Rationally-designed nucleoside antibiotics that inhibit siderophore biosynthesis of Mycobacterium tuberculosis

Article abstract of DOI:10.1021/jm051060o

A rationally designed nucleoside inhibitor of Mycobacterium tuberculosis growth (MIC₉₉ = 0.19 μM) that disrupts siderophore biosynthesis was identified. The activity is due to inhibition of the adenylate-forming enzyme MbtA which is involved in

Full text of DOI:10.1021/jm051060o

J. Med. Chem. 2006, 49, 31-34
31
Rationally Designed Nucleoside Antibiotics That
Inhibit Siderophore Biosynthesis of
Mycobacterium tuberculosis
Ravindranadh V. Somu,† Helena Boshoff,‡ Chunhua Qiao,†
Eric M. Bennett,† Clifton E. Barry III,‡ and
Courtney C. Aldrich†,*
Figure 1. Biosynthesis of the mycobactins and carboxymycobactins.
Salicylic acid (1) is converted to mycobactin T (2) and carboxymy-
cobactin (3).4 The depicted lipid side chain is a representative as both
2 and 3 are isolated as mixtures with various length lipid residues.
Center for Drug Design, Academic Health Center, UniVersity of
Minnesota, Minneapolis, Minnesota 55455, and Tuberculosis
Research Section, National Institute of Allergy and Infectious
Diseases, RockVille, Maryland 20852-1742
ReceiVed October 20, 2005
Abstract: A rationally designed nucleoside inhibitor of Mycobacterium
tuberculosis growth (MIC99 ) 0.19 µM) that disrupts siderophore
biosynthesis was identified. The activity is due to inhibition of the
adenylate-forming enzyme MbtA which is involved in biosynthesis of
the mycobactins.
Tuberculosis (TB) is the leading bacterial cause of infectious
disease mortality. The current WHO-approved treatment for TB,
known as directly observed therapy short-course (DOTS),
involves a three- or four-drug regimen comprising isoniazid,
rifampin, pyrazinamide, and/or ethambutol for a minimum of
6 months.1 While these first-line agents remain useful in treating
susceptible Mycobacterium tuberculosis strains, the emergence
of multidrug resistant tuberculosis demands the development
of new drugs.
Iron acquisition is an indispensable process for many
pathogens since this essential nutrient is sequestered by mam-
malian host proteins such as the transferrins.2 Pathogenic
microorganisms have evolved a number of mechanisms to
overcome this iron restriction, but the most common mechanism
involves the synthesis and secretion of low molecular weight,
high-affinity iron chelators termed siderophores.3 M. tuberculosis
produces two series of structurally related peptidic siderophores
known as the mycobactins and carboxymycobactins that vary
by the appended lipid residue (Figure 1).4 Because mycobactins
are critical for growth and virulence of M. tuberculosis, they
have emerged as attractive targets for the development of anti-
TB agents.5-7
Elucidation of the biosynthetic pathway of the mycobactins
has aided this approach. Biosynthesis begins with an adenylate-
forming enzyme, MbtA, which activates and loads salicylic acid
onto a mixed nonribosomal peptide synthetase polyketide
synthase (NRPS-PKS) assembly line comprised of five other
enzymes (MbtB-MbtF) that sequentially build the mycobactin
core scaffold (Figure 1).8 Additional post NRPS-PKS modifica-
tions through lipidation and N-hydroxylations afford the my-
cobactins (2) and carboxymycobactins (3). MbtA is an ideal
target since MbtA has no mammalian homologues. Further,
Marahiel and co-workers demonstrated that inhibitors developed
for the functionally related aminoacyl tRNA synthetases, which
activate amino acids to the corresponding aminoacyl adenylates,
can be utilized to inhibit adenylate forming enzymes from NRPS
systems despite a lack of either structure or sequence similar-
ity.9,10 The cocrystal structure of a homologous adenylating
Figure 2. Enzyme reaction mechanism catalyzed by MbtA. (A) First
half-reaction. (B) Second half-reaction. (C) Intermediate mimics.
enzyme (DhbE) with its adenylated substrate (2,3-dihydrox-
benzoic acid) has been solved, providing both structural and
mechanistic insight into the reaction catalyzed by MbtA.11 Last,
the catecholate-class of siderophores are characterized by a
2-hydroxyphenyl or 2,3-dihydroxyphenyl moiety at the ‘N-
terminus’; consequently, inhibitors toward the adenylation
protein MbtA are expected to be useful against the biosynthesis
of a wide array of structurally diverse siderophores.3
MbtA incorporates salicylic acid into the mycobactin core
scaffold, using a two-step reaction (Figure 2) that is mechanisti-
cally similar in all members of the adenylate-forming enzyme
superfamily and the functionally related aminoacyl tRNA
synthetases.12,13 In the first half-reaction, binding of both the
substrate acid (1) and ATP is followed by nucleophilic attack
of the substrate carboxylate on the R-phosphate of ATP to
generate a tightly bound acyl adenylate (4) and the release of
pyrophosphate (Figure 2, part A). In the second half-reaction,
the enzyme binds the phosphopantetheine (ppan) cofactor (5)
of the thiolation domain of MbtB and transfers the acyl adenylate
(4) onto the nucleophilic sulfur atom of this cofactor moiety to
provide salicyl-bound-MbtB (6) (Figure 2, part B). The rationale
for inhibitor development is the tight binding of the acyl
adenylate intermediate, which typically binds 3-5 orders of
magnitude more tightly that the substrate acid.14,15 Therefore,
analogues incorporating stable linkers as bioisosteres of the
labile acyl phosphate function provide potent enzyme inhibitors.
These inhibitors are classified as bisubstrate inhibitors16 or
intermediate mimics14 and not as mechanism-based inhibitors
as recently suggested.17 The salicyl adenylate scaffold provides
* To whom correspondence should be addressed. Phone: 612-625-7956.
Fax: 612-626-5173. E-mail: aldri015@umn.edu.
† University of Minnesota.
‡ National Institute of Allergy and Infectious Diseases.
10.1021/jm051060o CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/13/2005

32 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Letters
Scheme 1a
Scheme 2a
a Reaction conditions: (a) NH2SO2Cl, NaH, 82%; (b) 22, 15, Cs2CO3:
80% (16); (c) 23, 18, DBU: 36% (19); (d) 24, 18, DBU: 44% (21); (e)
H2, Pd/C: 95% (17); 41% (20) (f) 80% aq. TFA: 74% (7); 64% (8); 100%
(9).
a Reaction conditions: (a) CBr4, PPh3, NaN3, 48%; (b) H2, Pd/C, 70%;
(c) NH2SO2Cl, NaH, 34%,; (d) salicylic acid, CDI, DBU, 40%; (e) 80% aq
TFA, 81%.
Scheme 3a
many opportunities for inhibitor development. In this paper we
report our preliminary evaluation of several bioisosteric replace-
ments of the labile acyl phosphate function and investigate the
role of the salicyl moiety for activity.
The first inhibitor examined was based on the acylsulfamate
linker inspired from the natural product ascamycin, wherein the
phosphate moiety is mimicked by the isosteric sulfamate
group.18 The synthesis of 7 began with sulfamoylation of N6-
Boc-2′,3′-O-isopropylidene adenosine 1427 to provide sulfamate
15 (Scheme 1). Coupling to the N-hydroxysuccinimide (NHS)
ester of O-benzyl salicylic acid 22 mediated by Cs2CO3 afforded
16.19 Sequential deprotection of the benzyl ether by catalytic
hydrogenation to 17 followed by the isopropylidene acetal and
Boc carbamate with aqueous TFA yielded salicyl-sulfamate 7.
An alternative five step synthesis of 7 was recently reported by
Ferreras et al.17 Both anthraniloyl-sulfamate 8 and benzoyl-
sulfamate 9 were prepared in an analogous fashion.
a Reaction conditions: (a) NaBH4, 98%; (b) CBr4, PPh3, 88%; (c)
Cs2CO3, 48%; (d) H2, Pd/C, 83%; (d) 80% aq TFA, 81%.
Scheme 4a
The acylsulfamate linkage of inhibitor 7 is extremely acidic
with a calculated pKa of 0.6, while the pKa of 9 was experi-
mentally determined to be 2.8 (calculated 3.4), demonstrating
that the o-hydroxy function of the salicyl moiety modulates the
acidity of the NH proton through resonance delocalization.20
We have observed that the acylsulfamate linkage is intrinsically
unstable as the free acid but considerably more stable as the
conjugate base since the negative charge on the nitrogen atom
deters nucleophilic attack on the carbonyl function. This is
expected to be a principal mechanism of decomposition in
analogy to the acylsulfonamides.21 Significantly, we found that
8 decomposed rapidly over several days at 25 °C in CD3OD
but was unchanged after 30 days as the triethylammonium salt.
Accordingly, all inhibitors were subsequently converted to the
triethylammonium salts after purification while conversion to
alkali salts is readily achieved through ion-exchange.
Substitution of the sulfamate oxygen with a nitrogen affords
the acylsulfamide linkage of 10 and raises the pKa of the central
NH by approximately 2 units to a calculated value of 2.7.
Additionally, the acylsulfamide function was expected to have
improved stability relative to the acylsulfamate linkages of 7-9,
as 5′-O-sulfamoyladenosine is precedented to decompose with
expulsion of the 5′-O-sulfamoyl moiety through both hydrolysis
and cyclonucleoside formation.22,23 Synthesis of 10 was ac-
complished by treatment of 14 under Appel conditions to afford
the intermediate 5′-bromide that was converted in-situ to the
corresponding azide 25 (Scheme 2). Catalytic hydrogenation
gave amine 26, which was sulfamoylated to provide sulfamide
27. Coupling to salicylic acid mediated by CDI and DBU
yielded 28 that was deprotected with aqueous TFA to furnish
acylsulfamide 10.
a Reaction conditions: (a) 35, LiCH2P(O)(OMe)2, 88%; (b) (i) TMSBr,
(ii) pyr., 100%; (c) TrisylCl, 2′,3′-O-isopropylidene adenosine, 6%; (d) H2,
Pd/C, 40%; (e) 80% aq TFA, 90%; (f) (i) 34, (COCl)2, (ii) Me(OMe)NH‚HCl,
80%; (g) TMS-acetylene, n-BuLi, 76%; (h) TBAF, 79%; (i) 25, CuSO4,
sodium ascorbate, 89%; (j) H2, Pd/C; (k) 80% aq TFA, (100%, 2 steps).
To explore the significance of the linkage carbonyl group
for activity, analogue 11 was synthesized (Scheme 3). Removal
of the carbonyl function also serves to raise the pKa of the
sulfamate NH to a calculated value of 7.5 with an attendant
decrease in overall polarity. This was succinctly carried out by
NaBH4 reduction of 29 to provide alcohol 30 that was converted
to bromide 31. Alkylation of 5′-O-sulfamoyl adenosine 18 with
bromide 31 employing Cs2CO3 yielded sulfamate 32 which was
deprotected to furnish target 11.
The â-ketophosphonate linkage, which is the closest mimic
of the acyl phosphate moiety, was examined next (Scheme 4).
Synthesis was initiated by addition of the lithium anion of
methyl dimethylphosphonate to methyl benzoate derivative 35
to provide 36.24 Treatment with TMSBr afforded phosphonic
acid 37 that was coupled with 2′,3′-O-isopropylidene adenosine
employing 2,4,6-trimethylbenzenesulfonyl chloride to yield 38.24
Stepwise deprotection of the benzyl ether to 39 and isopropy-
lidene acetal furnished target inhibitor 12.

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 33
Table 1. Inhibition of M. tuberculosis H37Rva
inhibitor
MIC99 (µM)
MIC50 (µM)b
isoniazid
0.18
ndc
7
8
9
10
11
12
13
0.29
>100
12.5
0.091 ( 0.05
30.9 ( 2.5
4.5 ( 1.0
0.077 ( 0.022
>100
0.19
>100
>100
>100
>100
>100
a Grown in GAST Media w/out Fe3+; see Supporting Information for
details. b MIC50 value ( SD (N ) 4) was obtained from the dose-response
curves and corresponds to the drug concentration, which resulted in 50%
inhibition of growth. c Not determined.
The novel acyltriazole inhibitor 13, which still maintains the
critical four-atom spacer and possesses multiple H-bonding
acceptor sites, was the last analogue examined. This was
prepared from O-benzylsalicylic acid 34 by conversion to
Weinreb amide 40 (Scheme 4). Addition of the lithium anion
of TMS-acetylene to 40 afforded 41 that was desilylated with
TBAF to furnish ynone 42. The copper(I)-catalyzed Hu¨isgen
1,3-dipolar cycloaddition between ynone 42 and azide 25
provided exclusively the regioisomeric 1,4-disubstituted triazole
43, which was deprotected to generate the target inhibitor 13.25
The inhibitors were tested directly against whole-cell M.
tuberculosis H37Rv under iron-limiting conditions since this
allows the concurrent evaluation of binding affinity, stability,
and membrane permeability.26 The minimum inhibitory con-
centrations (MIC) that inhibited >99% of growth are shown in
Table 1. The MIC50 values were calculated from the dose-
response plots and are also shown in Table 1. Salicyl-sulfamide
10 displayed the highest activity with an MIC99 of 0.19 µM,
which rivals the first-line antitubercular isoniazid. Salicyl-
sulfamate 7 displayed slightly attenuated activity with an MIC99
of 0.29 µM. The importance of the hydroxy function of the
salicyl moiety was examined by deletion using the simple
benzoyl analogue 9 which displayed a 66-fold decrease in
MIC99, while substitution by an amine in anthraniloyl analogue
8 led to greater than 500-fold loss of activity (MIC99). Inhibitors
using either the sulfamate- (11), â-ketophosphonate- (12), or
acyltriazole-linker (13) showed no inhibition of growth up to
100 µM.
Recently, the MIC50 of analogue 7 against M. tuberculosis
H37Rv was reported as 2.2 µM. However, these authors
measured the MIC50 value using a much higher cell density
(OD620 ) 0.01 which approximates 107 bacteria/mL) than is
typically used, which can lead to underestimation of compound
potency due to drug inactivation by the high cell density as
well as increased likelihood of resistant mutants.17
The most potent inhibitors 7 and 10 were evaluated for their
ability to inhibit mycobactin biosynthesis utilizing a modified
radioassay originally developed by DeVoss and co-workers.6,17
M. tuberculosis H37Rv (initial OD620 ) 0.2) was treated with
radiolabeled [7-14C]-salicylic acid and inhibitors or DMSO
(0.25%) as a control.27 In the absence of inhibitor, synthesis of
both mycobactin and carboxymycobactin was robust while in
the presence of either 7 (20 µM) or 10 (10 µM) complete
inhibition of mycobactin and carboxymycobactin biosynthesis
was observed.
Figure 3. Model of 10 bound to MbtA. Legend: Compound 10
(colored by atom) was modeled into the DhbE active site after making
several amino acid changes to reflect the sequence of MbtA. Dihy-
droxybenzoate from the DhbE X-ray structure is overlaid in green. Key
distances in the model of 10 are shown using purple lines with distances
in Å. Only key residues affecting salicyl and linker binding are shown.
For comparison with the DhbE structure, residues are numbered
according to that sequence. Hydrogens removed for clarity.
where ED50 is defined as the concentration of compound that
inhibits 50% cell growth), demonstrating the pronounced
selectivity of the inhibitors and further validating the inhibitor
design strategy. The therapeutic index of the most potent
inhibitor 10 (ED50/MIC50) is greater than 900.
Next, the cocrystal structure of DhbE with an adenylated
dihydroxybenzoate in the active site was used to construct a
model of MbtA and compounds 7-13 were docked into the
model.11,27 Some activity differences among 7-13 may be due
to the geometry of the salicyl/linker region. Dihydroxybenzoate
adopts a flat conformation in the DhbE X-ray structure, as do
the most active docked compounds, acylsulfamate 7 and
acylsulfamide 10. Both form an internal hydrogen bond between
the salicyl o-hydroxy and the sulfamate amine. In contrast,
quantum calculations show that analogues 8 and 9, which have
lower activity, are inherently unable to adopt the preferred planar
conformation because of steric conflicts involving the aryl ortho
substituent and the sulfamate amino hydrogen. However, this
explanation is only valid if the active site environment changes
the pKa of the nitrogen atom, which is deprotonated in solution.
Additional selectivity may arise from interactions of the ortho
substituent with Asn235: unlike the more active compounds,
9 lacks the hydroxy necessary to maintain the Asn235 hydrogen
bond observed in the DhbE X-ray structure. In the case of 8,
further selectivity could be provided if the Asn235 side chain
presents its amino group to the ligand, which would create a
preference for hydroxy (7) rather than amino (8) ortho substit-
uents. However, it is not possible to infer from the DhbE
structure which rotamer this side chain adopts in MbtA.
Modeling of 11 confirms that reduction of the salicyl carbonyl
to a methylene causes the loss of a hydrogen bond to Lys519.
No structural explanation for the inactivity of â-ketophos-
phonate 12 was observed. The lack of activity is likely a result
of hard-ionized phosphate moiety that limits membrane perme-
ability, and this underscores the usefulness of the acylsulfamate
and acylsulfamide linkage as phosphate bioisosteres. Although
these linkages too are ionized, the charge is highly delocalized.
Modeling of triazole 13 showed that hydrogen bonds involving
the phosphate of the natural substrate (or sulfamate of 7) are
To obtain preliminary data about toxicity for future in vivo
studies, the cytotoxicity of inhibitors 7-13 was evaluated against
the P388 murine leukemia cell line that has been shown to be
sensitive to nucleoside derivatives.27 None of the inhibitors
displayed any toxicity up to the maximum concentration tested
(ED50 > 100 µg/mL that corresponds to 100-200 µM for 7-13,

34 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
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See Supporting Information for details.
lost when a triazole replaces the phosphate. In addition, the
planar geometry of the triazole was a poor fit for the binding
site, requiring out of plane bending of the ring substituents.
In conclusion, we have designed, synthesized, and evaluated
a series of bisubstrate inhibitors of the adenylate-forming
enzyme MbtA. These studies focused on the crucial linker
domain of the inhibitor scaffold and resulted in the identification
of acylsulfamide 10, which is a potent inhibitor of M. tuber-
culosis growth under iron-limiting conditions. Molecular model-
ing provided insight into the observed SAR profile, while
preliminary studies support the proposed mechanism of action.
This strategy represents a promising approach for the develop-
ment of a new class of antibiotics effective for the treatment of
TB.
Acknowledgment. This research was supported by the
Center for Drug Design in the Academic Health Center of the
University of Minnesota. We thank the Minnesota Supercom-
puting Institute VWL lab for computer time and Ms. Christine
Dreis for performing the cytotoxicty assay.
Supporting Information Available: Experimental details (1H,
13C NMR, HRMS, HPLC) for intermediates and final products,
growth inhibition assay of M. tuberculosis H37Rv under iron-
deficient conditions, dose-response curves for inhibitors 7-10,
cytotoxicity assay, molecular modeling of MbtA, siderophore
radioassay, pKa calculations and measurements. This material is
available free of charge via the Internet at http://pubs.acs.org.
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