Design, synthesis, and biological evaluation of β-ketosulfonamide adenylation inhibitors as potential antitubercular agents

Article abstract of DOI:10.1021/ol0617289

(Chemical Equation Presented) The antitubercular nucleoside antibiotics 1 and 2 were recently described that inhibit the adenylate-forming enzyme MbtA and disrupt biosynthesis of the virulence-conferring siderophore known as mycobactin in Mycobacterium tu

Full text of DOI:10.1021/ol0617289

ORGANIC
LETTERS
2006
Vol. 8, No. 21
4707-4710
Design, Synthesis, and Biological
Evaluation of -Ketosulfonamide
â
Adenylation Inhibitors as Potential
Antitubercular Agents
Jagadeshwar Vannada,† Eric M. Bennett,† Daniel J. Wilson,† Helena I. Boshoff,‡
Clifton E. Barry, III,‡ and Courtney C. Aldrich*,†
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
aldri015@umn.edu
Received July 13, 2006
ABSTRACT
The antitubercular nucleoside antibiotics 1 and 2 were recently described that inhibit the adenylate-forming enzyme MbtA and disrupt biosynthesis
of the virulence-conferring siderophore known as mycobactin in Mycobacterium tuberculosis. Herein, we report efforts to refine this inhibitor
scaffold by replacing the labile acylsulfamate linkage (highlighted) with the more chemically robust
â-ketosulfonamide linkage of 3 and 4.
Mycobacterium tuberculosis, the etiological agent of tuber-
culosis (TB), is the leading bacterial cause of infectious
disease mortality.1 The development of M. tuberculosis
strains which are resistant to all of the current front-line
antitubercular drugs has prompted worldwide efforts to
discover new antibiotics to treat this notorious pathogen. Iron
acquisition is an essential process for M. tuberculosis as well
as almost all other microorganisms.2 However, this essential
micronutrient is highly sequestered in a mammalian host.
Bacteria have evolved a variety of mechanisms to obtain this
vital nutrient, but the most common mechanism involves the
synthesis of small-molecule iron chelators, termed sidero-
phores.3 M. tuberculosis produces a pair of related peptidic
siderophores known as mycobactin-T 8 and carboxymyco-
bactins 9 that vary by the appended lipid residue and will
hereafter be referred to collectively as the mycobactins
(Figure 1).4 The critical role of the mycobactins for growth
and virulence of M. tuberculosis is supported by substantial
in vitro and in vivo evidence.5 Therefore, inhibition of
mycobactin biosynthesis6 or antagonism of mycobactin
function7 represents an attractive strategy for the development
of a new class of antitubercular agents.
The biosynthesis of the mycobactins is initiated by MbtA,
an adenylate-forming enzyme, which activates salicylic acid
5 at the expense of ATP to form salicyl-adenylate 6 that
remains tightly bound to the enzyme with expulsion of
pyrophosphate (Figure 1).8 MbtA then catalyzes the transfer
of 6 onto a carrier domain of MbtB to provide 7, which is
ultimately extended to the mycobactins through the activity
of almost a dozen additional proteins.8,9
Nucleoside analogues 1 and 2 were designed as stable in-
termediate mimetics of 6 and found to inhibit MbtA
(4) (a) Vergne, A. F.; Walz, A. J.; Miller, M. J. Nat. Prod. Rep. 2000,
17, 99. (b) Snow, G. A. Bacteriol. ReV. 1970, 34, 99.
(5) (a) De Voss, J. J.; Rutter, K.; Schroeder, B. G.; Su, H.; Zhu, Y.;
Barry, C. E., III. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1252. (b) Luo,
M.; Fadeev, E. A.; Groves, J. T. Nat. Chem. Biol. 2005, 1, 149. (c) Wagner,
D.; Maser, J.; Lai, B.; Cai, Z.; Barry, C. E., III; zu Bentrup, K. H.; Russell,
D. G.; Bermudez, L. E. J. Immunol. 2005, 174, 1491.
(6) (a) Ferreras, J. A.; Ryu, J.-S.; Lello, F. D.; Tan, D. S.; Quadri, L. E.
N. Nat. Chem. Biol. 2005, 1, 29. (b) Somu, R. V.; Boshoff, H.; Qiao, C.;
Bennett, E. M.; Barry, C. E., III; Aldrich, C. C. J. Med. Chem. 2006, 49,
31. (c) Miethke, M.; Bisseret, P.; Beckering, C. L.; Vignard, D.; Eustache,
J.; Marahiel, M. A. FEBS J. 2006, 273, 409.
† University of Minnesota.
‡ National Institute of Allergy and Infectious Diseases.
(1) Tuberculosis Handbook; World Health Organization: Geneva, 1998
(WHO/TB/98.253).
(2) Ratledge, C.; Dover, L. G. Annu. ReV. Microbiol. 2000, 54, 881.
(3) Crosa, J. H.; Walsh, C. T. Microbiol. Mol. Biol. ReV. 2002, 66, 223.
10.1021/ol0617289 CCC: $33.50
© 2006 American Chemical Society
Published on Web 09/16/2006

Figure 1. Biosynthesis of the mycobactins.
potently.6 These compounds also disrupted mycobactin bio-
synthesis and inhibited growth of M. tuberculosis H37Rv
under iron-limiting conditions.6a,b We recognized that hy-
drolysis of the acylsulfamate and acylsulfamide linkages of
1 and 2 would release 5′-O-(sulfamoyl)adenosine 10 or its
amino congener 11, which are among the most potent cyto-
toxic compounds yet reported (Figure 2).10 The potential for
suitable for these demands. Our retrosynthetic analysis
involves disconnection of 3 by Mitsunobu reaction to N-Boc-
â-ketosulfonamide 12 (Figure 3).13 Further retrosynthesis
leads to N-Boc-methylsulfonamide 13.
Figure 3. Retrosynthetic analysis.
Synthesis commenced with addition of LDA to 1314 to
afford dianion 14 (Scheme 1).15 Next, a solution of MOM-
Figure 2. Hydrolysis to cytotoxic sulfamoyladenosines.
such a reaction through in vivo metabolism could lead to
severe toxicity of these antibacterial agents. Furthermore,
both 1 and 2 are ionized at physiological pH, which may
limit oral bioavailability.6b To simultaneously address these
potential shortcomings, we replaced the central nitrogen atom
of the acylsulfamide linker of 2 with a carbon atom, which
was expected to prevent undesirable hydrolysis as well as
to increase the lipophilicity. The change of the sulfamide
moiety to a sulfonamide function was further motivated by
the prevalance of the sulfonamide group in numerous thera-
peutic agents including antiinfectives, antidiabetics, and
diuretics.11
Scheme 1. Synthesis of â-Ketosulfonamide
Herein, we report the synthesis of â-ketosulfonamide
analogues 3 and 4 featuring a novel Claisen-like condensation
to assemble the â-ketosulfonamide followed by Mitsunobu
coupling to attach the adenosyl subunit. The in vitro enzyme
inhibition of 3 and 4 and in vivo activity against whole cell
M. tuberculosis have been evaluated. Further, molecular
modeling provided important insight into the binding of these
adenylation inhibitors to MbtA.
protected salicylate derivative 15 was added to provide
â-ketosulfonamide 12. The third equivalent of LDA was
necessary to enolize the resultant â-ketosulfonamide to 16
preventing overaddition. This simple reaction represents a
new entry into the â-ketosulfonamide function.
We next turned our attention to the key Mitsunobu reaction
to couple a suitably protected adenosine derivative with
Several methods to synthesize â-ketosulfonamides have
been reported including reaction of enamines with N-
alkylsulfamoyl chlorides,12a treatment of silyl enol ethers with
N-methylsulfamoyl imines,12b coupling of primary amines
with N-aroylmethylsulfonyl derivatives,12c and condensation
of N-alkyl sulfonamides with a nitrile.12d We sought an
efficient synthesis of the â-ketosulfonamide targets that
would enable the facile introduction of the nucleoside subunit
and allow the rapid synthesis of the â-ketosulfonamide
moiety; however, none of the aforementioned methods were
(7) Xu, Y.; Miller, M. J. J. Org. Chem. 1998, 63, 4314.
(8) Quadri, L. E. N.; Sello, J.; Keating, T. A.; Weinreb, P. H.; Walsh,
C. T. Chem. Biol. 1998, 5, 631.
(9) Krithika, R.; Marathe, U.; Saxena, P.; Ansari, M. Z.; Mohanty, D.;
Gokhale, R. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2069.
(10) Bloch, A.; Coutsogeorgopoulos, C. Biochemistry 1971, 10, 4394.
(11) Lednicer, D.; Mitscher, L. A. The Organic Chemistry of Drug
Synthesis; Wiley: New York, 1998; Vol. 6.
(12) (a) Bender, A.; Gu¨nther, D.; Willms, L.; Wingen, R. Synthesis 1985,
66. (b) Vega, J. A.; Molina, A.; Alajar´ın, R.; Vaquero, J. J.; Garc´ıa-Navio,
J. L.; Alvarez-Builla, J. Tetrahedron Lett. 1992, 33, 3677. (c) Hendrickson,
J. B.; Bergeron, R. Tetrahedron Lett. 1970, 5, 345. (d) Thompson, M. E.
Synth. Commun. 1988, 733.
4708
Org. Lett., Vol. 8, No. 21, 2006

â-ketosulfonamide 12.13 Initial attempts to couple 12 and
2,3-isopropylidene adenosine 17 were unsuccessful (not
shown). We speculated that this failure might be due to the
competitive cyclization between N-3 and C-5′ of the activated
species as we observed consumption of the alcohol with
formation of an extremely polar product.16 Installation of a
Boc-carbamate at N6 of the adenine served to attenuate the
nucleophilicity of N-3 and suppress this undesired reaction.
Successful Mitsunobu coupling to afford 19 was achieved
by addition of DIAD via syringe pump to a stirring solution
of the â-ketosulfonamide 12, N6-Boc-adenosine 18, and TPP
at 0 °C (Scheme 2). Optimization of these conditions revealed
the pure product from a crude reaction mixture containing
excess and spent reagents. The fluorous approach employing
fluorous DIAD (named as F-DIAD) along with fluorous TPP
(named as F-TPP) enables the rapid purification of Mit-
sunobu reactions by fluorous solid-phase extraction (FSPE).18
Utilizing F-TPP and F-DIAD and our optimized reaction
conditions, we were able to obtain 19 in 85% yield by simple
filtration of the crude reaction mixture through a fluorous
SiO2 cartridge (Table 1, entry 5).
The synthesis of â-ketosulfonamide 3 was completed by
simultaneous deprotection of the MOM acetal, isopropylidene
ketal, and Boc carbamates employing aqueous TFA (Scheme
3). Fluorination of the active methylene of 19 with Selectfluor
Scheme 2. Mitsunobu Coupling
Scheme 3. Synthesis of Final Targets
that the DIAD addition time was the most crucial parameter,
thus the yield improved from 23% to 82% by increasing the
addition time of DIAD from 5 to 30 min (Table 1, entries
Table 1. Optimization of Mitsunobu Reaction Conditions
provided 20, which was deprotected to yield R,R-difluoro-
â-ketosulfonamide 4.19
Next, inhibition of recombinant MbtA by the bisubstrate
inhibitors was measured using a [32P]PPi-ATP exchange
assay to determine KIapp values (Table 2).6a,20 In addition to
addition time
of DIAD
(min)
yield
time 19
phosphine azodicarboxylate
(equiv) (equiv)
entry
(h)
(%)
1
2
3
4
5
TPP (2.0) DIAD (2.0)
TPP (1.5) DIAD (1.5)
TPP (1.5) DIAD (1.3)
TPP (1.5) DIAD (1.3)
F-TPP (1.5)F-DIAD (1.3)
5
10
30
30
30
12
12
12
48
12
23
46
82
0
85
Table 2. MIC99 and KIapp Values of Bisubstrate Inhibitors
inhibitor
KIapp (µM)
MIC99 (µM)
1-3). Attempts to further improve the yield by increasing
the total reaction time were unsuccessful (Table 1, entry 4),
which we speculate is due to the condensation with dicar-
boalkoxyhydrazine (DCH) to afford an enamine adduct (not
shown).17 Thus, the optimal reactions identified herein
involve the slow addition of DIAD over 30 min to a cooled
(0 °C) solution of the substrates and TPP followed by stirring
for 12 h at 23 °C.
1
2
3
4
10
21
0.00507 ( 0.00106
0.00375 ( 0.00058
3.30 ( 0.57
>100
>100
>100
0.29a
0.19a
25
>100
50
>100a
a See ref 6b.
Although these optimized conditions worked well, isola-
tion of 19 was challenging requiring multiple chromato-
graphic separations. Indeed, the application of the Mitsunobu
reaction is often limited by the difficulty involved in isolating
3 and 4 whose synthesis is described in this paper, we also
evaluated 1, 2, 10, and 21 (see Figure 4B)6b that we had
previously synthesized but not examined for enzyme
inhibition.6b These additional analogues provided important
SAR data. â-Ketosulfonamide 3 displayed modest inhibition
of MbtA with a KIapp of 3.30 ( 0.57 µM, and 4 showed no
(13) Henry, J. R.; Marcin, L. R.; McIntosh, M. C.; Scola, P. M.; Harris,
G. D., Jr.; Weinreb, S. M. Tetrahedron Lett. 1989, 30, 5709.
(14) Neustadt, B. R. Tetrahedron Lett. 1994, 35, 379.
(15) (a) Harter, W. G.; Albrecht, H.; Brady, K.; Caprathe, B.; Dunbar,
J.; Gilmore, J.; Hays, S.; Kostlan, C. R.; Lunney, B.; Walker, N. Bioorg.
Med. Chem. Lett. 2004, 14, 809. (b) Thompson, M. E. J. Org. Chem. 1984,
49, 1700. (c) Johnson, D. C., II; Widlanski, T. S. J. Org. Chem. 2003, 68,
5300.
(17) HRMS calcd C41H59N8O15S [M + H]+ 935.3815, found 935.3717
(error 10.5 ppm).
(18) Dandapani, S.; Curran, D. P. J. Org. Chem. 2004, 69, 8751.
(19) (a) Lal, G. S. J. Org. Chem. 1993, 58, 2791. (b) Ladame, S.; Willson,
M.; Pe´rie´, J. Eur. J. Org. Chem. 2002, 2640.
(16) Mitsunobu, O. Synthesis 1981, 1.
Org. Lett., Vol. 8, No. 21, 2006
4709

significantly from planarity, with energy differences of 6.6
kcal/mol or larger relative to the docked conformation.
Interestingly, these results held only when the compounds
were modeled with the aryl hydroxyl donating a hydrogen
bond to the R atom (in the case of the acylsulfamate and
acylsulfamide linkers, the R-nitrogen is expected to be
deprotonated6b). When the hydroxyl hydrogen was instead
oriented in the opposite direction to donate a hydrogen bond
to the side chain of Asn235, all compounds became less
planar during the QM optimization (data not shown; residues
are numbered as in DhbE).
Because 4 and 21 were inactive while the KIapp of 3 was
reduced approximately 1000-fold relative to 2, we hypothe-
size that linker planarity, stabilized by an internal hydrogen
bond (Figure 4A), may be important for inhibition of MbtA.
In this case, the Asn235 side chain must present its amino
group to the inhibitor, a conformation which is consistent
with the 150-fold loss of activity observed when the aryl
hydroxyl is replaced with an amino group (data not shown).5a
The minimum concentrations of 3, 4, and 10 that inhibited
>99% of growth of M. tuberculosis H37Rv under iron-
deficient conditions5a were determined and are shown in
Table 2. Compound 3 exhibited an MIC99 of 25 µM, and 4
was inactive. The correlation of KIapp and MIC99 values of
1-4 under iron-deficient conditions provides support for the
designed mechanism of action. Additionally, 5′-O-(sulfa-
moyl)adenosine 10 displayed an MIC99 of 50 µM, thus the
potent activity of 1 and 2 is not due to hydrolysis of these
to release 10 or 11, respectively.
Figure 4. Proposed hydrogen-bonding arrangements. Highly active
compounds (A) have linkers which readily adopt a planar geometry
stabilized by an internal hydrogen bond, whereas some inactive
compounds (B) have linkers which do not readily adopt a planar
geometry.
inhibition at the maximum concentration evaluated (KIapp
>
100 µM). By contrast, the parent acylsulfamide inhibitor 2
exhibited potent inhibition with a KIapp of 0.0038 ( 0.0006
µM. Additionally, â-ketophosphonate 21 was inactive (KIapp
> 100 µM).
To investigate the structural basis for activity, compounds
2-4, the natural acyl phosphate intermediate 6, and â-ke-
tophosphonate 216b (Figure 4B) were docked into our
homology model based on the X-ray structure of DhbE,
which performs the analogous adenylation of 2,3-dihydroxy-
benzoic acid.6b Molecular mechanics simulations of the
ligands free from the constraints of the protein binding site
showed that some compounds did not prefer the planar linker
conformation observed in the X-ray structure. Although many
enzymes must release their products quickly and therefore
may have reduced affinity for their products, MbtA must
retain the acyl phosphate for transfer to MbtB. Therefore,
the acyl phosphate’s conformational preference may be
relevant for inhibitor design.
To confirm the molecular mechanics results, the docked
conformations were truncated at the 5′ carbon and reopti-
mized free from the constraints of the active site at the
B3LYP/6-311G++(d,p) level. The sums of the absolute
values of the deviation from planarity of the two bonds about
the â-carbonyl are summarized in Table 3 (see Figure 4A:
arrows denote bonds of interest). The natural acyl phosphate
6 and the highly potent inhibitor 2 adopted a nearly planar
geometry similar to that observed for the acyl phosphate in
the X-ray structure.21 In contrast, 3, 4, and 21 deviated
In summary, we have developed an efficient synthesis of
â-ketosulfonamide adenylation inhibitors featuring a newly
developed Claisen-type condensation and the fluorous version
of the Mitsunobu reaction to install the nucleoside subunit
of the bisubstrate inhibitors. In this study, we have extended
our investigation of the structure-activity relationships of
the nucleoside bisubstrate inhibitors. The compromise in
potency of 3 is offset by its anticipated improved ADMET
profile. Modification of the nucleoside subunit of the inhibitor
scaffold to regain potency and further improve upon desirable
pharmacological properties is the focus of current efforts.
Acknowledgment. We thank Prof. Robert Vince for
invaluable advice and the Minnesota Supercomputing Insti-
tute VWL lab for computer time. This research was supported
by grants from the NIH (R01AI070219) and the Center for
Drug Design in the Academic Health Center of the University
of Minnesota to C.C.A.
Table 3. Total Torsional Deviation from Planarity around the
Inhibitor â-Keto Group
Supporting Information Available: Experimental pro-
deviation from planarity deviation from planarity
1
cedures, compound characterization data and H and 13C
compound
(dockeda in protein)
(QM-optimized)b
NMR of compounds reported herein, details of the [32P]PPi-
ATP exchange assay to determine KIapp values, molecular
modeling, and growth inhibition assay of M. tuberculosis
H37Rv under iron-deficient conditions. This material is
available free of charge via the Internet at http://pubs.acs.org.
2
3
4
31°
18°
17°
29°
19°
1°
67°
52°
14°
140°
6c
21
OL0617289
a Docked to an MbtA homology model using MacroModel. b Optimized
in the absence of binding site constraints using Jaguar. c In the X-ray
structure of DhbE, the bound phosphate product deviates from planarity
by 7°.
(20) Linne, U.; Marahiel, M. A. Methods Enzymol. 2004, 388, 293.
(21) May, J. J.; Kessler, N.; Marahiel, M. A.; Stubbs, M. T. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 12120.
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