Probing binding requirements of type I and type II isoforms of inosine monophosphate dehydrogenase with adenine-modified nicotinamide adenine dinucleotide analogues

Article abstract of DOI:10.1021/jm070568j

Novel tiazofurin adenine dinucleotide (TAD) analogues 25-33 containing a substituent at C2 of the adenine ring have been synthesized as inhibitors of the two isoforms of human IMP-dehydrogenase. The 2-ethyl TAD analogue 33 [K _i = 1 nM (type I),

Full text of DOI:10.1021/jm070568j

J. Med. Chem. 2007, 50, 5743-5751
5743
Probing Binding Requirements of Type I and Type II Isoforms of Inosine Monophosphate
Dehydrogenase with Adenine-Modified Nicotinamide Adenine Dinucleotide Analogues
Liqiang Chen,† Guangyao Gao,† Krzysztof Felczak,† Laurent Bonnac,† Steven E. Patterson,† Daniel Wilson,† Eric M. Bennett,†
Hiremagalur N. Jayaram,‡ Lizbeth Hedstrom,§ and Krzysztof W. Pankiewicz*,†
Center for Drug Design, UniVersity of Minnesota, Minneapolis, Minnesota 55455, Department of Biochemistry and Molecular Biology, Indiana
UniVersity School of Medicine and Richard Roudebush Veterans Affairs Medical Center, 1481 West Tenth Street, Indianapolis, Indiana 46202,
and Departments of Biochemistry and Chemistry, MS009, Brandeis UniVersity, Waltham, Massachusetts 02454
ReceiVed May 15, 2007
Novel tiazofurin adenine dinucleotide (TAD) analogues 25-33 containing a substituent at C2 of the adenine
ring have been synthesized as inhibitors of the two isoforms of human IMP-dehydrogenase. The 2-ethyl
TAD analogue 33 [Ki ) 1 nM (type I), Ki ) 14 nM (type II)] was found to be the most potent. It did not
inhibit three other cellular dehydrogenases up to 50 µM. Mycophenolic adenine bis(phosphonate)s containing
a 2-phenyl (37) or 2-ethyl group (38), were prepared as metabolically stable compounds, both nanomolar
inhibitors. Compound 38 [Ki ) 16 nM (type I), Ki ) 38 nM (type II)] inhibited proliferation of leukemic
K562 cells (IC50 ) 1.1 µM) more potently than tiazofurin (IC50 ) 12.4 µM) or mycophenolic acid (IC50
7.7 µM).
)
Introduction
Inosine monophosphate dehydrogenase (IMPDHa), a nicoti-
namide adenine dinucleotide (NAD)-dependent enzyme, is a key
enzyme in de noVo synthesis of purine nucleotides and a major
therapeutic target.1 It catalyzes the oxidation of IMP to
xanthosine monophosphate (XMP), which is further converted
into guanosine monophospahte (GMP) by GMP synthase. The
mechanism of action of the enzyme has been recently reviewed.2
Inhibition of IMPDH results in depletion of guanine nucleotides,
including GTP and dGTP. This G-restriction affects DNA and
RNA metabolism as well as the activity of GTP-binding proteins
involved in transduction pathways.3 IMPDH inhibitors have
antiproliferative activity and are used as immunosuppressive,
anticancer, and antiviral agents.
Figure 1.
Two isoforms of the human enzyme, type I and type II, are
known.4,5 Their genes are located on different chromosomes,
they are regulated differently, and their products (isoforms) are
not redundant.6-8 This raises the question of the role of the
isoforms in intracellular processes and their importance as
potential targets for therapeutic intervention. Most tissues
express both isozymes, although type I predominates in thymus,
brain, retina, and lung while type II is the major form in liver
and testis.8 IMPDH is a tetramer;9 the type II protein contains
514 residues and is 84% identical to type I. The canonical form
of type I is also 514 residues, although recently higher molecular
alternative splice forms have been discovered in the retina.
Knockout of the IMPDH2 gene in mice is embryonic lethal,
indicating that type I isoform cannot replace the function of
Figure 2.
the type II.7 Deletion of the type I gene has no apparent effect
on mouse development but decreases activation of lymphocytes.8
With the exceptions mentioned above, the type I isoform is
expressed in normal cells at a lower concentration than type
II.8 The type II enzyme is up-regulated in neoplastic cells while
the level of expression of type I is not affected. For example,
the level of IMPDH2 in myelogenous leukemia is considerably
higher (15-42 fold) as compared to normal leukocytes.10
Induction of both isoforms has been observed in normal
replicating lymphocytes.11 It was discovered very recently that
mycophenolic acid (MPA) showed inhibition of endothelial cell
proliferation in Vitro and inhibition of tumor-induced angio-
genesis in ViVo.12 These effects were credited to inhibition of
the type I isoform as proved by RNA interference knockdown
of its gene. It was therefore suggested that IMPDH1 might be
an attractive antiangiogenic drug target. In contrast to the type
II isoform, inhibition of type I would not lead to undesired
suppression of the patients’ immune system. Thus, selective
inhibitors of the type I or the type II isoform would be of interest
for studies of antiproliferative and/or antiangiogenic effects in
cancer, particularly in hematological cancers.
* Corresponding author. Tel. 612-625-7968, fax 612-625-8252, e-mail
panki001@umn.edu.
† University of Minnesota.
‡ Indiana University School of Medicine and Richard Roudebush
Veterans Affairs Medical Center.
§ Brandeis University.
a Abbreviations: MPA, mycophenolic acid; IMPDH, inosine monophos-
phate dehydrogenase; IMP, inosine monophosphate; XMP, xanthosine
monophosphate; GMP, guanosine monophospahte; CML, chronic myelog-
enous leukemia; BC, blast crisis; NMN, nicotinamide mononucleotide; TR,
tiazofurin; TAD, tiazofurin adenine dinucleotide; MAD, mycophenolic
adenine dinucleotide; NAD, nicotinamide adenine dinucleotide; ADH,
alcohol dehydrogenase; MDH, malate dehydrogenase; LDH, lactate dehy-
drogenase.
Four inhibitors of IMPDH are currently used clinically and
none show significant selectiVity against the IMPDH isoforms.
10.1021/jm070568j CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/24/2007

5744 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Chen et al.
Scheme 1a
a Reaction conditions: (i) P(O)Cl3, (EtO)3P(O), 0 °C overnight; (ii) DMF, rt, 20-24 h; (iii) Dowex 50 (H+ form), water, and then Dowex 50 (Na+ form);
(iv) Pd/C, MeOH or Lindlar catalyst, MeOH.
MPA (in the form of prodrug mycophenolic mofetil) and
mizoribine are used as immunosuppressants, ribavirin is a broad
spectrum antiviral agent, and tiazofurin is approved as an orphan
drug for treatment of patients in blast crisis of chronic
myelogenous leukemia (CML).13 Tiazofurin is not active and
requires an unusual metabolic activation. It is phosphorylated
by adenosine kinase to the tiazofurin-5′-monophosphate which
is coupled with ATP by NMN adenylyltransferase to give
tiazofurin adenine dinucleotide (TAD, Figure 1), an analogue
of NAD, in which tiazofurin replaces the nicotinamide riboside
moiety of NAD.14 TAD mimics the natural cofactor but cannot
participate in the hydride transfer, resulting in potent inhibition
of IMPDH (Ki ) 100 nM). MPA is one of the most potent and
specific inhibitors of IMPDH with higher activity against the
type II (Ki ) 7 nM) than the type I isoform (Ki ) 33 nM).15
MPA does not require metabolic activation and binds at the
cofactor (NAD) binding domain mimicking the interaction of
nicotinamide mononucleotide (NMN) moiety of NAD with the
enzyme.16 MPA’s therapeutic potential is limited by its undesir-
able metabolism. In humans, the compound is rapidly metabo-
lized into the inactive glucuronide, and as much as 90% of the
drug circulates in this inactive form.17,18 In recent years we
synthesized a number of NAD derivatives in which the
mycophenolic moiety replaces nicotinamide riboside (Figure 2)
to give mycophenolic adenine dinucleotides (MAD) which are
remarkably attractive as potential anti CML agents.19-26 C2-
MAD inhibited IMPDH at nanomolar levels (Ki ) 250-330
nM), was resistant to glucuronidation, and showed activity in
tiazofurin resistant cell line.22 It was more potent than tiazofurin
(8 fold) as a differentiation inducer of K562 cells and less toxic
and more efficient than tiazofurin in a murine model of CML.25
Several other IMPDH inhibitors are under development at major
pharmaceutical companies27-32 and in academia.24 Our group
is evaluating cofactor type inhibitors such as TAD and MAD
analogues as potential anticancer agents.22,24,26
In this paper we report the synthesis of TAD and MAD
analogues containing a substituent at C2 of the adenine ring as
probes for studies of binding requirements of IMPDH isoforms.
Our computer modeling based on crystal structures of type II
isoform33 in a complex with our TAD or MAD analogue
suggested that substitution at C2 position would create isozyme-
selective inhibitors. The TAD analogue containing an ethyl
group at C2 (38) was found to be 14-fold more selective against
the type I (Ki ) 1 nM) than type II isoform (Ki ) 14 nM). It is
also the most potent cofactor inhibitor of the IMPDH1 ever
reported.
Results and Discussion
Synthesis of TAD Analogues. A key intermediate, 2′,3′-O-
isopropylidene-2-iodoadenosine 134 was prepared from gua-
nosine35 and used for further conversion into the C2-substituted
adenine analogues 2, 4-7 (Scheme 1). Thus, palladium coupling
of 1 with phenylboronic acid afforded 2′,3′-O-isopropylidene-
2-phenyladenosine (2) in 60% yield. Isopropylidenation of
commercially available 2,6-diaminopurine riboside under condi-
tions similar to those reported by Matsuda for isopropylidenation
of 2-iodoadenosine34 afforded the protected nucleoside 3.
Palladium coupling of 1 with aniline gave the corresponding
2-phenylamino derivative 4. Treatment of 1 with benzylamine
or phenylethylamine in a sealed tube at 125 °C led to the
formation of 2-benzylamino- or 2-phenylethylamino derivatives
4 and 5 in high yield. Finally, coupling of 1 with ethynyltrim-

Isoforms of Inosine Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5745
Scheme 2a
a Reaction conditions: (i) pyridine, DIC, 2 or 7, H2O/Et3N; (ii) 35 or 36, H2, Pd/C, and then Dowex 50 (H+ form).
Table 1. Inhibition of IMPDH Type I and Type II, Alcohol, Malate, and Lactic Dehydrogenase, as Well as Inhibition of K562 Cell Proliferation, by
TAD and TAD Analogues, as well as by MPA and MAD Analogues
IMPDH type I
IMPDH type II
ADH
MDH
LDH
K562 cell proliferation
inhibitors
Ki (nM)
Ki (nM)
IC50 (µM)
IC50 (µM)
IC50 (µΜ)
IC50 (µΜ)
TAD
110
110
12.4a
25, X ) I
19 ( 2
20 ( 6
7 ( 9
84 ( 8
143 ( 22
27 ( 3
153 ( 9
169 ( 34
255 ( 28
49 ( 2
94 ( 6
14 ( 2
7
ND
ND
>50
>50
ND
>50
>50
>50
>50
30.0
12.7
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
26, X ) Ph
27, X ) NH2
28, X ) NHPh
29, X ) NHCH2Ph
30, X ) NHCH2CH2Ph
31, X ) CtCH
32, X ) CHdCH2
33, X ) CH2CH3
MPA
45 ( 5
45 ( 4
73 ( 5
10 ( 2
96 ( 39
1 ( 4
ND
>50
39.0
>50
33
7.7
5.7
1.1
1.0
C2-MAD
37, MAD; X ) Ph
38, MAD; X ) CH2CH3
330
66 ( 16
16 ( 4.4
250
108 ( 19
38 ( 15
>50
>50
14.3
>50
>50
>50
a This IC50 value is based on TR, which is converted into TAD through enzymatic reactions.
ethylsilane afforded the 2′-3′-O-isopropylidene-2-ethynylad-
enosine (7). The reaction conditions for preparation of 4, 5, and
7 were similar to those reported for the corresponding unpro-
tected nucleosides.36,37
their methylenebis(phosphonate) linkers. We synthesized two
MAD analogues 37 and 38 (Scheme 2) containing a phenyl and
an ethyl group, respectively, based on the fact that a TAD
analogue with either of these two substituents (Table 1) showed
highly potent inhibition of isoforms of IMPDH. We used our
earlier reported C2-mycophenolic bis(phosphonate) 3422 for a
DIC-mediated coupling with nucleosides 2 and 7 to give
intermediate 35 and 36, respectively. Hydrogenation (Pd/C) of
the protected intermediate 35 (X ) Ph) followed by treatment
with Dowex 50 (H+ and then Na+ form) resulted in removal of
the 7-benzyl group and deisopropylidenation to give MAD
analogue 37. A similar treatment of 36 (X ) CtCH) afforded
analogue 38.
Molecular Modeling. We modeled 37 and 38 using the X-ray
structure of C2-MAD bound to type II IMPDH. As expected,
the 2-substituents packed against the CG2 atom of Thr45 (Figure
3). Because of the close proximity of the Thr45 side chain, the
phenyl group was not coplanar with the adenine ring but was
30° out of plane. A QM energy scan of this torsion angle showed
that deviation from planarity by 45° carries an energy penalty
of 1 kcal/mol, but a deviation of 30° carries only a minimal
energy penalty of ∼0.3 kcal/mol, and therefore the adjustment
observed in the docking is not significantly unfavorable. In both
cases, the modeling suggests the hydrophobic C2 substituents
may be beneficial because they are sterically of reasonable size
and they prefer interaction with the hydrophobic portion of
Thr45 over interaction with the solvent.
Compounds 1-7 were phosphorylated using Yoshikawa’s
procedure38 to give the corresponding 5′-monophosphates 8-14
in moderate to good yield. Reaction of tiazofurin-5′-monophos-
hate imidazolide (15)39 with monophosphates 8-14 afforded
the corresponding isopropylidene-protected dinucleotides 16-
22. Under mild hydrogenolytic conditions (Pd/C, MeOH),
compound 22 was converted into a mixture of 2-ethenyl 23 and
2-ethyl 24-substituted adenosines. These were separated by
HPLC and deprotected to give TAD analogues 32 and 33,
respectively.
Synthesis of MAD Analogues. Even though TAD analogues
showed highly potent inhibition of human IMPDH enzymes
(Table 1), such pyrophosphate-linked molecules are readily
cleaved by cellular enzymes such as pyrophosphatases and
phosphodiesterases. Therefore, we chose to synthesize MAD
analogues in which two moieties are connected by a methyl-
enebis(phosphonate) linker. Previous studies by us and other
groups have clearly demonstrated that methylenebis(phospho-
nate)-linked TAD and MAD analogues are remarkably stable
either in the presence of phosphodiesterases or in serums. They
also exhibited potent antiproliferative activities against human
cancer cell lines, indicating that these TAD and MAD analogues
can penetrate the cell membrane in spite of the ionic nature of

5746 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Chen et al.
specific modification of the adenine ring of TAD or MAD
affects binding affinity in the similar manner. Indeed, substitu-
tion at the C2 position with ethyl or phenyl groups afforded
more potent compounds in both TAD and MAD series. The
MAD analogue 38 was found to be 20-fold more potent inhibitor
of type I and 6-fold more potent inhibitor of the type II isoform
than parent C2-MAD. As expected, MAD-38 did not inhibit
other cellular dehydrogenases up to 50 µM, showing a 3.1 ×
103 selectivity index. It is also a 4-fold more potent inhibitor
of proliferation of K562 cells and serves as a promising lead
compound for our future studies of MAD analogues as potential
therapeutic agents against CML.
Experimental Section
General Methods. All commercial reagents (Sigma-Aldrich,
Acros) were used as provided unless otherwise indicated. An
anhydrous solvent dispensing system (J. C. Meyer) using two
packed columns of neutral alumina was used for drying THF, Et2O,
and CH2Cl2, while two packed columns of molecular sieves were
used to dry DMF. Solvents were dispensed under argon. Flash
chromatography was performed with Ultra Pure silica gel (Silicycle)
with the indicated solvent system. Analytical HPLC was performed
on a Varian Microsorb column (C18, 5 µm, 4.6 × 250 mm) with
a flow rate of 0.5 mL/min while preparative HPLC was performed
on a Varian Dynamax column (C18, 8 µm, 41.4 × 250 mm) with
a flow rate of 40 mL/min. An isocratic or linear gradient of 0.04
M Et3N H2CO3 (TEAB) and aqueous MeCN (70%) was used.
Nuclear magnetic resonance spectra were recorded on a Varian 300
or 600 MHz with Me4Si, DDS, or signals from residual solvent as
Figure 3. Compound 37 docked to IMPDH2 (PDB entry 1NF7).
Residues Thr45, His253, and Phe282 of IMPDH2 are shown in green,
with a transparent green surface. Docking shows that both ethyl atoms
of 38 also pack against Thr45, whose hydroxyl is indicated by the arrow.
IMPDH1 (PDB entry 1JCN) is superimposed with the corresponding
residues Ile45, Arg253, and Tyr282 colored by atom and labeled in
parentheses. A red mesh surface for the IMPDH1 residues emphasizes
the increased hydrophobic surface area of IMPDH1 near the C2-
substituent.
Biological Evaluation and Conclusions
All new compounds reported herein were evaluated as
inhibitors of the two isoforms of human IMPDH. The obtained
Ki values (nM) are presented in the first two columns of Table
1. All these compounds were also tested against three other
cellular dehydrogenases, namely horse liver alcohol dehydro-
genase (ADH), porcine heart malate dehydrogenase (MDH), and
bovine heart lactate dehydrogenase (LDH). The corresponding
IC50 values are provided (in µM) in Table 1. Finally, inhibition
of proliferation of human leukemic K562 cells by metabolically
stable compounds was also examined. All modifications at the
2 position of adenine ring resulted in more potent inhibitors of
IMPDH1 than the parent compounds. The majority (25, 27, 31-
33, 37, 38), but not all, also showed higher affinity for the type
II isoform. In general, the inhibitory activity against the type I
enzyme was higher than that against the type II enzyme. The
two most potent inhibitors of IMPDH, compounds 33 and 38
in TAD and MAD series, respectively, were found to be as
potent as MPA, one of the most potent inhibitors of IMPDH.
As expected, compounds reported herein were much less
effective (3-4 orders of magnitude) as inhibitors of three other
cellular dehydrogenases. We have previously reported that
C-NAD (5-â-D-ribofuranosylnicotinamide),40 an isosteric ana-
logue of NAD, binds ADH (Ki ) 4 nM) 3-5 orders of
magnitude more potently than other cellular dehydrogenases
1
the internal standard for H or 13C, and external H3PO4 for 31P.
Chemical shifts are reported in ppm, and signals are described as
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), brs
(broad singlet), and dd (double doublet). Values given for coupling
constants are first order. High-resolution mass spectra were recorded
on an Agilent TOF II TOF/MS instrument equipped with either an
ESI or APCI interface.
Preparation of Nucleosides 2, 4-7. 2′,3′-O-Isopropylidene-
2-phenyladenosine (2). To 2′,3′-O-isopropylidene-2-iodoadenosine
(1, 109 mg, 0.25 mmol) in a sealed tube were added phenylboronic
acid (47 mg, 0.38 mmol), palladium acetate (6.0 mg, 0.027 mmol),
(2-biphenyl)dicyclohexylphosphine (13.1 mg, 0.037 mmol), and
potassium phosphate (106 mg, 0.50 mmol). After addition of
anhydrous dioxane (1.5 mL), the sealed tube was evacuated and
then flushed and filled with nitrogen. The tube was sealed and
heated at 100 °C for 24 h. After cooling to rt, the mixture was
diluted with EtOAc (10 mL) and filtered through a pad of Celite.
The filtrate was concentrated, and the resulting residue was purified
on a Chromatotron (1 mm, 0-6% MeOH/CH2Cl2) to give 2′,3′-
O-isopropylidene-2-phenyladenosine (2) as a pale solid (57.4 mg,
1
60%). H NMR (CD3OD, 300 MHz) δ 8.40-8.29 (m, 2H), 8.26
(s, 1H), 7.48-7.37 (m, 3H), 6.23 (d, J ) 2.7 Hz, 1H), 5.51 (dd, J
) 6.3, 3.0 Hz, 1H), 5.13 (dd, J ) 6.2, 2.8 Hz, 1H), 4.33 (ddd, J )
4.6, 4.6, 2.9 Hz, 1H), 3.75 (dd, J ) 11.8, 4.4 Hz, 1H), 3.69 (dd, J
) 11.8, 5.2 Hz, 1H), 1.62 (s, 3H), 1.40 (s, 3H). HRMS calcd for
C19H22N5O4 384.1671 (M + H)+, found 384.1665.
such as malate, lactate, and glutamate dehydrogenase.41-43
A
number of examples of specific or highly selective NAD-like
molecules44,45 or compounds bound at the NAD pocket could
be found in literature.46,47 Collectively, it is reasonable to believe
that an NAD analogue can be designed to selectively inhibit a
particular NAD-dependent enzyme, regardless of the conserva-
tion of the cofactor binding domain of NAD dependent enzymes.
In this paper we demonstrated that TAD analogues could be
used as models for probing binding requirements of MAD
analogues for type I and type II isoforms of IMPDH. TAD
analogues as pyrophosphates are more readily accessible by
chemical synthesis than MAD analogues; however, they are
metabolically unstable and cannot be used as potential drugs.
The adenine ring of TAD and MAD analogues is positioned at
the same place at the AMP subdomain of isoforms. Therefore,
2′,3′-O-Isopropylidene-2-phenylaminoadenosine (4). A similar
reaction of 1 (325 mg, 0.75 mmol) with palladium acetate (17 mg,
0.075 mmol), rac-BINAP (70 mg, 0.11 mmol), cesium carbonate
(370 mg, 1.1 mmol), and aniline (103 µL, 1.1 mmol) in anhydrous
1,4-dioxane (2 mL) in a sealed tube at 100 °C for 18 h afforded
after filtration and evaporation a residue, which was purified by
flash chromatography with CH2Cl2/MeOH (2-6%) as the eluent
1
to give 4 (53 mg, 53%) as a pale solid. H NMR (CD3OD, 300
MHz) δ 8.01 (s, 1H), 7.67 (d, J ) 8.1 Hz, 2H), 7.26 (t, J ) 8.0
Hz, 2H), 6.94 (t, J ) 7.5 Hz, 1H), 6.08 (d, J ) 3.0 Hz, 1H), 5.44
(dd, J ) 6.2, 2.8 Hz, 1H), 4.96 (dd, J ) 6.2, 2.8 Hz, 1H), 4.32-
4.23 (m, 1H), 3.70-3.58 (m, 2H), 1.60 (s, 3H), 1.39 (s, 3H). HRMS
calcd for C19H23N6O4 399.1780 (M + H)+, found 399.1774.
2′,3′-O-Isopropylidene-2-benzylaminoadenosine (5). A solution
of 1 (167 mg, 0.39 mmol) and benzylamine (0.43 mL, 3.9 mmol)

Isoforms of Inosine Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5747
in 2-methoxyethanol (0.50 mL) was heated in a sealed tube at
125 °C for 24 h. After cooling to rt, the mixture was directly purified
on a Chromatotron [1 mm, CH2Cl2/MeOH (0-6%)] to give 2′,3′-
O-isopropylidene-2-benzylaminoadenosine (5) as a tan solid (136
ine with 2,2-dimethoxypropane (3.0 equiv) in acetone in the
presence of perchloric acid (70% solution, 5.5 equiv) afforded 2′,3′-
O-isopropylidene-2-aminoadenosine (3). Compound 3 (323 mg,
1.00 mmol) was phosphorylated as described above to give
1
1
mg, 85%). H NMR (CD3OD, 300 MHz) δ 7.86 (d, J ) 0.6 Hz,
mononucleotide 10 (303 mg, 60%, Et3N salt) as a white solid. H
1H), 7.40-7.14 (m, 5H), 5.96 (d, J ) 2.7 Hz, 1H), 5.26 (dd, J )
6.0, 3.0 Hz, 1H), 4.86-4.78 (m, 1H), 4.62 (d, J ) 15.6 Hz, 1H),
4.53 (d, J ) 15.6 Hz, 1H), 4.24-4.16 (m, 1H), 3.84 (s, 1H), 3.67-
3.52 (m, 2H), 1.54 (s, 3H), 1.27 (s, 3H). HRMS calcd for
C20H25N6O4 413.1937 (M + H)+, found 413.1937.
2′,3′-O-Isopropylidene-2-phenethylaminoadenosine (6). In a
similar manner 1 (218 mg, 0.50 mmol) under treatment with
phenylethylamine (0.32 mL, 2.54 mmol) afforded 6 (62.2 mg, 74%)
as a pale solid. 1H NMR (CD3OD, 300 MHz) δ 7.90 (s, 1H), 7.32-
7.22 (m, 4H), 7.22-7.12 (m, 1H), 6.03 (d, J ) 3.0 Hz, 1H), 5.39
(dd, J ) 6.0, 3.0 Hz, 1H), 5.03 (dd, J ) 6.0, 2.7 Hz, 1H), 4.33-
4.27 (m, 1H), 3.79-3.42 (m, 4H), 3.36 (s, 1H), 2.90 (t, J ) 7.4
Hz, 2H), 1.59 (s, 3H), 1.36 (s, 3H). HRMS calcd for C21H27N6O4
427.2093 (M + H)+, found 427.2098.
NMR (D2O, 300 MHz) δ 8.11 (brs, 1H), 6.08 (d, J ) 3.0 Hz, 1H),
5.34 (dd, J ) 6.2, 2.8 Hz, 1H), 5.16 (dd, J ) 6.0, 2.1 Hz, 1H),
4.61 (pseudo brs, 1H), 4.12-3.98 (m, 2H), 3.18 (q, J ) 7.3 Hz,
(CH3CH2)3NH+), 1.63 (s, 3H), 1.42 (s, 3H), 1.26 (t, J ) 7.2 Hz,
(CH3CH2)3NH+). 31P NMR (D2O, 121 MHz) δ 1.06. HRMS calcd
for C13H18N6O7P 401.0974 (M - Et3N - H)-, found 401.0976.
2′,3′-O-Isopropylidene-2-phenylaminoadenosine 5′-Mono-
phosphate (11). Phosphorylation of nucleoside 4 (136 mg, 0.34
mmol) under the above conditions gave monophosphate 11 (87.4
1
mg, 44%) as a white solid. H NMR (CD3OD, 300 MHz) δ 8.12
(s, 1H), 7.77-7.66 (m, 2H), 7.30-7.18 (m, 2H), 6.97-6.86 (m,
1H), 6.12 (d, J ) 3.0 Hz, 1H), 5.41 (dd, J ) 6.0, 3.3 Hz, 1H), 5.09
(dd, J ) 6.3, 2.1 Hz, 1H), 4.49-4.40 (m, 1H), 4.09-3.90 (m, 2H),
3.00 (q, J ) 7.1 Hz, (CH3CH2)3NH+), 1.62 (s, 3H), 1.39 (s, 3H),
1.21 (t, J ) 7.2 Hz, (CH3CH2)3NH+). 31P NMR (CD3OD, 121 MHz)
δ 2.38. HRMS calcd for C19H22N6O7P 477.1293 (M - Et3N -
H)-, found 477.1297.
2′,3′-O-Isopropylidene-2-ethynyladenosine (7). A solution of
compound 1 (879 mg, 2.03 mmol), PdCl2(PPh3)2 (142 mg, 0.20
mmol), CuI (79 mg, 0.41 mmol), and triethylamine (0.60 mL, 4.30
mmol) in DMF (3 mL) in a sealed tube was evacuated and
backfilled with nitrogen five times. After addition of ethynyltri-
methylsilane (2.0 mL, 14.4 mmol), the resulting mixture was heated
at 80 °C for 18 h. The mixture was cooled to rt, diluted with toluene
(200 mL), and washed with water (2 × 50 mL). The aqueous layer
was extracted with Et2O (2 × 50 mL), and the combined organic
layer was stirred with Celite and activated carbon. After filtration,
the filtrate was concentrated and the residue was passed through a
silica gel column. The combined fractions was concentrated, and
the residue was dissolved in CH3CN (6 mL) and treated with TBAF
(645 mg, 2.04 mmol) for 1.5 h. The reaction mixture was then
concentrated, and the residue was purified by flash chromatography
with CH2Cl2/MeOH (0-6%) as the eluent to give 7 (518 mg, 77%)
2′,3′-O-Isopropylidene-2-benzylaminoadenosine 5′-Monophos-
phate (12). As above the reaction of nucleoside 5 (116 mg, 0.28
mmol) with P(O)Cl3 afforded nucleotide 12 (59.1 mg, 35%) as a
white powder. 1H NMR (CD3OD, 300 MHz) δ 7.96 (s, 1H), 7.42-
7.32 (m, 2H), 7.32-7.22 (m, 2H), 7.21-7.12 (m, 1H), 5.99 (d, J
) 2.7 Hz, 1H), 5.24 (dd, J ) 6.0, 3.0 Hz, 1H), 4.63 (d, J ) 15.3
Hz, 1H), 4.51 (d, J ) 15.3 Hz, 1H), 4.42-4.33 (m, 1H), 4.10-
3.90 (m, 2H), 3.10 (q, J ) 7.3 Hz, (CH3CH2)3NH+), 1.52 (s, 3H),
1.30-1.18 (m, isopropylidene methyl and (CH3CH2)3NH+). 31P
NMR (CD3OD, 121 MHz) δ 1.99. HRMS calcd for C20H24N6O7P
491.1449 (M - Et3N - H)-, found 491.1452.
2′,3′-O-Isopropylidene-2-phenethylaminoadenosine 5′-Mono-
phosphate (13). In a similar manner compound 6 (82.5 mg, 0.19
mmol) was converted into nucleotide 13 (49.5 mg, 42%, Et3N salt)
1H NMR (CD3OD, 300 MHz) δ 8.07 (brs, 1H), 7.32-7.21 (m,
4H), 7.21-7.10 (m, 1H), 7.21-7.12 (m, 1H), 6.08 (d, J ) 3.0 Hz,
1H), 5.40 (dd, J ) 6.0, 3.0 Hz, 1H), 5.11 (dd, J ) 5.8, 2.2 Hz,
1H), 4.48-4.38 (m, 1H), 4.04-3.92 (m, 2H), 3.64-3.50 (m, 2H),
2.96-2.78 (m, NHCH2CH2Ph and (CH3CH2)3NH+), 1.58 (s, 3H),
1.36 (s, 3H), 1.16 (t, J ) 7.4 Hz, (CH3CH2)3NH+). 31P NMR (CD3-
OD, 121 MHz) δ 2.99. HRMS calcd for C21H26N6O7P 505.1606
(M - Et3N - H)-, found 505.1612.
2′,3′-O-Isopropylidene-2-ethynyladenosine 5′-Monophosphate
(14). As above nucleoside 7 (389 mg, 1.17 mmol) was phospho-
rylated to give mononucleotide 14 (365.3 mg, 61%, Et3N salt)) as
off-white solid. 1H NMR (D2O, 600 MHz) δ 8.56 (s, 1H), 6.25 (d,
J ) 3.0 Hz, 1H), 5.48 (dd, J ) 5.4, 3.6 Hz, 1H), 5.23 (d, J ) 4.2
Hz, 1H), 4.68 (pseudo brs, 1H), 4.08-3.98 (m, 2H), 3.56 (s, 1H),
3.22 (q, J ) 7.2 Hz, (CH3CH2)3NH+), 1.72 (s, 3H), 1.50 (s, 3H),
1.30 (t, J ) 7.2 Hz, (CH3CH2)3NH+). 31P NMR (D2O, 243 MHz)
δ 3.02. HRMS calcd for C15H17N5O7P 410.0865 (M - Et3N -
H)-, found 410.0872.
Coupling of Mononucleotides (8-14) with 2′,3′-O-Isopropy-
lidenetiazofurin-5′-monophosphate Imidazolide (15). P1-(2′,3′-
O-Isopropylidenetiazofurin-5′-yl)-P2-(2′,3′-O-isopropylidene-2-
iodoadenosin-5′-yl) Pyrophosphate (16). Tiazofurin 5′-
phosphoimidazolide (15, 60.6 mg, 0.099 mmol) and nucleotide 8
(27.6 mg, 0.057 mmol) were dissolved in DMF-d7 (0.9 mL). The
resulting mixture was kept at rt, and the progress of reaction was
monitored by 31P NMR. After 8 days, the mixture was lyophilized.
The residue was dissolved in a TEAB solution, and the resulting
solution was purified by RP-HPLC to give pyrophosphate 16 (14.5
mg, 24%, Et3N salt) as a white solid. 1H NMR (D2O, 300 MHz) δ
8.29 (brs, 1H), 8.04 (s, 1H), 6.13 (d, J ) 3.3 Hz, 1H), 5.38 (dd, J
) 6.2, 3.4 Hz, 1H), 5.22 (dd, J ) 6.2, 2.0 Hz, 1H), 5.04 (d, J )
4.5 Hz, 1H), 4.88 (dd, J ) 6.2, 3.0 Hz, 1H), one signal buried
under D2O, 4.62 (pseudo brs, 1H), 4.38-4.30 (m, 1H), 4.22-4.05
(m, 2H), 3.96 (pseudo brs, 2H), 3.18 (q, J ) 7.3 Hz, (CH3CH2)3-
NH+), 1.66 (s, 3H), 1.61 (s, 3H), 1.44 (s, 3H), 1.37 (s, 3H), 1.26
1
as a pale solid. H NMR (CDCl3/CD3OD, 300 MHz) δ 8.02 (s,
1H), 5.90 (d, J ) 4.8 Hz, 1H), 5.12 (pseudo t, J ) 5.2 Hz, 1H),
5.03 (dd, J ) 5.7, 1.5 Hz, 1H), 4.48-4.40 (m, 1H), 3.90 (dd, J )
12.6, 2.1 Hz, 1H), 3.74 (dd, J ) 12.6, 2.4 Hz, 1H), 3.34 (s, 1H),
1.59 (s, 3H), 1.33 (s, 3H). HRMS calcd for C15H18N5O4 332.1353
(M + H)+, found 332.1371.
Preparation of Mononucleotides (8-14). 2′,3′-O-Isopropy-
lidene-2-iodoadenosine 5′-Monophosphate (8). To a solution of
compound 1 (217 mg, 0.50 mmol) in triethyl phosphate (1 mL) at
0 °C was added a solution of phosphorus oxychloride (0.10 mL,
0.11 mmol) in triethyl phosphate (1 mL, precooled with ice-water).
The resulting solution was kept in a refrigerator for 20 h. The
mixture was then added to a solution of TEAB (0.5 M, 20 mL,
cooled with ice-water). After stirring for 10 min, the mixture was
extracted with EtOAc (3 × 20 mL). The aqueous layer was
concentrated and lyophilized. The residue was dissolved in water
(5 mL) and purified by RP-HPLC to give 8 (165.8 mg, 54%, Et3N
1
salt) as a white solid. H NMR (D2O, 300 MHz) δ 8.46 (s, 1H),
6.18 (d, J ) 3.6 Hz, 1H), 5.36 (dd, J ) 5.8, 3.4 Hz, 1H), 5.18 (dd,
J ) 6.0, 1.8 Hz, 1H), 4.60 (pseudo brs, 1H), 4.00-3.82 (m, 2H),
3.08 (q, J ) 7.4 Hz, (CH3CH2)3NH+), 1.66 (s, 3H), 1.44 (s, 3H),
1.21 (t, J ) 7.4 Hz, (CH3CH2)3NH+). 31P NMR (CD3OD, 121 MHz)
δ 4.23. HRMS calcd for C13H16N5O7PI 511.9832 (M - Et3N -
H)-, found 511.9838.
2′,3′-O-Isopropylidene-2-phenyladenosine 5′-Monophosphate
(9). In a similar manner compound 2 (48.8 mg, 0.13 mmol) was
phosphorylated to give 9 (25.0 mg, 35%) as a white solid. 1H NMR
(CD3OD, 300 MHz) δ 8.41 (s, 1H), 8.39-8.31 (m, 2H), 7.50-
7.37 (m, 3H), 6.32 (d, J ) 2.7 Hz, 1H), 5.48 (dd, J ) 6.0, 3.0 Hz,
1H), 5.18 (dd, J ) 6.2, 2.2 Hz, 1H), 4.55-4.46 (m, 1H), 4.08-
3.98 (m, 2H), 3.09 (q, J ) 7.2 Hz, (CH3CH2)3NH+), 1.64 (s, 3H),
1.40 (s, 3H), 1.23 (t, J ) 7.2 Hz, (CH3CH2)3NH+). 31P NMR (CD3-
OD, 121 MHz) δ 1.89. HRMS calcd for C19H21N5O7P 462.1184
(M - Et3N - H)-, found 462.1173.
2′,3′-O-Isopropylidene-2-aminoadenosine 5′-Monophosphate
(10). Isopropylidenation of commercially available 2-aminoadenos-

5748 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Chen et al.
(t, J ) 7.4 Hz, (CH3CH2)3NH+). 31P NMR (D2O, 121 MHz) δ
-10.61. HRMS calcd for C25H31IN7O14P2S 874.0169 (M - Et3N
- H)-, found 874.0168.
pound 22 (227.3 mg, 42%)) was obtained as a white powder by
conversion of nucleotide 14 (295 mg, 0.55 mmol) as described
above. H NMR (D2O, 600 MHz) δ 8.46 (s, 1H), 8.07 (s, 1H),
1
P1-(2′,3′-O-Isopropylidenetiazofurin-5′-yl)-P2-(2′,3′-O-isopro-
pylidene-2-phenyladenosin-5′-yl) Pyrophosphate (17). In a similar
manner coupling of mononucleotide 9 (17.5 mg, 0.033 mmol) with
imidazolide 15 (23.1 mg, 0.041 mmol) afforded pyrophopshate 17
6.19 (d, J ) 3.6 Hz, 1H), 5.42 (dd, J ) 6.3, 3.6 Hz, 1H), 6.26 (dd,
J ) 6.0, 2.4 Hz, 1H), 5.06 (d, J ) 4.8 Hz, 1H), 4.91 (dd, J ) 6.3,
2.7 Hz, 1H), 4.88-4.84 (m, 1H), 4.78 (brs, 1H), 4.66 (brs, 1H),
4.36 (brd, J ) 3.6 Hz, 1H), 4.20 (ddd, J ) 11.4, 4.2, 4.2 Hz, 1H),
4.15 (ddd, J ) 11.4, 3.0, 3.0 Hz, 1H), 4.04-3.94 (m, 2H), 3.19 (q,
J ) 7.2 Hz, (CH3CH2)3NH+), 1.70 (s, 3H), 1.64 (s, 3H), 1.48 (s,
3H), 1.39 (s, 3H), 1.29 (t, J ) 7.2 Hz, (CH3CH2)3NH+). 31P NMR
(D2O, 243 MHz) δ 10.49 (d, J ) 20.4 Hz), 10.61 (d, J ) 20.9 Hz).
HRMS calcd for C27H32N7O14P2S 772.1197 (M - Et3N - H)-,
found 772.1157.
1
(10.6 mg, 31%) as a white powder. H NMR (D2O, 300 MHz) δ
8.28 (s, 1H), 7.98-7.88 (m, 2H), 7.84 (s, 1H), 7.50-7.36 (m, 3H),
6.26 (d, J ) 2.7 Hz, 1H), 5.41 (dd, J ) 6.0, 2.7 Hz, 1H), 5.26 (dd,
J ) 6.2, 2.6 Hz, 1H), 4.77-4.70 (m, 2H), 4.65-4.54 (m, 2H),
4.20-4.04 (m, 3H), 3.85 (pseudo brs, 2H), 3.17 (q, J ) 7.3 Hz,
(CH3CH2)3NH+), 1.67 (s, 3H), 1.55 (s, 3H), 1.46 (s, 3H), 1.29 (s,
3H), 1.25 (t, J ) 7.4 Hz, (CH3CH2)3NH+). 31P NMR (D2O, 121
MHz) δ -10.63. HRMS calcd for C31H36N7O14P2S 824.1516 (M
- Et3N - H)-, found 824.1519.
Deprotection of TAD Analogues 16-22. General Procedure.
To a solution of isopropylidene-protected pyrophosphate derivative
(0.030 mmol, Et3N form) in water (2 mL) was added Dowex 50
WX8-200 (H+ form, ca. 500 mg). The mixture was stirred for
15-20 h at rt and then passed through a column of Dowex 50
WX8-200 (Na+ form). After elution with water, UV active
fractions were collected and lyophilized to give deprotected
pyrophosphate (sodium salt) as a white powder in yields 35-96%.
P1-(Tiazofurin-5′-yl)-P2-(2-iodoadenosin-5′-yl) Pyrophosphate
P1-(2′,3′-O-Isopropylidenetiazofurin-5′-yl)-P2-(2′,3′-O-isopro-
pylidene-2-aminoadenosin-5′-yl) Pyrophosphate (18). Tiazofurin
5′-phosphoimidazolide (15, 50.2 mg, 0.10 mmol) and mononucle-
otide 10 (35.7 mg, 0.067 mmol) were coupled as described above
1
to give 18 (43.8 mg, 67%) as a white solid. H NMR (D2O, 300
MHz) δ 8.03 (s, 1H), 8.01 (s, 1H), 6.02 (d, J ) 3.6 Hz, 1H), 5.30
(dd, J ) 6.3, 3.6 Hz, 1H), 5.19 (dd, J ) 6.0, 2.4 Hz, 1H), 5.14-
5.08 (m, 1H), 4.90-4.86 (m, 2H), 4.58-4.50 (m, 1H), 4.38 (pseudo
brs, 1H), 4.13 (pseudo brs, 2H), 4.00 (pseudo brs, 2H), 2.71 (q, J
) 7.2 Hz, (CH3CH2)3NH+), 1.63 (s, 3H), 1.59 (s, 3H), 1.41 (s,
3H), 1.35 (s, 3H), 1.06 (t, J ) 7.2 Hz, (CH3CH2)3NH+). 31P NMR
(D2O, 121 MHz) δ -10.54. HRMS calcd for C25H33N8O14P2S
763.1312 (M - Et3N - H)-, found 763.1317.
1
(25). White powder (96%). H NMR (D2O, 300 MHz) δ 8.31 (s,
1H), 7.98 (s, 1H), 5.97 (d, J ) 5.7 Hz, 1H), 4.99 (d, J ) 4.8 Hz,
1H), 4.68 (pseudo t, J ) 5.2 Hz, 1H), 4.53-4.46 (m, 1H), 4.38
(pseudo brs, 1H), 4.30-4.02 (m, 7H). 31P NMR (D2O, 121 MHz)
δ -10.24. HRMS calcd for C19H23IN7O14P2S 793.9543 (M - H)-,
found 793.9546.
P1-(Tiazofurin-5′-yl)-P2-(2-phenyladenosin-5′-yl) Pyrophos-
1
P1-(2′,3′-O-Isopropylidenetiazofurin-5′-yl)-P2-(2′,3′-O-isopro-
pylidene-2-phenylaminoadenosin-5′-yl) Pyrophosphate (19). As
described above coupling of nucleotide 11 (68.1 mg, 0.13 mmol)
with 15 (91.7 mg, 0.16 mmol) gave pyrophosphate 19 (40.2 mg,
phate (26). White powder (60%). H NMR (D2O, 300 MHz) δ
8.37 (s, 1H), 8.04-7.94 (m, 2H), 7.79 (s, 1H), 7.56-7.42 (m, 3H),
6.15 (d, J ) 5.4 Hz, 1H), 4.87-4.83 (m, 1H), 4.77-4.72 (m, 1H),
4.57-4.50 (m, 1H), 4.42-3.90 (m, 8H). 31P NMR (D2O, 121 MHz)
δ -10.24. HRMS calcd for C25H28N7O14P2S 744.0890 (M - H)-,
found 744.0892.
1
30%) as a white powder. H NMR (D2O, 300 MHz) δ 7.92 (brs,
1H), 7.90 (s, 1H), 7.43 (d, J ) 8.1 Hz, 2H), 7.23 (t, J ) 7.6 Hz,
2H), 6.96 (t, J ) 7.2 Hz, 1H), 5.98 (d, J ) 2.1 Hz, 1H), 5.25 (dd,
J ) 5.8, 2.2 Hz, 1H), 4.99 (dd, J ) 6.0, 2.4 Hz, 1H), 4.77-4.68
(m, 2H), 4.52-4.42 (m, 1H), 4.26-4.16 (m, 1H), 4.04 (pseudo
brs, 2H), 3.88 (pseudo brs, 2H), 3.14 (q, J ) 7.3 Hz, (CH3CH2)3-
NH+), 1.60 (s, 3H), 1.52 (s, 3H), 1.39 (s, 3H), 1.28 (s, 3H), 1.23
(t, J ) 7.4 Hz, (CH3CH2)3NH+). 31P NMR (D2O, 121 MHz) δ
-10.62. HRMS calcd for C31H37N8O14P2S 839.1625 (M - Et3N
- H)-, found 839.1629.
P1-(Tiazofurin-5′-yl)-P2-(2-aminoadenosin-5′-yl) Pyrophos-
1
phate (27). White powder (64%). H NMR (D2O, 300 MHz) δ
8.05 (s, 1H), 7.96 (s, 1H), 5.86 (d, J ) 5.7 Hz, 1H), 5.03 (d, J )
4.5 Hz, 1H), 4.65 (pseudo t, J ) 5.6 Hz, 1H), 4.44 (dd, J ) 4.8,
3.9 Hz, 1H), 4.31 (pseudo brs, 1H), 4.26-4.02 (m, 7H). 31P NMR
(D2O, 121 MHz) δ -10.27. HRMS calcd for C19H25N8O14P2S
683.0686 (M - H)-, found 683.0693.
P1-(Tiazofurin-5′-yl)-P2-(2-phenylaminoadenosin-5′-yl) Pyro-
phosphate (28). White powder (64%). 1H NMR (D2O, 300 MHz)
δ 8.04 (s, 1H), 7.87 (s, 1H), 7.49-7.41 (m, 2H), 7.34-7.24 (m,
2H), 7.06-6.97 (m, 1H), 5.90 (d, J ) 5.1 Hz, 1H), 4.93 (d, J )
5.4 Hz, 1H), 4.66 (pseudo t, J ) 5.2 Hz, 1H), 4.44 (pseudo t, J )
4.6 Hz, 1H), 4.33-3.93 (m, 8H). 31P NMR (D2O, 121 MHz) δ
-10.24. HRMS calcd for C25H29N8O14P2S 759.0999 (M - H)-,
found 759.1007.
P1-(2′,3′-O-Isopropylidenetiazofurin-5′-yl)-P2-(2′,3′-O-isopro-
pylidene-2-benzylaminoadenosin-5′-yl) Pyrophosphate (20). The
similar coupling of nucleotide 12 (35.1 mg, 0.066 mmol) with
imidazolide 15 (50.7 mg, 0.085 mmol) afforded pyrophosphate 20
1
(19.4 mg, 28%) as a white powder. H NMR (D2O, 300 MHz) δ
7.87 (s, 1H), 7.79 (s, 1H), 7.40-7.24 (m, 4H), 7.18 (t, J ) 6.9 Hz,
1H), 5.94 (d, J ) 2.4 Hz, 1H), 5.10-5.00 (m, 2H), 4.90-4.84 (m,
1H), 4.56-4.32 (m, 5H), 4.16-3.94 (m, 5H), 3.16 (q, J ) 7.2 Hz,
(CH3CH2)3NH+), 1.57 (s, 3H), 1.55 (s, 3H), 1.32 (s, 3H), 1.30-
1.18 (m, isopropylidene methyl and (CH3CH2)3NH+). 31P NMR
(D2O, 121 MHz) δ -10.57. HRMS calcd for C32H39N8O14P2S
853.1781 (M - Et3N - H)-, found 853.1787.
P1-(Tiazofurin-5′-yl)-P2-(2-benzylaminoadenosin-5′-yl) Pyro-
phosphate (29). White powder (40%). 1H NMR (D2O, 300 MHz)
δ 7.96 (s, 1H), 7.88 (s, 1H), 7.42-7.21 (m, 5H), 5.85 (d, J ) 5.7
Hz, 1H), 5.02 (d, J ) 3.3 Hz, 1H), 4.60 (pseudo t, J ) 5.2 Hz,
1H), 4.50 (pseudo brs, 2H), 4.38 (pseudo t, J ) 4.4 Hz, 1H), 4.31-
3.99 (m, 8H). 31P NMR (D2O, 121 MHz) δ -10.26. HRMS calcd
for C26H31N8O14P2S 773.1155 (M - H)-, found 773.1162.
P1-(Tiazofurin-5′-yl)-P2-(2-phenethylaminoadenosin-5′-yl) Py-
rophosphate (30). White powder (35%). 1H NMR (D2O, 300 MHz)
δ 7.98 (s, 1H), 7.91 (s, 1H), 7.37-7.14 (m, 5H), 5.87 (d, J ) 5.4
Hz, 1H), 5.03-4.98 (m, 1H), 4.68-4.60 (m, 1H), 4.47 (pseudo t,
J ) 4.4 Hz, 1H), 4.34-3.98 (m, 8H), 3.54 (t, J ) 6.8 Hz, 2H),
2.85 (t, J ) 6.8 Hz, 2H). 31P NMR (D2O, 121 MHz) δ -10.28.
HRMS calcd for C27H33N8O14P2S 787.1312 (M - H)-, found
787.1319.
P1-(2′,3′-O-Isopropylidenetiazofurin-5′-yl)-P2-(2′,3′-O-isopro-
pylidene-2-phenethylaminoadenosin-5′-yl) Pyrophosphate (21).
As above nucleotide 13 (36.7 mg. 0.069 mmol) was converted into
1
the corresponding pyrophosphate 21 (16.6 mg) in 22% yield. H
NMR (D2O, 300 MHz) δ 7.95 (s, 1H), 7.90 (s, 1H), 7.30-7.10
(m, 5H), 6.00 (s, 1H), 5.26-5.12 (m, 2H), 5.04 (d, J ) 4.8 Hz,
1H), 4.88-4.82 (m, 1H), 4.76-4.70 (m, 1H), 4.61 (pseudo brs,
1H), 4.37 (pseudo brs, 1H), 4.15 (pseudo brs, 2H), 3.98 (pseudo
brs, 2H), 3.40 (t, J ) 6.8 Hz, 2H), 3.16 (q, J ) 7.2 Hz, (CH3CH2)3-
NH+), 2.82-2.66 (m, 2H), 1.58 (s, 3H), 1.54 (s, 3H), 1.35 (s, 3H),
1.29 (s, 3H), 1.25 (t, J ) 7.4 Hz, (CH3CH2)3NH+). 31P NMR (D2O,
121 MHz) δ -10.40 (d, J ) 20.7 Hz), -10.81 (d, J ) 20.7 Hz).
HRMS calcd for C33H41N8O14P2S 867.1938 (M - Et3N - H)-,
found 867.1944.
P1-(Tiazofurin-5′-yl)-P2-(2-ethynyladenosin-5′-yl) Pyrophos-
1
phate (31). White powder (88%). H NMR (D2O, 600 MHz) δ
8.59 (s, 1H), 8.03 (s, 1H), 6.06 (d, J ) 6.0 Hz, 1H), 5.02 (d, J )
6.0 Hz, 1H), 4.73 (pseudo t, J ) 5.4 Hz, 1H), 4.53 (pseudo t, J )
4.2 Hz, 1H), 4.42 (brd, J ) 1.8 Hz, 1H), 4.32-4.22 (m, 5H), 4.20-
4.14 (m, 1H), 4.14-4.08 (m, 1H), 3.78 (s, 1H). 31P NMR (D2O,
P1-(2′,3′-O-Isopropylidenetiazofurin-5′-yl)-P2-(2′,3′-O-isopro-
pylidene-2-ethynyladenosin-5′-yl) Pyrophosphate (22). Com-

Isoforms of Inosine Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5749
(t, 2H, benzyl), 7.52 (d, 2H, benzyl), 8.66 (s, 1H, H8-adenine). 31
NMR (D2O): δ 16.82 (d, J ) 24.6 Hz), 17.50 (d, J ) 24.6 Hz).
LC-MS (ESI): m/z 899 (M - H), 901 (M + H).
121 MHz) δ -10.27. HRMS calcd for C21H24N7O14P2S 692.0582
(M - H)-, found 692.0521.
P
Hydrogenolysis and Deprotection of TAD Analogue 22. P1-
(Tiazofurin-5′-yl)-P2-(2-ethyladenosin-5′-yl) Pyrophosphate (33).
A solution of pyrophosphate 22 (54.5 mg, 0.056 mmol, Et3N salt)
in MeOH (3 mL) was hydrogenated (1 atm) in the presence of
10% Pd/C (10.2 mg) for 5 h. After the reaction was complete, the
reaction mixture was filtered through a pad of Celite, and the filtrate
was concentrated. The residue was dissolved in water (3 mL), and
Dowex 50WX8-200 (H+ form, ca. 850 mg) was added. The
mixture was stirred for 18 h at rt and filtered through a pad of
Celite. After the filtrate was lyophilized, the residue was dissolved
in water (2 mL) and then passed through a column of Dowex
50WX8-200 (Na+ form). After elution with water, UV active
fractions were collected and lyophilized to give 33 (35.4 mg, 86%
P1-[7-Hydroxy-6-(hydroxyethyl)-5-methoxy-4-methylphthta-
lan-1-one-2-yl]-P2-(2-phenyladenosin-5′-yl)methylenebis(phos-
phonate) (37). Compound 35 (53 mg, 0.05 mmol, as the triethy-
lammonium salt) was dissolved in MeOH (10 mL) containing a
catalytic amount of Pd(OH)2 (20% Pd/C, 5-10 mg). The mixture
was stirred at room temperature under H2 for 36 h. Methanol was
removed in vacuo, and the residue was mixed with small amount
of Dowex 50WX8-200 (H+) overnight. This mixture was directly
pass a column of Dowex 50WX8-200 (Na+) to give the final
product as sodium salt (15 mg, 42%). 1H NMR (D2O): δ 1.88 (s,
3H, CH3), 2.23 (t, 2H, PCH2-P, J ) 20 Hz), 2.79 (m, 2H), 3.71
(s, 3H, OCH3), 3.98 (m, 1H), 4.21-4.30 (m, 2H), 4.39 (d, 1H, J )
3.0 Hz), 4.61 (t,1H, J ) 4.8 Hz), 4.78 (t, 1H, J ) 4.8 Hz), 4.93 (d,
2H, J ) 1.6 Hz), 5.21 (d, 1H, J ) 7.8 Hz), 6.17 (d, 1H, J ) 4.8
Hz, 1′-Η), 7.58 (m, 3H, phenyl), 8.10 (d, 2H, benzyl), 8.43 (s, 1H,
H8-adenine). 31P NMR (D2O): δ 18.0 (d, J ) 27.6 Hz), 18.80 (d,
J ) 27.6 Hz). HRMS (ESI-): calcd for C29H32N5O13P2 (M - H)
720.1471, found 720.1418.
P1-[7-Hydroxy-6-(hydroxyethyl)-5-methoxy-4-methylphthta-
lan-1-one-2-yl]-P2-(2-ethyladenosin-5′-yl)methylenebis(phospho-
nate) (38). Compound 36 (50 mg, 0.05 mmol, as the triethylam-
monium salt) was dissolved in MeOH (10 mL) and deprotected as
described above to give 38 as a sodium salt (15 mg, 42%). 1H NMR
(D2O): δ 1.29 (t, 3H, J ) 7.2, 7.8 Hz, CH3), 2.13 (s, 3H, CH3),
2.24 (t, 2H, PCH2-P, J ) 19.8 Hz), 2.74 (dd, 2H, CH2 J ) 7.2,
7.8 Hz), 2.86 (m, 2H), 3.75 (s, 3H, OCH3), 4.03 (m, 1H), 4.23(m,
1H), 4.28 (m, 1H), 4.36 (brs, 1H), 4.53 (t,1H, J ) 4.8 Hz), 4.64 (t,
1H, J ) 4.8 Hz), 5.02 (s, 2H), 5.23 (s, 1H), 6.03 (d, 1H, J ) 4.8
Hz, 1′-Η), 8.36 (s, 1H, H8-adenine). 31P NMR (D2O): δ 18.16 (d,
J ) 29 Hz), 18.55 (d, J ) 29 Hz). HRMS (ESI-): calcd for
C25H32N5O13P2 (M - H) 672.1471, found 672.1398.
1
for two steps) as the white powder sodium salt. H NMR (D2O,
600 MHz) δ 8.53 (s, 1H), 8.03 (s, 1H), 6.15 (d, J ) 5.4 Hz, 1H),
5.07 (d, J ) 5.4 Hz, 1H), 4.57 (pseudo t, J ) 4.5 Hz, 1H), 4.42
(brd, J ) 3.0 Hz, 1H), 4.36-4.24 (m, 6H), 4.24-4.10 (m, 2H),
2.93 (q, J ) 7.2 Hz, 1H), 2.90 (q, J ) 7.8 Hz, 1H), 1.36 (t, J ) 7.5
Hz, 3H). 31P NMR (D2O, 121 MHz) δ -10.28. HRMS calcd for
C21H28N7O14P2S 696.0895 (M - H)-, found 696.0844. Alterna-
tively, the similar hydrogenation of 22 (57.0 mg, 0.058 mmol) in
the presence of Lindlar catalyst (10.5 mg) afforded two compounds
33 (14.8 mg, 34%) and 32 (8.8 mg, 20%). They were separated on
the RP-HPLC column and converted into their corresponding
sodium salts by passing through a column of Dowex 50WX8-
200 (Na+ form). The pyrophosphate 32 was obtained as a white
1
powder. H NMR (D2O, 600 MHz) δ 8.54 (s, 1H), 8.00 (s, 1H),
6.71 (dd, J ) 17.4, 10.8 Hz, 1H), 6.59 (d, J ) 17.4 Hz, 1H), 6.15
(d, J ) 5.4 Hz, 1H), 5.99 (d, J ) 10.8 Hz, 1H), 5.03 (d, J ) 5.4
Hz, 1H), 4.56 (pseudo t, J ) 4.5 Hz, 1H), 4.42 (brd, J ) 3.0 Hz,
1H), 4.35-4.23 (m, 6H), 4.22-4.17 (m, 1H), 4.15-4.09 (m, 1H).
31P NMR (D2O, 121 MHz) δ -10.28. HRMS calcd for C21H26N7O14-
P2S 694.0739 (M - H)-, found 694.0703.
Molecular Modeling. The X-ray structure of human IMPDH2
with C2-MAD bound (PDB entry 1NF7) was used for modeling in
MacroModel48 with the MMFFs force field and GB/SA solvation
model. The structure was first minimized with only hydrogens free
to move and then with heavy atoms restrained by a 1.0 kcal/mol-
Å2 force. Residues with no atoms within 16 Å of the ligand were
deleted, and truncated portions of the backbone were methyl-capped.
Modeling was validated by docking mycophenolic acid (MPA) into
the structure using the MacroModel MCMM and LMCS methods,
resulting in a 1.2 Å ligand rmsd relative to the X-ray structure of
MPA bound to Chinese hamster IMPDH (PDB entry 1JR1). Simple
models for the binding of 37 and 38 were generated by modifying
the 2 position of the minimized C2-MAD and carrying out
conformational sampling on the adenosine portion of the ligand
(atoms up to the 5′ OH of adenosine were free to move, and the
remainder were not allowed to move). The glycosidic bond and
the C2-phenyl or C2-ethyl torsion were selected for sampling. The
simulations had converged by 1000 search steps. A relaxed torsion
energy scan around the phenyl group/adenine torsion angle was
calculated at 15° increments at the B3LYP/6-311G(d,p) level in
Jaguar 6.5 using the SCRF model for water solvation and was
performed on a model compound consisting of adenine, the
2-substituent, and an N9 methyl cap.
IMPDH Type I and Type II. Human IMPDH type I and type
II was expressed and purified as previously described.49,50 Inhibition
assays were performed as previously described.51 Briefly, assays
were set up in duplicate using two different concentrations of
IMPDH type I (87 and 155 nM) and type II (33 and 66 nM) and
varying concentrations of inhibitor. IMPDH and inhibitors were
added to 1 mL of reaction buffer (50 mM Tris, pH 8.0, 100 mM
KCL, 1 mM DTT, 100 µM IMP, 100 µM NAD) at 25 °C and mixed
gently while the production of NADH was monitored by following
changes in absorbance at 340 nm on a Hitachi U-2000 spectro-
photometer. Steady-state velocities were used to determine Ki (app)
values by fitting the velocities vs inhibitor concentration to a simple
binding model with Dynafit.52
Synthesis of C2-MAD Analogues. P1-(2-Phenyladenosin-5′-
yl)-P2-[7-O-benzyl-6-(2-hydroxyethyl)-5-methoxy-4-meth-
ylphthalan-1-one-2-yl]methylenebis(phosphonate) (35). To a
solution of 34 (338 mg, 0.50 mmol) in anhydrous pyridine (5 mL)
was added DIC (310 µL, 2 mmol), and the mixture was left
overnight until the intermediate was formed (multisignal resonances
in 31P NMR). Compound 2 (230 mg, 0.6 mmol) was then added,
and the reaction mixture was heated at 55-65 °C for 60 h. At that
time, the 31P NMR of the reaction mixture showed two broad signals
at 8 and 25 ppm. Then, a mixture of water (9 mL) and Et3N (1
mL) was added, and the reaction mixture was kept at 55-65 °C
for 27 h. After removal of the solvent, the residue was dissolved
in MeOH and purified on HPLC (50% to 100% buffer B in 20
min, gradient). Compound 35 (92 mg, 22%) obtained as the
triethylammonium salt. 1H NMR (D2O): δ 1.25 (t, CH3 from Et3N),
1.41 and 1.64 (two s, 3H each, isopropylidene), 2.14 (s, 3H, CH3),
2.96 (m, 2H, PCH2-P-), 3.02 (m, CH2- from Et3N), 3.80 (m,
OCH3), 4.02 (m, 2H), 4.15 (m, 2H), 4.46 (brs, 1H), 5.22 (s, 4H),
5.23 (m, 1H), 5.45 (m, 1H), 6.31 (d, 1H, J ) 3.0 Hz, 1′-Η), 7.31
(t, 1H, benzyl), 7.35 (t, 2H, benzyl), 7.43 (m, 3H, phenyl), 7.54 (d,
2H, benzyl), 8.38 (d, 2H, phenyl), 8.54 (s, 1H, H8-adenine). 31P
NMR (D2O): δ 16.58 (d, J ) 24.0 Hz), 17.46 (d, J ) 24.0 Hz).
LC-MS (ESI): m/z 850 (M - H).
P1-[2′,3′-O-Isopropylidene(2-ethynyladenosin-5′-yl)- P2-[7-O-
benzyl-6-(2-hydroxyethyl)-5-methoxy-4-methylphthalan-1-one-
2-yl]methylenebis(phosphonate) (36). In a similar manner 34 (338
mg, 0.50 mmol) was converted into 36 (112 mg, 25%, triethylam-
1
monium salt). H NMR (D2O): δ 1.25 (t, 9H, Et3N adduct on
ethynyl formed a 2-vinyl salt), 1.35 (t, 9H, Et3N), 1.38 and 1.59
(two s, 3H each, isopropylidene), 2.17 (t, 2H, PCH2-P, J ) 18.6
Hz), 2.19 (s, 3H, CH3), 3.02 (m, 8H, CH2 and Et3N), 3.85 (s, 3H,
OCH3), 4.02 (m, 8H, OCH2 and Et3N adduct on ethynyl formed a
2-vinyl salt), 4.08 (m, 2H), 4.16 (brs, 2H), 4.48 (brs, 1H), 5.21 (s,
2H), 5.23 (m, 1H), 5.24 (s, 2H), 5.31 (m, 1H), 6.05 (d, 1H, J )
10.6 Hz, 2-vinyl Et3N salt), 6.14 (d, 1H, J ) 2.4 Hz, 1′-Η), 6.80
(d, 1H, J ) 10.6 Hz, 2-vinyl Et3N salt), 7.29 (t, 1H, benzyl), 7.34

5750 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Chen et al.
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Cellular Dehydrogenases: Horse Liver Alcohol Dehydroge-
nase (ADH) Malate (MAD) and Lactate Dehydrogenase (LAD).
Inhibition studies were modified from previously described continu-
ous assays (Goldstein 1994). Enzymes and substrates were pur-
chased from Sigma. Enzymes were diluted in 100 mM sodium
phosphate buffer pH 7.8 to give a final concentration of 50 µU/µL
as determined by monitoring the generation of NADH at A360 under
the assay conditions used. For ADH reactions, 180 mM EtOH and
100 µM NAD were used as substrates. LDH and MDH reactions
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400 µM NAD. Enzymes (1 µL) were added to 100 µL of reaction
buffer (enzyme specific substrates in 100 mM sodium phosphate
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Cytotoxicity of MAD Analogues 37 and 38 to Human
Myelogenous Leukemia K562 Cells. Logarithmically growing
K562 cells in RPMI medium supplemented with 10% fetal bovine
serum were plated in 96-well plates at a density of 2 × 103 cells/
0.1 mL and incubated at 37 °C in an atmosphere of air and 5%
CO2. Twenty-four hours later, various concentrations of the
compounds were added in 3 µL volume, mixed, and further
incubated for 72 h. At the end of the incubation period, 20 µL of
cellTiter 96 reagent (Promega, Madison, WI) containing tetrazolium
compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was added
and incubated at 37 °C in an atmosphere of air and 5% CO2, the
color developed was read at 490 nm using SpectraMax software in
a microplate spectrophotometer (Molecular Devices Corp., Sunny-
vale, CA), and the data were analyzed by GraphPad Prism 4
software (GraphPad Software, San Diego, CA) as described
earlier.53
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Acknowledgment. This research was supported by USDOD
Army grant W81XWH-05-01-0216 and by the Center for Drug
Design in the Academic Health Center of the University of
Minnesota. We thank Drs. Takashi Tsuji and Kunisuke Izawa
of Ajinomoto Co., Inc., Japan, for a gift of MPA.
Supporting Information Available: HPLC profiles of com-
pounds 25-33, 37, and 38. This material is available free of charge
via Internet at http://pubs.acs.org.
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