Alkyl chain substituted 1,9-pyrazoloanthrones exhibit prominent inhibitory effect on c-Jun N-terminal kinase (JNK)

Article abstract of DOI:10.1039/c4ob00548a

N-Alkyl substituted pyrazoloanthrone derivatives were synthesized, characterized and tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). Among the tested molecules, a few derivatives showed significant inhibitory activity against JNK with minimal off-target effect on other mitogen-activated protein kinase (MAP kinase) family members such as MEK1/2 and MKK3,6. These results suggested that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders. This journal is

Full text of DOI:10.1039/c4ob00548a

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Alkyl chain substituted 1,9-pyrazoloanthrones
exhibit prominent inhibitory effect on c-Jun
N-terminal kinase (JNK)†
Cite this: Org. Biomol. Chem., 2014,
12, 4656
Karothu Durga Prasad,a Jamma Trinath,b Ansuman Biswas,c Kanagaraj Sekar,*d
Kithiganahalli N. Balaji*b and Tayur N. Guru Row*a
N-Alkyl substituted pyrazoloanthrone derivatives were synthesized, characterized and tested for their
in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). Among the tested molecules, a few deriva-
tives showed significant inhibitory activity against JNK with minimal off-target effect on other mitogen-
activated protein kinase (MAP kinase) family members such as MEK1/2 and MKK3,6. These results
suggested that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising thera-
peutic inhibitors for JNK in regulating inflammation associated disorders.
Received 12th March 2014,
Accepted 2nd May 2014
DOI: 10.1039/c4ob00548a
www.rsc.org/obc
disorders.6–8 JNK (Jun N-terminal kinase), a stress activated
protein kinase (SAPK), is one of the important members of the
1. Introduction
Cellular physiological processes are critically tailored by MAP kinase family along with MEK1/2, MEK3,6, MKK4,7, etc.
numerous signalling pathways. Regulated activation of that mediates the activation of the key transcription factor
Mitogen Activated Protein Kinase (MAP kinase) family AP-1 (Activator Protein-1 complex composed of c-Fos and
members determine the extent of mammalian gene c-Jun).9–13 Activated JNK phosphorylates c-Jun at the Ser63
expression.1 Lipopolysaccharide (LPS), chief constituent of position and eventually leads to formation of a functional AP-1
endotoxin, is the major cause of sepsis. Endotoxin mediated transcription factor complex.14,15 To date, three JNKs were
septic shock is mainly characterised by drastic and sudden identified in humans (JNK1, JNK2 and JNK3). In addition to
augmentation of several inflammatory genes like IL-12, IL-1β, these, 10 subsidiary isoforms arise from these JNKs as splice
and TNF-α.2 Exaggerated levels of these cytokines derails the variants.16 JNKs with several isoformic variants control crucial
host immune homeostasis, leading to inflammation associ- cellular processes like apoptosis and cell proliferation and are
ated risks like cardiac arrest, arthritis, etc.3 Activation of the also implicated in disorders associated with inflammation like
host cellular signalling pathways comprised of MAP kinases septic shock, arthritis, inflammatory bowel disease, etc.17,18
such as ERK1/2, p38 and JNK predominantly at the ground Therapeutic inhibition of JNK activity by small molecule
level is the key regulatory step in fine-tuning the immune cell inhibitors has proven to be advantageous in the treatment of
associated functions.4,5 Design and characterization of novel diseases coupled with derailed inflammation.19,20
small molecules on the one hand and potentiating the exiting
Even though several inhibitors of JNK have already been
target specific inhibitors on the other attain crucial reported21–23 with varied efficacies, the role of anthracyclines
importance in the treatment of inflammation associated identified in the screening of anti-cancer drugs24 had not been
focused in terms of kinase inhibitors until the introduction of
1,9-pyrazoloanthrone (SP600125) by Brydon et al.19 SP600125,
a flat molecule25 with a free NH group in the pyrazole ring,
acts as a reversible ATP competitive inhibitor and has proved
to be a small molecule inhibitor of JNK among several tested
kinases.26–28 But the methyl and ethyl derivatives of pyrazo-
loanthrone exhibited loss of inhibitory activity19 and hence
other higher alkyl derivatives were not tested. From a chem-
istry perspective, the tautomerism in 1,9-pyrazoloanthrone
(anthra[1,9-c,d]pyrazol-6-one, Fig. 1a) was confirmed by the
existence of two positional isomers after alkylation (Fig. 1b).29
According to Scapin and co-workers,30 the crystal structure of
JNK3 with 1,9-pyrazoloanthrone (PDB code: 1PMV) showed
aSolid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore,
India. E-mail: ssctng@sscu.iisc.ernet.in; Fax: +91 80 23601310;
Tel: +91 80 22932796
bDepartment of Microbiology and Cell Biology, Indian Institute of Science, Bangalore,
India. E-mail: balaji@mcbl.iisc.ernet.in; Fax: +91-80-23602697;
Tel: +91-80-22933223
cDepartment of Physics, Indian Institute of Science, Bangalore, India
dSupercomputer Education and Research Centre, Indian Institute of Science,
Bangalore, India
†Electronic supplementary information (ESI) available: Crystal structures, NMR
and ligplots. CCDC SPE2-990062, SPP1-990060, SPP2-990061, SPB1-990059,
SPB2-990535 and 983911. For ESI and crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/c4ob00548a
4656 | Org. Biomol. Chem., 2014, 12, 4656–4662
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 1 Chemical structures of (a) 1,9-pyrazoloanthrone and (b) N-alkyl-
ated pyrazoloanthrones. (c) Ligplot representation of interactions in the
active site of JNK3 with SP600125 (1PMV).
Fig. 2 ORTEP diagram of (a) SPB1 and (b) SPB2 with 50% probability
displacement ellipsoids.
higher selectivity towards inhibition compared to the other
molecules. It was shown that SP600125 interacts with the
protein via hydrogen bonds with the carbonyl oxygen of
Glu147 and the main chain nitrogen of Met149. In addition,
several hydrophobic contacts with Ile70, Ala91, Met146,
Leu148, Asp150, Asn152, Val196 and Leu206 (Fig. 1c) were
identified in the enzyme–inhibitor complex.
In this context, it may be surmised that an increase in the
alkyl chain length on the nitrogen atom might invoke
additional hydrophobic contacts in the active site of JNK, a
feature which needs to be explored. In the present study the
tautomerism in 1,9-pyrazoloanthrone was substantiated by the
positional isomers of alkyl derivatives and the structures have
been established by single crystal X-ray diffraction studies.
Based on calculated binding abilities with JNK structure gene-
rated from the in silico screening, 10 molecules were selected
for initial screening of their kinase inhibitory activity. Among
the tested molecules, a few of them show specific inhibition of
JNK among the several MAP kinases with favourable concen-
trations in the range of 1–20 μM. These molecules deserve
further evaluation in terms of their pharmacokinetic pro-
perties as well as biological functions to be potent therapeutics
in resolving inflammation.
Table 1 Crystallographic data for compounds SPB1 and SPB2
Compound
SPB1
SPB2
Formula
Formula weight
System
Space group
a (Å)
b (Å)
c (Å)
α
C
18H16N2O
C18H16N2O
276.3324
Monoclinic
P21/c
4.933(2)
15.036(7)
18.387(9)
90
276.3324
Orthorhombic
P212121
4.909(5)
14.694(2)
19.031(2)
90
β
γ
90
90
91.93(5)
90
1363.07(2)
4
1.35
0.085
Volume (Å3)
Z
1372.93(3)
4
1.34
0.084
583.9
Density (g cm−3
)
μ (mm−1
F (000)
)
583.9
No. of measured reflections
No. of unique reflections
No. of reflections used
R_all, R_obs
9877
3001
1861
0.120, 0.067
0.193, 0.152
−0.216, 0.257
21 681
2671
2173
0.058, 0.045
0.109, 0.102
−0.226, 0.234
wR2_all, wR2_obs
Δρmin,max (e Å−3
)
the butyl derivative of 1,9-pyrazoloanthrone (designated as
SPB1 and SPB2) were crystallized from a mixture of 30%
EtOAc–hexane and their crystal structures were determined
(Fig. 2; Table 1). Similarly, several alkyl derivatives of pyrazolo-
anthrone (SPM1, SPE2, SPP1 and SPP2; ESI†) were crystallized
by slow evaporation at ambient temperatures and their crystal
structures were determined.
2. Results and discussion
2.1. Synthesis
Based on preliminary results obtained with pyrazoloanthrones
in in silico screening, N-alkyl substituted pyrazoloanthrones
were synthesized according to modified procedures (ESI†). The
2.3. Virtual screening
The binding efficiencies of the alkyl derivatives were tested
in silico using docking simulations around the active site of
the protein. The known inhibitor SP600125 (537) was docked
into the active site of JNK3 to obtain an estimate of its binding
energy (−8.05 kcal mol−1). The predicted binding energies of
the other molecules (listed in Table 2 according to increasing
length of the alkyl group) were compared with this value. Com-
1
compounds were characterized by H & 13C NMR, and HR-MS,
and the purity was assessed by the analytical HPLC method
and found to be >98%. The crystal structures of these com-
pounds were determined by single crystal X-ray diffraction.
2.2. Crystal structure determination
Substitution of an alkyl group on the pyrazole ring in 1,9-pyra- pared to SP600125, all the derivatives, except SPM1 and SPE2,
zoloanthrone forms two isomers. For example, two isomers of showed enhanced hydrophobic contacts. With the increase in
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 4656–4662 | 4657

View Article Online
Paper
Organic & Biomolecular Chemistry
been analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide) assays. All the tested small molecules
exhibited cytotoxic effects greater than 20–30% only beyond
30 μM concentration (Fig. 4). DMSO is utilized in the study as
a solvent control as it shows no significant cytotoxic effect and
helps in confirming the specificity of the alkyl derivatives of
1,9-pyrazoloanthrone in dampening the cell survival. Based on
these results, the maximum concentration of the pyrazolo-
anthrone derivatives that can be tested for target specific inhi-
bition of JNK has been narrowed down to a range of 1 μM to
30 μM in further studies.
The kinase inhibitory effect of alkyl substituted pyrazolo-
anthrones was studied by monitoring the phosphorylation of
c-Jun, an immediate downstream target of activated JNK in
macrophages by the immunoblotting technique. All the alkyl
substituted derivatives of pyrazoloanthrone exhibited the inhi-
bition of JNK over a range of concentrations tested in LPS acti-
vated mouse macrophages. Of the compounds tested, SPP1,
SPB1 and SPEN1 (propyl, butyl and pentyl derivatives of pyra-
zoloanthrone) demonstrated the inhibition of JNK at lower
concentrations ranging from 1 µM to 5 µM whereas 1,9-pyrazo-
loanthrone showed inhibition with the minimum requirement
of 10 µM in LPS activated macrophages as shown in Fig. 5D–F
and H respectively. As reported by Brydon et al., the methyl
and ethyl derivatives of pyrazoloanthrone showed significant
loss in inhibitory activity;19 however, compounds with propyl
and butyl substitutions (SPP1, SPB1 and SPB2) showed signifi-
cant inhibitory function starting from 1 µM whereas SPEN1
showed inhibition from 5 µM onwards. Intriguingly, all these
obtained concentrations after the initial screen are far lower as
compared to 1,9-pyrazoloanthrone which initiates inhibition
only beyond 10 µM (Fig. 5H).
Table 2 Binding energies of 1,9-pyrazoloanthrone derivatives
Hydrogen bond
between protein
atom and ligand hydrophobic
atom
Binding
energy
No. of
Protein Ligand
(kcal mol−1
)
contacts
9
1PMV
537
−8.05
Met149 N⋯N
Glu147 O⋯N
Met149 N⋯O
Met149 N⋯O
Met149 N⋯O
Met149 N⋯O
Met149 N⋯O
Met149 N⋯O
Met149 N⋯O
Met149 N⋯O
Met149 N⋯O
Met149 N⋯O
SP600125
SPM1
SPM2
SPE1
−7.70
−7.80
−7.87
−7.90
−7.88
−8.09
−8.11
−8.27
−8.61
−8.69
7
11
9
11
11
13
13
14
11
13
SPE2
SPP1
SPP2
SPB1
SPB2
SPEN1
SPEN2
Fig. 3 Ligplot representation of interactions in active site of JNK3 with
(a) SPB1 and (b) SPB2.
length of the substituting alkyl group, improved binding ener-
gies were observed. It is of interest to note that the binding
energies of SPB1 and SPB2 are comparable to those of the
parent compound and are enhanced in the case of SPEN1 and
SPEN2 respectively.
As described in Fig. 1b the parent molecule 1,9-pyrazoloan-
throne in the protein–inhibitor complex interacts with the
protein via hydrogen bonds involving the carbonyl oxygen of
Glu147 and the main chain nitrogen of Met149. The second
hydrogen bonding is conserved in the interactions of all the
N-alkylated pyrazoloanthrones (ESI†) with the JNK protein
(1PMV), as observed from the docking simulations. Fig. 3a and
3b show the binding characteristics of SPB1 and SPB2 with
conserved hydrogen bonding involving MET149. The Ligplot
representations of interactions of the other derivatives are
presented in ESI.†
Thus, the fact that the substitution of alkyl chains of
increasing length enhances the hydrophobic interaction poten-
tial is quite evident from the extent of inhibitory activity with
SPP1 ≈ SPB1 > SPEN1 > SPM1 > SPE1. On the other hand, pyra-
zoloanthrone isomers (derivatives with alkyl substituents on
the left side of the pyrazole ring) such as SPP2 (5 μM) and
SPB2 (1 μM) also showed significant inhibition of JNK
activity as well as phosphorylation of c-Jun in comparison with
1,9-pyrazoloanthrone. It is noteworthy that on further exten-
sion of chain length like in the case of the pentyl derivative of
1,9-pyrazoloanthrone, there is a sudden drop in the inhibitory
activity and this may be attributed to the requirements of con-
served hydrogen bonding at the binding site as demonstrated
by the auto dock studies. Interestingly, SPP1 and SPB1 exhibi-
ted the inhibitory effect at a concentration lower than 1,9-
pyrazoloanthrone.
In order to evaluate the specific inhibitory activity of SPB1
and SPP1 molecules over JNK compared to LPS induced acti-
2.4. Biological activity
2.4.1. Evaluation of the biological efficacies of alkyl substi- vation of other MAP kinases such as ERK1/2 and p38, immu-
tuted pyrazoloanthrones. Sometimes the inhibitor activity of noblot analysis of the active state of ERK1/2 and p38 was
compounds could also be a result of their toxic effects and carried out. SPP1 and SPB1 block LPS induced activation of
consequently might cause a flawed conclusion. Hence, prior to ERK1/2 and p38 only at concentrations beyond 30 μM with
utilization of these small molecules as inhibitors, the cytotoxic marginal off target effects (Fig. 6). These results suggest that
effect of these on macrophages at various concentrations has alkyl substituted pyrazoloanthrone scaffolds need to be further
4658 | Org. Biomol. Chem., 2014, 12, 4656–4662
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 4 Analysis of cytotoxic effect of small molecules by the MTT assay. Mouse peritoneal macrophages were treated with respective molecules at
various concentrations for 12 hours and cell viability was analyzed by the MTT assay.
explored to treat inflammatory disorders with higher speci-
ficity at lower concentrations.
4. Experimental section
4.1. Materials and methods
Isolation of mouse peritoneal macrophages. Macrophages
utilized in the study were isolated from C57/BL6J mice. In
brief, mice were intraperitoneally injected with thioglycolate
(2 mL of 2X concentration per mice). After 4 days of injection,
3. Conclusions
In conclusion, the synthesis, isolation and structural determi-
nation of alkyl isomers of pyrazoloanthrone derivatives have
mice were sacrificed. Peritoneal cavities were flushed with ice
cold PBS and centrifuged to obtain the macrophages. Thus,
tration of these small molecule inhibitors was found to be less
the obtained cells were resuspended in DMEM containing
been achieved. The minimum required inhibitory concen-
than 10 μM in comparison with 1,9-pyrazoloanthrone to
inhibit JNK. The structural correlations appear to support the
inhibitor evaluation. The alkyl substituted scaffolds lead to
molecules with potent and selective inhibition of JNK. Criti-
cally, our lead candidates SPP1 and SPB1 display specific inhi-
bition of JNK among other LPS activated MAP kinases like
ERK1/2 and p38. Our results suggest that these two scaffolds
of inhibitors SPP1 and SPB1 would be promising leads for the
10% FBS (Sigma Aldrich) and seeded for further experiments.
The experiments with mouse macrophages were carried out
after the approval from the Institutional Ethics Committee for
animal experimentation as well as from the Institutional Bio-
safety Committee.
4.2. Cell viability assay
future development of more potent and selective inhibitors to Mouse macrophages were seeded in 96 well plates (75 000 cells
treat disorders associated with inflammation.
per well) in 200 μl of DMEM complete medium and incubated
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 4656–4662 | 4659

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 5 Optimum inhibitory concentrations of 1,9-pyrazoloanthrone derivatives over phosphorylation of c-Jun. B–F and J–N show the immunoblots
for phospho c-Jun in LPS (100 ng ml−1) activated macrophages at different concentrations of inhibitors tested. A, G and I represent the structures of
1,9-pyrazoloanthrone and its derivatives.
Fig. 6 Elucidation of the off-target effect of SPB1 and SPP1 over LPS induced MAP kinase activation. (A, B and C) Immunoblot analysis of LPS
induced activation of p38, ERK1/2 in the presence of various concentrations of SP600125, SPB1 and SPP1 respectively in mouse macrophages.
4660 | Org. Biomol. Chem., 2014, 12, 4656–4662
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
overnight. Later, the cells were treated with small molecules HBPLUS34 module in Ligplot with hydrogen bonding para-
reconstituted in DMSO at various concentrations as mentioned meters (D⋯A distance ≤3.35 Å, H⋯A ≤2.7 Å).
for 12 hours. Post 12 hour treatment, the medium was
removed carefully and fresh medium (100 μl per well) was
added. 20 μl of 5 mg ml−1 of MTT reagent was added to each
well and incubated for 4 hours at 37 °C aseptically. The
medium was removed and DMSO (100 μl per well) was added
to the cells and left on an orbital shaker (150 rpm) for 15 min.
After incubation, absorbance was read at 590 nm. Untreated
cells served as the control in all the cell viability assays.
Abbreviations
c-JNK
c-Jun N-terminal kinase
MAP Kinase Mitogen activated protein kinase
LPS
Lipopolysaccharide
ERKs
Extracellular signal-regulated kinases
4.3. Treatment with small molecule inhibitors
Acknowledgements
All the small molecules utilized in the study were reconstituted
in sterile DMSO (Sigma-Aldrich, USA) and used at various con-
centrations as mentioned. DMSO at 0.1% concentration was
used as the vehicle control. In all experiments with inhibitors,
a tested concentration was used after careful titration experi-
ments assessing the viability of the macrophages using the
MTT assay. In experiments with inhibitors, the cells (4 × 106
per well) were treated with a given inhibitor for 60 min before
experimental treatment.
Authors thank the Indian Institute of Science for infrastructure
facilities and financial support. We thank the Central Animal
facility, Indian Institute of Science for providing mice for
experimentation. KDP and JT acknowledge fellowships from
CSIR (Council of Scientific Industrial Research). TNG thanks
DST for a J C Bose fellowship.
4.4. Immunoblotting
References
Macrophages were treated with respective small molecule
inhibitors as mentioned and then stimulated with LPS (Sigma-
Aldrich, USA), 100 ng ml−1, for an additional 60 min. Cells
were washed twice with PBS, scrapped off the culture dish and
collected by centrifugation. Cell lysates were prepared in RIPA
buffer constituting 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25%
sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM
PMSF, 1 μg ml−1 of each aprotinin, leupeptin, pepstatin, 1 mM
Na3VO4, and 1 mM NaF and incubated in ice for 30 min.
Whole cell lysates were collected by centrifuging lysed cells at
13 000 rpm for 10 min at 4 °C. Equal amounts of protein from
each cell lysate were subjected to SDS-PAGE and transferred
onto PVDF membranes (Millipore, USA) by the semidry
western blotting (Bio-Rad, USA) method. Nonspecific binding
was blocked with 5% nonfat dry milk powder in TBST (20 mM
Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20) for
60 min. The blots were probed with anti-phospho Ser63 c-Jun
for 12 hours at 4 °C and then washed with TBST thrice fol-
1 D. C. Angus and T. V. Poll, N. Engl. J. Med., 2013, 369,
2062–2063.
2 N. A. Ali, Crit. Care, 2010, 14, 160.
3 J. Moreira, N. Engl. J. Med., 2013, 369, 2062–2063.
4 W. J. Lin and W. C. Yeh, Shock, 2005, 24, 206–209.
5 N. Mukaida, Y. Ishikawa, N. Ikeda, N. Fujioka,
S. Watanabe, K. Kuno and K. Matsushima, J. Leukoc. Biol.,
1996, 59, 145–151.
6 K. Watanabe, H. Nakagawa and S. Tsurufuji, Agents Actions,
1986, 17, 472–477.
7 R. M. Pinheiro and J. B. Calixto, Inflamm. Res., 2002, 51,
603–610.
8 M. S. Inayat, I. S. El-Amouri, M. Bani-Ahmad, H. L. Elford,
V. S. Gallicchio and O. R. Oakley, J. Inflamm., 2013, 7, 43.
9 T. Kallunki, B. Su, I. Tsigelny, H. K. Sluss, B. Derijard,
G. Moore, R. Davis and M. Karin, Genes Dev., 1994, 8, 2996–
3007.
lowed by anti-rabbit IgG HRP conjugated secondary antibody 10 B. Derijard, M. Hibi, I. H. Wu, T. Barrett, B. Su, T. Deng,
for 2 hours at 4 °C. Blots were washed and developed using the M. Karin and R. J. Davis, Cell, 1994, 76, 1025–1037.
enhanced chemiluminescence detection system (Perkin Elmer, 11 M. A. Bogoyevitch and P. G. Arthur, Biochim. Biophys. Acta,
USA) as per the manufacturer’s instructions. Blots were probed 2008, 1784, 76–93.
with anti-β-actin HRP (Sigma-Aldrich, USA) to ensure equal 12 M. Karin, J. Biol. Chem., 1995, 270, 16483–6.
loading of protein.
13 G. McMahon, L. Sun, C. Liang and C. Tang, Curr. Opin.
Drug Discov. Devel., 1998, 1, 131–146.
14 V. C. Foletta, D. H. Segal and D. R. Cohen, J. Leukoc. Biol.,
1998, 63, 139–152.
4.5. Docking
The three-dimensional (3D) structures of all ten small mole-
cules were modelled and minimised using the PRODRG 15 J. Jain, V. E. Valge-Archer and A. Rao, J. Immunol., 1992,
server31 and single crystal structures. AutoDock (version 4.2)32
148, 1240–1250.
was used for the ligand–protein docking. The Lamarckian 16 S. Gupta, T. Barrett, A. J. Whitmarsh, J. Cavanagh,
Genetic Algorithm was used with a population of 200 dock-
ings. The docking output was analysed using Pymol and
H. K. Sluss, B. Derijard and R. J. Davis, EMBO J., 1996, 15,
2760–2770.
Ligplot.33 Hydrogen bonds were determined using the in-built 17 P. P. Graczyk, Future Med. Chem., 2013, 5, 539–551.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 4656–4662 | 4661

View Article Online
Paper
Organic & Biomolecular Chemistry
18 Z. Xia, M. Dickens, J. Raingeaud, R. J. Davis and 25 K. D. Prasad, N. Venkataramaiah and T. N. Guru Row,
M. E. Greenberg, Science, 1995, 270, 1326–1331. Cryst. Growth Des., 2014, 14, 2118–2122.
19 B. L. Bennett, D. T. Sasaki, B. W. Murray, E. C. O’Leary, 26 K. Assi, R. Pillai, A. Gomez-Munoz, D. Owen and B. Salh,
S. T. Sakata, W. Xu, J. C. Leisten, A. Motiwala, S. Pierce, Immunology, 2006, 118, 112–121.
Y. Satoh, S. S. Bhagwat, A. M. Manning and 27 Y. T. Ip and R. J. Davis, Curr. Opin. Cell Biol., 1998, 10, 205–
D. W. Anderson, Proc. Natl. Acad. Sci. U. S. A., 2001, 98,
13681–13686.
20 A. Cerbone, C. Toaldo, S. Pizzimenti, P. Pettazzoni,
C. Dianzani, R. Minelli, E. Ciamporcero, G. Roma,
219.
28 N. Miyakoshi, C. Richman, Y. Kasukawa, T. A. Linkhart,
D. J. Baylink and S. Mohan, J. Clin. Invest., 2001, 107,
73–81.
M. U. Dianzani, R. Canaparo, C. Ferretti and G. Barrera, 29 L. Havlíčková, A. Koloničný, A. Lyčka, J. Jirman and I. Kolb,
PPAR Res., 2012, 269751. Dyes Pigm., 1989, 10, 1–11.
21 T. Kamenecka, R. Jiang, X. Song, D. Duckett, W. Chen, 30 G. Scapin, S. B. Patel, J. Lisnock, J. W. Becker and
Y. Y. Ling, J. Habel, J. D. Laughlin, J. Chambers, P. V. LoGrasso, Chem. Biol., 2003, 10, 705–712.
M. Figuera-Losada, M. D. Cameron, L. Lin, C. H. Ruiz and 31 A. W. Schuttelkopf and D. M. F. Van Aalten, PRODRG - a
P. V. LoGrasso, J. Med. Chem., 2010, 53, 419–431.
22 N. Kwiatkowski, N. Jelluma, P. Filippakopoulos,
M. Soundararajan, M. S. Manak, M. Kwon, H. G. Choi,
tool for high-throughput crystallography of protein-ligand
complexes, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2004,
60, 1355–1363.
T. Sim, Q. L. Deveraux, S. Rottmann, D. Pellman, J. V. Shah, 32 G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
G. J. Kops, S. Knapp and N. S. Gray, Nat. Chem. Biol., 2010,
6, 359–368.
23 M. A. Siddiqui and P. A. Reddy, J. Med. Chem., 2010, 53,
3005–3012.
24 H. D. Showalter, J. L. Johnson, J. M. Hoftiezer,
W. R. Turner, L. M. Werbel, W. R. Leopold, J. L. Shillis,
R. K. Belew, D. S. Goodsell and A. J. Olson, AutoDock4 and
AutoDockTools4: automated docking with selective recep-
tor flexibility, J. Comput. Chem., 2009, 2785–2791.
33 A. C. Wallace, R. A. Laskowski and J. M. Thornton,
LIGPLOT: A program to generate schematic diagrams of
protein-ligand interactions, Prot. Eng., 1995, 8, 127–134.
R. C. Jackson and E. F. Elslager, J. Med. Chem., 1987, 30, 34 I. K. McDonald and J. M. Thornton, J. Mol. Biol., 1994, 238,
121–131.
777–793.
4662 | Org. Biomol. Chem., 2014, 12, 4656–4662
This journal is © The Royal Society of Chemistry 2014