Design, synthesis, and biological evaluation of fluoroneplanocin A as the novel mechanism-based inhibitor of S-adenosylhomocysteine hydrolase

Article abstract of DOI:10.1021/jm025557z

Fluoroneplanocin A (12) was designed as a novel mechanism-based inhibitor of S-adenosylhomocysteine hydrolase (SAH) and efficiently synthesized via an electrophilic vinyl fluorination reaction (n-BuLi, N-fluorobenzenesulfonimide at -78 °C). Fluoroneplanoc

Full text of DOI:10.1021/jm025557z

© Copyright 2003 by the American Chemical Society
Volume 46, Number 2
J anuary 16, 2003
Letters
effective SAH inhibitors have been sought as useful
broad-spectrum antiviral agents.2,3
Design , Syn th esis, a n d Biologica l
Eva lu a tion of F lu or on ep la n ocin A a s th e
Novel Mech a n ism -Ba sed In h ibitor of
S-Ad en osylh om ocystein e Hyd r ola se
A number of compounds have been synthesized and
evaluated as SAH inhibitors.4 These inhibitors are
largely classified into two types. Type I mechanism-
based inhibitors, like neplanocin A, inactivate SAH by
converting cofactor-bound NAD+ into NADH, resulting
in the depletion of NAD+ while the tight binding
intermediate remains at the active site. Such inhibition
of SAH can be reversed after incubation with NAD+ or
dialysis.5 Type II mechanism-based inhibitors, on the
other hand, in addition to causing NAD+ depletion, are
able to bind covalently at the active site of the enzyme,
causing permanent and irreversible inhibition not re-
coverable by the addition of NAD+ or dialysis. Type II
mechanism-based inhibitors are represented by a group
of 4′,5′-modified nucleosides.6
Lak Shin J eong,*,† Su J eong Yoo,† Kang Man Lee,†
Mi J eong Koo,† Won J un Choi,† Hea Ok Kim,‡
Hyung Ryong Moon,† Min Young Lee,†
J ae Gyu Park,† Sang Kook Lee,† and
Moon Woo Chun§
Laboratory of Medicinal Chemistry, College of Pharmacy,
Ewha Womans University, Seoul 120-750, Korea, and
Division of Chemistry and Molecular Engineering and
College of Pharmacy, Seoul National University,
Seoul 151-742, Korea
Received J une 18, 2002
Neplanocin A has been recognized as one of the most
potent inhibitors of SAH.7-9 As shown in Scheme 1, the
SAH-catalyzed oxidation of neplanocin A by cofactor-
bound NAD+ mechanistically leads to the formation of
its keto form 1, which by virtue of being a dead end
product maintains the cofactor permanently in its
reduced form (NADH).4,5 In addition to this well-known
cofactor depletion mechanism, it has been hypothesized
that the keto form 1 is well poised to form a covalent
complex with the enzyme via Michael addition with an
appropriate enzyme nucleophile. However, formation of
this intermediate has never been demonstrated. One
reason for this is probably the expected reversibility of
the Michael adduct 2 due to the acidity of the R-keto
hydrogen (H4). On the basis of this reversible Michael
addition hypothesis, we wanted to demonstrate the
likelihood of this mechanism by designing an appropri-
ate substrate that would provide the enzyme with an
alternative leaving group for the addition-elimination
reaction. The substrate designed for this purpose was
the 6′-fluoro analogue of neplanocin A, which after
Michael addition would have the option of eliminating
the fluoride ion to trap intermediate 3 by forming an
irreversible covalent complex. In this communication,
we report the synthesis of fluoroneplanocin A, a more
Abstr a ct: Fluoroneplanocin A (12) was designed as a novel
mechanism-based inhibitor of S-adenosylhomocysteine hydro-
lase (SAH) and efficiently synthesized via an electrophilic vinyl
fluorination reaction (n-BuLi, N-fluorobenzenesulfonimide at
-78 °C). Fluoroneplanocin A exhibited 2-fold more potent SAH
inhibitory activity than the parent neplanocin A. A new
mechanism of irreversible inhibition discovered in this work
might provide new alternatives in the design of a different
class of antiviral agents operating via SAH inhibition.
In tr od u ction . S-Adenosylhomocysteine hydrolase
(SAH) catalyzes the hydrolysis of S-adenosylhomocys-
teine to adenosine and L-homocysteine. Inhibition of this
enzyme results in the accumulation of S-adenosyl-
homocysteine, which in turn inhibits cellular S-adeno-
syl-L-methionine dependent transmethylation reactions.
Inhibition of methylation, among other things, interferes
with the formation of the 5′-terminal-methylated N7-
guanosine mRNA cap of most animal infecting viruses,
which is necessary for viral replication.1 Therefore,
* To whom correspondence should be addressed. Phone: 82-2-3277-
3466. Fax: 82-2-3277-2851. E-mail: lakjeong@ewha.ac.kr.
† Ewha Womans University.
‡ Division of Chemistry and Molecular Engineering, Seoul National
University.
§ College of Pharmacy, Seoul National University.
10.1021/jm025557z CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/11/2002

202 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Letters
Ta ble 1. Concentration-Dependent Inhibition of SAH by 12
and Neplanocin A
Sch em e 1. Proposed Mechanism for the Reversible
(Neplanocin A, X ) H) and Irreversible
(Fluoroneplanocin A, X ) F) Reactions at the Active Site
of SAH
% activity remaining
compounds 0.2 µM 0.4 µM 0.5 µM 0.6 µM 0.8 µM 1.0 µM
12
100
100
99
99
36
98
0
97
0
54
0
0
neplanocin A
addition of n-BuLi at -78 °C, afforded an inseparable
mixture of 8a and 8b in a 12:1 ratio as determined by
1H NMR. Desilylation of the 8a /8b mixture with n-Bu4-
NF gave both the desired fluoro derivative 9a (63% from
7) and the reduced compound 9b (5.2% from 7) after
purification by silica gel chromatography. In the same
manner as for the synthesis of neplanocin A, the
mesylate ester 10 was successfully condensed with the
adenine anion to give the corresponding protected
carbocyclic nucleoside 11, which was deblocked with
BBr3 to give the target fluoroneplanocin A (12).13
En zym e In h ibition Assa y. Inhibition of SAH by
neplanocin A and its fluoro analogue 12 was measured
using pure recombinant enzyme from human placenta
(Table 1).
Both compounds were preincubated with the enzyme
at various concentrations ranging from 0.1 to 1.6 µM
for 5 min at 37 °C. The residual activity of the enzyme
was determined in the synthetic direction toward S-
adenosylhomocysteine using adenosine and L-homocys-
teine. The results showed that compound 12 (IC50 ) 0.48
µM) was about 2-fold more potent than the parent
neplanocin A (IC50 ) 0.82 µM) as shown in Figure 1 in
Supporting Information. Incubation of the enzyme with
fluoroneplanocin A (12) resulted in concentration- and
time-dependent inhibition of the enzyme, and the
inactivation by 12 was biphasic showing pseudo-first-
order kinetics only in the first period of inactivation
(about 1 min) as shown in Figure 2 in Supporting
Information, indicating that another mechanism, such
as competition between the inhibition process and
substrate activity and competition between reversible
and irreversible processes, might be possibly involved.
Similar biphasic kinetics have also been observed in
several inhibitions by 5′-S-alkynyl- and allenyl-5′-thio-
adenosines14 and (E)-5′,6′-didehydro-6′-deoxy-6′-fluoro-
homoadenosine.15 It was confirmed that fluoroneplano-
cin A (12) was an active-site-directed inhibitor by means
of protection experiments with adenosine (0-250 µM),
as shown in Figure 3 in Supporting Information.14 The
effects of fluoroneplanocin A (12) and neplanocin A on
the NAD+/NADH content of SAH were also determined
after complete inactivation of the enzyme, as shown in
Figure 4 in Supporting Information.14 Fluoroneplanocin
A (12) and neplanocin A resulted in similar conversions
(79% and 88%) in the initial NAD+/NADH ratio as
shown in Table 2 in Supporting Information, indicating
that 12 acts as substrate for the 3′-oxidative activity of
SAH like neplanocin A.
Sch em e 2a
a
Reagents and conditions: (a) I2, CCl4, pyridine, room temp;
(b) NaBH4, CeCl3, MeOH, 0 °C; (c) TBDPSCl, imidazole, 50 °C;
(d) n-BuLi, N-fluorobenzenesulfonimide, THF, -78 °C; (e) n-Bu4NF,
THF, room temp; (f) MsCl, pyridine, room temp; (g) adenine,
K2CO3, 18-crown-6, DMF, 80 °C; (h) (i) BBr3, CH2Cl2, -78 °C; (ii)
Ac2O, pyridine; (iii) NH3, MeOH.
potent inhibitor of SAH than its parent neplanocin A,
which appears to operate by the proposed, novel mech-
anism of inhibition. Synthesis of fluoroneplanocin A was
achieved via a critical electrophilic vinyl fluorination
step.
Resu lts a n d Discu ssion . Syn th esis. For the syn-
thesis of the desired fluoroneplanocin A (Scheme 2), the
known cyclopentenone derivative 410 was converted to
its iodo derivative 5 (I2, CCl4, pyridine),11 which was
selectively reduced to give allylic alcohol 6. After protec-
tion of alcohol 6 as the silyl ether 7, treatment with base
(n-BuLi or LDA) followed by reaction with electrophilic
fluorine (N-fluorobenzenesulfonimide, Selectfluor) gave
only the reduced compound 8b. However, the reverse
addition, consisting of a mixture of compound 7 and
N-fluorobenzenesulfonimide followed by the dropwise
The irreversible nature of the inhibition achieved with
12 was demonstrated in several ways. First, unlike
neplanocin A,5 the level of inhibition of SAH obtained
after preincubation of the enzyme with 0.60 µM of 12
(0% of SAH activity) remained unchanged after dialysis
as shown in Figure 5 in Supporting Information. Second,
and more importantly, unlike neplanocin A,5 the level
of inhibition remained unchanged after the mixture was

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 203
incubated with excess cofactor NAD+ or with excess
substrate adenosine. Third, 19F NMR spectra were
measured to determine the release of the fluoride ion
from compound 12 upon incubation with SAH, as shown
in Figure 6 in Supporting Information.15 Incubation of
either SAH or fluoroneplanocin A (12) alone did not
result in the appearance of a new fluoride ion peak
except for the fluoride peak (-129.1 ppm) derived from
12, as shown in the 19F NMR spectra. When a large
molar excess of 12 was incubated with SAH, a new
fluoride ion peak at -122.8 ppm matching that of
sodium fluoride, the external standard was observed in
addition to the peak from the unreacted 12, indirectly
confirming the formation of the covalently trapped
inhibitor. 19F NMR experiments revealed that the ratio
of moles of fluoride anion released to moles of enzyme
used was 0.80, indicating that the conversion from
intermediate 1 to intermediate 3 (Scheme 1) may not
be completely irreversible when the deprotonation
results in elimination of the enzyme instead of the
fluoride anion. Thus, additional type I reversible mech-
anism by intermediate 1 may also be involved in the
mechanism-based inhibition of SAH. This result might
explain the biphasic kinetic behavior of compound 12.
Overall, this lack of recovery of SAH activity clearly
demonstrates that fluoroneplanocin A (12) is a novel
mechanism-based inhibitor of SAH that possibly oper-
ates by the proposed mechanism described in Scheme
1, and thus, it can be regarded as a new form of type II
mechanism-based inhibitor not reported to date.
In Vitr o Biologica l Eva lu a tion . Antiviral assays
of fluoroneplanocin A (12) against human immunode-
ficiency virus type 1 (HIV-1, MT-4 cells), herpes simplex
virus type 1 (HSV-1, Vero cells), HSV-2 (Vero cells), and
vesicular stomatitis virus (VSV, HeLa cells) were per-
formed. Compound 12 exhibited more potent antiviral
activity (EC50 ) 0.43 µM) against VSV than the control,
ribavirin (EC50 ) 59.0 µM), without cytotoxicity up to
40 µM. However, fluoroneplanocin A (12) showed toxic-
ity-dependent antiviral activities against other viruses
such as HIV-1, HSV-1, and HSV-2.
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derivative followed by elimination by sodium methoxide), but
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available in this patent.
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Con clu sion . Fluoroneplanocin A was designed and
synthesized as a novel probe to investigate the mech-
anism-based inhibition of SAH. Fluoroneplanocin A was
efficiently synthesized via a novel electrophilic vinyl
fluorination reaction under mild conditions, which
contrasts with most conventional harsher fluorinating
procedures. Unlike neplanocin A showing reversible
inhibition of SAH, fluoroneplanocin A exhibited a new
type of irreversible inhibition that should serve as a new
template for the design of new antiviral agents operat-
ing via SAH inhibition.
Ack n ow led gm en t. This research was supported by
a grant from the Korea Science and Engineering Foun-
dation (Grant KOSEF 1999-2-21500-001-3). The authors
also thank Dr. Victor E. Marquez (NCI, NIH) for his
critical review of this manuscript.
Su p p or tin g In for m a tion Ava ila ble: Complete experi-
mental procedures and enzyme assay data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(15) Yuan, C.-S.; Wnuk, S. F.; Liu, S.; Robins, M. J .; Borchardt, R.
T. (E)-5′,6′-Didehydro-6′-deoxy-6′-fluorohomoadenosine: A sub-
strate that measures the hydrolytic activity of S-adenosylho-
mocysteine hydrolase. Biochemistry 1994, 33, 12305-12311.
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J M025557Z