Carbocyclic pyrimidine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase

Article abstract of DOI:10.1016/j.bmc.2006.07.052

The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase, EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained, 10 was fou

Full text of DOI:10.1016/j.bmc.2006.07.052

Bioorganic & Medicinal Chemistry 14 (2006) 7967–7971
Carbocyclic pyrimidine nucleosides as inhibitors
of S-adenosylhomocysteine hydrolase
Sylvester L. Mosley,a Brian A. Bakke,a Joshua M. Sadler,a Naresh K. Sunkara,a
Kathleen M. Dorgan,b Zhaohui Sunny Zhoub and Katherine L. Seley-Radtkea,*
aDepartment of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle,
Baltimore, MD 21250, USA
bDepartment of Chemistry, Washington State University, Pullman, WA 99164, USA
Received 15 March 2006; revised 14 July 2006; accepted 26 July 2006
Available online 10 August 2006
Abstract—The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase,
EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained,
10 was found to be active with a Ki value of 5.0 lM against SAHase.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
The significant inhibitory activity exhibited by the car-
bocyclic nucleosides against S-adenosylhomocysteine
hydrolase (SAHase) has, for the most part, been con-
fined to adenosine analogues (Fig. 1).1–3 One notable
exception4 was the unexpected inhibition exhibited by
a split guanosine nucleoside ‘fleximer.’ However, to
date, few non-adenosine analogues have been reported.
Inhibition of SAHase and several other enzymes critical
for viral replication has been suggested as strategic tar-
gets for the inhibition of the orthopox viruses.5 Given
the lack of effective chemotherapeutics against the pox-
viruses and their potential for use in bioweapons, the
search for new and potent inhibitors has increased in
intensity.
Figure 1. Potent carbocyclic SAHase inhibitors.
ceptible to recognition and phosphorylation by cellular
kinases, resulting in significant toxicity.10 In response
to this, strategic removal of the 40-hydroxymethyl group
of Ari and NpcA to afford the 40,50-saturated (2) and
40,50-unsaturated (4) derivatives proved to be successful,
as both were potent inhibitors of SAHase.11,12 Interest-
ingly, neither served as a substrate for adenosine kinase
or adenosine deaminase, thus did not exhibit the toxicity
associated with Ari and NpcA.11,12
It is well known that inhibition of SAHase also leads to
inhibition of S-adenosyl-L-methionine (SAM) mediated
biomethylations.1 As a result, methylation reactions in-
volved in viral replication become attractive targets for
inhibition.6 Two of the first naturally occurring carbocy-
clic nucleosides to exhibit activity were aristeromycin
(Ari, 1) and neplanocin A (NpcA, 3).7–9 Unfortunately
their close resemblance to adenosine rendered them sus-
Related to this, a report5 of potent activity by two car-
bocyclic pyrimidine nucleosides (Fig. 2) against cytidine
triphosphate (CTP) synthetase, in addition to numerous
substituted pyrimidines against thymidylate synthase,
prompted us to consider the design and synthesis of
additional carbocyclic pyrimidine analogues (Fig. 3),
incorporating the truncated scaffold. In that regard,
the first of the pyrimidine targets is described.
Keywords: Carbocyclic; Pyrimidine; Nucleoside; SAHase; MTAN.
*
Corresponding author. Tel.: +1 410 455 8684; fax: +1 410 455
2608; e-mail: kseley@umbc.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.052

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S. L. Mosley et al. / Bioorg. Med. Chem. 14 (2006) 7967–7971
Figure 2. Carbocyclic derivatives of uridine and cytidine.
Figure 3. Carbocyclic targets.
2. Chemistry
Traditional Mitsunobu coupling was envisioned as a
logical route to realize the targets. The cyclopentenyl
alcohol precursor13,14 was obtained in good yield, and,
as shown in Scheme 1, coupled to N3-Bz-uracil. The
benzoyl-protecting group was removed from the cou-
pled product by stirring with a 1% NaOH solution in
MeOH to afford analogue 13 in 45% yield for two steps.
This precursor is extremely important to this synthesis
because from it three different routes can be employed.
The first included deprotection of analogue 13 with a
mixture of TFA and H2O to result in the first final target
9. Second, in order to get the next immediate target,
analogue 13 was first reacted with TIPBSC1 then fol-
lowed with mild ammonolysis conditions to give ana-
logue 14 which was then deprotected in a similar
fashion as with analogue 9 to give final target 10.
Scheme 2. Reagents and conditions: (a) H2 (25 psi), Pd/C, EtOH, rt,
30 min, quant.; (b) TFA/H2O (2:1), rt, 3 h, 95%; (c) i—TIPBSCI, NEt3,
DMAP, CH3CN, rt, 4 h; ii—NH4OH, rt, 15 h, 65%.
using catalytic hydrogenation to give saturated cyclopen-
tyl analogue 15 (Scheme 2). The isopropylidene group of
analogue 15 was removed to give final target 11. The oxo
group of analogue 15 was also converted to amino 16,
which was then deprotected to give final target 12.
The four targets were then screened for their ability to
inhibit SAHase, DNA methyltransferase (MTase), and
CTP synthetase. No activity was seen for any of the ana-
logues against DNA methyltransferase or CTP synthe-
tase (data not shown). For SAHase, in the first
experiment the concentration of inhibitor was varied,
while the concentration of substrate (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 ab-
sence of inhibitors were measured (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 10 was found to
be 5.0 0.9 lM against SAHase in the hydrolysis direc-
tion, based on a Km value of 7.9 lM for the SAH sub-
strate.15 Ongoing studies suggest that the mechanism
may be more complicated than a simple competitive
inhibition mechanism, so that the ‘true’ inhibitory form
probably binds to the enzyme tighter than SAH. De-
tailed mechanistic studies will be reported in the future.
In order to synthesize the final two targets in this series, it
was necessary to reduce the double bond of the cyclopen-
tyl ring system and as a result analogue 13 was reduced
Scheme 1. Reagents and conditions: (a) i—N3-Bz-uracil, PPh3, DIAD,
CH3CN, 0 ꢁC to rt, 70%; ii—NaOH in MeOH (1%), rt, 15 h, 75%; (b)
TFA/H2O (2:1), rt, 3 h, 95%; (c) i—TIPBSCI, NEt3, DMAP, CH3CN,
rt, 4 h; ii—NH4OH, rt, 15 h, 65%.
The four compounds were also screened for their ability
to inhibit S-adenosylhomocysteine (SAH) nucleosidase

S. L. Mosley et al. / Bioorg. Med. Chem. 14 (2006) 7967–7971
7969
ate inhibitory activity, compound 10 may serve as a lead
compound for different approach to inhibit this
nucleosidase.
3. Experimental
3.1. General
Melting points are uncorrected. 1H and 13C spectra were
operated at 300 and 75 MHz, respectively, all referenced
to internal tetramethylsilane (TMS) at 0.0 ppm. The spin
multiplicities are indicated by the symbols s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), and br
(broad). Reactions were monitored by thin-layer chroma-
tography (TLC) using 0.25 mm Whatman Diamond silica
gel 60-F254 precoated plates. Column chromatography
Figure 4. Inhibition of SAHase by 10. 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 moni-
tored at 265 nm.
˚
was performed on Whatman silica, 200–400 mesh, 60 A
and elution with the indicated solvent system. Yields refer
to chromatographically and spectroscopically (1H and
13C NMR) homogeneous materials.
3.2. (10S, 20R, 30S)-1-[(20,30-O-Isopropylidene)-40-cyclo-
penten-10-yl]uracil (13)
To cyclopenten-ol (6.50 g, 41.67 mmol), N3-Bz-uracil
(18.02 g, 83.33 mmol), and PPh3 (21.88 g, 83.33 mmol)
in anhydrous CH3CN (500 mL) at 0 ꢁC was added drop-
wise DIAD (16.85 g, 83.33 mmol, 16.52 mL). After stir-
ring at rt for 15 h, the mixture was concentrated and
purified by column chromatography (EtOAc/hexanes,
3:1) to afford 9.74 g of 3-benzoyl-10-(20,20-dimethyl-
40,60-dihydro-30H-cyclopenta[10,30]dioxol-40-yl)- 1H-py-
Figure 5. Inhibition of SAHase by 10. In the assay solution, the
concentration of 10 was held constant at 45 lM, while the concentra-
tion of substrate was varied. Rates in the presence (data points at
bottom of plot) and absence (data points at top of plot) of inhibitors
were measured.
1
rimidine-2,4-dione as a white solid. H NMR (DMSO-
d6): d 1.23 (3H, s), 1.3 (3H, s), 4.71 (1H, d), 5.27 (1H,
s), 5.29 (1H, d), 5.81 (1H, d), 5.84 (1H, dd), 6.23 (1H,
dt), 4.71 (1H, d), 5.29 (2H, d), 5.84 (1H, d), 5.85 (1H,
d), 6.22 (1H, m), 7.51 (1H, d), 7.57 (2H, t), 7.78 (1H,
t), 7.97 (2H, m). 13C NMR (DMSO-d6): d 24.4, 26.2,
69.1, 83.2, 84.6, 101.1, 111.8, 128.9, 129.0, 130.1,
131.6, 135.0, 139.0, 143.0, 149.9, 163.0, 168.8.
(EC 3.2.2.9). SAH nucleosidase irreversibly cleaves SAH
and 50-methylthioadenosine (MTA) to form adenine
and the analogous thioribose. This enzyme plays an
important role in microbial pathways such as quorum
sensing, biological methylation, polyamine synthesis,
and methionine recycling. Inhibition of SAH nucleosi-
dase has been shown to lead to a buildup of SAH and
MTA, which are feedback inhibitors to various en-
zymes. This enzyme, encoded by the pfs gene, is present-
ed in about half of all bacterial species, including
Staphylococcus aureus, Enterococcus faecalis, Strepto-
coccus pyogenes, Streptococcus pneumoniae, Mycobacte-
rium tuberculosis, Salmonella typhimurium, Haemophilus
influenzae, Vibrio cholerae, and Bacillus anthracis; but is
not found in mammals.16,17 Knockout of the pfs gene
has been found to be lethal or to markedly impair cell
growth. All together, SAH nucleosidase represents an
attractive target for antimicrobial drug therapy. For
SAH nucleosidase inhibition, concentration of inhibitor
was varied, while the concentration of substrate (SAH)
was held constant at 10.8 lM. Consumption of substrate
was monitored at 265 nm. Compound 10 inhibited
about 50% of the activity at approximately a 13-fold ex-
cess (data not shown). Given the limited structural sim-
ilarity between the SAH substrate and compound 10,
the finding was unexpected. Thus, in spite of its moder-
NaOH (10 mL, 1% in MeOH) was added to the coupled
product and the mixture was allowed to stir at rt for
12 h. After this time, the mixture was neutralized with
HCl (1 N), the solvent removed, and the resulting resi-
due was dissolved in EtOAc (50 mL), washed with
H2O (25 mL), dried (MgSO4), filtered, and concentrated
to give a solid. This solid was purified by column chro-
matography (EtOAc/hexanes, 4:1) to afford 13 (7.50 g,
71%) as a white solid. 1H NMR (DMSO-d6): d 1.22
(3H, s), 1.31, (3H, s), 4.53 (1H, d), 5.27 (2H, m), 5.52
(1H, dd), 5.77 (1H, dd), 6.19 (1H, d), 7.23 (1H, d),
11.31 (1H, s).13C NMR (CD3OD): d 24.4, 26.2, 68.4,
83.4, 84.6, 101.2, 111.7, 129.3, 138.5, 142.5, 151.3,
165.0. Anal. Calcd for C12H14N2O4: C, 57.59; H, 5.64;
N, 11.19. Found: C, 57.92; H, 5.75; N, 11.18.
3.3. (10S, 20R,30S)-1-[(20,30-Dihydroxy)-40-cyclopenten-
10-yl]uracil (9)
A mixture of 13 (0.15 g, 0.60 mmol) in TFA/H2O (2:1,
4 mL) was allowed to stir for 12 h at rt upon which time

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S. L. Mosley et al. / Bioorg. Med. Chem. 14 (2006) 7967–7971
the TFA/H2O was removed by evaporation. The result-
ing residue was coevaporated with MeOH (3· 10 mL) to
remove trace amounts of TFA. The resulting residue
was then purified using C-18 HPLC eluting 90:10,
3.7. (10S,20R,30S)-1-[(20,30-Dihydroxy)-cyclopent-10-
yl]uracil (11)
See procedure for 9 for other reaction details. Compound
15 (0.25 g, 0.99 mmol) and TFA (15 mL) afforded 11
H2O/MeOH ! 50:50,
H2O/MeOH ! 10:90,
H2O/
MeOH afford 9 (0.106 g, 83 %) as a white solid. 1H
NMR (CD3OD): d 4.10 (1H, t), 4.55–4.60 (1H, dd),
5.45–5.50 (1H, dd), 5.70 (1H, d), 5.90 (1H, d), 6.20–
6.23 (1H, dd), 7.40 (1H, dd). 13C NMR (CD3OD): d
66.6, 73.0, 76.5, 101.6, 132.4, 136.7, 142.3, 151.8,
165.0. HRMS: calcd for C9H10N2O4 (M+H)+, 211.19;
found, 211.07.
(0.19 g, 91%) as a white solid. H NMR (CD3OD): d
1
1.69–1.79 (2H, m), 2.06–2.21 (2H, m), 4.06 (1H, m),
4.22–4.25 (1H, dd), 4.61–4.67 (1H, q), 4.86 (1H, s),
5.65–5.67 (1H, d), 7.62–7.64 (1H, d). 13C NMR (CD3OD):
d 19.9, 26.2, 59.5, 77.2, 85.9, 102.4, 141.3, 150.9, 163.6.
Anal. Calcd for C9H12N2O4 (0.6 H2O): C, 48.49, H,
5.96, N, 12.57. Found: C, 48.79, H, 5.51, N, 12.20.
3.4. (10S,20R,30S)-1-[(20,30-O-Isopropylidene)-40-cyclo-
3.8. (10S,20R,30S)-1-[(20,30-O-Isopropylidene)-cyclopent-
penten-10-yl]cytosine (14)
10-yl]cytosine (16)
To a solution of 13 (0.75 g, 3.00 mmol), DMAP
(1.47 g, 12.00 mmol), and Et3N (20 mL) in anhydrous
CH3CN (100 mL) at 0 ꢁC was added portionwise
TIPBSC1 (3.63 g, 12.00 mmol). After stirring at rt
for 3 h, NH4OH (50 mL) was added and the mixture
continued to stir for 12 h at rt. The solvent was then
removed and the resulting residue was purified via
column chromatography (EtOAc/acetone/EtOH/H2O,
6:1:1:0.5) to afford 14 (0.58 g, 78%) as a white solid.
1H NMR (CD3OD): d 1.27 (3H, s) 1.38 (3H, s),
4.53–4.54 (1H, d), 5.32–5.34 (1H, m), 5.43 (1H, m),
5.76–5.78 (1H, m), 5.81–5.83 (1H, d), 6.19–6.21
(1H, m), 7.30–7.32 (1H, m). 13C NMR (CD3OD): d
24.6, 26.3, 69.3, 83.8, 84.7, 94.8, 111.54, 130, 138.1,
See procedure for 14 for reaction details. Compound 15
(0.42 g, 1.67 mmol), DMAP (0.81 g, 6.66 mmol), Et3N
(12 mL), and CH3CN (75 mL) afforded 16 (0.34 g, 81%)
1
as a white solid. H NMR (DMSO-d6): d 1.18 (s, 3H),
1.36 (s, 3H), 1.64–1.80 (m, 2H), 1.94–2.15 (m, 2H),
4.40–4.50 (m, 1H), 4.60–4.68 (dd, 1H), 4.69 (m, 1H),
5.65 (d, 1H), 7.10 (br s, 1H), 7.30 (br s, 1H), 7.50 (d,
1H). 13C NMR (DMSO-d6): d 22.3, 23.8, 29.5, 30.5,
64.9, 80.6, 84.7, 110.7, 144.9, 163.0.
3.9. (10S,20R,30S)-1-[(20,30-Dihydroxy)-cyclopent-10-
yl]cytosine (12)
See procedure for 9 for other reaction details. Com-
pound 16 (0.10 g, 0.40 mmol) and TFA (5 mL) afforded
12 (0.07 g, 85%) as a white solid. 1H NMR (DMSO-d6):
d 1.4–1.6 (m, 2H), 1.8–2.1 (m, 2H), 3.85 (s, 1H), 4.05 (s,
1H), 4.3–4.6 (m, 2H), 4.61–4.8 (m, 1H), 5.65 (s, 1H),
6.95 (s, 2H), 7.55 (d, 1H). 13C NMR (DMSO-d6): d
39.4, 39.6, 63.1, 71.1, 75.5, 93.8, 144.7, 156.6, 165.8.
Anal. Calcd for C9H13N3O3(0.5 H2O): C, 49.08, H,
6.41, N, 19.08. Found: C, 49.21, H, 6.03, N, 18.73.
142.8,
157.4,
166.3.
Anal.
Calcd
for
C12H15N3O3(0.75 H2O): C, 54.88; H, 6.33; N,16.00.
Found: C, 54.64; H, 5.83; N, 15.73.
3.5. (10S, 20R,30S)-1-[(20,30-Dihydroxy)-40-cyclopenten-
10-yl]cytosine (10)
See procedure for 9 for other reaction details. Com-
pound 14 (0.15 g, 0.60 mmol) and TFA (4 mL) afforded
10 (0.11 g, 87%) as a white solid. 1H NMR (CD3OD): d
4.03–4.05 (1H, t), 4.58–4.59 (1H, m), 5.47–5.50 (1H, m),
5.90–5.98 (2H, dd), 6.19–6.21 (1H, m), 7.55–7.56 (1H,
d). 13C NMR (CD3OD): d 67.8, 73.7, 76.9, 95.1, 132.6,
136.4, 142.6, 157.9, 166.0. Anal. Calcd for C9H11N3O3:
C, 51.67, H, 5.30, N, 20.09. Found: C, 51.78, H, 5.39,
N, 19.70.
3.10. SAHase inhibition assay
A reported enzyme-coupled continuous assay in the
hydrolysis direction was employed. In this assay, SAH
is hydrolyzed to homocysteine and adenosine, which is
subsequently converted by adenosine deaminase into
ammonia and inosine, a process associated with an absor-
bance decrease around 265 nm.15 All assays were per-
formed in thermostatted 1 cm quartz cuvettes at 37 ꢁC
maintained by a Peltier unit on a Cary 100 ultraviolet–vis-
ible photospectrometer. In a total volume of 928 lL, a
typical enzyme assay solution contained 50 mM potassi-
um phosphate at pH 7.4, 0.39 units of adenosine deami-
nase (Worthington Biochemical, catalog number
LS009043), 132 nM of SAHase (final concentration; pro-
vided by Lynne Howell), 10 lM of SAH (final concentra-
tion), and various concentrations of inhibitors. The
reactions were initiated by the addition of SAH. Under
these conditions, SAH hydrolysis catalyzed by SAHase
was rate limiting (data not shown). The kinetic data were
analyzed using KaleidaGraph 4.0 (Synergy). Based on a
competitive inhibition mechanism, the Ki value was deter-
mined using the equation, v = kcat · [S] · [E]/
{Km · (1 + [I]/Ki) + [S]}; and v, kcat, [S], [E], Km, [I], and
3.6. (10S,20R,30S)-1-[20,30-(O-Isopropylidene)-cyclopent-
10-yl]uracil (15)
To 13 (0.25 g, 1 mmol) in MeOH (10 mL) was added Pd/
C (10 mol %, 0.025 g). This mixture was hydrogenated
at a pressure of 25 psi for 20 min. The Pd/C was re-
moved by filtration and the filtrate was concentrated
to afford 0.26 g of 15 in quantitative yield as a white sol-
1
id. H NMR (DMSO-d6): d 1.20 (3H, s), 1.35 (3H, s),
1.65–1.82 (2H, m), 1.95–2.15 (2H, m), 4.63 (1H, m),
4.64 (1H, m), 4.71 (1H, dd), 5.52 (1H, d), 7.58 (1H, d)
and 11.26 (1H, s). 13C NMR (DMSO-d6) d 24.4, 27.3,
29.1, 31.2, 63.9, 80.3, 84.4, 101.8, 110.0, 143.9, 152.6,
163.8. Anal. Calcd for C12H16N2O4(0.25 H2O): C,
56.30; H, 6.50; N, 10.95. Found: C, 56.42; H, 6.44;
N,10.88.

S. L. Mosley et al. / Bioorg. Med. Chem. 14 (2006) 7967–7971
7971
Ki stand for the initial reaction rates, rate constant, sub-
References and notes
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tively. The Km value for SAH in the hydrolysis direction
was assumed to be 7.9 lM as previously reported.15
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3.11. SAH nucleosidase assay
In this assay,18 SAH is hydrolyzed into S-ribosylhomo-
cysteine and adenine, which is subsequently converted
by adenine deaminase into ammonia and hypoxanthine,
a process associated with a decrease in absorbance around
265 nm. Assays were performed in thermostatted 1-cm
quartz cuvettes at 37 ꢁC maintained by a Peltier unit on
a Cary 100 ultraviolet–visible photospectrometer. In a to-
tal volume of 920 lL, a typical enzyme assay solution con-
tained 50 mM KPi (pH 7.4), 100 lM MnSO4, 30.0 nM
adenine deaminase, 4.2 nM SAH nucleosidase, 10.8 lM
SAH, and various concentrations of inhibitors. The reac-
tion was initiated by the additionof SAH. Under the assay
conditions, SAH hydrolysis catalyzed by the nucleosidase
was rate limiting (data not shown). Manganese or other
divalent ions (e.g., zinc) are required for adenine deami-
nase activity.19,20
14. Yang, M.; Ye, W.; Schneller, S. W. J. Org Chem. 2004, 69,
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Acknowledgments
This work was supported by the National Cancer Insti-
tute (NCI/NIH RO1 CA 97634 to K.S.R.), the National
Institute of Allergy and Infectious Diseases (NIAID/
NIH, 1R01AI058146 to Z.S.Z.), the National Institutes
of Health/NIGMS Initiative for Minority Student
Development (R25-GM55036 to S.L.M.), and the Her-
man Frasch Foundation (541-HF02 to Z.S.Z.). We are
grateful to Prof. George Carmen of Rutgers University
for the CTP synthetase assay, Prof. Moshe Szyf of
McGill University for the DNA methyltransferase as-
says, and Prof. Lynne Howell of the hospital for Sick
Children for providing the SAHase.
20. Dorgan, K. M.; Zhou, Z. S. unpublished results.