Synthesis and evaluation of multisubstrate bicyclic pyrimidine nucleoside inhibitors of human thymidine phosphorylase

Article abstract of DOI:10.1021/jm060428u

A series of novel, multisubstrate, bicyclic pyrimidine nucleoside inhibitors of human thymidine phosphorylase (TP) is described. Thymidine phosphorylase has been implicated in angiogenesis and plays a significant role in tumor progression and metastasis.

Full text of DOI:10.1021/jm060428u

J. Med. Chem. 2006, 49, 7807-7815
7807
Synthesis and Evaluation of Multisubstrate Bicyclic Pyrimidine Nucleoside Inhibitors of Human
Thymidine Phosphorylase
Amy L. Allan,*,† Patricia L. Gladstone,† Melissa L. P. Price,† Stephanie A. Hopkins,† Jose C. Juarez,† Fernando Don˜ate,†
Robert J. Ternansky,† David E. Shaw,‡,§ Bruce Ganem,# Yingbo Li,# Weiru Wang,# and Steven Ealick#
Attenuon, LLC, 11535 Sorrento Valley Road Suite 401, San Diego, California 92121, D. E. Shaw Research, LLC, 120 West 45th Street,
39th Floor, New York, New York 10036, Center for Computational Biology and Bioinformatics, IrVing Cancer Research Center,
Columbia UniVersity, 1130 St. Nicholas AVenue, New York, New York 10032, and Department of Chemistry and Chemical Biology,
Baker Laboratory, Cornell UniVersity, Ithaca, New York 14853-1301
ReceiVed April 11, 2006
A series of novel, multisubstrate, bicyclic pyrimidine nucleoside inhibitors of human thymidine phosphorylase
(TP) is described. Thymidine phosphorylase has been implicated in angiogenesis and plays a significant
role in tumor progression and metastasis. The presence and orientation of the phosphonate moiety (acting
as a phosphate mimic) in these derivatives were critical for inhibitory activity. The most active compounds
possessed a phosphonate group in an endo orientation. This was consistent with molecular modeling results
that showed the endo isomer protein-ligand complex to be lower in energy than the exo complex.
Introduction
Schramm determined through a study of kinetic isotope effects
that human TP most likely proceeds through an SN2-like
transition state, unlike the dissociative transition states previously
described for N-ribosyl transferases.30 They suggest that the
design of transition-state inhibitors would require mimics of the
three components to be present: pyrimidine, 2′-deoxyribose,
and the phosphate group.30
Relatively few TP inhibitors have been reported, and most
are analogues of uracil or thymidine or analogues of nucleosides
and acyclonucleosides.31-42 To date, the most potent inhibitor
of human TP is TPI (5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]-
uracil hydrochloride) (1) from Taiho Pharmaceutical Company
with a Ki of 35 nM (Figure 1).41,42 A combination of TPI and
Tumor growth and metastasis are processes dependent on
angiogenesis.1-3 Inhibition of the formation of new blood vessels
within a tumor has become one of the most active areas of
research for the discovery of new cancer therapeutics.4-6
Thymidine phosphorylase (TPa) has received considerable
attention from several research groups, as reviewed by Cole et
al. and others.7-10 Specifically, TP has been shown to be
identical to platelet-derived endothelial cell growth factor (PD-
ECGF),11-14 which has been implicated in angiogenesis and
chemotaxis in human tumors.15,16 Increased hypoxia correlates
with elevated TP activity, and increased levels of this enzyme
have been observed in colorectal, ovarian, pancreatic, and breast
tumors.10,17 It has been postulated that inhibition of TP would
reduce tumor growth and metastasis by interfering with angio-
genesis in tumor tissues.18,19 Overexpression of TP has also been
implicated in other hyperproliferative disease states including
rheumatoid arthritis20 and psoriasis.21
TP catalyzes the reversible phosphorolysis of thymidine to
thymine and 2-deoxyribose 1-phosphate (Scheme 1).22,23 The
phosphorylated sugar product is further converted to 2-deoxy-
D-ribose, which is thought to be responsible for the chemotactic
activity of TP in vitro and the angiogenic activity of TP in
vivo.12,13 In addition, TP recognizes and inactivates a variety
of 5-substituted pyrimidine 2′-deoxynucleoside derivatives with
antitumor/antivirus activities, including 5-fluoro-2′-deoxyuridine
and 5-trifluoromethyl-2′-deoxyuridine.24
X-ray crystallographic studies of the homologous pyrimidine
nucleoside phosphorylase (PyNP)25-27 and the functionally
similar purine nucleoside phosphorylase (PNP)28,29 suggest that
large-scale conformational changes are required to bring the
separate binding sites for the phosphate and the nucleoside into
proximity, which is required for catalysis. Recently, Birck and
Figure 1. Structure of Taiho Pharmaceutical inhibitor (TPI).
trifluorothymidine is currently in phase I clinical trials.43,44
Recently, Freeman et al. have reported some aminoimidazolyl-
methyluracil analogues that are potent inhibitors of E. coli and
human TP with IC50 values of about 20 nM.45 However, the
pharmacokinetic (PK) profiles were poor for these compounds
and the nitro prodrugs that could be reduced to the amino
inhibitors were also described.45
Multisubstrate inhibitors that bind to both the nucleoside and
phosphate binding sites have not been extensively explored.46,47
Multisubstrate inhibitors may have several advantages. There
are fewer degrees of translational and rotational freedom being
“frozen” upon binding of a multisubstrate inhibitor compared
to the binding of two independent substrates. This corresponds
to a lower entropic penalty for binding at the second site. In
addition, these compounds have more points of interaction with
the receptor compared to most single-substrate inhibitors, which
improves the enthalpic contribution to binding. To our knowl-
edge, all of the reported multisubstrate TP inhibitors have been
tested for activity in E. coli TP assays but none have been tested
against human TP.
* To whom correspondence should be addressed. Phone: (858) 720-
8797. Fax: (858) 720-1086. E-mail: allan@attenuon.com.
† Attenuon, LLC.
‡ D. E. Shaw Research, LLC.
§ Columbia University.
# Cornell University.
a Abbreviations: TP ) Thymidine phosphorylase, PD-ECGF ) Platelet-
derived endothelial cell growth factor, TPI ) Taiho Pharmaceutical Inhibitor
10.1021/jm060428u CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/06/2006

7808 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Scheme 1. Enzymatic Reaction Catalyzed by TP
Allan et al.
Table 1. Inhibition of TP by Multisubstrate Analogues
Li and Ganem have reported a novel multisubstrate TP
inhibitor 248 (Table 1) that demonstrates nanomolar activity
against human TP in an in vitro assay.49 This nucleoside
derivative contains an additional five-membered acetal ring
fused to the ribose moiety. The acetal portion of the inhibitor
bears an appended phosphonate group. In this report, we
describe the synthesis of a novel series of multisubstrate
analogues based on compound 2 and their activity against
recombinant human TP (Table 1). Novel compounds 3-8
incorporate a nucleoside framework along with an acidic or polar
group as the phosphate mimic. Since cell permeability is a
common problem for phosphonates, we also evaluated alterna-
tives to this moiety, including a carboxylic acid analogue 3 and
an amide analogue 4. In addition, concern over the potential
lability of the acetal functionality in 2 encouraged us to assess
the activity and stability of the 2′- and 3′-carbon analogues 5
and 7 and their corresponding diastereomers 6 and 8. Finally,
computational docking studies provided some insight into the
binding orientation of these compounds, as well as the observed
experimental trends in binding affinity.
compd
Ki (µM)a
2
3
4
5
6
7
8
0.236 ( 0.007
43.5b
no inhibition
8.03 ( 0.18
>500
1.05 ( 0.07
33.4 ( 4.0
a Values represent the mean of at least three experiments. Standard
deviations are also shown. b Sample set n ) 2. Average is reported.
3′,5′-disiloxane with TBAF gave the desired free diol 15 in
excellent yield. Subsequent treatment with sodium methoxide
in methanol induced the conjugate addition, affording the
cyclized phosphonate esters 16 in a 3:1 diastereomeric mixture
Results and Discussion
Synthesis. Analogues 3-8 were prepared from a common
intermediate, 5-methyluridine (9), which was synthesized in two
steps as previously reported.50,51 Carboxylic acid 3 and amide
4 were prepared in a manner analogous to that for phosphonate
2, as shown in Scheme 2.48 5′-Benzoyl protected 5-methyluridine
10 was treated with methyl 3,3-dimethoxypropionate in con-
centrated HCl to give ester 11. The ester intermediate 11 was
then converted to carboxylic acid 3 with LiOH or to amide 4
with ammonia in methanol. The endo diastereomer was the
major product in both instances.
In order to generate the 2′-carbon (5 and 6; see Scheme 3)
and 3′-carbon (7 and 8; see Scheme 4) analogues, a different
synthetic strategy was used. Distinct protecting group strategies
were employed in order to allow for the incorporation of a
carbon moiety at either the 2′ or 3′ position. The synthesis of
the 2′-carbon analogues (Scheme 3) began with the protection
of the 3′- and 5′-hydroxyl groups as the disiloxane 12 using
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane.52 This allowed the
2′-hydroxyl to be converted to the desired carbon moiety in a
manner previously described by De Mesmaeker et al.53
Compound 12 (Scheme 3) was converted to the corresponding
thioester with phenyl chlorothioformate, and the allyl group was
incorporated at the 2′ position utilizing allyltributyltin and AIBN
in benzene under photolysis conditions. A single diastereomer
of 13 was isolated, as indicated by NMR analysis.53 Allyl
compound 13 was treated with osmium tetroxide to give a
mixture of diols that underwent subsequent cleavage with
sodium periodate to provide aldehyde 14.53 The Horner-
Emmons-Wadsworth reaction followed by deprotection of the
1
as evidenced by the integration of the vinyl protons in the H
NMR spectra. Addition of TMSBr to the mixture gave the
phosphonic acids 5 and 6, which were separated by preparative
reverse-phase HPLC.
The synthesis of the 3′-carbon analogues (Scheme 4) began
with the conversion of 5-methyluridine (9) to the completely
protected glycoside 17 in two steps with di-tert-butylsilyl bis-
(trifluoromethanesulfonate) (DiTBSOTf),54 followed by TBSCl.
Removal of the di-tert-butylsilyl group followed by protection
of the 5′-alcohol gave 18, which was converted to the thiocar-
bamate 19 with diimidazolethiocarbonyl. Allylation with allyl-
tributyltin using photolytic conditions gave compound 20 in
moderate yield.53 Conversion to aldehyde 21 occurred smoothly
upon treatment with osmium tetroxide followed by oxidative
cleavage with sodium periodate.53 Assembly of the cyclized
phosphonate ester 22 was performed as described for the 2′-
carbon analogue and proceeded in moderate yield over three
steps. Finally, the ethyl groups of phosphonate ester 22 were
removed with TMSBr and the 3:1 mixture of the desired
diastereomeric products 8 and 7 was separated by preparative
HPLC.
Stereochemistry Assignment. The stereochemistry of com-
pound 2 was determined by a 2D ROESY NMR experiment,
as reported by Ganem.48 Cross-peaks were observed between
the methine H adjacent to the phosphonate and the H2 and H3
on the ribose ring, indicating that 2 is the endo isomer. The
2′-carbon (5 and 6) and 3′-carbon (7 and 8) analogues were

NoVel Inhibitors of Thymidine Phosphorylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7809
Scheme 2. Synthesis of Carboxylic Acid 3 and Amide 4a
a Reagents and conditions: (a) benzoic acid, Ph3P, DIAD, 1,4-dioxane (92%);48 (b) H(CH3O)2CCH2CO2Me, concentrated HCl, room temp, 16 h (43%);
(c) LiOH, 1,4-dioxane, room temp, 1.5 h (47%); (d) 7 M NH3/MeOH, room temp, 18 h (45%).
Scheme 3. Synthesis of 2′-Carbon Analogues 5 and 6a
indicating that 6, the major diastereomer of the 2′-carbon series,
is the exo isomer. The minor compound 5, which displayed no
enhancement of the anomeric proton signal upon irradiation of
the methane adjacent to the phosphonate, was assigned the endo
configuration. The 1D NOE difference, 2D NOESY, and 2D
ROESY experiments were performed on 8 (the major 3′-carbon
diastereomer), 7 (the minor 3′-carbon diastereomer), and 5 (the
minor 2′-carbon diastereomer), but unfortunately, an indicative
NOE was not observed in any of the experiments. Instead, the
structural assignments for 7 and 8 were made by analogy to
the 2′-carbon series. Therefore, the minor isomer 7 was assigned
as the endo isomer and 8 was assigned as the exo. We expected
the endo isomers 5 and 7 to be more active than the exo isomers
6 and 8 in the TP enzyme assay because of the orientation of
the phosphonates being similar to that of active compound 2.
This in fact was the case, and the values are shown in Table 1.
Human Thymidine Phosphorylase Inhibitory Activity. A
spectrophotometric assay described by Wataya and Santi49 was
used to determine the inhibitory effect of the synthesized
analogues 2-8 against human TP. Enzyme activity was
measured following the conversion of a chromagenic substrate,
5-nitro-2′-deoxyuridine, to 5-nitrouracil in the presence of excess
phosphate. The reaction was initiated by addition of recombinant
human TP (625 ng/mL), and the change in absorbance was
monitored at 350 nm at 25 °C. All the compounds were tested
against the known inhibitor 2.
Replacement of the phosphonate group with a carboxylic acid
(compound 3) reduced the inhibitory activity of the analogue,
and the substitution of an amide for the phosphonate (compound
4) rendered the compound inactive. For both series of carbon
analogues, the endo isomer was 30-60 times more active than
the exo isomer but significantly less active than 2.
Molecular Modeling. Conformational searches on the two
diastereomers (endo and exo) of each of the three sets of
phosphonate compounds 2 (endo and exo) (Figure 2), 5 and 6
(Table 1), and 7 and 8 (Table 1) were performed. The OPLS-
AA force field,55 with a distance-dependent dielectric of 4r, was
used with the exception that ligand charges were fit to the
electrostatic potential from ab initio calculations with the HF/
a Reagents and conditions: (a) 1,3-dichloro-1,1,3,3-tetraisopropyldisilox-
ane, pyr (91%);52 (b) phenyl chlorothioformate, NEt3, DMAP, CH2Cl2
(71%); (c) allyltributyltin, AIBN, benzene, hν (94%); (d) OsO4, NMO,
acetone/H2O (4:1); (e) NaIO4, dioxane/H2O (3:1) (72%, two steps); (f) NaH,
CH2(PO(OEt)2)2, THF (92%); (g) TBAF, THF (91%); (h) NaOMe, MeOH
(80%); (i) TMSBr, DMF, (quantitative).
both generated as a pair of diastereomers in a 3:1 ratio, with
opposite configurations at the carbon bearing the phosphonate
moiety. Assignment of the stereochemistry for the 2′-carbon
series was accomplished by NOE experiments. The anomeric
proton in diastereomer 6 (Scheme 3) showed a 7% NOE upon
irradiation of the methine proton adjacent to the phosphonate,

7810 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Scheme 4. Synthesis of 3′-Carbon Analogues 7 and 8a
Allan et al.
a Reagents and conditions: (a) t-Bu2SiOTf2, DMF (99%);54 (b) TBSCl, imidazole, DMF (84%); (c) HF‚pyr, THF (92%); (d) TBSOTf, 2,6-lutidine,
CH2Cl2 (88%); (e) diimidazolethiocarbonyl, CH2Cl2 (99%); (f) allyltributyltin, CH2Cl2/benzene (2:3), hν (53%); (g) OsO4, NMO, acetone/H2O (4:1); (h)
NaIO4, dioxane/H2O (3:1) (64%, two steps); (i) NaH, CH2(PO3Et2)2, THF (91%); (j) TBAF, THF; (k) NaOMe, MeOH (58%, two steps); (l) TMSBr, DMF
(quantitative).
Figure 3. Conformers of the (a) endo and (b, c) exo diastereomers.
The trans ring conformation is shown in parts a and b, whereas part c
shows the cis ring conformation. The labels “ax” and “eq” refer to
pseudoaxial and pseudoequatorial positions on the B-ring, respectively.
The word “base” has been substituted for the pyrimidine ring in this
depiction.
Figure 2. Diastereomers of compound 2.
6-31G* basis set. Five-thousand steps of the mixed MCMM/
LMOD searching method were performed with Macromodel.56
In each case, the global minimum for the endo diastereomer
was lower in energy than that of the exo isomer by 0.3-0.9
kcal/mol. Comparison of the three-dimensional conformations
of the endo and exo diastereomers highlights the factors that
contribute to the lower internal energy of the endo compound.
The two primary differences between these diastereomers are
the conformation of the B-ring in relation to the ribose A-ring,
as well as the orientation of the phosphonomethyl group
(pseudoaxial or pseudoequatorial) (Figure 3). On the basis of
model studies of the unsubstituted bicycle, the all-trans con-
formation of the B-ring is preferred over the folded conformation
(cf. Figure 3a vs Figure 3c). In addition, the pseudoequatorial
position is favored over the pseudoaxial. The endo diastereomer
permits a conformation where the B-ring is in the more stable
trans conformation and the phosphonomethyl group is pseudo-
equatorial (Figure 3a), whereas the exo diastereomer must
sacrifice either the ring conformation in order to allow the
phosphonomethyl group to occupy the pseudoequatorial position
(Figure 3c) or vice versa (Figure 3b).
While the endo diastereomer is lower in energy than the exo
diastereomer for all three sets of compounds, the intrinsic
rotational preference of the phosphonomethyl group differs
among the groups. In the compound pairs of 2 (endo and exo)
and 7 and 8, the phosphonate lies closer to the 5′-carbon of the
ribose ring than to the 1′-carbon in the lowest-energy structure;
the opposite is true for the diastereomeric pair of in the 2′-
carbon series of 5 and 6. Presumably, the orientational prefer-
ence of the phosphonate is influenced by electrostatic repulsion

NoVel Inhibitors of Thymidine Phosphorylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7811
suggest that the protein-ligand complex with the endo isomer
is lower in energy than the exo complex.
Experimental Section
General. All commercially available reagents were used without
further purification unless otherwise specified. All reactions were
performed under nitrogen atmosphere unless otherwise specified.
NMR spectra were obtained on a Bruker Ultrashield 300 MHz
spectrometer at 300 MHz for 1H NMR and at 75 MHz for 13C NMR
with tetramethylsilane as an internal standard. Mass spectra (MS)
were recorded on an AGI-150 mass spectrometer using an elec-
trospray technique. Elemental analyses were performed by Numega
Labs, San Diego, CA, on a Perkin-Elmer 2400-2 CHNS analyzer.
All final compounds were analyzed on an HPLC, equipped with a
Waters 1525 pump, a Waters 2487 dual λ Absorbance detector, a
Waters 717 Plus autosampler using a Phenomenex Synergi RP-18
250 mm × 4.6 mm reverse-phase column with a 5-15% methanol/
water gradient containing 0.1% TFA (method 1) and using a
Thermo Electron Aquasil C-18 250 mm × 4.6 mm reverse phase
column with a 5% isocratic acetonitrile, 95% water containing 0.1%
TFA system (method 2). Final compounds were shown to be at
least 95% pure based on the 254 nm UV wavelength under these
conditions. The flow rates for the analytical and semipreparative
experiments were 1 and 4 mL/min, respectively. The semiprepara-
tive separations were performed with a system equipped with a
GBC pump LC 1150, GBC LC 5100 photodiode array detector,
LC 1445 system organizer, and a Vydac C-18 column (10 mm ×
250 mm).
Figure 4. Plot of the % remaining (HPLC integration analysis) versus
time for the analogues 2, 5, 7, and 8 when incubated at 37 °C at pH
1.0: compound 2 ([), compound 5 (2), compound 7 (9), compound
8 (b).
between the lone 2′ or 3′ oxygen in compounds 7 and 8 and 5
and 6, respectively.
Docking studies were performed on compounds 2 and 5-8
in an effort to understand the differences in experimental binding
affinities. The compounds were docked to our homology model
of human thymidine phosphorylase using the methods we
previously reported.39 In each case, the protein-ligand complex
with the endo isomer was lower in energy than the exo complex,
which explains the tighter binding of 5 compared to 6 and of 7
compared to 8. In addition, the phosphate group was bound in
a pocket located closer to the 5′-carbon than the to the 1′-carbon
of the ribose ring. The fact that the rotational preference of the
phosphate differs from the uncomplexed molecule in both
compounds 5 and 6, as mentioned above, may provide some
insight into the reduced activity of these compounds compared
to analogues 7 and 8. Furthermore, there is a hydrogen-bond
interaction between the 2′-oxygen in 7 and 8 with His116 that
is lost in compounds 5 and 6 when the 2′-oxygen is replaced
by carbon. There were no obvious dissimilarities in the docked
structures of 2 and 5 that might explain their comparable
observed binding energy differences.
Stability in Acidic Buffer. To determine whether the carbon
analogues 5, 7, and 8 were more stable in acidic conditions
compared to lead compound 2, a kinetic study was performed.
The half-life of compound 2 was determined to be 98 h at 37
°C in pH 1.0 buffer, whereas the carbon analogues 5, 7, and 8
were unchanged under these same conditions (Figure 4). The
decomposition of compound 2 is presumed to occur through
hydrolysis of the acetal ring, although the products were not
isolated or characterized.
Synthesis of Pyrimidine-2,4-dione 14. N-Methylmorpholine
N-oxide (230 mg, 1.96 mmol) was added to a solution of
pyrimidine-2,4-dione 1353 (908 mg, 1.78 mmol) in 9.6 mL of
acetone and 2.4 mL of water, followed by the addition of a 0.0185
g/mL aqueous solution of osmium tetroxide (1.2 mL, 0.089 mmol).
The reaction mixture turned from colorless to orange immediately
and was stirred at room temperature for 105 min. A saturated,
aqueous solution of sodium thiosulfate (3 mL) was added, followed
by 3 mL of water, and the mixture was extracted twice with ethyl
acetate. The organic layers were washed twice with saturated
sodium bicarbonate solution and once with brine, dried (Na2SO4),
and concentrated to afford 963 mg (99%) of a mixture of diols as
1
an off-white foam: IR (film, cm-1) 3437, 1695; H NMR (300
MHz, CDCl3) δ 7.70 (s, 1H), 5.91 (s, 0.5H), 5.78 (s, 0.5H), 4.52-
4.40 (m, 1H), 4.28-4.18 (m, 2H), 4.01-3.85 (m, 3H), 3.76-3.45
(m, 3H), 2.72-2.62 (m, 0.5H), 2.44-2.35 (m, 0.5H), 1.93 (s, 3H),
1.14-0.96 (m, 28H); ES MS m/z (M + Na)+ 581.6. Sodium
periodate (730 mg, 3.41 mmol) was added to a solution of the crude
diols (954 mg, 1.71 mmol) in 19 mL of 1,4-dioxane and 6.5 mL of
water. The solution was stirred at room temperature for 35 min.
The reaction mixture was diluted with ethyl acetate, washed twice
with saturated sodium bicarbonate solution and once with brine,
dried (Na2SO4), and concentrated. The resulting residue was purified
by flash chromatography, eluting with 1:1 ethyl acetate/hexanes to
afford 656 mg (73%) of aldehyde 14 as a white foam: mp 60-65
1
°C; IR (film, cm-1) 3189, 1696; H NMR (300 MHz, CDCl3) δ
Conclusions
9.81 (s, 1H), 8.45 (s, 1H), 7.31 (d, J ) 1.2 Hz, 1H), 5.78 (d, J )
4.4 Hz, 1H), 4.59 (t, J ) 7.2 Hz, 1H), 4.05 (d, J ) 4.1 Hz, 2H),
3.93-3.88 (m, 1H), 3.03-2.93 (m, 1H), 2.91-2.82 (m, 1H), 2.63
(dd, J ) 17.3, 5.0 Hz, 1H), 1.93 (d, J ) 1.2 Hz, 3H), 1.12-0.97
(m, 28H); 13C NMR (75 MHz, CDCl3) δ 199.7, 163.9, 150.5, 134.6,
111.0, 87.8, 83.9, 69.7, 61.3, 43.0, 40.3, 17.4, 17.3, 17.20, 17.18,
16.89, 16.86, 16.82, 16.77, 13.3, 13.0, 12.8, 12.6 (2C); ES MS m/z
(M - H)- 525.8. Anal. (C24H42N2O7Si2) C, H, N.
A series of multisubstrate inhibitors of TP incorporating a
nucleoside framework and an acidic or polar group were
prepared. Replacement of the phosphonate with a carboxylic
acid reduced the activity of the compound against TP, and the
replacement of the phosphonate with an amide rendered the
compound inactive. When the five-membered acetal ring was
replaced with either the 2′-carbon ether or the 3′-carbon ether,
the chemical stability to low pH was increased but the activity
in the human TP enzymatic assay was reduced. The diastere-
omers of each series of the carbon analogues exhibited
significantly different activities, indicating that the orientation
of the phosphonate group is critical. The most active compounds
5 and 7 possess an endo phosphonate, which is consistent with
the phosphonate orientation in 2. Molecular modeling results
Synthesis of Phosphonic Acid Diethyl Ester 15. Tetraethyl
methylenediphosphonate (0.22 mL, 0.88 mmol) was added dropwise
to a mixture of sodium hydride (60% dispersion in oil, 35 mg, 0.88
mmol) in 4 mL of dry THF at 0 °C under nitrogen, and the reaction
mixture was stirred for 10 min and then stirred at room temperature
for 20 min. The reaction mixture was cooled to 0 °C, a solution of
aldehyde 14 (267 mg, 0.507 mmol) in 2 mL of dry THF was added,
and the resulting solution was stirred at room temperature for 19

7812 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Allan et al.
h. The reaction mixture was concentrated, and the residue was
purified by flash chromatography, eluting first with 1:1 ethyl acetate/
hexanes and then with 100% ethyl acetate to afford 312 mg (92%)
of the phosphonate ester as a white foam: IR (film, cm-1) 3169,
(m, 1H), 2.24-1.90 (m, 2H), 1.83-1.70 (m, 1H), 1.82 (s, 3H);
13C NMR (75 MHz, D2O, minor diastereomer) δ 166.9, 152.2,
138.3, 111.8, 92.2, 85.7, 84.3, 79.5, 62.1, 50.1, 36.4, 34.6 (d, J )
127 Hz), 11.9; ES MS m/z (M - H)- 361.4; MALDI FTMS m/z
(M + Na)+ calcd 385.0771, obsd 385.0773.
1
1710, 1694, 1682; H NMR (300 MHz, CDCl3) δ 8.89 (s, 1H),
6: mp >300 °C; 1H NMR (300 MHz, D2O, major diastereomer)
δ 7.56 (s, 1H), 5.87 (d, J ) 6.4 Hz, 1H), 4.45-4.34 (m, 1H), 4.07-
4.01 (m, 1H), 3.82-3.67 (m, 2H), 3.16-3.07 (m, 1H), 2.30-1.94
(m, 3H), 1.88-1.76 (m, 1H), 1.81 (s, 3H); 13C NMR (75 MHz,
D2O, major diastereomer) δ 166.8, 152.2, 138.1, 112.1, 90.4, 85.1,
83.4, 74.8, 62.0, 49.4, 36.3, 32.7 (d, J ) 133 Hz), 11.9; ES MS
m/z (M - H)- 361.4; MALDI FTMS m/z (M + Na)+ calcd
385.0771, obsd 385.0780.
7.34 (s, 1H), 6.92-6.73 (m, 1H), 5.85-5.73 (m, 2H), 4.55 (t, J )
7.3 Hz, 1H), 4.13-3.98 (m, 6H), 3.94-3.88 (m, 1H), 2.83-2.70
(m, 1H), 2.48-2.33 (m, 2H), 1.92 (s, 3H), 1.31 (t, J ) 7.1 Hz,
6H), 1.12-0.97 (m, 28H); 13C NMR (75 MHz, CDCl3) δ 163.7,
150.0, 149.9 (d, J ) 4.3 Hz), 134.9, 119.6 (d, J ) 186.5 Hz), 110.7,
88.2, 83.7, 69.5, 61.7 (dd, J ) 5.6, 2.9 Hz), 61.0, 47.4, 31.2 (d, J
) 23.2 Hz), 17.5, 17.4, 17.3, 17.2, 17.1, 17.0, 16.9, 16.44, 16.36,
13.4, 13.1, 12.8, 12.7, 12.6; ES MS m/z (M + H)+ 661.7; MALDI-
FTMS m/z (M + H)+ calcd 661.3100, obsd 661.3105. A 1.0 M
solution of tetrabutylammonium fluoride (1.40 mL, 1.40 mmol) was
added dropwise to a solution of the phosphonate ester (452 mg,
0.684 mmol) in 12 mL of dry THF, and the resulting solution was
stirred at room temperature under nitrogen for 25 min. The reaction
mixture was concentrated, and the resulting residue was purified
by flash chromatography, eluting first with 95:5 dichloromethane/
methanol and then with 9:1 dichloromethane/methanol to give 262
Synthesis of 19. To a solution of alcohol 18 (0.772 g, 1.59 mmol)
in CH2Cl2 (16 mL) was added DMAP (0.059 g, 0.48 mmol),
followed by 1,1′-thiocarbonyldiimidazole (0.369 g, 2.07 mmol) at
room temperature under nitrogen, and the reaction mixture was
stirred for 19 h. The solvent was removed in vacuo, and purification
by flash chromatography (elution with 40% ethyl acetate/hexanes)
gave 19 (0.943 g, 99%) as a white foam: mp 86-88 °C; IR (CCl4,
1
cm-1) 3179, 3127, 3060, 2956, 2933, 1696; H NMR (300 MHz,
mg (91%) of alcohol 15 as a sticky, white foam: IR (film, cm-1
)
CDCl3) δ 9.51 (s, 1H), 8.45 (s, 1H), 7.69 (bs, 1H), 7.47 (d, J )
1.2 Hz, 1H), 7.10 (bs, 1H), 6.21 (d, J ) 7.2 Hz, 1H), 5.79 (dd, J
) 5.3, 1.6 Hz, 1H), 4.45 (dd, J ) 7.1, 5.4 Hz, 1H), 4.40 (d, J )
1.6 Hz, 1H), 3.99 (q, J ) 9.7 Hz, 2H), 1.96 (d, J ) 1.0 Hz, 3H),
0.99 (s, 9H), 0.77 (s, 9H), 0.20 (s, 3H), 0.19 (s, 3H), -0.03 (s,
3H), -0.04 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 183.3, 163.6,
150.6, 137.4, 134.4, 131.1, 117.6, 111.7, 86.9, 82.7, 81.1, 73.9,
63.2, 25.9, 25.2, 18.3, 17.6, 12.4, -5.2, -5.23, -5.39, -5.4; ES
1
3371, 3063, 1688; H NMR (300 MHz, methanol-d4) δ 7.77 (s,
1H), 6.78-6.58 (m, 1H), 6.06 (d, J ) 9.0 Hz, 1H), 5.79 (dd, J )
21.6, 17.2 Hz, 1H), 4.28 (d, J ) 4.9 Hz, 1H), 4.08-3.96 (m, 5H),
3.76-3.73 (m, 2H), 2.70-2.58 (m, 1H), 2.53-2.30 (m, 2H), 1.88
(s, 3H), 1.29 (t, J ) 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ
166.2, 152.8, 152.6 (d, J ) 4.4 Hz), 138.0, 119.1 (d, J ) 186.8
Hz), 112.2, 89.3, 89.0, 74.0, 63.5, 63.4 (d, J ) 5.8 Hz), 30.7, 30.4,
16.7 (d, J ) 6.1 Hz), 12.5; ES MS m/z (M + Na)+ 441.3; MALDI-
FTMS m/z (M + H)+ calcd 419.1578, obsd 419.1582.
MS m/z (M + H)+ 597.9. Anal. (C26H44N4O6SSi ) C, H, N.
2
Synthesis of Pyrimidine-2,4-dione 20. A solution of 19 (0.911
g, 1.53 mmol) and AIBN (0.126 g, 0.765 mmol) in 26 mL of
CH2Cl2/benzene (2:3) was degassed with nitrogen for 5 min.
Allyltributyltin (2.37 mL, 0.765 mmol) was added, and the solution
was irradiated for 3 h with a high-pressure 450 W Hanovia mercury
lamp. The solvent was removed in vacuo, and purification by flash
chromatography (elution with a gradient of 10-30% ethyl acetate
in hexanes) afforded 20 (0.309 g, 40%), a white foam, as the major
product: IR (CCl4, cm-1) 2953, 2935, 2862, 1682; 1H NMR (300
MHz, CDCl3) δ 8.43 (bs, 1H), 7.71 (d, J ) 1.2 Hz, 1H), 5.69-
5.83 (m, 1H), 5.66 (d, J ) 1.6 Hz, 1H), 5.03-5.11 (m, 2H), 4.29
(d, J ) 4.3 Hz, 1H), 4.11 (d, J ) 11.9 Hz, 1H), 4.04 (d, J ) 8.9
Hz, 1H), 3.72 (dd, J ) 12, 2.5 Hz, 1H), 2.34-2.43 (m, 1H), 1.99-
2.18 (m, 2H), 1.92 (d, J ) 1.2 Hz, 3H), 0.95 (s, 9H), 0.90 (s, 9H),
0.21 (s, 3H), 0.14 (s, 3H), 0.12 (s, 3H), 0.10 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 164.0, 150.3, 135.7, 116.6, 109.6, 90.9, 84.6, 62.4,
40.8, 28.8, 26.0, 25.8, 18.6, 18.1, 12.7, -4.2, -5.2, -5.3, -5.5;
ESI MS m/z (M + Na)+ 533.8. Anal. (C25H46N2O5Si2) C, H, N.
Synthesis of Aldehyde 21. To a solution of 20 in 24 mL of
acetone/H2O (4:1) was added NMO (0.30 g, 2.58 mmol) and OsO4
(0.030 mg, 0.118 mmol) at room temperature, and the reaction
mixture was stirred for 3 h. Freshly prepared saturated aqueous
Na2S2O3 (4.6 mL) was added to the reaction mixture followed by
water (4.6 mL). The solution was extracted three times with ethyl
acetate, and the combined organic layers were washed with saturated
NaHCO3 and brine and dried over MgSO4. Purification by flash
chromatography (elution with a gradient of 50-100% ethyl acetate
in hexanes) gave a 1:1 mixture of diastereomeric diols (0.818 g,
67%) as a white foam: IR (CCl4, cm-1) 3429, 3185, 3063, 2958,
Synthesis of Phosphonic Acid Diethyl Ester 16. Sodium
methoxide (14 mg, 0.26 mmol) was added to a solution of alcohol
15 (88 mg, 0.21 mmol) in 3 mL of dry methanol, and the reaction
mixture was stirred at room temperature under nitrogen for 42 h.
Then 1 N HCl (0.6 mL) was added to achieve a pH of 2, and the
reaction mixture was concentrated. The residue was taken up in
acetone and filtered to remove the sodium salts, and the filtrate
was concentrated to give a colorless oil. The resulting residue was
purified by flash chromatography, eluting with 95:5 dichloro-
methane/methanol to afford 70 mg (80%) of mixture 16 as a sticky
white foam: IR (film, cm-1) 3421, 3062, 1690; 1H NMR (300 MHz,
CDCl3) δ 7.46 (d, J ) 1.2 Hz, 0.3H), 7.41 (d, J ) 1.2 Hz, 0.7H),
5.84 (d, J ) 6.5 Hz, 0.3H), 5.80 (d, J ) 6.5 Hz, 0.7H), 4.77 (dd,
J ) 7.5, 3.3 Hz, 0.7H), 4.55 (dd, J ) 6.9, 2.0 Hz, 0.3H), 4.49-
4.23 (m, 2H), 4.19-4.06 (m, 4H), 3.97-3.87 (m, 1H), 3.84-3.76
(m, 1H), 3.13-2.98 (m, 1H), 2.45-1.95 (m, 3H), 1.92 (s, 3H),
1.90-1.79 (m, 1H), 1.33 (t, J ) 7.1 Hz, 6H); 13C NMR (75 MHz,
CDCl3, major diastereomer) δ 163.6, 150.49, 136.5, 111.3, 91.5,
85.5, 83.4, 73.7, 62.8, 61.93 (dd, J ) 9.6, 6.2 Hz), 50.1, 34.6 (d,
J ) 284 Hz), 30.9, 16.4 (dd, J ) 5.9, 1.1 Hz), 12.53; 13C NMR
(75 MHz, CDCl3, minor diastereomer) δ 163.7, 150.53, 137.2,
111.1, 94.1, 85.4, 85.2, 73.7, 63.1, 61.89 (d, J ) 9.7, 6.6 Hz), 49.9,
34.7 (d, J ) 259 Hz), 31.1, 16.4 (dd, J ) 5.9, 1.1 Hz), 12.50; ES
MS m/z (M + Na)+ 441.4; MALDI-FTMS m/z (M + H)+ calcd
419.1578, obsd 419.1591.
Synthesis of Phosphonic Acids 5 and 6. Trimethylsilyl bromide
(1.55 mL, 11.7 mmol) was added dropwise to a stirred solution of
16 (249 mg, 0.594 mmol) in 7.5 mL of dry DMF under nitrogen at
-78 °C. The reaction mixture was allowed to warm to room
temperature and was stirred for 23 h. The solvent was removed in
vacuo, and the resulting residue was purified by ion-exchange
chromatography (Sephadex-CM, C25 resin), followed by lyo-
philization, providing 246 mg of 5 and 6 as a white solid and a
mixture of diastereomers in a 3:1 ratio (as determined by HPLC).
The diastereomers were separated by preparative HPLC. Fractions
containing the purified diastereomers were pooled, concentrated
in vacuo, triturated with ether, and dried under high vacuum.
5: mp >300 °C; 1H NMR (300 MHz, D2O, minor diastereomer)
δ 7.58 (s, 1H), 5.91 (d, J ) 5.5 Hz, 1H), 4.53-4.45 (m, 1H), 4.28-
4.17 (m, 2H), 3.79-3.63 (m, 2H), 3.18-3.08 (m, 1H), 2.50-2.38
1
2929, 2865, 1708; H NMR (300 MHz, CDCl3) δ 8.88 (bs, 1H),
8.66 (bs, 1H), 7.71 (d, J ) 1.2 Hz, 1H), 7.61 (d, J ) 1.2 Hz), 5.68
(d, J ) 1.6 Hz, 1H), 5.64 (s, 1H), 4.42 (d, J ) 4.0 Hz, 1H), 4.38
(d, J ) 4.8 Hz, 1H), 4.03-4.14 (m, 4H), 3.60-3.75 (m, 6H), 3.36-
3.49 (m, 2H), 3.01 (bs, 1H), 2.70 (bs, 1H), 2.30-2.40 (m, 1H),
2.18-2.28 (m, 3H), 1.91 (d, J ) 1.1 Hz, 3H), 1.88 (d, J ) 1.1 Hz,
3H), 1.30-1.39 (m, 4H), 0.95 (s, 9H), 0.94 (s, 9H), 0.91 (s, 9H),
0.90 (s, 9H), 0.24 (s, 3H), 0.19 (s, 3H), 0.14 (s, 3H), 0.138 (s, 3H),
0.13 (s, 3H), 0.12 (s, 3H), 0.11 (s, 6H); 13C NMR (75 MHz, CDCl3,
isomer A) δ 163.8, 150.3, 135.7, 109.7, 90.9, 84.7, 78.0, 70.8, 66.9,
62.3, 38.5, 27.8, 26.0, 25.8, 18.6, 18.1, 12.6, -5.1, -5.2, -5.3,
-5.4; 13C NMR (75 MHz, CDCl3, isomer B) δ 164.0, 150.4, 136.0,

NoVel Inhibitors of Thymidine Phosphorylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7813
109.3, 91.5, 85.2, 78.0, 69.8, 67.0, 62.7, 37.6, 27.4, 26.0, 25.8, 18.7,
18.1, 12.6, -4.0, -4.3, -5.0, -5.5; ES MS m/z (M + H)+ 511.7.
To a solution of the mixture of diols (0.818 g, 1.5 mmol) in 21 mL
of dioxane/water (3:1) was added sodium periodate (0.643 g, 3.0
mmol) at room temperature, giving a milky suspension. The mixture
was stirred for 2 h and then diluted with water and ethyl acetate.
The layers were separated, and the aqueous layer was extracted
twice with ethyl acetate. The combined organic layers were washed
with saturated aqueous NaHCO3 and brine and dried over MgSO4.
The solvent was removed in vacuo, and purification by flash
chromatography (elution with 15-25% ethyl acetate in hexanes)
afforded aldehyde 21 (0.679 g, 88%) as a white foam: mp 72-74
°C; IR (CCl4, cm-1) 3397, 3134, 2955, 2932, 2860, 1686; 1H NMR
(300 MHz, CDCl3) δ 9.82 (s, 1H), 8.18 (bs, 1H), 7.58 (d, J ) 1.2
Hz, 1H), 5.75 (d, J ) 2.2 Hz, 1H), 4.43 (dd, J ) 5.2, 2.2 Hz, 1H),
4.04 (dd, J ) 11.9, 2.4 Hz), 3.99 (dt, J ) 8.4 Hz, 1H), 3.75 (dd,
J ) 11.7, 2.4 Hz, 1H), 2.90 (dd, J ) 18, 8.3 Hz, 1H), 2.60-2.68
(m, 1H), 2.50 (dd, J ) 18, 5.2 Hz, 1H), 1.93 (d, J ) 1.1 Hz, 3H),
0.94 (s, 9H), 0.88 (s, 9H), 0.15 (s, 3H), 0.12 (s, 6H), 0.02 (s, 3H);
13C NMR (75 MHz, CDCl3) 199.9, 163.7, 150.2, 135.4, 110.1, 90.6,
84.2, 76.9, 62.5, 39.9, 36.0, 25.9, 25.7, 18.5, 17.9, 12.7, -4.5, -5.2,
-5.3, -5.5; ESI MS m/z (M - H)- 511.6. Anal. (C24H44N2O6Si2)
C, H, N.
Synthesis of Phosphonic Acid Diethyl Ester 22. To a suspen-
sion of sodium hydride (0.054 g, 2.26 mmol) in anhydrous THF
(18 mL) at 0 °C under nitrogen was added tetraethylmethylene
diphosphonate (0.65 g, 2.26 mmol) dropwise. The mixture was
stirred for 10 min at 0 °C and 20 min at room temperature. Aldehyde
21 (0.678 g, 1.33 mmol) in anhydrous THF (7 mL) was added to
the solution at 0 °C, and the reaction mixture was allowed to slowly
warm to room temperature and stirred overnight. The solvent was
removed in vacuo, and purification by flash chromatography (elution
with 60-90% ethyl acetate in hexanes) afforded the phosphonate
ester (0.781 g, 91%) as a white foam: mp 50-53 °C; IR (CCl4,
cm-1) 3171, 3051, 2950, 2854, 1694; 1H NMR (300 MHz, CDCl3)
δ 8.11 (bs, 1H), 7.63 (d, J ) 1.2 Hz, 1H), 6.58-6.76 (m, 1H),
5.66-5.80 (m, 2H), 4.31 (dd, J ) 4.2, 1.5 Hz, 1H), 4.02-4.12 (m,
4H), 3.70 (dd, J ) 12, 2.5 Hz, 1H), 2.50-2.64 (m, 1H), 2.14-
2.27 (m, 2H), 1.92 (d, J ) 1.2 Hz, 3H), 1.32 (t, J ) 8.6 Hz, 6H),
0.94 (s, 9H), 0.90 (s, 9H), 0.19 (s, 3H), 0.13 (s, 3H), 0.12 (s, 3H),
0.10 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 163.8, 150.3, 149.7
(d, J ) 4.8 Hz), 135.4, 119.4 (d, J ) 188 Hz), 109.9, 90.8, 84.2,
76.9, 62.2, 61.7 (dd, J ) 5.7, 1.8 Hz), 40.2, 29.2 (d, J ) 22.7 Hz),
26.0, 25.8, 18.5, 18.0, 16.3 (d, J ) 6.4 Hz), 12.7, -4.2, -5.2, -5.3,
-5.5; ESI MS m/z (M + Na)+ 669.8; MALDI-FTMS m/z (M +
H)+ calcd 647.3307, obsd 647.3309. Anal. (C29H55N2O8PSi2) C,
N. H: calcd, 8.57; found, 8.99. To a solution of the phosphonate
ester (0.905 g, 1.40 mmol) in anhydrous THF (23 mL) at room
temperature was added dropwise a 1.0 M TBAF solution in THF
(2.8 mL, 2.8 mmol), and the solution was stirred overnight. The
solvent was removed in vacuo, and purification by flash chroma-
tography (elution with a gradient of 2-10% methanol in methylene
chloride) gave an inseparable mixture of the diol and the diaster-
eomeric cyclized alcohols 22. To a solution of the mixture of diols
and 22 (0.385 g, 0.921mmol) in anhydrous MeOH (15 mL) was
added 0.5 M sodium methoxide in MeOH (2.22 mL, 1.11 mmol)
at room temperature, and the reaction mixture was stirred overnight.
The reaction was quenched with 1 N HCl, and the pH was adjusted
to 5. The solvent was removed in vacuo, the crude mixture was
dissolved in acetone, and the salts were removed by filtration. The
filtrate was concentrated in vacuo, and purification by flash
chromatography (elution with a gradient of 2-10% methanol in
methylene chloride) afforded 22 (0.339 g, 58%) as an inseparable
3:1 mixture of diastereomers (determined by NMR) and as a sticky
white foam: IR (CCl4, cm-1) 3400, 3056, 2987, 2935, 2822, 1685;
1H NMR (300 MHz, CDCl3) δ 8.91 (bs, 1H), 7.21 (d, J ) 1.2 Hz,
0.3H), 7.15 (d, J ) 1.2 Hz, 0.7H), 5.75 (d, J ) 2.4 Hz, 0.3H), 5.56
(d, J ) 2.4 Hz, 0.7H), 4.82 (dd, J ) 7.3, 2.2 Hz, 0.7H), 4.64 (dd,
J ) 7.3, 2.2 Hz, 0.3H), 4.23-4.49 (m, 1H), 4.07-4.17 (m, 4H),
3.91-3.97 (m, 2H), 3.68-3.74 (m, 1H), 3.28 (q, J ) 7.5 Hz, 1H),
2.87-2.90 (m, 1H), 2.21-2.43 (m, 1H), 1.98-2.10 (m, 3H), 1.90
(d, J ) 1.1 Hz, 3H), 1.33 (t, J ) 7.1 Hz, 6H); 13C NMR (75 MHz,
CDCl3, major diastereomer) δ 163.6, 150.2, 138.2, 111.0, 95.5, 88.0,
86.1, 74.3, 62.6, 61.9 (dd, J ) 13.3, J ) 6.2 Hz), 43.9, 36.9 (d, J
) 6.0 Hz), 31.8 (d, J ) 139 Hz), 16.4 (d, J ) 6 Hz), 12.3; 13C
NMR (75 MHz, CDCl3, minor diastereomer) δ 163.9, 150.6, 138.0,
110.9, 93.5, 88.9, 86.1, 74.3, 62.3, 61.9 (dd, J ) 13.3, 6.2 Hz),
44.3, 36.3 (d, J ) 6.0 Hz), 32.4 (d, J ) 139.7 Hz), 16.4 (d, J ) 6
Hz), 12.5; MALDI FTMS m/z (M + H)+ calcd 419.1578, obsd
419.1588.
Synthesis of Phosphonic Acids 7 and 8. To a solution of the
phosphonate esters 22 (0.300 g, 0.717 mmol) in anhydrous DMF
at -78 °C was added TMSBr dropwise, and the reaction mixture
was allowed to warm to room temperature and was stirred over-
night. The solvent was removed in vacuo, and purification by
ion-exchange chromatography (Sephadex-CM, C-25 resin) fol-
lowed by lyophilization provided 7 and 8 (114 mg, 44% yield) as
a white solid as a 3:1 mixture of diastereomers (determined by
HPLC). The diastereomers were purified by preparative HPLC.
Fractions containing the purified diastereomers were pooled,
concentrated in vacuo, triturated with ethyl ether, and dried under
high vacuum.
1
7: H NMR (300 MHz, D2O, minor diastereomer) δ 7.53 (s,
1H), 5.95 (s, 1H), 4.57-4.59 (m, 1H), 4.30 (bs, 1H), 4.04-4.08
(m, 1H), 3.81 (dd, J ) 13.1, 5.5 Hz, 1H), 3.66 (dd, J ) 13.1, 5.5
Hz, 1H), 2.92-3.00 (m, 1H), 2.36-2.46 (m, 1H), 2.12-2.25 (m,
1H), 1.93-2.04 (m, 1H), 1.83 (s, 3H), 1.59-1.69 (m, 1H); 13C
NMR (75 MHz, D2O, minor diastereomer) δ 166.9, 151.9, 138.5,
111.6, 90.8, 88.7, 88.4, 79.4, 62.0, 44.9, 35.9, 33.2 (d, J ) 111
Hz), 11.8; MALDI FTMS m/z (M + Na)+ calcd 385.0771, obsd
385.0763.
1
8: mp 295-298 °C (dec); H NMR (300 MHz, D2O, major
diastereomer) δ 7.48 (s, 1H), 5.83 (d, J ) 2.15 Hz, 1H), 4.40-
4.48 (m, 1H), 3.97-4.02 (m, 1H), 3.83 (dd, J ) 12.5, 3.0 Hz, 1H),
3.69 (dd, J ) 12.5, 5.6 Hz, 1H), 3.01 (q, J ) 7.4 Hz, 1H), 1.91-
2.22 (m, 3H), 1.83 (s, 4H); 13C NMR (75 MHz, D2O, major
diastereomer) δ 166.9, 151.9, 138.8 (d, J ) 16 Hz), 111.8, 92.2,
87.8, 85.8, 75.8, 62.3, 44.8, 36.4, 33.2 (d, J ) 220 Hz), 11.8; ESI
MS m/z (M - H)- 361.4; MALDI FTMS m/z (M + Na)+ calcd
385.0771, obsd 385.0777.
Acknowledgment. We thank Wayne Guida (Eckerd Uni-
versity) for valuable input on the computational studies.
Supporting Information Available: Elemental analysis of
selected compounds, experimental procedures and characterization
of compounds 3, 4, 11, 17, and 18, and HPLC analysis of traces of
compounds 4-8 under two diverse conditions. This material is
available free of charge via the Internet at http://pubs.acs.org.
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