Double click reaction for the acquisition of a highly potent and selective mPTPB inhibitor

Article abstract of DOI:10.1002/cmdc.201000348

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is a major worldwide threat to public health. Mycobacterium protein tyrosine phosphatase B (mPTPB) is a virulent phosphatase secreted by Mtb, which is essential for the survival and persistence of the bacterium in the host. Consequently, small-molecule inhibitors of mPTPB are expected to serve as anti-TB agents with a novel mode of action. Herein, we report the discovery of highly potent and selective mPTPB inhibitors using a novel, double Click chemistry strategy. The most potent mPTPB inhibitor from this approach possesses a Ki value of 160 nm and a >25-fold selectivity for mPTPB over 19 other protein tyrosine phosphatases (PTBs). Molecular docking study of the enzyme-inhibitor complex provides a rationale for the high potency and selectivity of the lead compound and reveals an unusual binding mode, which may guide further optimization effort.

Full text of DOI:10.1002/cmdc.201000348

DOI: 10.1002/cmdc.201000348
Double Click Reaction for the Acquisition of a Highly
Potent and Selective mPTPB Inhibitor
Rongjun He,[a] Zhihong Yu,[a] Yantao He,[a] Li-Fan Zeng,[a] Jie Xu,[a] Li Wu,[a, b]
Andrea M. Gunawan,[b] Lina Wang,[a] Zhong-Xing Jiang,[a] and Zhong-Yin Zhang*[a, b]
Tuberculosis (TB), which is caused by Mycobacterium tuberculo-
sis (Mtb), is a major worldwide threat to public health. Myco-
bacterium protein tyrosine phosphatase B (mPTPB) is a virulent
phosphatase secreted by Mtb, which is essential for the surviv-
al and persistence of the bacterium in the host. Consequently,
small-molecule inhibitors of mPTPB are expected to serve as
anti-TB agents with a novel mode of action. Herein, we report
the discovery of highly potent and selective mPTPB inhibitors
using a novel, double Click chemistry strategy. The most
potent mPTPB inhibitor from this approach possesses a Ki
value of 160 nm and a >25-fold selectivity for mPTPB over 19
other protein tyrosine phosphatases (PTBs). Molecular docking
study of the enzyme–inhibitor complex provides a rationale for
the high potency and selectivity of the lead compound and re-
veals an unusual binding mode, which may guide further opti-
mization effort.
Introduction
A vast array of biological phenomena, such as cell proliferation
and differentiation, motility, metabolism, apoptosis, and
immune responses, are regulated by protein tyrosine phos-
phorylation, a process that is controlled by the balanced action
of protein tyrosine phosphatases (PTPs) and protein tyrosine
kinases (PTKs).[1] Similar to the PTKs, unregulated PTP activity
causes aberrant dephosphorylation, which is associated with
many human diseases, including cancer, diabetes, and autoim-
mune disorders.[2] Strikingly, PTPs have also been used by
pathogenic bacteria to alter host defense mechanisms for their
own infectivity and/or survival in the host. For instance, Myco-
bacterium protein tyrosine phosphatase B (mPTPB) is a viru-
lence factor from Mycobacterium tuberculosis (Mtb), the causa-
tive agent of tuberculosis (TB). mPTPB is secreted by Mtb into
the cytoplasm of macrophages, where it mediates mycobacte-
rial survival in the host.[3] Deletion of mPTPB impairs the ability
of the mutant strain to survive in interferon-g (IFN-g)-activated
macrophages and severely reduces the bacterial load in a clini-
cally-relevant guinea pig model.[4]
TB, underscores the urgency for the development of more ef-
fective therapies against novel TB targets.[6]
Given the essential role of mPTPB for Mtb survival in the
host, there is increasing interest in developing mPTPB inhibi-
tors as novel anti-TB agents. Because mPTPB inhibitors have
no structural or mechanistic overlap with current drugs used
for TB treatment and function within the cytosol of host mac-
rophage, they have great potential to target the intracellular
pool and compliment/synergize with existing therapeutic strat-
egies. Moreover, since mPTPB is secreted into the cytosol of
host macrophages, drugs targeting mPTPB are not required to
penetrate the waxy mycobacterial cell wall, which is a major
barrier blocking translation of target inhibition to activity
against the intact pathogen. Consequently, specific mPTPB in-
hibitors may have therapeutic value with a unique mode of
action.
To date, a limited number of mPTPB inhibitors have been re-
ported. Those described in the literature include indoles dis-
covered by biology-oriented synthesis,[7a–c] indolizines synthe-
sized via solid phase synthesis,[7d] isoxazoles designed through
a substrate-based fragment approach,[7e] oxamic acid deriva-
tives discovered by a homogeneous plate assay,[7f] phenylisox-
TB is a major worldwide threat to public health, with ap-
proximately 9 million new cases reported and 1.8 million
deaths each year in the world.[5] No new anti-TB drugs have
been developed in close to 40 years. Traditional TB treatment
requires a 6–9 months administration of multiple antibiotics
targeting mycobacterial biosynthetic processes involved in cell
growth, including RNA transcription, protein translation, and
cell wall biogenesis. The limited effectiveness of current antibi-
otics and lengthy treatment lead to poor patient compliance,
which is responsible for high rates of treatment failure, relapse,
and emergence of multidrug-resistant (MDR) and extensively
resistant (XDR) TB. The prevalence of MDR-TB and XDR-TB, cou-
pled with the acquired immune deficiency syndrome (AIDS)
epidemic since AIDS patients are sometimes co-infected with
[a] Dr. R. He, Dr. Z. Yu, Dr. Y. He, Dr. L.-F. Zeng, Dr. J. Xu, L. Wu, L. Wang,
Dr. Z.-X. Jiang, Prof. Dr. Z.-Y. Zhang
Department of Biochemistry and Molecular Biology
Indiana University School of Medicine
635 Barnhill Drive, Indianapolis, IN 46202 (USA)
Fax: (+1)317-274-4686
E-mail: zyzhang@iupui.edu
[b] L. Wu, A. M. Gunawan, Prof. Dr. Z.-Y. Zhang
Chemical Genomics Core Facility
Indiana University School of Medicine
635 Barnhill Drive, Indianapolis, IN 46202 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000348.
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Z.-Y. Zhang et al.
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azole carboxylic acids,[7g] and most recently salicylic-acid-based
inhibitors synthesized via Click chemistry.[7h] Unfortunately, the
potency and selectivity for many of the reported compounds
are still modest, which is not surprising since PTPs share a con-
served active site, rendering the acquisition of highly selective
PTP inhibitors an extremely challenging endeavor. Moreover,
due to the complexity of the structures, these inhibitors re-
quire multistep synthesis (more than 7 steps for all reported in-
hibitors) with low yields, which could hinder their further de-
velopment. Herein, we present our discovery of a potent and
selective, yet easily accessible, mPTPB inhibitor synthesized via
a novel double Click chemistry strategy.
the inhibitory potencies were still modest, with IC50 values in
the low micromolar range.
As an initial attempt to further increase interactions with pe-
ripheral pockets surrounding the active site, we were interest-
ed in introducing two alkyne groups into the core structure,
which would enable a core to react with an azide in a 1:2 fash-
ion, thus creating a tridentate molecule (Scheme 1b). The addi-
tional fragment in the molecule would provide extra interac-
tions with the enzyme, hence the potency and selectivity
could be improved. Another advantage is that, since one more
fragment will be incorporated into the molecule, we could
choose cores that are easily accessible and structurally less
complicated for library assembly (Scheme 2a), which may save
a significant amount of time and effort for the synthesis of
cores.
Results and Discussion
To assess the effectiveness and feasibility of the double Click
reaction approach for PTP inhibitor design, we prepared five
benzoic- and naphthoic-acid-based cores with two alkyne func-
tional groups attached, as shown in Scheme 2b. The reasons
we chose these cores are that they represent the simplest pTyr
substrate analogues to fit into the positively charged active
site of the PTPs and that they can be easily synthesized in two
steps with high yields and large quantities from commercially
available and inexpensive precursors. The synthesis is com-
posed of alkylation followed by hydrolysis, which is illustrated
in Scheme 2b. Subsequently, these five cores were purified by
reversed-phase preparative HPLC and screened against mPTPB
for inhibitory activity. We found that L1, L2, and L4 showed no
inhibition of the enzyme at 200 mm concentration, while L3
and L5 inhibited mPTPB with an IC50 value of 50 and 75 mm, re-
spectively. As an initial proof-of-concept, we decided to focus
on core L5 for library construction using the double Click strat-
egy. The azides used in this study were prepared from 53
amines (Scheme 2c), as described previously.[7h,13]
To prepare the library, we coupled L5 with the azides using
Click chemistry. A stock solution of L5 in N,N-dimethylform-
amide (DMF) was introduced to plastic reaction vessels, fol-
lowed by the addition of two equivalents of various azides.
Then a catalytic (20 mol%) amount of tetrakis(acetonitrile)cop-
per(I) hexafluorophosphate was added as a solution in DMF.
After standing at room temperature for two days, representa-
tive reactions from ten vessels
It has been recognized that phosphotyrosine (pTyr) alone is
not sufficient for high-affinity binding, and residues flanking
pTyr are important for PTP substrate recognition.[8] Thus, an ef-
fective strategy for the acquisition of potent and selective PTP
inhibitors is by tethering a non-hydrolyzable pTyr mimetic to
an appropriately functionalized moiety in order to engage
both the active site and a unique nearby subpocket.[8,9] Click
chemistry refers to the 2+3 cycloaddition between an alkyne
and an azide,[10] which is ideally suitable for connecting two
fragments together. Because of its high yield and selectivity,
excellent functional group tolerance, and robust reaction con-
ditions, the Click reaction has been widely used in medicinal
chemistry for library construction, lead optimization, and cell-
based imaging.[11] Since Click reactions can be conducted in
aqueous solution in the absence of deleterious reagents, libra-
ries generated by Click reactions could be directly screened
in situ for enzyme inhibitors and protein ligands. In a conven-
tional Click reaction, an alkyne functional group is installed
into a core component, which then reacts with a set of azide-
containing fragments in a 1:1 mode to afford desired reaction
products (Scheme 1a). We have recently employed Click
chemistry for the construction of salicylic-acid-based focused li-
braries to target both the PTP active site and its adjacent, pe-
ripheral secondary binding sites.[7h,12,13] Although several biden-
tate inhibitors with excellent cellular activity were identified,
were monitored by liquid chro-
matography–mass spectrometry
(LC–MS), which indicated that
the reactions furnished products
in moderate-to-high conversion
with >70% purity. This was con-
sistent with published results
from both others and our own
group using Click reaction for li-
brary assembly.[7g,h,13] To avoid
possible false positive hits
caused by copper and other im-
purities, each reaction mixture
was diluted with water, and the
precipitate formed was filtered
Scheme 1. From a conventional to a double Click reaction: a) conventional Click reaction; b) double Click reac-
tion.
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mPTPB Inhibitors by a Double Click Reaction
Scheme 2. The Assembly of a compound
library by double Click reaction. a) Library
generation by double Click reaction.
b) The structures and synthesis of cores.
Reagents and conditions: a) Propargyl
bromide (3 equiv), K2CO3 (3 equiv), ace-
tone, reflux, overnight; b) 10% NaOH,
MeOH, RT, overnight. c) The structures
and synthesis of azides. Reagents and
conditions: a) RNH2; b) NaN3, 50–90%
yield (two steps).
and washed with water. The
solid we collected was dissolved
in DMSO to make a 20 mm stock
solution, which was ready for
the screening step.
To identify mPTPB inhibitors,
the ability of the library to inhib-
it the mPTPB-catalyzed hydroly-
sis of p-nitrophenyl phosphate
(pNPP) was assessed at pH 7 and
258C. Among the 212 members
in the library, two compounds
displayed excellent inhibitory ac-
tivity at ~10 mm concentration.
Resynthesis of the hits confirmed
that they were genuine inhibi-
tors of mPTPB with IC50 values in
the nanomolar range. Com-
pound L5B47 (Figure 1a) ap-
peared to be the most potent
inhibitor of mPTPB, with an IC50
value of 160Æ10 nm and was
selected for further characteriza-
tion. It should be noted that the
IC50 value for L5B47 is 469-fold
lower than that of the parent
core L5, indicating that the
linker and amine diversity ele-
ment contribute significantly to
mPTPB binding. The second hit
compound, L5D47 had an IC50
value of 270 nm for mPTPB (see
the Supporting Information).
Kinetic analysis revealed that
L5B47 is a reversible and non-
competitive inhibitor of mPTPB
with a Ki value of 162Æ10 nm
(Figure 1b). To determine the
specificity of L5B47, its inhibito-
ry activities against mPTPA and a
panel of mammalian PTPs, in-
cluding cytosolic PTPs, PTP1B,
TC-PTP, SHP2, Lyp and FAP1, the
receptor-like PTPs, CD45, LAR,
and PTPa, the dual specificity
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also prepared compound L5’B47, a single Click reac-
tion product from the same core and the same azide.
The IC50 value of L5’B47 against mPTPB (3.2 mm) is
20-fold higher (i.e., 20-fold less potent affinity) than
L5B47. Moreover, L5’B47 exhibits no selectivity
against the same panel of PTPs. Therefore, the
double Click chemistry strategy is indeed superior to
the conventional single Click reaction in producing
more potent and selective mPTPB inhibitors.
Given its exceptional potency and selectivity, we
performed a modeling study in order to aid the un-
derstanding of potential interactions between L5B47
and mPTPB. Docking of the inhibitor to mPTPB was
carried out using the AutoDock 4.01 software pack-
age[14] and the coordinates of mPTPB–OMTS complex
(PDB: 2OZ5).[7f] As shown in Figure 3, L5B47 is pre-
dicted to bind near the entrance of the active site,
with the carbon atom of the carboxyl group on the
naphthalene being 16.3 ꢁ from the sulfur atom on
the catalytic Cys160. This predicted binding pose
supports the theory that this inhibitor does not com-
pete with the artificial substrate pNPP at the catalytic
site, which explains the aforementioned kinetic ob-
servation that L5B47 is a reversible and noncompeti-
tive inhibitor of mPTPB. The carboxylate group could
Figure 1. a) Structure of L5B47; b) Lineweaver–Burk plot for L5B47-mediated mPTPB in-
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*
*
hibition. L5B47 concentrations were 0 ( ), 0.1 ( ), 0.2 ( ), 0.3 ( ), and 0.4 ( ) mm.
phosphatases, VHR, VHX, Cdc14, and the low molecular weight
PTP, were measured. As shown in Figure 2, L5B47 is highly se-
lective for mPTPB, exhibiting a 50-fold preference for mPTPB
over mPTPA and greater than 25-fold preference for mPTPB
over all mammalian PTPs examined. These results show that
L5B47 is among the most potent and specific mPTPB inhibi-
tors reported to date. Its easy synthesis (three linear chemical
steps) offers numerous benefits for further developments. We
Figure 2. IC50 values (mm) of L5B47 against a panel of PTPs.
Figure 3. A model for potential interactions between mPTPB and L5B47.
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mPTPB Inhibitors by a Double Click Reaction
254 nm. Mass spectra were obtained using an Agilent Technologies
6130 quadrupole LC–MS.
potentially form two hydrogen bonds with the h-nitrogen of
Arg210 and Arg63, which might be important for anchoring
the naphthalene core right at the entrance of the active site.
The piperidine and chlorine benzene from the carboxylate
proximal branch in L5B47 might sit in a hydrophobic pocket
interacting with Phe98, Leu102, Phe222, Val219 and Leu227.
The carboxylate distal branch of L5B47 extends to comple-
ment a neighboring groove on the surface of mPTPB; it is pre-
dicted to make Van der Waals contacts with the surrounded
residues, including Arg59, His94, Glu95, Thr96 and Phe98. Ad-
ditionally, weak polar interactions might also exist between the
chlorines on the naphthalene and benzene rings and the side
chains of Arg59 and Lys91, respectively. Interestingly, the ma-
jority of the residues implicated in the model of L5B47 binding
appear to be unique to mPTPB, which is consistent with the
observed selectivity of L5B47 for mPTPB.
Representative procedure for the synthesis of cores (L1–5): A so-
lution of methyl 4,7-dichloro-3,5-dihydroxy-2-naphthoate (0.287 g,
1 mmol) in acetone (3 mL) was treated with K2CO3 (0.42 g, 3 mmol)
and propargyl bromide (0.32 mL, 80% w/w in toluene, 3 mmol),
and the mixture was heated at reflux overnight. The reaction was
concentrated in vacuo, diluted with H2O (10 mL) and extracted
with EtOAc (3ꢂ10 mL). The combined organic layer was dried
(Na2SO4), filtered and concentrated in vacuo. Purification by
column chromatography (EtOAc/hexane; 1:5) gave the intermedi-
ate (methyl 4,7-dichloro-3,5-bis(prop-2-yn-1-yloxy)-2-naphthoate)
as a white solid (0.362 g, 99% yield). Subsequently, the intermedi-
ate was dissolved in MeOH (2 mL), and the solution was treated
with 20% aq NaOH (2 mL). The mixture was stirred at RT overnight.
This reaction was acidified with aq HCl (2m, 10 mL), and extracted
with EtOAc (3ꢂ10 mL). The combined organic layer was dried
(Na2SO4), filtered and concentrated in vacuo. HPLC purification
gave L5 as a white solid (0.312 g, 90%; >95% purity): 1H NMR
(500 MHz, CDCl3): d=8.39 (s, 1H), 7.80 (s, 1H), 7.74 (s, 1H), 5.07 (d,
J=2.3 Hz, 2H), 4.94 (d, J=2.4 Hz, 2H), 3.69 (t, J=2.3 Hz, 1H),
3.66 ppm (t, J=2.4 Hz, 1H); 13C NMR (500 MHz, CDCl3): d=166.5,
154.2, 147.8, 131.7, 127.7, 125.7, 125.6, 125.0, 124.1, 104.3, 99.5,
79.8, 79.3, 78.7, 78.3, 61.4, 56.4 ppm; MS (ESI+): m/z (%): 349.0
(100%) [M+H]+.
Conclusions
We demonstrate in this study that, by adding two fragments
into the core structure simultaneously, the double Click strat-
egy is superior to the conventional Click reaction for acquisi-
tion of more potent and selective enzyme inhibitors. We have
identified L5B47 as the most potent and selective, yet easily
accessible, mPTPB inhibitor through this double Click chemis-
try strategy. L5B47 inhibits mPTPB in a noncompetitive
manner with a Ki value of 160 nm and a selectivity of more
than 25-fold for mPTPB over 19 other PTPs. Molecular docking
analysis suggests a unique binding mode, in which L5B47
binds at the entrance of the active site with the two branches
occupying hydrophobic grooves on either side. We anticipate
the double Click chemistry approach to have broad applicabili-
ty in the development of multidentate inhibitors directed
against other enzymes, in addition to the protein phosphatas-
es.
L1: white solid (0.217 g, 94% yield; >95% purity): 1H NMR
(500 MHz, CDCl3): d=12.40 (s, 1H), 7.74 (d, , J=8.7 Hz, 1H), 7.67
(d, J=2.2 Hz, 1H), 6.69 (m, 1H), 4.87 (s, 4H), 3.59 (t, J=2.3 Hz, 1H),
3.56 ppm (t, J=2.3 Hz, 1H); 13C NMR (500 MHz, CDCl3): d=166.4,
161.2, 158.2, 133.2, 114.3, 106.5, 101.8, 78.9, 78.7, 78.7, 56.3,
55.9 ppm; MS (ESI+): m/z (%): 231.1 (100%) [M+H]+.
L2: white solid (0.220 g, 95% yield; >95% purity): 1H NMR
(500 MHz, CDCl3): d=13.1 (s, 1H), 7.19 (d, J=2.3 Hz, 2H), 6.86 (t,
J=2.3 Hz, 1H), 4.85 (d, J=2.3 Hz, 4H), 3.69 ppm (t, J=2.3 Hz, 2H);
13C NMR (500 MHz, CDCl3): d=166.9, 158.3, 133.0, 108.4, 107.0,
78.9, 78.6, 55.8 ppm; MS (ESI+): m/z (%): 231.1 (100%) [M+H]+.
L3: white solid (0.256 g, 91% yield; >95% purity): 1H NMR
(500 MHz, CDCl3): d=12.97 (s, 1H), 8.16 (s, 1H), 7.80 (d, J=9.1 Hz,
1H), 7.47 (m, 2H), 7.27 (dd, J=2.5, 9.0 Hz, 1H), 4.94 (d, J=2.3 Hz,
2H), 4.90 (d, J=2.3 Hz, 2H), 3.59 ppm (m, 2H); 13C NMR (500 MHz,
CDCl3): d=167.4, 154.3, 151.4, 130.6, 129.9, 128.4, 128.3, 124.2,
120.7, 109.0, 108.5, 79.2, 78.5, 78.4, 56.2, 55.6 ppm; MS (ESI+): m/z
(%): 281.1 (100%) [M+H]+.
Experimental Section
Materials: p-Nitrophenyl phosphate (pNPP) was purchased from
Fluke. Dithiothreitol (DTT) was provided by Fisher (Fair Lawn, NJ,
USA). For organic synthesis, reagents were used as purchased from
Aldrich, Acros, Alfa Aesar or TCI, except where noted. 1H and
13C NMR spectra were obtained on a Bruker Avance II 500 MHz
NMR spectrometer with trimethylsilane (TMS) or residual solvent as
an internal standard. All column chromatography was performed
using 230–400 mesh silica gel (SiO2; Dynamic Adsorbents) with the
solvent system indicated unless otherwise noted. Thin-layer chro-
matography (TLC) analysis was performed using 254 nm glass-
backed plates and visualized using UV light (254 nm). High-pres-
sure liquid chromatography (HPLC) purification was carried out on
a Waters Delta 600 equipped with a Sunfire Prep C18 OBD column
(30 mm ꢂ 150 mm, 5 mm) with MeOH/H2O (both containing 0.1%
TFA) as the mobile phase (gradient: 50–100% MeOH; flow rate:
10 mLminÀ1). The purity of all final tested compounds was estab-
lished to be >98% by reverse-phase HPLC on a Waters Breeze
HPLC system with a SunFire C18 analytical column (4.6 mm ꢂ
150 mm, 5 mm) using CH3CN/H2O (both containing 0.1% TFA) as
the mobile phase (gradient: 30–100% CH3CN; flow rate:
1.5 mLminÀ1), with UV monitoring at a fixed wavelength of
L4: white solid (0.253 g, 90% yield; >95% purity): 1H NMR
(500 MHz, CDCl3): d=13.1 (s, 1H), 8.26 (s, 1H), 7.69 (s, 1H), 7.58 (d,
J=8.2 Hz, 1H), 7.36 (t, J=7.8 Hz, 1H), 7.13 (d, J=7.7 Hz, 1H), 5.05
(s, 2H), 5.00 (s, 2H), 3.62 ppm (m, 2H); 13C NMR (500 MHz, CDCl3):
d=167.3, 152.6, 151.5, 131.0, 128.6, 126.7, 124.7, 124.3, 121.3,
108.2, 102.7, 79.2, 79.0, 78.8, 78.7, 56.2, 56.1 ppm; MS (ESI+): m/z
(%): 281.1 (100%) [M+H]+.
Azide synthesis: All of the azides were synthesized previously.[7h,13]
Library synthesis via double Click chemistry: L5 (0.05 mmol,
0.5 mL of 100 mm stock solution in DMF), azide (0.10 mmol,
2 equiv), and tetrakis(acetonitrile)copper(I) hexafluorophosphate
(0.01 mmol, 0.1 mL of 100 mmol stock solution in DMF, 20 mol%)
were added to a 2 mL plastic vessel. The vessel was allowed to
stand at RT for 2 days. The mixture was then poured into H2O
(6 mL), and the precipitate formed was collected by filtration and
washed with H2O (2ꢂ6 mL). The solid collected was dissolved in
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Z.-Y. Zhang et al.
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DMSO (2 mL) to give a stock solution at 20 mm (assuming the
product is obtained at 80% yield).
Keywords: Click chemistry · drug design · protein tyrosine
phosphatases · inhibitors · tuberculosis
L5B47: white solid (0.042 g, 85%, >95% purity after HPLC):
1H NMR (500 MHz, CDCl3): d=8.35 (s, 1H), 8.31 (s, 1H), 8.28 (s,
1H), 7.72 (m, 2H), 7.34 (m, 2H), 7.09 (m, 2H), 6.88 (m, 2H), 5.33 (s,
2H), 5.23 (s, 2H), 4.61 (m, 4H), 3.50 (m, 8H), 3.13 (m, 8H), 3.06 (t,
J=6.8 Hz, 2H), 2.98 ppm (t, J=6.8 Hz, 2H); 13C NMR (500 MHz,
CDCl3): d=206.5, 167.9, 167.8, 166.6, 155.0, 150.3, 150.3, 148.1,
142.0, 141.9, 131.8, 131.5, 131.5, 130.4, 130.4, 127.3, 127.2, 125.7,
125.5, 125.3, 125.2, 124.8, 124.0, 119.9, 116.5, 116.4, 115.4, 103.8,
99.5, 66.4, 62.1, 47.6, 47.3, 45.8, 44.1, 44.1, 40.6, 40.4, 32.7, 32.6,
30.7 ppm; MS (ESI+): m/z (%): 1001.1 (10%) [M+H]+; HRMS calcd.
for C45H43Cl6N8O6 (M+H+): m/z 1001.1431; found 1001.1556.
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L5’B47: white solid (0.028 g, 87%, >95% purity after HPLC):
1H NMR (500 MHz, CDCl3): d=8.63 (s, 1H), 8.31 (s, 1H), 7.65 (s,
1H), 7.42 (m, 1H), 7.31 (d, J=9.0 Hz, 1H), 7.08 (d, J=2.4 Hz, 1H),
6.88 (dd, J=8.9, 2.2 Hz, 1H), 5.15 (s, 2H), 4.63 (t, J=6.4 Hz, 2H),
3.56 (m, 4H), 3.14 (m, 4H), 3.02 ppm (t, J=6.4 Hz, 2H); 13C NMR
(500 MHz, CDCl3): d=167.9, 157.8, 150.3, 147.6, 141.9, 133.5, 131.4,
130.3, 128.6, 127.9, 125.5, 125.2, 124.6, 123.3, 119.9, 116.4, 115.4,
114.5, 106.7, 99.5, 66.2, 47.6, 47.2, 45.8, 44.2, 40.6, 40.4, 32.7 ppm;
MS (ESI+): m/z (%): 637.1 (10%) [M+H]+.
Inhibition study: Expression and purification of recombinant
mPTPB, and kinetic characterization of mPTPB inhibitors were per-
formed as described previously.[7h]
Docking analysis: The AutoDock 4.0 software package[14] was used
to build the binding model of L23B47 with mPTPB. The three-di-
mensional structure of L23B47 was modeled and energy mini-
mized in Chem3D program (ChemBio3D Ultra 12.0), and the coor-
dinates of mPTPB were taken from an mPTPB–OMTS complex
structure (PDB: 2OZ5).[7f] Both ligand and receptor were introduced
into AutoDockTools 1.4.6[15] for a series of preprocessing, such as
merging nonpolar hydrogen atoms, adding Gasteiger charges, set-
ting rotatable bonds for the ligand, adding solvation parameters
for the receptor, and so on. The docking space was visually set
around the active site, the energy-grid size was set to 66ꢂ60ꢂ80
points with 0.375 ꢁ spacing on each axis, then the energy-grid
maps for each atom type found in L23B47 (i.e., A, C, N, NA, OA
and Cl), as well as the electrostatic forces and desolvation maps
were calculated using the auxiliary program of AutoGrid 4. Based
on all of these prepared files, the automatic molecular docking
work was carried out using the AutoDock 4 program, the Lamarck-
ian genetic algorithm with local search (LGALS) was selected for
ligand conformational searching, the optimal binding conformation
was determined by the LGALS algorithm with the following impor-
tant parameters during each docking run: energy evaluations of
2500000, population size of 100, mutation rate of 0.02, crossover
rate of 0.8, Solis and Wets local search iterations of 300 with a
probability of 0.06. Finally, 256 separate docking runs were per-
formed, and the resulting 256 binding conformations were classi-
fied into different clusters and ranked according to the calculated
binding free energy. These cluster and energy information, togeth-
er with visual inspection in AutoDockTools 1.4.6, generate the most
possible binding mode.
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Acknowledgements
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This work was supported in part by the National Institutes of
Health (NIH; USA) (grants: CA69202 and CA126937).
Received: August 14, 2010
Published online on October 18, 2010
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