CHEMMEDCHEM
COMMUNICATIONS
To further increase the potency and selectivity of the opti-
mized hydroxyindole carboxylic acid 7a for mPTPB, we em-
ployed a focused library approach to introduce molecular func-
tionalities to its b-position in order to engage peripheral bind-
ing pockets adjacent to the active site (Figure 1). As discussed
above, the rationale for this stems from our earlier observa-
tions that diversity elements appended to the b-position of
the core enhance both inhibitor potency and selectivity,[16,23,24]
likely through added interactions with secondary binding
pockets adjacent to the PTP active site. However, the enhance-
ment in binding affinity through this approach has been rather
modest, likely due to the rigidity of the triazolidine linker
formed as a result of the click reaction. Indeed, no significant
contact was observed between the triazolidine linker and the
PTPs.[23,24] Here we chose amide chemistry because amide
bond formation is one of the most efficient and reliable meth-
ods for library constructions, and it allows the use of the most
common and commercially available amines and carboxylic
acids as reactants. In addition, amide chemistry can be carried
out in aqueous solution in the absence of deleterious reagents,
thus allowing direct screening and identification of hits from
the library. Finally, appropriately structured amide linker may
impart flexibility necessary for optimal interaction with the
enzyme. A short amine linker was attached to the b-position in
compound 7a for tethering a structurally diverse set of carbox-
ylic acids, aimed at capturing additional interactions with adja-
cent pockets surrounding the active site. In the interest of
keeping the library to a reasonable size, we selected 102 com-
mercially available carboxylic acids (figure S1, Supporting Infor-
mation) that vary by molecular weight, charge, polarity, hydro-
phobicity, sterics, etc., which therefore provide a reasonable
(albeit limited) structural diversity to increase the number and
strength of noncovalent interactions between mPTPB and the
inhibitor.
days to provide acid 9, which was treated with 20% trifluoro-
acetic acid in dichloromethane to produce compound 10, as
a result of exclusive conversion of alkyne to ketone under acid
condition. Condensation of compound 10 with the 102 acids
in the presence of HOBT, HBTU, and triethylamine in DMF over-
night furnished the amide library 11 in 96-well plates. The reac-
tions were assessed by LC–MS, which indicated that ~70% of
compound 10 were converted into target molecules.
This library was directly screened at 1 mm concentration for
inhibition of the mPTPB-catalyzed pNPP hydrolysis at pH 7 and
258C. Two of the library members, 11 a and 11 b, showed over
50% inhibition (81% for 11 a and 52% for 11 b) under these
conditions (table S1, Supporting Information). Resynthesis of
11 a and 11 b confirmed that they were genuine inhibitors for
mPTPB with IC50 values of 0.079Æ0.010 mm and 0.68Æ0.04 mm,
respectively. As negative controls, we also resynthesized and
HPLC purified compounds 11 c, 11 d and 11 e, which showed
<10% inhibition at 1 mm concentration (table S1, Supporting
Information). As expected, the IC50 values for these three com-
pounds were 3.3, 5.2, and >20 mm, much higher than those of
11 a and 11 b. The excellent agreement between the results
from the primary screen and those from the resynthesized
pure compounds indicated the success of the amide library ap-
proach. To determine the specificity of 11 a, its inhibitory activi-
ty toward a large panel of PTPs, including the bacterial PTPs
mPTPA and YopH, the mammalian cytosolic PTPs, SHP1, SHP2,
PTP1B, TC-PTP, Lyp, HePTP and FAP1, the receptor-like PTPs,
CD45, PTPe, PTPg, PTPm, and PTPs, and the dual specificity
phosphatases Laforin, VHR, VHX, VHZ, MKP3, and Cdc14A,
were measured. As shown in Table 2, compound 11 a exhibits
at least 100-fold selectivity for mPTPB over all PTPs examined.
To further characterize compound 11 a as a mPTPB inhibitor,
its IC50 value was determined under two different conditions:
1) 11 a was pre-mixed with pNPP, and the reaction was initiated
by the addition of mPTPB; and 2) 11 a was pre-mixed with the
enzyme for 30 min, and the reaction was initialized by the ad-
dition of pNPP. Irreversible, promiscuous nonspecific, or tight-
binding inhibitors would be expected to exhibit significantly
lower IC50 values when pre-incubated with the
To construct the 102-member library, iodide 6a was coupled
with tert-butyl prop-2-yn-1-ylcarbamate in the presence of Pd-
(PPh3)2Cl2 and copper(I) iodide to afford alkyne 8 (Scheme 2).
Alkyne 8 was hydrolyzed in 20% sodium hydroxide for two
enzyme. Similar IC50 values were obtained for com-
pound 11 a under these two conditions, suggesting
that 11 a is a reversible mPTPB inhibitor. Further ki-
netic analyses indicated that 11 a behaves as a non-
competitive inhibitor for mPTPB, with a Ki value of
50Æ2 nm (Figure 2). Thus, compound 11 a is the
most potent and selective mPTPB inhibitor reported
to date. Most importantly, compound 11 a also dis-
plays excellent cellular activity as shown below.
To facilitate the study of the cell biology of mPTPB
without having to directly work with major human
pathogens, we have established a murine macro-
phage Raw264.7 cell line ectopically expressing
mPTPB. Using this system, we delineated two path-
ways by which mPTPB enables survival of the Mtb
Scheme 2. Synthesis of hydroxyindole carboxylic acid based library 11. Reagents and con-
ditions: a) tert-butyl prop-2-yn-1-ylcarbamate, Pd(PPh3)2Cl2, CuI, Et3N, DMF, RT, 24 h, 68%;
b) 20% NaOH, CH3OH, THF/H2O, RT, 48 h, 87%; c) 20% TFA in CH2Cl2, RT, 24 h, 91.5%;
d) 102 acids, HOBT, HBTU, Et3N, DMF, RT, 24 h.
bacterium in the hostile environment of activated
host macrophages.[16] Firstly, mPTPB can subvert
innate immune responses by blocking ERK1/2-medi-
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 904 – 908 906
Zeng, Li-Fan
