Discovery of type II (Covalent) inactivation of S-adenosyl-L- homocysteine hydrolase involving its 'hydrolytic activity': Synthesis and evaluation of dihalohomovinyl nucleoside analogues derived from adenosine

Article abstract of DOI:10.1021/jm9801410

Treatment of the 5'-carboxaldehyde derived by Moffatt oxidation of 6-N- benzoyl-2',3'-Oisopropylideneadenosine (1) with the '(bromofluoromethylene)triphenylphosphorane' reagent and deprotection gave 9- (6-bromo-5,6-dideoxy-6-fluoro-β-D-ribo-hex-5-enofuran

Full text of DOI:10.1021/jm9801410

3078
J . Med. Chem. 1998, 41, 3078-3083
Discover y of Typ e II (Cova len t) In a ctiva tion of S-Ad en osyl-L-h om ocystein e
Hyd r ola se In volvin g Its “Hyd r olytic Activity”: Syn th esis a n d Eva lu a tion of
Dih a loh om ovin yl Nu cleosid e An a logu es Der ived fr om Ad en osin e1
Stanislaw F. Wnuk,†,# Yue Mao,†,∇ Chong-Sheng Yuan,§,| Ronald T. Borchardt,§ Graciela Andrei,‡ J an Balzarini,‡
Erik De Clercq,‡ and Morris J . Robins*,†
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700, Department of Biochemistry,
University of Kansas, Lawrence, Kansas 66045, and Rega Institute for Medical Research, Katholieke Universiteit of Leuven,
Leuven, Belgium
Received March 6, 1998
Treatment of the 5′-carboxaldehyde derived by Moffatt oxidation of 6-N-benzoyl-2′,3′-O-
isopropylideneadenosine (1) with the “(bromofluoromethylene)triphenylphosphorane” reagent
and deprotection gave 9-(6-bromo-5,6-dideoxy-6-fluoro-â-D-ribo-hex-5-enofuranosyl)adenine (4).
Parallel treatment with a “dibromomethylene Wittig reagent” and deprotection gave 9-(6,6-
dibromo-5,6-dideoxy-â-D-ribo-hex-5-enofuranosyl)adenine (7), which also was prepared by
successive bromination and dehydrobromination of the 6′-bromohomovinyl nucleoside 8.
Bromination-dehydrobromination of the 5′-bromohomovinyl analogue 11 and deprotection gave
(E)-9-(5,6-dibromo-5,6-dideoxy-â-D-ribo-hex-5-enofuranosyl)adenine (15). Compounds 4, 7, and
15 were designed as putative substrates of the “hydrolytic activity” of S-adenosyl-L-homocysteine
(AdoHcy) hydrolase. Enzyme-mediated addition of water across the 5,6-double bond could
generate electrophilic acyl halide or R-halo ketone species that could undergo nucleophilic attack
by proximal groups on the enzyme. Such type II (covalent) mechanism-based inactivation is
supported by protein labeling with 8-[3H]-4 and concomitant release of bromide and fluoride
ions. Incubation of AdoHcy hydrolase with 7 or 15 resulted in irreversible inactivation and
release of bromide ion. In contrast with type I mechanism-based inactivation, reduction of
enzyme-bound NAD+ to NADH was not observed. Compounds 4, 7, and 15 were not inhibitory
to a variety of viruses in cell culture, and weak cytotoxicity was observed only for CEM cells.
In tr od u ction
dehydro-6′-deoxy-6′-halohomoadenosines, EDDHHAs]
inhibit AdoHcy hydrolase and are enzymatically hydro-
lyzed to produce “homoadenosine-6′-carboxaldehyde”
which undergoes spontaneous decomposition.7 The
hydrolytic (C5′/C6′) and oxidative (C3′) activities of
AdoHcy hydrolase were differentiated most effectively
with the 6′-fluoro analogue (EDDFHA)7b which was
employed in a study that implicated Lys-426 as a crucial
residue for the hydrolytic activity of the enzyme.8 We
now report the design, synthesis, and bioactivity of the
geminal and vicinal (dihalohomovinyl)adenosines 4, 7,
and 15. These analogues were designed as putative new
substrates for the hydrolytic activity of AdoHcy hydro-
lase that might cause type II (covalent2b) mechanism-
based inhibition. Addition of enzyme-sequestered water
across the 5′,6′-double bond7 of such dihalohomovinyl
analogues followed by loss of hydrogen halide might
produce an electrophilic R-halomethyl ketone or acyl
halide (Figure 1). Nucleophilic attack by proximal
amino acid functionalities (e.g., an NH2 group on Lys-
426 or Arg-196) might cause type II inactivation of the
enzyme.
The cellular enzyme S-adenosyl-L-homocysteine
(AdoHcy) hydrolase (EC 3.3.1.1) effects cleavage of
AdoHcy to adenosine and L-homocysteine. Since AdoHcy
is a potent feedback inhibitor of crucial transmethyla-
tion enzymes, the design of inhibitors of AdoHcy hy-
drolase represents a rational approach for anticancer
and antiviral chemotherapy.2 The Z-isomer of 4′,5′-
didehydro-5′-deoxy-5′-fluoroadenosine3 (ZDDFA) and its
chloro analogue4 are potent mechanism-based inacti-
vators of AdoHcy hydrolase. The mechanism of inac-
tivation by ZDDFA involves addition of water at C5′ of
ZDDFA, elimination of hydrogen fluoride to give the
adenosine-5′-carboxaldehydes5a (nonlethal event), and
their oxidation to 3′-keto derivatives (lethal event) with
concomitant reduction of E‚NAD+ to E‚NADH5b (“co-
factor depletion” or type I inhibition2b). This indicated
that AdoHcy hydrolase has “hydrolytic activity” (addi-
tion of water at C5′) that functions independently of its
C3′-oxidation activity.5b
Our homologated halovinyl analogues6 [(E)-5′,6′-di-
* To whom correspondence should be addressed at Brigham Young
University.
Homologated 5′,5′-dibromomethylene-5′-deoxyuridine9
and adenosine10 analogues have been synthesized from
nucleoside 5′-carboxaldehydes (Corey-Fuchs proce-
dure,11 CBr4/PPh3/Zn) and have been converted into 5′-
deoxy-5′-methynyl nucleosides (4′-acetylenic deriva-
tives).9,10 The acetylenic analogue derived from adenosine
is a potent type II inhibitor of AdoHcy hydrolase6,12 with
† Brigham Young University.
§ University of Kansas.
‡ Katholieke Universiteit of Leuven.
#
Current address: Department of Chemistry, Florida International
University, Miami, FL.
∇ Current address: Abbott Laboratories, Abbott Park, IL.
| Current address: Tanabe Research Laboratories, Inc., San Diego,
CA.
S0022-2623(98)00141-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/25/1998

Dihalohomovinyl Nucleosides Derived from Adenosine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3079
Sch em e 2a
a
(a) Br2/CH2Cl2; (b) DBU/CH2Cl2; (c) NH3/MeOH; (d) TFA/H2O.
F igu r e 1. Possible generation of active intermediates from
5′,6′- and 6′,6′-dihalohomovinyl nucleoside analogues by the
“hydrolytic activity” of AdoHcy hydrolase.
Sch em e 1a
F igu r e 2. Time-dependent inactivation of AdoHcy hydrolase
with 4. AdoHcy hydrolase (20 nM) was incubated with 4 (0,
2.5 µM; ], 5 µM; O, 10 µM; 4, 20 µM) in buffer A at 37 °C. At
the indicated time points, residual enzyme activity was
determined in the synthetic direction5b as described in the
a
(a) DMSO/DCC/Cl2CHCO2H; (b) CBr3F/Ph3P/Zn/CH2Cl2; (c)
-1
Experimental Section. Inset: Plot of (kapp
)
versus [I]-1 from
NH3/MeOH; (d) TFA/H2O; (e) CBr4/Ph3P/Zn/CH2Cl2; (f) Br2/CH2Cl2;
(g) DBU/THF.
which the Ki and kinact values were calculated.17 Data are the
average of duplicate measurements.
antiviral6,10 and cytostatic activity.6 We also have
prepared 4′-acetylenic derivatives by oxidative destan-
nylation (lead tetraacetate) of vinyl 6′-stannanes.6
diazabicyclo[5.4.0]undec-7-ene (DBU) gave 6 (H6′ of 9
more acidic than H5′) which was deprotected to give the
6′,6′-dibromohomovinyl product 7 (73%).
Bromination of homovinyl derivative 106,15 gave 5′,6′-
dibromo diastereomers which were dehydrobrominated
(DBU) to give 5′-bromovinyl compound 116 (Scheme 2).
A second addition of bromine (with 11) gave 5′,5′,6′-
tribromo derivative 12 which decomposed during col-
umn chromatography. One-flask treatment of 11 with
bromine followed by dehydrobromination (DBU) gave
5′,6′-dibromohomovinyl derivative 13 (64%) as a single
diastereomer. The E configuration is expected from an
E2 (anti elimination) process involving the most stable
conformations of 12 with one bromine atom on C5′ anti
to the bromine on C6′. Debenzoylation of 13 and
deacetonization of 14 gave the crystalline 5′,6′-dibro-
mohomovinyl target 15 (64%).
Ch em istr y
Moffatt oxidation13 of 6-N-benzoyl-2′,3′-O-isopropyl-
ideneadenosine (1) and treatment of the 5′-carboxalde-
hyde (crude) with (bromofluoromethylene)triphen-
ylphosphorane (generated11 in situ with CBr3F/Ph3P/
Zn) gave the bromofluorovinyl diastereomers 2 (E/ Z,
∼3:2, 30%; Scheme 1). Stereochemistry was assigned
from J F-H5′ coupling constants [29.2 Hz (E) and 11.8 Hz
(Z)]. Analogous treatment of the purified 5′-carboxal-
dehyde13 gave 2 in similar yields. Debenzoylation of 2
(NH3/MeOH) gave 3 which was deprotected (TFA/H2O)
to give 4 (21% overall from 1). RP-HPLC purification
of 4 gave fractions enriched in E- or Z-isomers (71:29
or 44:56).
Oxidation13 of 1 and treatment of the 5′-carboxalde-
hyde (crude or purified) with (dibromomethylene)tri-
phenylphosphorane (CBr4/Ph3P/Zn)11 gave the 6′,6′-
dibromohomovinyl analogue 5 (55-64%) which was de-
benzoylated to give 6. Treatment of the bromohomovi-
nyl derivative 86 (prepared by halodestannylation of the
vinyl 6′-stannanes6,14) with bromine gave the somewhat
unstable 5′,6′,6′-tribromo diasteromers 9 (46% after
column chromatography). Treatment of 9 with 1,8-
In a ctiva tion of S-Ad en osyl-L-h om ocystein e
Hyd r ola se
Recombinant human placental AdoHcy hydrolase was
irreversibly inactivated by 4, 7, and 15. Inactivation
with 4 was shown to be both concentration- and time-
dependent (Figure 2) and resulted in covalent linkage
of the enzyme and inhibitor with concomitant release
of halide ions (F- and Br-). Ki values for 4, 7, and 15

3080 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Wnuk et al.
Ta ble 1. Kinetic Constants for Inhibition of AdoHcy
Hydrolase by 4, 7, and 15
An tivir a l a n d Cytotoxicity Eva lu a tion
Compounds 4, 7, and 15 were evaluated for their
inhibitory activity against a variety of viruses. These
included herpes simplex virus type 1 (HSV-1) (strains
KOS, F, and McIntyre), HSV-2 (strains G, 196, and
Lyons), thymidine kinase-deficient HSV-1/TK- (strains
B2006 and VMW 1837), vaccinia virus, and vesicular
stomatitis virus in E6SM cell cultures; varicella-zoster
virus (strains YS and Oka) and cytomegalovirus (strains
Davis and AD 196) in HEL cell cultures; Coxsackie virus
B4, parainfluenza virus-3, Sindbis virus, reovirus-1, and
Punta Toro virus in Vero cell cultures; and human
immunodeficiency virus type 1 (HIV-1) (strain IIIa) and
HIV-2 (strain ROD) in CEM cell cultures. No inhibitory
effects were observed with 4, 7, or 15 in any of the virus
cultures at subtoxic concentrations (EC50 >100-500
µM). These compounds had weak cytostatic activity
only against CEM cells (IC50 75-160 µM).
compd
Ki (µM)
kinact (min-1
)
kinact/Ki (min-1 M-1
)
4
7
15
3.9
3.1
3.6
0.040
0.036
0.081
1.02 × 104
1.16 × 104
2.25 × 104
Su m m a r y a n d Con clu sion s
We have designed and synthesized the dihalohomovi-
nyl nucleoside analogues 4, 7, and 15 (from adenosine
or hexofuranose precursors) that demonstrate type II
(covalent) mechanism-based inactivation of S-adenos-
ylhomocysteine hydrolase. These are the first examples
of type II inhibitors that are activated by the “hydrolytic
activity” of the enzyme without prior oxidation at C3′
(i.e., reduction of enzyme-bound NAD+ to NADH was
not observed). It is noteworthy that only moderate
cytotoxicity in one cell line (CEM) was observed with 4,
7, and 15. This is in harmony with our goal to minimize
cellular toxicity with these 5′-modified adenosine ana-
logues (which do not contain a 5′-hydroxyl group that
could be phosphorylated and undergo further metabolic
processing) that become inactivators only after mech-
anism-based interaction with the targeted enzyme. The
present compounds appear to be activated exclusively
by the 5′/6′-hydrolytic activity of AdoHcy hydrolase to
generate electrophilic species that undergo nucleophilic
attack by protein residues to give half of the sites
covalent binding, and the resulting functional tet-
rameric subunit complexes retained both oxidative and
hydrolytic activity.16 Further treatment of such par-
tially inactivated enzymes with a type I inhibitor caused
complete inactivation. Studies are in progress to further
probe these fascinating observations and utilize them
in the design of selective inhibitors for human versus
parasite AdoHcy hydrolases.
F igu r e 3. Stoichiometry of the covalent binding of 4 with
AdoHcy hydrolase. AdoHcy hydrolase (0.5 mg) was incubated
with 8-[3H]-4 (500 µM, 187 Ci/mol) in buffer A (250 µL) at 37
°C. At each time point, the reaction mixture was analyzed as
described in the Experimental Section (b, residual enzyme
activity; 9, covalently bound 8-[3H]-4).
were in the range of 3-4 µM, and the kinact value for 15
was about twice those for 4 and 7 (Table 1). Enzyme-
mediated addition of water at C6′ of 4 or 7 followed by
elimination of hydrogen halide could generate an elec-
trophilic acyl halide, whereas addition of water at C5′
of 15 could generate an R-halo ketone (Figure 1).
Nucleophilic attack by proximal group(s) on the enzyme
could result in type II mechanism-based enzyme inac-
tivation. Such type II inhibitors that were specifically
activated by the “hydrolytic activity” of AdoHcy hydro-
lase (i.e., without prior oxidation at C3′) would be
unique.
Incubation of 4 with the enzyme resulted in release
of Br- and F- ions and adenine (Ade). Approximately
2 mol equiv of 4 was covalently incorporated per mole
of the tetrameric enzyme at ∼80% inactivation (Figure
3). The rate constant for Br- release was greater than
those for release of F- and Ade (data not shown), and
the stoichiometry for release of Br-/F-/Ade was ap-
proximately 1:1:0.5 per inactivated enzyme subunit. The
mechanism for enzyme inactivation with 8-[3H]-4 is
proposed16 to involve enzyme-mediated addition of water
at C6′ followed by elimination of HBr to give the
homoAdo-6′-carboxylic acid fluoride (HACF). HACF is
partitioned by (a) attack by a protein nucleophile and
expulsion of F- to form a covalent bond or (b) de-
purination7b to release Ade and sugar byproducts that
release further fluoride ion. The partition ratio of
pathways a/b was approximately 1:1, and two enzymatic
turnovers result in one lethal event (enzyme inactiva-
tion). In contrast with type I mechanism-based inhibi-
tion (“cofactor depletion” in which enzyme-bound NAD+
is reduced to NADH), inactivation of AdoHcy hydrolase
with 4, 7, and 15 did not result in changes in the initial
NAD+/NADH ratios (data not shown).
Exp er im en ta l Section
Uncorrected melting points were determined with a capil-
lary apparatus. UV spectra were measured with solutions in
MeOH. 1H (200 or 500 MHz), 13C (50 or 125 MHz), and 19F
[470.3 MHz (CCl3F)] NMR spectra were determined with
solutions in CDCl3 unless otherwise specified. Mass spectra
(MS and HRMS) were obtained with electron impact (20 eV),
chemical ionization (CI, isobutane), or fast atom bombardment
(FAB, thioglycerol matrix) techniques. Merck Kieselgel 60-
F254 sheets were used for TLC, and products were detected with
254-nm light. Merck Kieselgel 60 (230-400 mesh) was used
for column chromatography. Preparative reversed-phase (RP)-
HPLC was performed with a Dynamax C18 column with a
Spectra Physics SP 8800 ternary pump system (gradient
solvent systems are noted). Elemental analyses were deter-
mined by M-H-W Laboratories, Phoenix, AZ. Reagent grade
chemicals were used, and solvents were dried by reflux over

Dihalohomovinyl Nucleosides Derived from Adenosine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3081
and distillation from CaH2 (except THF/potassium) under an
argon atmosphere. Sonication was performed with a 300
Ultrasonik unit.
6.04 (m, 1, H1′), 6.14 (dd, J 5′-F ) 12.4 Hz, 0.4, H5′Z), 8.26 (s,
1, H2), 8.33 (s, 1, H8); 13C NMR (Me2SO-d6) δ 72.75 and 72.85
(C3′), 73.89 and 74.06 (C2′), 77.72 and 81.34 (d, J C4′-F ) 7.7
Hz) (C4′), 87.66 and 87.72 (C1′), 110.53 (d, J C5′-F ) 16.0 Hz)
and 112.41 (d, J C5′-F ) 7.6 Hz) (C5′), 119.23 (C5), 134.29 (d,
J C6′-F ) 322.0 Hz) and 137.53 (d, J C6′-F ) 315.9 Hz) (C6′),
139.89 (C8), 149.23 (C4), 152.58 (C2), 156.06 (C6); 19F NMR
(Me2SO-d6/CCl3F) δ -71.08 (d, J F-H5′ ) 32.3 Hz, F6′E), -66.26
(d, J F-H5′ ) 12.9 Hz, F6′Z); HRMS m/z 361.0013 (94, M+
[C11H1181BrFN5O3] ) 361.0009), 359.0034 (100, M+ [79Br] )
359.029). Anal. [C11H11BrFN5O3‚0.5H2O (369.2)] C, H, N.
6-N-Ben zoyl-9-(6-br om o-5,6-d id eoxy-6-flu or o-2,3-O-iso-
p r op ylid en e-â-D-r ibo-h ex-5-en ofu r a n osyl)a d en in e (2). A
solution of 113 (1.23 g, 3 mmol) and N,N′-dicyclohexylcarbodi-
imide (DCC; 1.84 g, 9 mmol) in dried Me2SO (7.5 mL) was
cooled (∼10 °C) under argon, Cl2CHCO2H (0.12 mL, 193 mg,
1.5 mmol) was added, and stirring was continued for 90 min
at ambient temperature. The red-brown solution of 5′-car-
boxaldehyde was injected (syringe) into a mixture containing
(bromofluoromethylene)triphenylphosphorane [generated in
situ by stirring CBr3F (0.53 mL, 1.42 g, 5.25 mmol), Ph3P (1.37
g, 5.25 mmol), and activated Zn (dust; 343 mg, 5.25 mmol) in
dried CH2Cl2 (20 mL) for 5 h at ambient temperature under
Ar; sonication was applied intermittently for a total of 30 min],
stirring was continued for 16 h, and oxalic acid dihydrate (756
mg, 6 mmol) in MeOH (15 mL) was added. After 20 min the
reaction mixture was concentrated (to ∼1/3 volume), the
dicyclohexylurea was filtered and washed with cold MeOH,
and the combined filtrates were evaporated (in vacuo). The
residue was partitioned (NaHCO3/H2O//CHCl3), the organic
layer was washed (H2O, brine) and dried (MgSO4), and
volatiles were evaporated. Column chromatography of the
residue (EtOAc f 1% MeOH/EtOAc) gave 2. Repeated chro-
matography of this material gave 2 (E/ Z, ∼3:2; 360 mg,
30%): 1H NMR δ 1.39 and 1.67 (2 × s, 2 × 3, 2 × Me), 4.91
(ddd, J 4′-5′ ) 9.2 Hz, J 4′-3′ ) 2.2 Hz, J 4′-F ) 2.2 Hz, 0.4, H4′Z),
The preparation of 8-[3H]-4 was modeled by heating a
suspension of 4 (5 mg) in D2O (0.7 mL) at 85 °C for 6 h. The
resulting 8-[2H]-4 had 1H NMR (Me2SO-d6) δ 8.13 (s, 1, H2)
[but no peak at δ 8.37 (2H8)]; MS (FAB) m/z 363 (95, MH+
2
[81Br, H8]), 361 (100, MH+ [79Br,2H8]).
6-N-Ben zoyl-9-(6,6-d ibr om o-5,6-d id eoxy-2,3-O-isop r o-
p ylid en e-â-D-r ibo-h ex-5-en ofu r a n osyl)a d en in e (5). Com-
pound 1 (1.23 g, 3 mmol) was oxidized13 and the resulting
(crude) 5′-carboxaldehyde was treated with (dibromomethyl-
ene)triphenylphosphorane [generated in situ by stirring CBr4
(1.74 g, 5.25 mmol), Ph3P (1.37 g, 5.25 mmol), and activated
Zn (dust; 343 mg, 5.25 mmol) in dried CH2Cl2 (20 mL) for 5 h
at ambient temperature under Ar; sonication was applied
intermittently for a total of 15 min] for 4 h, as described for
the synthesis of 2. Analogous workup and purification gave
510 (1.09 g, 64%) as a light-tan solid: 1H NMR δ 1.40 and 1.55
(2 × s, 2 × 3, 2 × Me), 4.94 (dd, J 4′-5′ ) 8.1 Hz, J 4′-3′ ) 3.0 Hz,
1, H4′), 5.16 (dd, J 3′-2′ ) 6.0 Hz, 1, H3′), 5.56 (dd, J 2′-1′ ) 1.5
Hz, 1, H2′), 6.11 (d, 1, H1′), 6.59 (d, 1, H5′), 7.39-8.14 (m, 6,
Arom, NH), 7.97 (s, 1, H2), 8.74 (s, 1, H8); HRMS (CI) m/z
567.9854 (48, MH+ [C21H2081Br2N5O4] ) 567.9841), 565.9875
5.09-5.19 (m, 1.6, H3′, H4′E), 5.40 (dd, J 5′-4′ ) 9.2 Hz, J 5′-F
29.2 Hz, 0.6, H5′E), 5.58-5.67 (m, 1, H2′), 5.83 (dd, J 5′-F
)
)
11.8 Hz, 0.4, H5′Z), 6.12 (s, 1, H1′), 7.44-8.12 (m, 6, Arom,
NH), 8.84 (s, 1, H2), 9.03 (s, 1, H8); 19F NMR δ -68.11 (d,
J F-5′ ) 29.2 Hz, 0.6, F6′E), -63.62 (d, J F-5′ ) 11.8 Hz, 0.4,
F6′Z).
(100, MH+
563.9882).
[
81/79Br2] ) 565.9862), 563.9883 (56, MH+ [79Br2] )
Analogous treatment (24 h, ambient temperature, under Ar;
inverse Wittig reagent addition via a double-ended needle) of
the purified 6-N-benzoyl-2′,3′-O-isopropylideneadenosine-5′-
carboxaldehyde [prepared from the aldehyde hydrate (200 mg,
0.468 mmol) as described13] in dried CH2Cl2 (25 mL) with
(bromofluoromethylene)triphenylphosphorane [prepared from
CBr3F (0.092 mL, 254 mg, 0.937 mmol), Ph3P (245 mg, 0.937
mmol), and activated Zn (dust; 61 mg, 0.937 mmol) in dried
CH2Cl2 (5 mL)], evaporation, and chromatography (CH2Cl2 f
3% EtOH/CH2Cl2) gave 2 (71 mg, 30%) plus recovered aldehyde
hydrate (85 mg, 43%).
Analogous treatment of the 6-N-benzoyl-2′,3′-O-isopropyl-
ideneadenosine-5′-carboxaldehyde [prepared from the purified
aldehyde hydrate13 (200 mg, 0.47 mmol)] with (dibromometh-
ylene)triphenylphosphorane [prepared from CBr4/Ph3P/Zn (0.94
mmol)] gave 5 (145 mg, 55%).
9-(6,6-Dib r om o-5,6-d id eoxy-2,3-O-isop r op ylid en e-â-D-
r ibo-h ex-5-en ofu r a n osyl)a d en in e (6). Meth od A. DBU
(40 µL, 41 mg, 0.27 mmol) was added to a solution of 9 (50
mg, 0.092 mmol) in dried THF (25 mL) under argon at ∼0 °C.
Stirring was continued at ∼0 °C for 30 min and then at
ambient temperature for 2 h. Volatiles were evaporated, and
the residue was chromatographed (CH2Cl2 f 8% EtOH/CH2-
Cl2) to give 6 (42 mg, quantitative) as a white powder: 1H NMR
δ 1.44 and 1.65 (2 × s, 2 × 3, 2 × Me), 4.95 (dd, J 4′-5′ ) 8.2
Hz, J 4′-3′ ) 2.7 Hz, 1, H4′), 5.14 (dd, J 3′-2′ ) 6.1 Hz, 1, H3′),
5.54 (dd, J 2′-1′ ) 1.5 Hz 1, H2′), 6.08 (d, 1, H1′), 6.64 (d, 1,
H5′), 6.79 (br s, 2, NH2), 7.97 (s, 1, H2), 8.36 (s, 1, H8).
9-(6-Br om o-5,6-d id eoxy-6-flu or o-2,3-O-isop r op ylid en e-
â-D-r ibo-h ex-5-en ofu r a n osyl)a d en in e (3). A solution of 2
(71 mg, 0.14 mmol) in NH3/MeOH (10 mL) in a sealed flask
was stirred at ambient temperature for 24 h. Volatiles were
evaporated, and the residue was flash-chromatographed (CH2Cl2
f 8% EtOH/CH2Cl2) to give 3 (57 mg, quantitative) as a white
powder: 1H NMR δ 1.39 and 1.63 (2 × s, 2 × 3, 2 × Me), 4.87
(ddd, J 4′-5′ ) 9.2 Hz, J 4′-3′ ) 2.5 Hz, J 4′-F ) 2.5 Hz, 0.4, H4′Z),
Meth od B. A solution of 5 (145 mg, 0.26 mmol) in
saturated NH3/MeOH (50 mL) in a sealed flask was stirred at
ambient temperature for 24 h. Evaporation of volatiles and
flash chromatography of the residue (CH2Cl2 f 8% EtOH/CH2-
Cl2) gave 610 (118 mg, 100%) as a white powder with data
identical to that in method A.
5.07-5.19 (m, 1.6, H3′, H4′E), 5.46 (dd, J 5′-4′ ) 9.2 Hz, J 5′-F
)
29.7 Hz, 0.6, H5′E), 5.54-5.67 (m, 1, H2′), 5.85 (br s, 2, NH2),
5.89 (dd, J 5′-F ) 11.8 Hz, 0.4, H5′Z), 6.05 (s, 1, H1′), 7.84 (s, 1,
H2), 8.37 (s, 1, H8); 19F NMR δ -69.07 (d, J F-5′ ) 29.2 Hz,
0.6, F6′E), -64.52 (d, J F-5′ ) 11.8 Hz, 0.4, F6′Z); HRMS (CI)
m/z 402.0396 (96, MH+ [C14H1681BrFN5O3] ) 402.0400),
400.0414 (100, MH+ [79Br] ) 400.0421).
9-(6,6-Dibr om o-5,6-d id eoxy-â-D-r ibo-h ex-5-en ofu r a n o-
syl)a d en in e (7). A solution of 6 (44 mg, 0.095 mmol) in CF3-
CO2H/H2O (9:1, 7 mL) was stirred at ambient temperature for
1 h. Volatiles were evaporated immediately in vacuo, and the
slightly yellow residue was purified by preparative RP-HPLC
(20% CH3CN/H2O for 20 min, gradient of 20% f 50% for 25
min, flow rate 2.8 mL/min) to give 7 (35 mg, 90%) as an off-
white solid. Recrystallization (H2O) gave 710 (28 mg, 73%) as
9-(6-Br om o-5,6-dideoxy-6-flu or o-â-D-r ibo-h ex-5-en ofu r a-
n osyl)a d en in e (4). A solution of 3 (57 mg, 0.14 mmol) in CF3-
CO2H/H2O (9:1, 5 mL) was stirred at ambient temperature for
1 h. Volatiles were evaporated, and the slightly yellow residue
was purified by preparative RP-HPLC (30% CH3CN/H2O for
30 min followed by a gradient of 30% f 70% for 25 min at
flow rate of 2.8 mL/min) to give 4 (36 mg, 70%; early and late
fractions had E/ Z ratios of 71:29 and 44:56, respectively) as
an off-white solid. “Diffusion crystallization”17 (EtOAc/hexane)
gave 4 (18 mg, 35%) as white crystals: mp 100 °C dec; UV
max 261 nm (ꢀ 14 500), min 228 nm (ꢀ 2200); 1H NMR (CD3OD)
δ 4.28-4.39 (m, 1, H3′), 4.63 (ddd, J 4′-5′ ) 9.2 Hz, J 4′-3′ ) 4.5
Hz, J 4′-F ) 2.1 Hz, 0.4, H4′Z), 4.76-4.90 (m, 1.6, H2′, H4′E),
5.71 (dd, J 5′-4′ ) 9.4 Hz, J 5′-F ) 30.4 Hz, 0.6, H5′E), 5.98-
white crystals: mp 105 °C dec; UV max 258 nm (ꢀ 14 500),
1
min 237 nm (ꢀ 7000); H NMR (Me2SO-d6) δ 4.23 (dd, J 3′-4′
)
3.9 Hz, J 3′-2′ ) 5.0 Hz, 1, H3′), 4.56 (dd, J 4′-5′ ) 8.6 Hz, 1,
H4′), 4.70 (dd, J 2′-1′ ) 5.5 Hz, 1, H2′), 5.75 (br s, 2, OH2′,3′),
5.98 (d, 1, H1′), 7.11 (d, 1, H5′), 8.55 (br s, 2, NH2), 8.41 (s, 1,
H2), 8.63 (s, 1, H8); 13C NMR (Me2SO-d6) δ 73.06 (C3′), 74.00
(C2′), 84.58 (C4′), 88.20 (C1′), 93.45 (C6′), 119.16 (C5), 137.39
(C5′), 140.90 (C8), 149.64 (C4), 153.16 (C2), 156.26 (C6); MS

3082 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
m/z 424 (MH+ [81Br2]), 422 (MH+ 81/79Br2]), 420 (MH+ [79Br2]).
Wnuk et al.
[
were combined and assayed for remaining hydrolase activity
and then denatured with 8 M urea. Gel filtration with the
spin column was repeated to remove noncovalently bound
8-[3H]-4. The protein concentration and radioactivity were
determined, and the ratio of bound 8-[3H]-4 (mole) to inacti-
vated enzyme (mole of subunit) was calculated.
Anal. [C11H11Br2N5O3‚0.5H2O (430.1)] C, H, N.
9-(5,6,6-Tr ibr om o-5,6-d id eoxy-2,3-O-isop r op ylid en e-â-
D-r ibo-h exofu r a n osyl)a d en in e (9). Br2 (35 mg, 0.22 mmol)
in CH2Cl2 (2.5 mL) was added dropwise during 1 h to a solution
of 86 (78 mg, 0.20 mmol) in CH2Cl2 (10 mL) cooled to -10 °C,
and stirring was continued at ∼0 °C for 3.5 h. The solution
was washed [H2O (10 mL), NaHCO3/H2O (2 × 10 mL)] and
dried (MgSO4), and volatiles were evaporated. The residue
was chromatographed (EtOAc) to give 9 (50 mg, 46%) as an
off-white powder: 1H NMR δ 1.42 and 1.65 (2 × s, 2 × 3, 2 ×
CH3), 4.51 (d, J 4′-5′ ) 10.2 Hz, 1, H4′), 5.17 (d, 1, H5′), 5.35 (d,
J 3′-2′ ) 6.2 Hz, 1, H3′), 5.42 (d, 1, H2′), 5.82 (s, 1, H6′), 6.09 (s,
1, H1′), 6.26 (br s, 2, NH2), 7.87 (s, 1, H2), 8.36 (s, 1, H8).
(E)-6-N-Ben zoyl-9-(5,6-d ibr om o-5,6-d id eoxy-2,3-O-iso-
p r op ylid en e-â-D-r ibo-h ex-5-en ofu r a n osyl)a d en in e (13).
Br2 (60 mg, 0.38 mmol) in CH2Cl2 (4.3 mL) was added dropwise
during 1 h to a solution of 116 (180 mg, 0.37 mmol) in CH2Cl2
(15 mL) cooled at -10 °C. A slight orange color persisted, and
stirring was continued at -5 °C for 5 h. DBU (0.165 mL, 167
mg, 1.1 mmol) was added to crude 12, and stirring was
continued at ambient temperature for 10 h. Volatiles were
evaporated, and chromatography of the residue (CH2Cl2 f 3%
EtOH/CH2Cl2) gave 13 (133 mg, 64%) as a white powder: 1H
An tivir a l Assa ys. These assays were based on inhibition
of virus-induced cytopathicity in E6SM, Vero, and HEL or CEM
cell cultures following previously established procedures.19-21
Briefly, for all the viruses except HIV, confluent E6SM, Vero,
or HEL cell cultures in microtiter trays were inoculated with
100 CCID50 of virus (1 CCID50 is the virus dose required to
infect 50% of the cell cultures). After a 1-h virus-adsorption
period, residual virus was removed and the cell cultures were
incubated at 37 °C in a CO2-controlled, humidified incubator
in the presence of varying concentrations (400, 200, 80, 40, ...
µg/mL) of 4, 7, or 15. Viral cytopathicity was recorded
microscopically as soon as it reached completion in the control
virus-infected cell cultures. CEM cells were seeded in micro-
titer trays at 50 000 cells/200-µL well and infected with 100
CCID50 of HIV-1 or HIV-2 in the presence of different
concentrations of 4, 7, or 15. After 4 days of incubation, virus-
induced giant cell formation was recorded microscopically.
NMR δ 1.41 and 1.70 (2 × s, 2 × 3, 2 × Me), 5.07 (dd, J 3′-4′
)
Ack n ow led gm en t. We thank the American Cancer
Society (Grant No. DHP-34), Brigham Young University
development funds, the United States Public Health
Service (Grant No. GM-29332), the Belgian Fonds voor
Geneeskundig Wetenschappelijk Onderzoek (Project No.
3.0180.95), the Geconcerteerde Onderzoeksacties (Con-
ventie No. 95/5, Vlaamse Gemeenschap), and the Bio-
medical Research Programme of the European Com-
mission for financial support; Ann Absillis, Anita Camps,
Frieda De Meyer, and Anita Van Lierde for excellent
technical assistance; and Mrs. J eanny K. Gordon for
assistance with the manuscript.
4.1 Hz, J 3′-2′ ) 6.2 Hz, 1, H3′), 5.34 (dd, J 2′-1′ ) 2.8 Hz, 1,
H2′), 5.41 (d, 1, H4′), 6.31 (d, 1, H1′), 6.78 (s, 1, H6′), 7.60-
8.10 (m, 5, Arom), 8.30 (s, 1, H2), 8.81 (s, 1, H8), 9.28 (s, 1,
NH).
(E)-9-(5,6-Dibr om o-5,6-d id eoxy-2,3-O-isop r op ylid en e-
â-D-r ibo-h ex-5-en ofu r a n osyl)a d en in e (14). A solution of 13
(133 mg, 0.24 mmol) in saturated NH3/MeOH (50 mL) in a
sealed flask was stirred at ambient temperature for 24 h.
Volatiles were evaporated, and the residue was flash-chro-
matographed (CH2Cl2 f 8% EtOH/CH2Cl2) to give 14 (106 mg,
98%) as a white powder: 1H NMR δ 1.39 and 1.69 (2 × s, 2 ×
3, 2 × Me), 5.04 (dd, J 3′-4′ ) 4.1 Hz, J 3′-2′ ) 6.2 Hz, 1, H3′),
5.29 (dd, J 2′-1′ ) 3.0 Hz, 1, H2′), 5.36 (d, 1, H4′), 5.77 (br s, 2,
NH2), 6.23 (d, 1, H1′), 6.77 (s, 1, H6′), 8.06 (s, 1, H2), 8.37 (s,
1, H8).
Refer en ces
(E)-9-(5,6-Dibr om o-5,6-d id eoxy-â-D-r ibo-h ex-5-en ofu r a -
n osyl)a d en in e (15). A solution of 14 (30 mg, 0.065 mmol)
in CF3CO2H/H2O (9:1, 7 mL) was stirred at ambient temper-
ature for 1 h. Volatiles were evaporated in vacuo, and the
slightly yellow residue was purified by preparative RP-HPLC
(20% CH3CN/H2O for 40 min, gradient of 20% f 50% for 30
min, flow rate of 2.8 mL/min) to give 15 (23 mg, 85%) as an
off-white solid. “Diffusion crystallization”17 (MeOH/EtOAc)
gave 15 (18 mg, 66%) as white crystals: mp 115-118 °C, 125
(1) Nucleic Acid Related Compounds. 102. For the previous paper,
see: Robins, M. J .; Neschadimenko, V.; Ro, B.-O.; Yuan, C.-S.;
Borchardt, R. T.; Wnuk, S. F. Nucleic Acid Related Compounds.
101. S-Adenosyl-L-homocysteine Hydrolase Does Not Hydrate
(5′-Fluoro)vinyl or (6′-Halo)homovinyl Analogues Derived from
3′-Deoxyadenosine or 3′-(Chloro or Fluoro)-3′-deoxyadenosine.
J . Org. Chem., 1998, 63, 1205-1211.
(2) (a) Ueland, P. M. Pharmacological and Biochemical Aspects of
S-Adenosylhomocysteine and S-Adenosylhomocysteine Hydro-
lase. Pharmacol. Rev. 1982, 34, 223-253. (b) Wolfe, M. S.;
Borchardt, R. T. S-Adenosyl-L-homocysteine Hydrolase as a
Target for Antiviral Chemotherapy. J . Med. Chem. 1991, 34,
1521-1530. (c) Yuan, C.-S.; Liu, S.; Wnuk, S. F.; Robins, M. J .;
Borchardt, R. T. Design and Synthesis of S-Adenosylhomocys-
teine Hydrolase Inhibitors as Broad-Spectrum Antiviral Agents.
In Advances in Antiviral Drug Design; De Clercq, E., Ed.; J AI
Press: Greenwich, CT, 1996; Vol. 2, pp 41-88.
(3) McCarthy, J . R.; J arvi, E. T.; Matthews, D. P.; Edwards, M. L.;
Prakash, N. J .; Bowlin, T. L.; Mehdi, S.; Sunkara, P. S.; Bey, P.
4′,5′-Unsaturated 5′-Fluoroadenosine Nucleosides: Potent Mech-
anism-Based Inhibitors of S-Adenosyl-L-homocysteine Hydro-
lase. J . Am. Chem. Soc. 1989, 111, 1127-1128.
(4) Wnuk, S. F.; Dalley, N. K.; Robins, M. J . Nucleic Acid Related
Compounds. 76. Synthesis of 5′(E and Z)-Chloro-4′,5′-didehydro-
5′-deoxyadenosines via Chlorination and Thermolysis of Ad-
enosine 5′-Sulfoxides. Mechanism-Based Inhibition of S-Adenosyl-
L-homocysteine Hydrolase. J . Org. Chem. 1993, 58, 111-117.
(5) (a) Liu, S.; Wnuk, S. F.; Yuan, C.-S.; Robins, M. J .; Borchardt,
R. T. Adenosine-5′-carboxaldehyde: A Potent Inhibitor of S-
Adenosyl-L-homocysteine Hydrolase. J . Med. Chem. 1993, 36,
883-887. (b) Yuan, C.-S.; Yeh, J .; Liu, S.; Borchardt, R. T.
Mechanism of Inactivation of S-Adenosylhomocysteine Hydro-
lase by (Z)-4′,5′-Didehydro-5′-deoxy-5′-fluoroadenosine. J . Biol.
Chem. 1993, 268, 17030-17037.
1
°C dec; UV max 259 nm (ꢀ 15 700), min 233 nm (ꢀ 5300); H
NMR (Me2SO-d6) δ 4.48 (dd, J 3′-4′ ) 6.2 Hz, J 3′-2′ ) 5.5 Hz, 1,
H3′), 4.70 (dd, J 2′-1′ ) 3.5 Hz, 1, H2′), 5.03 (d, 1, H4′), 5.50-
5.90 (br s, 2, OH2′,3′), 5.99 (d, 1, H1′), 7.26 (s, 1, H6′), 7.58 (br
s, 2, NH2), 8.23 (s, 1, H2), 8.40 (s, 1, H8); 13C NMR (Me2SO-
d6) δ 72.99 (C2′,3′), 81.38 (C4′), 87.98 (C1′), 109.21 (C6′), 118.88
(C5), 124.19 (C5′), 139.93 (C8), 149.45 (C4), 152.21 (C2), 156.78
(C6); MS m/z 424 (MH+ [81Br2]), 422 (MH+ 81/79Br2]), 420 (MH+
[
[79Br2]). Anal. [C11H11Br2 N5O3 (421.1)] C, H, N.
Tim e-Dep en d en t In a ct iva t ion of S-Ad en osyl-L-h o-
m ocystein e Hyd r ola se w ith 4, 7, a n d 15. AdoHcy hydro-
lase [20 nM in 50 mM potassium phosphate buffer (pH 7.2)
containing 1 mM EDTA (buffer A)] was incubated with various
concentrations of inhibitors (2.5-20 µM) for different times
(0-20 min). The remaining enzyme activity at each time point
was determined in the synthetic direction as described
previously.5b The pseudo-first-order rate constant (kapp) was
obtained by plotting the logarithm of the remaining activity
versus time, and the Ki and kinact values were obtained by the
method of Kitz and Wilson.18
(6) Wnuk, S. F.; Yuan, C.-S.; Borchardt, R. T.; Balzarini, J .; De
Clercq, E.; Robins, M. J . Nucleic Acid Related Compounds. 84.
Synthesis of 6′-(E and Z)-Halohomovinyl Derivatives of Adenos-
ine, Inactivation of S-Adenosyl-L-homocysteine Hydrolase, and
Correlation of Anticancer and Antiviral Potencies with Enzyme
Inhibition. J . Med. Chem. 1994, 37, 3579-3587.
The stoichiometry of labeling of the enzyme (0.5 mg) with
8-[3H]-4 (500 µM, 187 Ci/mol) was determined by incubation
in buffer A (250 µL) at 37 °C for 2 h. The reaction mixture
was then applied to a Sephadex G-25 spin column to remove
nonbound 4. Fractions containing the enzyme-4 complex

Dihalohomovinyl Nucleosides Derived from Adenosine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3083
(7) (a) Yuan, C.-S.; Liu, S.; Wnuk, S. F.; Robins, M. J .; Borchardt,
R. T. Mechanism of Inactivation of S-Adenosylhomocysteine
Hydrolase by (E)-5′,6′-Didehydro-6′-Deoxy-6′-Halohomoadenos-
ines. Biochemistry 1994, 33, 3758-3765. (b) Yuan, C.-S.; Wnuk,
S. F.; Liu, S.; Robins, M. J .; Borchardt, R. T. (E)-5′,6′-
Didehydro-6′-Deoxy-6′-Fluorohomoadenosine: A Substrate That
Measures The Hydrolytic Activity of S-Adenosylhomocysteine
Hydrolase. Biochemistry 1994, 33, 12305-12311.
(8) Ault-Riche´, D. B.; Yuan, C.-S.; Borchardt, R. T. A Single
Mutation at Lysine 426 of Human Placental S-Adeonosylho-
mocysteine Hydrolase Inactivates the Enzyme. J . Biol. Chem.
1994, 269, 31472-31478.
logues from Uridine-derived Vinylsulfones via Vinyltin Inter-
mediates. Can. J . Chem. 1993, 71, 192-198.
(15) Wnuk, S. F.; Robins, M. J . Nucleic Acid Related Compounds.
63. Synthesis of 5′-deoxy-5′-methyleneadenosine and Related
Wittig-extended Nucleosides. Can. J . Chem. 1991, 69, 334-338.
(16) Yuan, C.-S.; Wnuk, S. F.; Robins, M. J .; Borchardt, R. T. A Novel
Mechanism-Based Inhibitor (6′-Bromo-5′,6′-didehydro-6′-deoxy-
6′-fluorohomoadenosine) That Covalently Modifies Human Pla-
cental S-Adenosylhomocysteine Hydrolase. J . Biol. Chem., in
press.
(17) Robins, M. J .; Mengel, R.; J ones, R. A.; Fouron, Y. Nucleic Acid
Related Compounds. 22. Transformation of Ribonucleoside 2′,3′-
O-Ortho Esters into Halo, Deoxy, and Epoxy Sugar Nucleosides
Using Acyl Halides. Mechanism and Structure of Products. J .
Am. Chem. Soc. 1976, 98, 8204-8213.
(9) Sharma, R. A.; Bobek, M. Acetylenic Nucleosides. 1. Synthesis
of 1-(5,6-Dideoxy-â-D-ribo-hex-5-ynofuranosyl)uracil and 1-(2,5,6-
Trideoxy-â-D-erythro-hex-5-ynofuranosyl)-5-methyluracil. J . Org.
Chem. 1978, 43, 367-369.
(10) Matsuda, A.; Kosaki, H.; Yoshimura, Y.; Shuto, S.; Ashida, N.;
Konno, K.; Shigeta, S. Nucleosides and Nucleotides. 142. An
Alternative Synthesis of 9-(5,6-Dideoxy-â-D-ribo-hex-5-ynofura-
nosyl)adenine and its Antiviral Activity. Bioorg. Med. Chem.
Lett. 1995, 5, 1685-1688.
(18) Kitz, R.; Wilson, I. B. Esters of Methanesulfonic Acid as
Irreversible Inhibitors of Acetylcholinesterase. J . Biol. Chem.
1962, 237, 3245-3249.
(19) De Clercq, E.; Descamps, J .; Verhelst, G.; Walker, R. T.; J ones,
A. S.; Torrence, P. F.; Shugar, D. Comparative Efficacy of
Different Antiherpes Drugs Against Different Strains of Herpes
Simplex Virus. J . Infect. Dis. 1980, 141, 563-574.
(20) De Clercq, E.; Holy´, A.; Rosenberg, I.; Sakuma, T.; Balzarini,
J .; Maudgal, P. C. A Novel Selective Broad-Spectrum Anti-DNA
Virus Agent. Nature 1986, 323, 464-467.
(21) Balzarini, J .; Naesens, L.; Slachmuylders, J .; Niphuis, H.;
Rosenberg, I.; Holy´, A.; Schellekens, H.; De Clercq, E. 9-(2-
Phosphonylmethoxyethyl)adenine (PMEA) Effectively Inhibits
Retrovirus Replication in vitro and Simian Immunodeficiency
Virus Infection in Rhesus Monkeys. AIDS 1991, 5, 21-28.
(11) Corey, E. J .; Fuchs, P. L. A Synthetic Method for Formyl f
Ethynyl Conversion (RCHO f RCtCH or RCtCR′). Tetrahe-
dron Lett. 1972, 3769-3772.
(12) Parry, R. J .; Muscate, A.; Askonas, L. J . 9-(5′,6′-Dideoxy-â-D-
ribo-hex-5′-ynofuranosyl)adenine, a Novel Irreversible Inhibitor
of S-Adenosylhomocysteine Hydrolase. Biochemistry 1991, 30,
9988-9997.
(13) Ranganathan, R. S.; J ones, G. H.; Moffatt, J . G. Novel Analogues
of Nucleoside 3′,5′-Cyclic Phosphates. I. 5′-Mono- and Dimethyl
Analogues of Adenosine 3′,5′-Cyclic Phosphate. J . Org. Chem.
1974, 39, 290-298.
(14) Wnuk, S. F.; Robins, M. J . Nucleic Acid Related Compounds.
78. Stereocontrolled Syntheses of 6′(E and Z)-Halovinyl Ana-
J M9801410