Synthesis of 5′-substituted fluoro-neplanocin A analogues: Importance of a hydrogen bonding donor at 5′-position for the inhibitory activity of S-adenosylhomocysteine hydrolase

Article abstract of DOI:10.1016/j.bmcl.2004.08.047

Four 5′-substituted fluoro-neplanocin A analogues 1a-d were designed and synthesized to study structure-activity relationship against SAH. The inhibitory activity against SAH was in the following order: NH₂ > SH > F, N₃, indicating a

Full text of DOI:10.1016/j.bmcl.2004.08.047

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5641–5644
Synthesis of 50-substituted fluoro-neplanocin A
analogues: importance of a hydrogen bonding donor
at 50-position for the inhibitory activity of
S-adenosylhomocysteine hydrolase
Hyung Ryong Moon,a Hyun Joo Lee,b Kyung Ran Kim,a Kang Man Lee,b
Sang Kook Lee,b Hea Ok Kim,b Moon Woo Chunc and Lak Shin Jeongb,*
aCollege of Pharmacy, Pusan National University, Pusan 609-753, Korea
bLaboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
cCollege of Pharmacy, Seoul National University, Seoul 151-742, Korea
Received 22 July 2004;revised 19 August 2004;accepted 19 August 2004
Available online 16 September 2004
Abstract—Four 50-substituted fluoro-neplanocin A analogues 1a–d were designed and synthesized, using cyclopentenone derivative
2 as a key intermediate. The inhibitory activity against SAH was in the following order: NH2 > SH > F, N3, indicating a hydrogen
bonding donor such as OH or NH2 was essential for inhibitory activity. All the final compounds showed much less decreased cyto-
toxicity in two cancer cell lines (Col2 and A549), implying that phosphorylation of the 50-hydroxyl group of fluoro-neplanocin A is
closely related to its high cytotoxicity.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
cysteine levels, which in turn inhibits virus-encoded
methyltransferases, resulting in the inhibition of matura-
tion of viral mRNA and thus exhibition of antiviral
activity against ( )-RNA viruses, (ꢀ)-RNA viruses,
and some DNA viruses.5–8 Therefore, SAH has been
considered as a promising target for the development
of antiviral agents.
A number of adenosine analogues including neplanocin
A exhibit a broad-spectrum antiviral activity against
DNA and RNA viruses. The antiviral spectrum of these
compounds results from a common mechanism of
action related to S-adenosylhomocysteine hydrolase
(SAH).
Neplanocin A, a naturally occurring carbanucleoside
has been known to be one of the most potent inhibitors
against SAH.9 Fluoro-neplanocin A, which was recently
developed in our laboratory, showed more potent inhib-
itory activity against SAH than neplanocin A and also a
significant antiviral activity.10 However, high cytotoxic-
ity of fluoro-neplanocin A hindered it from being fur-
ther developed as clinically useful antiviral agent. The
cytotoxicity appears to be derived from the conversion
of fluoro-neplanocin A into its triphosphate like neplano-
cin A, possibly interfering with DNA and/or RNA
polymerases.11
The cellular enzyme SAH catalyzes the hydrolysis of S-
adenosylhomocysteine to adenosine and L-homocys-
teine,1,2 which is the only known catabolism pathway
in eukaryotes.3,4 S-Adenosylhomocysteine is a product
of S-adenosylmethionine-dependent methylation reac-
tions by methyltransferases and its accumulation inhib-
its methyltransferases by a feedback mechanism.1,2
Viruses depend on the methylation reactions of their
mRNA at the 50-terminus for the stability and function-
ing of their mRNA. Inhibition of SAH causes a
significant increase in the intracellular S-adenosylhomo-
Therefore, if the 50-hydroxyl group of fluoro-neplanocin
A is removed, a significant decrease of the cytotoxicity
of fluoro-neplanocin A may be expected due to no phos-
phorylation at the 50-position, while maintaining the
Keywords: S-Adenosylhomocysteine hydrolase;Fluoro-neplanocin A;
Hydrogen bonding donor;Cytotoxicity;Phosphorylation.
*
Corresponding author. Tel.: +82 2 3277 3466;fax: +82 2 3277
2851;e-mail: lakjeong@ewha.ac.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.047

5642
H. R. Moon et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5641–5644
NH2
N
Cyclopentenone derivative 2, a key intermediate was
synthesized according to the short and efficient proce-
dure13 developed in our laboratory and converted to
its iodo derivative 3 via addition–elimination reaction
(I2, pyridine, CCl4).14 Iodo compound 3 was stereo
and regioselectively reduced to cyclopentenol 4, result-
ing from the 5-membered cerium chelation between car-
bonyl oxygen and a-oxygen of the isopropylidene
group.15–17 The hydroxyl group of 4 was protected as
TBDPS ether 5. Slow addition of n-BuLi to a mixture
of 5 and N-fluorobenzenesulfonimide afforded vinyl flu-
oride 6 in 51% yield through lithium–iodine exchange
reaction. For the coupling with adenine base, compound
6 was converted to mesylate 8 by desilylation followed
by mesylation of the resulting alcohol 7. Condensation
of 8 with adenine anion in DMF at 80°C afforded the
protected nucleoside 9 (63%). Trityl protecting group
of 9 was selectively removed under p-toluenesulfonic
acid in MeOH to give the corresponding alcohol 10 in
91% yield. Treatment of 10 with diethylaminosulfur tri-
fluoride (DAST) at 0°C produced its fluoro derivative
11, which was deprotected using 33% aqueous trifluoro-
acetic acid to afford the final 50-fluoro-fluoro-neplanocin
A (1a).
NH2
N
N
N
N
N
F
X
X
N
N
R
HO
OH
HO
OH
1a (X = F)
Neplanocin A (X = H, R = CH2OH)
Fluoro-neplanocin A (X = F, R = CH2OH)
Fluoro-DHCeA (X = F, R = H)
1b (X = N3)
1c (X = NH2)
1d (X = SH)
Figure 1. The rationale for the design of the desired nucleosides 1a–d.
inhibitory activity against SAH. Thus, we synthe-
sized fluoro-DHCeA, but it was found to be less potent
than fluoro-neplanocin A against SAH, indicating that
the 50-hydroxyl group might be essential for hydrogen
bonding.12
On the basis of these findings, it was interesting to de-
sign and synthesize 50-substituted fluoro-neplanocin A
derivatives 1a–d, which can serve as a hydrogen bonding
acceptor (F or N3), a hydrogen bonding donor (NH2),
or as a bioisostere (SH), without being phosphorylated
at the 50-position (Fig. 1). From this study, we discov-
ered that the 50-position of fluoro-neplanocin A should
be substituted with a hydrogen bonding donor such as
hydroxyl or amino group. Herein, we wish to report
the structure–activity relationship study of 50-substi-
tuted fluoro-neplanocin A derivatives 1a–d as S-adeno-
sylhomocysteine hydrolase inhibitors.
50-Azido- and 50-amino-fluoro-neplanocin A derivatives,
1b and 1c were synthesized, as depicted in Scheme 2. For
the introduction of a hydrogen bonding acceptor, azido
group at 50-position, the 50-hydroxyl group of 10 was
mesylated and then treated with sodium azide in DMF
at 60°C for 1h to give the azide 13. Treatment of 13
under acidic conditions gave the 50-azido-fluoro-neplano-
cin A (1b). For the synthesis of 50-amino derivative with
hydrogen bonding donor ability, compound 1b was con-
verted to the 50-amino-fluoro-neplanocin A (1c) using
LindlarÕs catalyst.18
2. Results and discussion
2.1. Synthesis
Finally, fluoro-neplanocin A analogue 1d with 50-sulf-
hydryl group was synthesized as a bioisostere of
fluoro-neplanocin A (Scheme 3). For the introduction
of thiol group, the same SN2 reaction was utilized, as
in the introduction of azido group in Scheme 2. Reac-
As shown in Scheme 1, fluoro-neplanocin A analogue
1a, whose 50-position was substituted with a hydrogen
bonding acceptor, fluorine was synthesized from D-
ribose.
Scheme 1. Reagents and conditions: (a) I2, pyridine, CCl4, rt, 12h;(b) NaBH 4, CeCl3, MeOH, 0°C, 30min;(c) TBDPSCl, imidazole, DMF, 40 °C,
overnight;(d) N-fluorobenzene sulfonimide, n-BuLi, THF, ꢀ78°C, 1h;(e) n-Bu4NF, THF, rt, 2h;(f) methanesulfonyl chloride, Et 3N, CH2Cl2, 0°C,
30min;(g) adenine, K 2CO3, 18-Crown-6, DMF, 80°C, overnight;(h) p-toluenesulfonic acid, MeOH, rt, 18h;(i) DAST, CH 2Cl2, 0°C, 30min;(j) 33%
aqueous CF3CO2H, THF, rt, 3d.

H. R. Moon et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5641–5644
5643
Table 1. Inhibitory activity of the final compounds 1a–d against SAH
NH2
N
N
F
X
N
N
HO
OH
Compound
IC50 (lM)a
Fluoro-neplanocin A (X = OH)b
1a (X = F)
1b (X = N3)
0.48
>100
>100
1c (X = NH2)
1d (X = SH)
12.68
97.27
a Determined using pure recombinant enzyme obtained from human
placenta.
b Data from Ref. 10.
Scheme 2. Reagents and conditions: (a) methanesulfonyl chloride,
Et3N, CH2Cl2, ꢀ5°C, 20min;(b) NaN 3, DMF, 60°C, 1h;(c) 33%
aqueous CF3CO2H, THF, rt, 3d;(d) Lindlar Õs catalyst, H2, MeOH, rt,
30min.
no enzyme inhibitory activity (IC50 > 100lM), while
introduction of a bioisosteric hydrogen bonding donor
(1c, X = NH2) at the 50-position of fluoro-neplanocin
A restored enzyme inhibitory activity (IC50 = 12.68lM)
dramatically. This trend was proved by compound 1d
(X = SH) with 50-thiol group, which possesses a hydro-
gen atom, but has no ability to form appropriate hydro-
gen bonding with the active site residues of the enzyme.
As expected, compound 1d showed very weak enzyme
inhibition (IC50 = 97.27lM). The inhibitory activity of
the tested compounds against SAH was in the following
order: OH > NH2 > SH > F, N3, indicating the intro-
duction of a hydrogen bonding donor such as OH or
NH2 at the 50-position of fluoro-neplanocin A is essen-
tial for enzyme inhibitory activity.
NH2
N
NH2
N
N
N
N
N
F
F
AcS
MsO
N
N
a
91%
O
O
O
O
12
14
b
71%
NH2
N
NH2
N
N
N
N
N
F
F
c
HS
AcS
N
N
65%
HO
OH
2.3. Cytotoxicity assay
HO
OH
1d
15
Cytotoxicity of the synthesized compounds 1a–d was
measured in human colon cancer cell lines (Col2) and
human lung cancer cell lines (A549) to determine if 50-
hydroxyl group of fluoro-neplanocin A is responsible
for its cytotoxicity.
Scheme 3. Reagents and conditions: (a) KSAc, DMF, rt, 1h;(b) 33%
aqueous CF3CO2H, THF, rt, 4d;(c) 28% NH 4OH, MeOH, rt, 20min.
tion of 12 with potassium thioacetate in DMF at room
temperature produced thioacetate 14 in a good yield.
Successive deprotection of the isopropylidene group
and the S-acetyl group of 14 afforded the 50-sulf-
hydryl-fluoro-neplanocin A (1d).
Table 2. Cytotoxicity of the final compounds 1a–d in two cancer cell
lines (Col2, A549)
NH2
N
N
2.2. Enzyme assay
F
X
N
SAH inhibitory activity of all synthesized 50-substituted
fluoro-neplanocin A analogues 1a–d was measured
using pure recombinant enzyme obtained from human
placenta (Table 1).
N
HO
OH
Compound
IC50 (lM)a
A549c
4.6
>100
53.0
42.8
>100
Col2b
1.4
>100
35.9
39.2
>100
All compounds were preincubated with the enzyme
SAH at various concentrations for 5min at 37°C. The
residual activity of the enzyme was measured in the syn-
thetic direction toward S-adenosylhomocysteine using
adenosine and L-homocysteine. As shown in Table 1,
the parent fluoro-neplanocin A (X = OH) exhibited
the most potent enzyme inhibitory activity
(IC50 = 0.48lM). Substitution of 50-hydroxyl group of
fluoro-neplanocin A with a bioisosteric hydrogen
bonding acceptor (1a, X = F or 1b, X = N3) resulted in
Fluoro-neplanocin A (X = OH)
1a (X = F)
1b (X = N3)
1c (X = NH2)
1d (X = SH)
a Indicative of 50% survival determined with serial dilutions of the test
compound.
b Human colon carcinoma cells.
c Human lung carcinoma cells.

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H. R. Moon et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5641–5644
As shown in Table 2, all compounds 1a–d exhibited
much less cytotoxicity than fluoro-neplanocin A in
human colon cancer cell lines (Col2) and human lung
cancer cell lines (A549). Azido and amino derivatives,
1b and 1c showed a 26- and 28-fold decreased cytotoxic-
ity in human colon cancer cell lines (Col2), respectively,
and fluoro and sulfhydryl compounds, 1a and 1d did not
exhibit any cytotoxicity up to 100lM. The similar trend
was observed in human lung cancer cell lines (A549).
This result indicates that conversion of 50-hydroxyl
group of fluoro-neplanocin A into its triphosphate
might be the major cause of high cytotoxicity.
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We synthesized novel 50-substituted fluoro-neplanocin A
derivatives to find out if the 50-hydroxyl group of fluoro-
neplanocin A was essential for hydrogen bonding with
the enzyme SAH and responsible for high cytotoxicity.
From this study, we revealed a hydrogen bonding donor
such as OH or NH2 was essential for enzyme inhibitory
activity and also discovered there was a close correlation
between cytotoxicity and 50-phosphorylation of fluoro-
neplanocin A like neplanocin A. These findings will pro-
vide great help for the design of potent and non-toxic
SAH inhibitors.
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This work was supported by the grant from the Korea
Health R&D Project, Ministry of Health & Welfare,
Korea (HMP-02-PJ2-PG10-21503-0004).