Novel methylenephosphophosphonate analogues of mycophenolic adenine dinucleotide. Inhibition of inosine monophosphate dehydrogenase

Article abstract of DOI:10.1021/jm060479r

Novel methylenephosphophosphonate analogues of mycophenolic adenine dinucleotide (MAD) have been prepared as potential inhibitors of IMP dehydrogenase. A coupling of the mycophenolic (hydroxymethyl)-phosphonate 6 with the phosphitylated adenosine analogue

Full text of DOI:10.1021/jm060479r

5018
J. Med. Chem. 2006, 49, 5018-5022
Novel Methylenephosphophosphonate Analogues of Mycophenolic Adenine Dinucleotide.
Inhibition of Inosine Monophosphate Dehydrogenase
Dominik Rejman,†,‡ Magda Olesiak,† Liqiang Chen,† Steven E. Patterson,† Daniel Wilson,† Hiramagalur N. Jayaram,§
Lizbeth Hedstrom,# and Krzysztof W. Pankiewicz*,†
Center for Drug Design, Academic Health Center, 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 Department of Biochemistry, MS009, Brandeis UniVersity, Waltham, Massachusetts 02454
ReceiVed April 24, 2006
Novel methylenephosphophosphonate analogues of mycophenolic adenine dinucleotide (MAD) have been
prepared as potential inhibitors of IMP dehydrogenase. A coupling of the mycophenolic (hydroxymethyl)-
phosphonate 6 with the phosphitylated adenosine analogue 11 followed by oxidation and deprotection afforded
the phosphophosphonate 8. A similar coupling between adenosine (hydroxymethyl)phosphonate 10 and
phosphitylated mycophenolic alcohol 5 gave the corresponding phosphophosphonate 13. Both 8 and 13 (Ki
) 20-87 nM) were found to be the most potent cofactor type inhibitors of IMP dehydrogenase.
Introduction
Nicotinamide adenine dinucleotide (NAD, Figure 1) serves
as a substrate in many biochemical processes, e.g., ribosylation
of proteins,1 cell signaling,2 DNA repair and recombination,
histone deacetylation (by the Sir2 family of NAD-dependent
deacetylases),3,4 and also plays a crucial role as a coenzyme in
numerous oxidation-reduction reactions. It was commonly
believed at one time that, since the NAD binding domain is
conserved among NAD-dependent enzymes, inhibitors that
mimic the cofactor would not bind selectively and therefore
would cause general toxicity. However, it is now evident that
some coenzyme analogues specifically inhibit a single NAD-
Figure 1.
dependent enzyme and therefore may be of interest as thera-
peutic agents.5-9 Some NAD mimics show potential as agents
for the treatment of cancer10,11 and infectious diseases12 or as
immunosuppressants.13
NAD-dependent IMPDHa emerged in recent years as a major
therapeutic target.14,15 IMPDH controls entry of purines into the
guanine nucleotide pool via salvage and de novo purine
synthesis pathways. It catalyzes oxidation of IMP to xanthosine
monophosphate, which is further converted into guanosine
monophosphate (GMP) by GMP synthase. Inhibition of IMPDH
leads to decreased GTP concentration, down-regulation of ras
and myc oncogenes,16 and impairment of signal transduction.17
Human IMPDH is well characterized, and because of its
importance in neoplasia, it is an exceptionally attractive target
for anticancer drug design.
Four inhibitors of the enzyme (Figure 2), ribavirin, mizoribine,
tiazofurin, and mycophenolic acid (in the form of the prodrug
mycophenolic mofetil) are currently used in the clinic. Ribavirin
Figure 2.
and mizoribine (in the form of their corresponding 5′-mono-
phosphates) bind to the enzyme at the substrate site. In contrast,
TR and MPA inhibit IMPDH at the cofactor site. As such TR
is a poor inhibitor of IMPDH and requires an unusual metabolic
Fax: 612-625-8252. E-mail: panki001@umn.edu.
* To whom correspondence should be addressed. Phone: 612-625-7968.
† University of Minnesota.
activation. In cells, TR is phosphorylated by adenosine kinase
and/or 5′-nucleotidase to the mononucleotide (TRMP), which
is further coupled with ATP by NMN-adenylyl transferase to
form tiazofurin adenine dinucleotide (TAD, Scheme 1). As an
analogue of NAD in which TR replaces the nicotinamide
riboside moiety of NAD, TAD mimics the natural cofactor but
cannot participate in hydride transfer resulting in potent inhibi-
tion of IMPDH. TAD inhibits the human enzyme with Ki )
‡ Current address: Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10
Prague 6, Czech Republic.
§ Indiana University School of Medicine and Richard Roudebush
Veterans Affairs Medical Center.
# Brandeis University.
a Abbreviations: MAD, mycophenolic adenine dinucleotide; IMPDH,
inosine monophosphate dehydrogenase; TR, tiazofurin; MPA, mycophenolic
acid; CML, chronic myelogenous leukemia.
10.1021/jm060479r CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/18/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 5019
Scheme 1a
a Reagents: (1) adenosine kinase; (2) NMN adenylyltransferase; (3)
TADase, phosphodiesterase; (4) phosphatases.
110 nM and at this concentration does not affect other cellular
dehydrogenases. The Km for NAD as an IMPDH substrate is
100 µM. The cofactor concentration usually required by other
cellular dehydrogenases is in the micromolar range.
MPA is a non-nucleoside and is a specific inhibitor of IMPDH
with a Ki of 7-33 nM.18 It is used as immunosuppressant but
is not active against cancer because of extensive glucuronidation
of the phenolic function.19,20 MPA binds at the nicotinamide
mononucleotide (NMN) subsite, leaving the adenosine mono-
phosphate (AMP) subsite empty.21,22 Thus, we synthesized MAD
(Figure 3) analogues in which the AMP was attached to the
MPA moiety through a methylenebis(phosphonate) linkage.23
24
The resulting analogues C2-MAD and C4-MAD (Figure 3)
Figure 3.
retained high specificity and potent inhibitory activity against
IMPDH (Ki ) 250-520 nM) regardless of number of atoms
between the phosphorus of adenosine phosphonate and the
aromatic ring of MPA. In contrast to MPA, the C2-MAD
analogue was resistant to glucuronidation in vitro under condi-
tions in which MPA was quickly and completely glucu-
ronidated.25
Scheme 2a
Encouraged by the above results, we evaluated the antileu-
kemic activity of C2-MAD in SCID mice xenotransplanted with
human K562 leukemia cells.11 We found that C2-MAD at 10,
30, or 60 mg/kg was well tolerated when administered daily
for 10 days by the intraperitoneal route. No deaths, no weight
loss, and no evidence of behavioral toxicity were observed. At
60 mg/kg, C2-MAD was as potent as TR at 200 mg/kg. TR is
approved by FDA as an orphan drug for treatment of patients
in blast crisis of CML. Since C2-MAD showed anticancer
activity in TR-resistant cell lines, our studies suggest that it may
be a promising drug candidate for treatment of CML.
In this paper we present the synthesis of new MAD analogues
in which the methylenebis(phosphonate) moiety (P-C-P) is
replaced with a methylenephosphophosphonate group (P-O-
C-P or P-C-O-P). Thus, the length of the linker of these
compounds is one atom longer than that of C2-MAD and one
atom shorter than that of C4-MAD.
a Reaction conditions: (a) pyridine, dimethoxytrityl chloride; (b) DAB-
CO, toluene, reflux, 8 h, then overnight at room temperature.
protected intermediates could be assembled and purified on a
silica gel column would be more efficient. The last step,
deprotection, would afford a pure final product or a product
that requires only HPLC purification. With this in mind we
designed a procedure depicted in Schemes 2 and 3, in which
new MAD analogues were obtained with an oxygen atom
inserted on either side of the methylene group as in 8 and 13.
We expected that insertion of the additional oxygen atom into
the C2-MAD linker would result in new analogues with binding
affinity similar to that of C2- and C4-MAD. Thus, dibenzyl-
(hydroxymethyl)phosphonate (1, Scheme 2) was prepared by
known reaction of paraformaldehyde with dibenzylphosphite and
then protected with dimethoxytrityl chloride to give 2. Depro-
tection with 1,4-diazabicyclo[2.2.2]octane (DABCO) in toluene
afforded monodebenzylated phosphonate 3 in good yield.
Coupling of 3 with a 7-O-benzyl protected C2-mycophenolic
alcohol 424 (Scheme 3) in the presence of 2,4,6-triisopropyl-
benzenesulfonyl chloride (TIPSCl) followed by detritylation with
acetic acid gave the corresponding benzyl protected C2-
mycophenolic (hydroxymethyl)phosphonate 6. Reaction of 6
Results and Discussion
1.Chemical Synthesis of Methylenephosphophosphonate
Analogues of C2-MAD. Our original synthesis of P1,P2
unsymmetrically disubstituted analogues of methylenebis(pho-
sphonic) acid is laborious and requires HPLC purification of a
monosubstituted methylenebis(phosphonate) intermediate and
the final disubstituted product.26 A synthetic route in which fully

5020 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Brief Articles
Scheme 3a
a Reaction conditions: (a) 3, 1-methylimidazole, TPSCl, CH3CN, overnight at room temperature, 80% acetic acid; (b) 4 or 9, [(iPr)2N]2POCH2CH2CN,
CH3CN; (c) 6 and 11, tetrazole, mCPBA; (d) 10 and 5, tetrazole, mCPBA; (e) H2, Pd/C, 8 M ethanolic methylamine, Dowex 50W (Na+ form).
Table 1. Inhibition of IMPDH by MPA, C2-MAD, and Its
pyrophosphates such as TAD, BAD, and their analogues.14
Methylenephosphophosphonate Analogues
These compounds are also superior to previously reported
inhibitors
KI (type I), nM
KI (type II), nM
Compound 13 was found to be as potent as an inhibitor of the
type I isoform of human IMPDH as MPA and is 10-fold more
potent than parent C2-MAD against both isoforms. It is the most
potent inhibitor among known cofactor type inhibitors of
IMPDH. Compound 8 showed approximately 4-fold better
inhibitory activity than its parent C2-MAD.
MPA
C2-MAD
C4-MAD
8
33 18
7 18
330 ( 9
520 ( 12
87 ( 18
20 ( 4
250 ( 11
380 ( 7
60 ( 6
37 ( 10
13
These results indicate that the characteristic pyrophosphate
(-P-O-P-) or bis(phosphonate) (-P-C-P-) geometry of
NAD analogues is not critical for preservation of the potent
inhibitory activity (or high affinity) of these compounds against
(toward) IMPDH. Thus, construction of other MAD analogues
with phosphorus atoms linked through more than one atom could
lead to even higher selectivity toward IMPDH. If other cellular
NAD-dependent enzymes are not inhibited significantly by new
P-O-C-P analogues, they should show much higher selectiv-
ity toward IMPDH than the parent MAD compounds. Vigorous
studies are underway in this laboratory to examine this
hypothesis and construct other inhibitors with two-atom linkers
replacing the pyrophosphate oxygen or bis(phosphonate) carbon.
with freshly prepared 2′,3′-O-benzoyl-N6-dibenzoyl-5′-cyano-
ethylphosphoroamidite (11) followed by oxidation with m-
chloroperbenzoic acid (mCPBA) afforded the fully protected
methylenephosphophosphonate analogue of C2-MAD 7 in 70%
yield after silica gel column purification. Hydrogenolysis (Pd/
C) of 7 with addition of 2 M triethylammonium bicarbonate
(TEAB) resulted in debenzylation and removal of the cyanoethyl
group. Further treatment of the partially deprotected derivative
with a solution of methylamine in EtOH afforded the desired
methylenephosphophosphonate analogue 8.
Similarly, TIPSCl coupling of 3 with the benzoyl protected
adenosine 9 followed by detritylation afforded the corresponding
2′,3′-O-dibenzoyl-N6-dibenzoyl-5′-O-(hydroxymethyl)phospho-
nate 10. Compound 10 was then reacted with freshly prepared
7-O-benzyl-C2-mycophenolic-cyanoethyl phosphoroamidite 5
to give the protected methylenephosphophosphonate 12, which
was deprotected in a similar manner as described for 7 to give
the desired C2-MAD analogue 13.
Thus, we developed a new and general procedure for the
synthesis of P1,P2 unsymmetrically substituted analogues of
methylenephosphophosphonates that is more efficient than our
original synthesis of the corresponding methylenebis(phospho-
nate)s. In addition, this new synthetic method can be used for
the efficient assembly of methylenephosphophosphonate ana-
logues of other cofactor analogues such as NAD, TAD, SAD,27
and BAD,28,29 and analogues of bis(phosphonic) acids used as
osteoporosis drugs.
2. Inhibition of Human IMPDH by Methylenephospho-
phosphonate Analogues of MAD. Our new methylenephos-
phophosphonate analogues of C2-MAD showed significantly
more potent inhibition of IMPDH (Table 1) than the parent
methylenebis(phosphonate)s of MAD reported by us earlier.24
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. HPLC
was performed on a Dynamax-60A C18-83-221-C column with
flow rate of 5 mL/min or Dynamax-300A C18-83-243-C column
with a flow rate of 20 mL/min of 0.1 M Et3NH2CO3 (TEAB)
followed by a linear gradient of 0.1 M TEAB-aqueous MeCN
(70%). Nuclear magnetic resonance spectra were recorded on a
Varian 500 or 600 MHz with Me4Si or DDS as the internal standard
for 1H 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), bs (broad singlet), and dd (double
doublet). J values given for coupling constants are first order and
in Hz. High-resolution mass spectra were recorded on a ZAB-EQ
(VG analytical spectrometer) using FAB or on an Agilent TOF II
TOF/MS instrument equipped with an ESI or APCI interface.

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 5021
Dimethoxytrityl Benzylhydroxymethylphosphonate (3). Dimeth-
oxytrityl chloride (4.4 g, 13 mmol) was added to the solution of
dibenzyl hydroxymethylphosphonate (1, 2.53 g, 8.66 mmol) in
pyridine (30 mL). The mixture was stirred at room temperature
overnight. The reaction was quenched with methanol (1 mL) and
evaporated. The residue was chromatographed on a silica gel
column using a linear gradient of ethyl acetate in toluene (containing
10% of Et3N) to give crude 2 ((4.71 g, 91% yield), which was
mixed with DABCO (0.89 g, 7.921 mmol) in toluene (60 mL),
refluxed for 8 h, and then left at room temp overnight. The crystals
that formed were filtered and washed with ethyl acetate followed
by petroleum ether to give the DABCO salt of 3 (4.1 g, 84% yield).
This salt was converted into the Et3N salt (3.0 g, 75% overall yield)
by passing a solution of 3 (DABCO) in ethanol through a column
of Dowex 50, Et3NH+ form.
7-Benzyloxy-6-(2-hydroxyethylbenzylhydroxymethylphos-
phonate)-5-methoxy-4-methylphatalan-1-one (6). Compound 3
(0.72 g, 1.19 mmol), 1-methylimidazole (0.1 mL, 1.19 mmol), and
TIPSCl (0.36 g, 1.19 mmol) were premixed in acetonitrile (10 mL),
and 7-benzyloxy-6-(2-hydroxyethyl)-5-methoxy-4-methylphatalan-
1-one (4,24 0.21 g, 0.638 mmol) dissolved in acetonitrile (2 mL)
was added after 4 min. The mixture was stirred overnight at room
temperature and concentrated in vacuo. The residue was purified
by column chromatography on silica gel, using a linear gradient of
ethyl acetate in toluene (containing 10% of Et3N), and DMTr
derivative was treated with 80% acetic acid (20 mL) for 3 h. Acetic
acid was evaporated in vacuo, and the mixture was coevaporated
with water (2×), ethanol (2×), and toluene (2×). A further
purification by chromatography on a silica gel column using a linear
gradient of ethyl acetate in toluene followed by a linear gradient
of ethanol in ethyl acetate gave 6 (0.254 g, 78% overall yield).
P1-(Cyanoethyl)-P1-(N6,N6,2′,3′-O-tetrabenzoyladenosin-5′-yl)-
methylenephospho-P2-(benzyl)-P2-[7-benzyloxy-6-(ethyl-2-yl)-5-
methoxy-4-methylphthalan-1-one]phosphonate (7). Tetrazole
(0.09 g, 1.25 mmol) was added to a mixture of tetrabenzoylad-
enosine cyanoethyl diisopropylphosphoroamidite 11 [(0.249 g, 0.28
mmol), prepared by phosphitylation of N6-dibenzoyl, 2′,3′,-O-
dibenzoyladenosine (9)30 with commercially available 2-cyanoethyl
diisopropyl-chlorophosphoramidite] and mycophenolic phosphonate
6 (0.13 g, 0.25 mmol) in acetonitrile (10 mL). After 1 h the reaction
mixture was cooled to 0 °C, and mCPBA (0.07 g, 0.4 mmol) was
added. Temperature was increased to r.t. and the mixture was stirred
for an additional 1 h. The mixture was diluted with ethyl acetate
(100 mL), and washed with a saturated solution of sodium
bicarbonate (2 × 80 mL). The organic phase was dried over sodium
sulfate and concentrated in vacuo. The residue was purified on a
silica gel column using a linear gradient of ethyl acetate in toluene
followed by a linear gradient of ethanol in ethyl acetate to give
compound 7 ((0.2303 g, 70% yield) as a mixture of 4 diastereo-
isomers.
(OCH3), 64.82 (d, JC,P ) 6, CH2-5′), 65.34 (d, JC,P ) 5, CH2OP),
70.41 (CH2-3′′), 70.56 (CH-3′), 75.33 (CH-2′), 84.07 (d, JC,P ) 9,
CH-4′), 87.94 (CH-1′), 107.02 (C-7′′a), 116.55 (C-3′′a), 118.71 and
118.74 (C-5, C-6′′), 139.92 (CH-8), 147.07 (C-4′′, C-7′′), 148.97
(C-4), 153.04 (CH-2), 155.64 (C-8), 163.50 (C-5′′), 173.68 (C-
1′′); 31P NMR (D2O) δ 1.91 (m, Jvic ) 38.0, JP,H ) 5.0, 4.8, 2.0
P-phosphate), 16.99 (dp, Jvic ) 38.0, JP,H ) 9.0, P-phosphonate);
HR FAB+ calcd (M+H)+ 706.090342, found 706.092567.
N6-Dibenzoyl-2′,3′-O-dibenzoyl-5′-yl(O-benzyl)hydroxymeth-
ylphosphonate (10). Monobenzyl dimethoxytrityloxymethanphos-
phonate (3, 0.82 g, 1.35 mmol), 1-methylimidazole (0.1 mL, 1.35
mmol) and TIPSCl (0.4 g, 1.35 mmol) were premixed in acetonitrile
(10 mL). Tetrabenzoyladenosine (9, 0.48 g, 0.7 mmol) dissolved
in acetonitrile (2 mL) was added after 4 min. The reaction mixture
was stirred overnight at r.t. Dimethoxytrityl derivative was obtained
by chromatography on silica gel, using a linear gradient of ethyl
acetate in toluene (containing 10% of Et3N) and immediately treated
with 80% acetic acid (20 mL) for 3 h. Acetic acid was evaporated
and the reaction mixture was coevaporated with water (2x), ethanol
(2x), and toluene (2x) to give 10 (0.254 g, 42% yield as a mixture
of two diastereoisomers A and B in the ratio of 4:3) after
chromatography on a silica gel column using a linear gradient of
ethanol in chloroform.
P1-(Benzyl)-P1-[7-benzyloxy-6-(ethyl-2-yl)-5-methoxy-4-meth-
ylphthalan-1-one]methylenephospho-P2-(cyanoethyl)-P2-
(N6,N6,2′,3′-O-tetrabenzoyladenosin-5′-yl)phosphonate (12). Tet-
razole (0.11 g, 1.55 mmol) was added to the mixture of phosphonate
10 (0.24 g, 0.28 mmol) and mycophenolic phosphoramidite 5
[(0.162 g, 0.315 mmol), prepared by phosphitylation of mycophe-
nolic alcohol 424 with commercially available 2-cyanoethyl diiso-
propyl-chlorophosphoramidite] in acetonitrile (5 mL). After 1 h the
reaction mixture was cooled to 0 °C, and mCPBA (0.2 g, 1.55
mmol) was added. Temperature was increased to r.t. and the mixture
was stirred for an additional 1 h. The mixture was diluted with
ethyl acetate (100 mL), and washed with saturated solution of
sodium bicarbonate (2 × 80 mL). Organic phase was dried over
sodium sulfate and concentrated in vacuo. The desired product (0.23
g, 64% yield, a mixture of 4 diastereoisomers) was obtained by
chromatography on silica gel using a linear gradient of ethanol in
chloroform.
P1-[7-Hydroxy-6-(ethyl-2-yl)-5-methoxy-4-methylphthalan-1-
one]methylenephospho-P2-(adenosin-5′-yl)phosphonate (13). Com-
pound 12 was dissolved in a mixture of ethanol (15 mL) and
dioxane (20 mL), and 2 M TEAB (0.1 mL) and Pd/C (70 mg) were
added. The mixture was hydrogenated overnight at room temper-
ature. The catalyst was filtered off on a Celite pad, and the filtrate
was concentrated. The obtained solid was dissolved in 8 M ethanolic
methylamine (3 mL) and stirred at room temperature for 2 h. The
mixture was evaporated, and the residue was purified by HPLC.
The triethylammonium salt was converted into the sodium salt by
passing through a column of Dowex 50 (Na+ form) giving 30%
P1-(Adenosin-5′-yl)methylenephospho-P2-[7-hydroxy-6-(ethyl-
2-yl)-5-methoxy-4-methylphthalan-1-one]phosphonate (8). Com-
pound 7 was dissolved in a mixture of ethanol (30 mL) and dioxane
(20 mL), the 2M TEAB (0.1 mL) and Pd/C (70 mg) were added
and the mixture was hydrogenated overnight at r.t. The catalyst
was filtered through a Celite pad and the filtrate was concentrated
in vacuo. The solid residue was dissolved in 8M ethanolic
methylamine (5 mL), and stirred at r.t. for 2 h. The mixture was
evaporated, and the residue was purified by HPLC to give the
desired product, which was converted into the sodium salt by
passing through a column of Dowex 50 (Na+ form) to give 8 in
1
overall yield (37.7 mg). H NMR (500 MHz, D2O) δ 2.01 (s, 3H,
CH3-4′′), 2.91 (m, 2H, CH2-6′′), 3.80 (s, 3H, OCH3), 3.92 (q, 2H,
JH,P ) 8.5, Jvic ) 7.7, CH2OP), 4.06 and 4.08 (2 × ddd, 2H, Jgem
) 14.1, JH,P ) 9.0, 5.0, P-CH2-OP), 4.31 (ddd, 1H, Jgem ) 11.7,
JH,P ) 4.8, J5′b,4′ ) 2.7, H-5′b), 4.35 (ddd, 1H, Jgem ) 11.7, JH,P
)
4.8, J5′a,4′ ) 2.6, H-5′a), 4.43 (dq, 1H, J4′,3′ ) 4.3, J4′,5′ ) 2.7, 2.6,
JH,P ) 2.0, H-4′), 4.56 (t, 1H, J3′,2′ ) 5.1, J3′,4′ ) 4.3, H-3′), 4.66
(t, 1H, J2′,1′ ) 5.1, J2′,3′ ) 5.1, H-2′), 5.10 (s, 2H, H-3′′), 6.03 (d,
1H, J1′,2′ ) 5.1, H-1′), 8.20 (s, 1H, H-2), 8.39 (s, 1H, H-8); 13C
NMR (125.8 MHz, D2O) δ 11.09 (CH3-4), 25.66 (d, JC,P ) 7, CH2-
6′′), 60.26 (dd, JC,P ) 163, 7, P-CH2-OP), 61.90 (OCH3), 64.64
(d, JC,P ) 5, CH2-5′), 65.38 (d, JC,P ) 6, CH2OP), 70.19 (CH2-3′′),
70.56 (CH-3′), 75.53 (CH-2′), 84.24 (d, JC,P ) 8, CH-4′), 88.06
(CH-1′), 107.04 (C-7′′a), 115.41 (C-3′′a), 118.63 and 118.81 (C-5,
C-6′′), 139.88 (CH-8), 147.03 (C-4′′, C-7′′), 148.82 (C-4), 153.01
(CH-2), 155.63 (C-8), 163.49 (C-5′′), 173.84 (C-1′′); 31P NMR
(202.5 MHz, D2O) δ 1.90 (dtt, Jvic ) 39.6, JP,H ) 9.0, 4.8,
P-phosphate), 17.61 (m, Jvic ) 39.6, JP,H ) 8.5, 5.0, 3.3, P-
phosphonate); HR FAB+ calcd (M + H)+ 706.090 342, found
706.093 022.
1
64% overall yield (80 mg, 0.1134 mmol). H NMR (D2O) δ 2.05
(s, 3H, CH3-4), 2.88 (m, 2H, CH2-6′′), 3.82 (s, 3H, OCH3), 3.98
(m, 2H, CH2OP), 4.09 (dd, 2H, JH,P ) 9.0, 5.0, P-CH2-OP), 4.24
(ddd, 1H, Jgem ) 11.7, JH,P ) 4.8, J5′b,4′ ) 2.8, H-5′b), 4.31 (ddd,
1H, Jgem ) 11.7, JH,P ) 4.8, J5′a,4′ ) 2.6, H-5′a), 4.42 (dq, 1H, J4′,3′
) 5.2, J4′,5′ ) 2.8, 2.6, JH,P ) 2.0, H-4′), 4.56 (t, 1H, J3′,4′ ) 5.2,
J3′,2′ ) 5.1, H-3′), 4.67 (t, 1H, J2′,1′ ) 5.1, J2′,3′ ) 5.1, H-2′), 5.15
(s, 2H, H-3′′), 6.04 (d, 1H, J1′,2′ ) 5.1, H-1′), 8.14 (s, 1H, H-2),
8.39 (s, 1H, H-8); 13C NMR (D2O) δ 11.14 (CH3-4), 26.20 (d, JC,P
) 4, CH2-6′′), 60.72 (dd, JC,P ) 161, 7, P-CH2-OP), 62.03

5022 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
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Enzyme Assays. Human type 1 and type 2 IMPDH was
expressed and purified as previously described.31,32 Inhibition assays
were performed as previously described.33 Briefly, assays were set
up in duplicate using two different concentrations of IMPDH type
1 (87 and 155 nM) and type 2 (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, mixed gently
while the production of NADH was monitored by following changes
in absorbance at 340 nm on a Hitachi U-2000 spectrophotometer.
Steady-state velocities were used to determine Ki(app) values by
fitting the velocities vs inhibitor concentration to a simple binding
model with Dynafit.34
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.
(21) Sintchak, M. D.; Fleming, M. A.; Futer, O.; Raybuck, S. A.;
Chambers, S. P.; et al. Structure and mechanism of inosine mono-
phosphate dehydrogenase in complex with the immunosuppressant
mycophenolic acid. Cell 1996, 85, 921-930.
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dinucleotide inhibition of Tritrichomonas foetus inosine 5′-mono-
phosphate dehydrogenase: implications on enzyme mechanism.
Biochemistry 1990, 29, 849-854.
Supporting Information Available: 1H, 13C, 31P NMR spectra
and HPLC profiles of compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
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