Inactivation of S-adenosyl-L-homocysteine hydrolase by amide and ester derivatives of adenosine-5'-carboxylic acid

Article abstract of DOI:10.1021/jm960313y

S-Adenosyl-L-homocysteine (AdoHcy) hydrolase has been shown to have (576') hydrolytic activity with vinyl (5') or homovinyl (6') halides derived from adenosine (Ado). This hydrolyric activity is independent of its 3'- oxidative activity. The vinyl (or hom

Full text of DOI:10.1021/jm960313y

4162
J . Med. Chem. 1996, 39, 4162-4166
In a ctiva tion of S-Ad en osyl-L-h om ocystein e Hyd r ola se by Am id e a n d Ester
Der iva tives of Ad en osin e-5′-ca r boxylic Acid
Stanislaw F. Wnuk,† Siming Liu,‡ Chong-Sheng Yuan,‡ Ronald T. Borchardt,*,‡ and Morris J . Robins*,†
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700, and Departments of
Medicinal Chemistry and Biochemistry, The University of Kansas, Lawrence, Kansas 66045
Received April 26, 1996X
S-Adenosyl-L-homocysteine (AdoHcy) hydrolase has been shown to have (5′/6′) hydrolytic activity
with vinyl (5′) or homovinyl (6′) halides derived from adenosine (Ado). This hydrolytic activity
is independent of its 3′-oxidative activity. The vinyl (or homovinyl) halides are converted into
5′(or 6′)-carboxaldehydes by the hydrolytic activity of the enzyme, and inactivation occurs via
the oxidative activity. Amide and ester derivatives of Ado-5′-carboxylic acid were prepared to
further probe the hydrolytic capability of AdoHcy hydrolase. The oxidative activity (but not
the hydrolytic activity) is involved in the mechanism of inhibition of the enzyme by the ester
and amide derivatives of Ado-5′-carboxylic acid, in contrast to the inactivation of this enzyme
by adenosine-derived vinyl or homovinyl halide analogues during which both activities are
manifested.
In tr od u ction
The cellular enzyme S-adenosyl-L-homocysteine (Ado-
Hcy) hydrolase (EC 3.3.1.1) effects cleavage of AdoHcy
to adenosine (Ado) and L-homocysteine (Hcy).1 Since
AdoHcy is a potent feedback inhibitor of crucial trans-
methylation enzymes, inhibitors of AdoHcy hydrolase
represent putative mechanism-based anticancer and
F igu r e 1.
antiviral agents.2,3 Cellular accumulation of AdoHcy
can cause inhibition of viral mRNA methyltransferases
bond (hydrolytic activity) independently of its 3′-oxida-
essential for viral replication.4 It has been shown that
tive activity rapidly with the homovinyl fluoride C
direct correlations exist between antiviral potency of
nucleosides and their ability to elevate cellular levels
(X ) F).13
of AdoHcy,5 and correlations between antiviral and
cytostatic potencies of Ado analogues and their inhibi-
tory effects on AdoHcy hydrolase have been demon-
strated.6 Inhibitors of AdoHcy hydrolase recently have
been shown to inhibit replication of the Ebola virus,
which causes hemorrhagic fever with mortalities of 40-
90% in sporadic human outbreaks.7
Other 5′-modified adenosine derivatives that inhibit
AdoHcy hydrolase include 5′-azido-5′-deoxyadenosine,14
5′-cyano-5′-deoxyadenosine,14 5′-deoxy-5′-methynylad-
enosine15 (the 4′-acetylenic derivative), and Ado-5′-
carboxaldehyde oxime derivatives.2b We now report
studies designed to further investigate the 5′-hydrolytic
potential of AdoHcy hydrolase with 5′-modified ana-
logues derived from Ado-5′-carboxylic acid. A number
of studies on the binding of Ado-5′-carboxylic (uronic)
acid ester16,17 and amide18-20 derivatives with adenosine
receptors have been reported.18-20
The “adenosine-5′-carboxaldehyde” mixture (e.g., 4′S-
isomer A; Figure 1) was shown to inactivate AdoHcy
hydrolase by reduction of NAD+ to NADH,8 the so-called
“cofactor depletion” or type I mechanism.3a NADH or
apoforms of the enzyme converted (Z)-4′,5′-didehydro-
5′-deoxy-5′-fluoroadenosine9 (B, ZDDFA) into the Ado-
5′-carboxaldehyde inhibitor(s).10 It had been postulated11a
that 5′-S-alkyl(or aryl)-5′-fluoro-5′-thioadenosines might
function as mechanism-based inhibitors of AdoHcy
hydrolase, and it was found11b,c that such R-fluoro
thioethers (thioacetal analogues) undergo chemical hy-
drolysis in situ to give the Ado-5′-carboxaldehyde in-
hibitor(s). We recently have synthesized (E)-5′,6′-
didehydro-6′-deoxy-6′-halohomoadenosine analogues (C,
EDDHHAs) which inhibit AdoHcy hydrolase.6b Amaz-
ingly, homovinyl halides C are hydrolyzed at the active
site of the enzyme to produce “homoadenosine-6′-car-
boxaldehyde” which decomposes spontaneously.12,13 The
enzyme catalyzes addition of water to the 5′,6′-double
Ch em istr y
Oxidation of 2′,3′-O-isopropylideneadenosine with
potassium permanganate21 gave more reliable yields of
2′,3′-O-isopropylideneadenosine-5′-carboxylic acid (1a ;
83%) than recently described procedures.22 Treatment
of 1a with diazomethane gave methyl ester 2a 23 (92%),
and treatment of 1a and 2a with aqueous trifluoroacetic
acid (TFA) gave Ado-5′-carboxylic acid23 (1b) and its
methyl ester16,17 2b (Scheme 1). Treatment of 1b with
thionyl chloride and the alcohol16,17 gave butyl (3b),
isobutyl (4b), benzyl (5b), and 2-chloroethyl (6b) esters.
Conversion of 1b to the acid chloride16 13b and its
treatment with isopropyl alcohol gave 7b.
Ado-5′-carboxamide (8a ) and N-substituted amides
9a -11a were prepared by ammonolysis of 2a . Dicyclo-
hexylcarbodiimide (DCC) effected coupling of 1a with
aniline to give N-phenylcarboxamide 12a . Treatment
of acid chloride 13a 16 with diethylamine gave 14a .
* Authors to whom correspondence should be addressed.
† Brigham Young University.
‡ University of Kansas.
X Abstract published in Advance ACS Abstracts, September 15, 1996.
S0022-2623(96)00313-5 CCC: $12.00 © 1996 American Chemical Society

Inactivation of AdoHcy Hydrolase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4163
Ta ble 1. 13C NMR Spectral Dataa,b
c
c
compd
C-2
C-4
C-5
C-6
C-8
C-1′
C-2′
C-3′
C-4′
C-5′
1b
152.84
152.50
152.30
151.63
152.85
152.66
152.51
152.91
153.06
149.55
149.19
148.90
148.99
148.95
149.01
148.68
149.30
149.98
118.93
119.17
118.30
119.09
119.52
119.74
119.64
119.68
118.72
156.13
156.31
155.50
155.80
156.13
156.34
156.19
156.24
156.03
139.54
140.93
138.40
141.33
141.08
141.09
141.19
140.72
139.38
87.35
87.62
86.40
88.03
88.03
88.09
88.33
88.20
87.18
73.93
73.99
73.10
73.42
73.11
73.31
73.29
73.31
74.89
73.44
73.24
72.70
73.22
72.15
72.09
71.95
72.66
73.06
82.80
82.85
81.90
84.23
84.75
84.89
84.98
84.64
80.70
172.09
170.05
169.70
171.16
170.36
169.61
169.95
168.49
168.67
2bd,e
5bf
8bd
9bg
10bh
11bi
12bj
14bk
a
b
d
δ (Me2SO-d6) at 50 MHz. Proton-decoupled singlets. c Assignments might be reversed. APT experiment. e δ 52.70 (Me). f δ 66.00
g
h
i
(CH2), 127.60, 127.80, 128.00, 135.00 (Ph). δ 25.56 (Me). δ 13.90, 19.76, 31.43, 39.78 (Bu). δ 42.27 (CH2), 127.38, 127.62, 128.74,
138.98 (Ph). δ 120.62, 124.68, 129.16, 137.88 (Ph). δ 13.05, 14.71, 41.52 (Et’s).
j
k
Sch em e 1a
Ta ble 2. Kinetic Constants for AdoHcy Hydrolase Inhibition
Activity of Ado-5′-carboxylic Acid (1b) and Its Esters 2b-7b
and Amides 8b-12b and 14ba
compd
k2 (min-1
)
KI (µΜ)
k2/KI (M-1 min-1
)
A (4′S)
1b
2b
3b
4b
5b
6b
7b
8b
0.65
0.045
0.78
0.039
0.41
1.7
0.71
0.57
0.36
0.32
0.77
2.6
1.93
19.8
43.4
48.5
0.57
1.7 × 107
1.1 × 105
4.6 × 105
1.6 × 105
1.4 × 105
4.9 × 104
1.1 × 106
8.4 × 104
3.8 × 104
4.9 × 104
1.5 × 103
6.9 × 102
5.8 × 102
8.8 × 104
0.11
0.077
0.017
0.35
0.064
0.1
0.095
0.03
0.03
9b
10b
11b
12b
14b
0.028
0.05
a
See the Experimental Section for details concerning the
procedures for conducting enzyme inactivation studies and data
analysis for determination of KI and k2 values.
a
(a) CH2N2/MeOH/dioxane; (b) SOCl2/ROH; (c) CF3CO2H/H2O;
In h ibition of Ad oHcy Hyd r ola se
(d) RNH2; (e) PhNH2/DCC; (f) SOCl2/DMF; (g) i-PrOH; (h)
(C2H5)2NH.
Ado-5′-carboxylic acid (1b), esters 2b-7b, and amides
8b-12b and 14b were evaluated for inhibition of
purified recombinant human placental AdoHcy hydro-
lase. Table 2 contains the dissociation constants (KI),
inactivation rate constants (k2), and derived second-
order rate constants (k2/KI). Figure 2 contains repre-
sentative Kitz and Wilson plots for compounds 2b and
8b from which the kinetic constants were obtained.
Deprotection (TFA/H2O) of 8a -12a and 14a and chro-
matography (short silica columns) and/or recrystalliza-
tion gave amides 8b-12b and 14b. All known es-
ters16,17 and amides18 had properties and 1H and 13C
(Table 1) NMR spectral data consistent with those
reported. Characterization data for 5b and 14b are
given in the Experimental Section.
F igu r e 2. Kitz and Wilson plots of compounds 8b (A) and 2b (B).

4164 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Wnuk et al.
It is apparent from Figure 2 and Table 2 that all of
the compounds evaluated showed time- and inhibitor
concentration-dependent inactivation of AdoHcy hydro-
lase with k2/KI values in the range of (5.8 × 102)-(1.1
× 106) M-1 min-1. Thus, Ado-5′-carboxylic acid (1b),
its esters 2b-7b, and amides 8b-12b and 14b are
much weaker inhibitors of AdoHcy hydrolase than Ado-
5′-carboxaldehyde (A), and amides 8b-12b and 14b are
generally weaker inhibitors than their ester analogues
2b-7b (k2/KI).
In summary, both ester (2b-7b) and amide (8b-12b,
14b) derivatives of Ado-5′-carboxylic acid (1b) produced
time-dependent enzyme inactivation. Observation of
the reduction of NAD+ to NADH indicates that they are
type I mechanism-based inhibitors. In contrast with
recent results with other mechanism-based inactivators,
no involvement of the hydrolytic activity of AdoHcy
hydrolase was detected. Thus, Ado-5′-carboxylic ester
and amide derivatives do not appear to form type II
covalent adducts with this enzyme.
The time dependency of inactivation of AdoHcy hy-
drolase upon incubation with 1b, 2b-7b, 8b-12b and
14b is consisent with a type I mechanism-based inhibi-
tion of AdoHcy hydrolase.3a This involves reduction of
the enzyme-bound NAD+ to NADH with concomitant
oxidization of the 3′-hydroxyl group of the inhibitors to
a ketone. The observation (UV spectral results not
shown) that NAD+ is converted to NADH upon incuba-
tion of 2b or 8b with the enzyme is in harmony with
the type I mechanism-based enzyme inhibition mech-
anism.
Exp er im en ta l Section
Uncorrected melting points were determined with a capil-
lary apparatus. UV spectra were determined with solutions
in MeOH. 1H (200 MHz) and 13C (50 MHz) NMR spectra were
recorded with solutions in Me2SO-d6 unless otherwise noted.
Low-resolution electron-impact (20 eV) (MS) and chemical
ionization (CI, CH4) mass spectra were obtained with direct
probe techniques. Elemental analyses were determined by
M-H-W Laboratories, Phoenix, AZ. Reagents and solvents
were of reagent quality, and solvents were purified and dried
before use. TLC was performed on Merck Kieselgel 60 F254
sheets with S1 (EtOAc/i-PrOH/H2O, 4:1:2; upper layer), S2
(i-PrOH/NH4OH/H2O, 7:1:2), or S3 (MeOH/CHCl3, 1:6) with
sample observation under 254-nm light. Column chromatog-
raphy was performed with Merck kieselgel 60 (230-400 mesh).
Adenosine-5′-carboxylic acid23 (1b) and its methyl ester 2b16,17
were prepared by deprotection (TFA/H2O; as described for 8b)
of the known 1a 21 and 2a .23
Ado-5′-carboxaldehyde (A) was recently shown to be
a potent AdoHcy hydrolase inhibitor (k2/KI ) 1.7 × 107
M-1 min-1).8,10 A also inactivates AdoHcy hydrolase by
the type I mechanism (reduction of cofactor NAD+), and
ZDDFA9 (B) was shown to be an enzyme-activated
prodrug of Ado-5′-carboxaldehyde (A).10 That mecha-
nism involves rapid addition of water at the C-5′ position
of ZDDFA, elimination of hydrogen fluoride to give Ado-
5′-carboxaldehyde (A), and slower oxidation of A to give
3′-keto-Ado-5′-carboxaldehyde with concomitant reduc-
tion of E‚NAD+ to E‚NADH. This mechanism indicates
that AdoHcy hydrolase has (C-5′) hydrolytic activity that
is independent of its (C-3′) oxidation capability.10 This
hydrolytic feature of AdoHcy hydrolase, functioning at
6′ as well as at C-5′ of the EDDHHAs (C), was recently
shown to be involved in the mechanism by which they
inactivate the enzyme.6b,12,13 Current knowledge of the
amino acid residues involved in the hydrolytic activity
of AdoHcy hydrolase is minimal. If this activity involves
nucleophilic catalysis, it is conceivable that a covalent
bond might be formed linking the enzyme and inhibitor
during hydrolysis of the esters 2b-7b and amides 8b-
12b and 14b. Unless this were merely a transitory
intermediate, covalent type II mechanism-based inhibi-
tion of AdoHcy hydrolase3a should occur in addition to
the cofactor depletion type I mechanism. The order of
potency of the present compounds (esters > amides) as
inhibitors of AdoHcy hydrolase is consistent with nu-
cleophilic catalysis, so this possibility was examined by
determinig if enzyme-facilitated hydrolysis of esters or
amides to acid 1b occurred. Compounds 2b and 8b
underwent spontaneous hydrolysis to 1b in phosphate
buffer, pH 7.2. The rate constant for spontaneous
hydrolysis of 2b was 0.0053 min-1, and spontaneous
hydrolysis of 8b was much slower. Incubation of 2b or
8b with AdoHcy hydrolase at 37 °C for 2 h did not
increase the rate of formation of 1b (data not shown),
indicating that these compounds are not substrates for
the hydrolytic activity of the enzyme. Thus, the ester
and amide derivatives most likely inhibit AdoHcy hy-
drolase by the cofactor depletion type I mechanism
without involvement of hydrolytic activity of the en-
zyme.
Ad en osin e-5′-ca r boxylic Acid Ben zyl Ester (5b). Thio-
nyl chloride (0.25 mL) was added slowly to a suspension of
Ado-5′-carboxylic acid16 (1b; 70 mg, 0.25 mmol) in benzyl
alcohol (3.5 mL) at 0 °C, and stirring was continued for 60 h.
The reaction mixture was cooled to -10 °C, and Et2O (40 mL)
was added. The precipitate was filtered, washed (Et2O), and
dissolved in cold H2O, and the solution was neutralized
(saturated NaHCO3/H2O, to pH ∼9). The precipitate was
filtered, washed with cold H2O, and dried to give 5b (16 mg,
1
17%): mp 135-137 °C; H NMR δ 4.42 (m, 1, H3′), 4.54 (d,
J 4′-3′ ) 2.8 Hz, 1, H4′), 4.62 (m, 1, H2′), 5.21 (s, 2, CH2), 5.66
(d, J OH-3′ ) 5.0 Hz, 1, OH3′), 5.84 (d, J OH-2′ ) 5.3 Hz, 1, OH2′),
6.05 (d, J 1′-2′ ) 6.3 Hz, 1, Hl′), 7.30 (br s, 2, NH2), 7.36 (m, 5,
Ph), 8.14 (s, 1, H2), 8.34 (s, 1, H8); HRMS (FAB) m/ z 372.1307
(MH+ [C17H18N5O5] ) 372.1308).
Oth er Ester s. Butyl ester 3b [13%; mp 169-171 °C (lit.16
mp 145-148 °C); MS m/ z 337 (M+)], isobutyl ester 4b [23%;
mp 181-183 °C (lit.17 mp 178-180 °C); MS m/ z 337 (M+)],
and 2-chloroethyl ester 6b [49%; mp 205-208 °C (lit.16 mp
208-210 °C); MS m/ z 343 (M+, 35Cl)] were prepared with the
respective alcohols by the procedure for 5b. Isopropyl ester
7b was prepared as described16 [mp ∼200 °C dec (lit.16 mp
222-223 °C, lit.17 mp 208 °C); MS (CI) m/ z 324 (MH+)].
Aden osin e-5′-car boxam ide (8b). (a) Am m on olysis: 2′,3′-
O-Isopropylideneadenosine-5′-carboxylic acid methyl ester23
(2a ; 100 mg, 0.3 mmol) was stirred with NH3/H2O (d ) 0.88
g/cm3, 5 mL) at ambient temperature for 2 h. The reaction
mixture [TLC (S1) Rf ∼0.5; 2a has Rf ∼0.8] was cooled (ice bath,
2 h), and the white solid was filtered and crystallized (MeOH)
to give 8a (84 mg, 87%): mp 223-226 °C (lit.18 mp 220-222
°C).
(b) Dep r otection : A solution of 8a (58 mg, 0.18 mmol) in
CF3CO2H/H2O (9:1, 5 mL) was stirred at 0 °C for 2 h,
evaporated, and coevaporated (EtOH), and the residue was
crystallized (MeOH) to give 8b (38 mg, 75%): mp 238-240 °C
dec (lit.18 mp 245-247 °C); MS (CI) m/ z 281 (100, MH+).
Ad en osin e-5′-(N-m eth ylca r boxa m id e) (9b). Ester 2a
(100 mg, 0.3 mmol) was stirred with MeNH2/H2O (40%, 1.5
mL) at ambient temperature for 4 h. Undissolved 2a (12 mg)
was filtered, the mother liquor was deprotected (as described
for 8b), and the residue was purified on a short silica column

Inactivation of AdoHcy Hydrolase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4165
1/Kapp ) 1/k2 + KI/k2(1/[I])
[EtOAc/MeOH (19:1) f S1] and recrystallized (MeOH) to give
9b (70 mg, 80%): mp 241-243 °C (lit.18 mp 240-241 °C); MS
m/ z 294 (7, M+).
Con ver sion of E‚NAD+ to E‚NADH. The inhibitor-
induced E‚NADH formation was determined by measuring the
increase in absorbance at 320 nm at different time intervals
after the enzyme was mixed with the inhibitor. To AdoHcy
hydrolase (44 µM) in 1 mL of 50 mM phosphate buffer (pH
7.2) containing 1 mM EDTA (buffer A) was added 100 µL of
Ado 5′-ester 2b (22 mM), with mixing for 10 s. The UV
spectrum (280-500 nm) was recorded periodically at 25 °C
using a HP 8452 diode array spectrophotometer. The reference
cell contained the same enzyme solution to which had been
added 100 µL of water. Spectra were recorded until no
increase in absorbance at 320 nm was observed (∼20 min).
Sp on ta n eou s Hyd r olysis of th e Ad o 5′-Ester 2b a n d
Ad o 5′-Am id e 8b. Hydrolyses of ester 2b and amide 8b were
studied by incubation in buffer A (with and without enzyme)
at 37 °C for various times. At each time point, an aliquot (30
µL) of the reaction solution was injected into a C-18 reversed-
phase HPLC column (Econosphere Alltech, 250 × 4.6 mm).
Chromatography was carried out with a linear gradient of
8-20% A in B for 0-15 min at a flow rate of 1 mL/min, where
mobile phase A was acetonitrile and B was 50 mM sodium
phosphate buffer (pH 3.2) containing 10 mM heptanesulfonic
acid. Peak areas of the product and remaining reactant were
monitored by UV at 258 nm. Kinetic data were fitted to a
first-order reaction equation.
Ad en osin e-5′-(N-bu tylca r boxa m id e) (10b). Ester 2a
(100 mg, 0.3 mmol) was suspended in dioxane (5 mL),
butylamine (1 mL) was added, and the mixture was stirred at
ambient temperature for 2 h. TLC (S1) showed a mixture of
10a /2a (∼3:2). MeOH (2 mL) and H2O (0.5 mmol) were added,
the reaction mixture was gently refluxed for 2 h, and volatiles
were evaporated. The residue was deprotected (as described
for 8b), chromatographed [EtOAc/MeOH (19:1) f S1], and
crystallized (MeOH/EtOAc, 2:1) to give 10b (62 mg, 62%): mp
122-124 °C (lit.18 mp 125 °C); MS m/ z 336 (10, M+).
Ad en osin e-5′-(N-ben zylca r boxa m id e) (11b). Ester 2a
(67 mg, 0.2 mmol) and benzylamine (0.22 mL, 214 mg, 2 mmol)
in dioxane/MeOH (1:1, 10 mL) were refluxed for 4 h. Depro-
tection, purification, and crystallization (MeOH/H2O, 1:1) (as
described for 10b) gave 11b (53 mg, 72%): mp 133-134 °C
(lit.18 mp 130-133 °C); MS m/ z 370 (20, M+).
Ad en osin e-5′-(N-p h en ylca r boxa m id e) (12b). A solution
of 1a (64 mg, 0.2 mmol) in MeOH/dioxane/H2O (3:3:1, 10 mL)
was added to a solution of aniline (45 µL, 46 mg, 0.5 mmol)
and dicyclohexylcarbodiimide (82 mg, 0.4 mmol) in THF (3
mL), and stirring was continued overnight at ambient tem-
perature. Volatiles were evaporated in vacuo, and the residue
was chromatographed [EtOAc/MeOH (19:1) f S1] to give 12a
(68 mg). This 12a (68 mg) was deprotected (as described for
8b) and crystallized (MeOH/EtOAc, 1:1) to give 12b (58 mg,
81%): mp 246-248 °C (lit.18 mp 252-254 °C); MS m/ z 356
(22, M+).
Ad en osin e-5′-(N,N-d ieth ylca r boxa m id e) (14b). Treat-
ment of 1a (160 mg, 0.5 mmol) with thionyl chloride (0.50 mL,
0.81 mg, 6.8 mmol) and DMF (2 drops) gave the acid chloride
13a as described.16 Et2NH (1 mL) was added dropwise at 0 °C
to this 13a in THF (2 mL), and stirring was continued at
ambient temperature for 2 h. The solution was evaporated,
the residue was partitioned (HCl/H2O/CHCl3), and the organic
layer was washed (NaHCO3/H2O, brine), dried (MgSO4), and
evaporated to give 14a (yellow oil). Deprotection of this 14a
(as described for 8b), chromatography [EtOAc/MeOH (19:1)
f S1], and crystallization (MeOH) gave 14b (104 mg, 62% from
1a ): mp 244-245 °C; UV max 259 nm (ꢀ 15 100), min 230 nm
(ꢀ 4100); 1H NMR 1.07 and 1.12 (t and t, J ) 7.0 Hz, 3 and 3,
CH3′s), 3.28-3.45 (m, 4, CH2′s), 4.28 (ddd, J 3′-4′ ) 2.9 Hz, J OH-3′
) 5.6 Hz, J 3′-2′ ) 4.4 Hz, 1, H3′), 4.54 (ddd, J 2′-1′ ) 5.8 Hz,
J OH-2′ ) 5.7 Hz, 1, H2′), 4.77 (d, 1, H4′), 5.65 (d, 1, OH3′), 5.68
(d, 1, OH2′), 6.07 (d, 1, H1′), 7.32 (br s, 2, NH2), 8.18 (s, 1,
H2), 8.59 (s, 1, H8); MS m/ z 336 (20, M+). Anal. (C14H20N6O4)
C,H,N.
Deter m in a tion of Ad oHcy Hyd r ola se In h ibition Con -
sta n ts. The purified recombinant human placental AdoHcy
hydrolase was prepared from Escherichia coli carrying the
expression vector (pPUCSAH) and grown in the presence of
isopropyl â-D-thiogalactopyranoside essentially as described.24
The enzyme activity was determined in the synthetic direction
by incubating AdoHcy hydrolase with 0.2 mM Ado and 5 mM
Hcy for 5 min at 37 °C in 50 mM potassium phosphate buffer
(pH 7.2) containing 1 mM EDTA and assaying the reaction
product, AdoHcy, by HPLC after quenching the reaction by
addition of perchloric acid (final concentration: 0.5 M). An
aliquot (100 µL) of the reaction mixture was injected into a
HPLC column (Econosphere Alltech, 25 cm × 4.6 mm, C-18
reversed-phase column) at a flow rate of 1 mL/min. The
elution gradient consisted of two sequential linear gradients:
6-15% B over 0-9 min and 15-50% B over 9-15 min, where
the mobile phase B was acetonitrile and A was 50 mM sodium
phosphate buffer (pH 3.2) containing 10 mM heptanesulfonic
acid. The peak of AdoHcy was detected by UV at 254 nm. For
the determination of inhibition constants, AdoHcy hydrolase
was preincubated with various concentrations of the inhibitors
for various time intervals and the remaining enzyme activity
Ack n ow led gm en t. We thank the American Cancer
Society (Grant No. DHP-34), Brigham Young University
development funds, and the United States Public Health
Service (Grant No. GM-29332) for financial support and
Mrs. J eanny K. Gordon for assistance with the manu-
script.
Su p p or tin g In for m a tion Ava ila ble: Kitz and Wilson
plots for AdoHcy hydrolase inhibition kinetic data in Table 2
and UV absorbance vs time plots for reduction of NAD+ to
NADH upon incubation of 2b with the enzyme (13 pages).
Ordering information is given on any current masthead page.
Refer en ces
(1) Ueland, P. M. Pharmacological and Biochemical Aspects of
S-Adenosylhomocysteine and S-Adenosylhomocysteine Hydro-
lase. Pharmacol. Rev. 1982, 34, 223-253.
(2) (a) De Clercq, E. S-Adenosylhomocysteine Hydrolase Inhibitors
as Broad-Spectrum Antiviral Agents. Biochem. Pharmacol. 1987,
36, 2567-2575. (b) Yuan, C.-S.; Liu, S.; Wnuk, S. F.; Robins, M.
J .; Borchardt, R. T. Design and Synthesis of S-Adenosylho-
mocysteine Hydrolase Inhibitors as Broad-Spectrum Antiviral
Agents. In Advances in Antiviral Drug Design; DeClercq, E., Ed.;
J AI Press: Greenwich, CT, 1996; Vol. 2, pp 41-88.
(3) (a) Wolfe, M. S.; Borchardt, R. T. S-Adenosyl-L-homocysteine
Hydrolase as a Target for Antiviral Chemotherapy. J . Med.
Chem. 1991, 34, 1521-1529. (b) Liu, S.; Wolfe, M. S.; Borchardt,
R. T. Rational Approaches to the Design of Antiviral Agents
Based on S-Adenosyl-L-homocysteine Hydrolase as a Molecular
Target. Antiviral Res. 1992, 19, 247-265.
(4) (a) Keller, B. T.; Borchardt, R. T. Metabolism and Mechanism
of Action of Neplanocin A - A Potent Inhibitor of S-Adenosyl-
homocysteine Hydrolase. In Biological Methylation and Drug
Design; Borchardt, R. T., Creveling, C. R., Ueland, P. M., Eds.;
Humana Press: Clifton, NJ , 1986; pp 385-396. (b) Keller, B.
T.; Borchardt, R. T. Inhibition of S-Adenosylmethionine-depend-
ent Transmethylation as an Approach to the Development of
Antiviral Agents. In Antiviral Drug Development: A Multidis-
ciplinary Approach; DeClercq, E., Walker, R. T., Eds.; Plenum:
New York, 1988; pp 123-138. (c) Ransohoff, R. M.; Narayan,
P.; Ayers, D. F.; Rottman, F. M.; Nilsen, T. W. Priming of
Influenza mRNA Transcription Is Inhibited in CHO Cells
Treated with the Methylation Inhibitor, Neplanocin A. Antiviral
Res. 1987, 7, 317-327.
(5) (a) Hasobe, M.; McKee, J . G.; Borchardt, R. T. Relationship
between Intracellular Concentration of S-Adenosylhomocysteine
and Inhibition of Vaccinia Virus Replication and Inhibition of
Murine L-929 Cell Growth. Antimicrob. Agents Chemother. 1989,
33, 828-834. (b) Cools, M.; De Clercq, E. Influence of S-
Adenosylhomocysteine Hydrolase Inhibitors on S-Adenosylho-
mocysteine and S-Adenosylmethionine Pool Levels in L929 Cells.
Biochem. Pharmacol. 1990, 40, 2259-2264.
was measured. The pseudo-first-order rate constants (Kapp
)
were obtained from the plot of log(% activity remaining) vs
time, and KI and k2 values (Table 1) were estimated from the
double-reciprocal plot of 1/Kapp vs 1/[inhibitor] using the
following equation:

4166 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Wnuk et al.
(6) (a) Cools, M.; De Clercq, E. Correlation Between the Antiviral
Activity of Acyclic and Carbocyclic Adenosine Analogues in
Murine L929 Cells and Their Inhibitory Effect on L929 Cell
S-Adenosylhomocysteine Hydrolase. Biochem. Pharmacol. 1989,
38, 1061-1067. (b) 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 Deriva-
tives of Adenosine, Inactivation of S-Adenosyl-L-homocysteine
Hydrolase, and Correlation of Anticancer and Antiviral Potencies
with Enzyme Inhibition. J . Med. Chem. 1994, 37, 3579-3587.
(7) Huggins, J . W.; Zhang, Z. X.; Davis, K.; Coulombe, R. A.
Inhibition of Ebola Virus by S-Adenosylhomocysteine Hydrolase
Inhibitors. Abstracts of the Eighth International Conference on
Antiviral Research, Santa Fe, NM; Antiviral Res. 1995, A301.
(8) Liu, S.; Wnuk, S. F.; Yuan, C.; 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.
(9) 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.
(15) 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.
(16) Prasad, R. N.; Fung, A.; Tietje, K.; Stein, H. H.; Brondyk, H. D.
Modification of the 5′ Position of Purine Nucleosides. 1. Synthesis
and Biological Properties of Alkyl Adenosine-5′-carboxylates. J .
Med. Chem. 1976, 19, 1180-1186.
(17) Fritz, H.-J .; Machat, R.; Schmidt, R. R. Nucleosid-5′-carbonsa¨ure-
ester als Modelle fu¨r die Konformationsanalyse von Nucleosiden
und Nucleotiden. (Esters of Nucleoside-5′-carboxylic Acids as
Model Compounds for Conformational Analysis of Nucleosides
and Nucleotides.) Chem. Ber. 1972, 105, 642-649.
(18) Prasad, R. N.; Bariana, D. S.; Fung, A.; Savic, M.; Tietje, K.;
Stein, H. H.; Brondyk, H.; Egan, R. S. Modification of the 5′
Position of Purine Nucleosides. 2. Synthesis and Some Cardio-
vascular Properties of Adenosine-5′-(N-substituted)carboxam-
ides. J . Med. Chem. 1980, 23, 313-319.
(19) (a) Hutchison, A. J .; Williams, M.; De J esus, R.; Yokoyama, R.;
Oei, H. H.; Ghai, G. R.; Webb, R. L.; Zoganas, H. C.; Stone, G.
A.; J arvis, M. F. 2-(Arylalkylamino)adenosin-5′-uronamides: A
New Class of Highly Selective Adenosine A2 Receptor Ligands.
J . Med. Chem. 1990, 33, 1919-1924. (b) Homma, H.; Watanabe,
Y.; Abiru, T.; Murayama, T.; Nomura, Y.; Matsuda, A. Nucleo-
sides and Nucleotides. 112. 2-(1-Hexyn-1-yl)adenosine-5′-urona-
(10) Yuan, C.-S.; Yeh, J .; Liu, S.; Borchardt, R. T. Mechanism of
Inactivation of S-Adenosylhomocysteine Hydrolase by (Z)-4′,5′-
Didehydro-5′-Deoxy-5′-Fluoroadenosine. J . Biol. Chem. 1993,
268, 17030-17037.
mides:
A New Entry of Selective A2 Adenosine Receptor
(11) (a) Robins, M. J .; Wnuk, S. F. Fluorination at C5′ of Nucleosides.
Synthesis of the New Class of 5′-Fluoro-5′-S-Aryl (Alkyl) Thio-
nucleosides from Adenosine. Tetrahedron Lett. 1988, 29, 5729-
5732. (b) Robins, M. J .; Wnuk, S. F.; Mullah, K. B.; Dalley, N.
K.; Borchardt, R. T.; Lee, Y.; Yuan, C.-S. Adenosine-Derived 5′-
R-Halo Thioether, Sulfoxide, Sulfone, and (5′-Halo)methylene
Analogues. Inhibition of S-Adenosyl-L-Homocysteine Hydrolase.
In Nucleosides and Nucleotides as Antitumor and Antiviral
Agents; Chu, C. K., Baker, D. C., Eds.; Plenum Press: New York,
1993; pp 115-126. (c) Robins, M. J .; Wnuk, S. F.; Mullah, K. B.;
Dalley, N. K.; Yuan, C.-S.; Lee, Y.; Borchardt, R. T. Nucleic Acid
Related Compounds. 80. Synthesis of 5′-S-(Alkyl and aryl)-5′-
fluoro-5′-thioadenosines with Xenon Difluoride or (Diethylami-
do)sulfur Trifluoride, Hydrolysis in Aqueous Buffer, and Inhi-
bition of S-Adenosyl-L-homocysteine Hydrolase by Derived
“Adenosine 5′-Aldehyde” Species. J . Org. Chem. 1994, 59, 544-
555.
(12) Yuan, C.-S.; Liu, S.; Wnuk, S. F.; Robins, M. J .; Borchardt, R.
T. Mechanism of Inactivation of S-Adenosylhomocysteine Hy-
drolase by (E)-5′,6′-Didehydro-6′-Deoxy-6′-Halohomoadenosines.
Biochemistry 1994, 33, 3758-3765.
(13) 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.
(14) Kim, I.-Y.; Zhang, C.-Y.; Cantoni, G. L.; Montgomery, J . A.;
Chiang, P. K. Inactivation of S-Adenosylhomocysteine Hydrolase
by Nucleosides. Biochim. Biophys. Acta 1985, 829, 150-155.
Agonists with Potent Antihypertensive Activity. J . Med. Chem.
1992, 35, 2881-2890.
(20) Gallo-Rodriguez, C.; J i, X.-D.; Melman, N.; Siegman, B. D.;
Sanders, L. H.; Orlina, J .; Fischer, B.; Pu, Q.; Olah, M. E.; Van
Galen, P. J . M.; Stiles, G. L.; J acobson, K. A. Structure-Activity
Relationships of N6-Benzyladenosine-5′-uronamides as A3-Selec-
tive Adenosine Agonists. J . Med. Chem. 1994, 37, 636-646.
(21) Harmon, R. E.; Zenarosa, C. V.; Gupta, S. K. Permanganate
Oxidation of Purine Nucleosides. Chem. Ind. (London) 1969,
1141.
(22) (a) Singh, A. K.; Varma, R. S. Ruthenium Tetraoxide: A Mild
Reagent for the Oxidation of 2′,3′-O-Isopropylidene Purine
Nucleosides. Tetrahedron Lett. 1992, 33, 2307-2310. (b) Varma,
R. S.; Hogan, M. E. Ruthenium Tetraoxide Catalyzed Oxidation
of Nucleosides: A Facile Synthesis of 5′-Carboxylic Acid Deriva-
tives. Tetrahedron Lett. 1992, 33, 7719-7720.
(23) Harper, P. J .; Hampton, A. Conversion of 2′,3′-O-Isopropylidene
Adenosine into Its 5′,5′-Di-C-Methyl Derivative. J . Org. Chem.
1970, 35, 1688-1689.
(24) Gomi, T.; Date, T.; Ogawa, H.; Fujioka, M.; Aksamit, R. R.;
Backlund, P. S., J r.; Contoni, G. L. Expression of Rat Liver
S-Adenosylhomocysteinase cDNA in Escherichia coli and Mu-
tagenesis at the Putative NAD Binding Site. J . Biol. Chem. 1989,
264, 16138-16142.
J M960313Y