Bis(sulfonamide) isosters of mycophenolic adenine dinucleotide analogues: Inhibition of inosine monophosphate dehydrogenase

Article abstract of DOI:10.1016/j.bmc.2008.06.003

Synthesis of novel inhibitors of human IMP dehydrogenase is described. These inhibitors are isosteric methylenebis(sulfonamide) analogues 5-8 of earlier reported mycophenolic adenine methylenebis(phosphonate)s 1-3. The parent bis(phosphonate) 1 and its bi

Full text of DOI:10.1016/j.bmc.2008.06.003

Bioorganic & Medicinal Chemistry 16 (2008) 7462–7469
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bis(sulfonamide) isosters of mycophenolic adenine dinucleotide analogues:
Inhibition of inosine monophosphate dehydrogenase
Liqiang Chen, Riccardo Petrelli, Magdalena Olesiak , Daniel J. Wilson, Nicholas P. Labello,
*
Krzysztof W. Pankiewicz
Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware Street S.E., Minneapolis, MN 55455, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of novel inhibitors of human IMP dehydrogenase is described. These inhibitors are isosteric
methylenebis(sulfonamide) analogues 5–8 of earlier reported mycophenolic adenine methylenebis(phos-
phonate)s 1–3. The parent bis(phosphonate) 1 and its bis(sulfonamide) analogue 5 showed similar sub-
Received 7 May 2008
Revised 3 June 2008
Accepted 5 June 2008
Available online 10 June 2008
micromolar inhibitory activity against IMPDH2 (Ki ꢀ 0.2
lM). However, the bis(sulfonamide) analogues 6
and 8 substituted at the position 2 of adenine were approximately 3- to 10-fold less potent inhibitors of
IMPDH2 (Ki = 0.3–0.4 lM) than the corresponding parent bis(phosphonate)s 2 and 3 (Ki = 0.04–0.11lM),
respectively.
Keywords:
Methylenebis(sulfonamide)
Methylenebis(phoshonate)
Nicotinamide adenine dinucleotide
Mycophenolic acid
Ó 2008 Elsevier Ltd. All rights reserved.
Inosine monophosphate dehydrogenase
NAD analogues
O
1. Introduction
NH2
In recent years, numerous studies of nicotinamide adenine
dinucleotide (NAD)-dependent biological processes have demon-
strated various significant roles of NAD in cell biology and medi-
cine.1 Consequently, molecules affecting interactions of NAD with
NAD-dependent or NAD-utilizing enzymes are now of great thera-
peutic interest. For example, mycophenolic acid (MPA, Fig. 1),
which targets the NAD-binding domain of IMP-dehydrogenase
(IMPDH), has been approved by the FDA as an immunosuppres-
sant.2 Tiazofurin, an antileukemic C-nucleoside, is converted in
the cell into tiazofurin adenine dinucleotide (TAD, Fig. 1), a mimic
of NAD in which the nicotinamide riboside moiety is replaced by
tiazofurin. TAD cannot participate in hydride transfer and selec-
tively inhibits IMPDH.3 Several other NAD-based potential thera-
peutics are now in Phase I/II clinical trials.1
NAD is usually bound at the characteristic Rossmann fold, a
conserved structural motif that also binds nucleotides.4–6 Large
portions of the Rossmann folds of different NAD-dependent
proteins superimpose well. Since this domain is conserved it
was believed that it would be difficult if not impossible to design
NAD-like molecules with good selectivity against a particular
S
N
OH
O
OH
HO
O
O
O
O
HO
OH
Mycophenolic acid
Tiazofurin
O
NH2
HO
OH
S
N
O
P
OH
O
P
OH
O
O
O
O
O
N
N
N
N
HO
OH
TAD
NH2
Figure 1. IMPDH inhibitors.
enzyme. However, dinucleotide-binding domains show very low
overall sequence homology. Taken together with the enormous
conformational flexibility of NAD the indication is that almost
countless binding patterns between NAD and NAD-utilizing
* Corresponding author. Tel.: +1 612 625 7968; fax: +1 612 624 825.
E-mail address: panki001@umn.edu (K.W. Pankiewicz).
Department of Chemistry and Biochemistry, University of Colorado at Boulders,
Boulder, CO 80309, USA.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.003

L. Chen et al. / Bioorg. Med. Chem. 16 (2008) 7462–7469
7463
proteins are possible.7 NAD has extraordinary hydrogen bonding
potential. It contains 19 polar oxygen and nitrogen atoms and
can form at least 16 hydrogen bonds with the NAD-binding pro-
teins; some of these hydrogen bonds can be mediated by water
molecules.8 Thus, selective recognition of divergent three-dimen-
sional arrangements of hydrogen bond acceptors and donors,
hydrophobic groups as well as steric interactions should lead to
selective or even specific inhibition of a single protein.
Indeed, NAD-dependent inosine monophosphate dehydroge-
nase (IMPDH) is a classic example of such selectively inhibited en-
zymes.9 With NAD bound in an extended conformation7 it is a
major therapeutic target for the design of potential immuno-sup-
pressants, anticancer, antiviral, and antimicrobial agents.10,11 It
catalyzes IMP to XMP conversion, the rate-limiting step in de novo
synthesis of guanine nucleotides that are crucial for cell growth
and proliferation. The mechanism of action of IMPDH has been re-
cently reported.12
Crystal structures of complexes of human IMPDH with sub-
strate and NAD analogues revealed that the dinucleotide-binding
pocket can be divided into three regions: (1) the nicotinamide ribo-
side sub-domain (N sub-site), (2) the adenosine sub-domain (A
sub-site), and (3) the pyrophosphate linker sub-site (P sub-do-
main). The contribution of these regions to the potential binding
of NAD-like inhibitors is not equal.
Mycophenolic acid (MPA) is a potent and specific inhibitor of
the two known isoforms of human IMPDH with higher activity
against IMPDH2 (Ki = 10 nM) than IMPDH1 (Ki = 40 nM). It binds
at the N sub-domain of IMPDH with its six-member ring roughly
mimicking the nicotinamide ring and its five-member ring
extending to additional space in the IMPDH pocket (PDB
1JR1).13,14 The side chain of MPA binds partially in the P sub-do-
main (groove) hydrogen bonding with one of the two serines
(Ser 276) present at the floor of the groove. The A sub-site is
empty.
The active metabolite of Tiazofurin, TAD, that inhibits IMPDH
(Ki = 100 nM) shows 3–4 orders of magnitude greater-binding
affinity to IMPDH than to alcohol, glutamate or lactate dehydro-
genases. A crystal structure of IMPDH2 in complex with SAD
(PDB 1B30), a selenium analogue of TAD, showed that the sele-
nazofurin moiety is bound at the N sub-domain.18 The selenazole
ring forms stacking interactions similar to that of MPA with the
substrate analogue aromatic base and the carboxyamide group
forming hydrogen bonds with the conserved Asn 303 residue. Only
one phosphorous group of SAD interacts with both serines (275
and 276) of the P-groove. SAD-binding interactions are very similar
to those of NAD with the adenine moiety located at the A sub-site.
SAD is a slightly more potent inhibitor of IMPDH (Ki = 40 nM) than
TAD.
We developed the mycophenolic adenine dinucleotide (MAD)
analogue as a hybrid of C2-mycophenolic alcohol and adenosine
linked through a metabolically stable bis(phosphonate) linker.19,20
This compound, C2-MAD (1, Fig. 3), mimics the entire NAD in
which nicotinamide riboside is replaced by a mycophenolic moi-
ety. Good selectivity against IMPDH is preserved as no inhibition
of cellular dehydrogenases (up to 50 lM) was determined. In a
mouse model of chronic myelegenous leukemia C2-MAD showed
lower toxicity and higher efficacy than tiazofurin.21 Indeed, our
crystal structure (PDB 1NF7) of the complex of IMPDH2 with C2-
MAD (Fig. 4, coded by blue) revealed that the mycophenolic moiety
is bound at the N sub-site of the cofactor-binding pocket whereas
the adenine ring of C2-MAD is in exactly the same position at the A
sub-domain as the adenine ring of NAD. It is sandwiched between
the His 253 and Phe 282 and the N3 of the adenine ring is in close
contact with Thr 45. This region around the adenine ring is not
conserved between the human isoforms of IMPDH and can be
HO
OH
Numerous inhibitors of IMPDH that take advantage of the
strong-binding interactions at the N sub-domain have been de-
signed by Vertex and then other major pharmaceutical compa-
nies.14–17 Some of these compounds contained a phenyl-oxazole
moiety [VX-497 (Merimepodib), VX-944 (now AVN-944), BMS-
337197, Fig. 2] that binds at the N sub-domain, stacking under
the covalently bound substrate hypoxanthine ring. AVN-944 is cur-
rently in Phase I/II clinical studies as an anticancer agent against
blood malignancies. These inhibitors extend neither to the A sub-
site nor to the P-groove.9
OH
O
O
P
O
P
O
O
O
O
X
N
N
N
OH OH
O
N
1
2
3
X = H, C2-MAD
X = Ph
NH2
X = Ethyl
Figure 3. C2-MAD and its methylenebis(phosphonate) analogues.
H
H
N
O
N
O
O
O
CN
N
N
O
O
O
O
O
Me
MeO
N
H
N
H
MeO
N
H
N
H
VX-497 (Merimepodib)
AVN944 (VX-944)
O
O
N
N
O
N
N
O
MeO
N
H
BMS-337197
Figure 2. Inhibitors of IMPDH developed at Vertex and Bristol-Myers-Squibb.

7464
L. Chen et al. / Bioorg. Med. Chem. 16 (2008) 7462–7469
O
N
NH2
HO
O
OH
S
O
S
O
O
S
O
H
N
H
N
O
N
N
N
N
HO
OH
4
NH2
Figure 5. Tiazofurin adenine methylenebis(sulfonamide).
or bis(phosphonate) moieties in the P-groove seems to be even less
important.
These results encouraged us to prepare NAD analogues with a
longer linker than the typical pyrophosphate (–P–O–P–) or methyl-
enebis(phosphonate) (–P–C–P–) group. Insertion of the oxygen
atom between the phosphorus and carbon atom of C2-MAD affor-
ded new inhibitors (–P–O–C–P– or P–C–O–P– linkers) that show 5-
to 10-fold more potent inhibitory activity against the enzyme than
the parent molecule (Ki’s in the range of 20.0–87.0 nM).23
The relative flexibility of binding patterns at the P-groove in-
spired us to replace the phosphorous atoms of TAD with isosteric
sulfur. In general, the similar geometry and shape of bis(sulfon-
amide) analogues of NAD may be well tolerated by NAD-depen-
dent enzymes. However, we found that tiazofurin adenine
methylenebis(sulfonamide) (4, Fig. 5) was about 200-fold less po-
Figure 4. Bis(sulfonamide) 5 (coded in blue) modeled in IMPDH and superimposed
with bis(phosphonate) 1 (C2-MAD, yellow). The starting conformation of each is
taken from the ligand (C2-MAD) in crystal structure 1NF7. Both compounds were
minimized. The protein is displayed in red ribbon while the ligands and several
important residues (green) are presented in a tube representation. Key hydrogen
bonds are shown with solid orange lines and distances measured for compound 5
are reported in angstroms.
tent against IMPDH1 (IC50 = 23.6
Table 1) than TAD (Ki = 0.1 M) or its bis(phosphonate) analogue
(Ki = 0.1
M).24
lM) and IMPDH2 (IC50 = 18.8 lM,
l
exploited for the design of isoform selective inhibitors. Both aden-
osine hydroxyl groups interact with Gln 469. We found that even
with the additional binding at the A sub-domain C2-MAD is less
potent inhibitor of IMPDH (Ki = 250–330 nM) than MPA.
l
2. Results and discussion
Recently, we found the C2-MAD analogue 2 (X = Ph, Fig. 3, Table
1) containing a phenyl group at the adenine 2 position is 5-fold
more potent (Ki = 70 nM) against IMPDH1 than the parent ana-
logue. Similarly, substitution with an ethyl group afforded ana-
logue 3 (X = Et), which demonstrated a 16-fold improvement in
potency (Ki = 20 nM) against IMPDH1.22
These studies indicate that binding interactions at the N sub-
domain of IMPDH are crucial and sufficient for high affinity of
inhibitors such as MPA or Vertex compounds. Additional MAD
binding at the A sub-site is neither synergistic nor significant. It
is likely that an entropy penalty for constructing the larger dinucle-
otide analogues negates any advantage of additional hydrogen
bonding at the A sub-domain. The binding of the pyrophosphate
In our bis(phosphonate) series of mycophenolic adenine dinu-
cleotides (MADs) the analogues 2 and 3 (Fig. 3) substituted at the
2 position of adenine were more potent than C2-MAD and TAD
analogue (Table 1).22 Therefore we expected that synthesis of novel
mycophenolic adenine bis(sulfonamide) isosteres of C2-MAD (5,
MABS, Fig. 6), and its substituted analogues, for example, MABS 6
(X = Ph), MABS 7 (X = C„CH), and MABS 8 (X = Et), might afford
more potent compounds than the parent molecules. With tetrahe-
dral sulfur atoms, the proposed methylenebis(sulfonamide) linker
should mimic the geometry of the methylenebis(phosphonate)
group present in C2-MAD. Indeed, bis(sulfonamide) 5 (Fig. 4, coded
in blue) modeled within the NAD-binding pocket of IMPDH2
showed very similar interactions (vide supra) as those observed
in the crystal complex of C2-MAD/IMPDH2 (Fig. 4, coded in yel-
low). We expect that the weakly acidic bis(sulfonamide) function-
ality will not ionize under physiological conditions. At the same
time, the partial negative charge delocalized onto the oxygen
atoms would sufficiently mimic the negative charges present in
Table 1
Inhibition of human IMPDH type 1 and type 2
Compound
IMPDH1 Ki (
l
M)
IMPDH2 Ki (lM)
MPA
0.04
0.01
1 (C2-MAD)
2 (X = Ph)
3 (X = Et)
4
5 (MABS)
6 (X = Ph)
7 (X = C„CH)
8 (X = Et)
14
0.33
0.07
0.25
0.11
HO
OH
0.02
0.04
23.6a
18.8a
OH
0.35 0.03b
0.66 0.11b
0.52 0.06b
0.82 0.08b
18.2 2.4b
15.4 2.4b
>100
0.17 0.02b
0.31 0.14b
0.18 0.03b
0.44 0.06b
4.0 0.2b
3.0 0.8b
>100
O
O
S
O
O
S
O
H
N
H
N
O
O
X
N
N
N
O
N
5
X = H
15 (MP-alcohol)
18
Adenosine
6
7
X = Ph
NH2
X = Ethynyl
>100
>100
8 X = Ethyl
a
These values were reported as IC50
The errors were given as 95% confidence intervals.
.
b
Figure 6. Mycophenolic adenine methylenebis(sulfonamide)s.

L. Chen et al. / Bioorg. Med. Chem. 16 (2008) 7462–7469
7465
which upon reduction with sodium borohydride afforded pro-
tected alcohol 12. It was subsequently used for the two sequential
Mitsunobu reactions. The first Mitsunobu reaction between
bis(sulfonamide) intermediate 9 and protected alcohol 12 pro-
duced a tri-substituted methylenebis(sulfonamide) 13.
We were also interested in assessing the contribution of indi-
vidual fragments, such as 14 and 15, to the overall binding pattern.
Thus, mildly acidic treatment of compound 13 led to the desired
fragment 14 (Scheme 2). The Mitsunobu reaction between bis(sul-
fonamide) intermediate 9 and 20,30-isopropylidene-adenosine (16)
gave tri-substituted methylenebis(sulfonamide) 17 (Scheme 3). A
subsequent acidic treatment of this compound afforded the second
desired fragment 18, in which a bis(sulfonamide) group is attached
to adenosine.
O
S
O
O
S
O
O
S
O
O
S
O
a
H
N
H
N
DMB
DMB
Cl
Cl
Methylenebis-
(sulfonyl chloride)
9
Scheme 1. Reagents and conditions: (a) 2,4-dimethoxybenzylamine, THF, 0 °C–rt.
the naturally occurring pyrophosphate linkage. Indeed, we found
that new analogues 5–8 showed significantly more potent inhibi-
tory activity against IMPDH than the corresponding bis(sulfon-
amide) analogue of TAD 4.
To prepare the fully assembled mycophenolic adenine bis(sul-
fonamide) analogue MABS 5, tri-substituted methylenebis(sulfon-
amide) intermediate 13 was coupled with 20,30-isopropylidene-
adenosine (16) via Mitsunobu reaction to give tetra-substituted
sulfonamide 21. Subsequent removal of SEM and DMB protective
groups afforded MABS 5. In a similar manner, the corresponding
MABS analogues 6 (X = Ph) and 7 (X = C„CH) were synthesized
from 20,30-isopropylidene-2-phenyladenosine (19) and 20,30-isopro-
pylidene-2-ethylnyladenosine (20), respectively. Finally, hydroge-
nation of compound 7 gave MABS analogue 8 (X = Et) (Scheme 4).
2.1. Chemistry
We recently reported that bis(sulfonamide) analogues could be
assembled by two sequential Mitsunobu reactions which linked
protected mycophenolic and adenosine derivative through a prop-
erly protected methylenebis(sulfonamide) linker intermediate.24 In
our initial attempt on the synthesis of TAD analogue 4, removal of
the benzyl protective groups had been difficult under hydrogenol-
ysis conditions.24 To circumvent this problem, we decided to use
2,4-dimethoxybenzyl (DMB) protective groups, which could be
cleaved under mild acidic conditions after the assembly of pro-
tected MABS analogues. To this end the key bis(sulfonamide) inter-
2.2. Biological evaluations
mediate
9 (Scheme 1) was readily prepared by reaction of
methylenebis(sulfonyl chloride)25 with 2,4-dimethoxybenzyl-
amine in high yield. For the protection of the 7-OH phenolic group
2-(trimethylsilyl)-ethoxymethyl (SEM) chloride was used based on
the fact that SEM group could be removed under acidic conditions.
Alternatively, SEM group could also be cleaved with a fluoride
source, such as tetrabutylammonium fluoride. Accordingly, the
known aldehyde 10 (Scheme 2) was converted into aldehyde 11,
The final MABS analogues 5–8, their fragments 14, 15, 18, and
adenosine were evaluated against human IMPDH1 and IMPDH2
(Table 1). Replacement of the bis(phosphonate) linker of C2-MAD
(1) with the isosteric bis(sulfonamide) moiety afforded compound
5, which showed as potent inhibitory activity against IMPDH1
(Ki = 0.35 lM) and IMPDH2 (Ki = 0.17 lM) as C2-MAD. The bis(sul-
fonamide) analogues 6–8 substituted at the 2-position showed
OR
OSEM DMB
O
OSEM
O
O
O
S
O
O
S
O
H
N
O
OH
c
b
N
DMB
O
O
O
H
O
O
O
10 R = H
12
13
a
11
R = SEM
OH
OH
O
O
O
S
O
O
S
O
H
OH
d
N
NH2
O
O
O
O
15
14
Scheme 2. Reagents and conditions: (a) SEMCl, Adogen 464, NaOH, CH2Cl2, H2O; (b) NaBH4, EtOH; (c) 9, DIAD, PPh3, THF, 0 °C–rt; (d) 40% (v/v) TFA, Et3SiH, CH2Cl2.
NH2
N
NH2
N
N
N
N
N
DMB
HN
DMB
N
O
S
O
O
S
O
N
O
S
O
O
S
O
a
N
b
H
N
9
H2N
O
O
O
O
OH OH
17
18
Scheme 3. Reagents and conditions: (a) 16, DIAD, PPh3, THF, 0 °C–rt; (b) 40% (v/v) TFA, Et3SiH, CH2Cl2; then 80% (v/v) TFA, H2O.

7466
L. Chen et al. / Bioorg. Med. Chem. 16 (2008) 7462–7469
NH2
N
N
OSEM DMB
DMB
N
O
O
S
O
O
S
O
N
N
X
b
5 X = H
a
N
6 X = Ph
O
O
13
7 X = Ethynyl
8 X = Ethyl
O
c
O
O
21 X = H
22 X = Ph
23 X = Ethynyl
Scheme 4. Reagents and conditions: (a) DIAD, PPh3, THF, 0 °C–rt, 16, 19 and 20 for compounds 21, 22 and 23, respectively; (b) 40% (v/v) TFA, Et3SiH, CH2Cl2; then 80% (v/v)
TFA, H2O; (c) H2, Pd/C, MeOH.
nanomolar inhibition in the range 0.18–0.84
were less potent than the corresponding bis(phosphonate)s
(0.02–0.11 M).
Among fragments alcohol 15 (Ki = 15.4
Ki = 3.0 M, IMPDH2) retained moderate inhibitory activity
l
M however, they
MAD series attachment of an ethyl group at C2 position improved
the potency by 21- and 6-fold for human IMPDH type 1 and type 2,
respectively.22
l
lM, IMPDH1 and
l
a
2.3. Computational modeling
against the human enzyme. Replacement of the hydroxyl group
with methylenebis(sulfonamide) as in compound 14 did not im-
prove the potency. On the other hand, neither adenosine nor its
methylenebis(sulfonamide) derivative 18 showed any activity
against IMPDH.
Bis(sulfonamide) 5 saw no loss in potency when compared to
the analogous bis(phosphonate) suggesting that our hypothesis is
correct and that a bis(sulfonamide) moiety can serve as an accept-
able linker between the MPA and nucleoside pharmacophores.
Interestingly, however, in the case of compound 4 identical substi-
tution of the bis(phosphonate) results in a 230-fold loss of activity
(IMPDH1).24 Previously, on the basis of a structural analysis of li-
gands crystallized in IMPDH (particularly 1B3O), it was determined
that at least one of the phosphate oxygen is protonated. In the case
of tiazofurin methlenebis(phosphonate) the 3-hydroxy is donating
a hydrogen bond paired with Asp 274 and accepting an internal
hydrogen bond with the protonated adenosine phosphate. With
an MPA head group, this internal hydrogen bond does not exist,
and there is no penalty for substituting the bis(sulfonamide). With
a tiazofurin head group, however, the internal hydrogen bond
plays an important role in maintaining an optimal ligand confor-
mation within the receptor. In the case of 4 the sulfonamide clearly
cannot maintain this internal hydrogen bond resulting in the ob-
served loss of potency when linking the tiazofurin and nucleoside
groups with a bis(sulfonamide) linkage.
During study of the related NAD analogues with a di-phosphate
linker it was noted that substitution of the adenine at the 2 posi-
tion with ethyl and phenyl groups resulted in an increase in activ-
ity as much as 16-fold. Docking studies utilizing both molecular
mechanics and QM/MM methods for geometry optimization are
in agreement that while the positioning of the bis(sulfonamide)
linker differs slightly from the (bis)phosphonate, displacement in
the MPA head group and adenine moiety is much less. The RMSD
of the heavy base atoms due to varying the linker is approximately
0.2 Å. Accordingly, a similar structure–activity relationship would
be expected for substitution of the position 2. Interestingly, the rel-
atively flat SAR suggests that no significant additional hydrophobic
interactions occurred. Indeed, evaluation of the interaction energy
for an ensemble of structures generated through molecular
dynamics simulations of MABS 4–7 indicates that there is no ener-
getic gain due to hydrophobic stacking for ligands in this series.
Additionally the QM/MM interaction energy calculated for a mini-
mum energy structure of each receptor–ligand complex indicates
only very minor differences in agreement with the results of the
enzyme assay.
This trend is not surprising, considering the different positions
these fragments occupy in the NAD-binding site. Alcohol 15 pre-
sumably binds at the N sub-site, which has a great degree of rigid-
ity. It is reasonable to expect that the functional groups on the
phthalide core of alcohol 15 can engage in hydrogen bond interac-
tions and aromatic stacking, as proposed for MPA. Therefore a por-
tion of activity is retained for alcohol 15. Nevertheless, the similar
potency of bis(sulfonamide) 14 indicates that methylenebis(sul-
fonamide) contribution to binding is negligible and cannot com-
pensate for strong binding of the crucial carboxylate group of
MPA. It is likely that a bis(sulfonamide) group cannot form the de-
sired hydrogen bonds needed for a high potency when adenosine is
not attached. In contrast to fragments 14 and 15, binding of aden-
osine and its bis(sulfonamide) derivative 18 to the A sub-site is
weak if any as this region is exposed to solvent. Therefore it is
energetically costly for adenosine or compound 18 to adopt a spa-
tial configuration that enables them to favorably interact with the
residues surrounding the A sub-site, resulting in no inhibition of
IMPDH.
Even though fragment 14 exhibited only modest potency, the
attachment of adenosine residue to compound 14 led to MABS ana-
logue 5 with dramatically enhanced potency against both human
IMPDH isoforms. When compared with fragment 14, compound
5 displayed activity 52- and 24-fold more potent against IMPDH
type 1 and type 2, respectively (Table 1). This dramatic gain of po-
tency suggests that the methylenebis(sulfonamide) linker most
likely facilitates the adenosine moiety to position itself so that it
engages in favorable interactions with the residues surrounding
the A sub-domain. Possibly, binding of the wholly assembled anal-
ogous such as MABS 5–8 is needed to trigger the conformational
change of the enzyme (such as ‘flap’ closing) resulting in tight
binding. This synergy also supports our current strategy for the de-
sign of IMPDH inhibitors based on linking two pharmacophores
that interact with both N and A sub-sites.
However, modifications on the C2 position of the adenine ring
resulted in bis(sulfonamide) analogues 6–8 with slightly reduced
inhibitory activity. This finding is clearly in contrast with the trend
we observed for the modifications made in the bis(phosphonate)
C2-MAD series. For instance, introduction of an ethyl group at C2
position of the adenine ring led to bis(sulfonamide) derivative 8,
which displayed inhibitory activities approximately 2-fold lower
than those of parent compound 5. On the contrary, in the C2-
3. Conclusions
It is well documented that the shape and physiochemical fea-
tures around the three major sub-domains of NAD-dependent en-
zymes are significantly diversified. Numerous studies of the

L. Chen et al. / Bioorg. Med. Chem. 16 (2008) 7462–7469
7467
Rossmann fold-NAD complexes showed that NAD-binding sites
share a common mode of binding to the P and A sub-sites, whereas
N sub-domain sites adopt a variety of conformations to fulfill dif-
ferent functions. The packing at the N sub-site is often strongly
substrate-dependent and it enforces the proper chemical activity
of NAD. It was believed that the substantial conservation of the A
sub-domain indicates that the adenosine interactions are indepen-
dent of an enzyme specificity. We found, however, that at least in
the case of human IMPDH, the differences in the amino acids se-
quences at the A sub-site could be exploited toward design of
inhibitors with some selectivity against the two isoforms of the hu-
man enzyme.22
The enzyme di-phosphate region (P-groove) appears to be
highly promiscuous. It is not crucial for enforcing potent binding
between ligands and their NAD-dependent enzymes. Therefore,
we expected that NAD analogues containing structural changes
at the pyrophosphate moiety would significantly affect neither
binding affinity nor selectivity toward their corresponding
proteins.
7.20 (d, J = 8.4 Hz, 2H), 6.54 (s, 2H), 6.51 (d, J = 8.4 Hz, 2H), 4.75
(s, 2H), 4.10 (s, 4H), 3.78 (s, 6H), 3.74 (s, 6H). 13C NMR (DMSO-
d6) d 160.1, 157.6, 129.5, 117.7, 104.4, 98.2, 67.2, 55.4, 55.2, 41.0.
C
19H30N3O8S2 492.1468 (M+NH4)+, found 492.1512.
4.2.2. Protected aldehyde (11)
To a mixture of CH2Cl2 (24 mL) and 0.5 N aqueous NaOH solu-
tion (24 mL) were added Adogen 464 (3.60 mL, 2.42 mmol) and
aldehyde 10 (2.82 g, 11.9 mmol). After stirring for 20 min, 2-(tri-
methylsilyl)ethoxymethyl chloride (SEMCl, 2.60 mL, 14.7 mmol)
was added dropwise. The resulting mixture was allowed to stir
at rt for 1 h, and the organic layer was separated and then concen-
trated. The residue was purified by silica gel gravity column chro-
matography (10–30% EtOAc/hexanes) to give protected aldehyde
11 as a clear syrup (2.78 g, 64%). 1H NMR (CDCl3) d 9.72 (s, 1H),
5.41 (s, 2H), 5.17 (s, 2H), 3.84 (s, 2H), 3.78 (t, J = 8.4 Hz, 2H), 3.75
(s, 3H), 2.21 (s, 3H), 0.94 (t, J = 8.4 Hz, 2H), 0.02 (s, 9H). 13C NMR
(CDCl3) d 198.8, 168.8, 163.0, 154.3, 148.3, 121.3, 120.2, 112.3,
99.6, 68.3, 67.8, 60.8, 39.7, 18.1, 11.6, ꢂ1.5. C18H26O6SiNa
389.1390 (M+Na)+, found 389.1388.
Herein, we replaced phosphorous atoms of methylenebis(phos-
phonate) analogues of MAD 1–3 with isosteric sulfur atoms that
led to new sulfonamide analogues MPBS 5–8 with potent inhibi-
tory activity against human IMPDH (Kis in the range of 0.17–
4.2.3. Protected alcohol (12)
To a solution of protected aldehyde 11 (2.78, 7.58 mmol) in
EtOH (80 mL) was added sodium borohydride (860 mg, 22.7 mmol)
in small portions. The resulting mixture was allowed to stir for
30 min and then concentrated. The residue was re-dissolved in
EtOAc (200 mL) and water (50 mL) and neutralized with 1 N HCl.
The organic layer was separated, and washed with water (2ꢁ
50 mL) and brine (2ꢁ 75 mL). After drying over Na2SO4, the organic
layer was filtered, and the filtrate was concentrated and dried in
vacuo to give protected alcohol 12 as clear syrup (2.72 g, 97%).
1H NMR (CDCl3) d 5.40 (s, 2H), 5.14 (s, 2H), 3.90–3.84 (m, 4H),
3.83 (s, 3H), 3.06 (t, J = 6.3 Hz, 2H), 2.30 (t, J = 5.7 Hz, 1H), 2.21 (s,
3H), 1.00 (t, J = 8.4 Hz, 2H), 0.03 (s, 9H). 13C NMR (CDCl3) d 169.1,
163.1, 154.6, 147.2, 126.8, 120.4, 112.2, 99.7, 68.2, 67.7, 62.5,
61.0, 27.8, 18.1, 11.7, ꢂ1.5. C18H28O6SiNa 391.1547 (M+Na)+, found
391.1535.
0.82 lM). Our report indicates that further studies of structural
diversity of the pyrophosphate moiety of NAD analogues on their
binding ability at the P-groove of NAD-dependent enzymes are jus-
tified. Indeed, we found and will report shortly that connecting of
pharmacophores binding at the N and A domains of IMPDH with a
linker, which is neither isosteric nor isopolar, resulted in potent
inhibition of the enzyme.
4. Experimental
4.1. General methods
All commercial reagents (Sigma–Aldrich, Acros) were used as
provided unless otherwise indicated. An anhydrous solvent dis-
pensing system (J.C. Meyer) using 2 packed columns of neutral alu-
mina was used for drying THF, Et2O, and CH2Cl2, while 2 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.
Nuclear magnetic resonance spectra were recorded on a Varian
600 MHz with Me4Si or signals from residual solvent as the inter-
nal standard for 1H or13C. Chemical shifts are reported in ppm, and
signals are described as s (singlet), d (doublet), t (triplet), q (quar-
tet), m (multiplet), br s (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 instru-
ment equipped with either an ESI or APCI interface.
4.2.4. Protected mycophenolic bis(sulfonamide) (13)
To a solution of protected alcohol 12 (923 mg, 2.50 mmol),
methylenebis(sulfonamide) 9 (3.56 g, 7.50 mmol), and triphenyl-
phosphine (2.02 g, 7.70 mmol) in anhydrous THF (30 mL) at 0 °C
was added via a syringe pump a solution of diisopropyl azodicar-
boxylate (DIAD, 1.50 mL, 7.74 mmol) in anhydrous THF (10 mL)
over 5 h. The mixture was concentrated and the residue was dis-
solved in Et2O (15 mL) and CH2Cl2 (5 mL). After an addition of hex-
anes (150 mL), a syrup formed on the wall of round-bottomed
flask. The organic solvents were decanted and this cleaning process
was then repeated for additional three times. The syrup obtained
was purified by silica gel gravity column chromatography (0–3%
MeOH/CH2Cl2) to give protected mycophenolic bis(sulfonamide)
13 as a white solid (1.24 g, 60%). 1H NMR (CDCl3) d 7.22 (d,
J = 8.4 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.48–6.40 (m, 3H), 6.38
(dd, J = 7.8, 2.4 Hz, 1H), 5.75 (t, J = 6.0 Hz, 1H), 5.39 (s, 2H), 5.12
(s, 2H), 4.35 (s, 2H), 4.23 (d, J = 6.0 Hz, 2H), 4.06 (s, 2H), 3.83 (s,
3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.78–3.73 (m, 5H), 3.72 (s, 3H),
3.38 (t, J = 7.2 Hz, 2H), 2.98 (t, J = 7.5 Hz, 2H), 2.16 (s, 3H), 0.90 (t,
4.2. Synthesis
4.2.1. Methylenebis(sulfonamide) (9)
To
a
solution of 2,4-dimethoxybenzylamine (33.0 mL,
220 mmol) in anhydrous THF (400 mL) at 0 °C was added dropwise
a solution of methanedisulfonyl dichloride (10.7 g, 50.2 mmol) in
anhydrous THF (100 mL). After stirring at 0 °C for 30 min, the reac-
tion mixture was allowed to warm to rt and stir at rt for 5 h. After
concentration, the residue was dissolved in EtOAc (600 mL) and
washed with 0.1 N HCl (220 mL), water (100 mL), saturated NaH-
CO3 (100 mL), and brine (2ꢁ 200 mL). The organic layer was dried
over Na2SO4 and filtered. The filtrate was concentrated and then
dried under high vacuum to give methylenebis(sulfonamide) 9 as
a yellow solid (21.7 g, 91%). 1H NMR (DMSO-d6) d 7.65 (br s, 2H),
J = 8.4 Hz, 2H), ꢂ0.03 (s, 9H).
C37H56N3O13S2Si 842.3018
(M+NH4)+, found 842.3030.
4.2.5. Mycophenolic bis(sulfonamide) (14)
To a solution of protected mycophenolic bis(sulfonamide) 13
(221 mg, 0.27 mmol) in dry CH2Cl2 (5.0 mL) and TFA (4.0 mL)
was added dropwise triethylsilane (0.50 mL). During this period
of time, the color of mixture changed from pink to light yellow.
After stirring for 1 h at rt, the reaction mixture was concentrated

7468
L. Chen et al. / Bioorg. Med. Chem. 16 (2008) 7462–7469
and the residue was triturated with hot CH2Cl2 (6 mL) to give
bis(sulfonamide) 14 as pale solid (53.6 mg, 51%). 1H NMR
(DMSO-d6) d 9.51 (s, 1H), 7.58 (t, J = 5.7 Hz, 1H), 7.20 (s, 2H), 5.24
(s, 2H), 4.70 (s, 2H), 3.74 (s, 3H), 3.16-3.08 (m, 2H), 2.81 (t,
J = 7.8 Hz, 2H), 2.08 (s, 3H). C13H18N2O8S2Na 417.0396 (M+Na)+,
found 417.0409.
4.3.2. Protected bis(sulfonamide) (22)
In
a
similar manner mycophenolic bis(sulfonamide) 13
(450 mg, 0.55 mmol) was coupled with 20,30-O-isopropylidene-2-
phenyladenosine (19, 70 mg, 0.18 mmol) to give a pale solid
(120 mg), which was used directly for the next reaction.
4.3.3. Protected bis(sulfonamide) (23)
4.2.6. Protected adenosine 50-methylenebis(sulfonamide) (17)
In a manner similar to that described for the preparation of pro-
tected bis(sulfonamide) 13, 20,30-O-isopropylideneadenosine (16,
986 mg, 3.21 mmol) was coupled with methylenebis(sulfonamide)
9 (2.53 g, 9.64 mmol) to give 17 as a pale solid (1.30 g, 53%). 1H
NMR (CDCl3) d 8.25 (s, 1H), 7.84 (s, 1H), 7.11 (d, J = 7.8 Hz, 1H),
6.96 (d, J = 7.8 Hz, 1H), 6.45 (s, 1H), 6.42–6.36 (m, 2H), 6.29 (d,
J = 8.4 Hz, 1H), 6.06 (s, 1H), 5.86 (s, 2H), 5.80 (t, J = 6.0 Hz, 1H),
5.40 (d, J = 6.0 Hz, 1H), 5.00–4.95 (m, 1H), 4.46–4.41 (m, 1H),
4.30 (d, J = 15.6 Hz, 1H), 4.22–4.16 (m, 3H), 4.11 (d, J = 15.0 Hz,
1H), 3.96 (d, J = 14.4 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.75 (s,
3H), 3.74 (s, 3H), 3.52 (dd, J = 15.6, 9.0 Hz, 1H), 3.39 (dd, J = 15.3,
3.3 Hz, 1H), 1.59 (s, 3H), 1.36 (s, 3H). 13C NMR (CDCl3) d 161.2,
161.0, 158.7, 158.5, 155.5, 153.0, 149.1, 140.1, 131.8, 130.6,
120.2, 116.3, 115.0, 114.5, 104.2, 103.9, 98.8, 98.4, 90.5, 86.1,
84.0, 82.3, 67.3, 55.3 (2C), 55.3, 55.1, 49.0, 46.5, 43.9, 27.1, 25.4.
In a similar manner mycophenolic bis(sulfonamide) 13 (1.64 g,
1.99 mmol) was coupled with 20,30-O-isopropylidene-2-ethynylad-
enosine (20, 223 mg, 0.67 mmol) to give 23 as a white solid
(129 mg, 11%). 1H NMR (CDCl3) d 7.87 (s, 1H), 7.26 (d, J = 7.8 Hz,
1H), 7.10 (d, J = 8.4 Hz, 1H), 6.42 (d, J = 8.4 Hz, 1H), 6.39 (s, 1H),
6.35 (s, 1H), 6.34 (d, J = 8.4 Hz, 1H), 6.04 (d, J = 1.2 Hz, 1H), 5.82
(br s, 2H), 5.40 (d, J = 6.6 Hz, 1H), 5.38 (d, J = 6.6 Hz, 1H), 5.29 (d,
J = 6.6 Hz, 1H), 5.10 (s, 2H), 4.96 (dd, J = 6.6, 4.2 Hz, 1H), 4.48–
4.40 (m, 4H), 4.35 (d, J = 15.0 Hz, 1H), 4.21 (d, J = 13.8 Hz, 1H),
4.13 (d, J = 14.4 Hz, 1H), 3.81–3.71 (m, 17H), 3.59 (dd, J = 15.6,
8.4 H, 1H), 3.54 (dd, J = 15.6, 4.8 Hz, 1H), 3.41 (t, J = 7.5 Hz, 2H),
2.99 (t, J = 7.8 Hz, 2H), 2.94 (s, 1H), 2.13 (s, 3H), 1.57 (s, 3H), 1.34
(s, 3H), 0.89 (t, J = 8.4 Hz, 2H), ꢂ0.03 (s, 9H). HRMS calcd for
C
52H68N7O16S2Si 1138.3927 (M+H)+, found 1138.3942.
4.4. Synthesis of mycophenolic adenine methylenebis
(sulfonamide)s (MABS) 5–7
C
32H42N7O11S2 764.2378 (M+H)+, found 764.2391.
4.2.7. Adenosine 50-methylenebis(sulfonamide) (18)
4.4.1. MABS (5) (X = H)
To a solution of protected adenosine 50-methylenebis(sulfon-
amide) 17 (285 mg, 0.37 mmol) in dry CH2Cl2 (6.0 mL) and TFA
(4.0 mL) was added dropwise triethylsilane (0.50 mL). The mixture
was allowed to stir for 1.5 h and then concentrated. The residue
was dissolved in TFA (8.0 mL) and water (2.0 mL) and the mixture
was allowed to stir overnight. After concentration, the residue was
co-evaporated with MeOH and purified by silica gel preparative
TLC (1000 microns, 20% MeOH/CH2Cl2) to give 18 as a pale solid
(58.5 mg, 37%). 1H NMR (DMSO-d6) d 8.56 (t, J = 5.7 Hz, 1H), 8.32
(s, 1H), 8.19 (s, 1H), 7.46 (s, 2H), 7.24 (s, 2H), 5.85 (d, J = 6.6 Hz,
1H), 5.50 (br s, 1H), 5.30 (br s, 1H), 4.80 (s, 2H), 4.69 (t,
J = 6.0 Hz, 1H), 4.12 (pseudo d, J = 2.4 Hz, 1H), 4.05 (pseudo d,
J = 2.4 Hz, 1H), 3.37 (dt, J = 14.4, 4.2 Hz, 1H), 3.34-3.26 (m, 1H).
HRMS calcd for C11H18N7O7S2 424.0703 (M+H)+, found 424.0672.
To a solution of protected mycophenolic adenine methylene-
bis(sulfonamide) 21 (134 mg, 0.12 mmol) in dry CH2Cl2 (5.0 mL)
and TFA (4.0 mL) was added dropwise triethylsilane (0.50 mL).
The mixture was allowed to stir for 1.5 h and then concentrated.
The residue was dissolved in TFA (4.0 mL) and water (1.0 mL)
and the mixture was allowed to stir overnight. After concentration,
the residue was co-evaporated with MeOH and purified by silica
gel preparative TLC (1000 microns, 10% MeOH/CH2Cl2) to give 5
as a pale solid (23.6 mg, 30%). 1H NMR (DMSO-d6) (two signals
overlap with the water signal) d 9.53 (br s, 1H), 8.59 (s, 1H), 8.30
(s, 1H), 8.16 (s, 1H), 7.60 (t, J = 6.0 Hz, 1H), 7.43 (br s, 2H), 5.83
(d, J = 6.6 Hz, 1H), 5.49 (br s, 1H), 5.24 (s, 2H), 4.86 (s, 2H), 4.68
(t, J = 6.0 Hz, 1H), 4.11 (pseudo s, 1H), 4.05 (pseudo s, 1H), 3.72
(s, 3H), 3,12 (dd, J = 15.0, 6.0 Hz, 1H), 2.80 (t, J = 7.8 Hz, 2H), 2.07
(s, 3H). HRMS calcd for C23H30N7O11S2 644.1439 (M+H)+, found
644.1415.
4.3. Synthesis of protected mycophenolic adenine
methylenebis(sulfonamide)s 21–23
4.4.2. MABS (6) (X = Ph)
4.3.1. Protected bis(sulfonamide) (21)
In similar manner compound 22 was deprotected to give myco-
phenolic 2-phenyladenine bis(sulfonamide) 6 as a pale solid
(14.8 mg, 11% for two steps). 1H NMR (DMSO-d6) d 9.51 (br s,
1H), 8.35 (s, 1H), 8.33 (d, J = 6.6 Hz, 2H), 7.74 (br s, 1H), 7.61 (br
s, 1H), 7.48–7.38 (m, 3H), 7.32 (br s, 2H), 5.97 (d, J = 6.6 Hz, 1H),
5.54 (d, J = 5.4 Hz, 1H), 5.33 (br s, 1H), 5.23 (s, 2H), 4.83 (d,
J = 14.4 Hz, 1H), 4.81-4.75 (m, 2H), 4.21 (pseudo s, 1H), 3.98 (dd,
J = 9.3, 5.7 Hz, 1H), 3.70 (s, 3H), 3,43 (dd, J = 13.5, 5.1 Hz, 1H),
3.27 (dd, J = 14.4, 6.6 Hz, 1H), 3.09 (t, J = 7.8 Hz, 2H), 2.78 (t,
To a solution of protected mycophenolic bis(sulfonamide) 13
(995 mg, 1.21 mmol), 20,30-O-isopropylideneadenosine (16,
123 mg, 0.40 mmol), and triphenylphosphine (430 mg, 1.64 mmol)
in anhydrous THF (10 mL) at 0 °C was added via a syringe pump a
solution of DIAD (0.32 mL, 1.65 mmol) in anhydrous THF (5 mL)
over 4.5 h. The mixture was allowed to slowly warm to rt and stir
overnight. After concentration, the residue was purified by silica
gel gravity column chromatography (0–6% MeOH/CH2Cl2) to give
protected bis(sulfonamide) 21 as a white solid (152 mg, 34%). 1H
NMR (CDCl3) d 8.25 (s, 1H), 7.83 (s, 1H), 7.25 (d, J = 8.4 Hz, 1H),
7.08 (d, J = 9.0 Hz, 1H), 6.42 (d, J = 9.0 Hz, 1H), 6.40 (s, 1H), 6.36
(d, J = 1.8 Hz, 1H), 6.33 (dd, J = 8.7, 2.1 Hz, 1H), 6.04 (d, J = 1.8 Hz,
1H), 5.69 (br s, 2H), 5.41 (d, J = 6.6 Hz, 1H), 5.39 (d, J = 6.6 Hz,
1H), 5.36 (dd, J = 6.6, 1.2 Hz, 1H), 5.10 (s, 2H), 4.97 (dd, J = 6.6,
4.2 Hz, 1H), 4.50–4.46 (m, 1H), 4.46–4.40 (m, 3H), 4.36 (d,
J = 15.0 Hz, 1H), 4.22 (d, J = 14.4 Hz, 1H), 4.10 (d, J = 14.4 Hz, 1H),
3.81–3.76 (m, 7H), 3.76–3.72 (m, 10H), 3.61 (dd, J = 15.3, 8.7 H,
1H), 3.51 (dd, J = 15.3, 4.5 Hz, 1H), 3.43–3.36 (m, 2H), 3.00 (t,
J = 7.8 Hz, 2H), 2.14 (s, 3H), 1.58 (s, 3H), 1.35 (s, 3H), 0.89 (t,
J = 8.4 Hz, 2H), ꢂ0.03 (s, 9H). HRMS calcd for C50H68N7O16S2Si
1114.3927 (M+H)+, found 1114.3964.
J = 8.1 Hz, 2H), 2.05 (s, 3H). HRMS calcd for
C29H34N7O11S2
720.1752 (M+H)+, found 720.1728.
4.4.3. MABS (7) (X = C„CH)
In similar manner compound 23 (115 mg, 0.10 mmol) was
deprotected to give mycophenolic 2-ethynyladenine methylene-
bis(sulfonamide) (7) as
a
pale solid (56 mg, 83%). 1H NMR
(DMSO-d6) (one signal overlaps with the water signal) d 9.52 (s,
1H), 8.40 (s, 1H), 7.79 (t, J = 5.7 Hz, 1H), 7.61 (t, J = 5.7 Hz, 1H),
7.49 (br s, 2H), 5.85 (d, J = 5.4 Hz, 1H), 5.53 (d, J = 5.4 Hz, 1H),
5.30 (d, J = 5.4 Hz, 1H), 5.24 (s, 2H), 4.84 (d, J = 14.4 Hz, 1H), 4.81
(d, J = 14.4 Hz, 1H), 4.56 (pseudo d, J = 5.4 Hz, 1H), 4.10 (pseudo

L. Chen et al. / Bioorg. Med. Chem. 16 (2008) 7462–7469
7469
4. Rossmann, M. G.; Moras, D.; Olsen, K. W. Nature 1974, 250, 194.
5. Carugo, O.; Argos, P. Proteins 1997, 28, 10.
6. Carugo, O.; Argos, P. Proteins 1997, 28, 29.
d, J = 3.0 Hz, 1H), 4.00–3.93 (m, 2H), 3.72 (s, 3H), 3.18–3.08 (m, 4H),
2.80 (t, J = 7.5 Hz, 2H), 2.07 (s, 3H). HRMS calcd for C25H30N7O11S2
668.1439 (M+H)+, found 668.1477.
7. Due to stacking abilities of its two aromatic moieties, adenine and
nicotinamide, NAD is folded in water solution (with the distance between
the bases of 4–5 Å). Several bacterial reductases bind NAD in such compact
(folded) conformation (see PDB entries 1RZ1, 1ZPT, 2BKJ). Other enzymes, such
as bacterial NAD kinases, including therapeutically important Mycobacterium
tuberculosis enzyme, unfold NAD partially; NAD is bent with the distance
around 12 Å between the bases. In majority of NAD-utilizing enzymes,
including IMPDH, the distance is around 16–17 Å. This means that NAD must
be unfolded into extended conformation to be able to bind.
4.4.4. MABS (8)
A mixture of MABS 7 (31 mg, 0.046 mmol) and 10% Pd/C
(30 mg) in MeOH (10 mL) was hydrogenated (1 atm) overnight.
The reaction mixture was filtered through a pad of Celite and the
filtrate was concentrated. The resulting residue was purified by sil-
ica gel preparative TLC (1000 microns, 10% and then 15% MeOH/
CH2Cl2) to give mycophenolic 2-ethyladenine methylenebis(sul-
fonamide) 7 as a pale solid (10.2 mg, 32%). 1H NMR (DMSO-d6) d
(one signal overlaps with the water signal) 8.22 (s, 1H), 7.80 (br
s, 1H), 7.13 (br s, 2H), 5.76 (d, J = 6.6 Hz, 1H), 5.47 (d, J = 5.4 Hz,
1H), 5.27 (br s, 1H), 5.24 (s, 2H), 4.83 (d, J = 14.4 Hz, 1H), 4.79 (d,
J = 14.4 Hz, 1H), 4.68 (pseudo d, J = 5.4 Hz, 1H), 4.15 (pseudo s,
1H), 3.96 (pseudo d, 2.4 Hz, 1H), 3.72 (s, 3H), 3.38 (dd, J = 13.8,
4.8 Hz, 1H), 3.11 (t, J = 7.5 Hz, 2H), 2.80 (t, J = 7.8 Hz, 2H), 2.66 (q,
J = 7.2 Hz, 2H), 2.07 (s, 3H), 1.21 (t, J = 7.8 Hz, 3H). HRMS calcd
for C25H34N7O11S2 672.1752 (M+H)+, found 672.1767.
8. Bottoms, C. A.; Smith, P. E.; Tanner, J. J. Protein Sci. 2002, 11, 2125.
9. Inosine Monophosphate Dehydrogenase:
A
Major Therapeutic Target;
Pankiewicz, K. W., Goldstein, B. M., Eds.; Oxford University Press:
Washington, 2003.
10. Chen, L.; Pankiewicz, K. W. Curr. Opin. Drug Discov. Dev. 2007, 10, 403.
11. Pankiewicz, K. W. Expert Opin. Ther. Pat. 2001, 11, 1161.
12. Hedstrom, L.; Gan, L. Curr. Opin. Chem. Biol. 2006, 10, 520.
13. Sintchak, M. D.; Fleming, M. A.; Futer, O.; Raybuck, S. A.; Chambers, S. P.; Caron,
P. R.; Murcko, M. A.; Wilson, K. P. Cell 1996, 85, 921.
14. Sintchak, M. D.; Nimmesgern, E. Immunopharmacology 2000, 47, 163.
15. Iwanowicz, E. J.; Watterson, S. H.; Guo, J.; Pitts, W. J.; Murali Dhar, T. G.; Shen,
Z.; Chen, P.; Gu, H. H.; Fleener, C. A.; Rouleau, K. A.; Cheney, D. L.; Townsend, R.
M.; Hollenbaugh, D. L. Bioorg. Med. Chem. Lett. 2003, 13, 2059.
16. Watkins, W. J.; Chen, J. M.; Cho, A.; Chong, L.; Collins, N.; Fardis, M.; Huang, W.;
Hung, M.; Kirschberg, T.; Lee, W. A.; Liu, X.; Thomas, W.; Xu, J.; Zeynalzadegan,
A.; Zhang, J. Bioorg. Med. Chem. Lett. 2006, 16, 3479.
17. Birch, H. L.; Buckley, G. M.; Davies, N.; Dyke, H. J.; Frost, E. J.; Gilbert, P. J.;
Hannah, D. R.; Haughan, A. F.; Madigan, M. J.; Morgan, T.; Pitt, W. R.; Ratcliffe,
A. J.; Ray, N. C.; Richard, M. D.; Sharpe, A.; Taylor, A. J.; Whitworth, J. M.;
Williams, S. C. Bioorg. Med. Chem. Lett. 2005, 15, 5335.
18. Colby, T. D.; Vanderveen, K.; Strickler, M. D.; Markham, G. D.; Goldstein, B. M.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3531.
19. Lesiak, K.; Watanabe, K. A.; Majumdar, A.; Powell, J.; Seidman, M.; Vanderveen,
K.; Goldstein, B. M.; Pankiewicz, K. W. J. Med. Chem. 1998, 41, 618.
20. Pankiewicz, K. W.; Lesiak-Watanabe, K. B.; Watanabe, K. A.; Patterson, S. E.;
Jayaram, H. N.; Yalowitz, J. A.; Miller, M. D.; Seidman, M.; Majumdar, A.;
Prehna, G.; Goldstein, B. M. J. Med. Chem. 2002, 45, 703.
21. Patterson, S. E.; Black, P. L.; Clark, J. L.; Risal, D.; Goldstein, B. M.; Jayaram, H. N.;
Schinazi, R. F.; Pankiewicz, K. W. In Developments in Nucleic Acids; Schinazi, R. F.
L. D., Ed.; IHL Press, 2004.
4.5. Computational chemistry
Model construction has been described elsewhere.22 All calcula-
tions were completed within the Schrodinger suite of modeling
programs, which includes MacroModel26 for molecular mechanics
minimization, conformational searching, and molecular dynamics
and Qsite27 for QM/MM calculations. The ligands were constructed
by modifying the ligand present in 1NF7 and subsequent minimi-
zation. MD simulations were run for no less than 150 ps following
minimization and equilibrium runs using the OPLS 200528 force
field. QM/MM optimizations and interaction energy calculations
were completed at the B3LYP29,30/6-31G* level of theory.31,32
22. Chen, L.; Gao, G.; Felczak, K.; Bonnac, L.; Patterson, S. E.; Wilson, D.; Bennett, E.
M.; Jayaram, H. N.; Hedstrom, L.; Pankiewicz, K. W. J. Med. Chem. 2007, 50,
5743.
23. Rejman, D.; Olesiak, M.; Chen, L.; Patterson, S. E.; Wilson, D.; Jayaram, H. N.;
Hedstrom, L.; Pankiewicz, K. W. J. Med. Chem. 2006, 49, 5018.
24. Chen, L.; Gao, G.; Bonnac, L.; Wilson, D. J.; Bennett, E. M.; Jayaram, H. N.;
Pankiewicz, K. W. Bioorg. Med. Chem. Lett. 2007, 17, 3152.
25. Castro, A.; Erickson, S. K.; Shechter, I.; Spencer, T. A. Bioorg. Chem. 1996, 24,
242.
Acknowledgment
These studies were founded by the Center for Drug Design, Aca-
demic Health Center, University of Minnesota, and USDOD ARMY
Grant W81XWH-05-01-0216.
26. MacroModel. Schrodinger, LLC: New York, NY, 2006.
27. Qsite, version 4.5, Schrodinger, LLC.: New York, 2007.
28. Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. J. Phys. Chem. B
2001, 105, 6474.
References and notes
29. Kaminski, G. A.; Stern, H. A.; Berne, B. J.; Friesner, R. A.; Cao, Y. X.; Murphy, R. B.;
Zhou, R.; Halgren, T. A. J. Comput. Chem. 2002, 23, 1515.
30. Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785.
31. Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
1. Chen, L.; Petrelli, R.; Gao, G.; Bonnac, L.; Yu, J. S.; Bennett, E. M.; Pankiewicz, K.
W. Curr. Med. Chem. 2008, 15, 650.
2. Bentley, R. Chem. Rev. 2000, 100, 3801.
3. Grifantini, M. Curr. Opin. Invest. Drugs 2000, 1, 257.
32. Ditchfield, R.; Hehre, W. J.; Pople, J. A. J. Chem. Phys. 1971, 54, 724.