Inhibition of siderophore biosynthesis by 2-triazole substituted analogues of 5′-O-[N-(salicyl)sulfamoyl]adenosine: Antibacterial nucleosides effective against Mycobacterium tuberculosis

Article abstract of DOI:10.1021/jm8008037

The synthesis, biochemical, and biological evaluation of a systematic series of 2-triazole derivatives of 5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) are described as inhibitors of aryl acid adenylating enzymes (AAAE) involved in siderophore biosynthesis by Mycobacterium tuberculosis. Structure-activity relationships revealed a remarkable ability to tolerate a wide range of substituents at the 4-position of the triazole moiety, and a majority of the compounds possessed subnanomolar apparent inhibition constants. However, the in vitro potency did not always translate into whole cell biological activity against M. tuberculosis, suggesting that intrinsic resistance plays an important role in the observed activities. Additionally, the well-known valence tautomerism between 2-azidopurines and their fused tetrazole counterparts led to an unexpected facile acylation of the purine N-6 amino group.

Full text of DOI:10.1021/jm8008037

J. Med. Chem. 2008, 51, 7495–7507
7495
Inhibition of Siderophore Biosynthesis by 2-Triazole Substituted Analogues of
5′-O-[N-(Salicyl)sulfamoyl]adenosine: Antibacterial Nucleosides Effective against Mycobacterium
tuberculosis
Amol Gupte,† Helena I. Boshoff,‡ Daniel J. Wilson,† Joa˜o Neres,† Nicholas P. Labello,† Ravindranadh V. Somu,†
Chengguo Xing,§ Clifton E. Barry III,‡ and Courtney C. Aldrich*,†
Center for Drug Design, Academic Health Center, UniVersity of Minnesota, Minneapolis, Minnesota 55455, Tuberculosis Research Section,
National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, and Department of Medicinal Chemistry, UniVersity of
Minnesota, Minneapolis, Minnesota 55455
ReceiVed July 3, 2008
The synthesis, biochemical, and biological evaluation of a systematic series of 2-triazole derivatives of
5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) are described as inhibitors of aryl acid adenylating enzymes
(AAAE) involved in siderophore biosynthesis by Mycobacterium tuberculosis. Structure-activity relationships
revealed a remarkable ability to tolerate a wide range of substituents at the 4-position of the triazole moiety,
and a majority of the compounds possessed subnanomolar apparent inhibition constants. However, the in
vitro potency did not always translate into whole cell biological activity against M. tuberculosis, suggesting
that intrinsic resistance plays an important role in the observed activities. Additionally, the well-known
valence tautomerism between 2-azidopurines and their fused tetrazole counterparts led to an unexpected
facile acylation of the purine N-6 amino group.
Introduction
Invasive pathogens are dependent on iron obtained from the
human host; however, the concentration of free iron in human
serum and body fluids is 10-24 M, a concentration that is too
low to support bacterial colonization and growth.4 In order to
fulfill their iron needs, many bacteria synthesize, secrete, and
reimport small molecule iron chelators known as siderophores
that abstract iron from host proteins.5,6 M. tuberculosis as well
as many other Gram-negative and some Gram-positive bacteria
synthesize structurally related aryl-capped siderophores, as
shown in Figure 1A.7,8 Installation of the aryl moiety during
the biosynthesis of these aryl-capped siderophores is performed
by stand-alone aryl acid adenylation enzymes (AAAE; see
Figure 1B). Given the documented importance of many sid-
erophores for virulence, lack of human AAAE homologues,
available structural information on AAAEs, and knowledge of
the AAAE enzyme mechanism, several groups including ours
have reported on the synthesis of potent AAAE bisubstrate
inhibitors.9-12 The initial lead compound 5′-O-[N-(salicyl)sul-
famoyl]adenosine (Sal-AMS, 4, Figure 1C) has emerged as a
promising inhibitor of diverse bacterial AAAEs and was shown
to possess promising whole-cell activity toward both M.
tuberculosis and Yersinia sp.9,13 Extensive structure-activity
relationships of Sal-AMS have systematically explored the
aryl,14 linker,10,15-17 glycosyl,13 and nucleobase18 domains
(Figure 1C). These results have provided a comprehensive
understanding of the minimal structural requirements to maintain
activity and also have served to define positions amenable to
modification of this promising series of antibacterial agents. In
general, the aryl, linker, and glycosyl domains only tolerated
conservative modifications, while the nucleobase domain ex-
hibited substantial flexibility and provides the greatest op-
portunity to modulate physiochemical and drug disposition
properties. Molecular dynamics simulations of the AAAE from
Mtb revealed substantial plasticity in the nucleoside binding
pocket, allowing binding of Sal-AMS derivatives with large
substituents at C-2 of the purine.18 The ability to tolerate these
bulky C-2 substituents was not evident on the basis of the
Tuberculosis (TBa) caused by the slow growing bacillus,
Mycobacterium tuberculosis (Mtb), is the leading cause of death
due to a bacterial pathogen.1 The World Health Organization
(WHO) estimates that at least one-third of the world’s population
is infected with a latent form of this organism and that about
5-10% of these individuals will progress to the active form of
the disease during their lifetime.2 Further, co-infection with HIV
is especially deadly and serves as a trigger to convert latent TB
into an active transmissible infection. The current drug therapy
known as DOTS (directly observed treatment short-course)
requires 6-9 months of drug treatment and results in an overall
cure rate of approximately 85% (global average).2 However,
the emergence of multidrug resistant TB (MDR-TB) and
extensively drug resistant TB (XDR-TB), coupled with the lack
of any new antitubercular agents in over 4 decades, provides a
clear motivation for the development of new chemotherapeutic
agents to treat drug-resistant strains, target latent, nonreplicating
bacilli, and shorten the duration of treatment.3
In almost all living organisms, iron is an essential cofactor
that is required for numerous essential biochemical processes.
* To whom correspondence should be addressed. Phone: 612-625-7956.
Fax: 612-626-5173. E-mail: aldri015@umn.edu.
†
Academic Health Center, University of Minnesota.
National Institute of Allergy and Infectious Diseases.
Department of Medicinal Chemistry, University of Minnesota.
‡
§
a Abbreviations: AAAE, aryl acid adenylating enzyme; AMP, adenosine
monophosphate; ATP, adenosine triphosphate; CuAAC, copper-catalyzed
azide alkyne cycloaddition; DOTS, directly observed therapy short-course;
DMA, dimethylacetamide; DME, dimethoxyethane; DMF, dimethylforma-
mide; HIV/AIDS, human immunodeficiency virus/acquired immune defi-
ciency syndrome; Kiapp, apparent inhibition constant; MDR-TB, multidrug
resistant tuberculosis; MIC, minimum inhibitory concentration; MIC99,
minimum inhibitor concentration at which 99% of organisms are inhibited
in growth; MOM, methoxymethyl; Mtb, Mycobacterium tuberculosis; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHS, N-
hydroxysuccinimide; PPi, pyrophosphate; p-TSA, p-toluenesulfonic acid;
SAR, structure-activity relationships; Sal-AMS, 5′-O-[N-(salicyl)sulfa-
moyl]adenosine; TB, tuberculosis; TFA, trifluoroacetic acid; XDR-TB,
extensively drug resistant tuberculosis.
10.1021/jm8008037 CCC: $40.75
2008 American Chemical Society
Published on Web 11/14/2008

7496 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Gupte et al.
Figure 1. (A) Structure of representative aryl-capped siderophores. (B) Enzyme mechanism catalyzed by AAAEs. AAAE binds aryl acid 1, and
ATP and catalyzes their condensation to form an intermediate acyladenylate 2 that remains tightly bound to the active site. In a second half-
reaction, the AAAE catalyzes the transfer of the acyl group (blue) onto a nucleophilic sulfur atom of an aryl carrier domain to provide 3 with the
release of AMP. (C) 5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS, 4) is a bisubstrate inhibitor that mimics the acyladenylate 2 but replaces the
labile acylphosphate moiety with a stabile acylsulfamate group (pink). The modular scaffold of Sal-AMS can be disconnected into four subunits:
aryl (blue), linker (pink), glycosyl (black), and nucleobase (black).
cocrystal structure of an AAAE with a bound acyladenylate.19
Significantly, 2-Ph-Sal-AMS 5 (Figure 1C) was the most potent
inhibitor yet identified with Kiapp of 0.27 nM and exceptionally
potent antitubercular activity under iron deficient conditions
(MIC99 ) 0.049 µM).18
tives influences adenosine acylation at N-6 and we have
provided a clear mechanistic rationale for this fascinating
reactivity.
Results
Herein, we report our continued efforts to further explore
modification of the C-2 position of the purine moiety of Sal-
AMS 4 with a systematic series of 4-substituted 1,2,3-triazoles,
inspired from the work of Van Calenbergh, as isosteres of the
C-2 phenyl ring of 5.20 These triazole analogues ideally position
the 4-substituent of the 1,2,3-triazole moiety into a flexible
channel in the AAAE, which is directed toward the solvent
exposed surface. Additionally, we show that the well-known
azide-tetrazole valence tautomerism of 2-azidoadenosine deriva-
Chemistry. The synthesis of the intermediate 2-azido-5′-O-
sulfamoyladenosine 10 began from guanosine 6, which was
elaborated to 2-azidoadenosine 7 as described by Hata and co-
workers (Scheme 1).21 Methanolysis of 7 followed by acetonide
protection furnished 9, which was sulfamoylated to provide 10.22
Copper catalyzed coupling of 2-iodoadenosine derivative 823
with sodium azide provided an alternative route to compound
9.24

Inhibition of Siderophore Biosynthesis
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7497
Scheme 1a
a Reaction conditions: (a) (i) 7 N NH3 in MeOH, (ii) p-TSA, dimethox-
ypropane, acetone, 48%; (b) NaN3 (1.2 equiv), CuSO4 ·5H2O (0.2 equiv),
sodium ascorbate (0.2 equiv), L-proline (0.2 equiv), H2O/t-BuOH (1:1), 65
°C, 16 h, 52%; (c) sulfamoyl chloride, DMA, 67%.
Scheme 2a
Figure 2. Relative nucleophilicity of a fused T1-tetrazole. Qualitatively,
the most nucleophilic regions are shown in blue while the least
nucleophilic are red. Additionally calculation of the local Fukui function
(f-) using atomic charges determined by Mulliken population was
completed. The values of the function for the four most nucleophilic
atoms are shown. N-10 possesses the highest nucleophilicity with a
f- value of 0.093. The figure is a surface representation generated from
the Fukui function (f-), plotted on the 0.01 isodensity surface of a
truncated derivative of T1 tetrazole, whereby the C5′ hydroxymethyl
group was replaced with a hydrogen atom.
distinguished by the diagnostic chemical shift of H-8.28 Proton
NMR of 10 showed an 83:17:0 mixture of A/T1/T3 in CD3OD.
Czarnecki demonstrated that the equilibrium is dependent on
pH, solvent polarity, and temperature; however, the most
dominant effect is pH with the tetrazole valence tautomer
favored almost exclusively at high pH (>11) such as under the
basic reaction conditions employed (Cs2CO3, DMF).26 Indeed,
the condensed Fukui function (f-) based on Mulliken population
analysis revealed that the most nucleophilic atom in fused
tetrazole T1 is N-10 (see Figure 2 and Experimental Section).
Acylation at N-10 of fused tetrazole T1, followed by an
intramolecular N-N acyl shift to the N-6 amino group, provides
a potential mechanism for the experimentally observed product
12.
a Reaction conditions: (a) 11 (2.2 equiv), Cs2CO3 (3.0 equiv), DMF, 12 h,
53%; (b) 7 N NH3 in MeOH, 60 °C, 3 h, 61%; (c) salicylic acid, CDI,
DBU, MeCN, 60 °C, 2 h, 93%; (d) 80% aqueous TFA, 62% (from 14) or
54% (over two steps from 12).
Coupling of 10 with the N-hydroxysuccinimidyl (NHS) ester
of salicylic acid 1113 using modified Castro-Pichel coupling
conditions employing Cs2CO3 afforded the bisacylated com-
pound 12 in 53% yield as a result of competitive acylation at
N-6.25 Regioselective ammonolysis of the N-6 salicyl group was
successively achieved with methanolic ammonia to provide 13
in a modest 61% yield. The stability of the acylsulfamate linkage
of 12 under these conditions was noteworthy. Deprotection of
the MOM acetal and isopropylidene protecting groups was
accomplished with 80% aqueous TFA to provide the key
intermediates 2-azido-Sal-AMS 15.
The observation that 10 undergoes acylation at N-6 was
intriguing, as we had not observed this side reaction in any of
our previously described nucleobase-modified analogues.18 The
well-known valence tautomerism between 2-azidopurines (A)
and the corresponding fused tetrazoles (T1 and T3) (Scheme
2B) accounts for this apparent unique reactivity.26 Tetrazole
(CH2N4) has been described as a nucleophilic catalyst for
the regio- and chemoselective N-6 acylation of adenosine; thus,
we propose that the tetrazole tautomer (T1) is responsible for
the undesired acylation.27 These valence tautomers are easily
On the basis of the aforementioned analysis, we hypothesized
that less basic conditions, which disfavor the tetrazole valence
tautomer, would circumvent the undesired N-6 acylation.
Consequently, we used the complementary conditions developed
by Tan and co-workers employing DBU and salicylic acid to
provide 14 in 93% yield without any observed acylation at N-6.9
Subsequent, TFA mediated deprotection afforded 15. Following
the successful optimized synthesis of the key 2-azido-Sal-AMS
intermediate 15, Cu(I)-catalyzed azide-alkyne cycloaddition in
methanol at room temperature with a panel of 30 alkynes
selected to systematically explore van der Waal and electrostatic
interactions provided exclusively the 1,4-substituted triazole
analogues 16-45 in moderate to high yields (Scheme 3).29,30
Enzyme Inhibition. Enzyme assays were performed at 37
°C with recombinant MbtA expressed in E. coli in a buffer of
75 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT, 250 µM
salicylic acid, 10 mM ATP, and 1 mM PPi. The initial rates of
pyrophosphate exchange (e10% reaction) were monitored using
an enzyme concentration (typically 5-10 nM) by measuring
the amount of [32P]ATP formed after addition of [32P]PPi. The
enzyme concentration was determined by active-site titration

7498 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Gupte et al.
Scheme 3a
solvent exposed surface. However, cyclohexyl 31, cyclohexenyl
32, and all phenyl derivatives 33-45 exhibited docked poses
with some degree of distortion in the Sal-AMS core. On the
basis of previous molecular dynamics studies, which show
modest plasticity in this interdomain region of the protein, we
expect a small conformation change in the protein to alleviate
ligand strain.18 The relatively flat SAR observed for aryl
derivatives 33-45 is consistent with the relatively nonpolar
binding pocket, and docking failed to show any H-bond
interactions between the protein and nitrogen or oxygen atoms
in pyridyl, hydroxyphenyl, or aminophenyl groups in 33-45.
Derivative 16 was the only one whose docked conformation
exhibited hydrogen bonds with the active site residue, through
its hydroxyl group to Lys332 and Glu470.
a Reaction conditions: (a) terminal alkyne (3.0 equiv), Cu(OAc)2 (0.1
equiv), sodium ascorbate (0.1 equiv), MeOH, 25 °C, 16 h, 44-92%.
with inhibitor 4. The apparent inhibition constants (Kiapp) were
determined by fitting the concentration-response plots to the
Morrison equation (eq 1; see Experimental Section), since the
inhibitor exhibited tight-binding behavior (Kiapp e 100[E]). This
class of inhibitors has been shown to exhibit reversible and
competitive inhibition toward MbtA with respect to both
salicylic acid and ATP. All of the Kiapp values reported are
uncorrected for the supersaturating substrate concentrations
(salicylic acid held at 250 µM or 120KM; ATP held at 10 mM
or 55KM) and represent an upper limit of the true dissociation
constants.
Initially, a small series of 4-substituted triazoles derivatives
with small substituents was investigated including hydroxym-
ethyl 16, methoxycarbonyl 17, ethoxycarbonyl 18, and n-propyl
19 (see Table 1). These compounds were 2-7 times more potent
than the parent Sal-AMS 4. Encouraged by these results, a
systematic series of linear and branched alkyl derivatives 20-28
was prepared and evaluated for enzyme inhibition with sub-
stituents ranging from C4 to C12. The activity followed a
parabolic relationship with potency increasing with chain length
from C3 to C6 and then decreasing from C6 to C12. Notably,
n-hexyl 22 was the most potent triazole derivative evaluated
and 23-fold more potent than 4. Cycloalkyl derivatives 29-31
were approximately 12-21 more potent than 4. Interestingly,
incorporation of unsaturation in cyclohexenyl 32 and phenyl
33 led to a 5- and 10-fold loss of potency, respectively, relative
to cyclohexyl 31.
A methyl scan of phenyl derivative 33 was performed with
34-36 to define the steric requirements in the C-2 binding
pocket. The SAR from this showed that substitution at all
positions was well tolerated and the additional methyl group
led to a 3- to 7-fold improvement in potency relative to phenyl
33. Next, three series of aryl derivatives were evaluated
incorporating a combination of hydrogen bond donor and/or
acceptor capabilities including aminophenyl derivatives 37-39,
pyridyl analogues 40-42, and hydroxyphenyl compounds
43-45. The SAR from these three series was relatively flat,
demonstrating that electrostatic interactions do not play a
significant role and that the SAR is primarily driven by steric
considerations.
Biological Activity. Compounds 15-45 were evaluated for
whole-cell activity against M. tuberculosis H37Rv under iron-
limiting and iron-rich conditions. The minimum inhibitory
concentrations (MIC99) that inhibited >99% of cell growth are
shown in Table 1. Despite a fairly flat SAR profile in the enzyme
assay, substantially greater differences in biological activity were
observed for this series of 2-triazole derivatives. Methoxycar-
bonyl 17 and ethoxycarbonyl 18 displayed equal MIC values
consistent with their equipotent enzyme activity; however,
hydroxymethyl 16 was 2-fold less active than these ester
derivatives despite being 3-fold more potent in the enzyme
assay. Linear and branched alkyl derivatives 19-28 showed a
clear trend with decreasing activity as chain length increased
from C3 to C12 with an optimal activity achieved with a C3
substituent and no observed activity at C12. On the other hand,
cycloalkyl derivatives 29-32 containing rings from C3 to C6
did not display any apparent trend in activity, although the
relative activities of these compounds only varied 8-fold overall.
Significantly, the cycloalkyl compounds 29-32 were all more
active than their linear chain homologues 19-22. Phenyl
derivative 33 (MIC99 ) 3.13 µM) was found to be equipotent
to ester derivatives 17 and 18, consistent with their nearly
identical Kiapp values. The methylphenyl-, aminophenyl-, hy-
droxyphenyl-, and pyridyl-series of derivatives 34-45 displayed
activities ranging from 0.78 to 3.13 µM, representing a mere
4-fold difference in relative activities.
As initially pointed-out by Quadri and co-workers, antimy-
cobacterial activity observed under iron-rich conditions is
indicative of off-target effects, since siderophore production is
only required under iron-deficient conditions.9 As a benchmark,
the parent compound Sal-AMS is 4-fold less active under iron
deficient conditions, representing a selectivity of 4. Several of
the triazole derivatives examined including n-propyl 19, phenyl
33, and 2-hydroxyphenyl 43, and 3-hydroxyphenyl 44 exhibited
selectivities equal to or greater than 16.
Cytotoxicity. All compounds described herein were evaluated
for inhibition of cell viability against Vero cells using the MTT
assay (see Experimental Section); however, no inhibition of cell
growth was observed at 100 µM, the maximum concentration
evaluated. Phenyltriazole 33 was selected for more extensive
evaluation against MEL, OCL-3, and REH human cancer cell
lines. Cell proliferation of OCL-3 and REH lines were not
affected with 100 µM 33, while the MEL line exhibited an
approximately 25% inhibition of growth at 100 µM.
Physiochemical Properties. The ClogP values of 4 and
15-45 were calculated using the QikProp software (Schro¨d-
inger), while the permeability of select compounds was mea-
sured using an artificial membrane permeability assay (PAMPA)
(Table 1). The ClogP of Sal-AMS 4 is -0.89, while the ClogP
values of 15-45 ranged from -2.06 for hydroxymethyl 16 to
Molecular Modeling. In order to gain insight into the
observed SAR, docking studies of all ligands were performed
using a previously described MbtA homology model (see
Experimental Section). The docked poses of most compounds
allowed favorable positioning of the Sal-AMS core in the active
site, retaining most of the significant interactions with the active
site while positioning the triazole group in the interdomain
region (Figure 3A).10,18 The docked conformations showed the
triazole was essentially coplanar with the purine ring with a
dihedral angle defined by N3-C2-N1′-N2′ very close to 0°
(see Figure 3B). Compounds 16-18 that contain relatively small
4-substituents on the triazole as well as n-alkyl derivatives
19-26 allowed excellent positioning of the Sal-AMS core,
maintaining all key hydrogen bonds observed for Sal-AMS.
Notably, the C8-C12 chains in 24-26 were able to reach the

Inhibition of Siderophore Biosynthesis
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7499
Figure 3. (A) Docked pose of compound 26 in the active site of MbtA in the predicted binding mode (Glide). The protein is reduced to a ribbon
diagram with the N-terminal in red (residues 1-443), C-terminal in blue (residues 455-558), and linker in gray (residues 444-454) to demonstrate
the location of the triazole substituents with respect to the tertiary structure of the protein. The active site region is expanded showing the residues
that surround the triazole and respective substituent. (B) Syn- and anti-coplanar conformations of the triazole moiety. Only the syn conformation
(φ ) 0°) was observed during docking studies with MbtA.
+2.44 for n-dodecyl 26. Overall, the majority of analogues
possessed negative ClogP values except for phenyl 33, meth-
ylphenyl 34-36, and analogues incorporating at least six
carbons in an aliphatic chain. To put these values in perspective,
the recent report by O’Shea and Moser shows us that most
antibacterial drugs are substantially more polar than drugs from
other therapeutic classes.31 From their study of 147 antibacterial
agents, the average ClogP value of antibacterial agents effective
against Gram-negative organisms was -0.1, while the corre-
sponding ClogP values for antibacterial agents active against
Gram-positive organisms was +2.1.31
The PAMPA assay was used to measure passive transport,
and the measurable permeabilities varied from 0.02 × 10-6 to
2.65 × 10-6 cm/s. Notably, many compounds failed to show
any permeability in artificial membranes and no consistent
overall trend in permeabilities was evident. For instance, the
homologues C5-C12 n-alkyl series of 21-26 showed no
permeability at C5, excellent permeability at C6-C8 ((1.61-2.65)
× 10-6 cm/s), and greatly diminished permeability from
C10-C12 ((0.03-0.05) × 10-6 cm/s). Similarly, for the
regioisomeric methylphenyl series 34-36, the permeabilities
followed the trend 4-methyl >3-methyl >2-methyl, but this
trend was inconsistently followed for the other aryl series of
compounds 37-45.
atic series of 1,2,3-triazole analogues of Sal-AMS from a
common intermediate, 2-azido-Sal-AMS 15.32 The moderate
yields obtained in the key CuAAC reaction did not reflect upon
the coupling efficiency but rather losses obtained on purification
of the very polar nucleoside products. Notably, the mild
conditions (25 °C, MeOH) of the CuAAC reaction were
compatible with the highly functionalized, polar, and fully
deprotected nucleoside analogues. By contrast, our previously
reported syntheses of C-2 modified analogues of Sal-AMS relied
on an early stage Suzuki coupling, which necessitated harsh
reaction conditions (100 °C, 16 h, toluene), a multistep synthesis,
and requirement for protecting groups.18
The 2-azidoadenine hetereocycle exists as a mixture of azide
(A) and fused tetrazole (T1 and T3) valence tautomers because
of cyclization at N-1 or N-3 of the purine moiety.26 Consistent
with earlier studies, only the azide tautomer A and tetrazole T1
tautomer were observed as an approximately 5:1 mixture under
neutral conditions in MeOH. The finding that 2-azido-5′-O-
(sulfamoyl)adenosine derivative 10 underwent mild acylation
at N-6 was reconciled by invoking the T1 tetrazole tautomer to
promote acylation at N-10 of the tetrazole followed by a rapid
intramolecular N-N acyl shift onto N-6. To our knowledge,
this is the first observation of the unique reactivity of 2-azi-
doadenosine derivatives, and we expect that this facile N-6
acylation can be exploited to rapidly prepare libraries of N-6-
acyl C-2 triazole derivatives of adenine as well as other
heterocycles that potentially exist as tetrazole valence tautomers.
Notwithstanding this interesting reactivity profile, less basic
conditions were successfully employed to prevent N-6 acylation,
enabling an efficient four-step synthesis of the key intermediate
Discussion
A primary goal of this study was to explore the SAR of the
C-2 position of Sal-AMS. For this we turned to the Hu¨isgen-
Meldal-Sharpless copper(I)-catalyzed azide-alkyne cycload-
dition (CuAAC), which enabled rapid preparation of a system-

7500 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Table 1. Biological and Physiochemical Properties
Gupte et al.
MIC99
MIC99
permeability
compd
R
Kiapp (nM)a
6.6 ( 1.5h
0.27 ( 0.07j
5.1 ( 0.7
(µM)b (Fe-rich)
(µM)c (Fe-deficient)
(×10-6 cm/s)d
ClogPe
4
5
na
1.56i
0.049j
1.56
12.5
25
12.5-25
25
50
50
0.39i
0.39j
0.39
6.25
3.13
3.13
1.56
6.25
12.5
25
ndg
-0.89
f
naf
naf
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
-2.16
-2.06
-1.78
-1.42
-0.50
-0.20
0.09
0.48
0.74
1.07
1.74
hydroxymethyl
methoxycarbonyl
ethoxycarbonyl
n-propyl
n-butyl
n-pentyl
n-hexyl
n-heptyl
n-octyl
n-decyl
0.90 ( 0.12
3.13 ( 0.25
3.16 ( 0.16
1.58 ( 0.20
1.90 ( 0.15
0.61 ( 0.18
0.29 ( 0.09
0.41 ( 0.11
1.28 ( 0.21
5.10 ( 0.49
2.65 ( 0.43
1.40 ( 0.23
0.96 ( 0.11
0.46 ( 0.04
0.54 ( 0.12
0.32 ( 0.05
1.50 ( 0.13
3.23 ( 0.28
0.59 ( 0.17
0.95 ( 0.20
0.48 ( 0.07
0.77 ( 0.09
0.63 ( 0.06
0.88 ( 0.18
0.95 ( 0.11
0.86 ( 0.07
1.47 ( 0.12
1.13 ( 0.14
0.57 ( 0.11
0.61 ( 0.10
<0.01
<0.01
50
50
50
2.07 ( 1.29
2.65 ( 0.52
1.61 ( 0.53
0.03 ( 0.17
0.05 ( 0.15
<0.01
>25
>25
50
>50
>50
12.5
12.5
12.5
12.5
50
>50
>50
12.5
25
n-dodecyl
isobutyl
tert-butyl
>25
6.25
6.25
3.13
1.56
12.5
6.25
3.13
3.13
3.13
ndg
2.44
-0.21
-0.23
-0.67
-0.18
0.15
0.06
0.07
0.24
0.33
<0.01
cyclopropyl
cyclopentyl
cyclohexyl
cyclohex-1-enyl
phenyl
2-methylphenyl
3-methylphenyl
4-methylphenyl
2-aminophenyl
3-aminophenyl
4-aminophenyl
pyrid-2-yl
pyrid-3-yl
pyrid-4-yl
2-hydroxyphenyl
3-hydroxyphenyl
4-hydroxyphenyl
<0.01
<0.01
0.39 ( 0.33
0.18 ( 0.05
0.84 ( 0.08
1.03 ( 0.13
0.03 ( 0.08
<0.01
0.33 ( 0.04
0.12 ( 0.02
0.46 ( 0.10
<0.01
25
3.13
0.33
0.78
-0.69
-0.78
-0.78
-0.46
-0.78
-0.78
-0.51
-0.62
-0.62
6.25
3.13
3.13
3.13
25
0.78
0.78
0.78
0.78
1.56
1.56
1.56
0.12 ( 0.01
0.09 ( 0.21
<0.01
25
6.25
a Assay performed with 7 nM MbtA, 10 mM ATP, 250 µM salicylic acid, 1 mM PPi. b Grown in normal pH 6.6 glycerol-alanine salts (GAS) medium
without ferric ammonium citrate. c Grown in normal pH 6.6 glycerol-alanine salts (GAS) medium supplemented with 200 µM ferric ammonium citrate.
d Permeability assayed using a parallel artificial membrane permeability assay. e ClogP values were calculated using the QikProp software (Schro¨dinger).
f Not applicable. g Not determined. h See ref 13. i See ref 10. j See ref 18.
2-azido-Sal-AMS 15 from 2-azidoadenosine 7. Although not
reported in this study, 15 can serve as a photoaffinity probe to
identify potential off-target receptors. The finding that 15
possesses an identical iron-dependent phenotype as Sal-AMS
4 suggests that 15 is an excellent candidate probe to elucidate
the putative secondary mechanism(s) of action of this promising
new class of antibacterial agents.
The overall SAR from these studies was remarkably flat with
a mere 18-fold difference in potency across the entire series
(n-hexyl 22 has a Kiapp of 0.29 nM, while n-dodecyl 26 possesses
a Kiapp of 5.10 nM). Despite the fairly level SAR of triazole
analogues 16-45 from in vitro studies, the calculated ClogP
values and experimentally determined permeabilities varied
significantly, providing opportunities to modulate physiochemi-
cal and drug disposition properties of these nucleoside deriva-
tives. For instance, although both 2-methylphenyl 34 and
3-methylphenyl 35 have equal antitubercular activity, the latter
compound possesses an approximately 5-fold enhancement in
membrane permeability.
despite having potent enzyme inhibition. The lack of close
correlation between the in vitro enzyme inhibition and whole-
cell activity is likely a result of other factors such as intrinsic
resistance of the organism due to limited permeability across
the mycobacterial cell envelope. These results were opposite
to our initial expectations, since we hypothesized that the long
lipid tails of 22-26 would actually promote membrane transport.
Indeed Mollmann, Miller, and co-workers have shown that long
lipid tails enhanced mycobacterial uptake of a synthetic sid-
erophore derivative.33 Furthermore, the mycobactin siderophores
possess extremely long lipid tails of 17-20 carbons and recent
evidence suggests that these lipid tails facilitate distribution of
the mycobactins within macrophages.34,35
Conclusion
A systematic series of 4-substituted triazole derivatives of
2-(1,2,3-triazol-1-yl)-Sal-AMS was prepared and evaluated for
inhibition of the aryl acid adenylating enzyme (AAAE) known
as MbtA and activity against whole-cell M. tuberculosis under
iron-deficient and iron-replete conditions. Remarkably, this
entire series of compounds displayed potent AAAE inhibition
with a majority of compounds displaying exceptional subna-
nomolar potency. The triazole moiety was found to exist in a
coplanar syn-conformation with respect to the purine ring
projecting the triazole 4-substituent into a binding pocket leading
The whole-cell antitubercular activity showed a 64-fold
variance between the most and least active compounds (ami-
nophenyl 37 and 39 as well as pyridyl 40-42 analogues each
have a MIC99 of 0.78 µM, while n-decyl 25 possesses a MIC99
of 50 µM). Significantly, the long chain n-alkyl derivatives
22-26 from C6-C12 showed weak activity (MIC99 g 25 µM)

Inhibition of Siderophore Biosynthesis
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7501
6.0, 2.6 Hz, 1H), 6.08 (d, J ) 3.0 Hz, 1H), 8.20 (br s, 1H); 13C
NMR (150 MHz, CD3OD) δ 25.5, 27.8, 63.4, 81.7, 83.2, 86.6, 93.6,
114.4, 118.4, 140.1, 150.2, 156.5, 156.9; HRMS (ESI+) calcd for
C13H15N8O4 [M - H]+ 347.1211, found 347.1230 (error 5.5 ppm).
Procedure B. Sodium ascorbate (18.3 mg, 0.09 mmol, 0.2 equiv)
and CuSO4 ·5H2O (23.0 mg, 0.09 mmol, 0.2 equiv) were added to
a mixture of 2-iodo-2′,3′-O-isopropylideneadenosine23 (200 mg,
0.46 mmol, 1.0 equiv), sodium azide (36.0 mg, 0.55 mmol, 1.2
equiv), L-proline (10.6 mg, 0.09 mmol, 0.2 equiv), and sodium
carbonate (9.7 mg, 0.09 mmol, 0.2 equiv) in H2O/tert-BuOH (1:1,
10 mL). The mixture was stirred 16 h at 65 °C. After cooling to
room temperature, the mixture was partioned between 0.02 M
NH4OH (50 mL) and EtOAc (50 mL). The aqueous layer was
further extracted with EtOAc (2 × 50 mL), and the combined
organic layers were washed with saturated aqueous NaCl (50 mL),
dried (MgSO4), and concentrated. Purification by flash chromatog-
raphy (95:5 EtOAc/MeOH) afforded the title compound (85 mg,
52%) as a light-yellow solid.
to the solvent exposed surface, formed at the interface of the
N- and C-terminal domains. Docking studies recapitulated the
observed SAR and provided support that binding is primarily
driven by van der Waals interactions while electrostatic interac-
tions are insignificant in the largely nonpolar C-2 binding pocket.
For the majority of compounds, the trend of antitubercular
activities paralleled enzyme inhibition; however, n-alkyltriazole
derivatives 20-26 deviated significantly from this behavior with
activity progressively decreasing with n-alkyl chain length.
Several compounds including phenyltriazole 33 displayed
enhanced selectivity with a 16-fold greater activity under iron-
deficient conditions relative to iron-replete conditions. The
triazole derivatives of Sal-AMS described herein were found
to be nontoxic, providing a therapeutic index of greater than
100 for the most active analogues 37, 39-42. On the basis of
the observed potency, selectivity, lack of cytotoxicity, and
enhanced lipophilicity, phenyltriazole 33 is the best candidate
of the triazole series and merits further preclinical evaluation.
In closing, our results highlight the utility of the copper-
catalyzed azide alkyne cycloaddition (CuAAC) to assemble a
diverse series of triazole analogues to rapidly establish detailed
structure-activity relationships. Moreover, our finding on the
ease of N-6 acylation of 2-azidopurines, via a fused tetrazole
tautomer, provides insight into the unique reactivity of these
heterocyclic azides.
2-Azido-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine (10).
To a solution of compound 9 (1.57 g, 4.5 mmol, 1.0 equiv) in DME
(150 mL) was added NaH (540 mg, 13.5 mmol, 3.0 equiv), and
the reaction mixture was stirred for 30 min at room temperature.
Next, sulfamoyl chloride37 (780 mg, 6.75 mmol, 1.5 equiv)
dissolved in DME (20 mL) was added and the reaction mixture
was stirred overnight at room temperature. The reaction mixture
was filtered and concentrated. Purification by flash chromatography
(5:95 hexanes/EtOAc) afforded the title compound (1.28 g, 67%)
1
as a white solid: Rf ) 0.6 (95:5 EtOAc/MeOH); H NMR (600
MHz, CD3OD) δ 1.38 (s, 3H), 1.59 (s, 3H), 4.25 (dd, J ) 10.2,
5.4 Hz, 1H), 4.29 (dd, J ) 10.2, 4.2 Hz, 1H), 4.45-4.55 (m, 1H),
5.12 (dd, J ) 6.0, 3.0 Hz, 1H), 5.43 (dd, J ) 6.0, 2.4 Hz, 1H),
6.17 (d, J ) 1.8 Hz, 1H), 8.10 (s, 1H); 13C NMR (150 MHz,
CD3OD) δ 25.5, 27.5, 70.0, 83.0, 85.6, 85.9, 91.7, 115.6, 118.0,
141.2, 151.6, 158.2, 158.5; HRMS (ESI+) calcd for C13H18N9O6S
[M + H]+ 428.1095, found 428.1098 (error 0.7 ppm).
Experimental Section
General Procedures. All commercial reagents (Sigma-Aldrich,
Acros, Fluka), 3,3-dimethyl-1-butyne (Alfa Aesar) were used as
provided unless otherwise indicated. 2-Ethynylphenol and 4-ethy-
nylphenol were synthesized as reported.36 An anhydrous solvent
dispensing system (J. C. Meyer) using two packed columns of
neutral alumina was used for drying THF and CH2Cl2, while two
packed columns of molecular sieves were used to dry DMF and
the solvents were dispensed under argon. Anhydrous grade DMA,
DME, MeOH, and MeCN were purchased from Aldrich. Flash
chromatography was performed using Combiflash Companion
equipped with Luknova flash column silica cartridges (www.
luknova.com) with the indicated solvent system. All reactions were
performed under an inert atmosphere of dry Ar or N2 in oven-
dried (150 °C) glassware. 1H and 13C NMR spectra were recorded
on a Varian 600 MHz spectrometer. Proton chemical shifts are
reported in ppm from an internal standard of residual methanol
(3.31 ppm), and carbon chemical shifts are reported using an internal
standard of residual methanol (49.1 ppm). Proton chemical data
are reported as follows: chemical shift, multiplicity (s ) singlet, d
) doublet, t ) triplet, q ) quartet, p ) pentet, m ) multiplet, hep
) heptet), coupling constant, integration. High resolution mass
spectra were obtained on Agilent TOF II TOF/MS instrument
equipped with either an ESI or APCI interface. Analytical HPLC
were obtained on an Agilent 1100 series HPLC system with a PDA
detector.
2-Azido-2′,3′-O-isopropylideneadenosine (9). Procedure A. A
solution of 721 (2.4 g, 5.32 mmol, 1.0 equiv) in 7 N NH3 in MeOH
(120 mL, 840 mmol NH3, 158 equiv) was stirred at 50 °C for 15 h.
The reaction was concentated in vacuo, then placed under high
vacuum for several hours to remove traces of ammonia. The
resulting residue was suspended in acetone (150 mL), and p-TSA
(1.6 g, 8.47 mmol, 1.6 equiv) and 2,2-dimethoxypropane (4.8 mL,
39.0 mmol, 7.3 equiv) were added, and the mixture was stirred
16 h at room temperature. The reaction was quenched by adding
solid NaHCO3 (5.0 g), and the resulting heterogeneous slurry was
stirred for 30 min, then filtered and concentrated. Purification by
flash chromatography (95:5 EtOAc/hexane) afforded the title
compound (1.57 g, 48%) as a light-yellow solid: Rf ) 0.55 (EtOAc);
1H NMR (600 MHz, CD3OD) δ 1.36 (s, 3H), 1.58 (s, 3H), 3.70
(dd, J ) 12.0, 4.8 Hz, 1H), 3.75 (dd, J ) 11.4, 3.6 Hz, 1H),
4.20-4.35 (m, 1H), 5.20 (dd, J ) 6.0, 2.4 Hz, 1H), 5.29 (dd, J )
2-Azido-2′,3′-O-isopropylidene-5′-O-{N-[2-(methoxymethoxy)-
benzoyl]sulfamoyl}-N6-[2-(methoxymethoxy)benzoyl]adenosine Tri-
ethylammonium Salt (12). To a solution of N-hydroxysuccinimdyl
ester of MOM protected salicylic acid 1113 (1.3 g, 4.7 mmol, 2.0
equiv) in DMF (50 mL) at 0 °C was added 10 (1.0 g, 2.3 mmol,
1.0 equiv) and Cs2CO3 (1.9 g, 6.9 mmol, 3.0 equiv). The reaction
mixture was warmed to room temperature and stirred for an
additional 16 h. The mixture was concentrated under reduced
pressure and the residue taken up in EtOAc and filtered. The solids
were washed with additional EtOAc (100 mL), and the combined
filtrate was concentrated. Purification by flash chromatography (10:
90:0.5 EtOAc/MeOH/Et3N) afforded the title compound (721 mg,
53%) as a thick oil: Rf ) 0.6 (19:1 EtOAc/MeOH); 1H NMR (600
MHz, CD3OD) δ 1.25 (t, J ) 7.2 Hz, 9H), 1.39 (s, 3H), 1.61 (s,
3H), 3.17 (q, J ) 7.2 Hz, 6H), 3.40 (s, 3H), 3.53 (s, 3H), 4.31 (dd,
J ) 10.8, 3.0 Hz, 1H), 4.40 (dd, J ) 11.4, 3.6 Hz, 1H), 4.59-4.69
(m, 1H), 5.13 (s, 2H), 5.22 (d, J ) 6.0 Hz, 1H), 5.43 (dd, J ) 6.0,
2.4 Hz, 1H), 5.48 (s, 2H), 6.27 (d, J ) 3.0 Hz, 1H), 6.91 (t, J )
7.2 Hz, 1H), 7.06 (d, J ) 7.8 Hz, 1H), 7.10 (t, J ) 7.2 Hz, 1H),
7.23 (t, J ) 8.4 Hz, 1H), 7.32 (d, J ) 8.4 Hz, 1H), 7.39 (d, J ) 7.2
Hz, 1H), 7.54 (t, J ) 6.6 Hz, 1H), 8.03 (d, J ) 7.8 Hz, 1H), 8.70
(s, 1H); 13C NMR (150 MHz, CD3OD) δ 9.3, 25.6, 27.6, 49.9,
56.7, 57.7, 70.2, 83.4, 85.9, 86.2, 90.0, 96.6, 96.8, 115.3, 116.4,
117.2, 121.3, 122.6, 123.8, 130.1, 131.4, 132.1, 132.6, 133.2, 135.7,
144.5, 151.2, 154.8, 155.8, 157.1, 157.9, 165.1, 176.6; HRMS
(ESI-) calcd for C31H32N9O12S [M - H]- 754.1897, found
754.1893 (error 0.5 ppm).
2-Azido-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2′,3′-O-isopropy-
lideneadenosine Triethylammonium Salt (14). A solution of sali-
cylic acid (389 mg, 2.8 mmol, 1.2 equiv) and CDI (456 mg, 2.8
mmol, 1.2 equiv) in MeCN (35 mL) was stirred at 60 °C for 2 h.
The solution was cooled to room temperature. A mixture of 10
(1.0 g, 2.3 mmol, 1.0 equiv) and DBU (0.5 mL, 3.5 mmol, 1.5
equiv) was then added and the mixture stirred for 16 h at room
temperature. The reaction mixture was concentrated in vacuo and
purified by flash chromatography (70:30:0.5 EtOAc/MeOH/Et3N)

7502 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Gupte et al.
to afford the title compound (1.4 g, 93%) as a white solid: Rf )
0.3 (9.5:0.5 EtOAc/MeOH); 1H NMR (600 MHz, CD3OD) δ 1.24
(t, J ) 7.2 Hz, 9H), 1.33 (s, 3H), 1.56 (s, 3H), 3.16 (q, J ) 7.2 Hz,
6H), 4.28-4.31 (m, 1H), 4.33-4.35 (m, 1H), 4.51-4.53 (m, 1H),
5.08-5.10 (m, 1H), 5.35-5.36 (m, 1H), 6.14 (d, J ) 2.4 Hz, 2H),
6.74-6.78 (m, 2H), 7.27 (t, J ) 7.8 Hz, 1H), 7.88 (d, J ) 7.8 Hz,
1H), 8.28 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 9.3, 25.5, 27.5,
47.9, 70.2, 83.2, 85.6, 85.9, 91.6, 115.4, 117.7, 118.0, 119.4, 120.5,
131.3, 134.5, 141.1, 151.6, 158.0, 158.2, 162.0, 174.9; MS (APCI-)
calcd for C20H20N9O8S [M - H]- 546.1, found 546.2.
2-Azido-2′,3′-O-isopropylidene-5′-O-{N-[2-(methoxymethoxy)-
benzoyl]sulfamoyl}adenosine Triethylammonium Salt (13). To
a 50 mL glass cylindrical pressure vessel at 0 °C was added a
methanolic ammonia solution (20 mL, 7.0 M) and compound 12
(40 mg, 0.05 mmol). The reaction vessel was sealed with a Teflon
bushing and heated at 60 °C for 8 h. The reaction mixture was
concentrated in vacuo and carried forward to the next step without
further purification.
MHz, CD3OD) δ 52.8, 69.6, 72.2, 76.5, 84.7, 89.9, 117.9, 119.3,
120.1, 120.7, 128.4, 131.4, 134.4, 140.7, 142.2, 150.4, 151.4, 158.2,
162.1, 162.4, 175.1; HRMS (ESI-) calcd for C21H20N9O10S [M -
H]- 590.1059, found 590.1053 (error 1.0 ppm).
2-(4-Ethoxycarbonyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxyben-
zoyl)sulfamoyl]adenosine Sodium Salt (18). Ethyl propiolate was
reacted with 15 using the general procedure for triazole synthesis
and converted to the sodium salt by ion exchange using Dowex
50WX2-Na+ to afford the title compound (12.8 mg, 55%) as a
white amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR
(600 MHz, CD3OD) δ 1.42 (t, J ) 7.2 Hz, 3H), 4.35-4.36 (m,
1H), 4.40-4.46 (m, 5H), 4.70 (t, J ) 5.4 Hz, 1H), 6.18 (d, J ) 5.4
Hz, 1H), 6.74-6.78 (m, 2H), 7.27 (t, J ) 7.2 Hz, 1H), 7.92 (d, J
) 7.8 Hz, 1H), 8.59 (s, 1H), 9.24 (s, 1H); 13C NMR (150 MHz,
CD3OD) δ 18.5, 58.4, 69.6, 72.2, 76.5, 84.7, 89.9, 117.9, 119.3,
120.1, 120.7, 128.4, 131.5, 134.4, 140.7, 142.2, 150.4, 151.4, 158.2,
162.1, 162.4, 175.2; HRMS (ESI-) calcd for C22H22N9O10S [M -
H]- 604.1216, found 604.1250 (error 5.6 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-(4-n-propyl-1,2,3-tria-
zol-1-yl)adenosine Triethylammonium Salt (19). 1-Pentyne was
reacted with 15 using the general procedure for triazole synthesis
to afford the title compound (11.2 mg, 50%) as a white amorphous
solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 1.00 (t, J ) 7.2 Hz, 3H), 1.27 (t, J ) 7.2 Hz, 9H), 1.76
(h, J ) 7.2 Hz, 2H), 2.75 (t, J ) 7.2 Hz, 2H), 3.17 (q, J ) 7.2 Hz,
6H), 4.32-4.34 (m, 1H), 4.38-4.40 (m, 1H), 4.44-4.48 (m, 2H),
4.72 (t, J ) 5.4 Hz, 1H), 6.14 (d, J ) 5.4 Hz, 1H), 6.73-6.77 (m,
2H), 7.26 (t, J ) 7.2 Hz, 1H), 7.91 (d, J ) 8.4 Hz, 1H), 8.51 (s,
1H), 8.55 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 9.3, 14.1, 23.8,
28.4, 48.0, 69.5, 72.2, 76.2, 84.6, 89.9, 117.9, 119.3, 119.8, 120.7,
122.1, 131.5, 134.4, 142.0, 149.6, 150.9, 151.5, 158.1, 162.1, 175.1;
HRMS (ESI-) calcd for C22H24N9O8S [M - H]- 574.1474, found
574.1464 (error 1.7 ppm).
2-(4-n-Butyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)sul-
famoyl]adenosine Sodium Salt (20). 1-Hexyne was reacted with
15 using the general procedure for triazole synthesis and converted
to the sodium salt by ion exchange using Dowex 50WX2-Na+ to
afford the title compound (9.5 mg, 47%) as a white amorphous
solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 0.97 (t, J ) 7.2 Hz, 3H), 1.43 (h, J ) 7.2 Hz, 2H), 1.73
(p, J ) 7.2 Hz, 2H), 2.79 (t, J ) 7.8 Hz, 2H), 4.33-4.35 (m, 1H),
4.39-4.41 (m, 1H), 4.45-4.49 (m, 2H), 4.74 (t, J ) 5.4 Hz, 1H),
4.76 (s, 2H), 6.15 (d, J ) 5.4 Hz, 1H), 6.74-6.78 (m, 2H), 7.27 (t,
J ) 7.2 Hz, 1H), 7.92 (d, J ) 7.8 Hz, 1H), 8.53 (s, 1H), 8.58 (s,
1H); 13C NMR (150 MHz, CD3OD) δ 14.1, 23.8, 24.2, 28.4, 69.5,
72.2, 76.2, 84.6, 89.9, 118.0, 119.3, 119.8, 120.7, 122.2, 131.5,
134.4, 142.0, 149.7, 150.9, 151.6, 158.1, 162.1, 175.1; HRMS
(ESI-) calcd for C23H26N9O8S [M - H]- 588.1631, found
588.1634 (error 0.5 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-(4-n-pentyl-1,2,3-tria-
zol-1-yl)adenosine Triethylammonium Salt (21). 1-Heptyne was
reacted with 15 using the general procedure for triazole synthesis
to afford the title compound (16.4 mg, 71%) as a white amorphous
solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 0.91 (t, J ) 6.6 Hz, 3H), 1.27 (t, J ) 7.2 Hz, 9H),
1.35-1.38 (m, 4H), 1.73 (p, J ) 7.2 Hz, 2H), 2.76 (t, J ) 7.8 Hz,
2H), 3.17 (q, J ) 7.2 Hz, 6H), 4.32-4.34 (m, 1H), 4.38-4.40 (m,
1H), 4.44-4.48 (m, 2H), 4.73 (t, J ) 5.4 Hz, 1H), 6.14 (d, J ) 5.4
Hz, 1H), 6.73-6.77 (m, 2H), 7.26 (t, J ) 7.2 Hz, 1H), 7.91 (d, J
) 7.8 Hz, 1H), 8.51 (s, 1H), 8.54 (s, 1H); 13C NMR (150 MHz,
CD3OD) δ 9.3, 14.4, 23.5, 26.3, 30.3, 32.6, 48.1, 69.5, 72.2, 76.2,
84.6, 89.9, 117.9, 119.3, 119.8, 120.7, 122.1, 131.5, 134.4, 142.0,
149.8, 150.9, 151.5, 158.1, 162.1, 175.2; HRMS (ESI-) calcd for
C24H28N9O8S [M - H]- 602.1787, found 602.1781 (error 1.0 ppm).
2-(4-n-Hexyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)sul-
famoyl]adenosine Triethylammonium Salt (22). 1-Octyne was
reacted with 15 using the general procedure for triazole synthesis
to afford the title compound (17.8 mg, 75%) as a white amorphous
solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 0.90 (t, J ) 7.2 Hz, 3H), 1.27 (t, J ) 7.2 Hz, 9H),
1.32-1.34 (m, 4H), 1.38-1.42 (m, 2H), 1.73 (p, J ) 7.2 Hz, 2H),
2-Azido-5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]adenosine Tri-
ethylammonium Salt (15). To a solution of 14 (1.3 g, 2.0 mmol)
or 13 (crude oil from above) was added 80% aqueous TFA (20
mL) at 0 °C. The resulting solution was stirred for 3 h at room
temperature and then concentrated under reduced pressure. Purifica-
tion by flash chromatography (70:30:0.5 EtOAc/MeOH/Et3N)
afforded the title compound (771.8 mg, 62%, from 14 or 16.2 mg,
54%, over two steps from 12 via 13) as a white solid: Rf ) 0.5
(85:15 EtOAc/MeOH); 1H NMR (600 MHz, CD3OD) δ 1.29 (t, J
) 7.2 Hz, 9H), 3.20 (q, J ) 7.2 Hz, 6H), 4.27-4.32 (m, 1H),
4.36-4.44 (m, 3H), 4.66 (t, J ) 6.0 Hz, 1H), 6.00 (d, J ) 5.4 Hz,
1H), 6.75-6.82 (m, 2H), 7.28 (t, J ) 7.8 Hz, 1H), 7.93 (d, J ) 7.8
Hz, 1H), 8.40 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 9.3, 48.0,
69.8, 72.5, 76.1, 84.7, 89.2, 117.7, 117.9, 119.4, 120.7, 131.5, 134.4,
140.7, 152.5, 158.1, 158.3, 162.2, 175.5; HRMS (ESI-) calcd for
C17H16N9O8S [M - H]- 506.0848, found 506.0847 (error 0.1 ppm).
General Procedure for Triazole Synthesis. To a solution of azide
15 (0.033 mmol, 1.0 equiv) in MeOH (2 mL) at room temperature
was added Cu(OAc)2 (0.0033 mmol, 0.1 equiv dissolved in 100
µL of H2O), sodium ascorbate (0.0033 mmol, 0.1 equiv dissolved
in 100 µL H2O), and the appropriate alkyne (0.099 mmol, 3.0
equiv). The resulting mixture was stirred for 16 h at room
temperature. The reaction mixture was concentrated under reduced
pressure onto silica gel. Purification was carried out using a
Combiflash Companion flash chromatography system fitted with
4 g of Luknova silica cartridges and a solvent system containing
70:30:0.5 EtOAc/MeOH/Et3N, pumped at a flow rate of 18 mL/
min. The separation of the title compounds was detected at a UV
wavelength of 254 nm.
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-(4-hydroxymethyl-1,2,3-
triazol-1-yl)adenosine Triethylammonium Salt (16). Propargyl
alcohol was reacted with 15using the general procedure for triazole
synthesis to afford the title compound (15.9 mg, 73%) as a white
amorphous solid: Rf 0.4 (8:2 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 3.17 (q, J ) 7.2 Hz, 6H),
4.32-4.34 (m, 1H), 4.38-4.41 (m, 1H), 4.46-4.48 (m, 2H), 4.73
(t, J ) 5.4 Hz, 1H), 4.76 (s, 2H), 6.13 (d, J ) 5.4 Hz, 1H),
6.73-6.77 (m, 2H), 7.26 (t, J ) 7.2 Hz, 1H), 7.91 (d, J ) 7.2 Hz,
1H), 8.51 (s, 1H), 8.72 (s, 1H); 13C NMR (150 MHz, CD3OD) δ
9.3, 48.0, 56.6, 69.5, 72.2, 76.1, 84.6, 89.9, 117.9, 119.4, 119.9,
120.7, 123.1, 131.4, 134.4, 142.1, 149.5, 150.8, 151.5, 158.1, 162.1,
175.1; HRMS (ESI-) calcd for C20H20N9O9S [M - H]- 562.1110,
found 562.1138 (error 5.0 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-(4-methoxycarbonyl-
1,2,3-triazol-1-yl)adenosine Sodium Salt (17). Methyl propiolate was
reacted with 15 using the general procedure for triazole synthesis
and converted to the sodium salt by ion exchange using Dowex
50WX2-Na+ to afford the title compound (13.8 mg, 61%) as a
white amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR
(600 MHz, CD3OD) δ 3.97 (s, 3H), 4.35-4.36 (m, 1H), 4.40-4.42
(m, 1H), 4.45-4.46 (m, 2H), 4.70 (t, J ) 5.4 Hz, 1H), 6.18 (d, J
) 5.4 Hz, 1H), 6.74-6.78 (m, 2H), 7.27 (t, J ) 7.8 Hz, 1H), 7.92
(d, J ) 7.8 Hz, 1H), 8.60 (s, 1H), 9.28 (s, 1H); 13C NMR (150

Inhibition of Siderophore Biosynthesis
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7503
2.77 (t, J ) 7.8 Hz, 2H), 3.17 (q, J ) 7.2 Hz, 6H), 4.32-4.35 (m,
1H), 4.38-4.42 (m, 1H), 4.46-4.48 (m, 2H), 4.74 (t, J ) 5.4 Hz,
1H), 6.15 (d, J ) 5.4 Hz, 1H), 6.74-6.78 (m, 2H), 7.27 (t, J ) 7.2
151.5, 158.1, 162.2, 175.1; HRMS (ESI-) calcd for C31H42N9O8S
[M - H]- 700.2883, found 700.2862 (error 3.0 ppm).
2-(4-Isobutyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)sul-
famoyl]adenosine Triethylammonium Salt (27). 4-Methyl-1-pen-
tyne was reacted with 15 using the general procedure for triazole
synthesis to afford the title compound (14.6 mg, 64%) as a white
Hz, 1H), 7.92 (d, J ) 7.8 Hz, 1H), 8.51 (s, 1H), 8.55 (s, 1H); 13
C
NMR (150 MHz, CD3OD) δ 9.2, 14.4, 23.6, 26.3, 30.0, 30.4, 32.7,
48.0, 69.4, 72.1, 76.0, 84.5, 89.8, 117.8, 119.2, 119.8, 120.6, 122.0,
131.4, 134.3, 141.9, 149.7, 150.7, 151.4, 158.0, 162.0, 175.0; HRMS
(ESI-) calcd for C25H30N9O8S [M - H]- 616.1944, found
616.1943 (error 0.1 ppm).
1
amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); H NMR (600
MHz, CD3OD) δ 0.96 (d, J ) 6.6 Hz, 6H), 1.27 (t, J ) 7.2 Hz,
9H), 2.00-2.04 (m, 1H), 2.65 (d, J ) 7.2 Hz, 2H), 3.17 (q, J )
7.2 Hz, 6H), 4.32-4.34 (m, 1H), 4.38-4.40 (m, 1H), 4.44-4.47
(m, 2H), 4.72 (t, J ) 4.8 Hz, 1H), 6.15 (d, J ) 5.4 Hz, 1H),
6.73-6.77 (m, 2H), 7.26 (t, J ) 7.2 Hz, 1H), 7.92 (d, J ) 7.8 Hz,
1H), 8.51 (s, 1H), 8.55 (s, 1H); 13C NMR (150 MHz, CD3OD) δ
9.3, 22.7 (2C), 29.8, 35.4, 48.0, 69.5, 72.2, 76.2, 84.6, 89.8, 117.9,
119.3, 119.8, 120.7, 122.7, 131.5, 134.4, 142.0, 148.6, 150.9, 151.5,
158.1, 162.1, 175.2; HRMS (ESI-) calcd for C23H26N9O8S [M -
H]- 588.1631, found 588.1634 (error 0.5 ppm).
2-(4-n-Heptyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)sul-
famoyl]adenosine Triethylammonium Salt (23). 1-Nonyne was
reacted with 15 using the general procedure for triazole synthesis
to afford the title compound (22.3 mg, 92%) as a white amorphous
solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 0.90 (t, J ) 7.2 Hz, 3H), 1.27 (t, J ) 7.2 Hz, 9H),
1.28-1.32 (m, 4H), 1.32-1.38 (m, 4H), 1.73 (p, J ) 7.2 Hz, 2H),
2.77 (t, J ) 7.2 Hz, 2H), 3.18 (q, J ) 7.2 Hz, 6H), 4.32-4.35 (m,
1H), 4.38-4.42 (m, 1H), 4.46-4.49 (m, 2H), 4.74 (t, J ) 5.4 Hz,
1H), 6.15 (d, J ) 5.4 Hz, 1H), 6.74-6.78 (m, 2H), 7.27 (t, J ) 7.2
2-(4-tert-Butyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)sul-
famoyl]adenosine Triethylammonium Salt (28). 3,3-Dimethyl-1-
butyne was reacted with 15 using the general procedure for triazole
synthesis to afford the title compound (15.2 mg, 67%) as a white
Hz, 1H), 7.92 (d, J ) 7.8 Hz, 1H), 8.51 (s, 1H), 8.55 (s, 1H); 13
C
NMR (150 MHz, CD3OD) δ 9.2, 14.4, 23.7, 26.3, 30.1, 30.2, 30.5,
32.9, 48.0, 69.4, 72.1, 76.0, 84.5, 89.8, 117.8, 119.2, 119.7, 120.6,
122.0, 131.4, 134.3, 141.9, 149.7, 150.8, 151.4, 158.0, 162.0, 175.1;
HRMS (ESI-) calcd for C26H32N9O8S [M - H]- 630.2100, found
630.2114 (error 2.2 ppm).
1
amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); H NMR (600
MHz, CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 1.42 (s, 9H), 3.17 (q,
J ) 7.2 Hz, 6H), 4.34-4.35 (m, 1H), 4.40-4.42 (m, 1H),
4.44-4.46 (m, 2H), 4.68 (t, J ) 5.4 Hz, 1H), 6.18 (d, J ) 5.4 Hz,
1H), 6.75-6.78 (m, 2H), 7.27 (t, J ) 7.2 Hz, 1H), 7.91 (d, J ) 7.8
Hz, 1H), 8.51 (s, 1H), 8.56 (s, 1H); 13C NMR (150 MHz, CD3OD)
δ 9.2, 30.5, 31.8, 48.0, 69.5, 72.2, 76.4, 84.6, 89.5, 117.8, 119.2,
119.6, 119.8, 120.6, 131.4, 134.3, 141.7, 150.9, 151.6, 158.0, 158.8,
162.0, 175.0; HRMS (ESI-) calcd for C23H26N9O8S [M - H]-
588.1631, found 588.1625 (error 1.0 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-(4-n-octyl-1,2,3-triazol-
1-yl)adenosine Triethylammonium Salt (24). 1-Decyne was reacted
with 15 using the general procedure for triazole synthesis to afford
the title compound (16.1 mg, 65%) as a white amorphous solid: Rf
) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz, CD3OD) δ 0.89
(t, J ) 7.2 Hz, 3H), 1.27 (t, J ) 7.2 Hz, 9H), 1.31-1.39 (m, 10H),
1.73 (p, J ) 7.2 Hz, 2H), 2.76 (t, J ) 7.2 Hz, 2H), 3.18 (q, J ) 7.2
Hz, 6H), 4.33-4.35 (m, 1H), 4.39-4.41 (m, 1H), 4.46-4.49 (m,
2H), 4.74 (t, J ) 5.4 Hz, 1H), 6.15 (d, J ) 5.4 Hz, 1H), 6.74-6.78
(m, 2H), 7.27 (t, J ) 7.2 Hz, 1H), 7.92 (d, J ) 8.4 Hz, 1H), 8.51
(s, 1H), 8.55 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 9.2, 14.4,
23.7, 26.3, 30.3, 30.3, 30.4, 30.5, 33.0, 48.0, 69.4, 72.1, 76.0, 84.5,
89.8, 117.8, 119.2, 119.7, 120.6, 122.0, 131.4, 134.3, 141.9, 149.7,
150.7, 151.4, 158.0, 162.0, 175.1; HRMS (ESI-) calcd for
C27H34N9O8S [M - H]- 644.2257, found 644.2250 (error 1.1 ppm).
2-(4-n-Decyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)sul-
famoyl]adenosine Triethylammonium Salt (25). 1-Dodecyne was
reacted with 15 using the general procedure for triazole synthesis
to afford the title compound (15.4 mg, 60%) as a white amorphous
solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 0.87 (t, J ) 7.2 Hz, 3H), 1.27 (t, J ) 7.2 Hz, 9H),
1.31-1.41 (m, 14H), 1.72 (p, J ) 7.2 Hz, 2H), 2.76 (t, J ) 7.2
Hz, 2H), 3.17 (q, J ) 7.2 Hz, 6H), 4.32-4.34 (m, 1H), 4.38-4.40
(m, 1H), 4.44-4.48 (m, 2H), 4.73 (t, J ) 5.4 Hz, 1H), 6.14 (d, J
) 5.4 Hz, 1H), 6.73-6.77 (m, 2H), 7.26 (t, J ) 7.2 Hz, 1H), 7.92
(d, J ) 8.4 Hz, 1H), 8.50 (s, 1H), 8.55 (s, 1H); 13C NMR (150
MHz, CD3OD) δ 9.3, 14.5, 23.8, 26.4, 30.4, 30.54, 30.55, 30.56,
30.78, 30.81, 33.2, 48.1, 69.5, 72.2, 76.2, 84.6, 89.9, 117.9, 119.3,
119.8, 120.7, 122.1, 131.5, 134.4, 142.0, 149.8, 150.9, 151.5, 158.1,
162.1, 175.2; HRMS (ESI-) calcd for C29H38N9O8S [M - H]-
672.2570, found 672.2558 (error 1.8 ppm).
2-(4-Cyclopropyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)-
sulfamoyl]adenosine Triethylammonium Salt (29). Cyclopropy-
lacetylene was reacted with 15 using the general procedure for
triazole synthesis to afford the title compound (15.8 mg, 71%) as
a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR
(600 MHz, CD3OD) δ 0.87-0.90 (m, 2H), 1.00-1.03 (m, 2H),
1.27 (t, J ) 7.2 Hz, 9H), 2.02-2.08 (m, 1H), 3.17 (q, J ) 7.2 Hz,
6H), 4.34-4.35 (m, 1H), 4.39-4.42 (m, 1H), 4.45-4.48 (m, 2H),
4.72 (t, J ) 5.4 Hz, 1H), 6.14 (d, J ) 5.4 Hz, 1H), 6.74-6.78 (m,
2H), 7.27 (t, J ) 7.8 Hz, 1H), 7.92 (t, J ) 7.8 Hz, 2H), 8.48 (s,
1H), 8.51 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 7.3, 8.27, 8.31,
9.2, 47.9, 69.4, 72.1, 76.1, 84.5, 89.7, 117.8, 119.2, 119.7, 120.6,
120.7, 131.4, 134.3, 141.9, 150.7, 151.4, 151.8, 158.0, 162.0, 175.1;
HRMS (ESI-) calcd for C22H22N9O8S [M - H]- 572.1318, found
572.1335 (error 3.0 ppm).
2-(4-Cyclopentyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)-
sulfamoyl]adenosine Triethylammonium Salt (30). Cyclopenty-
lacetylene was reacted with 15 using the general procedure for
triazole synthesis to afford the title compound (15.6 mg, 67%) as
a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR
(600 MHz, CD3OD) δ 1.26 (t, J ) 7.2 Hz, 9H), 1.70-1.76 (m,
4H), 1.80-1.82 (m, 2H), 2.12-2.17 (m, 2H), 3.17 (q, J ) 7.2
Hz,6H), 3.23 (p, J ) 7.8 Hz, 1H), 4.33-4.34 (m, 1H), 4.38-4.41
(m, 1H), 4.44-4.47 (m, 2H), 4.71 (t, J ) 5.4 Hz, 1H), 6.15 (d, J
) 5.4 Hz, 1H), 6.73-6.77 (m, 2H), 7.26 (t, J ) 7.8 Hz, 1H), 7.91
(d, J ) 7.8 Hz, 1H), 8.51 (s, 1H), 8.52 (s, 1H); 13C NMR (150
MHz, CD3OD) δ 9.3, 26.2, 34.20, 34.23, 37.9, 48.1, 69.6, 72.2,
76.3, 84.6, 89.8, 117.9, 119.3, 119.8, 120.7, 121.0, 131.5, 134.4,
141.9, 150.9, 151.6, 153.9, 158.1, 162.1, 175.2; HRMS (ESI-)
calcd for C24H26N9O8S [M - H]- 600.1631, found 600.1646 (error
2.5 ppm).
2-(4-n-Dodecyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)sul-
famoyl]adenosine Triethylammonium Salt (26). 1-Tetradecyne was
reacted with 15 using the general procedure for triazole synthesis
to afford the title compound (13.5 mg, 51%) as a white amorphous
solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 0.88 (t, J ) 7.2 Hz, 3H), 1.27 (t, J ) 7.2 Hz, 9H),
1.28-1.42 (m, 18H), 1.72 (p, J ) 7.2 Hz, 2H), 2.76 (t, J ) 7.2
Hz, 2H), 3.18 (q, J ) 7.2 Hz, 6H), 4.33-4.34 (m, 1H), 4.38-4.40
(m, 1H), 4.44-4.48 (m, 2H), 4.74 (t, J ) 5.4 Hz, 1H), 6.14 (d, J
) 5.4 Hz, 1H), 6.73-6.77 (m, 2H), 7.26 (t, J ) 7.2 Hz, 1H), 7.92
(d, J ) 7.2 Hz, 1H), 8.51 (s, 1H), 8.56 (s, 1H); 13C NMR (150
MHz, CD3OD) δ 9.3, 14.5, 23.8, 26.4, 30.4, 30.54, 30.56 (2C),
30.76, 30.83, 30.86, 30.87, 33.2, 48.1, 69.5, 72.2, 76.2, 84.6, 89.9,
117.9, 119.3, 119.8, 120.7, 122.1, 131.5, 134.4, 142.1, 149.8, 150.9,
2-(4-Cyclohexyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxybenzoyl)-
sulfamoyl]adenosine Triethylammonium Salt (31). Cyclohexy-
lacetylene was reacted with 15 using the general procedure for
triazole synthesis to afford the title compound (15.1 mg, 64%) as
a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR
(600 MHz, CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 1.30-1.34 (m,
1H), 1.45 (q, J ) 12.0 Hz, 2H), 1.52 (q, J ) 12.0 Hz, 2H),
1.74-1.76 (m, 1H), 1.83-1.85 (m, 2H), 2.07-2.09 (m, 2H),
2.77-2.84 (m, 1H), 3.18 (q, J ) 7.2 Hz, 6H), 4.34-4.35 (m, 1H),

7504 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Gupte et al.
4.39-4.42 (m, 1H), 4.46-4.48 (m, 2H), 4.72 (t, J ) 5.4 Hz, 1H),
6.16 (d, J ) 5.4 Hz, 1H), 6.74-6.78 (m, 2H), 7.27 (t, J ) 7.2 Hz,
1H), 7.92 (d, J ) 8.4 Hz, 1H), 8.51 (s, 1H), 8.53 (s, 1H); 13C
NMR (150 MHz, CD3OD) δ 9.2, 27.1, 27.2 (2C), 33.91, 33.95,
36.5, 48.0, 69.5, 72.1, 76.2, 84.5, 89.7, 117.8, 119.2, 119.7, 120.6,
120.7, 131.4, 134.3, 141.8, 150.8, 151.5, 154.8, 158.0, 162.0, 175.1;
HRMS (ESI-) calcd for C25H28N9O8S [M - H]- 614.1787, found
614.1799 (error 2.0 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(4-methylphenyl)-
1,2,3-triazol-1-yl]adenosine Triethylammonium Salt (36). 4-Ethy-
nyltoluene was reacted with 15 using the general procedure for
triazole synthesis to afford the title compound (10.5 mg, 44%) as
a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR
(600 MHz, CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 2.35 (s, 3H), 3.18
(q, J ) 7.2 Hz, 6H), 4.34-4.35 (m, 1H), 4.41-4.43 (m, 1H),
4.47-4.48 (m, 2H), 4.74 (t, J ) 5.4 Hz, 1H), 6.17 (d, J ) 5.4 Hz,
1H), 6.73-6.77 (m, 2H), 7.24-7.27 (m, 3H), 7.80 (d, J ) 7.8 Hz,
2H), 7.91 (d, J ) 7.8 Hz, 1H), 8.53 (s, 1H), 9.00 (s, 1H); 13C
NMR (150 MHz, CD3OD) δ 9.3, 21.4, 48.0, 69.6, 72.2, 76.3, 84.6,
89.8, 117.9, 119.3, 119.8, 120.3, 120.7, 127.0, 128.5, 130.7, 131.5,
134.4, 139.8, 142.0, 149.1, 150.8, 151.5, 158.1, 162.1, 175.1; HRMS
(ESI-) calcd for C26H24N9O8S [M - H]- 622.1474, found
622.1464 (error 1.6 ppm).
2-(4-Cyclohex-1-enyl-1,2,3-triazol-1-yl)-5′-O-[N-(2-hydroxyben-
zoyl)sulfamoyl]adenosine Sodium Salt (32). 1-Ethynylcyclohexene
was reacted with 15 using the general procedure for triazole
synthesis and converted to the sodium salt by ion exchange using
Dowex 50WX2-Na+ to afford the title compound (15.4 mg, 66%)
1
as a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); H
NMR (600 MHz, CD3OD) δ 1.70-1.73 (m, 2H), 1.79-1.83 (m,
2H), 2.23-2.25 (m, 2H), 2.44-2.47 (m, 2H), 4.34-4.35 (m, 1H),
4.39-4.42 (m, 1H), 4.44-4.48 (m, 2H), 4.71 (t, J ) 5.4 Hz, 1H),
6.17 (d, J ) 5.4 Hz, 1H), 6.62 (br s, 1H), 7.27 (t, J ) 8.4 Hz, 1H),
7.92 (d, J ) 7.8 Hz, 1H), 8.55 (s, 1H), 8.64 (s, 1H); 13C NMR
(150 MHz, CD3OD) δ 23.3, 23.6, 26.3, 27.3, 69.5, 72.2, 76.3, 84.6,
89.7, 117.8, 119.20, 119.22, 119.7, 120.6, 127.1, 128.1, 131.4,
134.3, 141.8, 150.5, 150.8, 151.5, 158.0, 162.1, 175.1; HRMS
(ESI-) calcd for C25H26N9O8S [M - H]- 612.1631, found
612.1630 (error 0.1 ppm).
2-[4-(2-Aminophenyl)-1,2,3-triazol-1-yl]-5′-O-[N-(2-hydroxyben-
zoyl)sulfamoyl]adenosine Triethylammonium Salt (37). 2-Ethyny-
laniline was reacted with 15 using the general procedure for triazole
synthesis to afford the title compound (10.6 mg, 45%) as a white
1
amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); H NMR (600
MHz, CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 3.16 (q, J ) 7.2 Hz,
6H), 4.35-4.37 (m, 1H), 4.42-4.44 (m, 1H), 4.44-4.48 (m, 2H),
4.72 (t, J ) 5.4 Hz, 1H), 6.20 (d, J ) 5.4 Hz, 1H), 6.73-6.78 (m,
3H), 6.85 (d, J ) 7.8 Hz, 1H), 7.11 (t, J ) 7.8 Hz, 1H), 7.27 (t, J
) 7.8 Hz, 1H), 7.59 (d, J ) 7.8 Hz, 1H), 7.93 (d, J ) 7.8 Hz, 1H),
8.56 (s, 1H), 9.01 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 9.3,
48.0, 69.6, 72.2, 76.5, 84.6, 89.8, 115.2, 117.9, 118.2, 118.9, 119.4,
119.8, 120.7, 120.9, 129.4, 130.5, 131.5, 134.4, 141.9, 146.8, 149.3,
150.9, 151.6, 158.1, 162.1, 175.2; HRMS (ESI-) calcd for
C25H23N10O8S [M - H]- 623.1427, found 623.1405 (error 3.5
ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-(4-phenyl-1,2,3-triazol-
1-yl)adenosine Sodium Salt (33). Phenylacetylene was reacted with
15 using the general procedure for triazole synthesis and converted
to the sodium salt by ion exchange using Dowex 50WX2-Na+ to
afford the title compound (9.1 mg, 46%) as a white amorphous
solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR (600 MHz,
CD3OD) δ 4.36-4.37 (m, 1H), 4.41-4.44 (m, 1H), 4.47-4.49 (m,
2H), 4.75 (t, J ) 5.4 Hz, 1H), 6.19 (d, J ) 4.8 Hz, 1H), 6.74-6.78
(m, 2H), 7.27 (t, J ) 7.2 Hz, 1H), 7.37 (t, J ) 7.2 Hz, 1H), 7.46
(d, J ) 7.2 Hz, 2H), 7.92 (d, J ) 8.4 Hz, 1H), 7.96 (d, J ) 7.2 Hz,
2H), 8.55 (s, 1H), 9.10 (s, 1H); 13C NMR (150 MHz, CD3OD) δ
69.5, 72.1, 76.2, 84.6, 89.8, 117.8, 119.2, 119.8, 120.6, 120.7, 127.0,
129.6, 130.0, 131.3, 131.4, 134.3, 142.0, 149.0, 150.7, 151.5, 158.1,
162.0, 175.2; HRMS (ESI-) calcd for C25H22N9O8S [M - H]-
608.1318, found 608.1310 (error 1.3 ppm).
2-[4-(3-Aminophenyl)-1,2,3-triazol-1-yl]-5′-O-[N-(2-hydroxyben-
zoyl)sulfamoyl]adenosine Triethylammonium Salt (38). 3-Ethyny-
laniline was reacted with 15 using the general procedure for triazole
synthesis to afford the title compound (15.2 mg, 63%) as a white
1
amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); H NMR (600
MHz, CD3OD) δ 1.24 (t, J ) 7.2 Hz, 9H), 3.14 (q, J ) 7.2 Hz,
6H), 4.35-4.37 (m, 1H), 4.41-4.44 (m, 1H), 4.47-4.49 (m, 2H),
4.74 (t, J ) 5.4 Hz, 1H), 6.17 (d, J ) 6.0 Hz, 1H), 6.70-6.77 (m,
3H), 7.15 (t, J ) 7.8 Hz, 1H), 7.22 (d, J ) 7.2 Hz, 1H), 7.26 (t, J
) 7.2 Hz, 1H), 7.29 (s, 1H), 7.91 (d, J ) 7.8 Hz, 1H), 8.52 (s,
1H), 8.93 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 9.3, 48.0, 69.6,
72.2, 76.2, 84.6, 89.9, 113.8, 116.8, 116.9, 117.9, 119.4, 119.9,
120.5, 120.7, 130.8, 131.5, 131.9, 134.4, 142.0, 149.4, 149.5, 150.8,
151.5, 158.1, 162.1, 175.2; HRMS (ESI-) calcd for C25H23N10O8S
[M - H]- 623.1427, found 623.1437 (error 1.6 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(2-methylphenyl)-
1,2,3-triazol-1-yl]adenosine Triethylammonium Salt (34). 2-Ethy-
nyltoluene was reacted with 15using the general procedure for
triazole synthesis to afford the title compound (12.3 mg, 51%) as
a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); 1H NMR
(600 MHz, CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 2.50 (s, 3H), 3.18
(q, J ) 7.2 Hz, 6H), 4.35-4.36 (m, 1H), 4.41-4.44 (m, 1H),
4.51-4.48 (m, 2H), 4.71 (t, J ) 5.4 Hz, 1H), 6.19 (d, J ) 5.4 Hz,
1H), 6.74-6.78 (m, 2H), 7.26-7.31 (m, 4H), 7.74 (d, J ) 8.4 Hz,
1H), 7.92 (d, J ) 7.8 Hz, 1H), 8.56 (s, 1H), 8.86 (s, 1H); 13C
NMR (150 MHz, CD3OD) δ 9.2, 21.4, 47.9, 69.5, 72.1, 76.4, 84.5,
89.7, 117.8, 119.2, 119.8, 120.6, 122.7, 127.2, 129.7, 130.1, 130.5,
131.4, 131.9, 134.3, 137.2, 141.8, 148.3, 150.8, 151.5, 158.0, 162.0,
175.0; HRMS (ESI-) calcd for C26H24N9O8S [M - H]- 622.1474,
found 622.1471 (error 0.5 ppm).
2-[4-(4-Aminophenyl)-1,2,3-triazol-1-yl]-5′-O-[N-(2-hydroxyben-
zoyl)sulfamoyl]adenosine Triethylammonium Salt (39). 4-Ethyny-
laniline was reacted with 15 using the general procedure for triazole
synthesis to afford the title compound (12.5 mg, 52%) as a white
1
amorphous solid: Rf ) 0.6 (80:20 EtOAc/MeOH); H NMR (600
MHz, CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 3.17 (q, J ) 7.2 Hz,
6H), 4.35-4.36 (m, 1H), 4.41-4.44 (m, 1H), 4.47-4.49 (m, 2H),
4.72 (t, J ) 5.4 Hz, 1H), 6.19 (d, J ) 5.4 Hz, 1H), 6.74-6.78 (m,
4H), 7.27 (t, J ) 7.2 Hz, 1H), 7.66 (d, J ) 8.4 Hz, 2H), 7.92 (d,
J ) 8.4 Hz, 1H), 8.55 (s, 1H), 8.86 (s, 1H); 13C NMR (150 MHz,
CD3OD) δ 9.3, 48.0, 69.6, 72.2, 76.4, 84.6, 89.8, 116.5, 118.0,
119.0, 119.4, 119.8, 120.6, 120.7, 128.1, 131.5, 134.4, 141.9, 149.8,
149.9, 150.9, 151.6, 158.1, 162.1, 175.2; HRMS (ESI-) calcd for
C25H23N10O8S [M - H]- 623.1427, found 623.1417 (error 1.6
ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(3-methylphenyl)-
1,2,3-triazol-1-yl]adenosine Triethylammonium Salt (35). 3-Ethy-
nyltoluene was reacted with 15 using the general procedure for
triazole synthesis to afford the title compound (10.5 mg, 44%) as
1
a white amorphous solid: Rf ) 0.6 (8:2 EtOAc/MeOH); H NMR
(600 MHz, CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 2.39 (s, 3H), 3.17
(q, J ) 7.2 Hz, 6H), 4.34-4.35 (m, 1H), 4.40-4.43 (m, 1H),
4.46-4.49 (m, 2H), 4.75 (t, J ) 5.4 Hz, 1H), 6.17 (d, J ) 6.0 Hz,
1H), 6.73-6.77 (m, 2H), 7.16 (d, J ) 7.8 Hz, 1H), 7.26 (t, J ) 7.8
Hz, 1H), 7.31 (t, J ) 7.8 Hz, 1H), 7.70 (d, J ) 7.2 Hz, 1H), 7.76
(s, 1H), 7.91 (d, J ) 7.2 Hz, 1H), 8.53 (s, 1H), 9.03 (s, 1H); 13C
NMR (150 MHz, CD3OD) δ 9.3, 21.6, 48.0, 69.6, 72.2, 76.3, 84.6,
89.8, 117.4, 117.9, 119.4, 119.9, 120.7, 124.2, 127.6, 130.0, 130.4,
131.2, 131.5, 134.4, 140.0, 142.1, 149.2, 150.8, 151.5, 158.1, 162.1,
175.1; HRMS (ESI-) calcd for C26H24N9O8S [M - H]- 622.1474,
found 622.1469 (error 0.8 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(pyrid-2-yl)-1,2,3-
triazol-1-yl]adenosine Triethylammonium Salt (40). 2-Ethynylpy-
ridine was reacted with 15 using the general procedure for triazole
synthesis to afford the title compound (11.6 mg, 50%) as a white
1
amorphous solid: Rf ) 0.3 (80:20 EtOAc/MeOH); H NMR (600
MHz, CD3OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 3.17 (q, J ) 7.2 Hz,
6H), 4.35-4.37 (m, 1H), 4.41-4.44 (m, 1H), 4.46-4.49 (m, 2H),
4.72 (t, J ) 4.8 Hz, 1H), 6.18 (d, J ) 4.8 Hz, 1H), 6.73-6.76 (m,
2H), 7.26 (t, J ) 7.2 Hz, 1H), 7.37 (m, 1H), 7.90-7.92 (m, 2H),
8.15 (d, J ) 7.8 Hz, 1H), 8.54 (s, 1H), 8.59 (br s, 1H), 9.19 (s,

Inhibition of Siderophore Biosynthesis
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7505
1H); 13C NMR (150 MHz, CD3OD) δ 9.3, 48.0, 69.6, 72.2, 76.5,
84.6, 89.9, 117.9, 119.3, 119.9, 120.7, 122.1, 122.7, 124.7, 130.0,
131.5, 132.5, 134.4, 139.0, 142.0, 148.7, 150.7, 151.5, 158.1, 162.1,
175.1; HRMS (ESI-) calcd for C24H21N10O8S [M - H]- 609.1270,
found 609.1261 (error 1.5 ppm).
MeOH); 1H NMR (600 MHz, CD3OD) δ 1.29 (t, J ) 7.2 Hz, 9H),
3.19 (q, J ) 7.2 Hz, 6H), 4.35-4.36 (m, 1H), 4.41-4.43 (m, 1H),
4.46-4.49 (m, 2H), 4.74 (t, J ) 5.4 Hz, 1H), 6.20 (d, J ) 5.4 Hz,
1H), 6.75-6.78 (m, 2H), 6.88 (d, J ) 8.4 Hz, 2H), 7.28 (t, J ) 7.2
Hz, 1H), 7.79 (d, J ) 9.0 Hz, 2H), 7.93 (d, J ) 7.8, 1H), 8.58 (s,
1H), 8.98 (s, 1H); 13C NMR (150 MHz, CD3OD) 9.3, 48.0, 69.6,
72.2, 76.4, 84.7, 89.7, 116.8, 117.9, 119.3, 119.6, 119.8, 120.7,
122.7, 128.5, 131.5, 134.4, 142.0, 149.3, 150.9, 151.6, 158.1, 159.4,
162.2, 175.2; HRMS (ESI-) calcd for C25H22N9O9S [M - H]-
624.1267, found 624.1197 (error 11.2 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(pyrid-3-yl)-1,2,3-
triazol-1-yl]adenosine Triethylammonium Salt (41). 3-Ethynylpy-
ridine was reacted with 15 using the general procedure for triazole
synthesis to afford the title compound (14.2 mg, 61%) as a white
1
amorphous solid: Rf ) 0.3 (80:20 EtOAc/MeOH); H NMR (600
Docking Studies. The MbtA structure obtained following QM/
MM studies of the complex formed between MbtA (homology
model) and 5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2-phenylad-
enosine was used for docking studies with Glide.18,38 Docking was
performed on a cubic box of 10 Å side, centered on the active site,
using the default XP (extra precision) settings. The core pattern
comparison option was used to match the heavy atoms of the
SalAMS core of the ligands with those of the SalAMS core of
minimized 5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2-phenyladenos-
ine in complex with MbtA, allowing a 2 Å tolerance in rmsd.18
Fukui Analysis. A geometry optimization was completed on the
neutral truncated tetrazole T1 (Scheme 2B) whereby the C5′
hydroxymethyl group was replaced with a hydrogen atom at the
B3LYP/6-31G* level of theory using the GAMESS electronic
structure package.39 By use of this geometry, a single point energy
calculation was completed on the cation (vertical ionization), and
the electron distribution and surface of each were plotted with
Molden.40 Subtracting the cation density from that of the neutral
species yields the Fukui function (a volumetric function indicating
the most nucleophilic regions of the molecule).41 Similiarly, the
atomic charges derived though Mulliken population analysis are
differenced for each atom to yield the local Fukui function.
Enzyme Kinetic Studies. ATP/PPi Exchange Assay. MbtA was
expressed in E. coli and purified as described.13 The inhibition
assays were performed as described in duplicate.13 In brief the
reaction was initiated by adding 10 µL of [32P]PPi with 7 nM MbtA
in 90 µL of reaction buffer (250 µM salicylic acid, 10 mM ATP,
1 mM PPi, 75 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT)
at 37 °C in the presence of eight different concentrations of the
inhibitor. The reaction was terminated by the addition of 200 µL
of quenching buffer (350 mM HClO4, 100 mM PPi, 1.8% w/v
activated charcoal). The charcoal was pelleted by centrifugation
and washed once with 500 µL of H2O and analyzed by liquid
scintillation counting. The KIapp values were calculated using the
Morrison equation:
MHz, CD3OD) δ 1.29 (t, J ) 7.2 Hz, 9H), 3.19 (q, J ) 7.2 Hz,
6H), 4.35-4.36 (m, 1H), 4.41-4.44 (m, 1H), 4.50-4.52 (m, 2H),
6.16 (d, J ) 6.6 Hz, 1H), 6.72-6.76 (m, 2H), 7.26 (t, J ) 8.4 Hz,
1H), 7.52 (t, J ) 8.4 Hz, 1H), 7.89 (d, J ) 7.2 Hz, 1H), 8.42 (d,
J ) 7.8 Hz, 1H), 8.51-8.53 (m, 2H), 9.15 (s, 1H), 9.27 (s, 1H),
H-2′ obscured by HOD; 13C NMR (150 MHz, CD3OD) δ 9.3, 48.0,
69.6, 72.3, 75.9, 84.7, 90.0, 117.9, 119.3, 120.1, 120.7, 121.9, 125.7,
128.4, 131.4, 134.4, 135.5, 142.4, 145.7, 147.6, 149.8, 150.7, 151.5,
158.1, 162.1, 175.1; HRMS (ESI-) calcd for C24H21N10O8S [M -
H]- 609.1270, found 609.1257 (error 2.1 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(pyrid-4-yl)-1,2,3-
triazol-1-yl]adenosine Triethylammonium Salt (42). 4-Ethynylpy-
ridine was reacted with 15 using the general procedure for triazole
synthesis to afford the title compound (12.4 mg, 64%) as a white
1
amorphous solid: Rf ) 0.3 (80:20 EtOAc/MeOH); H NMR (600
MHz, CD3OD) δ 1.26 (t, J ) 7.2 Hz, 9H), 3.16 (q, J ) 7.2 Hz,
6H), 4.33-4.35 (m, 1H), 4.41-4.44 (m, 1H), 4.51-4.52 (m, 2H),
4.87 (t, J ) 5.4 Hz, 1H), 6.14 (d, J ) 5.4 Hz, 1H), 6.71-6.75 (m,
2H), 7.25 (t, J ) 7.2 Hz, 1H), 7.88 (d, J ) 7.8 Hz, 1H), 7.98 (d,
J ) 5.4 Hz, 2H), 8.50 (s, 1H), 8.55 (d, J ) 4.8 Hz, 2H), 9.31 (s,
1H); 13C NMR (150 MHz, CD3OD) δ 9.3, 48.0, 69.6, 72.3, 75.8,
84.7, 90.1, 117.9, 119.3, 120.2, 120.7, 121.9, 123.1, 131.4, 134.4,
140.1, 142.6, 148.1, 150.6, 150.8, 151.4, 158.1, 162.1, 175.1; HRMS
(ESI-) calcd for C24H21N10O8S [M - H]- 609.1270, found
609.1308 (error 6.2 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(2-hydroxyphenyl)-
1,2,3-triazol-1-yl]adenosine Triethylammonium Salt (43). 2-Hy-
droxyphenylacetylene was reacted with 15 using the general
procedure for triazole synthesis to afford the title compound (12.2
mg, 51%) as a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/
MeOH); 1H NMR (600 MHz, CD3OD) δ 1.28 (t, J ) 7.2 Hz, 9H),
3.18 (q, J ) 7.2 Hz, 6H), 4.36-4.38 (m, 1H), 4.42-4.44 (m, 1H),
4.46-4.48 (m, 2H), 4.70 (t, J ) 5.4 Hz, 1H), 6.21 (d, J ) 4.8 Hz,
1H), 6.75-6.78 (m, 2H), 6.93-6.96 (m, 2H), 7.21 (t, J ) 7.2 Hz,
1H), 7.27 (t, J ) 7.2 Hz, 1H), 7.93 (d, J ) 7.2 Hz, 1H), 8.07 (d,
J ) 7.2 Hz, 1H), 8.57 (s, 1H), 9.13 (s, 1H); 13C NMR (150 MHz,
CD3OD) δ 9.3, 48.0, 69.7, 72.3, 76.5, 84.7, 89.7, 117.2, 117.4,
118.0, 119.4, 119.8, 120.7, 120.8, 122.6, 128.2, 130.6, 131.5, 134.4,
141.8, 145.9, 151.0, 151.6, 156.1, 158.1, 162.1, 175.1; HRMS
(ESI-) calcd for C25H22N9O9S [M - H]- 624.1267, found
624.1305 (error 6.1 ppm).
([E] - [I] - KIapp) + ([E] - [I] - K app)2 + 4[E][KIapp
]
√
I
νi ⁄ ν0 )
2[E]
(1)
PAMPA Assay. PAMPA assays were carried out according to
the manufacturer’s instructions in triplicate (BD Gentest no.
353015). Compound stock solutions (10 mM in DMSO) were
diluted to 100 µM in PBS (Dulbecco’s) to a final volume of 1.5
mL. The diluted stocks were added to the bottom donor plate (300
µL), and 200 µL of PBS was added to the top acceptor plate. The
plates were incubated for 5 h at room temperature. The equilibrium
plate was made by aliquoting 3 × 100 µL into a 96-well UV clear
half-area plate (Greiner) and integrating the absorbances at 280,
300, 320, 340, and 360 nm with the background subtracted using
PBS as a buffer blank on an M5e plate reader (Molecular Devices).
Wells were also read at 800 nm to check for light scattering. After
the incubation the acceptor plate was removed from the donor plate
and 100 µL was transferred from each well of the two plates to
two 96-well UV clear half-area 96-well plates. The plates were
read as described above, and the data were processed using the
Xcel analysis file available from BD Biosciences. Caffeine and
ketoprofin were included on the plate as positive controls, while
hydrochlorothiazide was used as a negative control.
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(3-hydroxyphenyl)-
1,2,3-triazol-1-yl]adenosine Triethylammonium Salt (44). 3-Hy-
droxyphenylacetylene was reacted with 15 using the general
procedure for triazole synthesis to afford the title compound (12.2
mg, 51%) as a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/
MeOH); 1H NMR (600 MHz, CD3OD) δ 1.26 (t, J ) 7.2 Hz, 9H),
3.15 (q, J ) 7.2 Hz, 6H), 4.34-4.36 (m, 1H), 4.41-4.43 (m, 1H),
4.45-4.47 (m, 2H), 4.72 (t, J ) 5.4 Hz, 1H), 6.18 (d, J ) 5.4 Hz,
1H), 6.73-6.77 (m, 2H), 6.78-6.80 (m, 1H), 7.26 (t, J ) 8.4 Hz,
2H), 7.38-7.39 (m, 2H), 7.91 (dd, J ) 7.8, 6.6 Hz, 1H), 8.55 (s,
1H), 9.00 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 9.3, 48.0, 69.6,
72.2, 76.4, 84.6, 89.8, 113.7, 116.8, 117.2, 118.0, 118.4, 119.4,
119.8, 120.7, 131.2, 131.5, 132.5, 134.4, 142.0, 149.1, 150.8, 151.6,
158.1, 159.2, 162.1, 175.1; HRMS (ESI-) calcd for C25H22N9O9S
[M - H]- 624.1267, found 624.1288 (error 3.4 ppm).
5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-[4-(4-hydroxyphenyl)-
1,2,3-triazol-1-yl]adenosine Triethylammonium Salt (45). 4-Hy-
droxyphenylacetylene was reacted with 15 using the general
procedure for triazole synthesis to afford the title compound (10.4
mg, 44%) as a white amorphous solid: Rf ) 0.6 (80:20 EtOAc/
Vero Cell Cytotoxicity Assay. African green monkey Cercop-
ithecus aethiops kidney cells (Vero, ATCC) cells were plated in

7506 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Gupte et al.
96-well plates at (2.5-5.0) × 104 cells per well (200 µL). Vero
cells were maintained in minimum essential medium (MEM)
supplemented with 5% fetal bovine serum (FBS), 100 U/mL
penicillin, and 100 µg/mL streptomycin. Compounds were prepared
as 20 mM stock solutions in DMSO, and 1 µL of the compound
stock solution was added to each well in 200 µL of MEM, yielding
a final compound concentration of 100 µM. Control wells contained
either 1% DMSO (negative control) or 100 µM 5′-O-(sulfamoy-
l)adenosine (positive control), and all reactions were done in
triplicate. The plate was incubated for 48 h at 37 °C in a 5% CO2/
95% air humidified atmosphere. Measurement of cell viability was
carried out using a modified method of Mosmann based on 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).42
MTT was prepared fresh at 1 mg/mL in serum-free, phenol red-
free RPMI 1640 media. MTT solution (200 µL) was added to each
well, and the plate was incubated as described above for 3 h. The
MTT solution was removed, and the formazan crystals were
solubilized with 200 µL of isopropanol. The plate was read on a
M5e spectrophotometer (Molecular Devices) at 570 nm for for-
mazan and 650 nm for background subtraction. Cell viability was
estimated as the percentage absorbance of sample relative to the
DMSO control.
MEL, OCL, and REH Cytotoxicity Assay. The three human
cancer cells (SK-MEL-2 cells, human melanoma; OCL-3 cells, OCI-
AML3, human acute myeloid leukemia; REH cells, human B cells
precursor leukemia) were cultured in RPMI-1640 media supple-
mented with 10% FBS at 37 °C and 5% CO2. The in vitro
cytotoxicity of the small molecules was assayed by determining
the GI50 values (the concentration of the small molecules to reduce
the cell growth by 50%). In brief, the cells were plated in a 96-
well plate (104 cells/well for OCI-AML3 and HEL and 2500 cells/
well for SK-MEL-2). The cells were treated with the small
molecules with a series of 3-fold dilution with 1% DMSO in the
final cell media (cells treated with media of 1% DMSO served as
a control). After a 48-h treatment, the relative cell viability in each
well was determined by using CellTiter-Blue Cell Viability Assay
kit (Promega, CA).
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M. tuberculosis H37Rv MIC Assay. All compound minimum
inhibitory concentrations (MICs) were experimentally determined
as previously described.10 MICs were determined in quadruplicate
in iron-deficient GAST and GAST supplemented with 200 µM
FeCl3 according to the broth microdilution method using compounds
from DMSO stock solutions or with control wells treated with an
equivalent amount of DMSO. Isoniazid was used as positive
controls, while DMSO was employed as a negative control. All
measurements reported herein used an initial cell density of 104-105
cells/assay, and growth was monitored at 10-14 days, with the
untreated and DMSO-treated control cultures reaching an OD620
of ∼0.2-0.3. Plates were incubated at 37 °C (100 µL/well), and
growth was recorded by measurement of optical density at 620 nm.
Acknowledgment. This research was supported by a grant
from the NIH (Grant R01AI070219) and funding from the
Center for Drug Design, Academic Health Center, University
of Minnesota to C.C.A. This research was supported in part by
the Intramural Research Program of the NIH, National Institute
of Allergy and Infectious Disease. We thank the Minnesota
Supercomputing Institute for computing time.
Supporting Information Available: Table of HPLC purities of
15-45. This material is available free of charge via the Internet at
http://pubs.acs.org.
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promoted CuI-catalyzed coupling reactions. Chem. Commun. 2004,
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