Methylenebis(sulfonamide) linked nicotinamide adenine dinucleotide analogue as an inosine monophosphate dehydrogenase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.03.035

A methylenebis(sulfonamide) linked NAD analogue has been designed to circumvent the metabolically unstable, ionic nature of the natural pyrophosphate linkage. This NAD analogue is assembled through two Mitsunobu reactions of a methylenebis(sulfonamide) li

Full text of DOI:10.1016/j.bmcl.2007.03.035

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3152–3155
Methylenebis(sulfonamide) linked nicotinamide
adenine dinucleotide analogue as an inosine
monophosphate dehydrogenase inhibitor
Liqiang Chen,a,* Guangyao Gao,a Laurent Bonnac,a Daniel J. Wilson,a Eric M. Bennett,a
Hiremagalur N. Jayaramb and Krzysztof W. Pankiewicza,*
aCenter for Drug Design, University of Minnesota, Minneapolis, MN 55455, USA
bDepartment of Biochemistry and Molecular Biology, Indiana University School of Medicine and Richard Roudebush Veterans
Affairs Medical Center, 1481 West Tenth Street, Indianapolis, IN 46202, USA
Received 15 February 2007; accepted 10 March 2007
Available online 15 March 2007
Abstract—A methylenebis(sulfonamide) linked NAD analogue has been designed to circumvent the metabolically unstable, ionic
nature of the natural pyrophosphate linkage. This NAD analogue is assembled through two Mitsunobu reactions of a methylene-
bis(sulfonamide) linker with two protected nucleosides. A 2,4-dimethoxybenzyl group is used as a sulfonamide protective group,
which allows facile removal under mildly acidic conditions. This NAD analogue inhibits IMPDH at low micromolar concentration.
Ó 2007 Elsevier Ltd. All rights reserved.
Nicotinamide adenine dinucleotide (NAD) contains a
pyrophosphate linkage which is present in numerous
biologically important molecules. In addition to NAD’s
key role in redox reactions in cells, it has recently been
found to be involved in various biological processes
including post-translational protein modification and
signal transductions.1 The pyrophosphate linkage is also
a crucial component in the activated form of glycosyl
donors used by numerous glycosyl transferases that par-
ticipate in oligosaccharide and glycoprotein syntheses.
Furthermore, exogenous nucleosides, such as tiazofurin
and benzamide riboside, can be metabolically activated
and converted into the corresponding NAD analogues
such as TAD (1, Fig. 1) through the formation of a
pyrophosphate linkage.2,3 These NAD analogues are
potent inhibitors of IMP-dehydrogenase (IMPDH),
which catalyzes the rate-limiting step in the de novo syn-
thesis of guanine nucleotides. However, such pyrophos-
phate linked molecules are readily cleaved by cellular
enzymes such as phosphodiesterases and their ionic nat-
ure prevents them from penetrating the cell membrane.
Therefore, there is a growing interest in design and syn-
O
N
NH2
HO OH
S
O
O
O
N
O P X P O
HO OH
O
N
N
HO OH
N
NH2
1 X = O
2 X = CH2
O
N
NH2
HO OH
S
O
O
H
O
N S CH2 S N
H
O
N
N
N
O
O
N
HO OH
3
NH2
Figure 1. TAD and analogues.
thesis of metabolically stable and preferably neutral
mimics of the pyrophosphate moiety of NAD and other
biologically important pyrophosphates.4–7
Our laboratory has been actively pursuing NAD ana-
logues as potent inhibitors of IMPDH, which in recent
years has emerged as a major therapeutic target for
the design of immunosuppressant, anticancer and antivi-
ral agents.8
Keywords: Inosine monophosphate dehydrogenase; IMPDH; NAD
analogue; Pyrophosphate mimics; Methylenebis(sulfonamide).
*
Corresponding authors. Tel.: +1 612 625 3264; fax: +1 612 625
8252; e-mail addresses: chenx462@umn.edu; panki001@umn.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.035

L. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3152–3155
3153
We and others have developed pyrophosphate mimics
NH2
N
O
O
N
N
such as methylenebis(phosphonate) analogues of TAD
(2),9,10 other bis(phosphonate)s,10–12 and phosphono-
phosphates13 Herein we report our design and synthesis
of NAD analogue 3 in which two nucleoside moieties
are connected via a methylenebis(sulfonamide) linker
(Fig. 1). We expect that the weakly acidic bis(sulfon-
amide) functionality will not ionize under physiological
conditions. At the same time, the partial negative charge
delocalized onto the oxygen atoms would sufficiently mi-
mic the negative charges present in the naturally occur-
ring pyrophosphate linkage. Furthermore, with
tetrahedral sulfur atoms, the proposed methylenebis(sul-
fonamide) linker is expected to closely resemble the
geometry of the methylenebis(phosphonate) linker
which mimics the pyrophosphate moiety well.
R
O
O
N
a
O
N S CH2 S NH
HO
O
N
N
O
O
R
N
N
O
O
NH2
9 R = phenyl
10 R = 2,4-dimethoxyphenyl
4
b
O
NH2
O
O
S
N
c
R
O
O
O
3
N S CH2 S N
O
O
N
N
N
O
R
N
O
O
NH2
11 R = phenyl
12 R = 2,4-dimethoxyphenyl
NAD analogue 3 was assembled by two sequential Mits-
unobu reactions (Scheme 2) between properly protected
nucleosides 4 and 5,14 and the sulfonamide moiety, fol-
lowed by removal of the protective groups (Fig. 2).
The key bis(sulfonamide) intermediate 7 (Scheme 1)
was readily prepared by reaction of bis(sulfonyl) chlo-
ride 615 with benzylamine in high yield. The first Mitsun-
obu reaction incorporated an isopropylidene protected
adenosine 4, while the second introduced an isopropy-
lidene protected tiazofurin 5 to give protected bis(sul-
fonamide) 11 albeit in low yields (Scheme 2). With two
nucleosides in place, hydrogenolysis was attempted to
remove the benzyl protective groups present in 11.
Unfortunately no cleavage of benzyl groups was ob-
served according to 1H NMR. This finding was not
unexpected considering the fact that cleavage of sulfon-
amide benzyl groups has been previously demonstrated
to be difficult.16,17
Scheme 2. Reagents and conditions: (a) DIAD, PPh3, 7 or 8, THF,
0 °C to rt, 20% for 9 and 53% for 10; (b) DIAD, PPh3, 5, THF, 0 °C to
rt, 15% for 11 and 27% for 12; (c) 40% (v/v) TFA, Et3SiH, CH2Cl2, and
then TFA, H2O, 54% starting from 12.
To circumvent this problem, we designed a new bis(sul-
fonamide) intermediate 8 (Scheme 1) which contains 2,4-
dimethoxybenzyl protective groups. The 2,4-dialkoxy-
benzyl moiety, which can be cleaved under mild acidic
conditions, has found broad applications in solid-phase
organic synthesis. For instance, acid sensitive methoxy
benzaldehyde linker (AMEBA) has been used for the so-
lid-phase syntheses of secondary amide, sulfonamide,
and carbamate derivatives.18 Recently fluorous-tagged19
and ionic liquid20 version of AMEBA have been devel-
oped. We expected that substitution of benzyl with
2,4-dimethoxybenzyl would allow facile cleavage under
mild acidic conditions after the assembly of a protected
dinucleotide analogue.17,21
3
Thus new bis(sulfonamide) intermediate 8 was prepared
in excellent yield under conditions similar to those de-
scribed above for 7. It was subjected to a sequence of
two Mitsunobu reactions to give 12 (Scheme 2). At least
three equivalents of acidic components involved in the
Mitsunobu reactions, for example, 8 and 10, should be
used. Slow addition of these acidic components was also
found to be crucial. A smooth removal of 2,4-dimeth-
oxybenzyl groups from 12 was accomplished as expected
under mild conditions. Subsequent one-pot acidic
hydrolysis of isopropylidene protective groups afforded
the desired NAD analogue 3.22
O
O
R
H
H
N S CH2 S N
Mitsunobu
NH2
R
Mitsunobu
O
O
O
N
NH2
N
N
S
N
N
HO
HO
O
O
O
O
O
O
5
4
The desired methylenebis(sulfonamide) NAD analogue
3 was evaluated against isoforms of human IMPDH.13
It showed IC50 values of 23.6 and 18.8 lM against the
type I and type II enzymes, respectively. However, this
NAD analogue did not show anti-proliferation activity
against the K562 cell line (IC50 > 100 lM).
Figure 2. Retrosynthetic scheme of 3.
O
O
O
O
H
a
R
H
Cl S CH2 S Cl
N S CH2 S N
R
O
O
O
O
6
7 R = phenyl
8 R = 2,4-dimethoxyphenyl
Analysis of the crystal structures of type II IMPDH with
two known inhibitors C2-MAD (13, Fig. 3),11 SAD (14),
and NAD (PDB entries 1NF7, 1B3O, and 1NFB)23 sug-
gests that in each case, one of the phosphate oxygen
Scheme 1. Reagents and conditions: (a) benzylamine or 2,4-dimeth-
oxybenzylamine, THF, 0 °C, 82% for 7 and 94% for 8.

3154
L. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3152–3155
may depend on the linker being adaptable to different
HO OH
O
O P
HO
hydrogen bonding situations as the protein structure
changes during the catalytic cycle,24 although the inter-
actions are not consistent among the X-ray structures.
Currently we are exploring alternative linkers which
have an enhanced ionic character and contain structural
features that can interact with the amino acid residues
surrounding the linker region.
O
P O
OH
H2
C
OH
O
O
N
N
O
O
N
N
NH2
13
O
NH2
HO OH
Se N
O
O P
HO
O
P O
OH
In summary, we have designed a methylenebis(sulfon-
amide) linked NAD analogue that shows inhibitory
activity against human type I and type II IMPDH. We
have devised a convergent synthesis that involves a se-
quence of two Mitsunobu reactions and facile removal
of 2,4-methoxybenzyl protective groups. Given the mild
conditions utilized in our synthesis, it is expected that
our approach will find broad application in the synthesis
of methylenebis(sulfonamide) as a mimic of pyrophos-
phate linkages which are present in various biologically
important molecules.
O
O
O
N
N
N
NH2
N
HO OH
14
Figure 3. Structures of C2-MAD (13) and SAD (14).
atoms must be protonated, although the specific interac-
tions are different in each case. For example, in C2-
MAD,11 the mycophenolic phosphate has one oxygen
˚
3.0 A away from water 73. Because water 73 is donating
one of its hydrogens to Gln 441 and its other hydrogen
is shared between the carboxylate oxygens of Asp 470,
the phosphate oxygen is likely protonated. In the
NAD cocrystal structure, the hydroxyl of Ser 275 is
within hydrogen bonding distance of the Gln 277 back-
bone carbonyl and an oxygen of the adenosine phos-
phate. Finally, in the structure of SAD (Fig. 4), a
close analogue of TAD (1), the 30-hydroxyl of the sele-
nazofurin sugar is within hydrogen bonding distance
of both the Asp 274 backbone carbonyl and one of
the selenazofurin phosphate oxygens.
Acknowledgements
These studies were funded by the Center for Drug De-
sign, University of Minnesota, and USDOD ARMY
Grant W81XWH-05-01-0216.
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Figure 4. Linker interactions of 14 with IMPDH (PDB entry 1B3O).
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L. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3152–3155
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22. Methylenebis(sulfonamide) linked NAD analogue 3 was
prepared from 12 according to the following procedures:
A solution of 12 (119.7 mg, 0.114 mmol) in dry CH2Cl2
(5 mL), TFA (4 mL), and Et3SiH (1 mL) was stirred at
rt for 7 h. After concentration, the residue was dissolved
in TFA (4 mL) and water (1 mL), and the solution was
allowed to stir at rt overnight. After concentration and
co-evaporation with MeOH several times, the residue
was then dissolved in MeOH (ca. 5 mL) and the solid
formed, after overnight, was filtered to give compound 3
(40.9 mg, 54%) as a white solid. 1H NMR (DMSO,
600 MHz) d8.65 (dd, J = 7.5, 4.5 Hz, 1H, exchangeable
with D2O), 8.30 (s, 1H), 8.22 (s, 1H), 8.17 (s, 1H), 7.65
(br s, 2H, exchangeable with D2O), 7.54 (br s, 1H,
exchangeable with D2O), 7.36 (br s, 2H, exchangeable
with D2O), 5.84 (d, J = 7.2 Hz, 1H), 5.48 (d, J = 6.6 Hz,
1H, exchangeable with D2O), 5.45 (d, J = 5.4 Hz, 1H,
exchangeable with D2O), 5.28 (d, J = 3.6 Hz, 1H,
exchangeable with D2O), 5.16 (d, J = 6.0 Hz, 1H,
exchangeable with D2O), 5.00–4.92 (m, 3H), 4.69 (dd,
J = 11.4, 6.0 Hz, 1H), 4.16–4.04 (m, 3H), 3.94 (dd,
J = 10.8, 5.4 Hz, 1H), 3.85 (dd, J = 10.5, 5.6 Hz, 1H),
3.40–3.31 (m, 3H), 3.26–3.19 (m, 1H). 13C NMR
(DMSO, 150 MHz) d 171.3, 162.2, 156.2, 152.5, 150.4,
148.7, 140.4, 124.4, 119.5, 88.3, 84.0, 82.7, 82.1, 76.4,
72.4, 72.0, 71.2, 67.1, 45.4, 45.0. HRMS calcd for
C20H28N9O11S3, 666.1064 (M+H)+; found, 666.1065.
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