Diversity-oriented synthesis for novel, selective and drug-like inhibitors for a phosphatase from Mycobacterium tuberculosis

Article abstract of DOI:10.1039/c4md00099d

Mycobacterium protein tyrosine phosphatase B (mPTPB) is a potential drug target of tuberculosis (TB). Using small molecule inhibitors of mPTPB could be a treatment to overcome emerging TB drug resistance. Using a Diversity-Oriented Synthesis (DOS) strateg

Full text of DOI:10.1039/c4md00099d

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Diversity-oriented synthesis for novel, selective
and drug-like inhibitors for a phosphatase from
Mycobacterium tuberculosis†
Cite this: Med. Chem. Commun., 2014,
5, 1496
Rongjun He, Yunpeng Bai, Zhi-Hong Yu, Li Wu, Andrea Michelle Gunawan
*
and Zhong-Yin Zhang
Mycobacterium protein tyrosine phosphatase B (mPTPB) is a potential drug target of tuberculosis (TB).
Using small molecule inhibitors of mPTPB could be a treatment to overcome emerging TB drug
resistance. Using a Diversity-Oriented Synthesis (DOS) strategy, we successfully developed a salicylic acid
based and drug-like mPTPB inhibitor with an IC50 of 2 mM and >20-fold specificity over many human
PTPs, making it an excellent lead molecule for anti-TB drug discovery. In addition, DOS generated
bicyclic salicylic acids are also promising starting points for acquiring inhibitors targeting other PTPs.
Received 5th March 2014
Accepted 21st July 2014
DOI: 10.1039/c4md00099d
www.rsc.org/medchemcomm
Protein tyrosine phosphatases (PTPs) have emerged as the next immune responses.9 Deletion of mPTPB impaired the ability of
generation drug targets due to their complementary roles to the mutant strain to survive in interferon-g (IFN-g) activated
protein tyrosine kinases in controlling protein tyrosine phos- macrophages and severely reduced the bacterial load in a clin-
phorylation levels.1 Small molecule PTP inhibitors thus possess ically relevant guinea pig model.10 Hence small molecules that
an enormous amount of therapeutic potential. However, there inhibit mPTPB possess great potential as anti-TB agents with
are two major challenges in PTP inhibitor development: speci- the nonoverlapping mechanism of action with existing drugs.4c
city and cell permeability, due to conserved and highly posi- Although a handful of mPTPB inhibitors have been reported,
tively charged active sites.2 Consequently, most existing PTP they oen have high molecular weight, high lipophilicity, and
inhibitors mimic the negatively charged pTyr substrate.1d,3 We exible structures, and are thus not ideal lead molecules.11
discovered that salicylic acid could serve as a novel pTyr Herein we report our DOS strategy for generating novel salicylic
mimetic, affording PTP inhibitors with excellent potency, acid based mPTPB inhibitors with good potency, specicity, cell
specicity, and cell permeability.4 To expand the salicylic acid activity, and more importantly, lean and compact structures
based chemistry, we sought to employ a Diversity-Oriented with drug-likeness.
Synthesis (DOS)5 strategy to develop salicylic acid based PTP
inhibitors with novel and diverse structures, and more impor- acid itself in inhibiting the PTP activity as a result of enhanced
tantly, drug-like properties.
interactions with the PTP active site,4 our target molecules in
Given that bicyclic salicylic acid is more active than salicylic
Tuberculosis (TB) is a worldwide threat to public health with the DOS strategy encompass a range of novel bicyclic hetero-
9 million new infections and 1.8 million deaths yearly.6 Existing cycles (Fig. 1). To this end, we aim to install heteroatoms in
TB treatments require administration of antibiotics targeting adjacent positions on the parent benzene ring of salicylic acid,
mycobacterial RNA transcription, protein translation, and cell which furnishes molecular handles for the generation of the
wall biogenesis for 6–9 months.7 The lengthy treatment oen second heterocycles. The substituents can be amino, hydroxyl,
leads to patient noncompliance, which contributes to the halogen, and other groups that are easily functionalized. As a
emergence of multidrug-resistant (MDR) and extensively resis- proof of concept, we synthesized protected salicylic acids with
tant (XDR) TB. Thus there is an urgent need to develop new and
more effective therapies against TB.8 Mycobacterium protein
tyrosine phosphatase B (mPTPB) was identied as a promising
target for novel anti-TB agents. It is secreted into host macro-
phages by Mtb and is a virulence factor to attenuate host
Department of Biochemistry and Molecular Biology, Chemical Genomics Core Facility,
Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN, 46202,
USA. E-mail: zyzhang@iu.edu; Fax: +1-317-274-4686; Tel: +1-317-274-4686
† Electronic supplementary information (ESI) available. See DOI:
Fig. 1 Structure of salicylic acid as pTyr mimetic and the design of
10.1039/c4md00099d
DOS for discovery of novel and drug-like mPTPB inhibitors.
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substituents of 4-hydroxy-5-amino (1), 4,5-diamino (2), 4-amino- salicylic acids (Scheme 2). Examples from intermediate 1
5-iodo (3), and 4-amino-3-iodo (4). The syntheses of these include isothiocyanates for aminooxazole 5, triphosgene for
intermediates are straightforward from commercially available cyclic carbamate 6, chloroacetic chloride and bromoacetic acid
starting materials (Scheme 1). 1 was obtained from 4-hydroxy methyl ester for six membered lactam 7 and lactone 8, and a
salicylic acid in a sequence of nitration, protection, and sequence of benzaldehyde and DDQ for oxazole 9.12 From
reduction with 46% overall yield. Similarly, 2 was prepared in intermediate 2, the method for synthesizing oxazole 9 failed to
5 steps including acetylation, nitration, hydrolysis, esterica- give cyclised product 10, however, treatment of 2 with benzal-
tion, and reduction, with an overall yield of 23%. Although dehyde in the presence of ammonium acetate successfully
iodine gave amino and iodo substituted 3 and 4 in low yields, afforded benzimidazole 10 in excellent yield, and oxygen in the
using 1.1 equivalents of iodine monochloride successfully air was the oxidant for cyclisation.13 Intermediates 3 and 4 were
delivered products 3 and 4 in excellent yield, which were easily reacted with phenyl isothiocyanate to give aminobenzothiazole
separated by ash chromatography.
compounds 11a and 11b, respectively.14,15 The reaction occurs
Intermediates 1–4 were treated with various cyclization in a sequence of thiourea formation and Cu(I) catalyzed S-ary-
reagents to make the second heterocyclic rings, which were then lation, and TBAB was proposed to play a role as a ligand and
hydrolyzed in 10% LiOH–MeOH (v/v ¼ 1 : 1) to afford bicyclic phase-transfer catalyst. All products generated by the DOS
strategy were obtained in moderate to good yields, and were
puried by reversed phase HPLC to give structurally diverse
bicyclic heterocycles with salicylic acid moiety. The ability of the
compounds to inhibit the mPTPB catalyzed para-nitrophenyl
phosphate hydrolysis was evaluated. Several structural motifs,
including benzoxazole 9, benzimidazole 10, and amino-
benzothiazole 11a, stand out as the most active among the DOS
generated bicyclic salicylic acids 5–11, with IC50 values from 20
to 30 mM, while bicycles such as 6, 7, and 8 did not exhibit
activity at 100 mM, likely due to the lack of aromaticity. Inter-
estingly, 11a is 3-fold more active than its close analogues 11b,
indicating that the hydroxyl group on the parent phenyl ring
contributes to the affinity, and its location on the benzene ring
inuences compound's activity against mPTPB. Compound
Scheme 1 The synthesis of 3,4 or 4,5-disubstituted intermediates as
11a's easy access, good activity, low molecular weight (MW ¼
precursors for DOS.
286), coupled with the prevalence of the aminothiazole motif in
FDA approved drugs such as Dasatinib and Norvir, make it ideal
for further elaboration to develop drug-like mPTPB inhibitors
with improved potency.
Thus, a focused library of aminothiazole-salicylic acids was
prepared by reacting precursor 3 with a set of aryl iso-
thiocyanates. Generally, o, m, or p-substituted phenyl iso-
thiocyanates with either electron donating or withdrawing
properties, as well as 1-naphthyl isothiocyanate, were tolerated
to afford products in moderate to good yields, except that alkyl
isothiocyanates did not react with 3 under the same reaction
conditions (Fig. 2). All hydrolyzed products were puried by
HPLC to ensure high purity. SAR analysis indicated that a
substituent at the m position of isothiocyanate is most bene-
cial, and compound 11h with m-bromo was found to be the
most potent mPTPB inhibitor with an IC50 of 2.0 mM, repre-
senting a 12-fold increase in comparison to 11a. It should be
noted that salicylic acid exhibits an IC50 value of 55 ꢀ 8 mM for
mPTPB. Thus, compound 11h improved the salicylic acid
mPTPB inhibitory activity by 27 500 fold. Subsequent kinetic
studies revealed that it is a noncompetitive inhibitor against
pNPP with a Ki of 2.2 ꢀ 0.1 mM (Fig. S1, ESI†). To determine the
specicity of 11h towards mPTPB, its inhibitory activity against
representative human PTPs including cytosolic PTPs, PTP1B,
SHP2, SHP1, PTP-Meg2, the receptor-like PTPs, CD45 and PTP3,
and the dual specicity phosphatases VHR and Laforin, was
measured. As shown in Table 1, 11h is highly selective for
Scheme 2 DOS of various bicyclic salicylic acids as mPTPB inhibitors.
Reaction conditions c: 10% LiOH–MeOH (v/v ¼ 1 : 1), 80 ꢁC, 1 h.
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such as PTP1B and VHR (Fig. S3, ESI†). mPTPB secreted by Mtb
down-regulates Erk1/2 activation in macrophage cells to block
IL-6 production, while it up-regulates Akt to promote survival,
and mPTPB inhibitors can rescue these processes.4c Gratify-
ingly, 11h increased Erk1/2 and decreased Akt phosphorylation
in mPTPB transfected Raw264.7 cells at 5 mM, 10 mM, in a dose
dependent manner. By contrast, 11a (IC50 ¼ 25 mM) has no such
effects at 10 mM, validating 11h's excellent cellular activity in
targeting mPTPB (Fig. S4, ESI†).
In summary, we have successfully applied a DOS strategy for
the discovery of a range of novel bicyclic salicylic acids as
mPTPB inhibitors. A subsequent focused library provided
compound 11h with improved potency and excellent specicity
for mPTPB. With advantages of efficient synthesis, low molec-
ular weight and excellent cell activity, it serves as a promising
lead molecule for anti-TB drug discovery targeting mPTPB.
Given the favourable pharmacological properties exhibited by
the salicylic acid pharmacophore, the bicyclic scaffolds gener-
ated by the DOS strategy should also be good starting points for
the development of drug-like inhibitors targeting other PTPs.
Fig. 2 Structures of aminothiazole salicylic acids and their activities
against mPTPB.
Table 1 Specificity studies of compound 11h against a panel of PTPs
Enzyme
IC50 (mM)
Enzyme
IC50 (mM)
mPTPB
PTP1B
SHP2
SHP1
PTP-Meg2
2 ꢀ 0.1
65 ꢀ 1
50 ꢀ 1
78 ꢀ 8
69 ꢀ 2
CD45
PTP3
VHR
[100
[400
43 ꢀ 3
56 ꢀ 3
Acknowledgements
Laforin
This work was supported in part by NIH GrantCA152194.
Notes and references
mPTPB, exhibiting over 20-fold preference against these PTPs.
The basis of the high specicity of 11h was also studied by
molecular modeling, which shows that the salicylic acid head
group in 11h strongly interacts with mPTPB active site P-loop
residues CFAGKDR (Fig. 3), however, 11h has weaker interac-
tions with cognate residues in the P-loop of PTP1B (highlighted
in green color). This has been validated by sequence alignment
showing that CFAGKDR is unique to mPTPB in comparison to
many human PTPs (Fig. S2 and S3, ESI†). In addition, benzo-
thiazole and 3-bromo benzene of 11h interact with helices a3A,
a7 and a8 of mPTPB, which are not present in human PTPs
1 (a) N. K. Tonks and B. G. Neel, Curr. Opin. Cell Biol., 2001, 13,
182–195; (b) R. H. van Huijisduijnen, A. Bombrun and
D. Swinnen, Drug Discovery Today, 2002, 7, 1013–1019; (c)
A. Alonso, J. Sasin, N. Bottini, I. Friedberg, I. Friedberg,
A. Osterman, A. Godzik, T. Hunter, J. Dixon and
T. Mustelin, Cell, 2004, 117, 699–711; (d) L. Bialy and
H. Waldmann, Angew. Chem., Int. Ed., 2005, 44, 3814–3839;
(e) A. Ostman, C. Hellberg and F. D. Bohmer, Nat. Rev.
Cancer, 2006, 6, 307–320; (f) N. K. Tonks, Nat. Rev. Mol. Cell
Biol., 2006, 7, 833–846.
2 Z.-Y. Zhang, Curr. Opin. Chem. Biol., 2001, 5, 416–423.
3 (a) A. P. Combs, J. Med. Chem., 2010, 53, 2333–2344; (b)
S. Thareja, S. Aggarwal, T. R. Bhardwaj and M. Kumar,
Med. Res. Rev., 2012, 32, 459–517.
4 (a) X. Yu, J.-P. Sun, Y. He, X.-L. Guo, S. Liu, B. Zhou,
A. Hudmon and Z.-Y. Zhang, Proc. Natl. Acad. Sci. U. S. A.,
2007, 104, 19767–19772; (b) X. Zhang, Y. He, S. Liu, Z. Yu,
Z.-X. Jiang, Z. Yang, Y. Dong, S. C. Nabinger, L. Wu,
A. M. Gunawan, L. Wang, R. J. Chan and Z.-Y. Zhang,
J. Med. Chem., 2010, 53, 2482–2493; (c) B. Zhou, Y. He,
X. Zhang, J. Xu, Y. Luo, Y. Wang, S. G. Franzblau, Z. Yang,
R. Chan, Y. Liu, J. Zheng and Z.-Y. Zhang, Proc. Natl. Acad.
Sci. U. S. A., 2010, 107, 4573–4578; (d) Y. He, L.-F. Zeng,
Z.-H. Yu, R. He, S. Liu and Z.-Y. Zhang, Bioorg. Med. Chem.,
2012, 20, 1940–1946.
Fig. 3 Proposed interactions between 11h and mPTPB. 11h (cyan) and
mPTPB residues (gray) within 5 A˚ distance are shown as sticks and
polar interactions are highlighted as a yellow dotted line. The P-loop of
PTP1B (green, side chains in blue) was superimposed on the
11h$mPTPB complex, showing that 11h has weaker interactions with
the P-loop of PTP1B.
5 (a) P. Arya, D. T. H. Chou and M.-G. Baek, Angew. Chem., Int.
Ed., 2001, 40, 339–346; (b) S. L. Schreiber, Science, 2000, 287,
1964–1969; (c) M. D. Burke and S. L. Schreiber, Angew. Chem.,
Int. Ed., 2004, 43, 46–58; (d) D. S. Tan, Nat. Chem. Biol., 2005,
1498 | Med. Chem. Commun., 2014, 5, 1496–1499
This journal is © The Royal Society of Chemistry 2014

View Article Online
Concise Article
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1, 74–84; (e) R. J. Spandl, A. Bender and D. R. Spring, Org.
Biomol. Chem., 2008, 6, 1149–1158.
6 WHO Report 2010 on Global TB Control, 2010.
10606–10611; (b) I. R. Correa, Jr, A. Noren-Muller,
H.-D. Ambrosi, S. Jakupovic, K. Saxena, H. Schwalbe,
M. Kaiser and H. Waldmann, Chem.–Asian J., 2007, 2,
1109–1126; (c) A. Noren-Muller, W. Wilk, K. Saxena,
H. Schwalbe, M. Kaiser and H. Waldmann, Angew. Chem.,
Int. Ed., 2008, 47, 5973–5977; (d) T. Weide, L. Arve,
H. Prinz, H. Waldmann and H. Kessler, Bioorg. Med. Chem.
Lett., 2006, 16, 59–63; (e) M. B. Soellner, K. A. Rawls,
C. Grundner, T. Alber and J. A. Ellman, J. Am. Chem. Soc.,
2007, 129, 9613–9615; (f) C. Grundner, D. Perrin,
R. H. V. Huijsduijnen, D. Swinnen, J. Gonzalez, C. L. Gee,
T. N. Wells and T. Alber, Structure, 2007, 15, 499–509; (g)
L. P. Tan, H. Wu, P.-Y. Yang, K. A. Kalesh, X. Zhang,
M. Hu, R. Srinivasan and S. Q. Yao, Org. Lett., 2009, 11,
5102–5105; (h) Y. He, J. Xu, Z.-H. Yu, A. M. Gunawan,
L. Wu, L. Wang and Z.-Y. Zhang, J. Med. Chem., 2013, 56,
832–842; (i) L.-F. Zeng, J. Xu, Y. He, R. He, L. Wu,
A. M. Gunawan and Z.-Y. Zhang, ChemMedChem, 2013, 8,
904–908.
7 (a) W. Fox and D. A. Mitchison, Lancet, 1976, 2, 1349–1350;
(b) M. Neff, Am. Fam. Physician, 2003, 68, 1854–1862.
8 (a) Y. Zhang, Annu. Rev. Pharmacol. Toxicol., 2005, 45, 529–
564; (b) A. E. Clatworthy, E. Pierson and D. T. Hung, Nat.
Chem. Biol., 2007, 3, 541–548; (c) Z. Ma, C. Lienhardt,
H. McIlleron, A. J. Nunn and X. Wang, Lancet, 2010, 375,
2100–2109; (d) A. D. Harries, R. Zachariah, E. L. Corbett,
S. D. Lawn, E. T. Santos-Filho, R. Chimzizi, M. Harrington,
D. Maher, B. G. Williams and K. M. De Cock, Lancet, 2010,
375, 1906–1919; (e) N. R. Gandhi, P. Nunn, K. Dheda,
H. S. Schaaf, M. Zignol, D. van Soolingen, P. Jensen and
J. Bayona, Lancet, 2010, 375, 1830–1843.
9 A. Koul, T. Herget, B. Klebl and A. Ullrich, Nat. Rev.
Microbiol., 2004, 2, 189–202.
10 (a) A. Koul, A. Choidas, M. Treder, A. K. Tyagi, K. Drlica,
Y. Singh and A. Ullrich, J. Bacteriol., 2000, 182, 5425–5432;
(b) R. Singh, V. Rao, H. Shakila, R. Gupta, A. Khera, 12 J. Chang, K. Zhao and S. Pan, Tetrahedron Lett., 2002, 43,
N. Dhar, A. Singh, A. Koul, Y. Singh, M. Naseema, 951–954.
P. R. Narayanan, C. N. Paramasivan, V. D. Ramanathan 13 H. Sharghi, O. Asemani and R. Khalifeh, Synth. Commun.,
and A. K. Tyagi, Mol. Microbiol., 2003, 50, 751–762. 2008, 38, 1128–1136.
11 (a) A. Noren-Muller, I. Reis-Correa, Jr, H. Prinz, 14 B. Zou, Q. Yuan and D. Ma, Angew. Chem., Int. Ed., 2007, 46,
C. Rosenbaum, K. Saxena, H. Schwalbe, D. Vestweber, 2598–2601.
G. Cagna, S. Schunk, O. Schwarz, H. Schiewe and 15 Y.-J. Guo, R.-Y. Tang, P. Zhong and J.-H. Li, Tetrahedron Lett.,
H. Waldmann, Proc. Natl. Acad. Sci. U. S. A., 2006, 103,
2010, 51, 649–652.
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