Synthesis of 5′-functionalized nucleosides: S-Adenosylhomocysteine analogues with the carbon-5′ and sulfur atoms replaced by a vinyl or halovinyl unit

Article abstract of DOI:10.1016/j.bmc.2008.04.017

Adenosine and uridine analogues functionalized with alkenyl or fluoroalkenyl chain at C5′ were prepared employing cross-metathesis, Negishi couplings, and Wittig reactions. Metathesis of the protected 5′-deoxy-5′-methyleneadenosine or uridine analogues wi

Full text of DOI:10.1016/j.bmc.2008.04.017

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5424–5433
Synthesis of 50-functionalized nucleosides:
S-Adenosylhomocysteine analogues with the carbon-50
and sulfur atoms replaced by a vinyl or halovinyl unit
Stanislaw F. Wnuk,a,* Pablo R. Sacasa,a Elzbieta Lewandowska,a,ꢀ Daniela Andrei,a
Sumin Caib and Ronald T. Borchardtb
aDepartment of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA
bDepartments of Molecular Biosciences and Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047, USA
Received 28 December 2007; revised 5 April 2008; accepted 9 April 2008
Available online 12 April 2008
Abstract—Adenosine and uridine analogues functionalized with alkenyl or fluoroalkenyl chain at C50 were prepared employing
cross-metathesis, Negishi couplings, and Wittig reactions. Metathesis of the protected 50-deoxy-50-methyleneadenosine or uridine
analogues with six-carbon amino acids (homoallylglycines) in the presence of Grubbs catalysts gave nucleoside analogues with
the C50–C60 double bond. Alternatively, the Pd-catalyzed cross-coupling between the protected 50-deoxy-50-(iodomethylene) nucleo-
sides and suitable alkylzinc bromides also provided analogues with alkenyl unit. Stereoselective Pd-catalyzed monoalkylation of 50-
(bromofluoromethylene)-50-deoxyadenosine with alkylzinc bromides afforded adenosylhomocysteine analogues with a 60-(flu-
oro)vinyl motif. The vinylic adenine nucleosides produced time-dependent inactivation of the S-adenosyl-L-homocysteine
hydrolases.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
(5-deoxy-5-fluoro-b-D-erythro-pent-4-enofuranosyl)ade-
nine],4 50-deoxy-50-(halomethylene)adenosine,5 50-(bromo-
fluoromethylene)-50-deoxyadenosine (i.e., A; Fig. 1),6
50-S-allenyl(or propynyl)-50-thioadenosine analogues,7a
and 6-fluoroneplanocin,7b among others.1c,7
The enzyme S-adenosyl-L-homocysteine (AdoHcy)
hydrolase (EC 3.3.1.1) effects the hydrolytic cleavage
of AdoHcy to adenosine (Ado) and L-homocysteine
(Hcy).1 The cellular levels of AdoHcy and Hcy are crit-
ical since AdoHcy is a potent feedback inhibitor of cru-
cial methylation enzymes,1,2 and elevated plasma levels
of Hcy in humans have been shown to be a risk factor
in coronary artery disease.3 Therefore, the design of
inhibitors of AdoHcy hydrolase provides a rational ap-
proach to mechanism-based chemotherapy of cancer
and viral diseases.1,2 Several inhibitors which function
as substrates for the ‘30-oxidative activity’ of AdoHcy
hydrolase and converts the enzyme from its active form
(NAD+) to its inactive form (NADH) have been pre-
pared.1 Inhibitors which function as substrates for the
‘50/60-hydrolytic activity’ include the vinyl fluorides [9-
Addition of an enzyme-sequestered water molecule
across the 50,60-double bond of A followed by the loss
of bromide was proposed to generate the homoAdo
60-carboxyl fluoride B at the active site of AdoHcy
hydrolase.6b The nucleophilic attack by proximal amino
acid functionalities caused the covalent binding inhibi-
tion of type C. Addition of water across the 50,60-triple
bond of haloacetylenes was postulated to generate sim-
ilar reactive electrophiles at the enzyme active site.8
The X-ray crystallographic analysis of the human Ado-
Hcy hydrolase inactivated with 9-(dihydroxycyclopentene)-
adenine,9a and neplanocin A,9b as well as AdoHcy
hydrolase from rat liver9c established the presence of a
water molecule in the active site of the enzyme. This
made it a priority to prepare analogues of AdoHcy that
closely resemble the natural substrate that bind tightly
to the enzyme. Such compounds should form ‘stable’
complexes with the enzyme that would help to identify
Keywords: S-Adenosylhomocysteine hydrolase; Cross-coupling; Cross-
metathesis; Nucleosides; Wittig reaction.
*
Corresponding author. Tel.: +1 305 348 6195; fax: +1 305 348
3772; e-mail: wnuk@fiu.edu
ꢀ
Faculty on leave from the Department of Chemistry, University of
Agriculture, Poznan, Poland.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.017

S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
5425
O
O
A
Br
A
Enz•H2O
Br
A
Arg-196
O
ArgNH
F
O
F
O
F
HO
OH
HO
OH
HO
OH
A
B
C
Figure 1. Generation of active intermediates from the (bromofluoro)vinyl analogue A by the ‘hydrolytic activity’ of AdoHcy hydrolase.6b
the key binding groups at the active site of the enzyme
Z
A
A
Cross
Y
that interact with the Hcy moiety and participate in
the subsequent elimination and ‘hydrolytic’ activity
steps. We now describe syntheses of the adenosine and
uridine10 nucleosides modified with alkenyl or haloalke-
nyl chain at C50. Adenosine analogues with the 50,60-vi-
nyl D or halovinyl E motif incorporated in place of the
carbon-50 and sulfur atoms (Fig. 2) are expected to be
the substrates for the ‘oxidative’ and/or ‘hydrolytic’
activity of AdoHcy hydrolase. Enzyme-mediated addi-
tion of water to E (X = F, Z = H or NH2) might occur
at C50 or C60. This would generate a new species bearing
hydroxyl or keto (after b-elimination of HF) binding
sites within the enzyme.
O
O
couplings
HO2C
X
X
HO OH
HO OH
E X = halogen
Z Z = H or NH2
F X and Y = halogen
Halogenation-
dehydrohalogenation
Z
A
A
5'
Y
O
O
Cross
couplings
HO2C
6'
HO OH
HO OH
2. Chemistry
G Y = halogen
D Z = H or NH2
The retrosynthetic analysis indicated that AdoHcy ana-
logue D can be targeted by the construction of new C50–
C60 double bond via either a Wittig reaction employing
adenosine 50-aldehyde H or cross-metathesis reaction
utilizing 50-deoxy-50-methyleneadenosine I as nucleoside
precursors (Fig. 3). The subsequent bromination–dehy-
drobromination of D might afford bromovinyl analogue
E (X = Br). It is also possible to use Pd-catalyzed cross-
coupling approaches to form a new C60–C70 bond in a
key step. For example, the alkylation of 50-deoxy-50-
(iodomethylene)adenosine G with the suitable alkylzinc
bromides should produce D. Alternatively, the selective
monoalkylation of 50-deoxy-50-(dihalomethylene)adeno-
sine F should afford E directly. Coupling approaches
were tested on model compounds11 and the reactions
were performed on the pyrimidine10 and purine nucleo-
sides to generalize the synthetic approaches.
Metathesis
Wittig
A
A
O
O
O
HO OH
OH
HO
I
H
Figure 3. Retrosynthetic analysis for AdoHcy analogues with the
carbon-50 and sulfur atoms replaced by a vinyl or halovinyl unit.
Moffatt oxidation12 of 20,30-O-isopropylideneuridine 1a
and treatment of the crude 50-aldehyde with Wittig re-
agent, generated from the commercially available
EtO2C(CH2)3PPh3ÆBr with lithium bis(trimethyl-
silyl)amide (LHMDS), gave ethyl 4(Z)-(50-deoxy-20,30-
O-isopropylideneuridin-50-ylidene)butanoate 2a in 16%
yield. Deprotections [trifluoroacetic acid (TFA)/H2O]
of 2a yielded 2b (Scheme 1). The vicinal coupling con-
stant (2b; J5 –6 ¼ 9:8 Hz) was diagnostic for the Z ste-
reochemistry, as expected for the Wittig product
derived from the non-stabilized ylide.13 Analogous Wit-
tig-treatment of 6-N-benzoyl-20,30-O-isopropylideneade-
nosine 1b gave 2c. Treatment of crude 2c with NH3/
MeOH removed the benzoyl group and converted the
ethyl ester into a methyl ester 2d (18% from 1b). Deacet-
onization of 2d and saponification of the resulting 2e
afforded 2f; a nine-carbon analogue of AdoHcy with
the vinyl unit between C50 and C60. The low yields
and notorious instability of 50-aldehydes14 toward the
experimental conditions, that is required for the genera-
NH2
NH2
0
0
N
N
N
N
N
N
5'
NH2
Z
N
O
N
O
6'
X
HO2C
S
HO2C
9'
HO OH
AdoHcy
HO OH
D X = H; Z = H or NH2
E X = Br or F; Z = H or NH2
Figure 2. S-Adenosyl-L-homocysteine (AdoHcy) and analogues with
the carbon-50 and sulfur atoms replaced by a vinyl or halovinyl unit.

5426
S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
R1O
As previously reported, metathesis of 6-N,N-dibenzoyl-
B
B
20,30-O-isopropylidene-50-deoxy-50-methyleneadenosine
with 4b in the presence of Hoveyda-Grubb’s catalyst
(o-isopropoxy-phenylmethylene-Ru) gave the protected
AdoHcy analogue D (Z = NH2) in 76% yield.17,22 How-
ever, the treatment of the latter with pyridinium tribro-
mide followed by the dehydrobromination with DBU
yielded 50-(bromo)vinyl AdoHcy analogue as a single
isomer, which was deprotected to ester 8a and acid
8b.17 Consequently, in order to prepare 60-(halo)vinyl
analogues E, we turned our attention to the Pd-cata-
lyzed selective monoalkylation of 50-deoxy-50-(dihalom-
ethylene)nucleoside precursors.
HO
O
O
a
O
O
O
RO OR
2a B = U; R,R = CMe2; R1 = Et
b
2b B = U; R = H; R1 = Et
1a B = U
1b B = ANHBz
2c B = ANHBz; R,R = CMe2; R1 = Et
2d B = A; R,R = CMe2; R1 = Me
2e B = A; R,R = H; R1 = Me
2f B = A; R = H; R1 = H
c
b
d
Scheme 1. Reagents: (a) i—DCC/DMSO/Cl2CHCO2H; ii—EtO2C-
(CH2)3PPh3Br/LHMDS/THF; (b) TFA/H2O; (c) NH3/MeOH; (d)
NaOH/H2O/MeOH.
Although Pd-catalyzed cross-coupling reactions are
powerful methods for the formation of carbon–carbon
bonds under conditions that are compatible with a
broad range of functional groups,23a couplings involving
Csp3 centers23b are less explored with the exception of
couplings between Csp2 as electrophiles and Csp3 as
nucleophiles.23a,24 Therefore among other ap-
proaches,25,26 we began with exploring Negishi cou-
pling.24 Treatment of the protected 50-deoxy-50-
(iodomethylene)adenosine5a (E)-10 with commercially
available EtO2C(CH2)3ZnBr produced ethyl 5-(50-
deoxy-20,30-O-isopropylideneadenosin-50-ylidene)pent-
tion of non-stabilized Wittig reagents, prompted us to
examine the metathesis approach for the synthesis of D.
In order to explore the metathesis route,15,16 ethyl 2-
amino-5-hexenoate (homoallylglycine) bearing a termi-
nal double bond was prepared by the alkylation of gly-
cine with 4-bromo-1-butene followed by protection of
the a-amino group with benzoyl or Boc group to give
racemic 4a or 4b, respectively.17 Cross-metathesis be-
tween 20,30-O-isopropylidene-50-deoxy-50-methyleneuri-
dine18 3 with 4a in a CH2Cl2 solution containing 2nd
generation (2-imidazolidinylidene-Ru) Grubbs cata-
lyst15 gave the desired product 5a (72%) in addition to
two dimers 7a (5%) and 6 (7%) resulting from the self-
metathesis of nucleoside16,19 3 and amino acid17,20 4b
substrates, respectively (Scheme 2). Deacetonization of
5a and HPLC purification yielded 5b (66%) as 50E iso-
0
0
anoate 12a (68%) as a single E isomer (J6 –5 ¼ 15:4 Hz;
Scheme 3). Deacetonization of 12a with TFA/H2O affor-
ded ester 12b (61%) and the subsequent saponification
gave 12c with a six-atom alkenyl chain attached to the
furanosyl C40 and the carboxylic group at C100 as in
AdoHcy. Subjection of the 50-deoxy-50-(iodomethyl-
ene)uridine18 (E)-9 to the analogous Negishi coupling
and deacetonization of the resulting 11a afforded 11b
(52% overall).
mers ðJ5 –6 ¼ 15:3 HzÞ of a 1:1 mixture of 90R/S diaste-
reomers. The ratio of diastereomers was determined
based on the NMR spectra where a double set of peaks
for several protons (1H) and carbons (13C) of similar
intensities were observed. Analogous metathesis of 3
and 4b gave 5c as the sole product (74%). Aqueous
TFA effected concomitant removal of Boc and isopro-
pylidene protection groups to produce 5d; a uracil ana-
logue of AdoHcy with the amino group at C90 and the
carboxylic group at C100.21
0
0
Encouraged by the results on the alkylation of iodo(vinyl)
nucleosides 9 and 10 with alkylzinc bromides, we have
explored a selective monoalkylation of dihalo(vinyl)
nucleoside substrates (e.g., 13), although the mono
cross-coupling reactions of 1,1-dihalovinyl electrophiles
with Csp3 nucleophiles are very scarce.24c,27 We recently
NHR1
U
U
NHR1
OEt
O
O
EtO2C
a
+
O
O
O
RO OR
5a B = U, R1 = Bz, R,R = CMe2
5b B = U, R1 = Bz, R,R = H
5c B = U, R1 = Boc, R,R = CMe2
5d B = U, R1 = H, R,R = H
4a R1 = Bz
b
b
3
4b R1 = Boc
O
U
Br
O
O
A
NHBz
OEt
RO
NH2
O
2
RO
OR
HO OH
2
6
8a R = Me/Et
8b R = H
7a R,R = CMe2
7b R,R = H
b
Scheme 2. Reagents and condition: (a) 2nd generation Grubbs catalyst/CH2Cl2/D; (b) TFA/H2O.

S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
5427
Table 1. Inhibition of AdoHcy hydrolase by 50,60-vinylic adenosine
derivatives
B
B
R1O
I
O
O
a
Compounda
% of enzyme inhibitionb,c
O
0.5 hd
4 hd
O
O
RO OR
2ee
2f
68.1
50.5
11.4
14.5
81
11a B = U; R,R = CMe2; R1 = Et
11b B = U; R,R = H; R1 = Et
12a B = A; R,R = CMe2; R1 = Et
12b B = A; R,R = H; R1 = Et
12c B = A; R,R = H; R1 = H
95.5
44.6
92.4
35.2
b
8ae
8b
9 B = U
10 B = A
b
c
12be
12c
14be
14c
28.5
14.1
15.2
Scheme 3. Reagents and condition: (a) EtO2C(CH2)3ZnBr/Pd2 (dba)3/
benzene/D; (b) TFA/H2O; (c) NaOH/H2O.
a Dienes, for example, ethyl (E/E)- and (Z/E)-4-(50-deoxyadenosin-50-
ylidene)but-2-enoate caused 96.4% and 98.2% of the enzyme inhibi-
tion under nearly identical conditions with 20 min incubation
time.33a
b AdoHcy hydrolase (204 nM) was incubated with the inhibitors
(281 lM) at 37 ꢁC for 12 min and the remaining enzyme activity was
assayed as described in Section 5.
c Data are the average of duplicate determinations.
d Preincubation time of the inhibitors with the enzyme.
e Esters were not tested with 4 h preincubation time because of the
chemical instability in the enzymatic assay.
A
A
R1O
Br
O
O
a
O
F
F
O
O
RO OR
14a R,R = CMe2; R1 = Et
14b R,R = H; R1 = Et
14c R,R = H; R1 = H
b
c
13
than the non-halogenated derivatives 12b and 12c per-
haps in accord with the fact that 50-deoxy-50-(fluorometh-
ylene)adenosine showed the lowest enzyme inactivation
efficiency among all 50-halomethylene derivatives.5b,c
The 50-(bromo)vinylic analogues 8a and 8b with the ami-
no group at C90 also showed a lower enzyme inhibition.
The 2f, 8b, 12c, and 14c produced similar time-dependent
inactivation of the human (Table 1) and T. cruzi (data not
shown) AdoHcy hydrolases. Since esters 2e and 12b are
less active than structurally related dienes33a (see Table
1, footnote a) and enynes,33b and 8a and 14b are signifi-
cantly less active no further enzymatic studies were
performed.
Scheme 4. Reagents and condition: (a) EtO2C(CH2)3ZnBr/Pd(PPh3)4/
benzene/D; (b) TFA/H2O; (c) NaOH/H2O.
developed Pd-catalyzed Negishi coupling of 1-fluoro-1-
(iodo, or bromo, or chloro)alkenes with alkylzincs, which
provided stereoselective access to the internal fluoroalk-
enes.11 We were gratified to find that treatment of the pro-
tected 50-(bromofluoromethylene)-50-deoxyadenosine6a
13 (E/Z, 3:2) with EtO2C(CH2)3ZnBr/Pd(PPh3)4 pro-
duced 14a (56%) as a mixture28 of the geometric isomers
(E/Z, 2:3; Scheme 4).29 Acid-catalyzed deprotection of
14a gave ester 14b, and saponification afforded 14c; a
10-carbon AdoHcy analogue with C50 and sulfur atoms
replaced by 60-fluoro(vinyl) unit. Attempted monoalkyla-
tion of 13 with the zinc salt of enatiomerically pure N-Boc-
c-bromohomoalanine-OMe,30 generated by Knochel’s
procedure31 utilizing a Zn/LiCl combination, failed to
give 60-(fluoro)vinyl analogue E (X = F, Z = NH2) bear-
ing amino group at C90.
4. Summary and conclusions
We have developed the synthesis of adenosine and uri-
dine analogues functionalized at C50 with alkenyl or
60-fluoroalkenyl unit employing metathesis, coupling,
and Wittig reactions. Cross-metathesis of the 50-deoxy-
50-methyleneadenosine or uridine analogues with homo-
allylglycines gave a 10-carbon nucleoside analogues with
the C50–C60 double bond. The Pd-catalyzed selective
monoalkylation of the 50-(bromofluoromethylene)-50-
deoxyadenosine with EtO2C(CH2)3ZnBr afforded Ado-
Hcy analogues with 60-(fluoro)vinyl unit. The vinylic
adenine nucleosides were tested against human and T.
cruzi AdoHcy hydrolases. The most active inhibitors
were found to be 10- and 9-carbon non-halogenated
vinylic analogues. The free carboxylic acids derivatives
produced time-dependent inactivation of the enzyme.
3. Interactions with AdoHcy hydrolase
The 50,60-vinylic adenine nucleosides were tested against
human AdoHcy hydrolase using a protocol involving a
0.5 or a 4 h preincubation with the enzyme followed by
a 12 min incubation that measures the residual enzyme
activity (Table 1). For esters 2e, 8a, 12b, and 14b only data
for the 0.5 h preincubation are included since these esters
are potentially unstable either chemically or enzymati-
cally under the assay conditions. The compounds had
similar activity toward the Trypanosoma cruzi AdoHcy
hydrolase32 (data not shown). The most active inhibitors
were found to be the 9-carbon 2e and 2f as well as the 10-
carbon 12b and 12c vinylic analogues. With a 30 min pre-
incubation, esters 2e and 12b were found to be more active
than the corresponding acids 2f and 12c. The 60-fluoro
vinylic analogues 14b and 14c showed a lower activity
5. Experimental
UV spectra were measured with solutions in MeOH. 1H
(400 or 600 MHz) and 13C (100 MHz) NMR spectra
were determined with solutions in CDCl3 unless other-

5428
S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
wise noted. Mass spectra (MS) were obtained with
atmospheric pressure chemical ionization (APCI) tech-
nique and HRMS in AP-ESI mode. TLC was performed
with Merck kieselgel 60-F254 sheets with MeOH/CHCl3
(1:19), EtOAc/hexane (2:1) or EtOAc/i-PrOH/H2O
(4:1:2, upper layer; S1) as developing systems and prod-
ucts were detected with 254 nm light. Merck kieselgel 60
(230–400 mesh) was used for column chromatography.
HPLC purifications were performed using XTerraꢂ pre-
parative RP18 OBDTM column (5 lm 19 · 150 mm) with
gradient program using CH3CN/H2O as a mobile phase.
Elemental analyses were determined by Galbraith Labo-
ratories, Knoxville, TN. Reagent grade chemicals were
used, and solvents were dried by reflux over and distilla-
tion from CaH2 (except THF/potassium) under argon.
J = 6.6,
8.2 Hz,
1,
H40),
5.64
(‘tt’,
J5 –4 =6 ¼ 9:8 Hz;J5 –7 =7 ¼ 1:2 Hz, 1, H50), 5.73–5.78
(m, 2, H5,60), 5.83 (d, J = 3.6 Hz, 1, H10), 7.63 (d,
J = 8.0 Hz, 1, H6); 13C NMR (MeOH-d4, 600 MHz) d
14.52 (CH3), 24.45 (C70), 35.08 (C80), 61.61 (CH2),
75.31 (C30), 75.81 (C30), 80.38 (C40), 92.17 (C10),
102.84 (C5), 129.41 (C50), 134.87 (C60), 142.57 (C6),
152.55 (C2), 166.21 (C4), 174.66 (C90); MS m/z 341
(100, MH+). Anal. Calcd for C15H20N2O7ÆH2O
(358.35): C, 50.28; H, 6.19; N, 7.82. Found: C, 49.91;
H, 5.82; N, 7.56.
0
0
0
0
0
00
5.3. Methyl 4(Z)-(50-deoxy-20,30-O-isopropylideneadeno-
sin-50-ylidene)butanoate (2d)
Oxidation of 1b14 (205 mg, 0.5 mmol) with DMSO
(2 mL)/DCC
5.1. Ethyl 4(Z)-(50-deoxy-20,30-O-isopropylideneuridin-50-
ylidene)butanoate (2a). Procedure A
(395 mg,
1.75 mmol)/Cl2CHCO2H
(0.044 mL, 65 mg, 0.5 mmol) and treatment with Wittig
reagent (274 mg, 0.6 mmol) by the procedure A gave 2c
(ꢀ90 mg, ꢀ36%; contaminated in ꢀ20%, 1H NMR): MS
m/z 508 (100, MH+). This material was dissolved in
NH3/MeOH and was stirred overnight at ambient tem-
perature. The volatiles were evaporated in vacuo and
the residue was partitioned (CHCl3//H2O/NaHCO3).
The organic layer was washed (brine), dried (MgSO4),
evaporated and purified on column chromatography
(50 ! 70% EtOAc/hexane) to give 2d (35 mg, 18% from
1b); 1H NMR d 1.41 (s, 3, CH3), 1.64 (s, 3, CH3), 2.42 (t,
J = 6.8 Hz, 2, H80,800), 2.47–2.52 (m, 2, H70,700), 3.68 (s,
3, CH3), 4.95 (dd, J = 3.3, 6.1 Hz, 1, H30), 5.06–5.09
(m, 1, H40), 5.52–5.68 (m, 3, H20,50,60), 5.81 (br s, 2,
NH2), 6.15 (d, J = 1.9 Hz, 1, H10), 7.92 (s, 1, H2), 8.37
(s, 1, H8); MS m/z 390 (100, MH+). HRMS calcd for
C18H24N5O5 [M+H]+ 390.1772, found 390.1782.
In flask A, Cl2CHCO2H (0.62 mL, 97 mg, 0.75 mmol)
was added dropwise via syringe to a stirred solution of
1a (426 mg, 1.5 mmol) and DCC (1.08 g, 5.25 mmol)
in DMSO (4 mL) at ambient temperature under N2.
The stirring was continued for an additional 1 h to pro-
duce the corresponding aldehyde. In flask B, lithium
bis(trimethylsilyl)amide (3.6 mL/1 M THF, 3.6 mmol)
was added to a stirred solution of [3-(ethoxycar-
bonyl)propyl]triphenylphosphonium bromide (823 mg,
1.8 mmol) in anhydrous THF (6 mL) at ambient tem-
perature under N2 and stirring was continued until the
reaction turned red (ꢀ2 h). The content of flask A was
then transferred to flask B via syringe and the resulting
mixture was stirred overnight (TLC analysis showed the
formation of a less polar product). Oxalic acid dihydrate
(378 mg, 3 mmol) dissolved in MeOH (3 mL) was added
and after 20 min the reaction mixture was concentrated
(to ꢀ1/3 volume). Dicyclohexylurea was filtered, washed
with cold MeOH, and the combined filtrates were evap-
orated. The residue was partitioned (CHCl3/NaHCO3/
H2O) and the organic layer was washed (3· H2O, brine),
dried (MgSO4), and was evaporated. Column chroma-
tography of the residue (40 ! 60% EtOAc/hexane) gave
2a (92 mg, 16%); 1H NMR d 1.23 (t, J = 7.1 Hz, 3, CH3),
1.32 (s, 3, CH3), 1.54 (s, 3, CH3), 2.34–2.48 (m, 2,
H70,700,80,800), 4.11 (q, J = 7.1 Hz, 2, CH2), 5.14 (dd,
J = 4.3, 6.3, Hz, 1, H30), 4.85 (dd, J = 4.2, 8.5 Hz, 1,
H40), 4.98 (dd, J = 1.8, 6.3 Hz, 1, H20), 5.51–5.68 (m,
3, H5,50,60), 5.81 (d, J = 2.0 Hz, 1, H10), 7.45 (d,
J = 8.1 Hz, 1, H6); MS m/z 381 (100, MH+).
5.4. Methyl 4(Z)-(50-deoxyadenosin-50-ylidene)butanoate
(2e)
Treatment of 2d (30 mg, 0.077 mmol) with TFA/H2O
(9:1, 5 mL) by procedure B gave 2e (15 mg, 56%); H
1
NMR (MeOH-d4) d 2.35–2.58 (m, 4, H70,700,80,800), 3.67
(s, 3, CH3), 4.22 (t, J = 5.3 Hz, 1, H30), 4.72 (‘t’,
J = 4.8 Hz, 1, H20), 4.82–4.92 (m, 1, H40), 5.62–5.78
(m, 2, H50,60), 6.02 (d, J = 4.3 Hz, 1, H10), 8.18 (s, 1,
H2), 8.21 (s, 1, H8); 13C NMR (MeOH-d4) d 23.34
(C70), 33.77 (C80), 51.09 (CH3), 74.27 (C30), 75.23
(C20), 80.19 (C40), 89.42 (C10), 119.63 (C5), 128.99
(C60), 132.93 (C50), 140.31 (C8), 149.57 (C4), 152.89
(C2), 156.33 (C6), 174.07 (C90); MS m/z 350 (100,
MH+). HRMS calcd for C15H20N5O5 [M+H]+
350.1464, found 350.1460.
5.2. Ethyl 4(Z)-(50-deoxyuridin-50-ylidene)butanoate (2b).
Procedure B
5.5. 4(Z)-(50-Deoxyadenosin-50-ylidene)butanoic acid (2f)
A solution of 2a (140 mg, 0.37 mmol) in TFA/H2O (9:1,
5 mL) was stirred at 0 ꢁC (ice bath) for 1 h. The volatiles
were evaporated under vacuum (<15 ꢁC) and co-evapo-
rated (3·) with toluene. The residue was column chro-
matographed (1 ! 6% MeOH/CHCl3) to give 2b
(48 mg, 38%): UV max 261 nm (e 8800), min 230
(2200); 1H NMR (MeOH-d4, 600 MHz) d 1.27 (t,
J = 7.1 Hz, 3, CH3), 2.41–2.58 (m, 4, H70,700,80,800),
3.95 (t, J = 5.8 Hz, 1, H30), 4.15 (q, J = 7.2 Hz, 2,
CH2), 4.22 (dd, J = 3.7, 5.4 Hz, 1, H20), 4.68 (dd,
NaOH/H2O (0.2 mL, 1 M) was added to a stirred solu-
tion of 2e (13 mg, 0.037 mmol) in MeOH (2 mL) at
ambient temperature. After 24 h, volatiles were evapo-
rated in vacuo and the residue was partitioned
[EtOAc/H2O/CH3COOH (pH ꢀ 4)]. Aqueous layer
was extracted four times with EtOAc and the combined
organic layer was dried (MgSO4), evaporated, and puri-
fied on a column chromatography (EtOAc ! 20% S1/
EtOAc) to give 2f (9 mg, 73%): mp 220–221 ꢁC (MeOH);

S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
5429
1H NMR (MeOH-d4, 600 MHz) d 1.92–2.18 (m, 4,
H70,700,80,800), 3.91 (t, J = 4.9 Hz, 1, H30), 4.46 (t,
J = 4.9 Hz, 1, H20), 4.53 (dd, J = 5.1, 8.0 Hz, 1, H40),
5.39 (dt, J = 10.5, 7.9 Hz, 1, H60), 5.44 (dd, J = 7.4,
10.6 Hz, 1, H50), 5.71 (d, J = 4.7 Hz, 1, H10), 7.99 (s,
1, H2), 8.08 (s, 1, H8); 13C NMR (Me2SO-d6) d 22.30
(C70), 33.51 (C80), 73.99 (C30), 75.45 (C20), 80.36
(C40), 88.64 (C10), 120.06 (C5), 130.05, 132.99 (C50
and 60), 140.78 (C8), 150.18 (C4), 153.50 (C2), 156.93
(C6), 175.89 (C90), MS m/z 336 (100, MH+). Anal. Calcd
for C14H17N5O5 (335.32): C, 50.15; H, 5.11; N, 20.89.
Found: C, 50.45; H, 5.47; N, 20.45.
0.015 mmol) with TFA/H2O (9:1, 1.0 mL) by procedure
B and HPLC purification (10 ! 40% CH3CN/H2O for
50 min, tR = 20 min) gave 7b (4 mg, 60%); 1H NMR
(MeOH-d4) d 4.02 (t, J = 5.8 Hz, 1, H30), 4.26 (dd,
J = 3.8, 5.3 Hz, 1, H20), 4.44 (‘ddd’, J = 1.8, 3.7,
5.8 Hz, 1, H40), 5.75 (d, J = 8.1 Hz, 1, H5), 5.83 (d,
J = 3.6 Hz, 1, H10), 6.08 (dd, J = 1.8, 3.7 Hz, 1, H50),
7.64 (d, J = 8.1 Hz, 1, H6); 13C NMR (MeOH-d4) d
74.93 (C20), 75.11 (C30), 84.68 (C40), 92.45 (C10),
102.95 (C5), 132.23 (C50), 142.80 (C6), 152.23 (C2),
166.15 (C4). HRMS calcd for C18H21N4O10 [M+H]+
453.1252, found 453.1260.
5.6. Ethyl 2(R/S)-benzamido-5(E)-(50-deoxy-20,30-O-iso-
5.7. Ethyl 2(R/S)-benzamido-5(E)-(50-deoxyuridin-50-yli-
dene)pentanoate (5b)
propylideneuridin-50-ylidene)pentanoate (5a). Procedure
C
Treatment of 5a (25 mg, 0.05 mmol) with TFA/H2O
(9:1, 1.5 mL) by procedure B [column chromatography
(CHCl3/MeOH; 95:5)] gave 5b (15 mg, 66%; R/S,
ꢀ1:1): UV max 260 nm (e 14,500), min 233 nm (e
9300); 1H NMR (MeOH-d4) d 1.28 (t, J = 7.1 Hz, 3,
CH3), 1.93–2.22 (m, 2, H80,80), 2.24–2.33 (m, 2,
H70,700), 3.92 (‘q’, J = 5.2 Hz, 1, H30), 4.15–4.20 (m, 1,
H20) 4.22 (‘dq’, J = 2.0, 7.0 Hz, 2, CH2), 4.35 (t,
J = 7.1, 1, H40), 4.62 (‘dd’, J = 4.9, 9.8 Hz, 1, H90),
5.62–5.76 (m, 2, H5,50), 5.81 (d, J = 3.4 Hz, 0.5, H10),
5.83 (d, J = 3.4 Hz, 0.5, H10), 5.90 (dt, J = 15.3,
6.7 Hz, 0.5, H60), 5.91 (dt, J = 15.3, 6.6 Hz, 0.5, H60),
7.51 (‘t’, J = 7.8 Hz, 2, Ar), 7.53–7.66 (m, 2, H6 and
Ar), 7.91 (d, J = 8.1 Hz, 2, Ar); 13C NMR (MeOH-d4)
d 13.53 (CH3), 28.82 and 28.89 (C70), 30.45 and 30.48
(C80), 52.69 (C90), 62.44 (CH2), 74.15 (C30), 74.22 and
74.25 (C20), 84.37 and 84.48 (C40), 90.69 (C10), 101.84
(C5), 127.54 and 127.56, 128.59, 131.95, 133.71 (Bz),
129.16 and 129.24 (C50), 134.22 and 134.24 (C60),
141.40 (C6), 151.25 (C2), 165.15 (C4), 169.51 (Bz),
172.85 (C100); MS m/z 474 (100, MH+). HRMS calcd
for C23H28N3O8 [M+H]+ 474.1871, found 474.1880.
Compounds 318 (90 mg, 0.3 mmol) and 4a17 (78 mg,
0.3 mmol) were added to a stirred solution of [1,3-bis-
(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro-
(phenylmethylene)(tricyclohexylphosphine)ruthenium (5 mg,
0.006 mmol) in dried CH2Cl2 (5 mL) at ambient temper-
ature under an Ar atmosphere. The reaction mixture
was stirred overnight at 55 ꢁC in the Teflon cap closed
vial, cooled down to ambient temperature, and was par-
titioned (CHCl3/H2O). The organic layer was washed
(brine), dried (MgSO4), and evaporated to give brown
foam. Column chromatography (1 ! 3% MeOH/
1
CHCl3) gave 5a (111 mg, 72%; R/S, ꢀ1:1); H NMR d
1.26 (t, J = 7.1 Hz, 3, CH3), 1.31 (s, 3, CH3), 1.36 (s,
3, CH3), 1.81–1.95 (m, 2, H80,800), 2.02–2.25 (m, 2,
H70,700), 4.23 (q, J = 7.1 Hz, 2, CH2), 4.47–4.52 (m, 1,
H40), 4.69–4.74 (m, 1, H30), 4.84 (‘q’, J = 6.5 Hz, 1,
H90), 5.01 (‘dm’, J = 6.4 Hz, 1, H20), 5.61 (d,
J = 2.8 Hz, 0.5, H10), 5.62 (d, J = 2.8 Hz, 0.5, H10)
5.64–5.75 (m, 2, H5,50), 5.79–5.87 (m, 1, H60), 6.84 (d,
J = 7.8 Hz, 0.5, NH). 6.91 (d, J = 7.8 Hz, 0.5, NH),
7.26 (d, J = 8.1 Hz, 0.5, H6), 7.28 (d, J = 8.1 Hz, 0.5,
H6), 7.45 (t, J = 7.7 Hz, 2, Ar), 7.52 (t, J = 7.9 Hz, 1,
Ar), 7.82 (d, J = 8.0, 2, Ar), 9.38 (br s, 1, NH);
13
C
5.8. Ethyl 2(R/S)-tert-butoxycarbonylamino-5(E)-(50-
deoxy-20,30-O-isopropylideneuridin-50-ylidene)pentanoate
(5c)
NMR d 13.18 (CH3), 24.27 and 26.11 (CMe2), 26.98
(C70), 30.75 (C80), 51.06 (C90), 60.72 (CH2), 83.15 and
83.24 (C30), 83.71, 83.78 (C20), 87.45 and 87.50 (C40),
93.51 and 93.38 (C10), 101.50 (C5), 113.46 (CMe2),
126.10, 127.59, 130.78, 133.03 (Bz), 127.12 and 127.13
(C50), 132.84 and 132.86 (C60), 141.36 (C6), 148.94
(C2), 162.28 (C4), 166.08 and 166.11 (Bz), 171.50 and
171.54 (C100); MS m/z 514 (100, MH+).
Metathesis of 318 (40 mg, 0.14 mmol) and 4b17 (35 mg,
0.14 mmol) by procedure C [column chromatography
(CHCl3 ! 2% MeOH)] gave 5c (73 mg, 74%, R/S, 1:1);
1H NMR d 1.28 (t, J = 7.1 Hz, 3, CH3), 1.36 (s, 3,
CH3), 1.45 (s, 9, t-Bu), 1.59 (s, 3, CH3), 1.69–2.19 (m,
4, H70,700,80,800), 4.22 (q, J = 7.1 Hz, 2, CH2), 4.27–4.34
(‘br q’, J = 8.0 Hz, 1, H90), 4.51 (dd, J = 4.2, 8.1 Hz,
0.5, H40), 4.52 (dd, J = 4.2, 8.1 Hz, 0.5, H40), 4.75–4.80
(m, 1, H30), 5.01–5.07 (m, 1, H20), 5.15 (‘t’, J = 9.0 Hz,
1, NH), 5.58-5.85 (m, 4, H10,5, 50,60), 7.25 (d,
J = 8.0 Hz, 0.5, H6), 7.27 (d, J = 8.0 Hz, 0.5, H6), 9.45
(br s, 1, NH); MS m/z 510 (100, MH+), 410 (97, MH+-
Boc). HRMS calcd for C24H36N3O9 [M+H]+ 510.2452,
found 510.2455.
Isolated also from column were self-metathesis dimers
diethyl 2,9-bis(benzamido)dec-5-enedioate 617 (10 mg,
7%, 14% consumption of 4a; less polar then 5a) and
7a (8 mg, 5%, 10% consumption of 3; more polar then
1
5a). Compound 7a had: H NMR: d 1.34 (s, 3, CH3),
1.56 (s, 3, CH3), 4.54 (‘dt’, J = 2.1, 3.8 Hz, 1, H40),
4.76 (dd, J = 4.0, 6.3 Hz, 1, H30), 5.05 (dd, J = 1.8,
6.4 Hz, 1, H20), 5.52 (d, J = 1.7 Hz, H10), 5.83 (d,
J = 8.0 Hz, 1, H5), 5.97 (‘dd’, J = 1.7, 3.7 Hz, 1, H50),
7.19 (d, J = 8.0 Hz, 1, H6), 9.73 (br s, 1, NH); 13C
NMR d 25.63 and 27.49 (CMe2), 84.88 (C20 and C30),
87.71 (C40), 95.92 (C10), 102.95 (C5), 114.88 (CMe2),
130.99 (C50), 143.45 (C6), 150.18 (C2), 164.66 (C4);
MS m/z 533 (100, MH+). Treatment of 7a (8 mg,
5.9. Ethyl 2(R/S)-amino-5(E)-(50-deoxyuridin-50-yli-
dene)pentanoate (5d)
Treatment of 5c (45 mg, 0.088 mmol) with TFA/H2O
(9:1, 3.5 mL) by procedure B [column chromatography

5430
S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
(5 ! 12% MeOH/CHCl3)] gave 5d (20 mg, 61%; R/S,
1:1) as a white hygroscopic solid: UV max 260 nm (e
10,500), min 233 nm (e 3800); H NMR (Me2SO-d6) d
4.04–4.10 (m, 3, H20,CH2), 4.15 (‘t’, J = 6.4 Hz, 1,
H40), 5.57–5.72 (m, 4, H10,5,50,60), 7.58 (d, J = 8.1 Hz,
1, H6); 13C NMR (Me2SO-d6) d 14.91 (CH3), 24.60
(C80), 31.75 (C70), 33.68 (C90), 60.60 (CH2), 73.73
(C30), 74.40 (C20), 84.81 (C40), 89.62 (C10), 102.79
(C5), 129.58 (C60), 134.44 (C50), 141.89 (C6), 151.49
(C2), 163.95 (C4), 173.61 (C100); MS m/z 355 (100,
MH+). Anal. Calcd for C16H22N2O7 (354.36): C,
54.23; H, 6.26; N, 7.91. Found: C, 54.34; H, 6.28; N,
7.68.
1
1.19 (t, J = 7.1 Hz, 3, CH3), 1.48–1.56 (m, 1, H80),
1.60–1.69 (m, 1, H800), 2.10 (‘q’, J = 7.2 Hz, 2, H70,700),
3.29–3.31 (m, 1, H90), 3.79 (m, 1, H30), 4.05–4.18 (m,
4, H20,40 and CH2), 5.21 (br s, 2, OH20 and OH30),
5.42–5.44 (m, 2, NH2), 5.63 (dd, J = 7.8, 15.2 Hz, 1,
H50), 5.65 (d, J = 8.0 Hz, 1, H5), 5.72 (d, J = 4.1 Hz,
1, H10), 5.74 (dt, J = 6.8, 15.2 Hz, 1, H60), 7.59 (d,
J = 8.0 Hz, 1, H6), 11.35 (br s, 1, NH); 13C NMR
(Me2SO-d6) d 15.00 (CH3), 28.78 (C70), 34.55 and
34.60 (C80), 54.22 (C90), 60.91 (CH2), 73.72 and 73.75
(C30), 74.37 and 74.39 (C20), 84.82 and 84.87 (C40),
89.60 (C10), 102.79 (C5), 129.31 (C50), 134.58 (C60),
141.88 (C6), 151.48 (C2), 163.96 (C4), 176.35 (C100);
MS m/z 370 (100, MH+). HRMS calcd for
C16H24N3O7 [M+H]+ 370.1609, found 370.1618.
5.12. Ethyl 5(E)-(50-deoxy-20,30-O-isopropylideneadeno-
sin-50-ylidene)pentanoate (12a)
Coupling of (E)-105a (41 mg, 0.1 mmol) with
EtO2C(CH2)3ZnBr (0.5 M/THF, 0.4 mL, 0.2 mmol) by
procedure
D
[column chromatography (1 ! 2%
MeOH/EtOAc)] gave 12a (27 mg, 68%); 1H NMR
(600 MHz) d 1.25 (t, J = 7.2 Hz, 3, CH3), 1.28 (s, 3,
CH3), 1.42 (s, 3, CH3), 1.68 (quint, J = 7.5 Hz, 2,
H80,800), 2.04 (q, J = 7.2 Hz, 2, H70,700), 2.28 (t,
J = 7.5 Hz, 2, H90,900), 4.08 (q, J = 7.1 Hz, 2, CH2),
4.65 (dd, J = 3.3, 7.5 Hz, 1, H40), 4.98 (dd, J = 3.5,
6.2 Hz, 1, H30), 5.51 (dd, J = 1.9, 6.3 Hz, 1, H20), 5.59
(ddt, J = 1.2, 7.6, 15.4 Hz, 1, H50), 5.72 (dt, J = 15.4,
6.6 Hz, 1, H60), 5.90 (br s, 2, NH2), 6.09 (d,
J = 1.9 Hz, 1, H10), 7.91 (s, 1, H2), 8.38 (s, 1, H8); MS
m/z 418 (100, MH+); HRMS calcd for C20H28N5O5
[M+H]+ 418.2090, found 418.2082.
5.10. Ethyl 5(E)-(50-deoxy-20,30-O-isopropylideneuridin-
50-ylidene)pentanoate (11a). Procedure D
Tris(dibenzylideneacetone)dipalladium
(9.2 mg,
0.01 mmol) was added to a solution of (E)-918 (40 mg,
0.1 mmol) in benzene (2 mL) followed by dropwise addi-
tion of EtO2C(CH2)3ZnBr (0.5 M/THF, 0.3 mL,
0.15 mmol) via syringe at ambient temperature under
N2. The resulting mixture was heated at 55 ꢁC for 8 h
[additional EtO2C(CH2)3ZnBr (0.1 mL, 0.05 mmol)
was added after 5 h] and progress of the reaction was
monitored (TLC) by the formation of slightly more po-
lar compound. The volatiles were evaporated and the
brown residue was partitioned (EtOAc/H2O). The or-
ganic layer was washed (brine), dried (Na2SO4), evapo-
rated, and the residue was column chromatographed
(EtOAc/hexane, 85:15) to give 11a (28 mg, 72%); 1H
NMR d 1.25 (t, J = 7.1 Hz, 3, CH3), 1.38 (s, 3, CH3),
1.58 (s, 3, CH3), 1.72 (quint, J = 7.6 Hz, 2, H80,800),
2.12 (q, J = 7.1 Hz, 2, H70,700), 2.32 (t, J = 7.6 Hz, 2,
H90), 4.14 (q, J = 7.1 Hz, 2, CH2), 4.50 (dd, J = 4.3,
7.8 Hz, 1, H40), 4.74 (dd, J = 4.4, 6.2 Hz, 1, H30), 5.02
(dd, J = 1.7, 6.5 Hz, 1, H20), 5.60 (d, J = 1.6 Hz, 1,
H10), 5.62 (dd, J = 8.0, 14.6 Hz, 1, H50), 5.73 (d,
J = 8.2 Hz, 1, H5), 5.82 (dt, J = 6.8, 14.6 Hz, 1, H60),
7.28 (d, J = 8.2 Hz, 1, H6), 9.40 (s, 1, NH); 13C NMR
d 14.25 (CH3), 24.03 (C80), 25.32 and 27.16 (CMe2),
31.53 (C70), 33.62 (C90), 60.35 (CH2), 84.22 (C30),
84.84 (C20), 88.63 (C40), 94.29 (C10), 102.45 (C5),
114.57 (CMe2), 128.57 (C60), 131.96 (C50), 142.23 (C6),
149.77 (C2), 163.03 (C4), 173.45 (C100); MS m/z 395
(100, MH+); HRMS calcd for C19H27N2O7 [M+H]+
395.1818, found 395.1811.
5.13. Ethyl 5(E)-(50-deoxyadenosin-50-ylidene)pentanoate
(12b)
Treatment of 12a (27 mg, 0.065 mmol) with TFA/H2O
by procedure B gave 12b (15 mg, 61%). Purification on
HPLC (10 ! 60% CH3CN/H2O for 1 h, tR = 43 min)
and recrystallization (MeOH) gave white crystals: mp
91–93 ꢁC dec; UV max 260 nm (e 12,700), min 230 nm
(e 1840); 1H NMR (MeOH-d6, 600 MHz) d 1.05 (t,
J = 7.2 Hz, 3, CH3), 1.55 (quint, J = 7.3 Hz, 2, H80,800),
1.98 (q, J = 7.0 Hz, 2, H70,700), 2.16 (t, J = 7.4 Hz, 2,
H90,900), 3.94 (q, J = 7.0 Hz, 2, CH2), 4.04 (t,
J = 5.2 Hz, 1, H30), 4.25 (dd, J = 5.4, 6.6 Hz, 1, H40),
4.59 (t, J = 4.8 Hz, 1, H20), 5.58 (dd, J = 6.8, 15.4 Hz,
1, H50), 5.65 (dt, J = 15.4, 6.4 Hz, 1, H60), 5.84 (d,
J = 4.5 Hz, 1, H10), 8.08 (s, 1, H2), 8.10 (s, 1, H8); 13C
NMR (Me2SO-d6) d 14.90 (CH3), 24.46 (C80), 31.57
(C70), 33.62 (C90), 60.74 (CH2), 73.54 (C30), 74.69
(C20), 85.40 (C40), 88.33 (C10), 119.76 (C5), 129.64
(C60), 133.98 (C50), 140.95 (C8), 150.11 (C4), 153.53
(C2), 156.56 (C6), 173.97 (C100); MS m/z 378 (100,
MH+). Anal. Calcd for C17H23N5O5ÆH2O (395.41): C,
51.64; H, 6.37; N, 17.71. Found: C, 51.53; H, 6.42; N,
17.37.
5.11. Ethyl 5(E)-(50-deoxyuridin-50-ylidene)pentanoate
(11b)
5.14. 5(E)-(50-Deoxyadenosin-50-ylidene)pentanoic acid
(12c)
Treatment of 11a (70 mg, 0.78 mmol) with TFA/H2O
(9:1, 2.5 mL) by procedure B gave 11b (45 mg, 72%):
mp 102–104 ꢁC dec; UV max 262 nm (e 9200), min
NaOH/H2O (1 M, 0.35 mL) was added to a solution of
12b (10 mg, 0.026 mmol) in MeOH (2.5 mL) and stirring
was continued at ambient temperature overnight. The
resulting mixture was neutralized with AcOH to
pH ꢀ 7. Volatiles were evaporated and the residue was
1
230 nm (e 2100); H NMR (Me2SO-d6/D2O) d 1.18 (t,
J = 7.1 Hz, 3, CH3), 1.62 (‘quint’, J = 7.3 Hz, 2,
H80,800), 2.08 (q, J = 6.8 Hz, 2, H70,700), 2.32 (t,
J = 7.4 Hz, 2, H90,900), 3.81 (t, J = 5.3 Hz, 1, H30),

S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
5431
purified on HPLC (10 ! 30% CH3CN/H2O for 50 min,
tR = 28 min) to give 12c (7.5 mg, 82%); 1H NMR
(MeOH-d4) d 1.74 (‘quint’, J = 7.4 Hz, 2, H80,800), 2.15
(q, J = 7.1 Hz, 2, H70,700), 2.32 (t, J = 7.4 Hz, 2,
H90,900), 4.21 (t, J = 5.2 Hz, 1, H30), 4.43 (dd, J = 5.5,
6.8 Hz, 1, H40), 4.72 (t, J = 4.9 Hz, 1, H20), 5.75 (dd,
J = 7.0, 15.4 Hz, 1, H50), 5.83 (dt, J = 15.5, 6.5 Hz, 1,
H60), 6.01 (d, J = 4.5 Hz, 1, H10), 8.16 (s, 1, H2), 8.22
NMR (MeOH-d4) d 1.24 (t, J = 7.1 Hz, 3, CH3), 1.86
(‘quint’, J = 7.4 Hz, 2, H80,800), 2.32 (‘dt’, J = 17.6,
7.3 Hz, 2, H70,700), 2.40 (t, J = 7.4 Hz, 2, H90,900), 4.13
(q, J = 7.1 Hz, 2, CH2), 4.23 (t, J = 4.7 Hz, 1, H30),
4.86 (‘t’, J = 4.4 Hz, 1, H20), 4.94 (dd, J = 4.5, 9.2 Hz,
1, H40), 5.20 (dd, J = 9.2, 36.5 Hz, 1, H50), 5.99 (d,
J = 4.8 Hz, 1, H10), 8.24 (s, 1, H2), 8.26 (s, 1, H8); 13C
NMR (MeOH-d4) d 14.50 (CH3), 22.34 (C80), 31.99 (d,
J = 27.2 Hz, C70), 33.87 (C90), 61.55 (CH2), 74.89
(C20), 76.29 (C30), 79.34 (d, J = 6.5 Hz, C40), 90.38
(C10), 106.01 (d, J = 11.3 Hz, C50), 120.82 (C5), 141.69
(C8), 150.62 (C4), 153.90 (C2), 157.37 (C6), 163.50 (d,
J = 260.9 Hz, C60), 174.86 (C100). (E)-14b had: 1H
NMR (MeOH-d4) d 1.24 (t, J = 7.1 Hz, 3, CH3), 1.84
(‘quint’, J = 7.4 Hz, 2, H80,800), 2.39 (t, J = 7.3 Hz, 2,
H90,900), 2.43–2.55 (m, 2, H70,700), 4.12 (q, J = 7.1 Hz,
2, CH2), 4.28 (t, J = 5.5 Hz, 1, H30), 4.60 (ddd, J = 1.9,
5.8, 9.8 Hz, 1, H40), 4.72 (dd, J = 4.0, 5.1 Hz, 1, H20),
5.44 (dd, J = 9.7, 19.9 Hz, 1, H50), 6.00 (d, J = 3.6 Hz,
1, H10), 8.25 (s, 1, H2), 8.23 (s, 1, H8); 13C NMR
(MeOH-d4) d 14.50 (CH3), 22.34 (C80), 28.56 (d,
J = 27.2 Hz, C70), 33.78 (C90), 61.55 (CH2), 75.21
(C20), 76.11 (C30), 81.03 (d, J = 14.4 Hz, C40), 90.57
(C10), 106.83 (d, J = 24.4 Hz, C50), 120.70 (C5), 141.31
(C8), 150.54 (C4), 153.94 (C2), 157.37 (C6), 165.42 (d,
J = 255.3 Hz, C60), 174.89 (C100).
13
(s, 1, H8); C NMR (MeOH-d4) d 25.37 (C80), 32.64
(C70), 34.32 (C90), 75.12 (C30), 75.70 (C20), 86.43
(C40), 90.14 (C10), 120.10 (C5), 129.76 (C60), 135.18
(C50), 141.28 (C8), 150.66 (C4), 153.93 (C2), 157.36
(C6), 177.59 (C100); MS m/z 350 (100, MH+); HRMS
calcd for C15H20N5O5 [M+H]+ 350.1464, found
350.1469.
5.15. Ethyl 5-fluoro-5-(50-deoxy-20,30-O-isopropylidenea-
denosin-50-ylidene)pentanoate (14a)
Pd(PPh3)4 (25.6 mg, 0.022 mmol) was added to a solu-
tion of 136a (E/Z, ꢀ60:40; 80 mg, 0.2 mmol) in benzene
(5 mL)
followed
by
dropwise
addition
of
EtO2C(CH2)3ZnBr (0.5 M/THF, 0.88 mL, 0.44 mmol)
via syringe at ambient temperature under N2. The result-
ing mixture was heated at 55 ꢁC for 8 h. The volatiles
were evaporated and the brown residue was partitioned
(EtOAc/NaHCO3/H2O). The organic layer was washed
(brine), dried (Na2SO4), evaporated, and the residue
was column chromatographed (1 ! 3% MeOH/CHCl3)
to give 14a (49 mg, 56%; E/Z, ꢀ40:60); 19F NMR d
5.17. 5-Fluoro-5(E)-(50-deoxyadenosin-50-ylidene)penta-
noic acid (14c)
0
0
00
ꢁ95.78 (‘q’, JF–H5 ;7 ;7 ¼ 21:8 Hz, 0.4, E), ꢁ101.80 (dt,
Treatment of 14b (8 mg, 0.020 mmol; E/Z, 35:65) with
NaOH as described for 12c and HPLC purification
(5 ! 50% CH3CN/H2O for 1 h, tR = 42 min) gave 14c
(6 mg, 82%; E/Z, 45:55); 19F NMR (MeOH-d4) d
0
0
00
JF–H5 ¼ 35:5 Hz; JF–H7 ;7 ¼ 17:3 Hz, 0.6, Z); MS m/z
436 (MH+); HRMS calcd for C20H27FN5O5 [M+H]+
436.1996, found 436.1991. (Z)-14a had: 1H NMR d
1.23 (t, J = 7.1 Hz, 3, CH3), 1.36 (s, 3, CH3), 161 (s, 3,
CH3), 1.84 (‘quint’, J = 7.3 Hz, 2, H80,800), 2.15 (dt,
J = 17.1, 7.7 Hz, 2, H70,700), 2.30 (t, J = 7.5 Hz, 2,
H90,900), 4.09 (q, J = 7.2 Hz, 2, CH2), 4.85 (dd, J = 9.2,
35.5 Hz, 1, H50), 4.97 (‘dd’, J = 3.3, 6.2 Hz, 1, H30),
5.15 (dd, J = 3.1, 9.2 Hz, 1, H40), 5.54 (dd, J = 1.9,
6.2 Hz, 1, H20), 5.88 (br s, 2, NH2), 5.98 (d,
J = 1.7 Hz, 1, H10), 7.86 (s, 1, H2), 8.34 (s, 1, H8). (E)-
0
0
00
ꢁ97.8 (‘q’, JF–H5 ;7 ;7 ¼ 23:9 Hz, 0.45, E), ꢁ105.0 (‘dt’,
0
0
00
JF–H5 ¼ 35:6 Hz; JF–H7 ;7 ¼ 17:6 Hz, 0.55, Z); MS m/z
368 (100, MH+); HRMS calcd for C15H19FN5O5
[M+H]+ 368.1370, found 368.1375. (Z)-14c had: 1H
NMR (MeOH-d4)
d 1.85 (‘quint’, J = 7.4 Hz, 2,
H80,800), 2.32 (dt, J = 17.3, 7.4 Hz, 2, H70,700), 2.38 (t,
J = 7.3 Hz, 2, H90,900), 4.23 (t, J = 4.8 Hz, 1, H30), 4.83
(‘t’, J = 5.0 Hz, 1H, H20), 4.92 (dd, J = 4.7, 9.3 Hz, 1,
H40), 5.20 (dd, J = 9.2, 36.0 Hz, 1, H50), 6.01 (d,
J = 5.2 Hz , 1, H10), 8.24 (s, 1, H2), 8.27 (s, 1H, H8);
1
14a had: H NMR d 1.21 (t, J = 7.1 Hz, 3, CH3), 1.35
(s, 3, CH3), 160 (s, 3, CH3), 1.74 (‘quint’, J = 7.2 Hz,
2, H80,800), 2.26 (t, J = 7.4 Hz, 2, H90,900), 2.35 (dt,
J = 23.3, 7.3 Hz, 2, H70,700), 4.11 (q, J = 7.2 Hz, 2,
CH2), 4.75 (ddd, J = 1.8, 3.2, 10.1 Hz, 1, H40), 4.97
(‘dd’, J = 3.3, 6.2 Hz, 1, H30), 5.30 (dd, J = 10.1,
19.6 Hz, 1, H50), 5.50 (dd, J = 1.7, 6.3 Hz, 1, H20),
5.88 (br s, 2, NH2), 5.97 (d, J = 1.5 Hz, 1, H10), 7.84
(s, 1, H2), 8.34 (s, 1, H8).
13C NMR (MeOH-d4)
d
23.02 (C80), 32.05 (d,
J = 27.4 Hz, C70), 33.82 (C90), 74.91 (C20), 76.27 (C30),
79.34 (d, J = 6.6 Hz, C40), 90.27 (C10), 105.90 (d,
J = 11.0 Hz, C50), 120.46 (C5), 141.36 (C8), 150.60
(C4), 153.88 (C2), 157.34 (C6), 163.30 (d,
J = 258.6 Hz, C60), 177.09 (C100). (E)-14c had: 1H
NMR (MeOH-d4) d 1.84 (‘quint’, J = 7.4 Hz, 2H,
H80,800), 2.39 (t, J = 7.3 Hz, 2, H90,900), 2.45 (dt,
J = 23.7, 7.3 Hz, 1, H70), 2.46 (dt, J = 23.7, 7.3 Hz, 1,
H700), 4.28 (t, J = 5.6 Hz, 1, H30), 4.60 (ddd, J = 1.8,
5.8, 9.7 Hz, 1, H40), 4.72 (dd J = 4.0, 5.2 Hz, 1, H20),
5.44 (dd, J = 9.8, 19.9 Hz, 1, H50), 6.00 (d, J = 3.7 Hz,
1, H10), 8.23 (s, 1, H2), 8.25 (s, 1, H8); 13C NMR
(MeOH-d4) d 22.45 (C80), 28.65 (d, J = 26.9 Hz, C70),
33.82 (C90), 75.17 (C20), 76.12 (C30), 81.04 (d,
J = 14.8 Hz, C40), 90.58 (C10), 106.73 (d, J = 24.7 Hz,
C50), 120.53 (C5), 141.68 (C8), 150.49 (C4), 153.91
(C2), 157.34 (C6), 165.88 (d, J = 258.6 Hz, C60), 177.09
(C100).
5.16. Ethyl 5-fluoro-5-(50-deoxyadenosin-50-ylidene)pent-
anoate (14b)
Treatment of 14a (40 mg, 0.092 mmol; E/Z, 40:60) with
TFA/H2O by procedure B and HPLC purification
(15 ! 50% CH3CN/H2O, tR = 49 min) gave 14b
(32 mg, 88%; E/Z, ꢀ35:65): 19F NMR (MeOH-d4) d
0
0
ꢁ94.73 (‘q’, JF–H5 ;7;7 ¼ 20:5 Hz, 0.35, E), ꢁ102.14 (dt,
0
0
00
JF–H5 ¼ 35:9 Hz; JF–H7 ;7 ¼ 17:7 Hz, 0.65, Z); MS m/z
396 (100, MH+); HRMS calcd for C17H23FN5O5
[M+H]+ 396.1678, found 396.1680. (Z)-14b had: 1H

5432
S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
Muzard, M.; Glapski, C.; Pilard, S.; De Clercq, E. J.
Med. Chem. 2006, 49, 1223.
5.18. Inactivation of AdoHcy hydrolase
8. (a) Robins, M. J.; Wnuk, S. F.; Yang, X.; Yuan, C.-S.;
Borchardt, R. T.; Balzarini, J.; De Clercq, E. J. Med.
Chem. 1998, 41, 3857; (b) Yang, X.; Yin, D.; Wnuk, S. F.;
Robins, M. J.; Borchardt, R. T. Biochemistry 2000, 39,
15234.
9. (a) Turner, M. A.; Yuan, C.-S.; Borchardt, R. T.;
Hershfield, M. S.; Smith, G. D.; Howell, P. L. Nat. Struct.
Biol. 1998, 5, 369; (b) Yang, X.; Hu, Y.; Yin, D. H.;
Turner, M. A.; Wang, M.; Borchardt, R. T.; Howell, P. L.;
Kuczera, K.; Schowen, R. L. Biochemistry 2003, 42, 1900;
(c) Hu, Y.; Komoto, J.; Huang, Y.; Gomi, T.; Ogawa, H.;
Takata, Y.; Fujioka, M.; Takusagawa, F. Biochemistry
1999, 38, 8323.
10. For recent examples on the importance and synthetic
challenges associated with the synthesis of the uracil-
nucleosides functionalized at C50 see: (a) Zhang, X.;
Bernet, B.; Vasella, A. Helv. Chim. Acta 2007, 90, 864; (b)
Kurosu, M.; Narayanasamy, P.; Crick, D. C. Heterocycles
2007, 72, 339; (c) Hirano, S.; Ichikawa, S.; Matsuda, A. J.
Org. Chem. 2008, 73, 569.
Recombinant human placental AdoHcy hydrolase
(204 nM) was incubated with 281 lM of 2e, 2f, 8a, 8b,
12b, 12c, 14b, and 14c in potassium-phosphate buffer
[50.5 lL; 50 mM, pH 7.2, containing 1 mM EDTA (buf-
fer A)] at 22 ꢁC for 0.5 or 4 h. The remaining enzyme
activity was assayed in the synthetic direction essentially
as previously described.8b An aliquot (5 lL) of the en-
zyme/inhibitor was added to 995 lL of buffer A contain-
ing 0.5 mM Ado and 0.5 mM Hcy in buffer A and then
incubated at 37 ꢁ C for 12 min followed by the addition
of HClO4 (25 lL, 5 M) to terminate the reaction. The
reaction product, AdoHcy, was quantitatively measured
using RP-HPLC with the detector monitoring at
258 nm.8b
Acknowledgments
11. Andrei, D.; Wnuk, S. F. J. Org. Chem. 2006, 71, 405.
12. Pfitzer, K. E.; Moffatt, J. G. J. Am. Chem. Soc. 1965, 87,
5661.
13. (a) Maryanoff, B. E.; Duhl-Emswiler, B. A. Tetrahedron
Lett. 1981, 22, 4185; (b) Maryanoff, B. E.; Reitz, A. B.
Chem. Rev. 1989, 89, 863.
This investigation was supported by grants from the Na-
tional Institutes of Health (S06GM08205 and
GM29332). The support of US ARO (W911NF-04-1-
0022) for the purchase of 600 MHz NMR spectrometer
is acknowledged.
14. (a) Wnuk, S. F.; Robins, M. J. Can. J. Chem. 1991, 69,
334; (b) Wnuk, S. F.; Yuan, C.-S.; Borchardt, R. T.;
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16. For application of metathesis towards synthesis of nucle-
oside analogues see: Amblard, F.; Nolan, S. P.; Agrofo-
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17. Andrei, D.; Wnuk, S. F. Org. Lett. 2006, 8, 5093.
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isopropylidene-a-D-ribo-hex-5-enofuranose) see: Hadwi-
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S. F. Wnuk et al. / Bioorg. Med. Chem. 16 (2008) 5424–5433
5433
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