 60-F254 precoated plates. Column chromatography was per-
To a solution of 7 (0.066 g, 0.23 mmol) in absolute EtOH
(50 mL) was added Pd/C (10%, 10 mg), and the mixture subjected
to hydrogenation at 25 psi for 20 min. The Pd/C was removed by
filtration over a pad of Celite, rinsed and the filtrate concentrated
to afford 0.060 g of 8 as a white solid (quantitative), mp 225–
227 °C. 1H NMR (DMSO-d6): d 1.36 (s, 3H), 1.41 (s, 3H), 1.51–
1.61 (m, 2H), 1.76–1.86 (m, 2H), 2.00 (br s, 2H), 3.88 (m, 1H),
3.93 (m, 1H), 4.50 (t, 1H), 4.70 (s, 1H), 11.00 (br s, 1H). 13C
NMR (DMSO-d6): d 20.2, 24.3, 25.9, 26.6, 52.5, 75.4, 81.9, 89.3,
110.7, 150.9, 153.1, 163.6. Anal. Calcd for C12H17N3O4 (0.75
H2O): C, 51.36; H, 6.65; N, 14.98. Found: C, 51.37; H, 6.59; N,
15.06.
4.2.4. Preparation of 6-amino-5-bromo-1-(20,30-O-isopropylide-
nedioxy-cyclopent-1-yl) uracil (9)
formed using silica gel (63–200 l) from Dynamic Adsorptions
To a solution of 8 (0.74 g, 2.77 mmol) and NaHCO3 (2.09 g,
24.93 mmol) in absolute EtOH (25 mL), was added dropwise, Br2
(5%, EtOH) until no starting material remained as observed by
TLC. After removing the EtOH under vacuum, EtOAc (50 mL) was
added, followed by washing with brine (3 ꢁ 50 mL). The organic
layer was dried (MgSO4), filtered, concentrated, and purified via
column chromatography eluting with n-hexanes/EtOAc (1:4) to af-
ford 0.77 g of 9 as a white solid (80%). Mp 214–216 °C. 1H NMR
(DMSO-d6): d 1.36 (s, 3H), 1.41 (s, 3H), 1.51–1.61 (m, 2H), 1.76–
1.86 (m, 2H), 2.00 (br s, 2H), 3.88 (m, 1H), 3.93 (m, 1H), 4.50 (t,
1H), 11.00 (br s, 1H). 13C NMR (DMSO-d6): d 20.2, 24.3, 25.9,
26.6, 51.8, 62.7, 81.9, 89.3, 110.7, 150.9, 159.9, 162.3. Anal. Calcd
for C12H16N3O4Br (0.20 EtOAc): C, 42.39; H, 4.89; N, 11.59. Found:
C, 42.78; H, 4.91; N, 11.73.
Inc. (Norcross, GA), and eluted with the indicated solvent system.
Yields refer to chromatographically and spectroscopically (1H and
13C NMR) homogeneous materials. Mass spectra were recorded at
the Johns Hopkins Mass Spectrometry Facility (Baltimore, MD).
Elemental analyses were recorded at Atlantic Microlabs, Inc. (Nor-
cross, GA).
4.2. Synthesis
4.2.1. Preparation of 6-iodo-1-(20,30-O-isopropylidenedioxycy-
clopent-1-yl)uracil (6)
To a flame dried flask under argon atmosphere was added
anhydrous THF (5 mL) and freshly distilled diisopropylamine
(0.23 g, 2.27 mmol). The temperature was lowered to ꢀ78 °C

5524
J. M. Sadler et al. / Bioorg. Med. Chem. 17 (2009) 5520–5525
4.2.5. Preparation of 3-(20,30-O-isopropylidenedioxy-cyclopent-
1-yl)xanthine (12)
from EtOH (3 ꢁ 50 mL). The crude product was purified using col-
umn chromatography eluting with hexanes/EtOAc (1:4) to give 15
as a sticky white solid (851 mg, 94%). 1H NMR (DMSO-d6): d 1.16 (s,
3H), 1.37 (s, 3H), 1.76 (m, 2H), 2.19 (m, 2H), 2.64 (d, 3H), 3.67 (s,
3H), 4.59 (s, 2H), 4.75 (m, 1H, H-30), 4.80 (t, 1H, H-10), 4.86 (dd,
1H, H-20), 6.46 (d, 1H, H-5), 6.81 (d, 2H), 7.15 (d, 2H). 13C NMR
(CDCl3): d 14.3, 24.7, 27.4, 28.6, 29.8, 30.2, 31.7, 43.2, 55.3, 60.5,
63.0, 81.2, 84.8, 112.0, 113.7, 130.2, 154.6, 158.9. HRMS calculated
for C21H27N3O5 [M+H]+ 402.2029; found, 402.2020.
A
solution of 9 (0.77 g, 2.22 mmol) and NaN3 (0.72 g,
11.10 mmol) in anhydrous DMF (20 mL) was stirred at rt for
30 min, at which point H2O (20 mL) was added and the mixture ex-
tracted with EtOAc (3 ꢁ 50 mL), washed with brine (50 mL), dried
(MgSO4), filtered, and concentrated to yield 0.54 g of 10, which was
used directly without further purification.
To a solution of 10 (0.54 g, 1.75 mmol) in absolute EtOH (50 mL)
was added Pd/C (10%, 0.054 g). This mixture was hydrogenated at a
pressure of 25 psi for 20 min. The Pd/C was removed by filtration
andthefiltratewasconcentratedtoafford0.49 gof11asawhitesolid,
which was used directly in the next step without further purification.
Diamino 11 was dissolved in triethylorthoformate, heated at re-
flux for 1 h and the reaction monitored by TLC. Upon completion,
the triethylorthoformate was removed by evaporation under re-
duced pressure and the resulting residue was purified by column
chromatography eluting with EtOAc/MeOH (9:1) to afford 0.30 g of
12 as a white solid (59%), mp 220–222 °C. 1H NMR (DMSO-d6): d
1.38 (s, 3H), 1.41 (s, 3H), 1.51–1.61 (m, 2H), 1.76–1.86 (m, 2H),
3.88 (m, 1H), 3.93 (m, 1H), 4.50 (t, 1H), 8.30 (s, 1H), 10.00 (s, 1H),
13.4 (s, 1H). 13C NMR (DMSO-d6): d 19.7, 24.2, 26.6, 27.1, 51.5,
81.8, 88.8, 110.7, 114.6, 144.6, 152.0, 154.9. HRMS: calcd for
C13H16N4O4 (M+H)+, 2931172; found, 293.1247.
4.2.9. Preparation of 4-[(20,30-O-isopropylidene)-cyclopent-10-yl]-
6-(4-methoxybenzyl)-thiazolo[4,5-d]pyrimidine-5,7-dione (16)
To a stirred solution of 15 (62.5 mg, 0.156 mmol) in pyridine
(1 mL) was added thionylchloride (5 mL) and refluxed for 3 h, then
cooled and additional pyridine (5 mL) was added and the mixture
evaporated under reduced pressure. The residue was co-evapo-
rated with pyridine (2 ꢁ 5 mL) and the residue purified by prepara-
tive TLC eluting with hexane/EtOAc (1:1) to afford 29.4 mg of 16
appeared as an off-white sticky solid (80%). 1H NMR (CDCl3): d
1.29 (s, 3H), 1.52 (s, 3H), 1.91–2.36 (m, 4H), 3.76 (s, 3H), 4.92 (m,
1H, H-30), 5.10 (t, 1H, H-10), 5.12 (s, 2H), 5.50 (td, 1H, H-20), 6.82
(d, 2H), 7.45 (d, 2H), 8.92 (s, 1H, H-thiazole). 13C NMR (CDCl3): d
14.3, 21.1, 25.0, 27.5, 29.4, 31.8, 44.6, 55.3, 60.5, 64.1, 81.4, 84.0,
111.6, 113.9, 128.9, 130.8, 151.2, 159.0, 159.3. HRMS calculated
for C21H23N3O5S [M+H]+ 430.1437; found, 430.1424.
4.2.6. Preparation of 3-(20,30-dihydroxy-cyclopent-1-yl)
xanthine (3)
4.2.10. Preparation of 4-[(20,30-O-diacetoxy)-cyclopent-10-yl]-6-
(4-methoxybenzyl)-thiazolo[4,5-d]pyrimidine-5,7-dione (17)
A solution of 16 (391 mg, 0.910 mmol) in TFA/H2O (2:1, 15 mL)
was stirred at rt for 18 h. The solvents were removed under re-
duced pressure and the resulting residue co-evaporated with EtOH
To a solution of 12 (0.30 g, 1.03 mmol) in TFA/H2O (2:1, 20 mL)
was allowed to stir for 3 h at rt upon which time the TFA/H2O was re-
moved by evaporation. The resulting residue was co-evaporated
with MeOH (3 ꢁ 10 mL) to remove trace amounts of TFA. The result-
ing residue was then purified first by column chromatography
(EtOAc/acetone/EtOH/H2O, 4:1:1:0.5), and lastly by using C-18 HPLC
eluting 90:10, H2O/MeOH?50:50, H2O/MeOH?10:90, H2O/MeOH
to give 0.25 g of 3 as an off-white solid (98%), mp 254–256 °C. 1H
NMR (CD3OD): d 1.74–1.77 (m, 1H), 2.11–2.24 (m, 3H), 4.14–4.16
(m, 1H), 4.81–4.83 (m, 1H), 5.19–5.26 (q, 1H), 7.88 (s, 1H). 13C
NMR (CD3OD): d 19.4, 26.1, 53.4, 77.1, 85.4, 114.6, 144.6, 150.2,
152.0, 154.9. Anal. Calcd for C10H12N4O4: C, 47.62; H, 4.80; N,
22.21. Found: C, 47.49; H, 4.89; N, 21.92.
(3 ꢁ 10 mL). The crude product was dissolved in pyridine (120
lL,
2.29 mmol) and dry CH2Cl2 (10 mL), acetic anhydride (250 mg,
2.45 mmol) and DMAP (20 mg, 0.16 mmol) added. The reaction
was stirred at rt for 12 h before cooling to 0 °C and quenched with
saturated NaHCO3 solution. The organic layer was washed with sat.
NaHCO3 (3 ꢁ 20 mL), 1 N HCl (3 ꢁ 20 mL), brine (20 mL), dried and
concentrated. The resulting yellow oil was purified using column
chromatography eluting with hexanes/EtOAc (1:4) to afford
354 mg of 17 (82%) as a white hygroscopic powder. 1H NMR
(CDCl3): d 1.94 (s, 3H), 2.10 (s, 3H), 2.17–2.44 (m, 4H), 3.76 (s,
3H), 5.15 (d, 2H), 5.52 (q, 1H, H-30), 5.69 (qd, 1H, H-10), 5.87 (dd,
1H, H-20), 6.82 (d, 2H), 7.47 (d, 2H), 8.95 (s, 1H, H-thiazole). 13C
NMR (CDCl3): d 20.8, 21.1, 24.6, 28.2, 29.8, 44.8, 55.3, 73.2, 75.1,
77.3, 113.8, 128.9, 130.8, 157.4, 159.1, 159.3, 170.3, 170.5. HRMS
calculated for C22H23N3O7S [M]+ 473.1257; found, 473.1254.
4.2.7. Preparation of 1-[(20,30-O-isopropylidene)-cyclopent-10-yl]
-3-(4-methoxybenzyl)-6-iodouracil (6d)
Freshly distilled diisopropylamine (145
(10 mL) was treated with n-butyllithium (414
l
L, 1.03 mmol) in THF
L, 2.5 M in hexanes)
l
at ꢀ78 °C and to this a stirred solution of 5d (192 mg, 0.517 mmol) in
THF (10 mL) was added dropwise. The mixture was stirred for
30 min at which point a solution of I2 (525 mg, 2.07 mmol) in THF
(10 mL) was added slowly and stirred for 3 h while maintaining
the temperature at ꢀ78 °C. The reaction was quenched with a few
drops glacial acetic acid, diluted with CHCl3 (100 mL), and the organ-
ic layer washed with saturated Na2S2O3 (2 ꢁ 25 mL), brine
(3 ꢁ 50 mL), dried and the solvent removed under reduced pressure.
The crude product was purified by column chromatography eluting
with hexanes/EtOAc (1:1) to give 200 mg of 6d as a yellow foam
(78%). 1H NMR (DMSO-d6): d 1.18 (s, 3H), 1.40 (s, 3H), 1.82 (m, 2H),
2.19 (m, 2H) 3.68 (s, 3H), 4.74 (m, 1H, H-30), 4.76 (t, 1H, H-10), 4.79
(s, 2H, CH2), 4.86 (dd, 1H, H-20), 6.46 (d, 1H, H-5), 6.83 (d, 2H), 7.18
(d, 2H). 13C NMR (CDCl3): d 25.0, 27.6, 29.6, 29.8, 31.9, 44.1, 55.3,
76.3, 81.9, 84.5, 111.3, 113.8, 113.9, 116.0, 128.6, 130.6, 159.3.
4.2.11. Preparation of 4-[(20,30-O-diacetoxy)-cyclopent-10-yl]-
thiazolo[4,5-d]pyrimidine-5,7-dione (18)
A stirred solution of 17 (354 mg, 0.748 mmol) in CH3CN/H2O
(10:1, 11 mL) was heated to 55 °C, ceric ammonium nitrate
(450 mg, 0.820 mmol) added and the mixture stirred for 3 h. The
reaction was cooled, quenched with saturated NH4Cl solution
(1 mL) and evaporated to dryness. The crude residue was purified
by column chromatography eluting with hexanes/EtOAc (1:4) to
afford 228.2 mg of 18 as a white foam (86%). 1H NMR (CDCl3): d
1.95 (s, 3H), 2.09 (s, 3H), 2.32 (m, 2H), 2.45 (m, 2H), 5.49 (q, 1H,
H-30), 5.64 (dt, 1H, H-10), 5.86 (dd, 1H, H-20), 9.01(s, 1H), 9.93 (s,
1H). 13C NMR (MeOH-d6): d 20.8, 21.1, 24.7, 28.2, 29.8, 31.0, 73.2,
75.0, 107.6, 151.2, 157.4, 157.7, 160.0, 170.4, 170.7.
4.2.8. Preparation of 1-[(20,30-O-isopropylidene)-cyclopent-10-yl]-
3-(4-methoxybenzyl)-6-methylaminouracil (15)
4.2.12. Preparation of 10-(thiazolo[4,5-d]-pyrimidine-5,7-dion-
4-yl)-cyclopentane-20,30-diol (4)
A stirred solution of 6d (1.12 g, 2.25 mmol) in NH2CH3 (30 mL,
33% EtOH) and stirred at rt for 1.5 h. The mixture was then con-
densed under reduced pressure and the residue co-evaporated
To a solution of 18 (107 mg, 0.304 mmol) in EtOH (0.6 mL) was
added concentrated NH4OH (1.0 mL) and stirred for 18 h. The
mixture was then concentrated under reduced pressure and the

J. M. Sadler et al. / Bioorg. Med. Chem. 17 (2009) 5520–5525
5525
crude residue purified by reverse phase C-18 HPLC eluting 90:10,
H2O/CH3CN?70:30, H2O/CH3CN?50:50, H2O/CH3CN to afford
61.4 mg of 4 as a white crispy foam (75%). 1H NMR (CD3OD): d
2.08 (m, 2H), 2.20 (m, 2H), 5.56 (q, 1H, H-30), 5.61 (dt, 1H, H-
10), 5.88 (q, 1H, H-20), 9.29 (s, 1H, H-thiazole). 13C NMR (CD3OD):
d 13.1, 19.5, 20.7, 29.4, 60.2, 74.5, 113.7, 127.5, 171.7, 175.1.
HRMS calculated for C10H11N3O4S [M+H]+ 270.0470; found,
270.0545.
Herman Frasch Foundation (541-HF02 to Z.S.Z.). We are grateful
of Professor Lynne Howell of the Hospital of Sick Children of pro-
viding SAHase and Dr. Phil Mortimer of the Johns Hopkins Mass
Spectrometry Facility for his invaluable assistance with HRMS
analysis. We also thank Ms. Sarah Zimmermann for her editorial
assistance.
References and notes
4.3. SAHase inhibition assay
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The enzyme-coupled continuous assay in the hydrolysis direc-
tion was performed as previously reported. Briefly, SAH is hydro-
lyzed to homocysteine and adenosine, which is subsequently
converted by adenosine deaminase into ammonia and inosine, a
process associated with a decrease of absorbance at 265 nm.26
Each assay was conducted in thermostatted 1 cm quartz cuvettes
at 37 °C maintained by a Peltier unit on a Cary 100 ultraviolet–vis-
ible photospectrometer. Enzyme assay solution typically contained
50 mM potassium phosphate at pH 7.4, 0.39 units of adenosine
deaminase
LS009043), 132 nM of SAHase (provided by Dr. Lynne Howell),
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l
l
SAH. In all cases, we ascertained that SAH hydrolysis catalyzed
by SAHase was rate limiting under testing conditions (data not
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{Km ꢁ (1 + [I]/Ki) + [S]}; and v, kcat, [S], [E], Km, [I] and Ki stand for
the initial reaction rates, rate constant, substrate concentration,
enzyme concentration, Michaelis–Menten constant, inhibitor con-
centration and dissociation constant of the enzyme–inhibitor com-
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plex, respectively. A previously reported Km value of 7.9 lM for
SAH in the hydrolysis direction was used for all calculation.26
23. Seley, K. L.; Zhang, L.; Hagos, A. Org. Lett. 2001, 3, 3209.
24. Seley, K. L.; Zhang, L.; Hagos, A.; Quirk, S. J. Org. Chem. 2002, 67, 3365.
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Acknowledgments
This work was supported by the National Institutes of Health
(NIH) (RO1 CA 97634 to KSR and 1R01AI58146 to Z.S.Z) and the
26. Elrod, P.; Zhang, J.; Yang, X.; Yin, D. H.; Hu, Y.; Borchardt, R. T.; Schowen, R. L.
Biochemistry 2002, 41, 8134.