Structural and biological evaluation of halogen derivatives of 1,9-pyrazoloanthrones towards the design of a specific potent inhibitor of c-Jun-N-terminal kinase (JNK)

Article abstract of DOI:10.1039/c8nj00852c

c-Jun N-terminal kinase (JNK), a member of the MAPK family, is associated with a variety of diseases and immune responses. To dissect the mechanistic role of JNKs in such processes, a specific inhibitor for JNKs holds great value. SP600125 is a widely used inhibitor of JNKs despite its non-specific activity. In an effort to obtain better specific inhibitors, three anthrapyrazolone halogenated derivatives have been synthesized and characterized. Among the three derivatives, 5-chloro-2-(2-chloroethyl)dibenzo[cd,g] indazol-6(2H)-one is clearly established as a specific inhibitor of JNK with augmented expression of chemokines in LPS-activated macrophages based on modelling studies followed by in vitro and ex vivo evaluation.

Full text of DOI:10.1039/c8nj00852c

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Structural and biological evaluation of halogen derivatives of 1,9-
pyrazoloanthrones towards the design of specific potent inhibitor of c-Jun-
N-terminal kinase (JNK)
Ramesh Ganduri,a Vikas Singh,b Ansuman Biswas,c Karothu Durga Prasad,a Kanagaraj Sekar,d Kithiganahalli N.
Balajib and Tayur N. Guru Row*a
a Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, Karnataka, India
Eꢀmail: gururow@iisc.ac.in; Fax:+91ꢀ80ꢀ23601310; Tel: +91ꢀ80ꢀ22932796
bDepartment of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, Karnataka,
India
c Department of Physics, Indian Institute of Science, Bangalore 560012, Karnataka, India
d Department of Computational and Data Sciences, Indian Institute of Science, Bangalore 560012, Karnataka,
India
Abstract
cꢀJun Nꢀterminal kinase (JNK), a member of MAPK family, is associated with a variety of
diseases and immune responses. To dissect the mechanistic role of JNKs in such processes,
specific inhibitor for JNKs holds great value. SP600125 is widely used inhibitor of JNKs
despite its nonꢀspecific activity. In an effort to obtain better specific inhibitors, three
anthrapyrazolone halogenated derivatives have been synthesized and characterized. Among
the three derivatives, 5ꢀchloroꢀ2ꢀ(2ꢀchloroethyl) dibenzo[cd,g] indazolꢀ6(2H)ꢀone is clearly
established as a specific inhibitor of JNK with augmented expression of chemokines in LPSꢀ
activated macrophages based on modelling studies followed by in vitro and ex vivo
evaluation.

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1. Introduction
JNKs are serine/threonine protein kinases which belong to the mitogenꢀactivated protein
kinases (MAPKs) family. Notably, there are three distinct genes that codes for JNK1, JNK2
and JNK3 which results in expression 10 isoforms of JNKs via alternative splicing. JNKs
play a central role in host inflammatory responses and they have been implicated in a wide
range of inflammation associated disorders such as arthritis and atherosclerosis.1–3 The host
immune responses to Gramꢀnegative bacteria cell wallꢀderived LPS is characterized by the
recruitment of immune cells such as macrophages and neutrophils at the site of infection.
Moreover, infiltrated immune cells induce inflammation which is mediated by localized
production of chemokines.4,5 Chemokines are the class of highly specialized cytokines that
are implicated in a wide range of biological processes including cellular stress, cancer,
angiogenesis and bacterial or viral pathogenesis.6–8 Growing body of evidences indicates a
significant contribution of cꢀJun Nꢀterminal kinase (JNK) in the regulation of chemokines
expression.9–11 JNKꢀinduced phosphorylation of cꢀJun at ser63 or ser73 facilitates its
interaction with cꢀFos which in turn form a complex with transcription factor activator
protein 1 (APꢀ1) and regulate host immune responses.12–14 In light of current literature it
becomes important to design and synthesize potent and JNKꢀspecific inhibitors with minimal
or no offꢀtarget effects to attain deeper understanding in JNK mediated signalling mechanism
and immune responses. Among existing JNKs inhibitors,15 SP600125 (anthra [1, 9] pyrazolꢀ6
(2H)ꢀone or 1,9ꢀpyrazoloanthrone),16 inhibits JNK1/2/3 with specificity and subsequently
inhibits activation of cꢀJun. Although SP600125 is utilized as a selective JNKs inhibitor, its
offꢀtarget effect on other kinases such as PI3K (p110δ), p70 ribosomal protein kinase (S6K1)
and mps1 invited new disputes in its utility as a selective inhibitor for JNKs.17–21 In an effort
to develop improved inhibitors with reduced or completely blocked off target effects, we
have shown that Nꢀalkyl substituted pyrazoloanthrone derivatives regulate inhibiting

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potential and JNKs activity with minimal offꢀtarget effect on other mitogen activated protein
kinases unlike the commercially available 1,9ꢀpyrazoloanthrone (SP600125).22 Several Nꢀ
alkyl and hydroxyl substituted derivatives of 1,9ꢀpyrazoloanthrone derivatives have been
designed, synthesised and tested in vitro for analysis of JNK inhibition at varied
concentrations. The results revealed that among all analogues of 1,9ꢀpyrazoloanthrone
(SP600125), propyl, butyl and hydroxyl substituted pyrazoloanthrone scaffolds confer better
binding energies along with notable inhibitory effect on JNKs activity.23 Such detailed
biochemical evaluation of selective JNK inhibition allows for the identification of specific
drugs to treat diseases like sepsis on one hand and on the other, provide complete
characterization of biological functions of JNK.23
Intermolecular interactions like hydrogen bonds, hydrophobic contacts and van der Waals
forces play a decisive role in both structural and functional aspects in chemical biology. In
recent years, halogen bonds (XB) which are demonstrated to be highly directional among
nonꢀcovalent interactions have been identified to provide well directed adhesive contacts for
proteinꢀligand interactions24,25 and are increasingly being explored based on structural and
theoretical estimates of energies.26,27 Experimental and theoretical charge density analysis
establish the highly directional and attractive nature of halogen bonded interactions in terms
of the so called σꢀhole concept.28–31 Moreover, halogen derivatives are preferred as enzyme
inhibitors since they improve the proteinꢀligand binding affinity, specificity along with tuning
ADME/T properties.32,33 Halogen bonds also provide sufficient binding energies to hold the
molecule/s in the binding pocket resulting in heightened activity of the inhibitor. An
examination of the Protein Data Bank (PDB) database34 reveals sufficiently large number of
structures with halogenated ligands suggesting the importance of halogen bonding for
rational design of selective and potent inhibitors against the therapeutic targets.35,36 The

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geometrical factors of halogen bonding for stabilizing interꢀ and intraꢀmolecular interactions
in biomolecules that can influence the ligand binding with protein have also been analyzed.37
Cl
O
O
Cl
N
NH
N
N
Br
K2CO3, DMF, rt
Cl
NH2-NH2.H2O
0
Pyridine, 115 C, 24h
X
O
Y
X
Y
X
O
Y
R1/R2/R3
X = H, Y = H
SP600125
X = H, Y = H
X = Cl, Y = H
X = H, Y = Cl
R1
R2
R3
Scheme 1 Synthetic scheme for the three anthrapyrazolone halogen derivatives.
In the present study, three chloro derivatives of SP600125, 2ꢀ(2ꢀchloroethyl)
dibenzo[cd,g]indazolꢀ6(2H)ꢀone, 7ꢀchloroꢀ2ꢀ(2ꢀchloroethyl)dibenzo[cd,g]indazolꢀ6(2H)ꢀone
and 5ꢀchloroꢀ2ꢀ(2ꢀchloroethyl)dibenzo[cd,g]indazolꢀ6(2H)ꢀone, (R1, R2 and R3
respectively; Scheme 1) are examined based on the fact that halogen interactions provide
highly directional and enhanced specificity towards JNK inhibitory binding.23,24 Even though
all three compounds depict halogen interactions in the binding pocket based on molecular
dynamics simulations, compounds R2 and R3 imply stable halogen interactions at optimal
distance for halogen bonding. Diffraction quality crystals could be grown only for R2 and its
crystal structure is reported. Indeed both R2 and R3 exhibit specific inhibition of JNK
activity at lower concentrations with no offꢀtarget effects based on in vitro biological studies.
However, compound R3 depicts highly specific inhibition with minimal off target effects at
significantly lower concentrations (<5 ꢁM).38

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2. Results and Discussion
2.1. Synthesis
The compounds 2ꢀ(2ꢀchloroethyl) dibenzo[cd,g]indazolꢀ6(2H)ꢀone, 7ꢀchloroꢀ2ꢀ(2 chloro
ethyl)dibenzo[cd,g]indazolꢀ6(2H)ꢀone and 5ꢀchloroꢀ2ꢀ(2ꢀchloroethyl) dibenzo[cd,g]indazolꢀ
6(2H)ꢀone, R1, R2, and R3 respectively (Scheme 1) have been synthesized. All three
1
13
compounds were characterized by HRꢀMS and purity was assessed by the H and CꢀNMR
spectroscopic techniques (Fig. S1aꢀS1i, ESI†).
2.2. Crystal Structure Description
Attempts to crystallize all three compounds resulted in diffraction quality crystals for only
R2. Single crystals of 7ꢀchloroꢀ2ꢀ(2ꢀchloroethyl)dibenzo[cd,g]indazolꢀ6(2H)ꢀone (R2), were
grown from solvent mixture of Methanol/Tetrahydrofuran (70/30%, v/v) by slow solvent
evaporation at ambient temperature. Xꢀray diffraction data were collected at 100K, structure
was solved and refined in a monoclinic space group P21/n with Z = 4 (Table 1). ORTEP with
thermal ellipsoids and crystal packing of R2 are shown in Fig. 1. It is of interest to note that
there are intermolecular interactions involving both chlorine atoms, Cl1 and Cl2 (two Cꢀ
H...Cl contacts; 2.913Å and 2.838Å and one Cl1...Cl2 halogen bond; 3.251Å; angles ∠C–
Cl1ꢂꢂꢂCl2 = 151.84°; ∠C–Cl2ꢂꢂꢂCl1 = 163.59° respectively suggesting a TypeꢀI geometry),30
which point out to the possibility of generating halogen atom involved interactions in the
binding pocket.

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Fig. 1 ORTEP diagram of R2 and crystal packing showing the three short interactions (two CꢀH...Cl
and one Cl...Cl).
Table 1 Crystallographic parameters of R2
Compound
R2
Formula
Formula weight
Crystal system
Space group
a (Å)
C16H10 Cl2N2O
317.16
Monoclinic
P21/n
6.7039(5)
14.5890(10)
13.9378(9)
90
b (Å)
c (Å)
α (°)
β (°)
96.283(6)
90
γ (°)
Volume (Å3)
Z
1354.97(16)
4
Temperature (K)
Density (g cm−3)
ꢁ (mm−1)
F (000)
100.08(2)
1.555
0.478
648
hmin, max
ꢀ8, 8
kmin, max
ꢀ18, 18

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lmin, max
ꢀ17, 18
No. of measured reflections
No. of unique reflections
No. of reflections used
R_all, R_obs
18436
3099
2314
0.1271,0.0964
0.2391, 0.2226
−0.506, 0.677
1.111
wR2_all, wR2_obs
ꢀ
ρ
min, max (e Å−3)
GOOF
CCDC No.
1560310
2.3. Optimization of JNK inhibitors through computational studies
The crystal structure of the protein JNK3 with the inhibitor SP600125, 1PMV (JNK3), along
with two other structures 2NO3 (JNK1) and 3E7O (JNK2) have been selected for the in silico
studies from the Protein Data Bank. The crystal structure 1PMV (JNK3) indicates that the
hydrophobic inhibitor (SP600125) interacts with the protein via hydrogen bonds with the
carbonyl oxygen of Glu147 and the main chain nitrogen of Met149, in addition to several
hydrophobic contacts with Ile70, Ala91, Met146, Leu148, Asp150, Asn152, Val196 and
Leu206 respectively39 (Fig. S2A, ESI†). It is noteworthy that on superposition of the active
site of AMPꢀPCP bound JNK3 with that of the inhibitorꢀbound JNK3 (1PMV), it is found
that SP600125 molecule binds to the hydrophobic adenine binding site via conserved
hydrogen bonds with the backbone (Met149[N] and Glu147[O]). Additionally, computational
alanineꢀscanning results show that Ile70, Leu206, Val196 are important for SP600125
recognition (Fig. S2B, ESI†).
Although the crystal structure of JNK3ꢀSP600125 complex (1PMV) is already available,39
docking calculations with the incorporation of the inhibitor at the active site were made to

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determine the least binding energy configuration (ꢀ8.05 Kcal molꢀ1).22,23 Similar calculations
were performed with the other JNK’s as well. It is observed that the binding of the inhibitors
exhibit similar trends for all three JNKs, with subtle variations. The studies were extended to
examine the propensity of binding with R1, R2 and R3 to evaluate the role of chlorine atoms
in the binding pocket. It is noteworthy that, as the number of chlorine atoms increases, a
decrease in binding energy is observed, suggesting favourable binding characteristics. The
binding energies of R1 and SP600125 are comparable in JNK2 and JNK3 (Table 2),23
whereas R2 and R3, have lower binding energies compared to R1 and SP600125. An
analysis of the proteinꢀligand interactions on the docked structures indicates that R2 and R3
exhibit several halogen contacts within the distance of 3.5Å (Table 2). In addition, conserved
hydrogen bonds are seen between the ligands and the active site of the corresponding protein.
However, it is of interest to note that in case of R2, the hydrogen bonding interaction is
disrupted when binding to JNK1. It may be concluded that the presence of halogen
interactions along with hydrogen bonds suggest better binding characteristics of R2 and R3
compared to that of R1(Table 2) (Fig. S3a–S3c, ESI†). A comprehensive analysis of the
binding characteristics (halogen bonding) will be obtained from the crystal structures of all
JNK’s with R1, R2 and R3 respectively and crystallization experiments are currently being
pursued.
Table 2 Analysis of halogen interactions among the JNKs (1PMV: JNK3, 3E7O: JNK2, 2NO3:
JNK1) and the ligand molecules. The halogen contacts were determined using PLIP server.

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Possible predictable
Binding
Energy
Hydrogen bond
with active site
residue
Protein, ligand
complex
halogen bonds between
the protein atom and
ligand atom
All halogen contacts*
(kcal/mol)
1PMV, R1
1PMV, R2
ꢀ8.08
ꢀ8.75
with Met149
with Met149
Cl...O(Gly76)
Cl...CG2(Val78)
Cl....NZ(LYS93)
Cl....HZ2(LYS93)
Cl....HZ3(LYS93)
Cl....N(GLY73)
ClX....O(Met149)
1PMV, R3
ꢀ8.44
with Met149
Not detected
Cl....CG2(VAL78)
ClY....O(MET149)
Cl....CD1(ILE86)
Cl....HN(MET111)
Cl....CG2(VAL158)
2NO3, R1
2NO3, R2
2NO3, R3
ꢀ6.69
ꢀ6.75
ꢀ6.73
with Asn 114
Not detected
with Met111
Not detected
Cl...O(Gly38)
Not detected
ClX....O(MET111)
Cl....HN(GLY35)
Cl....CG2(VAL40)
ClY..HN(MET111)
Cl....NZ(LYS55)
Cl....HZ2(LYS55)
Cl....HZ3(LYS55)
Cl....O(LEU168)
Cl....NZ(LYS55)
Cl....HZ2(LYS55)
Cl....HZ3(LYS55)
Cl....O(LEU168)
ClX....N(ALA113)
Cl....O(SER34)
3E7O, R1
3E7O, R2
3E7O, R3
ꢀ7.95
ꢀ8.64
ꢀ8.20
with Met111
with Met111
with Met111
Not detected
ClX....O(Met111)
Cl....CG2(VAL40)
ClY..CB(MET108)
Cl....O(GLU109)
ClY....O(Gly38)
* The halogen contacts (<3.5Å ) are only considered.
2.4. Molecular dynamics simulations

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Molecular dynamics simulations were carried out of ligand bound protein complexes (R1,
R2, R3 and PDB id:1PMV) to examine the stability of the protein molecules in the presence
of ligands. The ligand bound simulations showed overall stabilization of the protein molecule
compared to that of the apo protein as is evidenced by the lowering of RMSD (Fig. 2A). All
three ligands form an average of one hydrogen bond throughout the trajectory. The distances
between interacting atoms involved in halogen contacts in R1, R2 and R3 were computed
from the coꢀsimulations trajectory. It is noteworthy that the contact distances extracted from
Fig. 2 (A) RMSD plot (Black: apo form; Red: R1; Green: R2; Blue: R3) extracted from the
molecular dynamics simulations of apo and holo (R1, R2, R3) forms of JNK3 reveals that the protein
is stable in the presence of all three ligands at the active site. (B) The distribution of the length of the
halogen interactions are shown for all three holo simulations (Black: R1; Red: R2; Green: R3).
R2 and R3 simulations have a narrow distribution with peaks around 3.5Å, (which imply
stable interactions at an optimal distance for halogen contact), whereas R1 has a wider
distribution with a peak at 5Å, suggesting weaker propensity for halogen interactions (Fig.
2B).
2.5. Biological Evaluation
2.5.1. In vitro cytotoxic studies towards cell viability
The primary screen was performed to examine the cytotoxic effect of analogues of 1,9ꢀ
pyrazoloanthrone (SP600125, R1, R2, and R3) on macrophages. Careful titration with

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analogues displayed significant cytotoxicity for inhibitor only above 30µM concentration
(Fig. 3).
Fig. 3 MTT assay was performed to assess viability of mouse peritoneal macrophages upon treatment
with varied concentration of (A) SP600125, (B) R1, (C) R2, and (D) R3 for 12 hours.
Based on these observation, less than 30µM concentration was used to assess inhibitory
potential of analogues for JNK activity. Since, transcription factor, cꢀJun is the wellꢀ
characterized direct phosphorylation substrate of JNK, the activatoryꢀphosphorylation status
of cꢀJun was taken as read out of JNKs activity. Furthermore, other members of MAPKs
family such as ERK1/2 and p38 were analysed to assess the specificity of indicated inhibitors
(SP600125, R1, R2, and R3). In agreement with the existing literature, primary macrophages
treated with LPS were found to display elevated levels of phosphorylatedꢀERK1/2, p38 and
cꢀJun while upon treatment with inhibitors, macrophages showed a significant decrease in
LPSꢀinduced phosphorylation of cꢀJun in accordance with indicated concentration of
inhibitors. Importantly, R3 showed reduced phosphorylation of cꢀJun at 5µM concentration

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without affecting the phosphorylation status of other MAPKs, while R1 and R2 showed off
target effects on pERK1/2 and pp38 (Fig. 4).
Fig. 4 R3 specifically inhibits cꢀJun activity. Mouse peritoneal macrophages were treated with (A)
SP600125, (B) R1, (C) R2, and (D) R3 for 1h prior to 12h treatment with LPS and phosphorylation
status of cꢀJun, p38 and ERK1/2 was analysed by immunoblotting.
Moreover, inhibitor treatment significantly diminished LPS induced promoter activity of AP1
which is mediated by JNK signalling as depicted in (Fig. 5). Altogether, these results
establish R3 as a potent inhibitor of JNK activity at a dose as low as 1µM without offꢀtarget
effects on ERK1/2 and p38 (Fig. 6). Thus, the potential of R3 as a better inhibitor with
superior selectivity is not just in comparison with the other inhibitors R1 and R2 but
remarkably with that of the commercially utilized SP600125 is unequivocally established.
2.5.2. Ex vivo expression of LPS induced chemokines
At this stage, it is clear that R3 is the best possible choice available as a new superior
inhibitor of JNK and hence its contribution in modulating LPSꢀtriggered responses is carried

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out. Interestingly, the host immune response to LPS is characterized by elevated chemokines
expression in vitro as well in vivo40 and some of the chemokines are JNK dependent.
Fig. 5 Luciferase assay was performed on AP1 luciferase and βꢀgalactosidase coꢀtransfected murine
RAW264.7 macrophages and treatment with (A) SP600125, (B) R1, (C) R2, and (D) R3 for 1h was
given prior to LPS treatment for 12h.
It was observed that macrophages treated with 1µM R3 exhibited diminished expression of
LPSꢀinduced chemokines (Fig. 7). This data thus strongly suggests R3 as a potential
regulator of LPSꢀtriggered host immune response.

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Fig. 6 Activity of cꢀJun is specifically inhibited by R3 at lower concentrations. (A) Mouse peritoneal
macrophages were treated with R3 inhibitor for 1h prior to LPS treatment for 12h and
phosphorylation status of cꢀJun, p38, and ERK1/2 was analysed by immunoblotting. (B) Murine
RAW264.7 macrophages were transiently coꢀtransfected with AP1 luciferase and βꢀgalactosidase
luciferase followed by indicated inhibitors treatment for 1h prior to LPS treatment for 12h and
luciferase assay was performed.

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Fig. 7 R3 attenuates expression of LPSꢀinduced chemokines in macrophages. (A) Mouse peritoneal
macrophages were treated with LPS for 12h and expression of indicated chemokines were analysed
by quantitative real time RTꢀPCR. (B) Mouse peritoneal macrophages were treated with R3 inhibitor
1h prior to LPS treatment and incubated for 12h and expression of indicated chemokines were
analysed by quantitative real time RTꢀPCR.
3. Conclusions
In summary, we have designed and synthesized three halogenated derivatives of 1,9ꢀ
pyrazoloanthrone. The involvement of halogen bonding provides highly directional
interactions in the binding pocket and hence demonstrate improved inhibitory activity.
Among the three halogenated derivatives of 1,9ꢀpyrazoloanthrone, R3 exhibits the most
specific inhibition of JNKs activity at significantly lower concentrations (1ꢁM) compared to
R1, R2, and SP600125 with no offꢀtarget effects on other kinases such as p38 and ERK1/2.
Further, R3, being a potent and specific inhibitor of JNKs, regulates LPSꢀtriggered
expressions of chemokines in macrophages. R3 appears as the best designed inhibitor and
hence may facilitate the development of novel therapeutics to treat JNKsꢀassociated
disorders. Therefore, current study proposes R3 as a better alternative for SP600125 to inhibit
JNKs activity and may act as a promising tool to comprehend the physiological role of JNKs
for therapeutic benefits.
4. Experimental section
4.1. Chemical synthetic procedure
4.1.1. General information
All chemicals (Analytical grade) were purchased from SigmaꢀAldrich and were used without
1
additional purification. Solvents were purified according to standard methods. H and 13Cꢀ
NMR spectra (400 and 100 MHz) were recorded on Bruker avance IIΙꢀ400 MHz NMR

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spectrometer at 25°C, using tetramethylsilane (TMS) as the internal standard. Chemical shifts
were reported in ppm. HRꢀMS was obtained using a micromassꢀQTOF spectrometer using
electrospray ionization (ESI).
4.1.2. Synthesis of 2-(2-chloroethyl)dibenzo[cd,g]indazol-6(2H)-one (R1).
This compound is prepared according to the modified procedures.41–43 In 50 mL round
bottom flask, 1,9ꢀpyrazoloanthrone (220 mg, 1mmol) was dissolved in dimethylformamide
(DMF) (2 mL) and K2CO3 (138 mg, 1 mmol) was added and stirred for 30 min. To this
reaction mixture, 1ꢀbromoꢀ2ꢀchloroethane (142 mg, 1 mmol) was added slowly and stirred
overnight at 100oC. The formation of R1 was monitored by TLC. After completion of the
reaction, DMF was removed using high vacuum rotary evaporator. The reaction mixture was
dissolved in ethyl acetate and extracted with water. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. Further the
crude mixture was purified through silica gel column chromatography and recrystallized in
1
dichloromethane and methanol mixture gives light yellow solid. Yield: 76 %. H NMR (400
MHz, CDCl3):
δ 8.43 (dd, J = 8.0 Hz, 0.8 Hz, 1H), 8.18 (dd, J = 7.6 Hz, 0.8 Hz, 1H), 8.03 (d,
J = 6.8 Hz, 1H), 7.74ꢀ7.63 (m, 3H), 7.54 (dt, J = 8.0 Hz, 1.2 Hz, 1H), 4.81 (t, J = 6.4 Hz, 2H),
4.07 (t, J = 6.0 Hz, 2H). 13C NMR (100 MHz, CDCl3):
δ
183.5, 139.8, 133.3, 133.2, 131.4,
129.1, 128.6, 128.5, 126.3, 123.4, 122.7, 120.8, 114.8, 51.3, 42.9. HRMS: m/z calcd. for
C16H11ClN2O:calcd. 305.0458 [M+Na]+; found 305.0455
4.1.3. Synthesis of 7-chloro-2-(2-chloroethyl)dibenzo[cd,g]indazol-6(2H)-one (R2).
This compound was synthesized similar to R1, but 5ꢀchloroꢀ1,9ꢀpyrazoloanthrone was used.
The crude mixture was purified through silica gel column chromatography and recrystallized
in chloroform and methanol mixture gives brownish solid. Yield: 68 %. 1H NMR (400 MHz,
CDCl3):
δ
8.18 (dd, J =3.2 Hz, 2.8 Hz, 1H), 8.01 (d, J =6.8 Hz, 1H), 7.73 (d, J =8.0 Hz, 1H),
13
7.69ꢀ 7.65 (m, 1H), 7.56ꢀ 7.55 (m, 1H), 4.82 (t, J =6.4 Hz, 2H), 4.07 (t, J =6.0 Hz, 2H). C

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NMR (100 MHz, CDCl3):
δ 182.4, 139.7, 139.1, 137.2, 134.3, 132.9, 132.8, 129.1, 128.9,
126.9, 122.6, 121.8, 121.1, 114.6, 51.4, 42.9. HRꢀMS: m/z calcd. for C16H10Cl2N2O; calcd.
317.0248 [M+H]+; found 317.0248
4.1.4. Synthesis of 5-chloro-2-(2-chloroethyl) dibenzo[cd,g]indazol-6(2H)-one (R3).
This compound was synthesized as similar to R1, but 4ꢀchloroꢀ1,9ꢀpyrazoloanthrone was
used. The crude mixture was purified through silica gel column chromatography and
recrystallized in dichloromethane and methanol mixture gives light yellow solid. Yield: 62 %.
1H NMR (400 MHz, CDCl3):
δ 8.44 (dd, J =8.0 Hz, 0.8 Hz, 1H), 8.15 (dd, J =8.0 Hz, 0.9 Hz,
1H), 7.70 (td, J =8.0 Hz, 1.6 Hz, 1H), 7.69ꢀ7.48 (m, 3H), 4.79 (t, J =6.0 Hz, 2H), 4.07 (t, J
=6.0 Hz, 2H). 13C NMR (100 MHz, CDCl3):
δ
181.5, 139.2, 138.5, 133.3, 133.2, 131.6,
130.6, 129.3, 129.0, 128.8, 124.1, 122.4, 121.8, 115.3, 109.1, 51.5, 43.0. HRꢀMS: m/z calcd.
for C16H10Cl2N2O; calcd. 317.0248 [M+H]+; found 317.0248
4.2. Single crystal X-ray diffraction
Xꢀray diffraction data on suitable single crystals were collected on an Oxford Xcalibur Mova
E diffractometer equipped with an EOS CCD detector and a micro focus sealed tube using
Mo Kα radiation (λ = 0.71073 Å) at 100K using an Oxford Cobra open stream nonꢀliquid
nitrogen cooling device. Data collection and reduction were performed using CrysAlisPro
(version 1.171.36.32)44 and OLEX2 (version 1.2)45 was used to solve and refine the crystal
structures. All nonꢀhydrogen atoms were refined anisotropically and Hꢀatoms were located
geometrically and refined isotropically. The WinGX46 package was used for final refinement,
production of CIFs and crystallographic table and ORTEPꢀ347 for structure visualization and
making the molecular representations. Packing diagrams were generated by using
MERCURY.48

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4.3. In Silico studies
4.3.1. Ligand preparation, docking and analyses
The threeꢀdimensional (3D) structures of R1, R2, and R3 were modelled and minimised
using the PRODRG server49 since only one crystal structure was available. AutoDock
(version 4.2)50 was used for the ligandꢀprotein docking. The Lamarckian Genetic Algorithm
was used with a population of 200 dockings. The docking output was analysed using PLIP,50
Pymol and Ligplot.51 Hydrogen bonds were determined using the inꢀbuilt HBPLUS52 module
in Ligplot with hydrogen bonding parameters (DꢀA distance ≤3.35 Å, H...A ≤2.7 Å), while
halogen interactions were calculated using the Protein Structure Analysis Package (PSAP)53
with DꢀX distance ≤3.5 Å, where X is the halogen atom. Computational alanine scanning was
also performed on ligand bound structures obtained from docking and ABSꢀscan server54 was
used for that purpose.
4.3.2. Molecular dynamics
Molecular dynamics (MD) simulations were performed on JNK3 (PDBꢀid: 1PMV) in apo
form and with each of R1, R2, and R3 bound to it. Water molecules and hetero atoms were
removed from the PDBs, while the missing atoms and loops were modelled using SWISSꢀ
MODEL.55 SwissParam56 webꢀserver was used to generate parameter files for the small
molecules compatible with the CHARMM force field. MD simulation was run using the
GROMACS 4.5.3 package57 with CHARMM all atom force field58 and SPC/E water model.59
The protein was simulated in a dodecahedron box with a distance of 10Å between the box
and the protein. Clꢀ ions were added to neutralize unbalanced charges on the protein. Particle
Mesh Ewald (PME) method60 was used for treating the electrostatic interactions. The bond
lengths were constrained using the LINCS algorithm.61 The system was energy minimized
using the conjugate gradient algorithm with convergence criteria of 1 kJ mol−1 nm−1 to

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eradicate all short contacts. Thereafter, the system was coupled to a temperature bath using
the NoséꢀHoover thermostat.62,63 The pressure of the system was isotropically
(compressibility of 4.5 × 10−5 bar−1) coupled to a barostat at 1 bar using the Parrinelloꢀ
Rahman method.64 The position restrained dynamics was run for 100ps during both
temperature and pressure equilibration. Simulation was run with a time step of 2fs, while the
atomic coordinates and velocities were saved for every 2ps. Production simulation was run
for a period of 25ns.
4.4. Biological assays
4.4.1. Reagents and antibodies
General laboratory chemicals were obtained from SigmaꢀAldrich, Merck, HiMedia and
Promega. Tissue culture plasticware was purchased from Corning Inc. or Tarsons. LPS was
purchased from Sigma (USA). PhosphoꢀcꢀJun, PhosphoꢀERK1/2, and Phosphoꢀp38
antibodies were obtained from Cell Signaling Technology (USA).
4.4.2. Cells and mice
RAW 264.7 mouse macrophage cell line was obtained from the National Center for Cell
Sciences, Pune, India and was cultured in Dulbecco’s modified Eagle medium (DMEM)
(GibcoꢀLife Technologies) supplemented with 10% heatꢀinactivated FBS (GibcoꢀLife
Technologies), maintained at 37°C in 5% CO2 incubator. Primary macrophages were
obtained from peritoneal exudates of C57BL/6 WT and BALB/cJ mice. All strains of mice
were purchased from The Jackson Laboratory and maintained in the Central Animal Facility
(CAF), Indian Institute of Science (IISc).
4.4.3. MTT assay
10,000 cells were seeded to wells of 96 well microtiter plate. After 24 hours, when the
monolayer formed, the supernatant was flicked off and 100 µl of indicated supernatants were

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added to the cell in microtiter plate and kept for incubation at 37oC in 5% CO2 incubator for
12 hours. After 12 hours, supernatant was flicked off and 50 µl of MTT dye was added to
wells. The plates were gently shaken and incubated for 4 hours at 37oC in 5% CO2 incubator.
The supernatant was removed, 50 ꢁl of DMSO was added, and the plates were gently shaken
to solubilize the formed formazan. The absorbance was measured using a microplate reader at
a wavelength of 595nm and the percentage of cell survival was calculated.
4.4.4. Transfection studies and Luciferase assay
RAW 264.7 macrophages were transiently transfected with desired plasmids using low m.w.
polyehtylenimine (PEI). Transfection efficiency was more than 70% in all the experiments as
determined by counting the number of siGLO Lamin A/C positive cells in several
microscopic fields using fluorescent microscope. In all cases, 48h postꢀtransfection, the cells
were treated as indicated and processed for analysis. Treated cells were lysed in Reporter
Lysis Buffer (Promega) and assayed for luciferase activity using Luciferase Assay Reagent
(Promega) as per the manufacturer’s instructions. The results were normalized for
transfection efficiency measured by galactosidase activity. oꢀnitrophenol βꢀDꢀ
galactopyranoside (HiMedia) was utilized as substrate for the galactosidase assay.
4.4.5. RNA isolation and quantitative real-time RT-PCR
Macrophages were treated as indicated and total RNA from macrophages was isolated by
TRI reagent (SigmaꢀAldrich). 1.5 µg of total RNA was converted into cDNA using a First
Strand cDNA synthesis kit (Bioline). Quantitative realꢀtime RTꢀPCR was performed using
SYBR Green PCR mixture (KAPA Biosystems) for quantification of the target gene
expression. All the experiments were repeated at least 3 times independently to ensure the
reproducibility of the results. Gapdh was used as an internal control. The primers utilised for
quantitative realꢀtime RTꢀPCR amplification are listed (Table S1, ESI†).

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4.4.6. Immunoblotting
Treated cells were pelleted and lysed in RIPA buffer [50 mM TrisꢀHCl (pH 7.4), 1% NPꢀ40,
0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 ꢁg/ml each of
aprotinin, leupeptin, pepstatin, 1 mM Na3VO4, 1 mM NaF] on ice for 30 min. Whole cell
lysates were collected and an equal amount of protein from each cell lysate was subjected to
SDSꢀPAGE and transferred onto PVDF membranes (Millipore) by semiꢀdry Western blotting
method (BioꢀRad). Nonspecific binding was blocked with 5% nonꢀfat dry milk powder in
TBST [20 mM TrisꢀHCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20] for 60 min. The blots
were incubated overnight at 4ºC with primary antibody diluted in TBST with 5% BSA. After
washing with TBST, blots were incubated with antiꢀrabbit or antiꢀmouse IgG secondary
antibodies conjugated to HRP for 2h. The immunoblots were developed with enhanced
chemiluminescence detection system (PerkinElmer) as per manufacturer’s instructions and
βꢀACTIN was used as loading control. For probing another protein in the same region of
PVDF membrane, the blots were stripped in the stripping buffer [62.5 mM TrisꢀHCl (pH 6.8),
2% SDS and 0.7% βꢀmercaptoethanol] at 60oC on a shaker and followed by the same
procedure as mentioned above.
4.4.7. Statistical analysis
Levels of significance for comparison between samples were determined by the Student tꢀtest
distribution and oneꢀway ANOVA. The data in the graphs are expressed as the mean ± SE for
6 values from 3 independent experiments and P values < 0.05 were defined as significant.
GraphPad Prism 5.0 software (GraphPad Software) was used for all the statistical analysis.
†Electronic Supplementary Information (ESI)
NMR data, HRꢀMS, Computational alanine result, and Gene primers.

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Conflicts of interest
There are no conflicts to declare.
Acknowledgments
This work was supported by funds from the Department of Biotechnology (DBT),
Department of Science and Technology (DST), Council for Scientific and Industrial Research
(CSIR) and Indian Council of Medical Research (ICMR), Government of India and the Indoꢀ
French Center for Promotion of Advanced Research (CEFIPRA) (Reference number 4803ꢀ1).
We sincerely thank Dr. Gianluca Aguiari, Universita degli studi di Ferrara, Italy for 7XAP1
luciferase construct and Mr. Bharat Bhatt from KNB lab for useful discussions. RG and VS
thank CSIR for the Senior Research Fellowship. KNB and TNG thank DST for the J. C. Bose
Fellowship.
Abbreviations
LPS
Lipopolysaccharide
ERK
Extracellular signal regulated kinase
ADME/T
NMR
Absoption, Distribution, Metabolism, Excretion, and Toxicity
Nuclear Magnetic Resonance
ORTEP
MTT
Oak Ridge Thermal Ellipsoid Plot
3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀDiphenyltetrazolium Bromide
Reverse transcription polymerase chain reaction
RTꢀPCR
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Table of Contents and Graphical abstract
A specifically designed halogen substituted anthrapyrazolone derivatives for selective inhibition of JNKs
activity at lower concentrations with minimal off-target effects on other kinases such as ERK1/2 and p38.