A novel indigoid anti-tuberculosis agent

Article abstract of DOI:10.1016/j.bmcl.2013.11.024

The structure of a novel indigoid component was characterized by X-ray crystallography. This compound exhibited excellent anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv in whole cell culture showing a submicromolar minimum inhibitory concentration (MIC). A synthesis of this molecule was designed and carried out to produce sufficient material for further testing. The in vitro profile, structure, and first synthesis of this indigoid component is reported.

Full text of DOI:10.1016/j.bmcl.2013.11.024

Bioorganic & Medicinal Chemistry Letters 24 (2014) 268–270
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel indigoid anti-tuberculosis agent
Larry L. Klein a, , Valentina Petukhova , Baojie Wan , Yuehong Wang , Bernard D. Santasiero ,
⇑
a
a
a
b
c
a,c
a,c
David C. Lankin , Guido F. Pauli , Scott G. Franzblau
a
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago 60612, USA
Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, USA
Department of Medicinal Chemistry, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago 60612, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure of a novel indigoid component was characterized by X-ray crystallography. This compound
exhibited excellent anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv in whole cell
culture showing a submicromolar minimum inhibitory concentration (MIC). A synthesis of this molecule
was designed and carried out to produce sufficient material for further testing. The in vitro profile,
structure, and first synthesis of this indigoid component is reported.
Received 30 August 2013
Revised 8 November 2013
Accepted 11 November 2013
Available online 21 November 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Anti-tuberculosis
Indigoid
X-ray
There exists great demand for new agents capable of combating
infections associated with Mycobacterium tuberculosis (M.tb), the
causative bacterium of tuberculosis (TB). This organism afflicts
over a third of the world’s population with an annual death rate
P450 enzymes7 (20 isoforms) which inactivate functionalized
molecules.
1
in excess of 2 million. Current first-line therapy involves use of
O
O HO
a combination of isoniazid, rifampicin, pyrazinamide, and etham-
butol or streptomycin (Fig. 1). These drugs were identified decades
ago, and while a number of molecules are currently under study as
N
O
HN NH2
O
OH OH
HO
Isoniazid
2
3
O
new agents (e.g., PA-824, OPC-67683), only one (TMC-207) has
successfully completed phase III clinical-stage development.
O
O
N
O
NH
N
4
A standard therapeutic course of 6–8 months is required to kill
N
NH2
this slow-growing bacteria and to allow for sterilization of the per-
sistent phenotype of M.tb. Furthermore, the infected populations
are generally found in developing countries where such dosing
dynamics and logistics often lead to poor patient compliance. As
a result, the non-compliance can exacerbate development of drug
resistance; for example, in 2006, 500,000 cases of multi-drug resis-
tant TB (MTR-TB) were estimated with 6.6% of these cases carrying
HO
O
N
N
Pyrazinamide
Rifampin
H N
2
HO
HN
NH OH
HO
NH
NH2
H
N
HN
O
HO
HO
O
N
H
N
O
H
OH
5
HO
O
O
HO
the extensively-drug resistant TB (XTR-TB) strains. Considering all
OH
Ethambutol
infectious diseases, TB is the #1 cause of death of HIV-infected
populations, and the epidemic has now become an urgent global
health problem.
H
O2N
N
Streptomycin
O
N
High-throughput screening (HTS) of compound libraries in
search of new anti-TB actives is fraught with problems related to
O
6
N
O
the unique lipophilic cell wall of M.tb serving as a barrier to some
structural types, along with the relative abundance of cytochrome
PA-824
Br
N
OH
Ph
TMC207
OCF3
⇑
Corresponding author.
E-mail address: llk@uic.edu (L.L. Klein).
Figure 1. Anti-TB agents.
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.024

L. L. Klein et al. / Bioorg. Med. Chem. Lett. 24 (2014) 268–270
269
Table 1
In vitro profile of compound 1 (lM)
Assaya
(1)
Isoniazid
Rifampin
1
2
3
4
5
6
7
8
9
MIC H37Rv
Cytotoxicity Vero cell
LORA MIC
Protein shift MIC (4% BSA)
Protein shift MIC (10% FBS)
MIC M. smegmatis
MIC C. albicans
MIC S. aureus
MIC E. coli
0.57
>32
5.3
0.22
>100
>100
0.45
0.24
18.9
ND
0.038
>182
0.49
0.089
0.21
16.7
ND
2.1
0.57
>50
>50
>50
>50
ND
ND
ND
ND
a
Description of these assays are listed in Supplementary data section.
Figure 3. X-ray structure of indigoid 1 (CCDC 919159).
Recently, scientists from The Shaw Group, Inc. have successfully
O
employed genetic engineering to modify the active site of the oxi-
dative enzyme, toluene-4-monooxygenase(T4MO).8 The wild type
T4MO enzyme is able to hydroxylate a wide range of aromatic
and aliphatic chemicals, and through their efforts, the substrate
range has been expanded by creating new isoforms with different
substrate specificities and product distributions. This approach,
termed ‘combinatorial biocatalysis’,9 proceeds by incubating the
organism (Escherichia coli) expressing the cloned T4MO isoform,
with the substrate(s), and the resultant microbial processing of
these substrates is followed by HPLC analysis. Indole-based sub-
strates were selected for this experiment due to their status as a
privileged core in medicinal chemistry, their propensity for oxida-
tion and subsequent dimerization under these culture conditions.
Following the microbial processing of this modified oxidative sys-
O
4
O
N
H
Na2CO3
NH
N
CH3OH
N
Eq. 1
Eq. 2
H
OAc
O
Indirubin 2
O
O
O
N
H
O
O
3
5
N
Na2CO3
X
CH3OH
N
H
1
O
2
2
Scheme 1. Indoxyl route to indigoids.
tem, novel secondary metabolites were produced and isolated.
Upon screening these indigoid libraries from the aforementioned
bacterial culture for antimicrobial bioactivities, several structural
sub-sets typified by compound 1 were found to exhibit potent
O
O
1
0
anti-TB activity. Herein, we report the anti-TB profile, structural
PCl
5
O
Benzene,
reflux,
Cl
6a
12
characterization of 1, and the first chemical synthesis of this
N
H
N
1
1
1-2 h 81%
compound.
4
The in vitro profile of 1 is listed in Table 1 along with that of the
anti-TB agents, isoniazid and rifampin. The biocatalytic reaction
described above utilized indole and anthranil as the lone sub-
strates (Fig. 2) and produced multiple components from which 1
was isolated. Compound 1 exhibits good potency and is not greatly
affected by protein-shift assays 4 and 5 (Table 1) showing a 1- and
O
HO
HO
O
CH2Cl2
RT 1-2 h
32%
Cl
Cl
N
H
O
8
N
3
.7-fold increase in the MIC, respectively. This compound has
O
O
excellent selectivity in the breadth of spectrum assays (6–9) with
O
N
N
6
b
O
7
N
CH2Cl2
RT 1-2 h
1%
N
H
1
O
1
+
O
N
H
N
Scheme 2. Chemical synthesis of compound 1.
indole
anthranil
E. coli
all MICs >50 lM. The Low Oxygen Recovery Assay (LORA),
designed to test those mycobacteria in the non-replicating pheno-
O
O
N
type, shows approximately a 10-fold effect.
1
3
Single crystal X-ray analysis, of 1 confirmed its novel struc-
ture (Fig. 3). Compound 1 is structurally related to indirubin (2),
a dye-like indole dimer, such that both share a 3-indolone core;
N
H
14
however, as indirubin lacked anti-TB activity, both the structural
1
O
1
5
and biological novelty of 1 was established.
O
In order to determine the viability of compound 1 as an anti-TB
agent, we required quantities much greater than the scale-limited
cellular system could provide. Initially, synthetic routes were
NH
16
N
H
based upon the classical synthesis of indirubin utilizing 3-acet-
oxylindole (3) as the nucleophilic partner and isatin (4) as the
electrophile (Scheme 1; Eq. 1). In a similar manner, coupling of 3
with the putative quinone 5 (attempted prep of 5 via oxidation17
2
O
Indirubin
Figure 2. Biocatalytic formation of 1 in E. coli and structure of indirubin (2).

2
70
L. L. Klein et al. / Bioorg. Med. Chem. Lett. 24 (2014) 268–270
of 5-hydroxybenzisoxazole; Scheme 1; Eq. 2) led to a complex mix-
ture of reaction products none of which was the desired product.
Alternatively, through the use of the polarity inversion concept,
the 5-hydroxybenzisoxazole (7) was utilized as the nucleophilic
partner with the indole-related fragment serving as the electro-
phile via its corresponding 2-chloroindolen-3-one (6a/b)
References and notes
1
2
.
.
Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.
(a) Palomino, J. C.; Ramos, D. F.; da Silva, P. A. Curr. Med. Chem. 2009, 16, 1898;
(
b) Rivers, E. C.; Mancera, R. L. Drug Discovery Today 2008, 13, 1090; (c) Ma, Z.;
Lienhardt, C.; McIlleron, H.; Nunn, A. J.; Wang, X. Lancet 2010. http://dx.doi.org/
0.1016/50140-6736(10)60359-9 (online) May 19.
FDA to approve bedaquiline (TMC-207); http://www.sarpam.net/archives/
211, Dec. 11, 2012.
1
3.
(Scheme 2). As previously reported, treatment of isatin (4) with
2
phosphorus pentachloride actually leads to dimeric structure
4. Elzinga, G.; Raviglione, M. C.; Maher, D. Lancet 2004, 363, 814.
5. Haydel, S. E. Pharmaceuticals 2010, 3, 2268.
1
8
6
b. Although this dimer is isolable via rapid chromatographic
6
7
8
.
.
.
Brennan, P. J. Tuberculosis 2003, 83, 91.
purification techniques, its instability to moisture encouraged di-
rect use of the crude material in subsequent reactions. The addition
McKean, K. J.; Munro, A. W. Drug Metab. Rev. 2008, 40, 427.
McClay, K.; Boss, C.; Keresztes, I.; Steffan, R. J. Appl. Environ. Microbiol. 2005, 71,
5476.
of 6 to other aromatic systems has precedent; for example, reac-
tion of 6 with 2-naphthol afforded adduct 819 which did not exhibit
9. Rich, J. O.; Michels, P. C.; Khmelnitsky, Y. L. Curr. Opin. Chem. Biol. 2002, 6, 161.
0. Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
1
1
any anti-TB activity. Direct combination of 6 with 5-hydroxyben-
1
1. H NMR (400 MHz, DMSO-d
J = 7.38, 7.59 Hz), 7.49 (d, 1H, J = 7.79 Hz), 7.62 (dt, 1H, J = 7.38, 7.59 Hz), 7.70
d, 1H, J = 7.38 Hz), 7.90 (d, 1H, J = 9.85), 10.06 (s, 1H), 12.40 (br s, 1H).
6
) d ppm 6.76 (d, 1H, J = 9.85 Hz), 7.11 (dt, 1H,
20
zisoxazole (7) at 25 °C produced 1. Generally, these products
were obtained in a pure state by filtration from the dichlorometh-
ane (DCM) reaction mixtures. Further purification, if necessary,
was accomplished via silica gel chromatography using methano-
lic-DCM as eluting solvents.
(
1
3
C NMR (400 MHz, DMSO-d
6
) d ppm 100.59, 112.16, 114.08, 119.00, 122.82,
124.82, 128.14, 134.10, 137.27, 140.74, 151.59, 153.60, 156.66, 187.87, 188.89.
+
8 3 2
HRMS [ESI] calculated for C15H O N (M+H ) 265.0608; found 265.0603.
1
1
2. Several structures were proposed as possible matches for the 1-D and 2-D NMR
data for the isolate, which included the correct structure; G. Pauli, D. Lankin;
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois
at Chicago 833 S. Wood Street, Chicago, IL 60612.
These compounds exhibit poor water solubility and are unsta-
ble in the presence of secondary amines. The possibility of this
2
1
3. For X-ray data, see Supplementary data. The author has deposited atomic
chemical instability being related to the poor metabolic stability
coordinates for
1 with the Cambridge Crystallographic Data Centre and
is presently under investigation. Efforts to modify the solubility
and stability characteristics and expand the structure–activity
relationship is presently underway using new chemical approaches
for functionalization. The excellent potency, ease of access, and low
molecular weight of this novel anti-TB hit provides the encourage-
ment for these efforts.
allocated the deposition number: CCDC 919159. The coordinates can be
obtained, on request, from the Director, Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge, CB2 lEZ, U.K.
1
1
1
1
4. Indirubin, indigo, and isoindigo were tested under standard conditions and
found to show MICs of >32 lM, >32 lM, and 27 lM, respectively, against M.tb.
5. SciFinder (Chemical Abstracts Service) and Reaxys (Elsevier Properties) were
analyzed via substructure, term, and patent searches.
6. (a) Baeyer, A. Chem. Ber. 1881, 14, 1741; (b) Russell, G. A.; Kaupp, G. J. J. Am.
Chem. Soc. 1969, 91, 3851.
Acknowledgments
7. Attempted oxidation of 5-hydroxybenzoisoxazole afforded 76% of the putative
quinone. 1H NMR (CDCl
) d ppm 9.06 (s, 1H), 7.73 (d, 1H, J = 8.0 Hz), 6.74 (d,
H, J = 8.0 Hz). Mass spectral analysis showed only a dimer signal [M+H] = 299
3
1
The project described herein was supported by Grant Number
R21AI097670 from the National Institute of Allergy And Infectious
Diseases. The content is solely the responsibility of the authors and
does not necessarily represent the official views of the National
Institute of Allergy And Infectious Diseases or the National Insti-
tutes of Health. We are indebted to K. McClay and R. J. Steffan
rather than the parent [M+H] = 150.
18. Cornforth, J.; Hitchcock, P. B.; Rozos, P. J. Chem. Soc., Perkin Trans. 1 1996, 2787.
19. Begley, W. J.; Grimshaw, J. J. Chem. Soc., Perkin Trans. 1 1975, 1840.
2
2
2
0. A sample (25 mg) was purified for an analytical sample via silica gel
chromatography using DCM as eluent to give 9 mg (11%) of the desired
product, 1 whose 1H, 13C NMR data, HRMS and HPLC data agreed in total with
material obtained from the incubation.11
1. Unpublished results, L.L. Klein, V. Petukhova; Treatment of 1 with secondary
amines such as diethylamine, piperidine at room temperature led to slow loss
of starting material and formation of blue-green products which are, as yet,
unidentified.
2. (a) Baeyer, A. Chem. Ber. 1881, 14, 1741; (b) McClay, K.: Wan, B.; Wang, Y.; Cho,
S.; Yu, J.; Santarsiero, B.; Mehboob, S.; Johnson, M.; Franzblau, S.; Steffan, R. US
patent US2011/0082180 A1, 2013; Appl. Microbiol. Biotechnol. 2013, 97, 7151.
(
The Shaw Group) for their initial findings and collaboration in this
project.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
1
1.024.