Synthesis, biological evaluation, and molecular modeling of 11H-indeno[1,2-b]quinoxalin-11-one derivatives and tryptanthrin-6-oxime as c-Jun N-terminal kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.10.023

c-Jun N-terminal kinases (JNKs) play a central role in many physiologic and pathologic processes. We synthesized novel 11H-indeno[1,2-b]quinoxalin-11-one oxime analogs and tryptanthrin-6-oxime (indolo(2,1-b)quinazoline-6,12-dion-6-oxime) and evaluated their effects on JNK activity. Several compounds exhibited sub-micromolar JNK binding affinity and were selective for JNK1/JNK3 versus JNK2. The most potent compounds were 10c (11H-indeno[1,2-b]quinoxalin-11-one O-(O-ethylcarboxymethyl) oxime) and tryptanthrin-6-oxime, which had dissociation constants (K_d) for JNK1 and JNK3 of 22 and 76 nM and 150 and 275 nM, respectively. Molecular modeling suggested a mode of binding interaction at the JNK catalytic site and that the selected oxime derivatives were potentially competitive JNK inhibitors. JNK binding activity of the compounds correlated with their ability to inhibit lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) activation in human monocytic THP-1Blue cells and interleukin-6 (IL-6) production by human MonoMac-6 cells. Thus, oximes with indenoquinoxaline and tryptanthrin nuclei can serve as specific small-molecule modulators for mechanistic studies of JNK, as well as potential leads for the development of anti-inflammatory drugs.

Full text of DOI:10.1016/j.ejmech.2018.10.023

Accepted Manuscript
Synthesis, biological evaluation, and molecular modeling of 11H-
indeno[1,2-b]quinoxalin-11-one derivatives and tryptanthrin-6-oxime as c-Jun N-
terminal kinase inhibitors
Igor A. Schepetkin, Andrei I. Khlebnikov, Andrei S. Potapov, Anastasia R. Kovrizhina,
Vladislava V. Matveevskaya, Maxim L. Belyanin, Dmitry N. Atochin, Svitlana O.
Zanoza, Nadiya M. Gaidarzhy, Sergiy A. Lyakhov, Liliya N. Kirpotina, Mark T. Quinn
PII:
S0223-5234(18)30885-7
10.1016/j.ejmech.2018.10.023
EJMECH 10809
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 June 2018
Revised Date: 20 August 2018
Accepted Date: 9 October 2018
Please cite this article as: I.A. Schepetkin, A.I. Khlebnikov, A.S. Potapov, A.R. Kovrizhina, V.V.
Matveevskaya, M.L. Belyanin, D.N. Atochin, S.O. Zanoza, N.M. Gaidarzhy, S.A. Lyakhov, L.N. Kirpotina,
M.T. Quinn, Synthesis, biological evaluation, and molecular modeling of 11H-indeno[1,2-b]quinoxalin-11-
one derivatives and tryptanthrin-6-oxime as c-Jun N-terminal kinase inhibitors, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.10.023.
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ACCEPTED MANUSCRIPT
Anti-inflammatory Activity
1000
750
500
250
0
B
Compound 10c
(R=CH2COOCH2CH3)
Tryptanthrin
Compound 9b
N
Molecular Modeling
Tryptanthrin-6-oxime
Tryptanthrin-6-oxime
N
10c
Trp-Ox
Compound 10c
N
OR
Kd (nM)
0
10
20
30
40
50
IQ-1 Analogs
Concentration ( M)
JNK1
JNK3
22
76
150
275

ACCEPTED MANUSCRIPT
Synthesis, Biological Evaluation, and Molecular Modeling of 11H-indeno[1,2-b]quinoxalin-11-
one Derivatives and Tryptanthrin-6-Oxime as c-Jun N-terminal Kinase Inhibitors
Igor A. Schepetkin1, Andrei I. Khlebnikov2,3, Andrei S. Potapov2, Anastasia R. Kovrizhina2,
Vladislava V. Matveevskaya2,4, Maxim L. Belyanin2, Dmitry N. Atochin2,5, Svitlana O. Zanoza6,
Nadiya M. Gaidarzhy6, Sergiy A. Lyakhov6, Liliya N. Kirpotina1, and Mark T. Quinn1,*
1Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717,
USA;
2Kizhner Research Center, Tomsk Polytechnic University, Tomsk 634050, Russia;
3Scientific Research Institute of Biological Medicine, Altai State University, Barnaul 656049,
Russia;
4Department of Chemistry, Siberian State Medical University, Tomsk 634050, Russia;
5Cardiovascular Research Center, Cardiology Division, Massachusetts General Hospital,
Charlestown, MA 02129, USA;
6A.V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, Odessa,
Ukraine
Running title: Indenoquinoxalines and Tryptanthrin-6-oxime as JNK Inhibitors
*Address for Correspondence:
Mark T Quinn, Ph.D.
Department of Microbiology and Immunology
Montana State University, Bozeman, MT 59717
Phone: 406-994-4707; Fax: 406-994-4303
Email: mquinn@montana.edu
1

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Abstract
c-Jun N-terminal kinases (JNKs) play a central role in many physiologic and pathologic
processes. We synthesized novel 11H-indeno[1,2-b]quinoxalin-11-one oxime analogs and
tryptanthrin-6-oxime (indolo(2,1-b)quinazoline-6,12-dion-6-oxime) and evaluated their effects
on JNK activity. Several compounds exhibited sub-micromolar JNK binding affinity and were
selective for JNK1/JNK3 versus JNK2. The most potent compounds were 10c (11H-indeno[1,2-
b]quinoxalin-11-one O-(O-ethylcarboxymethyl) oxime) and tryptanthrin-6-oxime, which had
dissociation constants (Kd) for JNK1 and JNK3 of 22 and 76 nM and 150 and 275 nM,
respectively. Molecular modeling suggested a mode of binding interaction at the JNK catalytic
site and that the selected oxime derivatives were potentially competitive JNK inhibitors. JNK
binding activity of the compounds correlated with their ability to inhibit lipopolysaccharide
(LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) activation in human
monocytic THP-1Blue cells and interleukin-6 (IL-6) production by human MonoMac-6 cells.
Thus, oximes with indenoquinoxaline and tryptanthrin nuclei can serve as specific small-
molecule modulators for mechanistic studies of JNK, as well as potential leads for the
development of anti-inflammatory drugs.
Keywords: c-Jun N-terminal kinase; tryptanthrin; 11H-indeno[1,2-b]quinoxalin-11-one; oxime;
tropomyosin‐related kinase; inflammation
2

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1. Introduction
c-Jun N-terminal kinases (JNKs) belong to the family of mitogen-activated protein
kinases (MAPK) that are activated in response to various stress stimuli, such as ultraviolet
radiation, oxidative stress, heat and osmotic shock, and ischemia-reperfusion injury of the brain
and heart [1-4]. In mammals, 10 highly similar isoforms are expressed by alternative splicing of
three different genes: JNK1 (four isoforms), JNK2 (four isoforms), and JNK3 (two isoforms) [5,
6]. JNK1 and JNK2 are found in all cells and tissues of the body, while JNK3 is expressed
mainly in the brain, heart, and testicles [2].
The JNKs have been shown to play an important role in regulation of the signaling
pathways involved in apoptosis, necrosis, inflammation, and ischemia/reperfusion injury [7-10].
They are involved in a wide range of diseases, including rheumatoid arthritis, osteoarthritis,
multiple sclerosis, inflammatory bowel disease, insulin resistance, tumorigenesis, stroke, renal
ischemia, Alzheimer's and Parkinson's diseases [6, 11-17]. Upstream kinases of the MAPK
cascade (MKK4 and MKK7) phosphorylate and activate JNK [18], whereas transcription factors
such as c-Jun, specificity protein 1 (Sp1), activating transcription factor 2 (ATF2), and nuclear
factors of activated T-cells (NFATc2 and NFATc3) are substrates for phosphorylation-activated
JNKs [5, 7, 9, 19]. There are also numerous non-nuclear substrates of JNK that participate in the
degradation of proteins, signal transduction, and regulation of apoptotic cell death [3, 20]. For
example, JNK1 phosphorylates insulin receptor substrate 1 (IRS-1), a key molecule in the
insulin-sensing pathway, which down-regulates insulin signaling [21]. Recently Tudor-SN, a
multifunctional protein that is implicated in a variety of cellular processes, was identified as a
novel JNK target [22].
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A significant amount of pharmacological and genetic evidence suggests that inhibition of
JNK signaling may represent a promising therapeutic strategy [23], and numerous efforts have
focused on the development of selective and nontoxic JNK inhibitors. For example, selective
JNK1/3 inhibitors may have clinical benefit in treating neurodegenerative disorders [24].
However, it has been difficult to design selective JNK inhibitors because of the high sequence
identity among JNK isoforms (from 73 to 75%) and, specifically, sequence identity of their ATP
binding pockets (close to 98%) [25]. Recently, JNK2/3 inhibitors with an aminopyrazole scaffold
that have >30-fold selectivity over JNK1 were identified [25].
Although no JNK inhibitors have been approved for use in humans, a few small molecule
JNK inhibitors have entered clinical trials for various indications, including tanzisertib for the
treatment of lupus erythematous and idiopathic pulmonary fibrosis, bentamapimod for the
treatment of inflammatory endometriosis, and D-JNKi1 for the treatment of inflammation and
stroke (for review [26-28]). Previously, we identified a new class of JNK inhibitors based on the
11H-indeno[1,2-b]quinoxalin-11-one scaffold [29]. Specifically, compound IQ-1 (11H-
indeno[1,2-b]quinoxalin-11-one oxime) and its oxime analogs (Fig. 1) inhibited JNK activity
and, consequently, proinflammatory cytokine production by murine and human leukocytes [29].
We also found that IQ-1 reduced inflammation and cartilage loss associated with collagen-
induced arthritis (CIA) [30] and protected against cerebral ischemia–reperfusion injury in mice
[31]. These JNK inhibitors contain oxime (IQ-1) or O-acyl-oxime (IQ-2 through IQ-4 and IQ-6)
groups (Fig. 1) and exhibited some (~3.6-5.3 fold) selectivity for JNK3 versus JNK1/2 [29, 30].
Thus, we propose that modification of IQ-1 by introduction of various substituents could
Figure 1. Chemical structure of
previously identified JNK1-3 inhibitors
with an 11H-indeno[1,2-b]quinoxalin-11-
one scaffold.
4

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increase potency and/or a selectivity of the resulting analogs toward the JNK isoforms.
In the present studies, novel 11H-indeno[1,2-b]quinoxalin-11-one oxime analogs, such as
aza-analogues, O-substituted derivatives, and analogs with different substituents in the
indenoquinoxaline tetracyclic moiety, were synthesized and evaluated against JNK1-3. We
conducted molecular modeling for selected compounds and estimated their anti-inflammatory
potential using in vitro cell-based assays. We also report for the first time that tryptanthrin-6-
oxime, a derivative of the natural alkaloid tryptanthrin, has a relatively high binding affinity for
JNK1-3 and tropomyosin‐related kinases (TRK) A and B.
5

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2. Results and discussion
2.1. Chemistry
All new compounds were synthesized as reported in Scheme 1, and the structures were
confirmed on the basis of analytical and spectral data. As reported previously, 11H-indeno[1,2-
b]quinoxalin-11-one (compound 1) was synthesized by the condensation of ninhydrin with o-
phenylenediamine [32, 33]. We synthesized oximes of known [34-36] and commercially
available ketones 2a-i as described in Scheme 1A.
To synthesize indenoquinoxaline analogues 3a, 3b, and 5 containing an additional
nitrogen atom in the tetracyclic nucleus, we used the reaction of 2,3-diaminopyridine, its 5-
chloro derivative, and 3,4-diaminopyridine, with ninhydrin in EtOH (Scheme 1B). It has been
established that the use of H2O as a solvent instead of EtOH does not significantly affect yields
of the products and selectivity of the process. The existence of two isomers is possible for each
of the resulting aza-analogues. We determined the isomer ratios from integral intensities of the
1
signals in H-NMR spectra of the products and found that 3,4-diaminopyridine reacts
regiospecifically, with the sole formation of 5 (73% yield). To provide a rationale for
regioselectivity of this reaction, we performed density functional theory (DFT) calculations (see
the description and Supplementary Fig. S1). Previously, compound 5 was synthesized with
comparable yield at higher temperature in boiling isobutyl alcohol [37]. From the reaction of 2,3-
diaminopyridine with ninhydrin, a mixture of isomers 3a and 4a (90:10%) was obtained, with a
total yield of 82%, from which we isolated pure compound 3a. It should be noted that in boiling
MeOH, an inseparable mixture of ketones 3a and 4a was obtained [36]. Reaction of 2,3-diamino-
6-methylpyridine leads to a mixture of compounds 3b and 4b (83:17%). From this mixture, we
isolated pure isomer 3b by recrystallization from dimethylformamide.
6

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о
Scheme 1. Reagents and conditions: (a) NH2OH·HSO4, NaOH in EtOH, 60 C, 8 h, 80-
о
о
93%; (b,d) in EtOH, 60 C, 10 h, 81%; (c,e) NH2OH·HCl in EtOH, 60 C, 10 h, 80%; (f)
о
o
NH2OH·HCl in pyridine, 60 C, 2 h, 95%; (g) in EtOH, 78 C, 9 h, 88-92%; (h) KOH in
DMSO, r.t., 1 h (10a, 54%; 10c, Method A, 84%), or Na2CO3 in DMSO, r.t., 9-10 h (10b,
52%; 10c, Method B, 56%; 10d, 83%).
7

ACCEPTED MANUSCRIPT
Oximes were synthesized via ketone precursors through a reaction with hydroxylamine.
Treatment of compounds 2a-i with hydroxylamine in hot EtOH in presence of NaOH led to the
11H-indeno[1,2-b]quinoxalin-11-one oximes (6a-i) (Scheme 1A). Likewise, oximes 7a, 7b, 8,
and tryptanthrin-6-oxime were synthesized from ketones 3a, 3b, 5, and tryptanthrin, respectively,
using hydroxylamine hydrochloride in EtOH or pyridine (Scheme 1B). For the synthesized
oximes, the ratio of the Z- and E-isomers was determined from the integral intensities of the
1
signals in H-NMR spectra. We found that compounds 6a-d, 6f-i, 8, and tryptanthrin-6-oxime
were formed as individual isomers, while a mixture of Z- and E-isomers (90:10) was obtained for
oxime 7a. The Z- and E-isomers of oximes 6e and 7a,b exist in dynamic equilibrium in solution
and could not be isolated as individual forms. We speculate that the Z-isomer is predominant for
the synthesized oximes, since it must be stabilized by an intramolecular H-bond between the OH
group and the nitrogen atom of the pyrazine ring.
A convenient synthetic route to the O-substituted derivatives was synthesis from the
corresponding ketone 1 by an oximation reaction with O-R-hydroxylamines (Scheme 1C). In the
present work, we carried out the oximation of compound 1 using O-methyl, O-ethyl, O-benzyl,
1
and O-allyl hydroxylamine hydrochlorides. According to the H-NMR spectra, products 9a-d
synthesized according to Scheme 1C were isomerically pure individual compounds.
We also investigated the reactivity of IQ-1 towards alkylating reagents. IQ-1 has low
solubility in most organic solvents, thus alkylation was evaluated in dimethylsulfoxide (DMSO);
solubility of IQ-1 in this solvent is about 0.01 M at room temperature. KOH was used as a base.
Being an aprotic solvent, DMSO easily solvates the potassium cations, while the OH-anions are
solvated slightly, which leads to an extremely high basicity of the medium and activates the
alkylation process in the DMSO-KOH system [38]. We also used Na2CO3 as a base. O-alkylation
8

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of IQ-1 in DMSO was carried out according to Scheme 1D at room temperature and vigorous
stirring in the presence of a two-fold molar excess of a base (threefold on the synthesis of the
carboxylic acid 10b).
Compound 10a in CDCl3 solution exists as a mixture of Z- and E-isomers with respect to
1
the exocyclic C=N bond, as two sets of side-chain proton signals are observed in the H-NMR
spectrum. The ratio of isomers is approximately 1:2, as determined from the integral intensities
in each pair of signals. According to DFT calculations [B3LYP/6-31+G(d,p)] of the 10a isomers,
the E-isomer is thermodynamically more stable. The effect of the solvent (chloroform) was taken
into account within the polarizable continuum model (PCM). We determined that for the 10a(Z)
1
10a(E) equilibrium,
∆
Go is equal to 5.23 kJ/mol. The H-NMR results for compound 10c
298
showed two sets of the side chain proton signals of the isomers with 1:3 integral intensity.
Similarly to 10a, indenoquinoxaline 10c in chloroform has a more stable E-isomer. Thus, for the
10c(Z) 10c(E) process, ∆
Go evaluated by DFT is 10.38 kJ/mol. Obviously, the mixture of
298
Z- and E-isomers is formed on synthesis under the reaction conditions.
It should be noted that the use of DMSO-Na2CO3 instead of DMSO-KOH (Scheme 1D)
led to a longer reaction time: complete alkylation of IQ-1 was attained in 9-10 hours. However,
on the alkylation by ethyl chloroacetate, isomerically pure 10c was formed with only traces of
the minor isomer present, in contrast to the method using the superbasic medium DMSO-KOH.
1
Comparison of the H-NMR spectra of the isomer mixture and the individual isomer 10c
synthesized in DMSO-KOH and DMSO-Na2CO3 systems, respectively, showed that in the latter
case, the product consisted of the isomer that was predominant when DMSO-KOH medium was
used. According to results of our DFT calculations presented above, this isomer has an E-
configuration, and this product was used for further biological evaluation.
9

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2.2. Structure–activity relationship (SAR) analysis for JNK1-3 binding affinity
All compounds were evaluated for their ability to bind to the three JNK isoforms in
comparison with IQ-1, and the results presented in Table 1 demonstrated that the 11H-
indeno[1,2-b]quinoxalin-11-one nucleus is an appropriate scaffold for JNK inhibitor
development. Indenoquinoxalines 6e, 7a, and 10c exhibited Kd values in the nanomolar range for
all three JNKs, with the most potent being 10c, which had even lower Kd values for JNK1 and
JNK3 compared to IQ-1 [29]. Moreover, 10c had much higher specificity toward JNK1 and
JNK3 (Kd values of 22 nM and 76 nM, respectively) versus JNK2 (Kd = 735 nM). To further
evaluate the relative potency of 10c, we compared its binding affinity with that of a
commercially available JNK inhibitor, SP600125. As shown in Table 1, the Kd of 10c toward
JNK1 was even lower to that of SP600125.
As reported previously [29], we found that the side chain oxime R substituent was critical
for JNK binding and biological activities. The observation that oxime derivatives 9a-d and 10a,
which have hydrocarbon side chains, were inactive in the competition binding assay suggests
that the R oxime substituent is involved in H-bond donor/acceptor interactions with JNK. These
interactions occur possibly due to the presence of additional oxygen atoms in the carboxyl, ester,
or OH groups of molecules 10b-d, which can be anchored in the binding site in a favorable
conformation, as shown below for 10c. Although oxime groups may contribute important
interactions in the JNK binding site, the tetracyclic nucleus seems to be responsible for proper
ligand positioning. Indeed, substitution of an aromatic carbon atom at position 6 with nitrogen
led to less active compound 8 with all JNK isoforms. On the other hand, substitution of a carbon
atom at position 8 with nitrogen (7a) or introduction of a CH3 group as the R2 substituent (6f)
had little effect on binding affinity with all three JNK isoforms. Other modifications of the
10

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tetracyclic nucleus, including introduction of CH3 at R3 (6a), OCH2CH5 or NO2 at R2 (6f and 6g,
respectively),
11

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Table 1. Chemical structures of synthesized oxime derivatives, their binding affinity, and effect
on LPS-induced NF-κB/AP-1 transcriptional activity and interleukin-6 production.
O
R
3
R
N
N
N
N
N
R
R
N
N
2
N
N
N
1
N
OH
N
R
OH
N
OR
R = H (7a), Cl (7b)
R = O (Trp),
R= NOH (Trp-Ox)
NF-κB/
8
IQ-1, 6a-j, 9a-d, 10a-d
JNK1
JNK2
JNK3
IL-6
Compd
R
R1
R2
R3
AP-1
Kd (µM)
0.084 ± 0.023 0.022 ± 0.009
IC50 (µM)
SP600125a
IQ-1
6a
0.10 ± 0.043
0.24 ± 0.11
3.3 ± 0.9
N.A.
2.7 ± 0.4
2.1 ± 0.3
0.58 ± 0.035
2.3 ± 0.57
6.0 ± 0.21
N.A.
1.1 ± 0.2
0.24 ± 0.057
5.3 ± 1.3
0.95 ± 0.071
N.A.
3.4 ± 0.5
2.3 ± 0.4
3.0 ± 0.9
N.A.
5.2 ± 1.2
3.8 ± 0.8
0.5 ± 0.2
N.A.
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
CH3
H
CH3
OC2H5
NO2
CF3
H
H
CH3
Cl
H
H
H
H
H
H
H
0.36 ± 0.054
2.5 ± 0.7
N.A.
2.5 ± 0.6
6.0 ± 0.2
0.84 ± 0.17
2.0 ± 0.14
12.0 ± 1.41
N.A.
21.5 ± 7.8
0.36 ± 0.092
6.4 ± 0.1
1.4 ± 0.14
N.A.
0.10 ± 0.036
6.0 ± 1.1
N.A.
1.3 ± 0.2
3.9 ± 0.6
0.34 ± 0.014
0.91 ± 0.13
4.3 ± 0.28
N.A.
0.71 ± 0.11
0.39 ± 0.014
2.9 ± 0.1
0.28 ± 0.042
N.A.
6b
6c
6d
6e
6f
6g
6h
6i
7a
7b
8
9a
9b
9c
9d
CH3
COOH
2.9 ± 0.8
0.6 ± 0.2
24.6 ± 5.7 11.1 ± 2.3
H
H
H
H
Cl
3.9 ± 1.2
5.9 ± 1.7
2.7 ± 0.7
N.A.
0.9 ± 0.3
6.7 ± 2.2
3.0 ± 1.2
N.A.
5.5 ± 1.6
4.3 ± 1.1
3.3 ± 1.1
5.4 ± 0.4
N.A.
1.2 ± 0.4
3.5 ± 1.3
6.7 ± 2.2
2.1 ± 0.8
N.A.
CH3
CH2CH3
CH2Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
CH2CH=CH2
i-Bu
CH2COOH
CH2COOCH2CH3
(CH2)2OH
N.A.
N.A.
10a
10b
10c
10d
Trp
Trp-Ox
N.A.
N.A.
23.5 ± 12.0
0.022 ± 0.006
1.15 ± 0.071
23.0 ± 1.4
0.15 ± 0.081
16.5 ± 0.7
0.735 ± 0.19
1.25 ± 0.071
N.A.
28.5 ± 0.7
0.076 ± 0.006
0.31 ± 0.042
N.A.
N.A.
N.A.
2.5 ± 0.8
4.5 ± 1.8
N.A.
3.3 ± 0.7
5.9 ± 1.8
N.A.
1.0 ± 0.14
0.275 ± 0.21
3.8 ± 1.1
3.2 ± 1.2
aJNK binding data for known JNK inhibitors SP600125 (anthra[1–9-cd]pyrazol-6(2H)-one) and
IQ-1 are taken from [29]. N.A., no inhibition at concentrations <40
µM; N.T., nontoxic at
concentrations <40 M. Trp, tryptanthrin; Trp-Ox, tryptanthrin-6-oxime. For biological
µ
experiments, oximes 6b and 6h were used in the form of sodium salts.
COOH at R1 (6d), and two CH3 groups at R1 and R2 (6d), led to compounds with relatively low
JNK binding affinity. Furthermore, 6h containing a CF3 group at R2 and 6b with two Cl atoms at
R1/R3 were completely inactive. The most interesting modification of the tetracyclic nucleus was
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the introduction of Cl at position R1 (6i), as it led to an increase in relative specificity toward
JNK1/JNK3 versus JNK2.
The natural alkaloid tryptanthrin has an indolo(2,1-b)quinazoline-6,12-dion nucleus,
which is analogous to the 11H-indeno[1,2-b]quinoxalin-11-one scaffold. Indeed, charge
distributions in IQ-1 and tryptanthrin-6-oxime molecules are very similar (Fig. 2), although the
latter has a very polar carbonyl group, which results in lower hydrophobicity (LogP values are
4.04 and 2.92 for IQ-1 and tryptanthrin oxime, respectively). Thus, we also evaluated JNK
binding activity of this IQ-1 analog. Although tryptanthrin was inactive for JNK2/JNK3 and had
a very low binding affinity for JNK1 (Kd ~23.0 µM), tryptanthrin-6-oxime exhibited high
binding affinity for JNK1 and JNK3 (Table 1).
Figure 2. Electrostatic potential
maps for IQ-1 (11H-indeno[1,2-
b] quinoxalin-11- one oxime)
(left) and tryptanthrin-6-oxime
(right). Different colors
correspond to the potential
values between -0.020 (red) and
+0.002 (blue).
2.3. Kinase inhibition profile of tryptanthrin-6-oxime
Since tryptanthrin-6-oxime demonstratedd high affinity for JNKs, we evaluated its
specificity for various other kinases to evaluate its specificity compared to IQ-1. Specifically, it
was profiled in a competition binding assay for its ability to compete with an active-site directed
ligand for 97 different kinases (KINOMEscan, Eurofins Pharma Discovery, San Diego),
representing all known kinase families. The panel included 10 kinases that were reported
previously to be targets of SP600125 with similar or greater potency than the JNKs [39].
Tryptanthrin-6-oxime was screened at 10 µM, and the kinases for which >90% inhibition of
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ligand binding and kinase activity was observed were designated as “kinase targets of the
compound.” Five such kinase targets were identified, including casein kinase 1 σ (CK1σ, gene
symbol CSNK1D), tropomyosin‐related kinase A (TRK-A, gene symbol NTRK1), JNK1, JNK2,
and JNK3 (Fig. 3). Thus, similar to IQ-1 [30], tryptanthrin-6-
oxime had high specificity for
Figure 3. Kinase profile of
tryptanthrin-6-oxime.
Shown is the percentage
inhibition of human JNK
inhibition of binding to an
active-site directed ligand
for each of the indicated
kinases after treatment with
10 µM tryptanthrin-6-
oxime.
isoforms. Note however, that
IQ-1 and IQ-3 (both potent JNK
inhibitors
with
an
indenoquinoxaline scaffold) did
not bind TRK-A [29, 30]. Because TRKA-C are important
targets for treatment of several tumors [40-42], the parent
tryptanthrin and tryptanthrin-6-oxime were evaluated for their
binding affinities (Kd) to these 3 kinases. We found that
tryptanthrin-6-oxime had higher affinity toward TRKA-C in
comparison with the parent alkaloid (Table 2).
Table 2. Binding affinity of tryptanthrin and tryptanthrin-6-
oxime toward TRKA-C isoforms.
Compound
Kd (µM)
TRKA
9.2 ± 1.1 7.2 ± 2.6
TRKB
TRKC
N.A.
Tryptanthrin
Tryptanthrin-6-oxime 1.1 ± 0.2 3.8 ± 0.2 13.0 ± 2.1
Activity of TRK-family proteins (TRKA-C) is associated with poor survival in many
types of cancer [43]. For example, TRK-A, a high affinity receptor for nerve growth factor
(NGF) has been associated with the development of epithelial ovarian cancer [44]. Brain-derived
14

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neurotrophic factor (BDNF) is a potent neurotrophic factor that has been shown to stimulate
breast cancer cell growth and metastasis via TRK-A and TRK-B [45]. Several compounds,
including crizotinib and entrectinib, have been shown to inhibit the growth of tumor cells that
express TRK-family fusion proteins and have demonstrated remarkable clinical response in
patients with TRK-A fusion-positive tumors [46-48]. Sharma et al. [49] reported that some
oxime derivatives of tryptanthrin exhibited anticancer activity in vitro against a panel of human
cancer cell lines, but mechanisms of this activity are still non-identified. To our knowledge, this
is first report demonstrating co-activity of a kinase inhibitor toward TRK and JNK isoforms.
Using a selectivity score S(10), based on >90% inhibition of ligand binding at a single 10 µM
screen concentration [50], we found that the S(10) for tryptanthrin-6-oxime was much lower
(0.015 = 5/99) compared with the S(10) for SP600125 (0.328 = 39/119) [51], indicating much
higher target kinase selectivity for tryptanthrin-6-oxime.
2.4. Molecular modelling
To further characterize our most active analogs, we performed docking studies of 10c and
tryptanthrin-oxime into the binding sites of the three JNK isoforms. Since tryptanthrin was
inactive, we were also able to directly compare binding of the inactive parent and active oxime
derivative. According to our modelling, tryptanthrin formed a weak H-bond with Asn114 on
binding with JNK1. At the same time, the highest partial interaction energy of this molecule was
observed with Met111, which was due to van der Waals forces. The docking pose of
tryptanthrin-6-oxime (Fig. 4) was characterized by strong H-bonding between the oxygen atom
of the amide group and Met111. It should be noted that Met111 is considered as an important
residue for small molecule interactions with JNK [52, 53]. The calculated docking score for
15

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tryptanthrin-6-oxime was about 15 kcal/mol more negative than for tryptanthrin, which may
explain the higher binding affinity of the oxime derivative. Docking studies of these compounds
to JNK2 showed that the parent alkaloid did not form H-bonds with any of the residues of this
kinase, retaining in the binding center only by non-valent interactions. The highest attraction
with a score of 14 kcal/mol was obtained for His149. In contrast, tryptanthrin-6-oxime was H-
bonded with JNK2 through its oxime group with Gly171 (Fig. 4). Docking scores for
tryptanthrin and its oxime derivative differed by 16 kcal/mol in favor of the oxime. According to
the docking results obtained for JNK3, the low-energy pose for tryptanthrin formed a weak H-
bond through its amide oxygen with Asn152 and was fixed in the binding site mainly by van der
Waals interactions. On the other hand, tryptanthrin-6-oxime was anchored in the kinase cavity
through H-bonding of the oxime group with Asp207 (Fig. 4). The docking score of the oxime in
JNK3 was ~ 30 kcal/mol more negative than that of tryptanthrin. Thus, it can be assumed that, at
least for JNK2 and JNK3, the introduction of an oxime moiety into the molecule of tryptanthrin
caused the formation of a new H-bond with the kinase through participation of this moiety.
Docking of the highly active compound 10c in JNK1 gave a pose similar to tryptanthrin-
6-oxime, meaning that the molecule formed a strong H-bond with Met111 via the ester group of
the ligand (Fig. 4). It is important that such an arrangement of the ester group is achieved for the
Z-isomer of 10c. We also performed docking of the E-isomer, but another pose with a markedly
worse docking score was obtained in this case. In its unbound form, the E-isomer of 10c is more
stable; however, our DFT calculations show that the Z-isomer in solution is only slightly higher
in energy than the E-isomer of the substituted oxime 10c. Obviously, when interacting with the
kinase, 10c adopts the Z-configuration, which binds more effectively to the JNK1 active site, and
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in general, a gain in energy is achieved. When docking compound 10c in the JNK2 binding site,
a more energy-efficient pose was obtained for the E-isomer (by 20.4 kcal/mol better according to
Figure 4. Docking poses of tryptanthrin (left), tryptanthrin-6-oxime (middle), and 10c (right)
in JNK1 (PDB code 1UKI), JNK2 (PDB code 3NPC), and JNK3 (PDB code 1PMV). Strong
H-bonds are shown as darker dashed lines and indicated by red arrows. Weak H-bonds are
shown as light dashed lines and indicated by blue arrows. Residues within 4 Å from each pose
17
are shown.

ACCEPTED MANUSCRIPT
the docking score) than for the Z-isomer. Compound 10c forms two strong H-bonds with Lys55
and Leu168 of JNK2 with participation of two nitrogen atoms in the heterocycle. It should be
noted that the oxygen of the oxime group is located near one of these nitrogen atoms and forms
an H-bond with Gly171 located in the vicinity of Lys55 for the pose of tryptanthrin-6-oxime
(Fig. 4). We determined that 10c binds JNK3 in the form of the Z-isomer (by 60.2 kcal/mol
lower in docking score than the corresponding E-isomer), forming two H-bonds to Lys93 with
participation of the oxime and ethoxy oxygen atoms. In the pose of tryptanthrin-6-oxime, the
oxygen atom, although located in the same region of space, forms an H-bond with Asp207 (Fig.
4). Consequently, there is a similarity in the location of the most active compounds (10c and
tryptanthrin-6-oxime) by their location within the binding sites of the three JNK isoforms. Note
that these molecules occupy the same region of space where co-crystallized ligand SP600125 is
located. As shown in Supplementary Fig. 2S, the tetracyclic moieties of all three compounds
are approximately parallel within a narrow binding site of JNK3.
We also performed docking studies of tryptanthrin and tryptanthrin-6-oxime into the
TRK-A binding site. The major difference between their docking poses was the presence of H-
bonding between the oxime moiety of tryptanthrin-6-oxime and the kinase (Fig. 5). Specifically,
the oxygen atom of the oxime group is strongly H-bonded with Asp596 and Arg599.
Additionally,
a
weaker
H-bonding
Figure 5. Docking poses of tryptanthrin
(left) and tryptanthrin-6-oxime (right) in
the binding site of TRK-A (PDB code
4AOJ). Strong H-bonds are shown as
darker dashed lines and indicated by red
arrows. Weak H-bonds are shown as
light dashed lines and indicated by blue
arrows. Residues within 4 Å from each
pose are shown.
18

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interaction is possible between a nitrogen atom in the tetracyclic alkaloid derivative and Arg599.
In contrast, tryptanthrin interacted with the kinase via van der Waals forces only, although a
strong attraction of the ligand to Asp596 exists according to our calculations. The dissimilarity in
docking modes of tryptanthrin and tryptanthrin-6-oxime is likely responsible for the difference in
their binding affinities to TRPA-C (Table 2).
2.5. Evaluation of compound biological activity
All compounds were evaluated for their ability to inhibit LPS-induced NF-κB/AP-1
reporter activity and interleukin (IL)-6 production in human monocytic THP-1Blue and
MonoMac-6 cells, respectively. As shown in Table 1, the 13 oxime compounds inhibited LPS-
induced NF-κB/AP-1 activity and IL-6 production. As examples, the dose-dependent inhibitory
effects of 10c and tryptanthrin-6-oxime on NF-κB/AP-1 activity and IL-6 production are shown
in Fig. 6. As expected, these compounds also inhibited c-Jun phosphorylation in treated cells
Figure 6. Effect of selected compounds on NF-κB/AP-
1 activation and IL-6 production. Panel A. THP-1Blue
cells were pretreated with the indicated compounds or
DMSO for 30 min, followed by addition of 250 ng/ml
LPS or buffer for 24 h. NF-κB/AP-1 activation was
monitored by measuring secreted alkaline phosphatase
A
1.4
Tryptanthrin
1.2
1.0
Compound 9b
0.8
0.6
activity spectrophotometrically in the cell supernatants
0.4
Tryptanthrin-6-oxime
(absorbance at 655 nm). Panel B. MonoMac-6 cells
0.2
were pretreated with the indicated compounds or
0.0
Compound 10c
10 20
DMSO for 30 min, followed by addition of 250 ng/ml
LPS or buffer for 24 h. Production of IL-6 in the
supernatants was evaluated by ELISA. The data are
presented as the mean ± S.D. of triplicate samples from
one experiment that is representative of three
0
30
40
50
1000
750
500
250
0
B
Tryptanthrin
(examples shown in Fig. 7). Although compound 10c
Compound 9b
had higher JNK1/3 binding affinity than tryptanthrin-6-
Tryptanthrin-6-oxime
Compound 10c
19
0
10
20
30
40
50
Concentration ( M)

ACCEPTED MANUSCRIPT
oxime, they both inhibited c-Jun phosphorylation, NF-κB/AP-1 activity, and IL-6 production
over a similar concentration range (Figs. 6 and 7). This may be due to the higher cell
permeability of tryptanthrin-6-oxime. It is also possible that JNK2 may play a greater role in
these cellular responses and is targeted differently by the two inhibitors. Note that the oxime
Figure 7. Pharmacological inhibition of c-Jun (Ser73)
phosphorylation by selected compounds. Human
MonoMac-6 monocytic cells were pretreated with
indicated concentrations of 10c and tryptanthrin-6-oxime
for 30 min, followed by treatment with LPS (250 ng/ml)
or vehicle for another 30 min. The cells were lysed, and
the lysates were analyzed by Western blotting. Total JNK
(non-phosphoryalted) was used as loading control for the
lysates. A representative blot from two independent
experiments is shown (Panel A). The blots were analyzed
by densitometry, and the ratio of phospho-c-Jun/total c-
Jun is shown in Panel B.
derivatives (6a, 6c, 6e, 6f, 6i, 7a, 7b, 8, 10c, and tryptanthrin-6-oxime) all had IC50 values close
to that of SP600125 for inhibition of LPS-induced NF-κB/AP-1 activity and IL-6 secretion in
biological assays (Table 1). Consistent with the JNK binding assay, 6b, 6h, 9a-d, and 10a-b did
not inhibit NF-κB/AP-1 activity or IL-6 production (Table 1; examples are shown in Fig. 6),
supporting the specificity of our assays. In contrast to the active oximes, ketone derivatives (2a-
2j, 3a, 5) (data not shown), as well as tryptanthrin (Fig. 6), did not inhibit LPS-induced NF-
κB/AP-1 activity or IL-6 production, even at concentrations up to 50 µM.
To verify that the results were not influenced by possible toxicity, cytotoxicity of the
compounds was evaluated at concentrations up to 50 µM in MonoMac-6 and THP-1Blue cells
during a 24-h incubation with the compounds. None of the compounds affected cell viability,
even at the highest tested concentrations, thereby verifying that these compounds were not
cytotoxic during the 24-h incubation period of our assays (data not shown).
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It should be noted that many aryl oxime derivatives, including IQ-1, release nitric oxide
(NO) during their oxidoreductive bioconversion to ketones [31, 54, 55]. Thus, biological
activities of NO and these ketone precursors, including trypthantrin, should also be considered in
biological experiments. For example, although compound 1 (ketone corresponding to IQ-1) has
not been shown to be a DNA intercalator [56], the ketone precursors of compounds 9a, 6i,
and 7a have cytotoxicity against some cancer cell lines, probably because of their topoisomerase
I inhibitory activity [36]. Tryptanthrin is a natural alkaloid found in Polygonum tinctorium and
Isatis tinctoria [57, 58] and has been reported to have various pharmacological effects, such as
anti-inflammatory [59-61], antimicrobial [62], and anti-tumor activity [63, 64]. Tryptanthrin has
also been reported to suppress NO and prostaglandin E synthesis in macrophages exposed to
oxidative stress [65] and inhibit enzymatic activity of 5-lipoxygenase, cyclooxygenase-2, and
indoleamine 2,3-dioxygenase [66-68]. Previously, several tryptanthrin derivatives with different
substituents have been reported, including compounds with antiplasmodium and antitoxoplasma
activities, inhibitors of indoleamine 2,3-dioxygenase, and DNA triplex stabilizing agents [68-
73].
We synthesized novel 11H-indeno[1,2-b]quinoxalin-11-one oxime analogs and
tryptanthrin-6-oxime (indolo(2,1-b)quinazoline-6,12-dion-6-oxime) and evaluated their effects
on JNK activity. Several compounds exhibited sub-micromolar JNK binding affinity and were
selective for JNK1/JNK3 versus JNK2. The most potent compounds were 10c (11H-indeno[1,2-
b]quinoxalin-11-one O-(O-ethylcarboxymethyl) oxime) and tryptanthrin-6-oxime, which had
dissociation constants (Kd) for JNK1 and JNK3 of 22 and 76 nM and 150 and 275 nM,
respectively. Molecular modeling suggested a mode of binding interaction at the JNK catalytic
site and that the selected oxime derivatives were potentially competitive JNK inhibitors. JNK
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binding activity of the compounds correlated with their ability to inhibit lipopolysaccharide
(LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) activation in human
monocytic THP-1Blue cells and interleukin-6 (IL-6) production by human MonoMac-6 cells.
Thus, oximes with indenoquinoxaline and tryptanthrin nuclei can serve as specific small-
molecule modulators for mechanistic studies of JNK, as well as potential leads for the
development of anti-inflammatory drugs.
3. Conclusion
Synthesis and analysis of novel 11H-indeno[1,2-b]quinoxalin-11-one oxime analogs and
tryptanthrin-6-oxime demonstrated that several of these compounds had high affinity for JNK
and were selective for JNK1/JNK3 versus JNK2. These analogs also inhibited LPS-induced
nuclear NF-κB/AP-1 activation and IL-6 production in human monocytic cells. Our molecular
modeling showed that oxygen atoms of the oxime or ester groups participated in the formation of
strong H-bonds with residues in the JNK1/JNK3 binding sites. Thus, it is reasonable to suggest
that further modification of the O-substituent in the oxime moiety could lead to more specific
inhibitors with higher JNK selectivity. Our results also suggest that pan-JNK inhibition may be
suitable for suppression of the production of proinflammatory cytokines by human monocytic
cells. Finally, the identified oximes represent new chemical tools that may be useful in further
development of JNK and/or TRK inhibitors and could find application in the treatment of
inflammatory diseases, neurodegenerative pathologies, and cancer.
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4. Experimental section
4.1. Chemistry
4.1.1. Reagents and general procedures
Indenoquinoxaline ketones 2b, 2d, 2f, and 2g were purchased from Vitas-M Laboratory
(Moscow, Russia); 2e and 2h were from Maybridge (Cornwall, United Kingdom); and 2i was
from Specs (Delft, The Netherlands). Tryptanthrin was purchased from Combi-Blocks (San
Diego, CA). All other starting reagents were purchased from Sigma Aldrich. The chemicals were
analytical grade and used without further purification. Compounds 1 (11H-indeno[1,2-
b]quinoxalin-11-one) and IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) were synthesized,
as described previously [32]. Ketone 2a was synthesized according to [34, 35], and compound 2c
was synthesized according to [36]. Reaction progress was monitored by thin-layer
chromatography (TLC) with UV detection using pre-coated silica gel F254 plates (Merck) or a
Silufol UV-254. The synthesized structures were confirmed on the basis of analytical and
spectral data. The melting points (m.p.) were determined using an electrothermal Mel-Temp
capillary melting point apparatus. Elemental analysis was performed with a Carlo Erba
instrument. GC-MS analysis was performed on an Agilent 7890A GC combined with an Agilent
5975C mass detector (Agilent Technologies, USA); carrier gas was helium. LC-MS analysis was
performed on an Agilent 1260 Infinity combined with an Agilent 6530 Accurate Mass Q-TOF
detector. Compounds dissolved in 3-nitrobenzyl alcohol were subjected to fast atom
bombardment (FAB) ionization using a 10 kV argon beam, and the mass spectra were recorded
with a VG 70-70 EQ spectrometer. IR spectra were recorded on a FT-IR spectrometer Nicolet
1
5700 with KBr pellets. H NMR spectra were recorded on Bruker 400 or 600 MHz
23

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spectrometers. For atom numbering detaails, see Supplementary Fig. 3S. Representative NMR
spectra for compounds 8 and 10c are provided in supplementary material.
4.1.2. 6-Methyl-11H-indeno[2,3-b]quinoxalin-11-one oxime (6a) and general procedure for the
synthesis of 6b
-i
. A mixture of 2a (2.17 g, 9.45 mmol), hydroxylamine hydrosulfate (3.06 g, 23.6
o
mmol), and NaOH (1.0 g, 25 mmol) in EtOH (100 mL) was heated for 8 h at 60 C. After
cooling, the mixture was poured into water (600 mL), the precipitate was filtered out, dried, and
1
recrystallized from EtOH. Yield 2.26 g (92%). M.p. 303-304°. H NMR (600 MHz, DMSO-d6),
δ, ppm: 2.85 (s, 3H, CH3), 7.38-7.64 (m, 4H, H-2, H-3, H-7, H-8), 7.95 (d, 1H, 3J = 8 Hz, H-9),
3
3
8.20 (d, 1H, J = 7.6 Hz, H-4), 8.85 (d, 1H, J = 7.6 Hz, H-1), 13.31 (s, 1H, OH). M.w. 261.29.
C16H11N3O. LC-MS – m/z (I, %): 262.04377 (100) [MH]+; 244.03701 (83) [MH – H2O]+. FAB-
MS – m/z (I, %): 262 (100) [MH]+. A similar procedure was used for synthesis of the following
11H-indeno[2,3-b]quinoxaline-11-one oximes. To isolate sodium oximates of 6b and 6h, a 2-
fold excess of NaOH was added (with respect to hydroxylamine salt) after completion of the
reaction.
4.1.3. 6,8-Dichloro-11H-indeno[2,3-b]quinoxalin-11-one oxime (6b). Yield of sodium salt 93%.
M.p. 344о. 1H NMR (600 MHz, DMSO-d6), δ, ppm: 7.55-7.70 (m, 2H, H-2, H-3), 7.77 (s, 1H, H-
3
3
7), 7.92 (s, 1H, H-9), 8.2 (d, 1H, J = 8 Hz, H-4), 8.55 (d, 1H, J = 7.5 Hz, H-1). M.w. 338.13.
C15H6Cl2N3NaO. LC-MS – m/z (I, %): 316.91 (100) [MH]+; 298.84 (30) [MH – H2O]+.
4.1.4. 7,8-Dimethyl-11H-indeno[2,3-b]quinoxalin-11-one oxime (6c).Yield 89%. M.p. 323-324°.
1H NMR (600 MHz, DMSO-d6), δ, ppm: 2.46 (s, 3H, CH3), 2.50 (s, 3H, CH3), 7.66-7.73 (m, 2H,
24

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3
H-2, H-3), 7.90 (s, 1H, H-7 or H-9), 7.91 (s, 1H, H-7 or H-9), 8.14 (d, J = 8 Hz, H-4), 8.53 (d,
3
1H, J = 8 Hz, H-1), 13.26 (s, 1H, OH). M.w. 275.31. C17H13N2O. LC-MS – m/z (I, %):
276.0828 (100) [MH]+. FAB-MS – m/z (I, %): 276 (100) [MH]+; 258 (40) [MH – H2O]+.
4.1.5. 8-Carboxy-11H-indeno[2,3-b]quinoxalin-11-one oxime (6d). Yield 90%. M.p. 323-324°.
1H NMR (600 MHz, CDCl3), δ, ppm: 7.68-7.76 (m, 2H, H-2, H-3), 8.03 (d, 1H, 3J = 7 Hz, H-6),
8.20 (d, 1H, 3J = 8 Hz, H-4), 8.32 (d, 1H, 3J = 7 Hz, H-7), 8.58 (s, 1H, H-9), 8.60 (d, 1H, 3J = 7
Hz, H-1), 13.65 (s, 1H, =N-OH). M.w. 291.27. C16H9N3O3. LC-MS – m/z (I, %): 292.03225
(100) [MH]+; 274.02414 (50) [MH – H2O]+.
4.1.6. 7-Methyl-11H-indeno[2,3-b]quinoxaline-11-one oxime (6e). Yield 80%. M.p. 297-298°.
1H NMR (600 MHz, DMSO-d6), δ, ppm: 2.58 (s, CH3), 7.65-7.74 (m, 3H, H-2, H-3, H-8), 7.94
(s, 1H, H-6), 8.03 (d, 1H, 3J = 7.6 Hz, H-9), 8.17 (d, 1H, 3J = 7 Hz, H-4), 8.55 (1H, 3J = 7 Hz, H-
1), 13.30 (s, OH). M.w. 261.29. C16H11N3O. LC-MS – m/z (I, %): 262.27436 (100) [MH]+. FAB-
MS – m/z (I, %): 262 (100) [MH]+.
4.1.7. 7-Ethoxy-11H-indeno[2,3-b]quinoxalin-11-one oxime (6f). Yield 87%. M.p. 303°-304°. 1H
NMR (600 MHz, DMSO-d6), δ, ppm: 1.44 (t, 3H, 3J = 5 Hz, CH3), 4.26 (q, 2H, 3J = 5 Hz, CH2),
7.30-7.59 (m, 3H, H-2, H-3, H-8), 8.00 (d, 1H, 3J = 7.5 Hz, H-9), 8.06 (s, 1H, H-6), 8.15 (d, 1H,
3J = 7 Hz, H-4), 8.78 (d, 1H, 3J = 7 Hz, H-1), 13,17 (s, 1H, OH). M.w. 291.31. C17H13N3O2. LC-
MS – m/z (I, %): 292.07027 (100) [MH]+; 274.06221 (41) [MH – H2O]+. FAB-MS – m/z (I, %):
292 (100) [MH]+.
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1
4.1.8. 8-Nitro-11H-indeno[2,3-b]quinoxalin-11-one oxime (6g). Yield 84%. M.p. > 360°. H
NMR (600 MHz, DMSO-d6), δ, ppm: 7.42-7.67 (m, 2H, H-2, H-3), 8.18 (d, 1H, 3J = 7 Hz, H-4),
8.31 (d, 1H, 3J = 7 Hz, H-6), 8.87 (d, 3J = 8 Hz, H-1), 8.90 (d, 3J = 7 Hz, H-7), 8.99 (s, 1H, H-9),
13.47 (s, 1H, OH). M.w. 292.26. C15H8N4O3. LC-MS – m/z (I, %): 293.54 (100) [MH]+; 275.50
(41) [MH – H2O]+.
4.1.9. 8-Trifluoromethyl-11H-indeno[2,3-b]quinoxalin-11-one oxime (6h). Yield of sodium salt
81%. M.p. 303о. 1H NMR (600 MHz, DMSO-d6), δ, ppm: 7.60-7.75 (m, 2H, H-2, H-3), 8.02 (d,
1H, 3J = 7 Hz, H-6), 8.26 (d, 1H, 3J = 8 Hz, H-4), 8.34 (d, 1H, 3J = 7 Hz, H-7), 8.48 (s, 1H, H-9),
3
8.74 (d, 1H, J = 8 Hz, H-1). M.w. 337.24. C16H7F3N3NaO. LC-MS – m/z (I, %): 316.04372
(100) [MH]+.
4.1.10. 7-Chloro-11H-indeno[2,3-b]quinoxalin-11-one oxime (6i). Yield 89%. M.p. 323-324°.
1H NMR (600 MHz, DMSO-d6), δ, ppm: 7.47-7.64 (m, 2H, H-2, H-3), 7.76 (d, 1H, 3J = 8 Hz, H-
8), 8.13 (d, 1H, 3J = 8 Hz, H-9), 8.21 (d, 1H, 3J = 7 Hz, H-4), 8.85 (d, 1H, 3J = 7 Hz, H-1), 13.64
(s, 1H, OH). M.w. 281.70. C15H8ClN3O. LC-MS – m/z (I, %): 282.14929 (100) [MH]+.
4.1.11. 6H-Indeno[1,2-b]pyrido[3,2-e]pyrazin-6-one (3a). A mixture of 2,2-dihydroxyindane-
1,3-dione (ninhydrin, 0.39 g, 2.2 mmol) and 2,3-diaminopyridine (0.22 g, 2.0 mmol) in EtOH
(50 mL) was heated for 10 h (TLC monitoring) at 60 °C. The mixture was then cooled, and the
resulting precipitate (mixture of isomers 3a and 4a, 90:10 %) was filtered and recrystallized from
1
EtOH to give 3a (0.50 g, 81% yield) as a yellow solid. M.p. 266-268°С. H NMR (500 MHz,
CDCl3), δ, ppm: δ 7.68 (td, 1H, 3J = 7.5 Hz, 4J = 1 Hz, H-8), 7.73 (dd, 1H, 3J = 8 Hz, 4J = 4.5 Hz,
26

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3
4
3
H-3), 7.83 (td, 1H, J =7.5 Hz, J = 1 Hz, H-9), 7.97 (d, 1H, J = 7.5 Hz, H-7), 8.25 (d, 1H, J =
7.5 Hz, H-10), 8.60 (dd, 1H, 3J = 8 Hz, 4J = 2 Hz, H-4), 9.17 (dd, 1H, 3J = 4.5 Hz, 4J = 1.5 Hz, H-
13
2). NMR C (125 MHz, CDCl3), δ, ppm: 123.8 (C-10), 125.1 (C-3), 125.6 (C-7), 133.6 (C-8),
137.4 (C-6a), 137.4 (C-9), 138.3 (C-4a), 140.4 (C-4), 141.2 (C-10a), 150.6 (C-5a), 152.0 (C-
11a), 155.6 (C-2), 160.0 (C-10b), 188.8 (C-6). IR bands, cm-1: 1720 (C=O), 1612, 1602, 1502,
1153, 787. Found, %: C 72.24, H 3.18, N 18.30. C14H7N3O. Calculated, %: C 72.10, H 3.03, N
18.02.
4.1.12. 3-Chloro-6H-indeno[1,2-b]pyrido[3,2-e]pyrazin-6-one
(3b
).
Compound 3b was
synthesized, as described under 4.1.1.12 from ninhydrin and 3,4-diamino-5-chloropyridine.
Yield 58 %, M.p. 293-295. 1H NMR (400 MHz, CDCl3), δ, ppm: δ 7.72 (t, 1H, 3J = 7.2 Hz, H-8),
3
3
3
7.87 (t, 1H, J = 7.2 Hz, H-9), 8.00 (d, 1H, J = 7.6 Hz, H-7), 8.26 (d, 1H, J = 7.6 Hz, H-10),
8.55 (s, 1H, 4J = 2.4 Hz, H-4), 9.08 (s, 1H, 4J = 2.4 Hz, H-2). IR bands, cm-1: 1725 (C=O), 1610,
1596, 1481, 1134, 803, 752. Found, %: C 62.93, H 2.04, N 15.85. C14H6ClN3O. Calculated, %: C
62.82, H 2.26, N 15.70.
4.1.13. 10H-Indeno[1,2-b]pyrido[3,4-e]pyrazin-10-one (5). Compound 5 was synthesized, as
described under 4.1.1.12 from ninhydrin and 3,4-diaminopyridine. Yield 72%, M.p. 270-272°С.
1H NMR (600 MHz, CDCl3), δ, ppm: δ 7.72 (t, 1H, 3J = 7.8 Hz, H-8), 7.86 (t, 1H, 3J = 7.8 Hz, H-
3
3
3
7), 7.99 (d, 1H, J = 5 Hz, H-4), 8.00 (d, 1H, J = 7.2 Hz, H-9), 8.19 (d, 1H, J = 7.8 Hz, H-6),
13
8.89 (d, 1H, 3J = 5 Hz, H-3), 9.62 (s, 1H, H-1). NMR C (150 MHz, CDCl3), δ, ppm: 122.4 (C-
4), 123.6 (C-6), 125.3 (C-9), 134.0 (C-8), 137.4 (C-7), 137.7 (C-11a), 137.8 (C-9a), 140.7 (C-
5b), 146.5 (C-4a), 149.8 (C-3), 151.2 (C-10a), 155.8 (C-1), 160.4 (C-5a), 188.5 (C-10). IR bands,
27

ACCEPTED MANUSCRIPT
cm-1: 1728 (C=O), 1559, 1572, 1430, 1123, 744. Found, %: C 72.37, H 2.89, N 18.24.
C14H7N3O. Calculated, %: C 72.10, H 3.03, N 18.02.
4.1.14. 6H-Indeno[1,2-b]pyrido[3,2-e]pyrazin-6-one oxime (7a). A mixture of 3a (0.41 g, 1.74
mmol) and hydroxylamine hydrochloride (0.30 g, 4.33 mmol) in EtOH (50 mL) was heated for
10 h (TLC monitoring) at 60 °C. The mixture was then cooled and poured into H2O (500 mL).
The resulting precipitate was filtered, washed with water, and recrystallized from EtOH to give
7a (0.35 g, 80% yield) as a colorless solid. M.p. 291-293°С. 1H NMR (400 MHz, pyridine-d5), δ,
3
4
3
4
ppm: 7.65 (td, 1H, J = 7.6 Hz, J = 1.2 Hz, H-8), 7.71 (td, 1H, J = 7.6 Hz, J = 1.2 Hz, H-3),
3
3
8.06 (d, 1H, J = 5.6 Hz, H-9), 8.35 (d, 1H, J = 7.2 Hz, H-7), 8.92 (d, 1H, J = 5.6 Hz, H-10),
8.96 (d, 1H, 3J = 7.6 Hz, H-4), 9.78 (1H, H-2). NMR 13C (100 MHz, pyridine-d5), δ, ppm: 122.6,
123.5, 129.6, 132.1, 133.7, 135.4, 138.0, 145.9, 147.9, 148.2, 153.6, 155.9, 157.5. IR bands, cm-
1: 1631 (C=N), 1604, 1575, 1470, 1381 (O–H), 1096, 905 (N–O), 776. Found, %: C 67.47, H
3.02, N 22.69. C14H8N4O. Calculated, %: C 67.74, H 3.25, N 22.57.
4.1.15. 3-Chloro-6H-indeno[1,2-b]pyrido[3,2-e]pyrazin-6-one oxime (7b). Compound 7b was
obtained from 3b and hydroxylamine hydrochloride, as described under 4.1.1.15. Yield 56%,
M.p. 296-298°C. 1H NMR (400 MHz, DMSO-d6), δ, ppm: 7.77 (m, 2H, H-9, H-8), 8.25 (dd, 1H,
3J = 8 Hz, 4J = 4 Hz, H-10), 8.57 (dd, 1H, 3J = 8 Hz, 4J = 4 Hz, H-9), 8.77 (s, 1H, 4J = 2.8 Hz, H-
4
4), 9.13 (s, 1H, J = 2.4 Hz, H-2), 13.58 (s, 1H, OH). IR bands, cm-1: 1639 (C=N), 1559, 1572,
1475, 1371 (O–H), 1091, 951 (N–O), 784, 739 (C–Cl). Found, %: C 59.18, H 2.47, N 19.93.
C14H7ClN4O. Calculated, %: C 62.82, H 2.26, N 15.70.
28