Organocatalytic multicomponent reaction for the acquisition of a selective inhibitor of mPTPB, a virulence factor of tuberculosis

Article abstract of DOI:10.1039/c3cc38961h

Mycobacterium protein tyrosine phosphatase B (mPTPB) is essential for the survival and persistence of Mycobacterium in the host. Thus small molecule inhibitors of mPTPB are potential anti-TB agents. We developed an efficient organocatalytic multicomponent reaction (MCR) between pyrrole, formaldehyde and aniline, affording a potent and selective mPTPB inhibitor with an IC ₅₀ value of 1.5 μM and >50-fold specificity. Our studies provide a successful example of using organocatalysis as a discovery tool for the acquisition of PTP inhibitors. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc38961h

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Organocatalytic multicomponent reaction for
the acquisition of a selective inhibitor of mPTPB,
a virulence factor of tuberculosis†
Cite this: Chem. Commun., 2013,
49, 2064
Received 14th December 2012,
Accepted 24th January 2013
Rongjun He, Li-Fan Zeng, Yantao He, Li Wu, Andrea Michelle Gunawan and
Zhong-Yin Zhang*
DOI: 10.1039/c3cc38961h
www.rsc.org/chemcomm
Mycobacterium protein tyrosine phosphatase B (mPTPB) is essential for of them lack the required potency and selectivity, due to the
the survival and persistence of Mycobacterium in the host. Thus small challenge in acquiring selective PTP inhibitory agents targeting
molecule inhibitors of mPTPB are potential anti-TB agents. We the conserved active site.15 Moreover, these molecules were acquired
developed an efficient organocatalytic multicomponent reaction through multiple fragments appending procedures, which unavoid-
(MCR) between pyrrole, formaldehyde and aniline, affording a potent ably introduce high molecular weight and lipophilicity, and thus are
and selective mPTPB inhibitor with an IC50 value of 1.5 lM and >50-fold not appropriate as lead compounds.
specificity. Our studies provide a successful example of using organo-
catalysis as a discovery tool for the acquisition of PTP inhibitors.
Pyrroles are favourable substrates in organic chemistry due to
their high reactivity towards electrophilic aromatic substitutions
and Diels–Alder reactions.16 Pyrrole is also a privileged structure
Organocatalysis is a very useful tool for the preparation of various motif that exists in various biologically active molecules such as
chiral and nonchiral molecules, owing to the mild reaction drugs and natural products. Compound 1 and several analogues
conditions, low cost, and environmental consciousness.1–3 have been reported to inhibit PTP1B in the micromolar range
A recent trend in organocatalysis is organo-catalyzed multi- (Fig. 1).17 Unfortunately, this class of compounds exhibited no
component reactions (MCRs)4 affording novel and complex selectivity towards other PTPs, which is a common issue in the field
molecules with multiple stereocenters, which is highly desirable due to the highly conserved active sites in over 100 PTP family
in modern organic and medicinal chemistry.5 Examples of these members. In addition, compound 1 also exhibits poor stability. We
reactions include three-component domino condensations,6 envisaged that the poor stability is probably due to the high
Biginelli reactions,7 and Mannich reactions8 catalyzed by various reactivity of the pyrrole ring, and that substitutions at the pyrrole
organocatalysts to yield important novel amine building blocks reactive sites may mask its reactivity and hence increase its stability.
and heterocycles. We are interested in applying these advanced More importantly, fragments added through the substitution reac-
synthetic strategies to the discovery of protein tyrosine phos- tions may not only enhance its binding affinity to PTPs, but also
phatase (PTP) inhibitors, which possess enormous potential improve its specificity, as targeting both the PTP active site and the
therapeutic values in many human diseases.
nearby peripheral site by two or more fragments is a proven strategy
Tuberculosis (TB) is a major worldwide threat to public health, for acquiring potent and selective PTP inhibitors.15,18 To these
with approximately 9 million new cases and 1.8 million deaths each ends, we sought to develop a pyrrole Mannich type reaction that
year in the world.9 No new anti-TB drugs have been developed in couples the pyrrole, an amine and an aldehyde or a ketone, which
close to 40 years.10 The inadequate efficacy, lengthy treatment, and should be very useful for preparing pyrrole-based libraries that are
multi-drug resistant TB underscore the urgency of developing new potent PTP inhibitors with improved potency and specificity.
and more effective therapies.11 mPTPB has emerged as a novel
anti-TB target. It is secreted by Mtb into the cytoplasm of macro-
phages, where it mediates mycobacterial survival in the host and
serves as a virulence factor.12,13 Small molecules that inhibit mPTPB
hence possess great potential as novel anti-TB agents. Unfortunately,
only a handful of mPTPB inhibitors have been reported,14 and many
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@iupui.edu; Fax: +1-317-274-4686; Tel: +1-317-274-4686
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc38961h
Fig. 1 Structures and activities of N-phenyl-2,5-dimethyl pyrroles.
c
2064 Chem. Commun., 2013, 49, 2064--2066
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To begin our study, we designed 2 (Table 1) as the parent pyrrole
compound, which, after hydrolysis, afforded compound 3 with a
salicylic acid group serving as a nonhydrolyzable p-Tyr mimetic.19
3 is a moderately selective inhibitor against mPTPB with an IC50 of
2.9 mM.20 Subsequently, MCR Mannich reaction between 2, form-
aldehyde and aniline was studied as the model reaction to probe the
optimal conditions prior to the library generation. The reaction was
first carried out in a range of solvents using HOAc as a catalyst.
CH2Cl2 stands out as the most optimal solvent in affording both
mono- and di-alkylated products in a combined 74% conversion
(entry 1, Table 1). In exploring for alternative acids as catalysts, we
found that this reaction was very sensitive to the acidity of catalysts.
Fig. 2 Representative Mannich reaction products from 2, formaldehyde and
For example, TFA catalyzed reaction provided a complex mixture aromatic amines.
with the complete consumption of pyrrole (entry 2, Table 1), weaker
acids such as proline, PTSA and benzoic acid, and inorganic acid
HCl afforded products in zero to low conversions (entries 3–6, Thus we were able to obtain mono- and di-alkylated products by
Table 1). In contrast, methoxyacetic acid catalyzed the reaction fine-tuning the catalysts and the relative ratios of reactants.
slightly more efficiently than acetic acid, but with low selectivity
With reaction conditions towards either 4a or 5 in hand, we
for 4a (entry 7, Table 1). We also evaluated N,N-di[3,5-di(trifluoro- proceeded to expand the substrate scope by treating pyrrole 2 with
methyl)phenyl]thiourea, a frequently used organocatalyst,21 and it paraformaldehyde and various amines. Generally, reactions went
showed no capability to catalyze this reaction (entry 8, Table 1). well with aromatic amines, such as aniline derivatives and naphthyl
Increasing acetic acid from 20 mol% to 100 mol% did not show amines, and products were obtained in moderate to good yields. For
much improvement in total conversion, however, the selectivity for aniline derivatives, substituents at o, m, and p positions and with
product 4a was increased by 1.7-fold (entry 1 vs. entry 9, Table 1), either electron donating or withdrawing properties are tolerated
and a further increase of acetic acid in large excess resulted in a (Fig. 2). Unlike many similar reactions where substituents at the 2
complex mixture with a trace amount of the product. Finally, using position were not feasible,8 our studies indicate a broader opportu-
2 equiv. of pyrrole, 2 equiv. of HCHO, and 1 equiv. of aniline nity in choosing anilines for this reaction, and we attribute it to the
greatly improved the selectivity for 4a with 85% isolated yield small size of formaldehyde. Indeed, when we used larger aldehydes,
(entry 10, Table 1). And reaction using 1 equiv. of pyrrole, 3 equiv. of i.e. acetaldehyde and benzaldehyde, the reaction did not proceed,
HCHO, and 3 equiv. of aniline in the presence of methoxyacetic acid even at elevated temperature, extended time, and with stronger acid
after extended time affords 5 as the sole product in 75% isolated yield. catalysts. Aliphatic amines are usually not applicable in Mannich
reactions,8 due to the sluggish imine formation. Thus it is not
surprising that propyl amine, benzyl amine, diethyl amine and
piperidine did not afford any product under our reaction conditions.
Nevertheless, our goals are not limited to develop a Mannich
Table 1 Mannich MCR between pyrrole, paraformaldehyde and aniline under
various conditionsa
reaction, but more importantly, to use it as a tool to develop more
potent, selective, and stable PTP inhibitors. We anticipated that, after
hydrolysis, the N-salicylic acid moiety would occupy the active site,
while the added aniline moiety would target an adjacent secondary
site, with the methylene from formaldehyde serving as a linker.18
4a was converted to 6a under hydrolysis conditions (Scheme S1,
ESI†), which was observed in good yield by LC-MS analysis.
However, once the reaction mixture was acidified with 2 M HCl or
NH4Cl prior to extraction, we were not able to observe the product,
indicating that it decomposed rapidly even under weakly acidic
conditions. The reason could be that the basic aniline nitrogen is
easily protonated, and this internal proton donor in the right
orientation could promote the degradation of pyrrole. To reduce
the basicity of this nitrogen, we decided to protect it with a small
group with electron withdrawing ability. Hence compound 4a was
reacted with acetic anhydride to give compound 7a, and hydro-
lyzed to afford product 8a in excellent yield (Scheme S1, ESI†). As
expected, compound 8a indeed showed much improved stability.
Encouraged by these results, all of the Mannich adducts obtained
in Fig. 2 were protected by the acetyl group, hydrolyzed and purified
by reverse phase HPLC, to afford products in good yields and high
purities. All hydrolyzed products display much improved stability
Ratio of
Isolated
yield%
Entry
Catalyst
4a/5/2b%
1
2
3
4
5
6
7
8
HOAc (20 mol%)
TFA (20 mol%)
Proline (20 mol%)
PTSA (20 mol%)
PhCO2H (20 mol%)
2 M HCl (20 mol%)
MeOCH2CO2H (20 mol%)
(3,5-(CF3)2-PhNH)2CS (20 mol%)
HOAc (100 mol%)
HOAc (100 mol%)
MeOCH2CO2H (100 mol%)
52//22/26
NA
7/0/93
21/5/74
28/13/59
26/4/70
45/36/19
No reaction
59//17/24
45/9/46
0/100/0
9
10c
11d
85 (4a)e
75 (5)
a
The reaction was carried out at rt for 24 h in 1 mL of CH2Cl2 with 2
(0.1 mmol), paraformaldehyde (0.12 mmol), aniline (0.12 mmol). b The ratio
is based on UV absorption in LC-MS studies of crude reaction mixtures.
c
2 Equiv. of pyrrole, 2 equiv. of paraformaldehyde, and 1 equiv. of aniline
were used. d 1 Equiv. of pyrrole, 3 equiv. of formaldehyde, and 3 equiv. of
aniline were used, reaction time was 48 h. e Based on 1 equiv. of aniline.
c
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products in good yields. More importantly, these reactions enabled
us to identify PTP inhibitors, which have increased stability,
potency, and selectivity. In particular, compound 8f has an IC50
value of 1.5 mM against mPTPB, with >50-fold selectivity over a
large panel of PTPs. The low molecular weight and compact
structure render 8f a good lead molecule for anti-TB drug discovery
targeting mPTPB. Our studies provide a successful example of
using organocatalysis as a tool to discover enzyme inhibitors.
Given that a vast array of biologically active molecules containing
pyrrole, furan, and indole moieties can serve as substrates in
organocatalytic reactions, this study should have a broader impact
on the discovery of enzyme inhibitors beyond the PTP target class.
This work was supported in part by NIH Grant CA152194.
Fig. 3 Structure of hydrolyzed products from Mannich reactions and their
inhibition activities against mPTPB.20
Notes and references
Table 2 Specificity studies of compound 8f against a panel of PTPs20
1 (a) K. A. Ahrendt, C. J. Borths and D. W. C. MacMillan, J. Am. Chem.
Soc., 2000, 122, 4243–4244; (b) B. List, R. A. Lerner and C. F. Barbas III,
J. Am. Chem. Soc., 2000, 122, 2395–2396.
Enzyme
IC50 (mM)
2 (a) M. J. Gaunt, C. C. C. Johansson, A. McNally and N. T. Vo, Drug
Discovery Today, 2007, 12, 8–27; (b) S. Bertelsen and K. A. Jørgensen,
Chem. Soc. Rev., 2009, 38, 2178–2189.
3 (a) Enantioselective Organocatalysis, ed. P. I. Dalko, Wiley-VCH,
Weinheim, Germany, 2007; (b) H. Pellissier, Recent Developments
in Asymmetric Organocatalysis, Royal Society of Chemistry, 2010.
4 (a) D. B. Ramachary and C. F. Barbas III, Chem.–Eur. J., 2004, 10,
5323–5331; (b) D. B. Ramachary and S. Jain, Org. Biomol. Chem.,
2011, 9, 1277–1300.
mPTPB
mPTPA
PTP1B
SHP2
CD45
PTPa
1.5 Æ 0.2
180 Æ 30
200 Æ 30
86 Æ 7
78 Æ 7
c100
c100
MKP5
5 D. B. Ramachary and Y. V. Reddy, J. Org. Chem., 2010, 75, 74–85.
6 Z.-J. Xu, D. Zhu, X. Zeng, F. Wang, B. Tan, Y. Hou, Y. Lv and
G. Zhong, Chem. Commun., 2010, 46, 2504–2506.
7 N. Li, X.-H. Chen, J. Song, S.-W. Luo, W. Fan and L.-Z. Gong, J. Am.
Chem. Soc., 2009, 131, 15301–15310.
8 (a) J. M. M. Verkade, L. J. C. van Hemert, P. J. L. M. Quaedflieg and
F. P. J. T. Rutjes, Chem. Soc. Rev., 2008, 37, 29–41; (b) L. Cheng,
X. Wu and Y. Lu, Org. Biomol. Chem., 2007, 5, 1018–1020; (c) X. Fu,
W.-T. Loh, Y. Zhang, T. Chen, H. Liu, J. Wang and C.-H. Tan, Angew.
Chem., Int. Ed., 2009, 40, 7387–7390; (d) A. Kumar, M. Kumar Gupta
and M. Kumar, Green Chem., 2012, 14, 290–295.
compared to 3, and most of them exhibit good inhibitory activity
under the same assay conditions (Fig. 3).20 Although there is no
profound influence on activity, substituents at the meta position are
generally more beneficial than those at ortho and para positions.
Among them, 8f is the most potent mPTPB inhibitor, with an IC50
of 1.5 mM. Meanwhile bis-Mannich adduct 9 exhibited no activity at
100 mM (page 19, ESI†), suggesting that the second aniline fragment
may disrupt the binding to mPTPB. Additionally, we reasoned that
the introduction of one or two halogen atoms into the pyrrole ring
may be helpful in improving potency, specificity and stability.
9 WHO Report 2010 on Global TB Control, 2010.
10 (a) W. Fox and D. A. Mitchison, Lancet, 1976, 2, 1349–1350; (b) M. Neff,
Am. Fam. Physician, 2003, 68, 1854, 1857–1858, 1861–1852.
Though bromination using either Br2 or NBS gave a complex 11 Z. Ma, C. Lienhardt, H. McIlleron, A. J. Nunn and X. Wang, Lancet,
2010, 375, 2100–2109.
12 A. Koul, A. Choidas, M. Treder, A. K. Tyagi, K. Drlica, Y. Singh and
mixture, iodination successfully afforded mono-iodinated 10a
and di-iodinated 10b, using I2 and ICl, respectively. The hydro-
A. Ullrich, J. Bacteriol., 2000, 182, 5425–5432.
lyzed compounds 11a and 11b (Scheme S2, ESI†) indeed 13 R. Singh, V. Rao, H. Shakila, R. Gupta, A. Khera, N. Dhar, A. Singh,
A. Koul, Y. Singh, M. Naseema, P. R. Narayanan, C. N. Paramasivan,
V. D. Ramanathan and A. K. Tyagi, Mol. Microbiol., 2003, 50, 751–762.
14 (a) A. Noren-Muller, I. Reis-Correa, Jr., H. Prinz, C. Rosenbaum, K. Saxena,
exhibited improved stability over parent compound 3, unfortu-
nately they are several fold less potent than 8f obtained from
our MCR Mannich reactions as mPTPB inhibitors.
H. Schwalbe, D. Vestweber, G. Cagna, S. Schunk, O. Schwarz, H. Schiewe
and H. Waldmann, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 10606–10611;
(b) M. B. Soellner, K. A. Rawls, C. Grundner, T. Alber and J. A. Ellman,
J. Am. Chem. Soc., 2007, 129, 9613–9615; (c) 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; (d) 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.
Given the increased potency and stability, we proceeded to study
compound 8f’s specificity to mPTPB over selected members of the
PTP superfamily, including mPTPA, PTP1B, SHP2, CD45, PTPa,
and MKP5. As shown in Table 2, 8f is highly selective for mPTPB,
exhibiting a greater than 50-fold preference for mPTPB over all
PTPs examined, which indicates that compound 8f is one of the 15 (a) L. Bialy and H. Waldmann, Angew. Chem., Int. Ed., 2005, 44,
3814–3839; (b) A. P. Combs, J. Med. Chem., 2010, 53, 2333–2344.
most selective mPTPB inhibitors reported to date. In comparison,
compound 3 has only 4 and 5-fold selectivity for mPTPA and
´
´
16 V. Estevez, M. Villacampa and J. C. Menendez, Chem. Soc. Rev., 2010,
39, 4402–4421.
PTP1B, respectively. These results show that PTP inhibitors gener- 17 M. Manger, M. Scheck, H. Prinz, J. P. von Kries, T. Langer, K. Saxena,
H. Schwalbe, A. Frstner, J. Rademann and H. Waldmann, ChemBioChem,
2005, 6, 1749–1753.
18 Y. A. Puius, Y. Zhao, M. Sullivan, D. S. Lawrence, S. C. Almo and
ated from the MCR Mannich reaction not only exhibit increased
stability and potency, but also greatly improved specificity.
In conclusion, we have successfully developed an efficient
organocatalyzed MCR Mannich type reaction between pyrrole,
paraformaldehyde, and anilines. By fine-tuning the catalyst and
Z.-Y. Zhang, Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 13420–13425.
19 Y. He, L.-F. Zeng, Z.-H. Yu, R. He, S. Liu and Z.-Y. Zhang, Bioorg.
Med. Chem., 2012, 20, 1940–1946.
20 IC50 values were determined at pH 7 and 25 1C (for details, see ESI†).
reactant ratios, we were able to obtain mono- and di-alkylated 21 P. R. Schreiner and A. Wittkopp, Org. Lett., 2002, 4, 217–220.
c
2066 Chem. Commun., 2013, 49, 2064--2066
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