Structure-Based Design, Synthesis, and Antimicrobial Activity of Indazole-Derived SAH/MTA Nucleosidase Inhibitors

Article abstract of DOI:10.1021/jm0302039

The structure-based design, synthesis, and biological activity of a novel indazole-containing inhibitor series for S-adenosyl homocysteine/methylthioadenosine (SAH/MTA) nucleosidase are described. Use of 5-aminoindazole as the core scaffold provided a structure-guided series of low nanomolar inhibitors with broad-spectrum antimicrobial activity. The implementation of structure-based methodologies provided a 6000-fold increase in potency over a short timeline (several months) and an economy of synthesized compounds.

Full text of DOI:10.1021/jm0302039

J . Med. Chem. 2003, 46, 5663-5673
5663
Str u ctu r e-Ba sed Design , Syn th esis, a n d An tim icr obia l Activity of
In d a zole-Der ived SAH/MTA Nu cleosid a se In h ibitor s
Xiaoming Li,§ Sam Chu,§ Victoria A. Feher,† Mitra Khalili,§ Zhe Nie,§ Stephen Margosiak,‡ Victor Nikulin,§
J ames Levin,| Kelly G. Sprankle,§ Martina E. Tedder,§ Robert Almassy,† Krzysztof Appelt, and Kraig M. Yager*,§
Departments of Medicinal Chemistry, Protein Biochemistry, and Structural Biology, Quorex Pharmaceuticals,
Carlsbad, California 92008
Received April 29, 2003
The structure-based design, synthesis, and biological activity of a novel indazole-containing
inhibitor series for S-adenosyl homocysteine/methylthioadenosine (SAH/MTA) nucleosidase are
described. Use of 5-aminoindazole as the core scaffold provided a structure-guided series of
low nanomolar inhibitors with broad-spectrum antimicrobial activity. The implementation of
structure-based methodologies provided a 6000-fold increase in potency over a short timeline
(several months) and an economy of synthesized compounds.
In tr od u ction
from inspection of homology model: substrate com-
plexes, (ii) having molecular volumes less than that of
the homology model binding pocket, and (iii) conforming
to Lipinski5 guidelines (i.e., molecular weight < 500,
-2.0 < ClogP < 5.0). The resulting data set was clustered
for 2D fingerprint diversity and a representative selec-
tion of 1288 compounds identified using SELECTOR
(Tripos, Inc.).
X-ray structural determination of lead compounds
cocrystallized with SAH/MTA nucleosidase derived from
Escherichia coli and other pathogenic species revealed
the mode of inhibitor binding within the active site.
These co-structures provided the structural information
for design of individual compounds and focused librar-
ies.
Growing drug resistance among bacterial pathogens
drives the need for new agents effective against them.
Ideally, such antibacterial agents hit essential proteins
that (i) appear in as many bacterial pathogens as
possible but (ii) differ significantly from related mam-
malian proteins, to yield a broad spectrum of activity
with minimal mechanism-based toxicity.
S-adenosyl homocysteine/methylthioadenosine (SAH/
MTA) nucleosidase presents a potentially useful target,
for three reasons. First, as a product of the highly
conserved pfs gene,1 its active site is similarly highly
conserved across bacterial species, while differing from
that of the related mammalian proteins (methylthioad-
enosine phosphorylase and purine nucleotide phospho-
rylase2,3). Second, SAH/MTA nucleosidase participates
in synthesis of the quorum sensing autoinducer AI-2,4
which in turn stimulates expression of virulence factors.
Consequently, inhibiting SAH/MTA nucleosidase should
attenuate bacterial virulence. Third, and most impor-
tantly, such inhibition should kill bacteria because
accumulation of SAH and MTA inhibits certain essential
methyltransferase reactions and thereby impedes recy-
cling of adenine and methionine (necessary for DNA and
protein synthesis, respectively) (Figure 1).
Ch em ica l Meth od s
Syntheses of substituted indazole SAH/MTA nucle-
osidase inhibitors began from commercially available
5-nitroindazole (1) and from 2-chloro-5-nitro acetophe-
none (2) (Scheme 1). A solution of 1 in glacial AcOH
was exposed to chlorine gas providing 3-chloroindazole
(3) in good yield.6 Condensation of acetophenone 2 with
hydrazine afforded the corresponding 3-methyl deriva-
tive 4. Reduction of the nitro groups either by hydro-
genation over palladium or, in the case of the 3-chloro
derivative 3, by heating with an excess of stannous
chloride, avoided complications due to over-reduction
and loss of the 3-chloro substituent. Regardless of
method, the crude amino indazoles were typically used
without further purification and were allowed to react
with desired sulfonyl chlorides. Initial attempts to
selectively sulfonylate the C5 and C6 amino groups over
the indazole N1 nitrogen were problematic. Ultimately,
by using pyridine as both the base and the solvent, we
were able to cleanly and reproducibly modify only the
C5 and C6 amino groups.
These features provide an enzyme target well-suited
for structure-based design of a broad-spectrum antibac-
terial drug with low potential for mechanism-based
toxicity in mammals. We describe here the rationale and
results of this approach.
In Silico Lea d Id en tifica tion
Selection of compounds for screening in the SAH/MTA
nucleosidase activity assay began by filtering a virtual
library of ∼390 000 commercially available compounds
to approximately 2000 satisfying the following criteria:
(i) possessing three pharmacophoric features identified
Synthesis of 3,5,7-trisubstituted indazoles (Scheme 2)
began with nitration of 3 and 4 to provide 5,7-dinitroin-
dazoles 10 and 11 in good yield.7 Treatment with
aqueous ammonium sulfide8 cleanly and selectively
reduced the 7- but not the 5-NO2 group to provide the
7-amino-5 nitro indazoles 12 and 13. Initial attempts
to protect the C7 amino group, sulfonylate C5, and
* To whom correspondence should be addressed. Quorex Pharma-
ceuticals, 1890 Rutherford Drive, Carlsbad, CA 92008. Phone: (760)
494-6230. Fax (760) 602-1915. E-mail: kyager@quorex.com.
§ Department of Medicinal Chemistry.
‡ Department of Protein Biochemistry.
† Department of Structural Biology.
| Department of Preclinical Microbiology.
10.1021/jm0302039 CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/22/2003

5664 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Li et al.
F igu r e 1. Reaction catalyzed by MTA nucleosidase in vivo.
Sch em e 1a
a
Reagents: (a) Cl2, AcOH; (b) H2NNH2, DMF; (c) SnCl2, EtOH or H2, Pd/C, MeOH; (d) dansyl chloride or 4-chlorobenzenesulfonyl
chloride, pyr.
Sch em e 2a
a
Reagents: (a) HNO3, H2SO4; (b) S(NH4)2, EtOH; (c) R1CHO, NaBH(OAc)3, THF; (d) SnCl2, EtOH or H2, Pd/C, MeOH; (e) R2SO2Cl,
pyr.
deprotect the C7 amino group failed due to the proximity
of the latter to the N1 secondary amino group. Ulti-
mately, reductive amination9 with an appropriate al-
dehyde followed by reduction of the C5 nitro group and
sulfonylation afforded aliphatic and aryl aliphatic groups
at the C7 position (Scheme 2). Less reactive aldehydes
necessitated either reaction with the indazole prior to
reduction with NaBH(OAc)3, or introduction of the
aldehyde portionwise during the reaction, to minimize
consumption of the aldehyde by the reducing agent. This
approach, albeit tedious, proved effective and allowed
optimization of interactions between the enzyme and
the C7 substituent of the inhibitors.
The more highly elaborated C5 biphenyl sulfonamides
were incorporated by direct sulfonylation in pyridine
when the sulfonyl chloride was available, or by Suzuki10
type palladium-mediated arylation of bromo sulfamides
18k and 19 with commercially or synthetically derived
aryl boronates (Scheme 3).
Biologica l Meth od s
In a primary screen for SAH/MTA nucleosidase
inhibition, homocysteine from cleavage of residual S-
adenosyl homocysteine by SAH hydrolase was deter-
mined spectrophotometrically (at 405 nm) by monitoring
the 2-nitro-4-mercaptobenzoate generated upon addition
of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). Addition
of adenosine deaminase to assay mixtures removed the
inhibitory SAH/MTA nucleosidase product, adenine, and
thereby ensured rapid SAH turnover. A secondary assay
monitored the turnover of MTA by SAH/MTA nucleosi-
dase directly as the loss of MTA peak area in an HPLC
chromatogram.
Resu lts a n d Discu ssion
The co-structure of 9a with SAH/MTA nucleosidase
confirmed the binding mode predicted by docking in-
hibitors into the enzyme active site (Figure 2). Key

SAH/ MTA Nucleosidase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5665
Sch em e 3a
a
Reagents: (a) 3-Ar-C6H4SO2Cl, pyr.; (b) ArB(OR3)2, Pd(PPh3)4, aq. Na2CO3, DME.
F igu r e 2. Compound 9a (Ki ) 2.8 µM) bound in the active site of SAH/MTA nucleosidase as determined by X-ray crystallography.
The solvent-accessible surface of the active site (A) illustrates the general shape of the pocket. The hydrogen bonding pattern
comprised of protein backbone and side-chain interactions are illustrated in (B, C). Inhibitor hydrophobic interactions with a
cluster of phenylalanines and V102 are also illustrated; residues from the associated monomer are indicated by an asterisk (C).
Bound water molecules are indicated as yellow spheres (A, B).
features of the binding include those observed in the
adenine:SAH/MTA nucleosidase cocrystal structure1e: a
pair of hydrogen bonds between the heterocyclic core of
the inhibitor and I152 and edge-on π-π interactions
with F151 ( dist ) 4.0 Å). Here N1 of the inhibitor
donates a hydrogen bond to the carbonyl oxygen atom
of I152 while N2 accepts a hydrogen bond from the I152
backbone NH. Additional hydrogen bonding interactions
between the sulfonamide oxygen atoms and two serine
residues S76 and S196 further stabilize the inhibitor-
enzyme complex. The dansyl group makes a series of
hydrophobic interactions: a staggered π-π stacking
interaction with F207 ( dist ) 4.1 Å), a face-on π-π
stacking interaction with F105 ( dist ) 4.0 Å), and a
van der Waals contact between the methylamine and
V102; these latter two residues are contributed from the
neighboring monomer. Ab initio methods predict that
the cisoid sulfonamide bond geometries observed in the

5666 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Li et al.
Ta ble 2. Trisubstituted Indazole Inhibitors of SAH/MTA
Ta ble 1. Inhibitors of SAH/MTA Nucleosidase: Relative and
Absolute Affects of Substituents at C3, C5, and C6
Nucleosidase: C7 Substitutions
compound 9
R
R1
R2
Ki (µM)
a
b
c
d
e
f
H
H
Cl
Me
H
H
Cl
Me
dansyl
H
dansyl
dansyl
4-CPS
H
H
2.8
1
dansyl
H
H
H
4-CPS
H
H
22
64
27
17
5.2
4
g
h
4-CPS
4-CPS
crystal structure are among the lowest energy conform-
ers; thus there is not a significant energy penalty for
the compound to adopt the observed conformer. Inter-
estingly, despite the bulk of the dansyl group, moving
it from the 6- to the 5-position (as in 9b to 9a ) has little
effect on potency, as long as the 3-position remains
unsubstituted. Apparently 180° rotation about the
C(aryl)-N(sulfonamide) bond of 9a and a slight upward
shift within the pocket allows hydrogen bonding and
favorable hydrophobic interactions comparable with
those of 9b (binding as the 2H-indazole tautomer).
Substituents on the 3-position, however, preclude this
accommodation in 5-dansyl derivatives 9c (3-Cl) and 9d
(3-Me). Movement to mitigate the steric bulk of their
5-dansyl groups forces their 3-substituents too close to
the protein backbone. As a consequence, 3-substituted
derivatives 9c and 9d bind poorly (Ki ) 22 and 64 µM,
respectively).
Replacing the 5-dansyl substituent with the lesser
sterically demanding 4-chlorophenylsulfonamide group,
however, largely restores activity in the 3-Cl and 3-Me
derivatives 9g and 9h . Importantly, this replacement
therefore allows functionalization at C3 to address, e.g.,
metabolism issues that might otherwise arise from the
presence of a hydrogen atom at this position.
Examination of the 9a ‚MTA nucleosidase co-structure
revealed a hydrophobic pocket (referred to as the
“northern pocket”) adjacent to the 1- and 6-positions of
9a (Figure 2) that, although highly conserved, does not
participate in binding the native substrate. A suitably
designed inhibitor could therefore exploit this “northern
pocket” to improve its binding to the bacterial enzyme
not only in an absolute sense (increasing its potency
against bacteria), but also in a relative sense vis a vis
mammalian analogues (increasing its selectivity for the
bacterial enzyme and thereby minimizing its mecha-
nism-based toxicity in mammals).
Initial attempts to access the “northern pocket” with
the 7-acetamide derivative 18a (Scheme 2) decreased
inhibition activity 2-fold with respect to the 7-unsub-
stituted derivative 9g (Table 2). The structure of 18a
bound to MTA nucleosidase suggested that the rela-
tively poor affinity arose from a dramatic 1.6 Å upward
shift of inhibitor within the active site compared to the
position of bound 9a (Figure 3). This shift allows the
relatively small active site side pocket to accommodate
the larger C7-chloro group. Furthermore, this upward
a
∆G°solvation energies determined using SE-AM1 methods and
the Cramer/Truhlar 5.4 model12 in Spartan (Wavefunction, Inc.).
Compound conformations were generated by minimizing each
compound in the protein active site or by relaxing the cocrystal
ligand structures. The mean value for compounds 18b-18j is 15.1
( 1.0 kcal/mol.
shift within the active site pocket allows the inhibitor
to make a more direct face on π-π stacking interaction
between the 4-chlorophenyl group of 18a and the side-
chain phenyl of F207. The crystal structure illustrates
a second reason for the poor affinity of this inhibitor
(Figure 4). The orientation of the C7 acetyl group toward
the hydrophobic “northern” pocket clearly failed to
maximize favorable interactions. While the C7 amide
NH hydrogen bonds with the side-chain carboxyl group
of D197, formation of an internal hydrogen bond be-
tween the acetyl carbonyl oxygen atom and N1 brings
the former unfavorably close to the backbone carbonyl
group of I152 (dist. ) 2.9 Å). Reduction of the C7

SAH/ MTA Nucleosidase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5667
F igu r e 3. Superposition of 9a (blue-green) and 18a (atom-type color) cocrystal structures illustrating the 1.6 Å upward shift of
18a within the active site to accommodate the C3 chloro group in the small side pocket. The solvent-accessible surface is included
(A) to outline the overall shape of the protein active site represented as stick in (B).
F igu r e 4. Compound 18a (Ki ) 11.0 µΜ) in the SAH/MTA nucleosidase active site as determined by X-ray crystallography. This
derivative has a chloro group at C3 and an acetamide at the C7-positions of the indazole core. The acetamide N is 2.9 Å from the
D197 Oγ. The intramolecular hydrogen bond between the acetamide carbonyl group and the N1 hydrogen places the former
within 3.0 Å of I152 carbonyl creating an unfavorable (denoted with an asterisk) contact. The binding mode for 18a establishes
direct π-π stacking of the 4-chlorophenyl and F207 ( dist ) 3.55 Å). The solvent-accessible surface is included (A) to outline the
overall shape of the protein active site in stick representation (B).
acetamide group to the corresponding ethylamino group
(to yield 18e) eliminated this contact and thereby
improved potency 2-fold. This change also is favored by
relative desolvation energies for these two compounds.
The next objective was to find the C7 substituent that
best occupied the hydrophobic space afforded by the
enzyme’s northern pocket. The volume of this pocket
varies dramatically from completely occluded in the
formycin-MTA nucleosidase structure to a solvent-
accessible one in the adenine-MTA nucleosidase struc-
ture. This variation complicated modeling predictions
for the appropriate substituents at C7 and necessitated
a more traditional approach to establish the structure-
activity relationship. Results for relatively large aro-
matic groups, cf. 18i and 18j, and a variety of small,
branched, unsaturated, and cyclic aliphatic groups as
C7 substituents show that the larger aliphatic and
aromatic substituents are not accommodated in the
pocket as well as the smaller substituents (Table 2).
Although this may be a consequence of the relative
solvation energies (perhaps contributing ∼1 kcal/mol for
the aryl substitutions), the uncertainties in the calcu-
lated values are on the same order as the differences
between ∆G°
for these compounds over this lim-
solvation
ited Ki range (Table 2). The results are best understood
as demonstrating limitations in the size and shape of
the pocket, the ability of the flexible loop to accom-
modate these compounds, and limits in the conforma-
tional energies required for these compounds to adopt
the bound shape. Overall, the isobutyl and cyclopropy-
lmethyl derivatives 18b (Ki ) 760 nM) and 18c (Ki )
500 nM), offered the best activity (Ki ) 760 and Ki )
500 nM, respectively).
With the indazole core optimized, attention turned to
other sites for potentially improving binding affinities.
In the cocrystal structure for 18b, a hydrophobic chan-
nel near the dimer interface is observed that flanks the
area occupied by the 4-chlorophenyl group (Figure 5).
An appropriately substituted aryl sulfonamide could fill
this relatively solvent-accessible space, and X-ray struc-
ture analysis suggested that the 3-position of the aryl
sulfonamide offered the most promising way of accessing

5668 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Li et al.
An tiba cter ia l Activity. Determination of minimum
inhibitory concentrations for several Gram-positive and
-negative pathogenic bacteria showed that the most
potent inhibitor (20c; Ki ) 1.6 nM) has modest activity
against the Gram-negative bacterium Neisseria men-
ingitidis but little against Gram-positive strains. Com-
pounds 20a and 20b yielded the broadest spectrum of
activity, showing comparable, albeit unexceptional,
activity against both Gram-positive and -negative or-
ganisms. These and other indazole derivatives have
antimicrobial activity against the microorganisms listed
in Table 4. Poor activity against these bacteria, despite
strong inhibition of the enzyme in vitro, suggests that
the compounds fail to penetrate bacterial cell mem-
branes.
Con clu sion
Structure-based design of a series of indazole-based
SAH/MTA nucleosidase inhibitors led to the discovery
of 20c, a potent, low molecular weight inhibitor with
nanomolar potency in an enzyme inhibition assay. In
antimicrobial assays, this compound inhibits the growth
of three important pathogenic species, showing MIC
values less than 10 µM.
F igu r e 5. The solvent-accessible surface (green) for the active
site cocrystal structure with 18b (Ki ) 0.76 µM) illustrates
the replacement of the acetyl group at the C7 NH indazole
position. This hydrophobic substituent better fits into the
“northern pocket” and removes the intramolecular hydrogen
bond observed in 18a . The channel to the right of the
4-chlorophenyl group is indicated (see text).
Exp er im en ta l Section
this channel. It was also clear that an additional arene
directly linked to this position would yield maximal
interaction. The first meta-biphenyl derivative prepared
(20b, with a 4-chlorophenyl group meta to the sulfona-
mide linkage, Table 3) provided gratifying confirmation
of this suggestion by improving binding approximately
60-fold.
Gen er a l Ch em istr y Meth od s. Proton NMR spectra were
run at 300 MHz on a Bru¨ker Avance 300 spectrometer, and
chemical shifts are reported in parts per million (δ) downfield
from tetramethylsilane as internal standard. Atmospheric
pressure ionization electrospray mass spectra and LCMS were
recorded on an Agilent 1100 series LC/MSD-SL 1946D spec-
trometer equipped with an Agilent 100 series HPLC System.
Silica gel 60 (230-400 mesh) from EM Science was used for
column chromatography, and analytical or preparative thin-
layer chromatography was conducted using EM Science Kie-
selgel 60 F254 plates. An Agilent 1100 Series HPLC with an
Agilent Zorbax Eclipse XDB-C8 (4.6 X 150 mm) reversed phase
column was used for analytical HPLC analyses. Preparative
RP-HPLC was performed on a Gilson instrument with a
MetaSil AQ 10 m C18 column. The elution buffer was an A/B
gradient; A ) H2O-0.1% TFA, B ) AcCN-0.1% TFA. For
reactions performed under anhydrous conditions, glassware
was either oven- or flame-dried, and the reaction was run
under a positive pressure of nitrogen. Anhydrous solvents were
used as purchased from commercial sources. Except where
noted, reagents were purchased from commercial sources and
used without further purification. The reported yields are the
actual isolated yields of purified material and are not opti-
mized.
3-Meth yl-5-n itr oin d a zole (4). 2-Chloro-5-nitroacetophe-
none (2) (1.00 g, 5 mmol) was dissolved in dry DMF (50 mL).
To this solution was added anhydrous hydrazine (0.166 g, 165
µL, 5.2 mmol), and the mixture was heated at 110 °C for 16 h.
The reaction mixture was then poured into saturated brine
solution and extracted three times with diethyl ether. The
combined extracts were then dried over MgSO4, filtered, and
concentrated. The resulting oil was purified by silica gel
chromatography (20% ethyl acetate-hexane) providing (700
mg, 86%) of product 4: 1H NMR (CD3OD) δ 8.74 (d, J ) 0.02
Hz, 1 H), 8.24 (dd, J ) 0.02, 0.1 Hz, 1 H), 7.57 (d, J ) 0.1 Hz,
1 H), 2.62 (s, 3 H); LCMS (API-ES) m/z 176.0 [M - H].
5-Dim eth yla m in o-n a p h th a len e-1-su lfon ic a cid (1H-in -
d a zol-5-yl)-a m id e (9a ) r ep r esen ta tive p r oced u r e for 9b-
9h . To a solution of 1H-indazol-5-ylamine (5) (133 mg, 1.0
mmol) in dry pyridine (4 mL) was added dansyl chloride (297
mg, 1.1 mmol) portionwise with stirring at ambient temper-
ature. The reaction mixture was allowed to stir for 2 h; after
which time LCMS indicated complete consumption of starting
aniline. The pyridine was removed in vacuo, and the residue
A co-structure of 20f with the enzyme confirmed this
binding hypothesis (Figure 6). The hydrophobic residues
F105, F151, V102, P113, and M173 together form the
channel and provide key contacts between the inhibitor
and protein. The interaction between F151 and the
inhibitor appears to be especially important. Here, as
in earlier inhibitor-protein complexes, staggered π-π
stacking interaction ( dist ) 4.0 Å) between the inda-
zole core and the phenyl ring of F151 in combination
with a π-σ interaction with the proximal arene of the
biarylsulfonamide should promote binding of these and
all the 4-chlorophenylsulfonamide derivatives. However,
in the case of the biaryl analogues, additional affinity
(10-475-fold) results from π-π stacking of the distal
arene, in this case, the pyrimidine ring with the phenyl
group of F105. This key interaction essentially locks the
inhibitor in the active site. Substitution on the distal
arene yields two benefits: small groups in the meta
position occupy a small cavity created by the side-chains
of M173, V102, and P113 and provide ca. 2-3-fold
additional affinity, while halogens in this and other
positions on the distal arene withdraw electron density
from this and proximal arene, which, from the derived
SAR, appears to promote favorable ligand-protein
interactions. The dihedral angle of the biaryl system in
bound 20f (32.7°; cf. the theoretically and empirically
expected angle of 50-60°) allows maximal π-π interac-
tion with F105 without perturbing the edge-on interac-
tion between F151 and F105. Mitigation of the energetic
penalties for this dihedral angle may further improve
the binding of these inhibitors.

SAH/ MTA Nucleosidase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5669
Ta ble 3. Trisubstituted Indzole Inhibitors of SAH/MTA Nucleosidase: 5-Biarylsulfonamides
was purified by RPHPLC (AcCN/H2O, 1.0% TFA) to afford 9a
as a tan solid. H NMR (DMSO-d6) δ 13.30 (s, 1H), 10.63 (s,
1H), 7.58 (t, 1H), 7.45 (m, 2H), 7.25 (m, 2H), 6.68 (m, 1H),
1
2.86 (s, 6H); LCMS m/z 368.9, 367.0 [M + H+]; 365.0, 367.0
1H), 8.45 (d, 1H), 8.05 (d, 1H), 7.88 (s, 1H), 7.55 (t, 1H), 7.35
(t, 1H), 7.28 (m, 2H), 7.25 (t, 2H), 6.94 (m, 1H), 2.87 (s, 6H);
LCMS m/z 368.9, 367.0 [M + H+]; 365.0, 366.0 [M + H-].
5-Dim eth yla m in o-n a p h th a len e-1-su lfon ic a cid (1H-In -
d a zol-6-yl)-a m id e (9b). 1H NMR (DMSO-d6) δ 13.40 (s, 1H),
11.03 (s, 1H), 8.48 (d, 1H), 8.38 (d, 1H), 8.18 (d, 1H), 7.92 (s,
[M + H-].
5-Dim eth yla m in o-n a p h th a len e-1-su lfon ic a cid (3-ch lo-
r o-1H-in d a zol-5-yl)-a m id e (9c). 1H NMR (DMSO-d6) δ 13.39
(s, 1H), 10.82 (s, 1H), 8.60 (m, 2H), 8.32 (m, 1H), 7.81-7.73
(m, 2H), 7.55 (m, 1H), 7.42 (m, 1H), 7.34 (m, 2H), 2.98 (s, 6H);
LCMS m/z 380.9, 383.0 [M + H+]; 379.1, 381.0 [M + H-].

5670 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Li et al.
F igu r e 6. (A) Cocrystal structure of inhibitor 20f (Ki ) 0.036 µM) bound to SAH/MTA nucleosidase (solvent-accessible surface
of the active site illustrated in green). (B) Ribbon diagram of the active site channel located at the dimer interface that accommodates
the biarene portion of 20f. The channel is comprised of residues I50, F151, and M173 from one monomer (cyan) and V102, F105,
and P113 (green).
Ta ble 4. Minimum Inhibitory Concentrations (MIC) for Select
Indazole MTA Nucleosidase Inhibitors
The combined extracts were dried over MgSO4, concentrated,
and the crude product was purified by flash column chroma-
tography (20% EtOAc-hexanes) to afford (1.5 g, 86%) of 12
MIC (µM)
1
as an orange solid: mp: 264-266 °C; H NMR (DMSO-d6) δ
compound Ki (µM) N. meningitidis S. pneumoniae S. pyogenes
13.51 (s, H), 7.79 (d, J ) 2.0 Hz, 2H), 7.41 (d, J ) 2.0 Hz, 2H),
6.16 (s, 2H); LCMS (API-ES) m/z 212.9, 214.9 [M + H+]; 210.9,
222.9 [M + H-].
18a
18b
20a
20b
20c
20h
20i
21c
20l
21d
20o
0.76
0.5
5
5
5
-
3.75
2.5
7.5
-
7.5
-
-
-
-
7.5
>20
3.75
1.25
7.5
>20
7.5
>20
>20
>20
>20
0.0034
0.0125
0.0016
0.052
0.0039
0.02
0.013
0.1
0.094
1.875
2.5
1.875
10
7.5
20
5
20
2.5
7-Am in o-5-n itr oin d a zole (13). According to the procedure
for compound 12 above, 5,7-dinitroindazole (11) (1.50 g, 6.8
mmol) and aq S(NH4)2 (2.93 M, 9.2 mL, 27 mmol) provided 13
(0.50 g, 61% yield) as a pale brown solid after silica gel
chromatography (50% EtOAc-hexanes): 1H NMR (DMSO-d6)
δ 12.76 (s, 1 H), 7.95 (d, J ) 0.02 Hz, 1 H), 7.28 (d, J ) 0.02
Hz, 1 H,), 5.90 (s, 2 H), 2.50 (s, 3 H); MS (API-ES) m/z 191 [M
- H].
N-[3-Ch lor o-5-n itr o-1H-in d a zol-7-yl]-a ceta m id e (14a ).
To a solution of 3-chloro-5-nitro-7-amino-1(2)H-indazole (12)
(500 mg, 2.3 mmol) in glacial AcOH (10 mL) was added Ac2O
(287 mg, 2.8 mmol). The reaction mixture was heated at 80
°C with stirring for 2 h. After cooling of the sample, the
mixture was quenched (H2O) and extracted with EtOAc (3 ×
50 mL). The combined organic extracts were washed with
NaHCO3 and brine and dried (MgSO4). Purification by silica
gel chromatography (20% EtOAc-hexane) provided 14a (380
5-Dim eth ylam in o-n aph th alen e-1-su lfon ic Acid (3-m eth -
yl-1H-in d a zol-5-yl)-a m id e (9d ). 1H NMR (DMSO-d6) δ 12.54
(s, 1H), 10.36 (s, 1H), 8.40 (m, 2H), 8.07 (m, 1H),7.62-7.52
(m, 2H), 7.24 (m, 3H), 6.93 (m, 1H), 2.80 (s, 6H), 2.32 (s, 3H);
LCMS m/z 380.9, 383.0 [M + H+]; 379.1, 381.0 [M + H-].
4-Ch lor o-N-(1H-in dazol-5-yl)-ben zen esu lfon am ide (9e).
1H NMR (DMSO-d6) δ 13.20 (s, 1H), 11.42 (s, 1H), 8.18 (s, 1H),
7.84, 7.75, (AA′,BB′, 2 × 2H), 7.60 (m, 2H), 7.23 (m, 1H); LCMS
m/z 309.9, 307.0 [M + H+]; 307.0, 305.0 [M + H-].
4-Ch lor o-N-(1H-in d a zol-6-yl)-ben zen esu lfon a m id e (9f).
1H NMR (DMSO-d6) δ 12.89 (s, 1H), 10.48 (s, 1H), 7.96 (s, 1H),
7.74, 7.63, (AA′,BB′, 2 × 2H), 7.25 (s, 1H), 6.88 (m, 1H); LCMS
m/z 309.9, 307.0 [M + H+]; 307.0, 305.0 [M + H-].
1
mg, 63%) as a tan solid: mp: 186-188 °C; H NMR (DMSO-
d6) δ 13.47 (s, H), 10.24 (s, H), 8.37 (d, J ) 2.0 Hz, 1H), 8.36
(d, J ) 2.0 Hz, 1H), 2.20 (s, 3H); LCMS m/z 254.9, 256.9 [M +
H+]; 254.9, 252.9 [M + H-].
3-Ch lor o-5-n itr o-7-isobu tyla m in o-1(2)H-in d a zole (14b).
3-Chloro-5-nitro-7-amino-1(2)H-indazole (12) (200 mg, 0.94
mmol) in AcCN (10 mL) was allowed to react with isovaler-
aldehyde (80 mg, 1.1 mmol) and NaBH(OAc)3 (0.3 g, 1.4 mmol)
at ambient temperature for 18 h. Evaporation to dryness and
purification by silica gel chromatography (20% EtOAc-hexane)
afforded 14b (180 mg, 72%) as an orange-red solid: mp: 178-
4-Ch lor o-N-(3-m eth yl-1H-in dazol-5-yl)-ben zen esu lfon a-
1
m id e (9h ). H NMR (DMSO-d6) δ 7.95-7.82 (m, 4H), 7.54 (s,
1H), 7.41 (d, J ) 8.7 Hz, 1H), 7.15 (d, J ) 8.7 Hz, 1H), 2.61 (s,
3H); LCMS m/z 322.7 [M + H+]; 320.8 [M + H-].
3-Met h yl-5,7-d in it r o-1H-in d a zole (11). 3-Methyl-5-ni-
troindazole (4) (2.36 g, 13 mmol) was dissolved in concentrated
H2SO4 (75 mL) and cooled to 0° C. To this solution was added
concentrated HNO3 (2.70 mL, 53 mmol) and the reaction was
stirred for 3 h while warming to room temperature. The crude
solution was poured onto crushed ice (100 g), and the resulting
solid was collected by vacuum filtration and dried over P2O5.
The product 11 was isolated (3.75 g, 94%) as the sulfuric acid
salt: 1H NMR (DMSO-d6) δ 9.27 (d, J ) 1.8 Hz, 1 H), 8.94 (d,
J ) 1.8 Hz, 1 H), 2.67 (s, 3 H).
1
180 °C; H NMR (DMSO-d6) δ 13.32 (s, H), 7.64 (d, J ) 2.0
Hz, 1H), 6.96 (d, J ) 2.0 Hz, 1H), 5.99 (m, 1H), 2.98 (m, 2H),
1.88 (m, 1H), 0.91 (m, 6H); LCMS m/z 269.0, 271.0 [M + H+];
267.0, 269.0 [M + H-].
In ter m ed ia tes 14c-14j. Synthesized according to the
general procedure above using 3-chloro-5-nitro-7-amino-1(2)-
H-indazole (12) (1 equiv), the appropriate aldehyde (1.2 equiv)
and NaBH(OAc)3 (1.5 equiv) in AcCN. The products thusly
derived were used directly without purification.
3-Ch lor o-5-n itr o-7-a m in o-1(2)H-in d a zole (12). A solu-
tion of 3-chloro-5,7-dinitro-1(2)H-indazole (10) (2.0 g, 8.2 mmol)
in EtOH (100 mL) was treated with 20% aqueous (NH4)2S (15
mL, 44.0 mmol) and the mixture was heated at 80 °C for 0.5
h. The solution was filtered, concentrated to one-third volume,
and then poured into brine and extracted with EtOAc (3×).
3-Ch lor o-5-am in o-7-isobu tylam in o-1(2)H-in dazole (16b).
A mixture of 3-chloro-5-nitro-7-isobutylamino-1(2)H indazole
(14b) (200 mg, 0.94 mmol), SnCl2‚H2O (1.0 g, 4.7 mmol) in
EtOH (10 mL) was heated at reflux for 18 h. The mixture was
cooled to room temperature, diluted with saturated aq NaH-
CO3, and extracted with EtOAc. The combined organic extracts

SAH/ MTA Nucleosidase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5671
were dried (MgSO4) and concentrated, and the product was
purified by silica gel chromatography (20% EtOAc-hexane)
affording 16b (180 mg, 72%) as an orange solid: mp: 143-
N-(7-Ben zyla m in o-3-ch lor o-1H-in d a zol-5-yl)-4-ch lor o-
ben zen esu lfon a m id e (18i). Prepared from 16i according to
the general procedure above: 1H NMR (DMSO-d6) δ 12.90 (s,
1H), 10.18 (s, 1H), 7.65 (m, 4H), 7.48 (m, 5H), 6.61 (d, J ) 2.0
Hz, 1H), 6.36 (d, J ) 2.0 Hz, 1H), 4.43 (s, 2H); LCMS m/z 445.9,
447.9 [M + H+]; 443.9, 445.9 [M + H-].
4-Ch lor o-N-(3-ch lor o-7-p h en eth yla m in o-1H-in d a zol-5-
yl)-ben zen esu lfon a m id e (18j). Prepared from 16j according
to the general procedure above: 1H NMR (DMSO-d6) δ 12.63
(s, 1H), 9.96 (s, 1H), 7.13-7.62 (m, 9H), 6.42 (d, J ) 2.0 Hz,
1H), 6.13 (d, J ) 2.0 Hz, 1H), 3.18 (m, 2H), 2.72 (m, 2H); LCMS
m/z 460.8, 462.8 [M + H+]; 458.9, 460.8 [M + H-].
1
145 °C; H NMR (DMSO-d6) δ 13.11 (s, 1H), 10.01 (bs, 2H),
6.59 (d, J ) 2.0 Hz, 1H), 6.16 (d, J ) 2.0 Hz, 1H), 2.94 (m,
2H), 1.88 (m, 1H), 1.05 (s, 3H), 1.03 (s, 3H); LCMS m/z 238.9,
240.9 [M + H+]; 236.9, 238.9 [M + H-].
In ter m ed ia tes 16a , 16c-16j. Synthesized according to the
procedure above for 16b using crude 14a , 14c-14j (1 equiv),
SnCl2‚H2O (4.7 equiv) and EtOH. Extractive workup yielded
the crude 5-aminoindazoles which were used without further
purification.
3-Br om o-N-(3-ch lor o-7-isob u t yla m in o-1H -in d a zol-5-
yl)-ben zen esu lfon a m id e (18k ). Prepared from 16b accord-
ing to the general procedure above: mp; 243-245 °C; 1H NMR
(DMSO-d6) δ 12.84 (s, 1H), 10.11 (s, 1H), 7.88 (m, 2H), 7.76
(m, 1H), 7.74 (m, 1H), 6.56 (d, J ) 2.0 Hz, 1H), 6.16 (d, J )
2.0 Hz, 1H), 5.80 (m, 1H), 2.91 (m, 2H), 1.88 (m, 1H), 1.02 (s,
N-[3-Ch lor o-5-(4-ch lor o-ben zen esu lfon yla m in o)-1H-in -
d a zol-7-yl]-a ceta m id e (18a ). 3-Chloro-5-amino-7-acetamide-
1(2)H-indazole (16a ) (20 mg, 0.09 mmol) dissolved in dry
pyridine (5 mL) was allowed to react with 4-chlorobenzene
sulfonyl chloride (23 mg, 0.1 mmol) at ambient temperature
for 16 h. The mixture was concentrated to dryness, recovered
in EtOAc, and washed with saturated aq. NaHCO3 and brine.
Removal of solvent and purification by RP-HPLC (AcCN/H2O,
1.0% TFA) afforded 18a (26 mg, 75%) as a tan solid: 1H NMR
(DMSO-d6) δ 12.73 (s, 1H), 10.06 (s, 1H), 7.73, 7.63, (AA′,BB′,
2 × 2H), 6.54 (d, J ) 2.0 Hz, 1H), 6.12 (d, J ) 2.0 Hz,1H),
5.82 (m, 1H), 2.11 (s, 3H); LCMS m/z 398.9, 400.9 [M + H+];
396.8, 398.9 [M + H-].
4-Ch lor o-N-(3-ch lor o-7-isob u t yla m in o-1H -in d a zol-5-
yl)-ben zen esu lfon a m id e (18b). Prepared from 16b accord-
ing to the general procedure above: 1H NMR (DMSO-d6) δ
7.79, 7.68 (AA′,BB′, 2 × 2H) 6.58 (d, J ) 2.0 Hz, 1H), 6.14 (d,
J ) 2.0 Hz, 1H), 2.92 (m, 2H), 1.86 (m, 1H), 1.03 (s, 3H), 1.01
(s, 3H); LCMS m/z 412.9, 414.9 [M + H+]; 410.9, 412.9 [M +
H-].
4-Ch lor o-N-[3-ch lor o-7-(cyclop r opylm eth yl-a m in o)-1H-
in d a zol-5-yl]-ben zen esu lfon a m id e (18c). Prepared from
16c according to the general procedure above: 1H NMR
(DMSO-d6) δ 12.81 (s, 1H), 10.03 (s, 1H), 7.71, 7.61 (AA′,BB′,
2 × 2H),6.51 (d, J ) 2.0 Hz, 1H), 6.14 (d, J ) 2.0 Hz, 1H),
5.82 (m, 1H), 2.90 (d, 2H), 1.04 (m, 1H), 0.53 (m, 2H), 0.26 (m,
2H); LCMS m/z 410.9, 412.9 [M + H+]; 408.9, 410.9 [M + H-].
4-Ch lor o-N-[3-ch lor o-7-(2,2-d im et h yl-p r op yla m in o)-
1H-in dazol-5-yl]-ben zen esu lfon am ide (18d). Prepared from
16d according to the general procedure above: 1H NMR
(DMSO-d6) δ 12.60 (s, 1H), 9.99 (s, 1H), 7.70, 7.58 (AA′,BB′, 2
× 2H), 6.51 (d, J ) 2.0 Hz, 1H), 6.14 (d, J ) 2.0 Hz, 1H), 5.82
(m, 1H), 2.82 (s, 2H), 0.95 (s, 9H); LCMS m/z 427.0, 428.9 [M
+ H+]; 424.9, 427.0 [M + H-].
4-Ch lor o-N-(3-ch lor o-7-(eth yla m in o)-1H-in d a zol-5-yl)-
ben zen esu lfon a m id e (18e). Prepared from 16e according to
the general procedure above: 1H NMR (DMSO-d6) δ 12.85 (s,
1H), 10.06 (s, 1H), 7.71, 7.61 (AA′,BB′, 2 × 2H), 6.49 (d, J )
2.0 Hz, 1H), 6.15 (d, J ) 2.0 Hz, 1H), 5.65 (m, 1H), 3.07 (m,
2H),1.22 (t, 3H); LCMS m/z 384.9, 386.9 [M + H+]; 382.9, 384.9
[M + H-].
4-Ch lor o-N-[3-ch lor o-7-(3-m et h yl-b u t yla m in o)-1H -in -
d a zol-5-yl]-ben zen esu lfon a m id e (18f). Prepared from 16f
according to the general procedure above: 1H NMR (DMSO-
d6) δ 12.82 (s, 1H), 10.10 (s, 1H), 7.76, 7.67 (AA′,BB′, 2 ×
2H),0.98 (m, 6H), 1.52 (m, 2H), 1.76 (m, 1H), 3.10 (m, 2H),
5.82 (m, 1H), 6.17 (d, J ) 2.0 Hz, 1H), 6.56 (d, J ) 2.0 Hz,
1H); LCMS m/z 427.0, 428.9 [M + H+]; 424.9, 426.9 [M + H-].
4-Ch lor o-N-[3-ch lor o-7-(3,3-d im eth yl-bu tyla m in o)-1H-
in d a zol-5-yl]-ben zen esu lfon a m id e (18 g). Prepared from
16g according to the general procedure above: 1H NMR
(DMSO-d6) δ 12.73 (s, 1H), 10.06 (s, 1H), 7.73, 7.63 (AA′,BB′,
2 × 2H), 6.54 (d, J ) 2.0 Hz, 1H), 6.12 (d, J ) 2.0 Hz, 1H),
5.82 (m, 1H), 3.05 (m, 2H), 1.50 (m, 2H), 0.98 (s, 9H); LCMS
m/z 440.9, 443.0 [M + H+]; 438.9, 440.9 [M + H-].
4-Ch lor o-N-[3-ch lor o-7-(3-m eth yl-bu t-2-en ylam in o)-1H-
in d a zol-5-yl]-ben zen esu lfon a m id e (18h ). Prepared from
16h according to the general procedure above: 1H NMR
(DMSO-d6) δ 12.70 (s, 1 H), 10.07 (s, 1 H), 7.71 (m, 2 H), 7.61
(m, 2 H), 6.50 (s, 1 H), 6.15 (s, 1 H), 5.27 (m, 1H), 3.63 (d, J )
6.4, 2 H), 1.74 (s, 3 H), 1.70 (s, 3 H); LCMS (API-ES) m/z 425.0
and 427.0 [M + H+], 423.0 and 425.0 [M - H-].
3H),1.01 (s, 3H); LCMS m/z 457.0, calcd mass 457.78 (C17H18
-
BrClN4O2S).
3-Br om o-N-(7-isob u t yla m in o-3-m et h yl-1H -in d a zol-5-
yl)-ben zen esu lfon a m id e (19a ). To a solution of N-7-isobutyl-
3-methyl-1H-indazole-5,7-diamine hydrochloride (17) (367 mg,
1.44 mmol) in dry pyridine (5 mL) was added 3-bromobenze-
nesulfonyl chloride (370 mg, 1.44 mmol) portionwise with
stirring at ambient temperature. The reaction mixture was
allowed to stir for 2 h; after which time LCMS indicates
complete consumption of starting aniline. The pyridine was
removed in vacuo, and the residue was dissolved in EtOAc (30
mL) and washed with 1% aq HCl and brine and dried over
anhydrous Na2SO4. Removal of solvent and purification by
flash chromatography (40-60% EtOAc/hexanes) provided 19a
as a tan solid (460 mg, 73%): 1H NMR (DMSO-d6) δ 12.14 (s,
1 H), 9.80 (s, 1 H), 7.85 (s, 1 H), 7.80 (d, J ) 7.1 Hz, 1 H), 7.77
(d, J ) 7.1 Hz, 1 H), 7.46 (t, J ) 8.0 Hz, 1 H), 6.56 (s, 1 H),
5.92 (s, 1 H), 5.56 (m, 1H), 2.80 (t, J ) 6.1 Hz, 2 H), 2.31 (s, 3
H), 1.70 (m, 1 H), 0.93 (s, 3 H), 0.91 (s, 3 H); 13C NMR (75
MHz, DMSO-d6) δ 179.3, 165.4, 165.0, 156.7, 147.4, 132.3,
130.7, 129.1, 129.0, 108.0, 104.7, 102.5, 98.0, 54.3, 43.0, 30.3,
27.5; LCMS (API-ES) m/z 436.9, 439.0 [M + H+]; 434.9, 436.9
[M - H-].
Gen er a l P r oced u r e for th e P r ep a r a tion of Bia r yl
Su lfon a m id es 20 a n d 21 via P d -Med ia ted Cr oss Cou -
p lin g. A mixture of 3-bromobenzenesulfonamide derivative (1
equiv), Pd(PPh3)4 (3 mol %), aryl boronic acid or ester (1.5
equiv), 2 M aq Na2CO3 (3 equiv) in DME (to 0.5 M sulfonamide)
is heated at 85 °C under an inert atmosphere for 5 h or until
HPLC or TLC analysis indicates complete reaction. The
mixture is then concentrated in vacuo, dissolved in EtOAc (20
mL), and washed with H2O (2 × 5 mL) and brine (1 × 5 mL)
and dried over anhydrous Na2SO4. Removal of solvent affords
the crude product that is purified by RP-HPLC.
3′-Ch lor o-4′-flu or o-bip h en yl-3-su lfon ic Acid (3-ch lor o-
7-isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20a ). Synthe-
sized according to the general procedure using 18k (50 mg,
0.1 mmol), 3-chloro-4-fluorophenylboronic acid (28 mg, 0.16
mmol) and Pd(PPh3)4 (2.0 mg). The crude product was purified
by RP-HPLC (AcCN/H2O, 1.0% TFA), affording 20a (44 mg,
80%) as a tan solid: 1H NMR (DMSO-d6) δ 12.78 (s, 1H), 9.92
(s, 1H), 8.01 (s, 1H), 7.54-7.96 (m, 8H), 6.58 (d, J ) 2.0 Hz,
1H), 6.16 (d, J ) 2.0 Hz, 1H), 5.71 (m, 1H), 2.72 (d, 2H), 1.77
(m, 1H), 0.89 (s, 3H), 0.87 (s, 3H); LCMS m/z 506.9, 508.9 [M
+ H+]; 504.8, 506.9 [M + H-].
4′-Ch lor o-bip h en yl-3-su lfon ic Acid (3-ch lor o-7-isobu -
tyla m in o-1H-in d a zol-5-yl)a m id e (20b). Derived from 16b
according to the general sulfonamide coupling conditions
described for 18a : 1H NMR (DMSO-d6) δ 12.64 (s, 1H), 10.11
(s, 1H), 7.93 (s, 1H), 7.45-7.83 (m, 8H), 6.58 (d, J ) 2.0 Hz,
1H), 6.17 (d, J ) 2.0 Hz, 1H), 2.72 (d, 2H), 1.78 (m, 1H), 0.89
(s, 3H), 0.87 (s, 3H); LCMS m/z 488.9, 490.9 [M + H+]; 486.8,
488.9 [M + H-].
3′,4′-Dich lor o-b ip h en yl-3-su lfon ic Acid (3-ch lor o-7-
isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20c). Prepared ac-
cording to the general sulfonamide coupling procedure. 1H
NMR (DMSO-d6) δ 12.74 (s, 1 H), 9.93 (s, 1 H), 8.03 (m, 1 H),

5672 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Li et al.
7.94 (m, 1 H), 7.83 (m, 1 H), 7.74 (m, 2 H), 7.61 (m, 2 H), 6.55
(s, 1 H), 6.13 (s, 1 H), 5.70 (m, 1H), 2.78 (d, J ) 6.6 Hz, 2 H),
1.73 (m, 1 H), 0.84 (d, J ) 6.8 Hz, 6 H); LCMS (API-ES) m/z
523.0, 525.0 [M + H+], 521.0, 523.0 [M - H-].
3′,4′-Diflu or o-b ip h en yl-3-su lfon ic Acid (3-Ch lor o-7-
isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20d ). Prepared ac-
cording to the general Pd-mediated cross coupling procedure:
1H NMR (DMSO-d6) δ 12.74 (s, 1 H), 9.93 (s, 1 H), 8.01 (m, 1
H), 7.90 (m, 1 H), 7.74-7.43 (m, 5 H), 6.55 (s, 1 H), 6.14 (s, 1
H), 5.69 (m, 1H), 2.79 (d, J ) 6.6 Hz, 2 H), 1.84 (m, 1 H), 0.94
(d, J ) 6.6 Hz, 6 H); LCMS (API-ES) m/z 491.0 [M + H+], 489.0
[M - H-].
N-(3-Ch lor o-7-isobu tyla m in o-1H-in d a zol-5-yl)-3-qu in o-
lin -3-yl-ben zen esu lfon a m id e (20e). Prepared according to
the general Pd-mediated cross coupling procedure: 1H NMR
(DMSO-d6) δ 12.85 (s, 1H), 10.01 (s, 1H), 9.26 (s, 1H), 9.25 (s,
1H), 8.63 (s, 1H), 7.75-8.25 (m, 8H), 6.67 (d, J ) 2.0 Hz, 1H),
6.25 (d, J ) 2.0 Hz, 1H), 5.72 (m, 1H), 2.86 (d, 2H), 1.80 (m,
1H), 0.88 (s, 3H), 0.86 (s, 3H); LCMS m/z 506.0, 508.0 [M +
H+]; 503.9, 505.9 [M + H-].
N-(3-Ch lor o-7-isobu tylam in o-1H-in dazol-5-yl)-3-(2-m eth -
yl-p yr im id in -4-yl)-ben zen esu lfon a m id e (20f). Prepared
according to the general sulfonamide coupling procedure: 1H
NMR (DMSO-d6) δ 12.79 (s, 1H), 10.13 (s, 1H), 8.87 (d, 1H),
8.68 (s, 1H), 8.35 (d, 1H), 7.94 (m, 2H), 7.75 (m, 2H), 6.61 (d,
J ) 2.0 Hz, 1H), 6.18 (d, J ) 2.0 Hz, 1H), 3.53 (s, 3H), 2.86
(m, 2H), 1.89 (m, 1H), 0.92 (s, 3H), 0.89 (s, 3H); LCMS m/z
470.9, 473.0 [M + H+]; 469.0, 470.9 [M + H-].
N-(3-Ch lor o-7-isobu tylam in o-1H-in dazol-5-yl)-3-(2-m eth -
yl-p yr id in -4-yl)-ben zen esu lfon a m id e (20 g). Prepared ac-
cording to the general Pd-mediated cross coupling procedure:
1H NMR (DMSO-d6) δ 12.76 (s, 1 H), 9.99 (s, 1 H), 8.66 (m, 1
H), 8.12 (m, 1 H), 8.07 (m, 1 H), 7.85 (m, 1 H), 7.70 (m, 3 H),
6.67 (s, 1 H), 6.14 (s, 1 H), 5.70 (m, 1H), 2.80 (d, J ) 6.6, 2 H),
2.60 (s, 3 H), 1.75 (m, 1 H), 0.86 (d, J ) 6.3 Hz, 6 H); LCMS
(API-ES) m/z 470.0 [M + H+], 468.0 [M - H-].
N-(3-Ch lor o-7-isobu t yla m in o-1H-in d a zol-5-yl)-3-p yr i-
d in -4-yl-ben zen esu lfon a m id e (20h ). Prepared according to
the general Pd-mediated cross coupling procedure: 1H NMR
(DMSO-d6) δ 12.76 (s, 1 H), 10.03 (s, 1 H), 8.80 (d, J ) 5.3 Hz,
1 H), 8.19 (s, 1 H), 8.09 (d, J ) 7.8 Hz, 1 H), 7.85 (m, 1 H),
7.88 (d, J ) 6.0 Hz, 2 H), 7.83 (d, J ) 7.8 Hz, 1 H), 7.71 (t, J
) 7.8 Hz, 1 H), 6.54 (s, 1 H), 6.14 (s, 1 H), 5.71 (m, 1H), 2.80
(d, J ) 6.7, 2 H), 1.74 (m, 1 H), 0.85 (d, J ) 6.6 Hz, 6 H);
LCMS (API-ES) m/z 456.0 [M + H+], 454.0 [M - H-].
4′-Ch lor o-3′-m eth yl-bip h en yl-3-su lfon ic Acid (3-ch lor o-
7-isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20i). Prepared
according to the general Pd-mediated cross coupling proce-
dure: 1H NMR (DMSO-d6) δ 12.75 (s, 1H), 9.94 (s, 1H), 7.94
(s, 1H), 7.40-7.80 (m, 7H), 6.55 (d, J ) 2.0 Hz, 1H), 6.13 (d, J
) 2.0 Hz, 1H), 5.72 (m, 1H), 2.80 (d, 2H), 2.37 (s, 3H), 1.81
(m, 1H), 0.86 (s, 3H), 0.84 (s, 3H); LCMS m/z 502.9, 504.9 [M
+ H+]; 500.9, 502.9 [M + H-].
1.73 (m, 1 H), 0.83 (d, J ) 6.6 Hz, 6 H); LCMS (API-ES) m/z
523.1 [M + H+], 521.1 [M - H-].
3′-Meth ylsu lfa n yl-bip h en yl-3-su lfon ic Acid (3-ch lor o-
7-isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20m ). Prepared
according to the general Pd-mediated cross coupling proce-
dure: 1H NMR (DMSO-d6) δ 12.87 (s, 1H), 9.99 (s, 1H), 8.02
(s, 1H), 7.23-7.79 (m, 8H), 6.59 (d, J ) 2.0 Hz, 1H), 6.18 (d, J
) 2.0 Hz, 1H), 5.70 (m, 1H), 3.71 (s, 3H), 2.74 (d, 2H), 1.79
(m, 1H), 0.90 (s, 3H), 0.88 (s, 3H); LCMS m/z 500.9, 503.0 [M
+ H+]; 499.0, 500.9 [M + H-].
3′-Meth yl-bip h en yl-3-su lfon ic Acid (3-ch lor o-7-isobu -
tyla m in o-1H-in d a zol-5-yl)-a m id e (20n ). Prepared according
to the general Pd-mediated cross coupling procedure: 1H NMR
(DMSO-d6) δ 12.81 (s, 1H), 9.90 (s, 1H), 7.86 (s, 1H), 7.17-
7.80 (m, 8H), 6.60 (d, J ) 2.0 Hz, 1H), 6.19 (d, J ) 2.0 Hz,
1H), 5.70 (m, 1H), 2.74 (d, 2H), 2.28 (s, 3H), 1.81 (m, 1H), 0.90
(s, 3H), 0.88 (s, 3H); LCMS m/z 469.0, 471.0 [M + H+]; 467.0,
469.0 [M + H-].
Bip h en yl-3-su lfon ic Acid (3-ch lor o-7-isobu tyla m in o-
1H-in d a zol-5-yl)-a m id e (20o). Prepared according to the
general sulfonamide coupling procedure: 1H NMR (DMSO-
d6) δ 12.80 (s, 1H), 10.04 (s, 1H), 7.93 (s, 1H), 7.90 (d, 1H),
7.62 (d, 1H), 7.52 (m, 3H), 7.38 (m, 3H), 6.50 (d, J ) 2.0 Hz,
1H), 6.20 (d, J ) 2.0 Hz, 1H), 2.84 (m, 2H), 1.80 (m, 1H), 0.91
(s, 3H), 0.88 (s, 3H); LCMS m/z 455.0, 457.0 [M + H+]; 452.9,
454.9 [M + H-].
3′-(2-Hydr oxy-eth yl)-biph en yl-3-su lfon ic Acid (3-ch lor o-
7-isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20p ). Prepared
according to the general Pd-mediated cross coupling proce-
dure: 1H NMR (DMSO-d6) δ 12.74 (s, 1 H), 9.96 (s, 1 H), 7.97
(m, 1 H), 7.86 (m, 1 H), 7.68 (m, 1 H), 7.58 (m, 1 H), 7.39 (m,
3 H), 7.27 (m, 1 H), 6.56 (s, 1 H), 6.16 (s, 1 H), 5.67 (m, 1H),
3.63 (t, J ) 6.9, 2 H) 2.78 (m, 4 H), 1.76 (m, 1 H), 0.87 (d, J )
6.3 Hz, 6 H); LCMS (API-ES) m/z 499.1 [M + H+], 497.1 [M -
H-].
N-(7-Isobu tylam in o-3-m eth yl-1H-in dazol-5-yl)-3-(2-m eth -
yl-p yr im id in -4-yl)-ben zen esu lfon a m id e (21a ). Prepared
according to the general sulfonamide coupling procedure: 1H
NMR (DMSO-d6) δ 9.77 (s, 1 H), 8.73 (d, J ) 5.3 Hz, 1 H),
8.54 (s, 1 H), 8.30 (d, J ) 7.1 Hz, 1H), 7.80 (d, J ) 5.4 Hz, 1
H), 7.76 (d, J ) 7.9 Hz, 1 H), 7.60 (t, J ) 7.8 Hz, 1 H), 6.55 (s,
1 H), 5.89 (s, 1H), 2.65 (d, J ) 6.1 Hz, 2 H), 2.61 (s, 3H), 2.24
(s, 3 H), 1.62 (m, 1 H), 0.75 (s, 3 H), 0.73 (s, 3 H); LCMS (API-
ES) m/z 450.6, [M + H+] 451.0.
N-(7-Isobu tyla m in o-3-m eth yl-1H-in d a zol-5-yl)-3-p yr i-
d in -4-yl-ben zen esu lfon a m id e (21b). Prepared according to
the general Pd-mediated cross coupling procedure: 1H NMR
(300 MHz, DMSO-d6) δ 9.72 (s, 1 H), 8.70 (d, J ) 5.4 Hz, 2 H),
8.09 (s, 1 H), 8.05 (d, J ) 8.6 Hz, 1H), 7.78 (d, J ) 5.5 Hz, 2
H), 7.76 (d, J ) 7.9 Hz, 1 H), 7.62 (t, J ) 7.8 Hz, 1 H), 6.55 (s,
1 H), 5.90 (s, 1H), 2.67 (d, J ) 6.6 Hz, 2 H), 2.24 (s, 3 H), 1.64
(m, 1 H), 0.78 (s, 3 H), 0.76 (s, 3 H); LCMS (API-ES) m/z 435.5,
[M + H+] 436.0.
3′-Tr iflu or om eth yl-bip h en yl-3-su lfon ic Acid (3-ch lor o-
7-isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20j). Prepared
according to the general Pd-mediated cross coupling proce-
dure: 1H NMR (DMSO-d6) δ 12.73 (s, 1H), 9.96 (s, 1H), 8.07
(s, 1H), 7.60-7.80 (m, 8H), 6.56 (d, 1H, J ) 2.0 Hz), 6.12 (d,
1H, J ) 2.0 Hz), 5.69 (m, 1H), 2.77 (d, 2H), 1.72 (m, 1H), 0.90
(s, 3H), 0.88 (s, 3H); LCMS m/z 523.0, 524.9 [M + H+]; 520.8,
522.8 [M + H-].
4′-F lu or o-3′-m eth yl-bip h en yl-3-su lfon ic Acid (3-ch lor o-
7-isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20k ). Prepared
according to the general Pd-mediated cross coupling proce-
dure: 1H NMR (DMSO-d6) δ 12.75 (s, 1H), 9.92 (s, 1H), 7.99
(s, 1H), 7.51-7.93 (m, 7H), 6.55 (d, J ) 2.0 Hz, 1H), 6.13 (d, J
) 2.0 Hz, 1H), 5.71 (m, 1H), 3.44 (s, 3H), 2.78 (d, 2H), 1.74
(m, 1H), 0.85 (s, 3H), 0.83 (s, 3H); LCMS m/z 487.0, 489.0 [M
+ H+]; 484.9, 486.9 [M + H-].
3′-Tr iflu or om eth yl-bip h en yl-3-su lfon ic Acid (7-isobu -
tyla m in o-3-m eth yl-1H-in d a zol-5-yl)-a m id e (21c). Prepared
according to the general Pd-mediated cross coupling proce-
dure: 1H NMR (300 MHz, DMSO-d6) δ 9.74 (s, 1 H), 8.06 (s,
1 H), 7.75 (m, 7 H), 6.63 (s, 1 H), 5.95 (s, 1H), 2.70 (d, J ) 4.3
Hz, 2 H), 2.31 (s, 3 H), 1.71 (m, 1 H), 0.79 (m, 6 H); LCMS
(API-ES) m/z 503.0 [M + H+]; 501.0 [M - H-].
3′-Meth yl-bip h en yl-3-su lfon ic Acid (7-isobu tyla m in o-
3-m eth yl-1H-in d a zol-5-yl)-a m id e (21d ). Prepared according
to the general Pd-mediated cross coupling procedure: 1H NMR
(DMSO-d6) δ 9.69 (s, 1 H), 7.89 (s, 1 H), 7.64 (d, J ) 7.7 Hz, 1
H), 7.33 (m, 4 H), 6.61 (s, 1 H), 5.98 (s, 1H), 2.70 (d, J ) 5.7
Hz, 2 H), 2.31 (m, 6 H), 1.71 (m, 1 H), 0.82 (m, 6 H); LCMS
(API-ES) m/z 449.0 [M + H+]; 447.0 [M - H-].
Bip h en yl-3-su lfon ic Acid (7-isobu tyla m in o-3-m eth yl-
1H-in d a zol-5-yl)-a m id e (21e). Prepared according to the
general sulfonamide coupling procedure: 1H NMR (DMSO-
d6) δ 9.72 (s, 1 H), 7.96 (s, 1 H), 7.85 (d, J ) 7.6 Hz, 1 H), 7.65
(d, J ) 7.7 Hz, 1 H), 7.56 (t, J ) 7.5 Hz, 1 H), 7.42 (m, 5 H),
6.61 (s, 1 H), 6.00 (s, 1H), 2.74 (t, J ) 6.8 Hz, 2 H), 2.30 (s, 3
H), 1.71 (m, 1 H), 0.83 (s, 3 H), 0.81 (s, 3 H); 13C NMR (75
MHz, DMSO-d6) δ 179.3, 165.4, 165.0, 156.7, 147.4, 132.3,
4′-Tr iflu or om eth yl-bip h en yl-3-su lfon ic Acid (3-ch lor o-
7-isobu tyla m in o-1H-in d a zol-5-yl)-a m id e (20l). Prepared
according to the general sulfonamide coupling procedure: 1H
NMR (DMSO-d6) δ 12.76 (s, 1 H), 10.00 (s, 1 H), 8.07 (m, 1 H),
7.96 (m, 1 H), 7.83 (m, 4 H), 7.75 (m, 1 H), 7.64 (m, 1 H), 6.55
(s, 1 H), 6.14 (s, 1 H), 5.70 (m, 1H), 2.78 (d, J ) 5.7 Hz, 2 H),

SAH/ MTA Nucleosidase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5673
130.7, 129.1, 129.0, 108.0, 104.7, 102.5, 98.0, 54.3, 43.0, 30.3,
27.5; LCMS (API-ES) m/z 435.0 [M + H+]; 433.0 [M - H-].
3′-Eth yl-bip h en yl-3-su lfon ic Acid N-(7-Isobu tyla m in o-
3-m eth yl-1H-in d a zol-5-yl)-3-a m id e (21f). Prepared accord-
ing to the general Pd-mediated cross coupling procedure: 1H
NMR (300 MHz, DMSO-d6) δ 9.65 (s, 1 H), 7.85 (s, 1H), 7.80
(d, J ) 7.8 Hz, 1 H), 7.59 (d, J ) 7.8 Hz, 1H), 7.50(t, J ) 7.8
Hz, 1 H), 7.30 (d, J ) 5.5 Hz, 2 H), 7.24 (s, 1 H), 7.22 (m, 1 H),
6.56 (s, 1H), 5.93 (s, 1H), 2.68 (d, J ) 6.7 Hz, 2 H), 2.55 (q, J
) 7.5 Hz, 2H), 2.25 (s, 3 H), 1.65 (m, 1 H), 1.12 (t, J ) 7.6 Hz,
3H), 0.77 (s, 3 H), 0.75 (s, 3 H); LCMS (API-ES) m/z 462.6, [M
+ H+] 463.1.
analyzed by RP-HPLC using a Zorbax 80A extend C8 column
developed with TFA/AcCN/water mobile phase. Peaks corre-
sponding to MTA were integrated, turnover calculated, and
plotted to determine IC50 and Ki. A Km of 1.0 µM for MTA
and E. coli SAH/MTA nucleosidase was used to calculate Ki.
Ack n ow led gm en t. The authors thank Dr. Steven
Cooper for invaluable assistance in the preparation of
this manuscript and Dr. Carol Dammel of our Preclini-
cal Microbiology Department for MIC determinations
and fruitful discussions.
Com p u ta tion a l Ch em istr y Meth od s. Homology modeling
for MTA nucleosidase utilized the published E. coli MTA
nucleosidase sequence, the homology program COMPOSER
(Tripos, Inc. St. Louis, MO), and the published crystal struc-
tures for human MTA phosphorylase (1CG6.pdb),3 human PNP
(1A9Q.pdb)2, and E. coli PNP (1A69.pdb).11 Protein and small
molecule modeling visualization were accomplished with Sybyl
v. 6.7 (Tripos, Inc., St. Louis, MO) and Macromodel v. 4.1
(Schro¨dinger, Inc., Portland, OR). Handling of the small
molecule virtual library, calculations of Lipinski properties,
conversion of the library to 3D structures, calculation of 2D
fingerprints, diversity clusters and selection were accom-
plished by the Unity 4.2, CONCORD, and SELECTOR mod-
ules in the Tripos suite (Tripos, Inc., St. Louis, MO). The
virtual library of commercially available compounds was
comprised of the Available Chemical Directory (MDL, San
Leandro, CA) and libraries from three other vendors. Protein-
small molecule docking experiments were performed using
FlexX. Conformational energies and solvation free energies
were calculated using Spartan (Wavefunction, Inc., Irvine, CA).
Exp r ession a n d P u r ifica tion of SAH/MTA Nu cleosi-
d a se. A C-terminally His-tagged, full-length E. coli pfs gene
encoding 238 amino acids was generated by PCR, inserted into
the pET 11a expression vector, and transformed into the E.
coli strain BL21-Gold (DE3) pLysS for protein expression.
Protein expression was induced in the transformed E. coli cells
using IPTG in the presence of ampicillin. Cells were harvested
by centrifugation at 4 °C and frozen before lysis. Cells were
thawed at ambient temperatures and resuspended in ice-cold
buffer A (25 mM Tris pH 8.5/ 5 mM DTT/ 5% v/v glycerol) with
20 mM NaCl and lysed by microfluidization using Microfluidics
L-110 per manufacturers recommended protocol. Crude lysate
was fractionated by centrifugation at 12000g for 30 min. The
tagged SAH/MTA nucleosidase protein was purified from the
supernatant fraction by IMAC. Eluted protein was dialyzed
against 20 mM KH2PO4, pH 7.0/100 mM NaCl, concentrated
to 15 mg/mL, and used for crystallization and enzymatic
assays. Cloning, purification, and assay of pathogenic SAH/
MTA nucleosidase proteins were performed similarly. All other
clones had an N-terminal histidine tag.
En zym a tic Assa ys. Spectrophotometric SAH detection
assays were performed in 96-well microtiter plates as follows;
in a final volume of 200 µL, 1.4 nM E. coli MTA nucleosidase
was incubated for 30 min at room temperature with 25 µM
SAH and 50 µM inhibitor in 50 mM Hepes pH 7.5/2.5% DMSO.
The SAH/MTA nucleosidase reaction was terminated by the
addition of 20 µL of SAH hydrolase and adenosine deaminase
mixture at final concentrations of 0.01 mg/mL SAH hydrolase;
4.1 µg/mL adenosine deaminase and incubated for an ad-
ditional 30 min. Color development was achieved by adding
DTNB to 100 µM, incubating for 10 min and then reading at
A405. The absorbance level was proportional to the amount
of SAH that did not get turned over during the SAH/MTA
nucleosidase reaction.
Direct HPLC assays were performed in 96-well microtiter
plates as follows: in a final volume of 200 µL, 50 pM E. coli
SAH/MTA nucleosidase was incubated for 10 min at room
temperature with 2.5 µM MTA, and inhibitor ranging in
concentration from 0.005 to 100 µM. The reaction was termi-
nated by the addition of 10 µL of 8% TFA. Stopped assays were
Refer en ces
(1) (a) Della Ragione, F.; Porcelli, M.; Carteni-Farina, M.; Zappia,
V.; Pegg, A. E. Escherichia coli S-Adenosylhomocysteine/5′-
Methylthioadenosine Nucleosidase. Biochem. J . 1985, 232, 335-
341. (b) Cornell, K. A.; Swarts, W. E.; Barry, R. D.; Riscoe, M.
K. Characterization of Recombinant Escherichia coli S-Adeno-
sylhomocysteine/5′-Methylthioadenosine Nucleosidase: Analysis
of Enzymatic Activity and Substrate Specificity. Biochem. Bio-
phys. Res. Comm. 1996, 228, 724-732. (c) Allart, B.; Gatel, M.;
Guillerm, D.; Guillerm, G. The Catalytic Mechanism of Adeno-
sylhomocysteine/Methylthioadenosine Nucleosidase from Es-
cherichia coli. Chemical Evidence for a Transition State with a
Substantial Oxocarbenium Character. Eur. J . Biochem. 1998,
256, 155-162. (d) Cornell, K. A.; Riscoe, M. K. Cloning and
Expression of Escherichia coli 5′-Methylthioadenosine/S-Adeno-
sylhomocysteine Nucleosidase: Identification of the pfs Gene
Product. Biochim. Biophys. Acta 1998, 1396, 8-14. (e) Lee, J .
E.; Cornell, K. A.; Riscoe, M. K.; Howell, P. L. Structure of E.
coli 5′-Methylthioadenosine/S-Adenosylhomocysteine Nucleosi-
dase Reveals Similarity to the Purine Nucleoside Phosphory-
lases. Structure 2001, 9, 941-953.
(2) Mao, C.; Cook, W. J .; Zhou, M.; Federov, A. A.; Almo, S. C.;
Ealick, S. E. Calf spleen purine nucleoside phosphorylase
complexed with substrates and substrate analogues. Biochem-
istry 1998, 37, 7135-46.
(3) Appleby, T. C.; Erion, M. D.; Ealick, S. E. The structure of human
5′-deoxy-5′-methylthioadenosine phosphorylase at 1.7 Å resolu-
tion provides insights into substrate binding and catalysis.
Struct. Fold Des. 1999, 7, 629-41.
(4) (a) Surette, M. G.; Miller, M. B.; Bassler, B. L. Quorum Sensing
in Escherichia coli, Salmonella typhimurium, and Vibrio har-
veyi: A New Family of Genes Responsible for Autoinducer
Production. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1639-1644.
(b) Schauder, S.; Shokat, K.; Surette, M. G.; Bassler, B. L. The
LuxS Family of Bacterial Autoinducers: Biosynthesis of a Novel
Quorum-Sensing Signal Molecule. Mol. Biol. 2001, 41, 463-476.
(5) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. F.
Experimental and computational approaches to estimate solubil-
ity and permeability in drug discovery and development setting.
Adv. Drug Del. Rev. 1997, 23, 3-25.
(6) V. Auwers, D. J ustus Lieb. Ann. Chem. 1927, 451, 285-302.
(7) Bergman, J .; Bergman, S.; Brimert, T. Tetrahedron 1999, 55,
10447-10466.
(8) Cai, S. X.; Huang, J .-C.; Espitia, S. A.; Tran, M.; Ilyin, V. I.;
Hawkinson, J . E.; Woodward, R. M.; Weber, E.; Keana, J . F. W.
5-(N-Oxya za )-7-su bst it u t ed-1,4-dih ydroqu in oxa line-2,3-di-
ones: Novel, Systemically Active and Broad Spectrum Antago-
nists for NMDA/glycine, AMPA, and Kainate Receptors. J . Med.
Chem. 1997, 40, 3679-3686.
(9) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C.
A.; Rekha, D.; Shah, R. D. Reductive Amination of Aldehydes
and Ketones with Sodium Triacetoxyborohydride. Studies on
Direct and Indirect Reductive Amination Procedures. J . Org.
Chem. 1996, 61, 3849-3862.
(10) (a) Hassan, J .; Sevignon, M.; Gozzi, C.; Schulz, E.; Lemaire, M.
Chem. Rev. 2002, 102, 1359-1470. (b) Miyaura, N.; Suzuki, A.
Chem. Rev. 1995, 2457-2483.
(11) Koellner, G.; Luic, M.; Shugar, D.; Saenger, W.; Bzowska, A.
Crystal structure of the tenary complex of E. coli purine
nucleoside phosphorylase with formycin B, a structural analogue
of the substrate inosine, and phosphate (Sulphate) at 2.1 Å
resolution. J . Mol. Biol. 1998, 280, 153-166.
(12) Chambers, C. C.; Hawkins, G. D.; Cramer, C. J .; Truhlar, D. G.
Model for aqueous solvation based on class IV atomic charges
and first solvation shell effects. J . Phys. Chem. 1996, 100,
16385-16398.
J M0302039