Synthesis and in vitro evaluations of 6-(hetero)-aryl-imidazo[1,2-b]pyridazine-3-sulfonamide's as an inhibitor of TNF-α production

Article abstract of DOI:10.1016/j.bmcl.2017.11.026

Tumor necrosis factor-α is an important pro-inflammatory cytokine having a key role in hosts defensive process of immune systems and its over expression led to a diverse range of inflammatory diseases such as Rheumatoid arthritis, Cronh's disease, psoriasis, etc. This paper describes our medicinal chemistry efforts on imidazo[1,2-b]pyridazine scaffold: design, synthesis and biological evaluation. By the introducing sulfonamide functionality at 3 positions and substituting 6 positions with (hetero)-aryl groups’, a small library of compounds was prepared. All synthesized compounds were screened for lipopolysaccharide (LPS) mediated TNF-α production inhibitory activity. Biological data revealed that the majority of the compounds of this series showed moderate to potent TNF-α production inhibitory activity. Compound 5u and 5v are the most potent compounds from the series with activity of IC₅₀ = 0.5 μM and 0.3 μM respectively. A short SAR demonstrates that 3-sulfonyl-4-arylpiperidine-4-carbonitrile moiety on imidazo[1,2-b]pyridazine showed better activity compared to the 3-(4-aryllpiperazin-1-yl) sulfonyl) in hPBMC assay. The molecular modeling studies revealed that the potent TNF-α production inhibitory activity 5v due to the extra stability of complex because of an extra pi-pi (π-π) stacking, hydrogen-bonding interactions.

Full text of DOI:10.1016/j.bmcl.2017.11.026

Accepted Manuscript
Synthesis and in-vitro evaluations of 6-(hetero)-aryl-imidazo[1,2-b]pyridazine
-3- sulfonamide’s as an inhibitor of TNF-α production.
Shivaji.S. Pandit, Mahesh R. Kulkarni, Yashwant B. Pandit, Nitin P. Lad, Vijay
M. Khedkar
PII:
S0960-894X(17)31112-5
https://doi.org/10.1016/j.bmcl.2017.11.026
BMCL 25432
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
31 July 2017
1 November 2017
13 November 2017
Please cite this article as: Pandit, Shivaji.S., Kulkarni, M.R., Pandit, Y.B., Lad, N.P., Khedkar, V.M., Synthesis and
in-vitro evaluations of 6-(hetero)-aryl-imidazo[1,2-b]pyridazine -3- sulfonamide’s as an inhibitor of TNF-α
production., Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.11.026
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Synthesis and in-vitro evaluations of 6-(hetero)-aryl-imidazo[1,2-
b]pyridazine -3- sulfonamide’s as an inhibitor of TNF-α
production.
Shivaji. S. Pandita*, Mahesh R. Kulkarnia,b, Yashwant B. Pandita, Nitin P. Lada, Vijay M.
Khedkarc
aPost Graduate and Research centre, Department of Chemistry, Padmashri Vikhe Patil
College of Arts, Science and Commerce, Pravaranagar, A/P Loni, Tal. Rahata, Dist.
Ahmednagar - 413713. India.
bDepartment of Medicinal chemistry, Piramal Enterprises Limited 1, Nirlon Complex,
Off Western Exp. Highway, Near NSE Complex, Goregaon East, Mumbai, Maharashtra - 400
063. India.
cDepartment of Pharmaceutical Chemistry, Shri Vile Parle Kelavani Mandal's Institute of
Pharmacy, Dhule, Maharashtra 424001, India.
.
*Authors for correspondence
Dr. Shivaji. S. Pandit, M. Sc., Ph. D.,
Post Graduate and Research centre, Department of Chemistry, Padmashri Vikhe Patil College
of Arts, Science and Commerce, Pravaranagar, A/P Loni, Tal. Rahata, Dist. Ahmednagar –
413713. India.
E-mail: akankshapandit2002@yahoo.com
Tel.; +91-9766959195

Abstract
Tumor necrosis factor-α is an important pro-inflammatory cytokine having a key role in hosts
defensive process of immune systems and its over expression led to a diverse range of
inflammatory diseases such as Rheumatoid arthritis, Cronh’s disease, psoriasis, etc. This
paper describes our medicinal chemistry efforts on imidazo[1,2-b]pyridazine scaffold: design,
synthesis and biological evaluation. By the introducing sulfonamide functionality at 3
positions and substituting 6 positions with (hetero)-aryl groups’, a small library of
compounds was prepared. All synthesized compounds were screened for lipopolysaccharide
(LPS) mediated TNF-α production inhibitory activity. Biological data revealed that the
majority of the compounds of this series showed moderate to potent TNF-α production
inhibitory activity. Compound 5u and 5v are the most potent compounds from the series with
activity of IC50 = 0.5 µM and 0.3 µM respectively. A short SAR demonstrates that 3-
Sulfonyl-4-arylpiperidine-4-carbonitrile moiety on imidazo[1,2-b]pyridazine showed better
activity compared to the 3-(4-aryllpiperazin-1-yl) sulfonyl) in hPBMC assay. The molecular
modeling studies revealed that the potent TNF-α production inhibitory activity 5v due to the
extra stability of complex because of an extra pi-pi (π- π) stacking, hydrogen-bonding
interactions.
Keywords
Imidazo [1,2-b]pyridazine, Sulfonamide, Tumor Necrosis Factor-Alpha (TNF-α),
hPBMC (human peripheral blood mononuclear cells), RA (Rheumatoid arthritis).
Inflammation is self-protective process of the immune system to toxic stimuli, infection,
tissue injury, etc. The pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) is having
an important role in the body’s defensive process of immune systems.1,2 It was produced by
the body as a natural physiological response to infections, tissue injury.3-4 The excessive
production of TNF-α led to the development of several inflammatory diseases such as
rheumatoid arthritis (RA), psoriasis, inflammatory bowel disease, Crohn’s disease and
multiple sclerosis, etc.4-10 An effective anti TNF biological therapies were available in the
market, but the use of these biological agents was associated with several disadvantages.10-15
A number of research groups devoted their considerable efforts to find out orally active small
molecules as TNF-α production inhibitors with the same level of efficacy as biological agents
without undesirable side effect; as a result, during the last few years quite a lot of classes of
heterocyclic compound have been reported in literature as, TNF-α production inhibitor.10, 16-17

The structure of representative TNF-α inhibitor is illustrated in Figure1; compound (I)
SPD304 having 1-(3-(trifluoromethyl)phenyl)-1H-indole moiety linked to a 6,7-dimethyl-4H-
chromen-4-one by a dimethylethanamine spacer inhibits TNF-α production (IC50 = 22 µM),18
the compound C87 (II) having thiazole and pyrazolidin-3-one core inhibits TNF-α production
with IC50 = 9 µM19 while the compound BIHC (III) having chromene and imidazole rings
inhibits TNF-α production with IC50 = 16.5µM.20 The rolipram (IV) is the most potent TNF-α
production inhibitor ever reported with an IC50 = 0.2 µM.21
Figure 1. Molecular structure of representative TNF-α production inhibitor.
The imidazo[1,2-b]pyridazine have been identified as one of the promising scaffold in
medicinal chemistry. Which have been known to possess a wide range of biological
activities, including studied for treatment of inflammatory diseases such as, treatment of
rheumatoid arthritis, JAK inhibitors (VI)23, TNF-α inhibitors (V)22, 25-26 treatment of dermal
allergic inflammation24 treatment of inflammatory diseases caused by dysfunctions of
kinases,27 etc. The compound V (Figure 2), reported by H. Shimizu et.al inhibits TNF-α
production with IC50 = 1.7 µM.22
Figure 2. Molecular structure of representative developmental imidazo[1,2-
b]pyridazine for treatment of rheumatoid arthritis (RA).
Similarly sulfonamide is an important pharmacophore having attractive
pharmacological properties. It is present in many drug molecules from different therapeutic

category, such as antibacterial, diuretic, anti-viral, anti-inflammatory, anti-HIV, etc. More
than 112 marketed drug molecules such as celecoxib, meloxicam, piroxicam, sulfasalazine,
etc. have sulfonamide functionality.28 Shen et. al. discovered the anti-TNF small molecule by
the virtual screen (IX) also having sulfonamide motifs (Figure 3).29 Further, upon additional
SAR studies Shen and co-worker found out that most potent compound (X) contains two
sulfonamide functional groups. They have reported on their investigations, the sulfonamide
makes extra hydrogen bonds in the binding pocket of TNF with polar residues and the extra
hydrogen-bonds increase TNF-α inhibition ability of compounds.
Figure 3. Molecular structure marketed sulfonamide derivatives for the treatment of
inflammatory diseases and biologically active TNF-α inhibitor
In recent years we have been engaged in the design, synthesis of novel heterocyclic
compounds and evaluate their TNF-α production inhibitory potential.30 In persistent of our
work and motivated by aforementioned literature, we envisioned our approach toward design
and synthesis of 6-(hetero)-aryl-imidazo[1,2-b]pyridazine -3- sulfonamide’s for evaluation of
TNF-α production inhibitory potential. Therefore the single molecule which contains
pharmacologically active sulfonamide functionality and biologically important imidazo[1,2-
b]pyridazine core (Figure 4) could be more beneficial for inhibiting TNF- α production. We
are reporting for the first time, 6-(hetero)-aryl-imidazo[1,2-b]pyridazine-3-sulfonamide
derivatives as TNF-α production inhibitor.

Figure 4. Designed Modification on imidazo[1,2-b]pyridazine core
Scheme 1. Reagents and conditions: a) Chloroacetaldehyde dimethyl acetal, NaOAc, Conc.
HCl, 60% aqueous ethanol, 80 °C, 16 h, 87%; (b) i) ClSO3H, Ethylenedichloride, 80 - 85 °C,
3 h, ii) Cool to 50 °C, triethylamine, 10 min.; iii) POCl3, 100 - 105 °C , 16 h, 35%; (c)
Triethylamine, Ethylenedichloride, 25-30 °C, 1 - 3 h, 28 – 73%; (d) ArB(OH)2/ArB(pin),
K2CO3, Pd(PPh3)2Cl2, DMF-H2O, 120 °C, 3-5 h, 12 – 76%.
Syntheses of the designed derivatives of imidazo[1,2-b]pyridazine,(5) was achieved by the
reaction sequence as depicted in Scheme 1. The reaction of 3-amino-6-chloropyridazine (1)
with 2-chloro-1,1-dimethoxyethane in the presence of sodium acetate and concentrated
hydrochloric acid in 60% aqueous ethanol at reflux produced core 6-chloroimidazo[1,2-
b]pyridazine (2) as an off-white solid in 87% yield. The chlorosulfonation of the 2 to 6-
chloroimidazo[1,2-b]pyridazine-3-sulphonylchloride (3) was achieved by sequential one pot
reaction with chlorosulphonic and phosphorous oxychloride.31 The reaction of 6-
chloroimidazo[1,2-b]pyridazine-3-sulfonyl chloride (3) with suitable amine in the presence of
triethylamine in ethylenedichloride produced sulfonamides (4a-h) in 29 – 73% yield. Finally,
the Suzuki-Miyaura coupling of compound 4 with respective boronic acid or its pinacol ester
in the presence of catalytic amount of bis-(triphenylphosphine)palladium(II) dichloride,
potassium carbonate, and DMF-water provided desired inhibitors 5a-ag in (12 - 76%) yield.

The purity of compounds synthesized was confirmed by TLC and HPLC. All newly
synthesized compounds (Table 1, 4a-h and Table 2, 5a to 5ag) were in full agreement with
their spectral data 1H-NMR and MS.
Table 1. Inhibition of TNF-α production by imidazo[1,2-b]pyridazines
Sr. Compound
No. no.
X
TNF-α
Toxicity
IC50 in
µM
Docking
Score
Glide
inhibition
(IC50 in µM)a
28
energy
(kcal/mol)
-32.196
>30
-6.052
-5.895
1
2
4a
4b
30
>30
>30
-31.565
-26.117
>30
-5.495
3
4c
>30
>30
>30
-5.551
-26.473
-30.849
4
4d
30
-5.944
5
4e
30
25
>30
>30
-5.759
-6.099
-31.451
-37.758
6
7
4f
4g

>30
>30
-5.549
-28.137
8
4h
a
IC50: the concentration that affords 50% inhibition of TNF-α, values are mean of three
independent experiments. Assay conditions listed in the reference section, ref. no.34.
The TNF-α production inhibitory activity of the analogs of 4 was ascertained using
LPS treated hPBMC assay in comparison with standard rolipram and activities are
summarized in Table 1. The sulfonamide 4a, 4b, 4e, 4f, 4g showed moderate TNF-α
inhibitory activity ranging from 25 to 30 µM and other sulfonamides 4c, 4d, 4h were
inactive. The sulfonamide 4g with 4-phenylpiperidine-4-carbonitrile moiety is the most active
compound among the derivatives of 4. The un-substituted sulfonamide 4a showed TNF-α
production inhibition with IC50 = 28 µM. Table 1 depicts that, the designed sulfonamide
derivatives of 4 showed moderate activity for TNF-α production inhibition.
Table 2. Inhibition of TNF-α production by imidazo[1,2-b]pyridazines(5)
Sr.
No.
Compound
No.
X
Ar
TNF-α
Toxicity
IC50
Docking
in Score
Glide
energy
inhibition
(IC50 inµM)a µM
(kcal/mol)
-43.711
-34.981
-30.045
-38.719
-33.022
-33.185
-34.372
01
02
03
04
05
06
07
I
D
A
B
C
E
F
4
15
>30
9
20
20
15
>30
-7.082
5a
5b
5c
5d
5e
5f
K
K
K
K
K
K
>30
>30
>30
>30
>30
>30
-6.458
-5.740
-6.695
-6.355
-6.342
-6.413
H
5g

08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
N
N
N
L
L
L
L
L
L
M
M
M
M
M
O
O
O
O
O
O
J
A
B
H
A
B
C
D
E
H
A
B
C
H
G
A
B
C
D
E
18
>30
12
11
28
11
11
>30
12
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
-6.396
-5.597
-6.526
-6.652
-6.321
-6.621
-6.632
-5.487
-6.500
-6.853
-5.685
-6.346
-6.739
-7.430
-7.773
-6.953
-5.711
-5.325
-5.266
-6.334
-7.064
-7.063
-5.727
-7.045
-7.025
-5.411
-8.669
-33.992
-29.525
-35.293
-36.490
-32.144
-36.751
-36.365
-29.725
-34.7438
-40.894
-29.985
-33.606
-39.912
-46.240
-48.595
-41.034
-27.652
-25.439
-24.405
-32.384
-42.785
-42.741
-27.296
-41.604
-41.842
-25.265
-50.378
5h
5i)
5j
5k
5l
5m
5n
5o
5p
5q
5r
7
>30
20
5s
5t
5u
8
0.5
0.3
6
>30
>30
>30
28
4
4
>30
6
5v
5w
5x
5y
5z
H
A
B
C
E
5aa
5ab
5ac
5ad
5ae
5af
5ag
Rolipram
J
J
J
J
F
H
4
J
>30
0.5 µM
a
IC50: the concentration that affords 50% inhibition of TNF-α, values are mean of three
independent experiments. Assay conditions listed in the reference section, ref. no.34.
The in-vitro potencies of the 6-(hetero-)aryl-imidazo[1,2-b]pyridazine -3-
sulfonamide (Table - 2, entry 1 - 33) are illustrated in the table 2. To begin with, we planned
to synthesize the compound bearing simple sulfonamide group and synthesized compound 5a
by Suzuki-Muryia coupling of 4a with 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-
(trifluoromethyl)pyridin-2-amine. Compound 5a showed TNF-α production inhibitory
activity with IC50 = 4 µM with 7 fold improvement in activity over the parent 4a. This results
were inspiring to us and showed that designed modification have potential for TNF-α
production inhibition. Further, we explored SAR by synthesizing compounds by modification
of simple sulfonamides to substituted sulfonamides. For this purpose, we synthesized
compounds with substituted piperazine, substituted piperidine, 3-methoxybenzylamine and
(tetrahydrofuran-2-yl)methanamine. If we see the table 2, some interesting trends in SAR
were observed with the nature of substituents on sulfonamide and type of substituent at 6
positions of imidazo[1,2-b]pyridazines. Among the derivatives 5b – 5g having 4-

ethylpiperazine-1-sulfonamide: 5d having 5-(trifluoromethyl)pyridine group showed 3 fold
more activity (IC50 = 9 µM) over its chloro analog. Whereas upon changing substituent from
5-(trifluoromethyl)pyridine to less lipophilic 5-fluoropyridine (compound 5c) loss in activity
was observed. The rest of the analogs from this series (4-ethylpiperazine-1-sulfonamide)
bearing, pyridine (5b), 6-methoxypyridine (5e), 4-methoxyphenyl (5f) and 4-
methanesulfonylphenyl (5g) showed moderate activity (IC50 = 15 - 20 µM). Further, we
synthesized compound with a more lipophilic benzyl group on piperazine. If we compare the
activity of inhibitors bearing benzylpiperazine with 4-ethylpiperazine-1-sulfonamide
derivatives, the activity is almost same with little difference (Table-2, compound 5h - j). The
compound 5h, 5j having 3-pyridyl and 4-methanesulfonylphenyl substitutions showed
moderate activity for TNF-α inhibitions with IC50 = 18 and 12 µM respectively and the other
compounds from this series were inactive. From the series of compounds 5k-p with N-
pyridylpiperazine substitutions, 5m and 5n showed moderate activity with IC50 = 11 µM,
inhibitor 5p having 4-(methylsulfonyl)phenyl substitutions showed IC50 = 12 µM and the
compound 5l with 5-fluoropyride derivative showed TNF- α inhibition with IC50 =28 µM.
The compound 5o having 6-methoxy-3-pyridyl substitution is inactive from the series. Most
of the compounds from the piperazine series (5b - p) shows moderate activity. Further, we
were curious to see the effect of changing piperazine to piperidine and we synthesized the
compounds 5q – 5u bearing 4-phenylpiperidine-4-carbonitrile. The compound 5u with 3,4-
dimethoxyphenyl substitutions showed promising TNF-α production inhibitory activity (IC50
= 0.5 µM). The 5u is 50 fold more active over its chloro analog 4g. This indicates the
replacement of chloro with the lipophilic 3,4-dimethoxyphenyl is favorable for activity. The
5q, with 3-pyridyl substitutions showed good TNF-α activity IC50 = 7 µM and the compound
5s with 5-(trifluoromethyl)pyridine substitutions showed moderate activity (IC50 = 7 µM). All
the compounds of this series bearing 4-phenylpiperidine-4-carbonitrile showed very well to
moderate activity indicating piperidine substitutions are very well tolerated. Additionally, we
explored the SAR by synthesizing compounds with 3-methoxybenzylamine and
(tetrahydrofuran-2-yl)methanamine sulfonamide derivatives (Compound 5v - 5ag).
Compound 5v with 3-pyridyl substitutions showed most potent TNF-α production inhibitory
activity throughout the synthesized derivatives of 5 with IC50 = 0.3 µM. 5v showed 100 fold
improvement in activity over its chloro analog 4h. 5w showed moderate activity (IC50 = 6
µM) and the compound 5x, 5y, 5z were inactive. Among (tetrahydrofuran-2-yl)methanamine
sulfonamide derivatives 5ad, 5ag were inactive, 5ab, 5ac, 5ae and 5af showed good activity
(IC50 = 4 – 6 µM).

If we see the results illustrated in Table1 and Table 2, some interesting trends in SAR
are observed with the nature and type of substitutions on sulfonamide and 6th position of
imidazo[1,2-b]pyridazine. The replacement of chloro with (hetero)-aryl results in
improvement in activity. However, in some cases there is no change or little change in
activity is observed after replacement of chloro to corresponding (hetero)-aryl. Form the SAR
one can conclude that the (hetero)-aryl group as well as the substitutions on sulfonamide have
contributed towards the activity. 3-Sulfonyl-4-arylpiperidine-4-carbonitrile moiety on
imidazo[1,2-b]pyridazines showed better activity compared to the 3-(4-aryllpiperazin-1-
yl)sulfonyl) in hPBMC assay. Compound 5u with substituted piperidine sulfonamide and 3,4-
dimethoxyphenyl substitution at 6th position of imidazo[1,2-b]pyridazines inhibit production
of TNF- α with IC50 = 0.5 µM. Also the compound 5v having N-(3-methoxybenzyl)-
sulfonamide with pyridine substitutions 6-positions of imidazo[1,2-b]pyridazines showed
TNF-α production inhibition with IC50 = 0.3 µM. Compound 5u and 5v are the most potent
compounds among the series. The more lipophilic 3-trifluoromethyl group on pyridine
showed better activity rather than fluoro substituted pyridine. The 4-(methylsulfonyl)phenyl
is well tolerated as compared to 4-methoxyphenyl or 6-methoxypyridine only exception in
case of (tetrahydrofuran-2-yl)methanamine (inhibitors 5ae and 5af inhibits TNF-α production
with IC50 6 µM and 4 µM while the 5ag bearing 4-(methylsulfonyl)phenyl is inactive).
We were further interested in understanding the binding mode of these inhibitors with
binding pocket of TNF-α in complex. The molecular docking study was performed using
Glide (Grid-Based Ligand Docking with Energetics)32-33 module incorporated in the
Schrödinger molecular modeling software (Schrödinger, Inc., USA, 2016) to investigate the
binding modalities of sulfonamide analogues into the active site of toward possible targets
comprising of TNF-α . The crystal structure of the target TNF-α in complex with its inhibitor
(pdb code: 2az5) was retrieved from the Protein Data Bank (www.rcsb.org) and further
optimized and minimized to obtain a low energy and structural correct target protein using
the Protein Preparation Wizard. The protein structure was prepared for docking by adding
essential hydrogen atoms, eliminating the cocrystallized water molecules (since none of them
were found to be conserved in the ligand interaction) and assigning all atom force field
(OPSL-2005) charges and atom types. Finally, the prepared structure was subjected to energy
minimization until the average root mean square deviation of non-hydrogen atoms reached
0.3 Å in order to relieve the steric clashes among the residues due to addition of hydrogen
atoms. The 3D structures of the ligands were sketched under investigation were sketched
using the build panel in Maestro and refined using LigPrep module. This involved addition of

hydrogens, adjusting realistic bond lengths and bond angles, correction of chirality’s and
ionization states, generation of tautomer’s, and ring conformations and assignment of partial
charges to the structure using the OPLS-2005 force-field. Thereafter, each of them was
subjected to energy minimization setting a RMSD cutoff of 0.001Å. After ensuring that the
enzyme and ligands were in the correct form, target grid was generated using the position of
the native ligand as the reference co-ordinates to define the active site for docking. The
Receptor Grid Generation panel in Glide generates two cubical boxes having a common
centroid to organize the calculations: a larger enclosing and a smaller binding box. With the
non-covalently bound native ligand in place, the active site was defined by a box that has
dimensions of 14x14x14Å centered on the centroid of native ligand in the crystal complex
which was large enough to allow ligands to explore a large portion of the enzyme. All the
sulfonamide molecules under study were docked into the binding site of TNF-α using the
extra precision mode (XP) and Glide Score has been used to predict the binding affinity and
ranking of the ligands. The Glide algorithm adopts a funnel approach which involves
performing a systematic search of positions, orientations, and conformations of the ligand in
the receptor binding site. The search begins with a random positioning of ligand in the active
site and scoring phase that significantly limits the search space and reduces the number of
poses to be selected for minimization on the precomputed OPLS-2005 van der Waals and
electrostatic grids for the protein. The lowest-energy poses (5–10 ) obtained at this stage are
then subjected to Monte Carlo simulations in which the ligand-receptor complex is subjected
to a full molecular mechanics energy minimization (OPLS–AA force field) in order to
optimize flexible side-chain residues of TNF-α that may interact with the ligands. Finally the
energy minimized poses accepted are further rescored using the GlideScore function, which
is a more sophisticated version of ChemScore.32, 33 This force field includes additional terms
accounting for solvation and repulsive interactions. The output file in terms of the docking
poses of the ligands was visualized and quantitatively analyzed for the key elements of
interaction with the target enzyme using the Maestro’s Pose Viewer utility.
The molecular docking study revealed that all the sulfonamide analogues could optimally
fit into the active site of TNF-α with varying degree of affinities adopting a very similar
orientation and at co-ordinates close to that of the native ligand in the crystal structure. Their
complexation was stabilized through a network of significant steric and electrostatic
interactions. Their docking scores ranged from -5.266 to -7.773 while the minimum energy
for the formation of the complex between the TNF-α and each of these analogues (Glide
energy) was also observed to be negative, ranging from -24.405 kcal/mol to -48.595 kcal/mol

while the docking score and binding energy for the reference ligand-Rolipram (Figure 1S)
(found in ESI section) was found to be -8.669 and -50.378 kcal/mol respectively. These
findings are in agreement with the experimentally observed anti-inflammatory activity
wherein the active compounds showed higher docking scores while those with relatively low
inhibition were also predicted to have a lower score (Table 2). A higher negative value for
the docking score and the binding energy (Glide energy) signifies a good binding affinity of
the ligand towards the target and vice versa. The binding energy represents the energy
required for a ligand to cover the entire enzyme surface and its potential interactions with
residues lining the active site.
Figure 5. Binding mode of 5v into the active site of TNF-α (on right side: green lines signify
pi-pi stacking interactions while the pink lines represent the hydrogen bonding interactions)
A detailed quantitative analysis of the per-residue interaction between the residue
lining the active site of the TNF-α and these sulfonamide derivatives was carried out to
identify the most significantly interacting the residues and the type of thermodynamic
elements (bonded and non-bonded interactions) governing the binding of these molecules to
the target. However, in order to maintain the brevity of text, this analysis is elaborated herein
only for the most active compound 5v while the results for the remaining active molecules
(5a, 5d, 5q, 5u, 5w and 5ab) are summarized in Table 03 (found in ESI section) and their
binding modes are provided in the supplementary material as Figures (2S-7S) (found in ESI
section).
The lowest energy docked conformation of the most active compound 5v, having N-
(3-methoxybenzyl)-sulfonamide with pyridine substitutions at 6th positions of imidazo[1,2-
b]pyridazine, into the active site of TNF-α (Figure 5) showed that the inhibitor binds at the
same co-ordinates as the native ligand and the reference ligand- Rolipram (Figure 1S) with
an excellent binding affinity producing a docking score of -7.773 with a binding energy -
48.595 kcal/mol. This higher binding affinity is attributed to the specific bonded and non-

bonded per-residue interactions with the residues lining the active site of TNF-α. A detailed
analysis of the per-residual interactions revealed that the compound is stabilized within the
active site TNF-α through an extensive network of significant van der Waals interactions with
Tyr:A151(-3.735 kcal/mol), Tyr:A119(-7.651 kcal/mol), SerA60(-1.812 kcal/mol) through
the imidazo[1,2-b]pyridazine scaffold. Similarly the 3-methoxy benzylsulfonamide
component is also engaged in favorable van der Waals interactions with Gln:B61(-1.576
kcal/mol), Gln:A61(-2.886 kcal/mol), Tyr:A59(-6.049 kcal/mol) and His:A15(-2.091
kcal/mol) while pyridine ring substituted at the 6th position of the scaffold showed van der
Waals interactions with Tyr:B151(-1.922 kcal/mol), Leu:B120(-1.968 kcal/mol), Tyr:B119(-
3.917), Ser:B60(-1.996 kcal/mol), Tyr:B59(-2.852 kcal/mol), Gly:A121(-2.643 kcal/mol),
Leu:A120(-2.293 kcal/mol). The enhanced binding affinity of 5v can also be attributed to
favorable electrostatic contacts observed with Gln:A149(-3.867 kcal/mol), Gly:B148(-1.392
kcal/mol), Glu:A116(-4.865 kcal/mol), Ser:A60(-1.884 kcal/mol), Ile:A58(-1.146 kcal/mol)
through imidazo[1,2-b]pyridazine scaffold; with Tyr:A151(-5.506 kcal/mol), Val:A62(-
1.666 kcal/mol),
Gln:A61(-7.236 kcal/mol) via the 3-methoxy benzylsulfonamide
component and with Tyr:B151(-5.019 kcal/mol), Gly:B122(-1.564 kcal/mol), Leu:B120(-
2.145 kcal/mol), Glu:B116(-5.990 kcal/mol), Gln:B61(-3.531 kcal/mol), Ser:B60(-3.761
kcal/mol), Gly:A122(-1.453 kcal/mol), Gly:A121(-3.549 kcal/mol) residues through the
pyridine ring respectively. Three prominent pi-pi (π-π) stacking interaction was also observed
between the 3-methoxy benzylsulfonamide and Tyr:A59(2.812Å); between the imidazole
ring of imidazo[1,2-b]pyridazine scaffold and Tyr:A119 (2.816Å) and finally between the
pyridine ring and Tyr:B119 (2.669Å) residues lining the active site. Furthermore the
compound is also held in the active site through three crucial hydrogen bonding interactions:
with Tyr:B151 (2.54Å) and Gly:A121( (2.73Å) through the pyridine ring and with
Tyr:A151(1.99) through the amino component of sulfonamide moiety. These types of the pi-
pi (π- π) stacking and hydrogen-bonding interactions not only serve as an "anchor",
determining the 3D orientation of the ligand in the active site but also facilitate the steric and
electrostatic interactions adding to the stability of the enzyme-inhibitor complex. A similar
binding mode and set of interactions was observed for the 5a, 5d, 5q, 5u, 5w and 5ab as well
but decreasing gradually with their observed anti-inflammatory activity.
Overall, it is evident from the molecular docking simulations and specifically from
the per-residue interaction analysis that these sulfonamide derivatives have a promising
affinity for the TNF-α qualifying them as pertinent starting points for structure-based lead
optimization.

In summary, we have designed modifications on imidazo[1,2-b]pyridazines by the
introduction of the sulfonamide functionality and synthesized a small library of 6-(hetero)-
aryl-imidazo[1,2-b]pyridazines-3-sulfonamide series of compounds of biological interests.
Synthesized compounds were evaluated for TNF-α production inhibitory potential, results are
presented in Table 1 and 2. The detailed SAR was described; from the SAR it was concluded
that, the (hetero)-aryl group as well as the substitutions on sulfonamide have contributed
towards the activity and the designed modification has the potential for TNF-α production
inhibition, The compound 5u and 5v are the most potent compounds from the series with IC50
= 0.5 and 0.3µM respectively. Furthermore, the molecular docking study of the compounds
was carried out for the better understanding of the drug-receptor interactions at the active site
TNF-α. The docking studies of molecules shed some light on the binding modes, and
subsequently the binding potency of the SAR molecules on to active site of TNF-α
compared to the standard rolipram. The promising findings observed herein provides a
good starting point for an extensive medicinal chemistry program, which may lead to further
identification of more potent TNF-α production inhibitor.
Acknowledgement
The author MRK thanks the Medicinal Chemistry, Analytical Chemistry and Department of
Pharmacology of Piramal Enterprises Ltd. Goregaon, Mumbai for analytical data and
biological studies. Authors would like to thank Schrödinger Inc. and especially Craig Coel
for providing the Demo license of Schrödinger Molecular Modeling Suite that has
significantly helped in this study. Authors are also thankful to Management and Principal of
Padmashri Vikhe Patil College Pravaranagar, India. SSP thanks to UGC, New Delhi, INDIA
for financial assistance wide file no. 47-618/13 (WRO) dated 20/05/2014.
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34. a) hPBMC assay (to check the cytotoxic effect of the compounds on mononuclear
blood cells): Blood was collected from healthy donors. It was then diluted in the ratio
1:1 with sterile endotoxin free PBS (w/o calcium and magnesium). Blood was then
loaded onto histopaque (by Sigma Chemicals) in a 50 mL centrifuge (15 mL
histopaque and 30 mL of blood). These tubes were centrifuged at 1500 rpm for 40
min with acceleration and break at 1. With a pipette, the upper plasma layer is gently
removed and discarded leaving approximately 1 cm of buffy coat above the interface.
With a pipette, the interface layer containing the PBMCs was gently collected in
another 50 mL centrifuge tube and diluted with PBS in the ratio 1:1. The cells were
the spun at 1800 rpm for 10 min. The pellet obtained was broken and resuspended in
PBS, and centrifuged at 1000 rpm for 10 min. Above step was repeated once again,

this time cells were resuspended in Roswell Park Memorial Institute (RPMI) 1640
medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10%
fetal bovine serum (FBS). The cells were plated in 96-well tissue culture plates at a
concentration of 1 x 106 cells/ml. The viability, as determined by trypan blue dye
exclusion, was uniformly ≥98%. After plating, different concentrations e.g. (0.03, 0.1,
0.3, 1, 3, 10, 30, and 100 µM) of the compound dissolved in dimethylsulfoxide
o
(DMSO) were added to the cells and incubated at 37 C for 30 min in 5% CO2
atmosphere. The final concentration of DMSO was maintained at 0.5%. The vehicle
(0.5% DMSO) was used as control. Rolipram (300 µM) was used as a standard. The
cells were then stimulated with lipopolysaccharide (LPS, Escherichia coli serotype
0127:B8) at a final concentration of 1 µg/mL and the incubation was continued for 5
h. The culture plate was centrifuged (2500 rpm for 10 min) and the supernatants
collected and stored at -70 oC. The amount of TNF-α in the supernatants were assayed
using the ELISA protocol recommended by the manufacturers (OptiEIA ELISA sets,
BD BioSciences, Pharmingen). The percentage inhibition was calculated by using
pg/mL levels of Vehicle control by the formula. % inhibition= (X-Y)/ X *100, Where
X = Mean cytokine level of Vehicle control, Y = Mean cytokine level of Test.
b) Evaluation of cytotoxicity: The cytotoxicity of the test sample in hPBMC assay
was assessed by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfonyl)-2H-tetrazolium) staining. 40ul of MTS-PMA solution was added
to the cells and incubated for 5-6 hours till the absorbance reaches 0.5.
Absorbance was measured at 490 nm. Percent cytotoxicity was calculated by the
equation: % Cytotoxicity = (A-B)/A x100, Where X = Mean Absorbance of
Vehicle control, Y = Mean Absorbance of Test.
† Electronic Supplementary Information (ESI) available: Chemistry protocols and
experimental data, scan spectral, Quantitative per-residue interaction analysis and Molecular
docking images.

Synthesis and in-vitro evaluations of 6-(hetero)-aryl-imidazo[1,2-
b]pyridazine -3- sulfonamide’s as an inhibitor of TNF-α
production.
Shivaji. S. Pandita*, Mahesh R. Kulkarnia,b, Yashwant B. Pandita, Nitin P. Lada, Vijay M.
Khedkarc
aPost Graduate and Research centre, Department of Chemistry, Padmashri Vikhe Patil
College of Arts, Science and Commerce, Pravaranagar, A/P Loni, Tal. Rahata, Dist.
Ahmednagar - 413713. India.
bDepartment of Medicinal chemistry, Piramal Enterprises Limited 1, Nirlon Complex,
Off Western Exp. Highway, Near NSE Complex, Goregaon East, Mumbai, Maharashtra - 400
063. India.
cDepartment of Pharmaceutical Chemistry, Shri Vile Parle Kelavani Mandal's Institute of
Pharmacy, Dhule, Maharashtra 424001, India.
*Authors for correspondence
Dr. S. S. Pandit, M. Sc., Ph. D.,
Post Graduate and Research centre, Department of Chemistry, Padmashri Vikhe Patil College
of Arts, Science and Commerce, Pravaranagar, A/P Loni, Tal. Rahata, Dist. Ahmednagar –
413713. India.
E-mail: akankshapandit2002@yahoo.com
Tel.; +91-9766959195
Graphical Abstract

Unit from biologically active
TNF- inhibitor
Sulfonamide unit
from drug molecule/
TNF- inhibitor
N
N
N
N
N
N
N
O
O
S
N
S
O
O
N
Ar/Het
N
MeO
HN
O
N
S
O
OMe
HN
R
CN
MeO
Control of Liphophilic
hydrophilic properties
5u
TNF- : IC50 = 0.5
5v
TNF- : IC50 = 0.3
M
M