Structure activity relationship (SAR) study identifies a quinoxaline urea analog that modulates IKKβ phosphorylation for pancreatic cancer therapy

Article abstract of DOI:10.1016/j.ejmech.2021.113579

Genetic models validated Inhibitor of nuclear factor (NF) kappa B kinase beta (IKKβ) as a therapeutic target for KRAS mutation associated pancreatic cancer. Phosphorylation of the activation loop serine residues (S¹⁷⁷, S¹⁸¹) in IKKβ

Full text of DOI:10.1016/j.ejmech.2021.113579

European Journal of Medicinal Chemistry 222 (2021) 113579
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Structure activity relationship (SAR) study identifies a quinoxaline
urea analog that modulates IKK
cancer therapy
b
phosphorylation for pancreatic
,
,
,
, 1
Satish Sagar a 1, Sarbjit Singh a 1, Jayapal Reddy Mallareddy a 1, Yogesh A. Sonawane a
,
John V. Napoleon a, Sandeep Rana a, Jacob I. Contreras a, Christabelle Rajesh a,
Edward L. Ezell a, Smitha Kizhake a, Jered C. Garrison c, Prakash Radhakrishnan a
,
, b, d, e, *
Amarnath Natarajan a
, c, d, e, *
a Eppley Institute for Cancer Research, Omaha, NE, USA
b Department of Biochemistry and Molecular Biology, Omaha, NE, USA
c Department of Pharmaceutical Sciences, Omaha, NE, USA
d Department of Genetics Cell Biology and Anatomy, Omaha, NE, USA
e Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Genetic models validated Inhibitor of nuclear factor (NF) kappa B kinase beta (IKK
target for KRAS mutation associated pancreatic cancer. Phosphorylation of the activation loop serine
residues (S177, S181) in IKK
is a key event that drives tumor necrosis factor (TNF) induced NF-
mediated gene expression. Here we conducted structure activity relationship (SAR) study to improve
potency and oral bioavailability of a quinoxaline analog 13e197 that was previously reported as a NF
inhibitor for pancreatic cancer therapy. The SAR led to the identification of a novel quinoxaline urea
analog 84 that reduced the levels of p-IKK in dose- and time-dependent studies. When compared to 13
e197, analog 84 was ~2.5-fold more potent in TNF -induced NF B inhibition and ~4-fold more potent in
b) as a therapeutic
Received 5 April 2021
Received in revised form
19 May 2021
Accepted 22 May 2021
Available online 30 May 2021
b
a
kB
k
B
b
Keywords:
Quinoxaline urea
a
k
inhibiting pancreatic cancer cell growth. Analog 84 exhibited ~4.3-fold greater exposure (AUC0-∞)
resulting in ~5.7-fold increase in oral bioavailability (%F) when compared to 13e197. Importantly, oral
IKK
NF
b
kB
administration of 84 by itself and in combination of gemcitabine reduced p-IKKb levels and inhibited
Pancreatic cancer
pancreatic tumor growth in a xenograft model.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
the antibacterial agent Quinacillin [5], macrocyclic anti-infectives
Voxilaprevir, Grazoprevir and Glecaprevir that target hepatitis C
virus NS3/4A protease [6,7], and the anticancer agent Erdafitinib
that inhibits fibroblast growth factor receptor [8,9]. Although rare
the quinoxaline core is also found in macrocyclic DNA-binding
quinomycin antibiotics, echinomycin and triostin A [10,11]. The
Nitrogen containing heterocycle quinoxaline derivatives are
associated with varied applications [1], including being part of
several approved drugs, such as the smoking cessation agent Var-
enicline [2], Brimonidine that exhibits antiglaucoma activity [3,4],
differential
p electron density determined by molecular orbital
calculations among the three sets of (5,8 > 6,7 > 2,3) carbon atoms
on the quinoxaline core, allows extensive derivatization of the core.
Quinoxaline analogs have also been explored as antiangiogenic
agents, DNA targeting agents, topoisomerase II inhibitors and ki-
nase inhibitors [12]. Our high throughput screening campaigns
identified a quinoxaline analog as a hit [13,14]. Follow-up iterative
synthesis and screening led us to a quinoxaline analog (13e197), an
* Corresponding author. Eppley Institute for Cancer Research, University of
Nebraska Medical Center, 986805 Nebraska Medical Center, BCC 5.12.390, Omaha,
NE 68198, USA.
* Corresponding author. Eppley Institute for Cancer Research, University of
Nebraska Medical Center, 986805 Nebraska Medical Center, BCC 8.12.321, Omaha,
NE 68198, USA.
E-mail addresses: pradhakr@unmc.edu (P. Radhakrishnan), anatarajan@unmc.
edu (A. Natarajan).
orally bioavailable IKKb inhibitor that exhibited anti-cancer activity
1
Equal contribution.
https://doi.org/10.1016/j.ejmech.2021.113579
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
in multiple cancer models [15e21].
was synthesized by condensation of 2,3-di(furan-2-yl)quinoxalin-
IKK
activated canonical NF
inflammatory diseases and cancer [22e24]. The presence of TNF
the tumor microenvironment of ~50% of surgically resected sam-
ples suggests constitutive activation (ca) of the canonical NF
pathway in these tumors [25]. In Kras mutation-driven cancers,
tissue-specific elimination of IKK resulted in stalling of the disease
in the premalignant state resulting in increased survival of mice
[26e28]. In contrast, the introduction of ca-IKK in the presence of
b
is a member of the IKK complex that regulates the TNF
B pathway, which is implicated in chronic
in
a
6-amine 7 with phenyl isothiocyanate in DCM [49].
k
a
2.2. Synthesis of mono-substituted quinoxaline analogs
kB
Scheme 2 summarizes the synthesis of key common in-
termediates 2-chloro-7-nitroquinoxaline (13) and 2-chloro-6-
nitroquinoxaline (22a) required for the synthesis of the mono-
substituted quinoxaline analogs 54e84 and 86e91. Regioisomers
13 and 22a were synthesized in 2-steps from commercially avail-
able 2-quinoxalinol [50]. Under weakly acidic conditions, the 7-
position of 2-quinoxalinol (11) is susceptible to electrophilic
nitration to yield 7-nitroquinoxalin-2-ol (12). The regioselectivity is
dictated by the nitrogen atom at the 4-postion of 2-quinoxalinol.
Nitration of 11 using the strong acid H2SO4 results in protonation of
the tautomer quinoxalin-2(1H)-one resulting in nitration at the 6-
position to yield 6-nitroquinoxalin-2-ol (22). Here the regiose-
lectivity is dictated by the OH group at the 2-position. The OH in 12
and 22 were replaced with Cl using POCl3/PCl5 to yield the key
intermediates 2-chloro-7-nitroquinoxaline (13) and 2-chloro-6-
nitroquinoxaline (22a), respectively. The regioselective nitration
was confirmed by X-ray crystal structures of compounds 13 and 22a
(Figure S1).
b
b
oncogenic insults such as mutant Apc or Kras enhances tumori-
genesis resulting in a dramatic reduction in the median survival of
mice, suggesting that ca-IKK
Activation of IKK results in the rapid phosphorylation of its
endogenous substrate I , resulting in its ubiquitination by beta-
transducin repeat containing E3 ubiquitin ligase ( -TrCP) followed
by proteasomal degradation [32,33]. The primary role of I in
cells is to sequester NF B in the cytoplasm, therefore TNF induced
degradation of I results in nuclear translocation and expression
of NF B regulated genes [34,35].
Dysregulation of TNF -induced IKK
b supports oncogenesis [29e31].
b
kBa
b
kBa
k
a
kBa
k
a
b-mediated NFkB activation
in pathological conditions led to the development of an array of
small molecules and biologics against proteins in this pathway,
including five TNF
block the degradation of I
majority of these inhibitors target NF
second in that list are IKK and I phosphorylation inhibitors, such
as ML-120B, TPCA1 and BMS345541 developed by pharmaceutical
companies [42e44]. The development of ML-120B and TPCA1
stalled due to unexpected toxicity upon systemic administration
[28,45,46]. This provides the impetus for the development of non-
a
blockers and three proteasome inhibitors that
approved by the FDA [36e41]. The
B-DNA binding and the
Scheme 3 summarizes the synthesis of mono-substituted qui-
noxaline analogs 54e84, 86e91 and a quinoline analog 85. Suzuki
coupling of 13 and 22a with different boronic acids/esters resulted
in analogs 14e17 and 23e26 respectively [51]. Palladium catalyzed
hydrogenation of 14e17 and 23e26 yielded the corresponding
amines 18e21 and 27e30, respectively. Quinoxaline urea analogs
54e61, 70, 72, 75e84 and 86 were synthesized either by conden-
sation of quinoxaline amines 18e21 with different isocyanates or
by generating quinoxaline isocyanates from 18e21 followed by the
addition of the corresponding amines. Addition of various iso-
cyanates to the quinoxaline amines 27e30 yielded the quinoxaline
urea analogs 62e69, 71 and 73e74. Fused quinoxaline urea analogs
(88e92) were generated by the addition of corresponding iso-
cyanates to analog 21. A Skraup-type reaction of 4-nitroaniline with
2,2,3-tribromopropanal gave 3-bromo-6-nitroquinoline (32).
Suzuki coupling of 32 followed by reduction yielded the pyrazole
derivative 34. The quinoline isocyanate was generated from 34 and
condensed with 3-bromo-4-fluoroaniline to yield the pyrazolo-
quinoline analog 85.
kBa
k
kBa
toxic small molecules that modulate IKK
TNF -induced NF B activation.
Here we used a cell-based luciferase reporter assay that spe-
cifically responds to TNF stimulation to induce IKK -mediated
NF B activity to conduct hit-to-lead optimization of a quinoxaline
b function and inhibit
a
k
a
b
k
analog 13e197. Despite the remarkable in vivo anti-cancer effects
exhibited by 13e197 it suffers from poor oral bioavailability
[15e18]. We addressed the above with a structure activity rela-
tionship (SAR) study that resulted in the identification of analog 84
that was ~2.5-fold more potent in TNFa-induced NFkB inhibition.
Analog 84 was ~4-fold more potent in inhibiting pancreatic cancer
cell growth compared to 13e197. We also observed that 84 had
~4.3-fold greater exposure (AUC0-∞) resulting in ~5.7-fold increase
in oral bioavailability (%F) when compared to 13e197. Importantly,
oral administration of 84 by itself and in combination of gemcita-
2.3. Structure activity relationship study with quinoxaline and
quinoline analogs
bine reduced p-IKK
b
levels and inhibited pancreatic tumor growth
The library of 55 quinoxaline and quinoline analogs were
in a xenograft model.
screened in a cell-based luciferase reporter assay that specifically
reports on TNF
were screened at 10
a
-induced NF
k
B activation (Fig. 1A). The compounds
in-
2. Results and discussion
mM with 13e197 and the allosteric IKKb
hibitor BMS345541 as controls. The substitutions on the 2,3
2.1. Synthesis of 2,3 disubstituted symmetrical quinoxaline analogs
disubstituted quinoxaline analogs (38e53) was well tolerated and
changes did not have a significant effect on the inhibition of TNF
a-
The synthesis of quinoxaline analogs 38e53 are summarized in
Scheme 1. Symmetrical diones (1) were refluxed with 4-
nitrobenzene-1,2-diamine (2) in ethanol to generate 2,3-
substituted nitro quinoxaline analogs 3e6. Reduction of com-
pounds 3e6 under hydrogen atmosphere over Pd/C yielded the
corresponding amines 7e10 [47]. Condensation of the 2,3-
substituted quinoxaline amines with substituted phenyl iso-
cyanates resulted in quinoxaline urea analogs 38e39, 42e45 and
47e53 in moderate to excellent yields. Furan and phenyl
substituted quinoxaline amines 7 and 10 were condensed with 4-
bromophenylacetic acid to generate 2,3-substituted quinoxaline
amide analogs 40 and 46, respectively [48]. The thiourea analog 41
induced IKK -mediated NF activity. All of them exhibited
b
kB
~15e45% inhibition of the activity, which was comparable to that of
13e197. Among the mono-substituted quinoxaline analogs, the N-
methyl pyrazole substituted analogs 76e78, showed significant
improvement in activity. Replacing the electron donating eOCH3
and OCF3 at the meta-position of 77 and 78 with CF3 in analog 79
resulted in further improvement of activity. The meta-Cl and para-F
disubstituted analog 80 was ~3-fold more potent than 13e197.
Replacing the Cl in analog 80 with a CH3 in analog 81 dramatically
reduced the activity. The activity was restored when an OCH3 in 82
replaced the CH3 in 80 and further improved with a OCF3 substi-
tution in 83. Incorporating a F atom to the ortho position in analog
2

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
Scheme 1. Reagents and conditions: (a) Ethanol, reflux, 85e99%; (b) H2/PdeC, EtOH, rt, 85e96%; (c) Isocyanate derivatives, CH2Cl2, rt, 62e83%; (d) Phenyl isothiocyanate, DIPEA,
CH2Cl2, rt, 70%; (e) 4-Bromophenylacetic acid, EDC, DIPEA, CH2Cl2, rt, 62e83%.
Scheme 2. Reagents and conditions: a) Acetic acid, conc. HNO3, rt, 24 h, 85%; (b) NaNO3, H2SO4, 0oC-rt, 2 h, 88%; (c) PCl5, POCl3, 110 ꢀC, 12 h, 90%.
79 yielded the most potent inhibitor in analog 84. When compared
to 84, changing the core to a quinoline in 85 or replacing the N-
methylpyrazole with an N-methylpyrrole in 86 resulted in loss of
activity. The three most potent inhibitors were the disubstituted
analogs 79, 80 and 84; however, incorporating a cyclic structure to
replace the di-substitution in analogs 87e91 resulted in a signifi-
cant loss of activity. Together our SAR identified the quinoxaline
urea, N-methylpyrazole and an electron-deficient phenyl as the key
The absolute configuration of 84 was assigned using 1D and 2D
NMR spectroscopy. 1H NMR taken in DMSO‑d6 showed a singlet at
3.9 ppm corresponding to H-6 of NeCH3. Three singlets at 9.1, 8.6
and 8.3 ppm correspond to the protons adjacent to nitrogen atoms
of the quinoxaline (9.1 ppm) and pyrazole rings (8.6 and 8.2 ppm).
The doublets appearing at 8.2 ppm (J ¼ 2.2 Hz) and 7.9 ppm
(J ¼ 8.9 Hz) correspond to H-10 and H-13 due to their respective
meta and ortho couplings with H-12. The doublet of doublets at
7.6 ppm correspond to H-12 (J ¼ 9.0, 2.3 Hz) due its coupling with
H-13 and H-10. The multiplets at 8.4 and 7.3 ppm are of H-22 and
H-23/24, respectively. Whereas, the exchangeable protons at 9.6
and 9.0 correspond to amidic NH-17 and NH-20, respectively. The
quantitative 13C NMR of 84 taken in DMSO‑d6 showed a doublet at
149.9 and 148.2 ppm corresponding to C-26 due to its coupling
with an attached fluorine atom (JCF ¼ 252 Hz). On the other hand, C-
25 appears as a quartet of doublets at 116 ppm due to its long-range
couplings with fluorine atoms present on C-26 and C-28.13C-HMBC
NMR showed interactions of amidic NH-17 with C-10 and C-12.
Whereas, another amidic NH-20 showed interactions with carbonyl
C-18 as well as with C-22 and C-26 confirming that fluorine atom is
substituted ortho to NH-20. The appearance of C-25 as doublet of
quartet suggests C-25 and C-26 are adjacent to each other (Fig. 1D).
elements required for inhibiting TNF
NF B activity. Analog 84 with an o-F, m-CF3 substituted phenyl
quinoxaline urea was the most potent inhibitor.
a-induced IKKb-mediated
k
The activation of caspases, a class of cysteine proteinases, is
routinely used by us and others as indicators for the induction of
apoptosis [20,40,41,52e55]. We assessed the ability of N-methyl-
pyrazole quinoxaline analogs (78e81, 83 and 84) along with
13e197 and BMS345541 to activate effector caspases 3/7 in
pancreatic cancer (MiaPaCa2) cells (Fig. 1B). The cells were sub-
jected to 10 mM of the 8 compounds and incubated for 24 h and
caspase 3/7 activation was assessed using a luminescence assay.
The results showed that analog 84 most potently induced caspase
activation. This is consistent with the inhibition of TNFa-induced
NFkB activity and 84 was found to be ~4-fold more potent than
13e197. Moreover, we observed remarkable correlation (R2 ¼ 0.74)
between the ability of the compounds to induce caspase 3/7 and
inhibit TNFa-induced NFkB activity (Fig. 1C).
3

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
Scheme 3. Reagents and conditions: a) Boronic acids or esters, Pd(PPh3)4, Na2CO3, DMF:Dioxane (1:1), 100 ꢀC, 20 h, 48e88%; (b) Pd/C, H2, EtOH, 18 h, 88e92%; (c) either (i) BTC,
DIPEA, amines, CH2Cl2:THF (4:1), rt or (ii) Isocyanates, CH2Cl2, rt, 1e3 days, 55e74%; (d) 2,2,3,-tribromopropanal, Glacial acetic acid, 110 ꢀC, 38%; (e) 3-Bromo-4-fluoroaniline,
triphosgene, DIPEA, DCM, rt, 32%.
2.4. Characterization of 84 in cell-based assays
manner with low-
analog 84 inhibited the growth of pancreatic cancer cell lines
(MiaPaCa2 and T3M4) with low- M potency (Fig. 2C). Consistent
mM potency. In a 3-day cell growth assay
In a follow-up experiment, we show that analog 84 inhibited
m
TNF
manner and had an IC50 value (3.3 0.5
greater than the reported value for 13e197 (8.4
(Fig. 2A) [16]. Next, we conducted dose-response and time-course
studies to assess the ability of analog 84 to modulate IKK func-
tion (Fig. 2B). We found that analog 84 reduced the levels of
phosphorylated IKK in a dose-dependent and a time-dependent
a
-induced IKK
b
-mediated NF
k
B activity in a dose-dependent
M) that was ~2.5-fold
1.2 M)
with caspase 3/7 activation, in a live/dead assay, analog 84 induced
MiaPaCa2 cell death in a dose-dependent manner (Fig. 2D). The
ability of cancer cells to form colonies is assessed by crystal violet
staining in clonogenic assays [56]. Analog 84 inhibited MiaPaCa2
colony forming ability by > 50% when compared to DMSO treated
cells (Fig. 2E). Pancreatic cancer is highly metastatic and wound
healing assays are used to assess the ability of compounds to inhibit
m
m
b
b
4

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
Fig. 1. SAR study identifies 84 as an inhibitor of TNFa-induced NFkB. (A) Quinoxaline and quinoline analogs screened for inhibition of TNFa-induced NFkB activity in a cell-based
(A549) luciferase reporter assay. The data is normalized to viability and reported as Ave SEM (n ¼ 4), *p-value < 0.05, **p-value < 0.005. (B) MiaPaCa2 cells were treated with
analogs 78e81, 83 and 84 along with 13e197 and BMS345541 for 24 h. Apoptosis assessed by measuring the induction of caspase 3/7 activity using Caspase-Glo® 3/7 system
(n ¼ 3). (C) Correlation plot derived from the inhibition of TNF
a-induced NFk
B activity and induction of caspase 3/7 activity by quinoxaline analogs. (D) 13C-HMBC spectra of 84
(blue) overlapped on 13C-HSQC (red).
migration of cells [57]. In a time course study, when compared to
DMSO treated cells analog 84 was more effective inhibiting the
migration of PDAC cells (Fig. 2F). Together our data shows that
furan groups and is ~3e4 fold more potent than 13e197 in multiple
cell-based assays.
analog 84 reduced the levels of phosphorylated IKK
b which results
2.5. Pharmacokinetic studies with 13e197 and analog 84
in the inhibition of TNF -induced NF B activity. Moreover, analog
a
k
84 inhibited cancer cell growth, induced apoptosis, inhibited colony
formation and migration of PDAC cells. Our SAR studies have
identified analog 84 that does not possess the metabolically labile
Furan rings are susceptible to oxidation resulting in the gener-
ation of electrophilic intermediates which could lead to toxic and
carcinogenic effects [58]. We previously showed that 13e197 had
5

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
Fig. 2. Evaluation of 84 in PDAC cells. (A) Dose-response study with analog 84 in a dual luciferase reporter assay to assess the inhibition of TNF
activation (n ¼ 3). (B) Dose-response and time-course studies to assess the effect of analog 84 on the activated IKK in pancreatic cancer cells. Cell lysates were subjected to Western
blotting and probed using the indicated antibodies. -actin serves as loading control (C) Growth inhibition studies with analog 84 in pancreatic cancer cell lines. Cell viability was
M for 24 h) MiaPaCa2 cells (n ¼ 3).
M at 24 and 48 h) MiaPaCa2 cells (n ¼ 3). All data
a-induced IKKb mediated NFkB
b
b
determined using PrestoBlue following incubation of compounds for 72 h (n ¼ 3). (D) Live and dead cell assay with analog 84 treated (5 and 10
m
(E) Colony formation assay with analog 84 treated (5
m
M) MiaPaCa2 cells (n ¼ 3). (F) Scratch assay with analog 84 treated (5
m
represented as Ave SEM.
oral bioavailability's (%F) of 3 and 16% in mice and rats, respectively.
This was attributed to the identification of >20 metabolites and
excretion of <1% of intact 13e197 suggesting extensive metabolism
of 13e197 [17]. Like 13e197, analog 84 obeys the Lipinski's rule of 5,
which suggested > 90% chance that 84 would be orally bioavailable
[59]. To determine if replacing the furan rings in 13e197 with a
metabolically stable N-methylpyrazole resulted in improved %F we
conducted PK studies with 13e197 and analog 84. Mice were orally
dosed with 20 mg/kg of 13e197 or 84 and sampled at 0.083, 0.25,
0.5, 1, 2, 4, 8, and 24 h. PK parameters summarized in Table 1 shows
that 13e197 and 84 had similar T1/2, however the Tmax and Cmax for
84 was ~2.5 times greater than that of 13e197. Although this did not
have a significant effect on the mean residence time of 84, it
resulted in ~4.3-fold greater exposure (AUC0-∞) when compared to
13e197. To assess oral bioavailability, mice were intravenously
dosed with 10 mg/kg of 84 and sampled at the times described
above. At half the oral dose the intravenous exposure (AUC0-∞) of
84 was ~2.8-fold higher resulting in an oral bioavailability of 17.6%.
In summary, replacing the metabolically labile furan rings in
13e197 with N-methylpyrazole and replacing the bromine atom
with a eCF3 and fluorine on the phenyl ring in 84 resulted in a ~5.7-
fold increase in oral bioavailability (%F).
bioavailability exhibited by analog 84 prompted us to explore 84 as
an anticancer agent in a pancreatic tumor model. Gemcitabine is
used as a first line therapy for pancreatic cancer patients therefore
we also assessed 84 in a combination setting with gemcitabine.
Briefly, MiaPaCa2 cells (2.5ꢁ 106 cells with Matrigel, 2:1 ratio) were
subcutaneously implanted in the flanks of athymic nude mice. 20
days after implantation, mice were randomized and distributed
into the following treatment groups (n ¼ 6/group): Vehicle (100
ml);
84 (40 mg/kg); Gemcitabine (15 mg/kg) and 84 þ Gemcitabine. 84
was dosed orally once a day, Gemcitabine was administered
intraperitoneally (i.p.) every 3 days. Tumor growth was monitored
every 3-days and mice were sacrificed after 4-week treatment
(Fig. 3A). At the end of the 4-weeks, when compared to vehicle
treated mice gemcitabine alone showed ~45% reduction in tumor
volume and tumor weight while 84 alone showed ~75% reduction
in tumor volume and tumor weight (Fig. 3B). More importantly
mice treated with combination of gemcitabine and 84 showed a
>90% reduction in tumor volume and tumor weight when
compared to the vehicle treated mice. We observed reduction in
Ki67 staining in treated mice indicating a reduction in the prolif-
eration index (Fig. 3C). We also observed reduction in p-IKKb levels
in the treated samples indicating modulation of the target by 84 in
an in vivo setting (Fig. 3D). Together these results show that 84 is an
orally bioavailable anticancer agent, combined with gemcitabine is
a novel therapeutic option for pancreatic cancer.
2.6. In vivo efficacy studies with analog 84 in pancreatic cancer
model
The cellular efficacy data along with the improved oral
6

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
Table 1
Pharmacokinetic studies with 13e197 and analog 84.
3. Conclusion
4. Experimental methods
Phosphorylation mediated constitutive activation of IKK
b
has
4.1. Chemistry
been implicated in enhancing tumorigenesis in the presence of
oncogenic insults in multiple cancer models. Phosphorylated IKK
has also been associated with ~50% of all cancers; therefore, agents
that reduce the levels of phosphorylated IKK offer a novel thera-
peutic option. Here, we used a cell-based mechanism specific assay
to conduct hit-to-lead optimization studies that identified a novel
b
4.1.1. General procedures
All reagents were purchased from commercial sources and were
used without further purification. Pre-coated ANALTECH uniplate
was used for Thin layer chromatography (TLC) and monitored un-
der UV light at 254 nm and with potassium permanganate stain.
Silica gel (230e400 mesh, grade 60, Fisher scientific, USA) was used
for Column chromatography. 1H NMR and 13C NMR spectra were
recorded in chloroform-d or DMSO‑d6 on a Bruker-400, Bruker-500
and Bruker-600 spectrometer. The purity of final compounds was
determined by analytical HPLC and was found to be ꢂ 95% pure.
Analysis of sample purity was performed on a Waters Alliance 2695
b
quinoxaline analog 84 as an inhibitor of TNF
mediated NF B activity. Among quinoxaline analogs we show an
excellent correlation between their ability to inhibit TNF -induced
IKK -mediated NF B activity and the induction of caspase 3/7 ac-
tivity. In pancreatic cancer cells, analog 84 in a dose- and time-
dependent manner reduces p-IKK levels, induced apoptosis, in-
hibits cell growth, colony formation and migration, at low-
a-induced IKKb-
k
a
b
k
b
mM
HPLC
system
Phenomenex
Luna-2
RP-C18
(5
mm,
concentrations. Replacing the metabolically labile furan groups in
13e197 with N-methylpyrazole in 84 resulted in ~5.7-fold increase
in orally bioavailability. Most importantly analog 84 alone and in
4.6 mm ꢁ 250 mm, 120 Å, Torrance, CA) column. HPLC conditions:
solvent A, H2O containing 0.1% formic acid (FA); solvent B, CH3CN
containing 0.1% FA; gradient, 10% B to 100% B over 15 min followed
by 100% B over 4 min with flow rate, 1 mL/min. Mass spectrometric
data was acquired using Quattro Micro triple quadrupole mass
spectrometer with electron-spray ionization (ESI) technique and as
TOF mass analyzer. HRMS data was generated on an Agilent 6230
LC/TOF system with UV detector (254 nm).
combination with gemcitabine reduced p-IKKb levels resulting in a
>90% reduction of tumor volume and tumor growth in a xenograft
pancreatic mouse model. In summary, we present 84 as a suitable
pre-therapeutic anticancer agent that is ideal for lead optimization
studies.
4.1.2. Synthesis
4.1.2.1. General methods
7

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
Fig. 3. Tumor growth studies with 84 and gemcitabine. (A) MiaPaCa2 cells were subcutaneously implanted in nude mice and treated with 84 (40 mg/kg, P$O.) or gemcitabine
(15 mg/kg i.p.) or the combination and tumor growth monitored for 4-weeks (n ¼ 6). (B) Tumor weights measured at the end of the study. (C) Ki67 staining indicates proliferation
index in tumor tissue (n ¼ 5). (D) Levels of p-IKK
and ***P < 0.0005.
b
in the tumor tissue indicates in vivo target modulation (n ¼ 5). All the data represented as average SEM. *P < 0.05, **P < 0.005
4.1.2.1.1. General procedure of Suzuki coupling (Method A).
Respected halide (1 eq), Na2CO3 (4 eq) in DMF:Dioxane (1:1) and
water (0.2 mL) were taken a round bottom flask. The mixture was
sonicated and degassed for 5 min by passing nitrogen through it.
Degassing was followed by addition of Pd(Ph3)4 (0.05 eq) and
corresponding boronic acids or esters (1 eq). The reaction mixture
was stirred under inert atmosphere overnight at 100 ꢀC and
quenched by addition of water. The precipitates formed were
filtered and triturated in THF and hexane (70e88%).
4.1.2.1.2. General procedure of reduction of nitro compounds to
amines (Method B). Respected quinaxoline-nitro compound (1 eq)
was dissolved in EtOH followed by the addition of Pd/C (10% w/w)
under inert conditions. The reaction mixture was stirred under H2
atmosphere for 4e12 h at room temperature. After completion of
reaction, the mixture was filtered through Celite and solvent was
evaporated under reduced pressure to yield corresponding
quinaxoline-amine which were used without purification for next
transformations (85e99%).
diisopropylethylamine (2 eq) and added slowly to a solution of BTC
(0.35 eq) in dichloromethane at 0 ꢀC. The mixture was further
stirred for 3e6 h until the starting material disappears. A mixture of
corresponding amine (1.5 eq) and diisopropylethylamine (2 eq) was
added to this mixture and reaction was stirred for next 10e24 h at
room temperature. After completion of reaction, the crude was
purified by column chromatography using MeOH and DCM as
eluting system (55e88%).
Compounds 3e10, 12e13, 22, 22a, 38e39 and 41e45 have been
reported previously [19,20,50]. Compounds 14e17a, 23e26 were
synthesized according to Method A and compounds 18e21a,
27e30 were synthesized according to Method B described in gen-
eral methods. Compounds 47e81, 84, 86 and 88e92 were synthe-
sized according to Method C described in general methods.
Compounds 82, 83 and 87 were synthesized according to Method D
described in general methods.
4.1.2.1.5. 7-Nitro-2-(thiophen-2-yl)quinoxaline
(15).
9.36 (s, 1H), 8.96
Yield ¼ 51%; 1H NMR (500 MHz, Chloroform-d)
d
4.1.2.1.3. General procedure of synthesis of urea analogs (Method
C). To a suspension of respected quinaxoline amine (1 eq) in DCM
was added corresponding isocyanate (1.5 eq). The reaction mixture
was stirred for 24e48 h at room temperature. Product was filtered
and washed with DCM, hexane and dried under vacuum (55e88%).
4.1.2.1.4. General procedure of synthesis of urea analogs (Method
D). Compound 21, 21a, or 34 was dispersed in THF and
(d, J ¼ 2.6 Hz, 1H), 8.46 (dd, J ¼ 2.5, 9.1 Hz, 1H), 8.21 (d, J ¼ 9.1 Hz,
1H), 7.95 (dd, J ¼ 1.1, 3.8 Hz, 1H), 7.66 (dd, J ¼ 1.1, 5.0 Hz, 1H),
7.34e7.16 (m, 1H); 13C NMR (125 MHz, CDCl3)
d 149.5, 148.6, 145.1,
143.8, 141.5, 141.3, 131.7, 131.0, 129.0, 128.6, 125.5, 122.6; HRMS (ESI-
MS) calcd for C12H8N3O2Sþ m/z (M þ H)þ 258.0332, found:
258.0338.
8

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
4.1.2.1.6. 7-Nitro-2-(thiophen-3-yl)quinoxaline
(16).
J ¼ 8.3, 13.2 Hz, 7H), 7.28 (t, J ¼ 8.0 Hz, 1H), 7.19 (d, J ¼ 7.9 Hz, 1H);
Yield ¼ 49%; 1H NMR (500 MHz, DMSO‑d6)
d
9.72 (s, 1H), 8.84 (d,
13C NMR (125 MHz, DMSO‑d6)
d 153.1, 152.3, 150.8, 141.4, 141.0,
J ¼ 2.6 Hz, 1H), 8.77 (dd, J ¼ 1.3, 2.9 Hz, 1H), 8.49 (dd, J ¼ 2.5, 9.0 Hz,
1H), 8.31 (d, J ¼ 9.1 Hz, 1H), 8.04 (dd, J ¼ 1.2, 5.0 Hz, 1H), 7.83 (dd,
J ¼ 2.8, 5.1 Hz, 1H); HRMS (ESI-MS) calcd for C12H8N3O2Sþ m/z
(M þ H)þ 258.0332, found: 258.0342.
138.9, 138.9, 136.8, 130.7, 129.6, 129.6, 129.2, 128.6, 128.4, 128.0,
128.0, 124.7, 123.7, 121.7, 120.7, 117.3, 113.4. HRMS (ESI-MS) calcd for
C
27H20BrN4Oþ m/z (M þ H)þ 495.0815, found: 495.0817.
4.1.2.1.16. 1-(2-bromophenyl)-3-(2,3-diphenylquinoxalin-6-yl)
4.1.2.1.7. 2-(1-Methyl-1H-pyrazol-4-yl)-7-nitroquinoxaline (17).
urea (48). Yield ¼ 69%; 1H NMR (500 MHz, DMSO‑d6)
d 10.02 (s,
Yield ¼ 78%; 1H NMR (400 MHz, DMSO‑d6)
d
9.47 (s, 1H), 8.74e8.67
1H), 8.42 (d, J ¼ 2.4 Hz, 1H), 8.36 (s, 1H), 8.10 (t, J ¼ 8.2 Hz, 2H), 7.80
(dd, J ¼ 2.5, 9.0 Hz, 1H), 7.65 (d, J ¼ 8.0 Hz, 1H), 7.53e7.42 (m, 4H),
7.42e7.26 (m, 7H), 7.03 (t, J ¼ 7.7 Hz, 1H); 13C NMR (125 MHz,
(m, 2H), 8.42 (dd, J ¼ 9.1, 2.6 Hz, 1H), 8.34 (s, 1H), 8.25 (d, J ¼ 9.1 Hz,
1H), 3.97 (s, 3H); HRMS (ESI-MS) calcd for C12H10N5Oþ2 m/z
(M þ H)þ 256.0829, found: 256.0838.
DMSO‑d6) d 153.2, 152.1, 150.8, 141.5, 141.1, 138.9, 136.8, 136.7, 132.6,
4.1.2.1.8. 2-(1-Methyl-1H-pyrrol-3-yl)-7-nitroquinoxaline (17a).
129.7, 129.6, 129.4, 128.7, 128.5, 128.2, 128.0, 128.0, 124.6, 123.6,
122.6, 113.5, 113.3; HRMS (ESI-MS) calcd for C27H20BrN4Oþ m/z
(M þ H)þ 495.0815, found: 495.0821.
Yield ¼ 48%; 1H NMR (400 MHz, CDCl3)
d 9.20 (s, 1H), 9.02 (d,
J ¼ 2.5 Hz, 1H), 8.40 (dd, J ¼ 9.1, 2.5 Hz, 1H), 8.17 (d, J ¼ 9.1 Hz, 1H),
7.73 (t, J ¼ 2.0 Hz, 1H), 6.95 (dd, J ¼ 2.9, 1.8 Hz, 1H), 6.80e6.77 (m,
1H), 3.82 (s, 3H); HRMS (ESI-MS) calcd for C13H11N4Oþ2 m/z
(M þ H)þ 255.0877, found: 255.0882.
4.1.2.1.17. 1-(2,3-Diphenylquinoxalin-6-yl)-3-(4-fluorophenyl)
urea (49). Yield ¼ 64%; 1H NMR (500 MHz, DMSO‑d6)
d 9.30 (s, 1H),
8.95 (s, 1H), 8.36 (d, J ¼ 2.3 Hz, 1H), 8.06 (d, J ¼ 9.0 Hz, 1H), 7.83 (dd,
J ¼ 2.4, 9.1 Hz, 1H), 7.53 (dd, J ¼ 4.9, 8.8 Hz, 2H), 7.45 (dd, J ¼ 7.0,
11.0 Hz, 4H), 7.35 (h, J ¼ 6.7 Hz, 6H), 7.16 (t, J ¼ 8.7 Hz, 2H); 13C NMR
4.1.2.1.9. 6-Nitro-2-phenylquinoxaline (23). Yield
¼
58%; 1H
NMR (400 MHz, DMSO‑d6)
d
9.83 (s, 1H), 8.94 (d, J ¼ 2.5 Hz, 1H),
8.59 (dd, J ¼ 9.2, 2.6 Hz, 1H), 8.44 (dd, J ¼ 6.7, 2.9 Hz, 2H), 8.37 (d,
J ¼ 9.2 Hz, 1H), 7.66 (m, 3H); HRMS (ESI-MS) calcd for C14H10N3O2þ
m/z (M þ H)þ 252.0768, found: 252.0778.
(125 MHz, DMSO‑d6) d 158.5, 156.6, 154.1, 153.1, 152.5, 150.7, 141.5,
141.4, 139.0, 139.0, 136.7, 135.7, 129.7, 129.6, 129.2, 128.7, 128.4,
128.0, 128.0, 123.7, 120.4, 120.3, 115.5, 115.4, 115.3, 115.2, 113.1;
HRMS (ESI-MS) calcd for C27H20FN4Oþ m/z (M þ H)þ 435.1616,
found: 436.1623.
4.1.2.1.10. 6-Nitro-2-(thiophen-2-yl)quinoxaline
(24).
Yield ¼ 62%; 1H NMR (500 MHz, CDCl3)
d 9.38 (s, 1H), 8.98 (d,
J ¼ 2.5 Hz, 1H), 8.53 (dd, J ¼ 2.5, 9.2 Hz, 1H), 8.24 (d, J ¼ 9.2 Hz, 1H),
8.04 (d, J ¼ 3.7 Hz, 1H), 7.68 (dd, J ¼ 1.2, 5.1 Hz, 1H), 7.27 (dd, J ¼ 3.8,
5.0 Hz, 1H); HRMS (ESI-MS) calcd for C12H8N3O2Sþ m/z (M þ H)þ
258.0332, found: 258.0326.
4.1.2.1.18. 1-(4-chlorophenyl)-3-(2,3-diphenylquinoxalin-6-yl)
urea (50). Yield ¼ 68%; 1H NMR (500 MHz, DMSO‑d6)
d 9.34 (s, 1H),
9.06 (s, 1H), 8.36 (d, J ¼ 2.3 Hz, 1H), 8.07 (d, J ¼ 9.0 Hz, 1H), 7.84 (dd,
J ¼ 2.4, 9.1 Hz, 1H), 7.54 (d, J ¼ 8.6 Hz, 2H), 7.51e7.42 (m, 4H), 7.36
4.1.2.1.11. 6-Nitro-2-(thiophen-3-yl)quinoxaline
(25).
(dt, J ¼ 6.7, 9.7 Hz, 8H); 13C NMR (125 MHz, DMSO‑d6)
d 153.1, 152.3,
Yield ¼ 54%; 1H NMR (500 MHz, CDCl3)
d
9.38 (s, 1H), 8.98 (d,
150.7, 141.5, 141.2, 139.0, 138.9, 138.3, 136.8, 129.7, 129.6, 129.3,
128.7, 128.7, 128.5, 128.0, 128.0, 125.8, 123.7, 120.0, 113.3; HRMS
(ESI-MS) calcd for C27H20ClN4Oþ m/z (M þ H)þ 451.1320, found:
451.1327.
J ¼ 2.5 Hz, 1H), 8.53 (dd, J ¼ 2.5, 9.2 Hz, 1H), 8.24 (d, J ¼ 9.2 Hz, 1H),
8.04 (d, J ¼ 3.7 Hz, 1H), 7.68 (dd, J ¼ 1.2, 5.1 Hz, 1H), 7.27 (dd, J ¼ 3.8,
5.0 Hz, 1H); HRMS (ESI-MS) calcd for C12H8N3O2Sþ m/z (M þ H)þ
258.0332, found: 258.0342.
4.1.2.1.19. 1-(2,3-Diphenylquinoxalin-6-yl)-3-(4-methoxyphenyl)
4.1.2.1.12. 2-(1-Methyl-1H-pyrazol-4-yl)-6-nitroquinoxaline (26).
urea (51). Yield ¼ 81%; 1H NMR (500 MHz, DMSO‑d6)
d 9.23 (s, 1H),
1H NMR (400 MHz, DMSO‑d6)
d
9.49 (s, 1H), 8.82 (d, J ¼ 2.4 Hz, 1H),
8.72 (s, 1H), 8.36 (s, 1H), 8.05 (d, J ¼ 9.0 Hz, 1H), 7.83 (d, J ¼ 9.2 Hz,
8.74 (s, 1H), 8.50 (dd, J ¼ 9.2, 2.5 Hz, 1H), 8.37 (s, 1H), 8.16 (d,
J ¼ 9.2 Hz,1H), 3.98 (s, 3H); HRMS (ESI-MS) calcd for C12H10N5Oþ2 m/
z (M þ H)þ 256.0829, found: 256.0839.
1H), 7.50e7.32 (m, 12H), 6.91 (d, J ¼ 8.4 Hz, 2H), 3.73 (s, 3H); 13C
NMR (125 MHz, DMSO‑d6)
d 154.8, 153.1, 152.6, 150.5, 141.5, 139.0,
136.7, 132.3, 129.7, 129.6 (3C), 129.2, 128.6, 128.4, 128.0, 128.0 (2C),
123.7, 120.4 (2C), 114.1 (2C), 112.8, 55.2; HRMS (ESI-MS) calcd for
4.1.2.1.13. 2-(4-bromophenyl)-N-(2,3-di(furan-2-yl)quinoxalin-6-
yl)acetamide (40). A mixture of Compound 7 (1 eq) and DIPEA (3
eq) was dissolved in DCM. To this mixture was added EDC (2 eq)
and was stirred for 10 min at 0 ꢀC followed by addition of 4-
bromophenylacetic (3eq). The reaction mixture was further stir-
red for 8 h at room temperature and then extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4 and
evaporated under reduced pressure. Then crude was purified on
silica gel chromatography using hexane/EtOAc as eluting solvents
to yield 40 as pure compound; Yield ¼ 85%; 1H NMR (500 MHz,
C
28H23N4Oþ2 m/z (M þ H)þ 447.1816, found: 447.1817.
4.1.2.1.20. 1-(2,3-Diphenylquinoxalin-6-yl)-3-(4-(trifluoromethyl)
phenyl)urea (52). Yield ¼ 83%; 1H NMR (500 MHz, DMSO‑d6)
d 9.43
(s, 1H), 9.34 (s, 1H), 8.38 (s, 1H), 8.08 (d, J ¼ 9.0 Hz, 1H), 7.85 (d,
J ¼ 9.1 Hz, 1H), 7.77e7.61 (m, 4H), 7.46 (dd, J ¼ 7.3, 11.5 Hz, 4H), 7.36
(h, J ¼ 6.9 Hz, 6H); 13C NMR (125 MHz, DMSO‑d6)
d 153.2, 152.2,
150.9,143.1,141.4,141.0,138.9,138.9,136.9,129.7,129.6,129.3,128.7,
128.5, 128.0, 128.0, 126.1, 125.6, 123.7, 123.4, 122.0, 118.1, 117.8, 113.5,
67.0; HRMS (ESI-MS) calcd for C28H20F3N4Oþ m/z (M þ H)þ
485.1584, found: 485.1587.
Chloroform-d)
d
8.24 (d, J ¼ 2.3 Hz, 1H), 8.04 (d, J ¼ 9.1 Hz, 1H), 7.88
(dd, J ¼ 9.1, 2.4 Hz, 1H), 7.75 (s, 1H), 7.60 (d, J ¼ 1.7 Hz, 2H),
7.56e7.50 (m, 2H), 7.25 (s, 1H), 6.65 (dd, J ¼ 14.8, 3.4 Hz, 2H), 6.55
(dd, J ¼ 3.5, 1.8 Hz, 2H), 3.77 (s, 2H); HRMS (ESI-MS) calcd for
4.1.2.1.21. 1-(2,4-bis(trifluoromethyl)phenyl)-3-(2,3-
diphenylquinoxalin-6-yl)urea (53). Yield ¼ 59%; 1H NMR (500 MHz,
DMSO‑d6)
d
9.61 (d, J ¼ 8.9 Hz, 2H), 8.38 (d, J ¼ 2.3 Hz, 1H), 8.19 (s,
C
24H17BrN3Oþ3 m/z (M þ H)þ 474.0448, found: 474.0155.
2H), 8.07 (d, J ¼ 9.0 Hz, 1H), 7.89 (dd, J ¼ 2.3, 9.0 Hz, 1H), 7.67 (s, 1H),
4.1.2.1.14. 2-(4-bromophenyl)-N-(2,3-diphenylquinoxalin-6-yl)
7.53e7.42 (m, 4H), 7.42e7.24 (m, 6H); 13C NMR (125 MHz,
acetamide (46). Same method as used for the synthesis of 40
DMSO‑d6) d 153.1, 152.4, 151.0, 141.5, 141.3, 140.7, 138.9, 136.9, 131.1,
130.9, 130.6, 130.3, 129.7, 129.6, 129.2, 128.7, 128.5, 128.0, 124.4,
123.9, 122.2, 118.3, 114.7, 114.1; HRMS (ESI-MS) calcd for
starting from compound 10; Yield ¼ 90%; 1H NMR (500 MHz,
DMSO‑d6)
d
10.74 (s, 1H), 8.56 (d, J ¼ 2.3 Hz, 1H), 8.10 (d, J ¼ 9.0 Hz,
1H), 7.94 (dd, J ¼ 9.0, 2.3 Hz, 1H), 7.55 (d, J ¼ 8.1 Hz, 2H), 7.49e7.42
(m, 4H), 7.42e7.31 (m, 8H), 3.78 (s, 2H); HRMS (ESI-MS) calcd for
C
29H19F6N4Oþ m/z (M þ H)þ 553.1458, found: 553.1462.
4.1.2.1.22. 1-(4-fluorophenyl)-3-(3-phenylquinoxalin-6-yl)urea
C
24H17BrN3Oþ3 m/z (M þ H)þ 474.0448, found: 474.0452.
(54). Yield ¼ 54%; 1H NMR (500 MHz, DMSO‑d6)
d 9.39 (s, 1H), 9.27
4.1.2.1.15. 1-(3-bromophenyl)-3-(2,3-diphenylquinoxalin-6-yl)
(s, 1H), 8.94 (s, 1H), 8.36 (d, J ¼ 2.3 Hz, 1H), 8.34e8.27 (m, 2H), 8.02
urea (47). Yield ¼ 72%; 1H NMR (500 MHz, DMSO‑d6)
d 9.37 (s, 1H),
(d, J ¼ 9.0 Hz, 1H), 7.78 (dd, J ¼ 2.4, 9.0 Hz, 1H), 7.64e7.46 (m, 5H),
9.11 (s, 1H), 8.43e8.29 (m, 1H), 8.07 (d, J ¼ 9.0 Hz, 1H), 7.90 (d,
7.16 (t, J ¼ 8.8 Hz, 2H); 13C NMR (125 MHz, DMSO‑d6)
d 158.7, 156.8,
J ¼ 1.8 Hz, 1H), 7.88e7.76 (m, 1H), 7.52e7.42 (m, 4H), 7.37 (dt,
152.7, 151.3, 142.7, 141.7, 141.4, 137.6, 136.4, 135.8, 130.4, 129.4, 129.2,
9

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
127.5, 123.3, 120.5, 120.4, 115.6, 115.4, 113.8; HRMS (ESI-MS) calcd
for C21H16FN4Oþ m/z (M þ H)þ 359.1303, found: 359.1313.
4.1.2.1.23. 1-(4-chlorophenyl)-3-(3-phenylquinoxalin-6-yl)urea
(s, 1H), 8.93 (s, 1H), 8.35 (d, J ¼ 2.3 Hz, 1H), 8.32e8.24 (m, 2H), 8.06
(d, J ¼ 9.0 Hz, 1H), 7.83 (dd, J ¼ 2.4, 9.2 Hz, 1H), 7.64e7.47 (m, 5H),
7.16 (t, J ¼ 8.7 Hz, 2H). 13C NMR (125 MHz, CDCl3)
d 158.7, 156.8,
(55). Yield ¼ 61%; 1H NMR (500 MHz, DMSO‑d6)
d
9.40 (s, 1H), 9.32
152.7,149.0,144.0,142.3,141.1, 137.8,136.5,135.8, 130.1,129.8,129.2,
127.2, 124.0, 120.6, 120.5, 115.6, 115.4, 113.6; HRMS (ESI-MS) calcd
for C21H16FN4Oþ m/z (M þ H)þ 359.1303, found: 359.1310.
4.1.2.1.31. 1-(4-chlorophenyl)-3-(2-phenylquinoxalin-6-yl)urea
(s, 1H), 9.06 (s, 1H), 8.36 (d, J ¼ 2.4 Hz, 1H), 8.35e8.29 (m, 2H), 8.03
(d, J ¼ 9.0 Hz, 1H), 7.79 (dd, J ¼ 9.0, 2.4 Hz, 1H), 7.64e7.52 (m, 5H),
7.40e7.34 (m, 2H); 13C NMR (125 MHz, CDCl3)
d 152.6, 151.4, 142.7,
141.6, 138.6, 137.7, 136.5, 130.5, 129.5, 129.3, 129.0, 127.6, 126.0,
123.4, 120.3, 114.0; HRMS (ESI-MS) calcd for C21H16ClN4Oþ m/z
(M þ H)þ 375.1007, found: 375.1017.
(63). Yield ¼ 54%; 1H NMR (500 MHz, DMSO‑d6)
d 9.49 (s, 1H), 9.32
(s, 1H), 9.04 (s, 1H), 8.35 (d, J ¼ 2.3 Hz, 1H), 8.33e8.26 (m, 2H), 8.07
(d, J ¼ 9.0 Hz, 1H), 7.83 (dd, J ¼ 2.4, 9.1 Hz, 1H), 7.63e7.50 (m, 5H),
4.1.2.1.24. 1-(4-bromophenyl)-3-(3-phenylquinoxalin-6-yl)urea
7.41e7.31 (m, 2H); 13C NMR (125 MHz, CDCl3)
d 152.3, 148.9, 143.8,
(56). Yield ¼ 83%; 1H NMR (500 MHz, DMSO‑d6)
d
9.40 (s, 1H), 9.32
142.0, 140.8, 138.3, 137.7, 136.3, 129.9, 129.6, 129.0, 128.7, 127.0,
125.7, 123.8,120.0, 113.5; HRMS (ESI-MS) calcd for C21H16ClN4Oþ m/
z (M þ H)þ 375.1007, found: 375.1014.
(s, 1H), 9.06 (s, 1H), 8.40e8.27 (m, 3H), 8.03 (d, J ¼ 9.0 Hz, 1H), 7.79
(dd, J ¼ 2.4, 9.1 Hz,1H), 7.59 (dd, J ¼ 7.1, 11.5 Hz, 3H), 7.50 (s, 4H); 13
C
NMR (125 MHz, DMSO‑d6)
d
152.3, 151.1, 142.5, 141.3, 141.2, 138.8,
4.1.2.1.32. 1-(4-bromophenyl)-3-(2-phenylquinoxalin-6-yl)urea
137.5, 136.2, 131.5, 131.5, 130.2, 129.2, 129.0, 127.3, 123.1, 120.4,
120.2, 113.8, 113.6; HRMS (ESI-MS) calcd for C21H16BrN4Oþ m/z
(M þ H)þ 419.0502, found: 419.0507.
(64). Yield ¼ 83%; 1H NMR (500 MHz, DMSO‑d6)
d 9.49 (s, 1H), 9.34
(s, 1H), 9.06 (s, 1H), 8.35 (d, J ¼ 2.3 Hz, 1H), 8.30 (dd, J ¼ 1.7, 7.6 Hz,
2H), 8.06 (d, J ¼ 9.0 Hz, 1H), 7.83 (dd, J ¼ 2.4, 9.1 Hz, 1H), 7.63e7.53
4.1.2.1.25. 1-(3-bromophenyl)-3-(3-phenylquinoxalin-6-yl)urea
(m, 3H), 7.50 (s, 4H); 13C NMR (125 MHz, DMSO‑d6)
d 152.3, 148.9,
(57). Yield ¼ 63%; 1H NMR (500 MHz, DMSO‑d6)
d
9.39 (d,
143.8, 142.0, 140.7, 138.8, 137.7, 136.3, 131.6, 129.9, 129.6, 129.0,
127.0, 123.8, 120.4, 113.6, 113.5; HRMS (ESI-MS) calcd for
J ¼ 8.3 Hz, 2H), 9.13 (s, 1H), 8.39e8.27 (m, 3H), 8.03 (d, J ¼ 9.0 Hz,
1H), 7.91 (t, J ¼ 2.0 Hz, 1H), 7.79 (dd, J ¼ 2.4, 9.0 Hz, 1H), 7.64e7.50
(m, 4H), 7.39 (dd, J ¼ 1.9, 8.2 Hz, 1H), 7.28 (t, J ¼ 8.0 Hz,1H), 7.20 (dd,
C
21H16BrN4Oþ m/z (M þ H)þ 419.0502, found: 419.0506.
4.1.2.1.33. 1-(3-bromophenyl)-3-(2-phenylquinoxalin-6-yl)urea
J ¼ 1.8, 7.9 Hz, 1H); 13C NMR (125 MHz, DMSO‑d6)
d
152.3, 151.1,
(65). Yield ¼ 64%; 1H NMR (500 MHz, DMSO‑d6)
d 10.55 (s, 1H),
142.4, 141.3, 141.2, 141.0, 137.5, 136.2, 130.7, 130.3, 129.2, 129.1, 127.3,
124.7, 123.2, 121.7, 120.7, 117.3, 113.9; HRMS (ESI-MS) calcd for
10.28 (s, 1H), 9.42 (s, 1H), 8.41 (d, J ¼ 2.3 Hz, 1H), 8.25 (d, J ¼ 7.4 Hz,
2H), 7.96e7.87 (m, 3H), 7.54 (dq, J ¼ 13.5, 7.2 Hz, 4H), 7.20e7.07 (m,
C
21H16BrN4Oþ m/z (M þ H)þ 419.0502, found: 419.0505.
2H); 13C NMR (125 MHz, DMSO‑d6)
d 152.7, 148.5, 143.5, 142.1, 141.8,
4.1.2.1.26. 1-(2-bromophenyl)-3-(3-phenylquinoxalin-6-yl)urea
141.5, 137.5, 136.4, 130.4, 129.8, 129.1, 129.0, 127.0, 124.2, 121.6,
120.6, 117.2, 113.4; HRMS (ESI-MS) calcd for C21H16BrN4Oþ m/z
(M þ H)þ 419.0502, found: 419.0503.
(58). Yield ¼ 64%; 1H NMR (500 MHz, DMSO‑d6)
d 9.19 (s, 1H), 8.56
(s, 1H), 7.58 (d, J ¼ 2.3 Hz, 1H), 7.53 (s, 1H), 7.50e7.45 (m, 2H), 7.27
(dd, J ¼ 1.5, 8.3 Hz, 1H), 7.20 (d, J ¼ 8.9 Hz, 1H), 6.90 (dd, J ¼ 2.3,
9.1 Hz, 1H), 6.81 (dd, J ¼ 1.3, 8.0 Hz, 1H), 6.74 (dq, J ¼ 7.1, 13.9 Hz,
3H), 6.55 (t, J ¼ 7.7 Hz, 1H), 6.18 (t, J ¼ 7.5 Hz, 1H); 13C NMR
4.1.2.1.34. 1-(2-bromophenyl)-3-(2-phenylquinoxalin-6-yl)urea
(66). Yield ¼ 59%; 1H NMR (500 MHz, DMSO‑d6)
d 10.08 (s, 1H),
9.48 (s, 1H), 8.50e8.36 (m, 2H), 8.29 (d, J ¼ 7.5 Hz, 2H), 8.07 (t,
J ¼ 8.3 Hz, 2H), 7.91e7.77 (m, 1H), 7.64 (d, J ¼ 8.0 Hz, 1H), 7.56 (dt,
J ¼ 7.4, 24.5 Hz, 3H), 7.38 (t, J ¼ 7.8 Hz, 1H), 7.03 (t, J ¼ 7.7 Hz, 1H);
(125 MHz, DMSO‑d6)
d 152.1, 151.1, 142.5, 141.3, 141.2, 137.5, 136.7,
136.2, 132.5, 130.3, 129.4, 129.1, 128.1, 127.3, 124.5, 123.0, 122.6,
113.8, 113.5; HRMS (ESI-MS) calcd for C21H16BrN4Oþ m/z (M þ H)þ
419.0502, found: 419.0507.
13C NMR (125 MHz, DMSO‑d6)
d 152.2, 148.9, 143.8, 142.1, 140.8,
137.7, 136.8,136.3,132.5,129.9,129.7, 129.1, 128.1, 127.0, 124.6,123.7,
122.8, 113.8, 113.5; HRMS (ESI-MS) calcd for C21H16BrN4Oþ m/z
(M þ H)þ 419.0502, found: 419.0505.
4.1.2.1.27. 1-(4-methoxyphenyl)-3-(3-phenylquinoxalin-6-yl)urea
(59). Yield ¼ 59%; 1H NMR (500 MHz, DMSO‑d6)
d 9.38 (s, 1H), 9.21
(s, 1H), 8.71 (s, 1H), 8.35 (d, J ¼ 2.3 Hz, 1H), 8.34e8.26 (m, 2H), 8.01
(d, J ¼ 9.0 Hz, 1H), 7.77 (dd, J ¼ 2.4, 9.0 Hz, 1H), 7.66e7.52 (m, 3H),
7.46e7.34 (m, 2H), 6.97e6.85 (m, 2H), 3.73 (s, 3H); 13C NMR
4.1.2.1.35. 1-(4-methoxyphenyl)-3-(2-phenylquinoxalin-6-yl)urea
(67). Yield ¼ 51%; 1H NMR (500 MHz, DMSO‑d6)
d 10.18 (s, 1H), 9.66
(s, 1H), 9.47 (s, 1H), 8.40 (m, 2H), 8.30 (d, J ¼ 7.6 Hz, 2H), 8.04 (d,
J ¼ 8.0 Hz, 1H), 7.91 (dd, J ¼ 8.0 Hz, 1H), 7.63e7.50 (m, 6H), 3.71 (s,
(125 MHz, CDCl3)
d 155.0, 152.8, 151.4, 142.8, 142.0, 137.6, 136.5,
132.5, 130.5, 129.4, 129.3, 127.6, 123.4, 120.7, 114.3, 113.6, 55.4;
HRMS (ESI-MS) calcd for C22H19N4Oþ2 m/z (M þ H)þ 371.1503,
found: 371.1509.
3H). 13C NMR (126 MHz, CDCl3)
d 154.7, 152.5, 148.7, 143.7, 142.1,
141.1, 137.5, 136.3, 132.2, 129.8, 129.6, 129.0, 127.0, 123.8, 120.4,
114.0, 113.1, 55.2; HRMS (ESI-MS) calcd for C22H19N4Oþ2 m/z
(M þ H)þ 371.1503, found: 375.1511.
4.1.2.1.28. 1-(3-Phenylquinoxalin-6-yl)-3-(4-(trifluoromethyl)
phenyl)urea (60). Yield ¼ 53%; 1H NMR (500 MHz, DMSO‑d6)
d
8.60
4.1.2.1.36. 1-(2-Phenylquinoxalin-6-yl)-3-(4-(trifluoromethyl)
(s, 1H), 8.57 (s, 1H), 8.53 (s, 1H), 7.55 (d, J ¼ 2.3 Hz, 1H), 7.52e7.45
(m, 2H), 7.20 (d, J ¼ 9.0 Hz, 1H), 6.97 (dd, J ¼ 2.4, 9.0 Hz, 1H), 6.89 (d,
J ¼ 8.6 Hz, 2H), 6.84 (d, J ¼ 8.6 Hz, 2H), 6.79e6.68 (m, 3H); 13C NMR
phenyl)urea (68). Yield ¼ 63%; 1H NMR (500 MHz, DMSO‑d6)
d 9.50
(s, 1H), 9.43 (s, 1H), 9.34 (s, 1H), 8.37 (d, J ¼ 1.9 Hz, 1H), 8.35e8.25
(m, 2H), 8.08 (d, J ¼ 9.0 Hz, 1H), 7.92e7.80 (m, 2H), 7.73 (d,
J ¼ 8.5 Hz, 2H), 7.68 (d, J ¼ 8.5 Hz, 2H), 7.64e7.48 (m, 3H); 13C NMR
(125 MHz, DMSO‑d6)
d 152.2, 151.1, 143.1, 142.4, 141.4, 141.1, 137.5,
136.2, 130.3, 129.3, 129.1, 127.3, 126.1, 123.4, 123.2, 118.1, 114.0;
HRMS (ESI-MS) calcd for C22H16F3N4Oþ m/z (M þ H)þ 409.1271,
found: 409.1277.
(125 MHz, DMSO‑d6) d 152.2, 149.0, 143.8, 143.1, 142.0, 140.5, 137.8,
136.2, 129.9, 129.7, 129.0, 127.0, 126.1, 123.9, 118.1, 113.8; HRMS (ESI-
MS) calcd for
409.1279.
C
22H16F3N4Oþm/z (M
þ
H)þ 409.1271, found:
4.1.2.1.29. 1-(2,4-bis(trifluoromethyl)phenyl)-3-(3-
phenylquinoxalin-6-yl)urea (61). Yield ¼ 82%; 1H NMR (500 MHz,
4.1.2.1.37. 1-(2,4-bis(trifluoromethyl)phenyl)-3-(2-
DMSO‑d6)
d
9.64 (s, 1H), 9.61 (s, 1H), 9.43 (s, 1H), 8.40 (d, J ¼ 2.3 Hz,
phenylquinoxalin-6-yl)urea (69). Yield ¼ 66%; 1H NMR (500 MHz,
1H), 8.37e8.30 (m, 2H), 8.20 (s, 2H), 8.05 (d, J ¼ 9.0 Hz, 1H), 7.85
DMSO‑d6) d 10.62 (s, 1H), 10.48 (s, 1H), 9.38 (s, 1H), 8.31 (d,
(dd, J ¼ 2.4, 9.0 Hz, 1H), 7.69 (s, 1H), 7.64e7.50 (m, 3H); 13C NMR
J ¼ 2.1 Hz, 1H), 8.28e8.19 (m, 2H), 8.16 (s, 2H), 7.93e7.76 (m, 2H),
(125 MHz, DMSO‑d6)
d
152.4, 151.2, 142.5, 141.5, 137.6, 136.2, 130.3,
7.59e7.35 (m, 4H); 13C NMR (125 MHz, DMSO‑d6)
d
152.5, 148.7,
129.2, 129.1, 127.3, 123.3, 118.3, 114.6; HRMS (ESI-MS) calcd for
C
143.5, 142.0, 141.9, 140.8, 137.6, 136.3, 130.6, 130.4, 130.1, 129.8,
129.2, 129.0, 127.0, 124.3, 124.0, 122.2, 117.9, 114.1, 114.0; HRMS (ESI-
MS) calcd for C23H15F6N4Oþ m/z (M þ H)þ 477.1145, found:
477.1149.
23H15F6N4Oþ m/z (M þ H)þ 477.1145, found: 477.1151.
4.1.2.1.30. 1-(4-fluorophenyl)-3-(2-phenylquinoxalin-6-yl)urea
(62). Yield ¼ 68%; 1H NMR (500 MHz, DMSO‑d6)
d 9.48 (s, 1H), 9.28
10

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
4.1.2.1.38. 1-(4-bromophenyl)-3-(3-(thiophen-2-yl)quinoxalin-6-
d
159.77, 152.41, 147.36, 142.77, 141.40, 141.28, 140.61, 138.01, 136.72,
yl)urea (70). Yield ¼ 81%; 1H NMR (500 MHz, DMSO‑d6)
d
9.39 (s,
130.98, 129.69, 129.30, 121.89, 120.28, 113.21, 110.82, 107.70, 104.29,
55.01, 41.39; HRMS (ESI-MS) calcd for C20H19N6Oþ2 m/z (M þ H)þ
375.1564, found: 375.1564.
1H), 9.29 (s, 1H), 9.06 (s, 1H), 8.26 (d, J ¼ 2.4 Hz,1H), 8.20 (dd, J ¼ 1.1,
3.8 Hz, 1H), 7.99 (d, J ¼ 9.0 Hz, 1H), 7.86 (dd, J ¼ 1.0, 5.0 Hz, 1H), 7.72
(dd, J ¼ 2.4, 9.1 Hz, 1H), 7.50 (s, 4H), 7.30 (dd, J ¼ 3.7, 5.0 Hz, 1H);
HRMS (ESI-MS) calcd for C19H14BrN4OS þ m/z (M þ H)þ 425.0066,
found: 425.0066.
4.1.2.1.46. 1-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)-3-
(3-(trifluoromethoxy)phenyl)urea (78). Yield
(500 MHz, DMSO‑d6)
¼
55%; 1H NMR
9.41 (s, 1H), 9.20 (s, 1H), 9.13 (d, J ¼ 7.3 Hz,
d
4.1.2.1.39. 1-(4-bromophenyl)-3-(2-(thiophen-2-yl)quinoxalin-6-
1H), 8.62 (d, J ¼ 5.6 Hz, 1H), 8.30e8.23 (m, 2H), 7.96 (dd, J ¼ 15.0,
9.0 Hz, 1H), 7.74 (dd, J ¼ 5.7, 3.1 Hz, 1H), 7.68 (dd, J ¼ 9.2, 2.4 Hz, 1H),
7.47e7.29 (m, 2H), 6.98 (t, J ¼ 9.6 Hz, 1H), 3.95 (d, J ¼ 4.9 Hz, 3H);
yl)urea (71). Yield ¼ 57%; 1H NMR (500 MHz, DMSO‑d6)
d 9.48 (s,
1H), 9.30 (s, 1H), 9.05 (s, 1H), 8.31 (d, J ¼ 2.3 Hz, 1H), 8.15 (d,
J ¼ 3.6 Hz, 1H), 7.97 (d, J ¼ 9.0 Hz, 1H), 7.84e7.71 (m, 2H), 7.50 (s,
4H), 7.28 (dd, J ¼ 3.7, 5.0 Hz, 1H); 13C NMR (125 MHz, DMSO‑d6)
13C NMR (125 MHz, DMSO‑d6)
d 152.84, 149.21, 147.85, 143.15,
141.99, 141.71, 141.47, 138.45, 137.25, 131.43, 130.95, 129.82, 129.75,
122.34, 120.69, 117.54, 114.51, 113.99, 110.89, 40.26. MS calcd for
d
152.3, 145.3, 142.9, 142.0, 141.7, 140.5, 138.8, 137.4, 131.6, 130.1,
129.0, 128.8, 127.6, 123.9, 120.4, 113.9, 113.7; HRMS (ESI-MS) calcd
for C19H14BrN4OS þ m/z (M þ H)þ 425.0066, found: 425.0068.
4.1.2.1.40. 1-(4-bromophenyl)-3-(3-(thiophen-3-yl)quinoxalin-6-
C
20H15F3N6O2 m/z 428.12, found: 429.09 (Mþ1).
4.1.2.1.47. 1-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)-3-
(3-(trifluoromethyl)phenyl)urea (79). Yield
(500 MHz, DMSO‑d6) 9.44 (s, 1H), 9.36 (s, 1H), 9.12 (s, 1H), 8.61 (s,
¼
71%; 1H NMR
yl)urea (72). Yield ¼ 52%; 1H NMR (500 MHz, DMSO‑d6)
d
9.36 (s,
d
1H), 9.30 (s, 1H), 9.05 (s, 1H), 8.61 (dd, J ¼ 1.3, 2.8 Hz, 1H), 8.33 (d,
J ¼ 2.4 Hz, 1H), 8.03e7.96 (m, 2H), 7.78 (dd, J ¼ 2.9, 5.1 Hz, 1H), 7.73
(dd, J ¼ 2.4, 9.0 Hz, 1H), 7.50 (s, 4H); 13C NMR (125 MHz, DMSO‑d6)
1H), 8.29e8.24 (m, 2H), 8.06 (d, J ¼ 2.5 Hz, 1H), 7.94 (d, J ¼ 9.0 Hz,
1H), 7.71 (dd, J ¼ 9.0, 2.3 Hz, 1H), 7.65 (d, J ¼ 8.2 Hz, 1H), 7.55 (t,
J ¼ 8.0 Hz, 1H), 7.35 (d, J ¼ 7.7 Hz, 1H), 3.95 (s, 3H); 13C NMR
d
151.8, 147.4, 142.0, 141.2, 140.7, 138.8, 138.3, 136.6, 131.1, 131.0,
(125 MHz, DMSO‑d6) d 152.95, 147.81, 143.12, 141.99, 141.45, 140.74,
128.7, 127.2, 126.4, 125.9, 122.1, 119.8, 113.1, 113.1; HRMS (ESI-MS)
calcd for C19H14BrN4OS þ m/z (M þ H)þ 425.0066, found: 425.0071.
4.1.2.1.41. 1-(4-bromophenyl)-3-(2-(thiophen-3-yl)quinoxalin-6-
138.44, 137.24, 131.40, 130.48, 129.73, 122.61, 122.40, 120.67, 114.88,
114.06, 79.59, 41.80; MS calcd for C20H15F3N6O m/z 412.13, found:
413.17 (Mþ1).
yl)urea (73). Yield ¼ 59%; 1H NMR (500 MHz, DMSO‑d6)
d
9.43 (s,
4.1.2.1.48. 1-(3-Chloro-4-fluorophenyl)-3-(3-(1-methyl-1H-pyr-
1H), 9.29 (s, 1H), 9.04 (s, 1H), 8.52 (d, J ¼ 2.9 Hz, 1H), 8.30 (d,
J ¼ 2.3 Hz, 1H), 8.00 (d, J ¼ 9.0 Hz, 1H), 7.95 (d, J ¼ 5.0 Hz, 1H), 7.80
azol-4-yl)quinoxalin-6-yl)urea (80). Yield
¼
72%; 1H NMR
(500 MHz, DMSO‑d6) 9.44 (s, 1H), 9.24 (s, 1H), 9.12 (s, 1H), 8.61 (s,
d
(dd, J ¼ 2.4, 9.1 Hz, 1H), 7.76 (dd, J ¼ 2.9, 5.1 Hz, 1H), 7.49 (s, 4H); 13
C
1H), 8.28e8.22 (m, 2H), 7.93 (d, J ¼ 9.0 Hz, 1H), 7.85 (dd, J ¼ 6.8,
2.4 Hz, 1H), 7.70 (dd, J ¼ 9.0, 2.4 Hz, 1H), 7.43e7.30 (m, 2H), 3.95 (s,
NMR (125 MHz, DMSO‑d6)
d 152.3, 145.9, 144.3, 141.7, 140.4, 139.3,
138.8, 137.6, 131.6 (2C), 129.3, 127.7, 126.3, 126.1, 123.7, 120.4 (2C),
113.8,113.7; HRMS (ESI-MS) calcd for C19H14BrN4OS þ m/z (M þ H)þ
425.0066, found: 425.0074.
3H); 13C NMR (125 MHz, DMSO‑d6)
d 153.50, 152.46, 151.58, 147.32,
142.67, 141.45, 137.96, 136.78, 130.92, 129.24, 121.85, 120.22, 119.82,
118.84, 117.02, 116.94, 116.84, 116.77, 113.45, 42.10; HRMS (ESI-MS)
calcd for C19H15ClFN6Oþ m/z (M þ H)þ 397.0974, found: 397.0979.
4.1.2.1.49. 1-(4-Fluoro-3-methylphenyl)-3-(3-(1-methyl-1H-pyr-
4.1.2.1.42. 1-(4-bromophenyl)-3-(2-(1-methyl-1H-pyrazol-3-yl)
quinoxalin-6-yl)urea (74). Yield
¼
80%; 1H NMR (500 MHz,
DMSO‑d6)
d
9.35 (s, 1H), 9.30 (s, 1H), 9.06 (s, 1H), 8.34 (d, J ¼ 2.3 Hz,
azol-4-yl)quinoxalin-6-yl)urea (81). Yield
¼
57%; 1H NMR
1H), 8.05 (d, J ¼ 9.0 Hz, 1H), 7.85 (dd, J ¼ 2.4, 9.1 Hz, 1H), 7.61 (d,
(500 MHz, DMSO‑d6) 9.20 (s, 1H), 9.11 (s, 1H), 8.83 (s, 1H), 8.60 (s,
d
J ¼ 2.0 Hz, 1H), 7.50 (s, 4H), 7.19 (d, J ¼ 2.0 Hz, 1H), 4.30 (s, 3H); 13
C
1H), 8.25 (d, J ¼ 13.3 Hz, 2H), 7.92 (d, J ¼ 9.0 Hz, 1H), 7.66 (d,
J ¼ 9.1 Hz, 1H), 7.44e7.38 (m, 1H), 7.34e7.29 (m, 1H), 7.08 (t,
J ¼ 9.1 Hz, 1H), 3.95 (s, 3H), 2.24 (s, 3H); 13C NMR (125 MHz,
NMR (125 MHz, DMSO‑d6)
d 151.7, 144.7, 141.9, 140.9, 140.6, 138.2,
137.6, 137.5, 136.2, 131.1, 129.0, 123.4, 119.9, 113.2, 113.1, 107.4; HRMS
(ESI-MS) calcd for C19H16BrN6Oþ m/z (M þ H)þ 423.0563, found:
423.0571.
DMSO‑d6)
d 157.74, 155.84, 153.10, 147.87, 143.30, 141.89, 138.53,
137.22, 135.87, 131.49, 129.80, 124.97, 122.41, 122.15, 120.79, 118.35,
115.66, 113.72, 39.51, 14.98; MS calcd for C20H17FN6O m/z 376.14,
found: 377.10 (Mþ1).
4.1.2.1.43. 1-(4-fluorophenyl)-3-(3-(1-methyl-1H-pyrazol-4-yl)
quinoxalin-6-yl)urea (75). Yield
¼
61%; 1H NMR (500 MHz,
DMSO‑d6) 9.20 (s, 1H), 9.12 (s, 1H), 8.91 (s, 1H), 8.62 (s, 1H),
d
4.1.2.1.50. 1-(4-Fluoro-3-methoxyphenyl)-3-(3-(1-methyl-1H-
8.34e8.18 (m, 2H), 7.94 (d, J ¼ 9.0 Hz, 1H), 7.68 (dd, J ¼ 9.0, 2.3 Hz,
pyrazol-4-yl)quinoxalin-6-yl)urea (82). Yield
¼
65%; 1H NMR
1H), 7.61e7.46 (m, 2H), 7.17 (t, J ¼ 8.7 Hz, 2H), 3.96 (s, 3H) ppm; 13
C
(500 MHz, DMSO‑d6) 9.24 (s, 1H), 9.11 (s, 1H), 8.96 (s, 1H), 8.61 (s,
d
NMR (100 MHz, CDCl3)
d
151.8, 144.2, 142.1, 141.2, 141.0, 138.2, 137.6,
1H), 8.31e8.24 (m, 2H), 7.93 (d, J ¼ 9.0 Hz, 1H), 7.67 (dd, J ¼ 9.0,
2.4 Hz, 1H), 7.46 (dd, J ¼ 8.0, 2.5 Hz, 1H), 7.14 (dd, J ¼ 11.4, 8.7 Hz,
137.4, 136.2, 131.1 (2C), 128.8, 122.9, 120.0 (2C), 113.2, 113.1, 107.9,
38.5; HRMS (ESI-MS) calcd for C19H15FN6Oþ m/z (M þ H)þ 362.1291,
found: 362.1299.
1H), 6.94 (ddd, J ¼ 8.8, 3.7, 2.5 Hz, 1H), 3.95 (s, 3H), 3.84 (s, 2H); 13
C
NMR (125 MHz, DMSO‑d6)
d 152.24, 147.04, 146.73, 145.83, 142.45,
4.1.2.1.44. 1-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)-3-
141.10, 137.71, 136.43, 135.94, 135.91, 130.68, 128.97, 121.58, 119.97,
115.53, 112.95, 110.00, 104.44, 55.55, 40.20; HRMS (ESI-MS) calcd
for C20H18FN6Oþ2 m/z (M þ H)þ 393.1470, found: 393.1482.
4.1.2.1.51. 1-(4-Fluoro-3-(trifluoromethoxy)phenyl)-3-(3-(1-
methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)urea (83).
(p-tolyl)urea (76). Yield ¼ 70%; 1H NMR (500 MHz, CDCl3)
d 9.16 (s,
1H), 9.11 (s, 1H), 8.76 (s, 1H), 8.62 (s, 1H), 8.27 (s, 1H), 8.24 (d, 1H,
J ¼ 2.0 Hz), 7.93 (d, 1H, J ¼ 9.0 Hz), 7.66 (dd, 1H, J ¼ 9.0, 2.0 Hz), 7.40
(d, 2H, J ¼ 8.5 Hz), 7.13 (d, 2H, J ¼ 8.5 Hz), 3.96 (s, 3H), 2.27 (s, 3H);
13C NMR (100 MHz, CDCl3)
d
151.9, 166.8, 142.2, 140.9, 140.7, 137.4,
Yield ¼ 60%; 1H NMR (400 MHz, DMSO‑d6)
d 9.32 (s, 1H), 9.20 (s,
136.3, 136.1, 130.6, 130.4, 128.7 (2C), 121.3, 119.7, 118.0 (2C), 117.7,
112.5, 38.5, 19.8; HRMS (ESI-MS) calcd for C20H19N6O þ m/z
(M þ H)þ 358.1542, found: 358.1551.
1H), 9.12 (s, 1H), 8.61 (s, 1H), 8.29e8.21 (m, 2H), 7.97e7.86 (m, 2H),
7.69 (dd, J ¼ 9.0, 2.4 Hz, 1H), 7.46 (t, J ¼ 9.6 Hz,1H), 7.39 (ddd, J ¼ 9.1,
4.2, 2.6 Hz,1H), 3.95 (s, 3H); 13C NMR (125 MHz, DMSO‑d6)
d 153.00,
4.1.2.1.45. 1-(3-methoxyphenyl)-3-(3-(1-methyl-1H-pyrazol-4-yl)
138.59, 137.45, 131.62, 127.54, 124.95, 123.03, 120.08, 119.96, 119.90,
117.27, 117.09, 114.24, 108.03; MS calcd for C20H14F4N6O2 m/z 446.11,
found: 447.02 (Mþ1).
quinoxalin-6-yl)urea (77). Yield
¼
61%; 1H NMR (400 MHz,
DMSO‑d6) 9.17 (s, 1H), 9.10 (s, 1H), 8.87 (s, 1H), 8.60 (s, 1H),
d
8.28e8.21 (m, 2H), 7.92 (d, J ¼ 9.0 Hz, 1H), 7.65 (dd, J ¼ 9.0, 2.4 Hz,
1H), 7.26e7.16 (m, 2H), 6.98 (dd, J ¼ 7.8, 2.0 Hz,1H), 6.59 (dd, J ¼ 8.2,
2.5 Hz, 1H), 3.94 (s, 3H), 3.75 (s, 3H); 13C NMR (125 MHz, DMSO‑d6)
4.1.2.1.52. 1-(4-Fluoro-3-(trifluoromethyl)phenyl)-3-(3-(1-
methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)urea (84).
Yield ¼ 64%; 1H NMR (600 MHz, DMSO‑d6)
d 9.63 (s, 1H), 9.11 (s,
11

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
1H), 9.01 (s, 1H), 8.60 (s, 1H), 8.46 (m, 1H), 8.27 (d, J ¼ 2.9 Hz, 1H),
8.26 (s, 1H), 7.94 (d, J ¼ 8.9 Hz, 1H), 7.62 (dd, J ¼ 9.0, 2.3 Hz, 1H),
7.41e7.37 (m, 2H), 4.01e3.82 (s, 3H); 13C (125 MHz, DMSO‑d6)
(M þ H)þ 440.0517, found: 440.0518.
4.1.2.1.58. 1-(benzo[d] [1,3]dioxol-5-yl)-3-(3-(1-methyl-1H-pyr-
azol-4-yl)quinoxalin-6-yl)urea (87). Yield
(500 MHz, DMSO‑d6)
¼
70%; 1H NMR
d
151.97, 149.89, 148.23, 147.39, 142.66, 141.61, 140.64, 137.98,
d
9.13 (d, J ¼ 26.8 Hz, 2H), 8.77 (s, 1H), 8.60 (s,
136.82, 130.97, 129.41, 128.67, 128.61, 125.36, 125.01, 124.98, 123.56,
121.76, 121.64, 120.17, 119.95, 119.45, 116.83, 116.76, 116.61, 116.54,
1H), 8.33e8.13 (m, 2H), 7.92 (d, J ¼ 9.0 Hz, 1H), 7.65 (dd, J ¼ 8.9,
2.4 Hz, 1H), 7.25 (d, J ¼ 2.1 Hz, 1H), 6.93e6.80 (m, 2H), 5.97 (d,
J ¼ 16.7 Hz, 2H), 3.95 (s, 3H); 13C NMR (125 MHz, DMSO‑d6)
113.43, 38.89; 19F NMR (564 MHz, DMSO‑d6)
d
ꢃ119.00 (3F), ꢃ60.68
(1F); HRMS (ESI-MS) calcd for C20H15F4N6Oþ m/z (M þ H)þ
d 152.99,147.75,147.73,143.21,142.84,141.85,138.42,137.09,134.14,
431.1238, found: 431.1240.
131.39, 129.69, 122.29, 120.70, 113.50, 111.89, 108.64, 101.67, 101.37,
40.53, 39.36; MS calculated for C20H16N6O3 m/z 388.13, found:
389.15 (Mþ1).
4.1.2.1.53. 3-Bromo-6-nitroquinoline (32). 4-nitroaniline (5.0 g,
36.2 mmol) in acetic acid (35 mL) was treated with 2,2,3 tri-
bromopropanal (11.7 g, 39.8 mmol) and the mixture was heated at
110 ꢀC for 2 h. AcOH was evaporated in vacuum followed by
extraction of crude with ethyl acetate and water. Organic layer was
washed with sat. NaHCO3 and brine, respectively and dried over
Na2SO4. Crude product was purified by column chromatography
using ethyl acetate and hexane as eluting solvents; Yield ¼ 38.2%
4.1.2.1.59. 1-(2,3-dihydrobenzo[b] [1,4]dioxin-6-yl)-3-(3-(1-
methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)urea (88). Yield ¼ 61%; 1H
NMR (500 MHz, DMSO‑d6)
d
9.12 (d, J ¼ 15.1 Hz, 2H), 8.69 (s, 1H),
8.61 (s, 1H), 8.31e8.14 (m, 2H), 7.92 (d, J ¼ 9.0 Hz, 1H), 7.65 (dd,
J ¼ 9.0, 2.4 Hz, 1H), 7.14 (d, J ¼ 2.5 Hz, 1H), 6.94e6.70 (m, 2H),
4.37e4.14 (m, 4H), 3.95 (s, 3H); 13C NMR (125 MHz, DMSO‑d6)
(3.5 g); 1H NMR (400 MHz, DMSO‑d6)
d
9.23 (dt, J ¼ 17.1, 8.6 Hz, 1H),
d 152.91, 147.75, 143.60, 143.22, 141.89, 141.71, 139.20, 138.41, 137.08,
9.14 (d, J ¼ 2.4 Hz, 1H), 8.89e8.73 (m, 2H), 7.86 (dd, J ¼ 8.3, 4.2 Hz,
1H); HRMS Calculated for C9H6BrN2O2þ m/z 252.9607, found
mass ¼ 252.9611.
133.41, 131.38, 129.68, 122.28, 120.71, 117.38, 113.42, 112.40, 108.27,
64.71, 64.37, 40.38; MS calculated for C21H18N6O3 m/z 402.14,
found: 403.13 (Mþ1).
4.1.2.1.54. 3-(2-Methyl-1H-2l4-pyrazol-4-yl)-6-nitroquinoline
(33). Compound 32 (250 mg, 0.987 mmol), Na2CO3 (209.4 mg,
1.97 mmol), DMF (3 mL), Dioxane (3 mL) and water (0.2 mL) were
mixed together in a round bottom flask. The mixture was sonicated
and degassed for 5 min by passing nitrogen through it. Degassing
was followed by addition of Pd(Ph3)4 (57.0 mg, 0.04 mmol) and 1-
Methylpyrazole-4-boronic acid pinacol ester (246.6 mg,
1.185 mmol). The reaction mixture was stirred under inert atmo-
sphere at 100 ꢀC overnight and quenched by addition of water to
the reaction mixture. Precipitates formed were filtered and tritu-
rated in THF and hexane; Yield ¼ 47.8% (120 mg); 1H NMR
4.1.2.1.60. 1-(benzo[b]thiophen-4-yl)-3-(3-(1-methyl-1H-pyr-
azol-4-yl)quinoxalin-6-yl)urea (89). Yield
¼
52%; 1H NMR
(500 MHz, DMSO‑d6) 9.28 (s, 1H), 9.11 (s, 1H), 9.02 (s, 1H), 8.61 (s,
d
1H), 8.27 (d, J ¼ 2.0 Hz, 2H), 8.15 (d, J ¼ 2.0 Hz,1H), 7.93 (t, J ¼ 9.0 Hz,
2H), 7.75 (d, J ¼ 5.4 Hz, 1H), 7.70 (dd, J ¼ 9.0, 2.4 Hz, 1H), 7.46e7.39
(m, 2H), 3.95 (s, 3H); 13C NMR (125 MHz, DMSO‑d6)
d 152.69,147.34,
142.78, 141.47, 141.38, 140.15, 138.04, 136.72, 136.35, 133.14, 131.00,
129.31, 128.25, 124.05, 122.79, 121.93, 120.27, 117.13, 113.16, 112.76,
41.40; MS calculated for C21H16N6OS m/z 400.11, found: 401.02
(Mþ1).
4.1.2.1.61. 1-(2,3-Dihydro-1H-inden-4-yl)-3-(3-(1-methyl-1H-
(400 MHz, CDCl3)
d
9.15 (dd, J ¼ 4.1, 1.8 Hz, 1H), 8.65 (dd, J ¼ 12.2,
pyrazol-4-yl)quinoxalin-6-yl)urea (90). Yield
¼
56%; 1H NMR
2.5 Hz, 2H), 8.56 (s, 1H), 8.38 (dd, J ¼ 8.3, 1.7 Hz, 1H), 8.23 (s, 1H),
(500 MHz, DMSO‑d6) 9.16 (s, 1H), 9.10 (s, 1H), 8.74 (s, 1H), 8.60 (s,
d
7.62 (dd, J ¼ 8.3, 4.2 Hz, 1H), 4.07 (s, 3H); 13C NMR (100 MHz, CDCl3)
1H), 8.28e8.21 (m, 2H), 7.92 (d, J ¼ 8.9 Hz, 1H), 7.66 (dd, J ¼ 9.0,
2.4 Hz, 1H), 7.42 (d, J ¼ 1.9 Hz, 1H), 7.20 (dd, J ¼ 8.1, 2.1 Hz, 1H), 7.15
(d, J ¼ 8.1 Hz, 1H), 3.95 (s, 3H), 2.83 (dt, J ¼ 22.5, 7.4 Hz, 4H), 2.54 (s,
d
152.6, 147.1, 139.3, 138.4, 134.2, 132.0, 122.7, 121.8, 119.5, 39.2;
HRMS Calculated for
C
14H13N3Oþ2 m/z 255.1002, found
mass ¼ 255.0891.
2H), 2.01 (p, J ¼ 7.4 Hz, 2H); 13C NMR (125 MHz, DMSO‑d6)
d 152.45,
4.1.2.1.55. 3-(1-Methyl-1H-pyrazol-4-yl)quinolin-6-amine (34).
Compound 34 was synthesized according to Method B;
147.25, 144.27, 142.74, 141.44, 141.21, 137.92, 137.48, 137.41, 136.59,
130.88, 129.19, 124.24, 121.75, 120.22, 116.75, 114.78, 112.92, 40.43,
32.55, 31.70, 25.21; MS calculated for C22H20N6O m/z 384.17, found:
385.13 (Mþ1).
Yield ¼ Quantitative; 1H NMR (400 MHz, DMSO‑d6)
d 8.59e8.53 (m,
1H), 8.50 (s, 1H), 7.99 (s, 1H), 7.94 (d, J ¼ 8.3 Hz, 1H), 7.42 (d,
J ¼ 2.4 Hz, 1H), 7.30 (dd, J ¼ 8.3, 4.1 Hz, 1H), 6.70 (d, J ¼ 2.3 Hz, 1H),
5.51 (s, 2H), 3.93 (s, 3H); HRMS Calculated for C13H13Nþ4 m/z
225.1135, found mass ¼ 225.1208.
4.1.2.1.62. 1-(2,3-Dihydrobenzofuran-4-yl)-3-(3-(1-methyl-1H-
pyrazol-4-yl)quinoxalin-6-yl)urea (91). Yield
(400 MHz, DMSO‑d6)
¼
66%; 1H NMR
d
9.11 (d, J ¼ 14.7 Hz, 2H), 8.62 (d, J ¼ 13.3 Hz,
4.1.2.1.56. 1-(3-Bromo-4-fluorophenyl)-3-(3-(1-methyl-1H-pyr-
azol-4-yl)quinolin-6-yl)urea (85). Compound 86 was synthesized
according to Method D; Yield ¼ 32%; 1H NMR (400 MHz, CDCl3)
2H), 8.30e8.18 (m, 2H), 7.91 (d, J ¼ 9.0 Hz, 1H), 7.66 (dd, J ¼ 9.0,
2.4 Hz, 1H), 7.41 (d, J ¼ 2.2 Hz, 1H), 7.13 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.71
(d, J ¼ 8.5 Hz, 1H), 4.50 (t, J ¼ 8.6 Hz, 2H), 3.95 (s, 3H), 3.18 (t,
d
8.77 (d, J ¼ 2.1 Hz, 2H), 8.72 (s, 1H), 8.34 (s, 1H), 8.07e7.90 (m, 3H),
J ¼ 8.6 Hz, 2H); 13C NMR (125 MHz, DMSO‑d6)
d 155.71, 153.15,
7.76 (dd, J ¼ 6.0, 2.4 Hz, 1H), 7.73 (d, J ¼ 2.0 Hz, 1H), 7.36 (m, 1H),
147.69, 143.19, 142.09, 141.64, 138.42, 137.03, 132.56, 131.38, 129.66,
128.01, 122.28, 120.68, 119.36, 117.33, 113.27, 109.06, 71.29, 40.37,
29.92; MS calculated for C21H18N6O2 m/z 386.15, found: 387.07
(Mþ1).
7.33e7.21 (m, 1H), 6.96 (t, J ¼ 8.5 Hz, 1H), 3.95 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d 153.2, 147.5, 141.6, 138.7, 137.0, 136.3, 136.0,
131.7, 131.6, 129.7, 123.7, 121.3, 120.9, 119.6, 118.9, 116.3116.1, 113.2,
108.8, 108.6, 46.3; HRMS (ESI-MS) calcd for C20H16BrFN5Oþ m/z
(M þ H)þ 440.0517, found: 440.0511.
4.2. Biology
4.1.2.1.57. 1-(3-Bromo-4-fluorophenyl)-3-(3-(1-methyl-1H-pyr-
rol-3-yl)quinoxalin-6-yl)urea (86). Yield ¼ 65%; 1H NMR (400 MHz,
4.2.1.
kB luciferase reporter assay
DMSO‑d6)
d
9.27 (s, 1H), 9.09 (s, 1H), 9.06 (s, 1H), 8.18 (d, J ¼ 2.3 Hz,
The following modifications were made to the previously re-
ported method [16]. Cells were pre-incubated with compounds for
30 min, which was followed by a 6 h TNFa stimulation.
1H), 7.98 (dd, J ¼ 6.3, 2.6 Hz, 1H), 7.89 (d, J ¼ 9.0 Hz, 1H), 7.78 (t,
J ¼ 2.0 Hz, 1H), 7.63 (dd, J ¼ 9.0, 2.4 Hz, 1H), 7.42 (ddd, J ¼ 9.0, 4.5,
2.7 Hz, 1H), 7.34 (t, J ¼ 8.8 Hz, 1H), 6.90 (t, J ¼ 2.5 Hz, 1H), 6.84 (t,
J ¼ 2.0 Hz, 1H), 3.73 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
158.21,
4.2.2. Caspase 3/7 assay
157.98,154.38,152.79,152.40,149.32,140.80,136.96,136.26,129.09,
124.12, 123.77, 122.55, 121.35, 120.93, 119.35, 116.77, 116.62, 113.53,
MiaPaCa2 cells (2.5 ꢁ 104) were plated in 96-well white walled
clear bottom plate and allowed to attach overnight. DMSO solutions
of the inhibitors were added (final concentration of DMSO 0.1%)
107.24, 36.15; HRMS (ESI-MS) calcd for
C
20H16BrFN5Oþ m/z
12

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
and incubated for 24 h. 21 h post drug addition, alamarBlue®
(Biorad, BUF012B) was added at a volume of 1/10 total volume per
well (10 mL) and cells were allowed to incubate for 3 h. The plate
was then read for fluorescence at 560ex/590em on a Spectrmax
m5e. The plate was then allowed to equilibrate to room tempera-
ture for 15 min. 100 mL of Caspase-Glo (Promega G8093) reagent
(0.083, 0.25, 0.5, 1, 2, 4, 8, 24 h) mice were sacrificed and the plasma
was subjected to LC/MS/MS (AB Sciex Triple Quad 5500) and the
compound levels determined using an internal standard method
(Carbamazepine). The PK parameters were calculated using non-
compartmental approach with PhoenixTM WinNonlin®, version
8.3 (Pharsight, CA).
was added and 2 h later the plate was read for luminescence at
1000 ms. Values were calculated by: ([Luminescence*100]/Fluo-
rescence)/DMSOavg [60].
4.2.9. Pancreatic tumor model
To assess in vivo efficacy of analog 84, MiaPaCa2 cells (2.5 ꢁ
106 cells and Matrigel, 2:1 ratio) were subcutaneously implanted in
left flank of athymic nude mice (Crl: NU-Foxn1nu, Charles River
laboratories, USA). After 20 days of implantation, mice were ran-
domized and grouped into the following treatment groups:
4.2.3. Immunoblotting
T3M4 cells were treated with analog 84 at indicated doses (0.5,
1.0, 2.0 and 5.0
0.5, 3.0, 6.0, 12 and 24 h) and harvested. Equal amounts of protein
(40 g) were separated on 4e20% gradient SDS-PAGE gels (Bio rad,
USA) and transferred to 0.45 m PVDF membranes (Millipore, USA).
After blocking in 5% nonfat skim milk powder for 1 h, the mem-
branes were probed with the following primary antibodies p-IKK
IKK and
-actin (Cell Signaling Technology, USA) overnight at 4 ꢀC
mM for 6 h) and the indicated time points, (5 mM at
Wehicle (100 ml daily orally), analog 84 (40 mg/kg body weight
m
daily orally), gemcitabine (15 mg/kg body weight every 72 h
intraperitoneally), and analog 84 plus gemcitabine (n ¼ 6, per
group). The tumor volume was measured every 3-days for 4-weeks
by using digital calibers. Tumor volumes were determined by
measuring (1/2 ꢁ length ꢁ width2) in cubic millimeters. After the
27th day of treatment, the mice were sacrificed, and the tumor
weight was determined.
m
b,
b
b
and secondary antibody (Horse anti-mouse and Goat anti-rabbit,
Cell Signaling Technology, USA) at room temperature for 1 h.
Membranes were visualized with chemiluminescent detection kits
(Bio rad, USA).
4.3. Proliferation index
4.2.4. Live/dead cell assay
MiaPaCa2 cells were treated with 5 and 10
24 h and subjected to live/dead cell assay as per the manufacturer's
instruction (Invitrogen, USA). Briefly, vehicle and compound 84
m
M of analog 84 for
Vehicle and analog 84 treated subcutaneous tumor tissue were
formalin-fixed, paraffin-embedded sections (5 mm) were stained
with anti-Ki-67 antibody (Abcam, USA) in Tissue Science Facility,
UNMC, USA. Results were expressed as percentage of Ki-
67epositive cells mean SEM per original magnification. A total of
4 fields per tumor were examined and counted from vehicle and
treated groups (n ¼ 5). The values were subjected to unpaired
Student t-test.
treated cells were washed with 1x PBS. 20
1 (EthD-1, 2 mM) was dissolved in 10 mL PBS. To this solution, 5
of calcein acetoxymethyl ester (calcein-AM, 4 mM) was added.
500 l of this mixture was added to the well and incubated for
ml ethidium homodimer-
ml
m
30 min. The numbers of live and dead cells were detected with
Nikon fluorescent microscopy (Nikon, Japan). The ratio of dead cells
to total cells was calculated for quantitative comparisons using
ImageJ (NIH, USA) software.
4.4. Immunohistochemistry
The paraffin-embedded vehicle and treated slides were depar-
affinized with xylene, re-hydrated with series of alcohol, antigen
retrieval in citrate buffer (pH ¼ 6.0) for 10 min heating chamber and
quenched with hydrogen peroxide. After blocking with a universal
blocker (Thermo Fisher Scientific, USA), the slides were incubated
4.2.5. Colony formation assay
MiaPaCa2 cells (1.0 ꢁ 103) were seeded in 6-well plates. Cells
were treated with 5 mM of analog 84 for 24 h. Next day cells were
washed with 1x PBS and allowed to grow for 8e10 days, with a
medium change every 3 days. The vehicle and compound 84
treated cells were fixed with 4% paraformaldehyde for 15 min,
washed and stained with 0.5% crystal violet for 2 h at room tem-
perature. The colony number was determined in triplicate after
capturing the image in 10x bright field microscope.
with antibody p-IKK
4
b (Cell Signaling Technology, USA) overnight at
ꢀC. Next day, the slides were washed and incubated with HRP
conjugated secondary antibody (Dako, USA) for 1 h at room tem-
perature. Further, the slides were washed and developed using
3,30-diaminobenzidine tetrahydrochloride (DAB, Vector Labora-
tories, USA) substrate. The slides were counterstained with hema-
toxylin, washed with water, dehydrated with alcohol series, and
after xylene washes, the slides were mounted with the coverslip.
Image were captured by bright field microscope (Nikon, Japan). The
histological scoring was performed based on staining intensity (0,
negligible; 1, low; 2, moderate; 3, high) and proportion (0%e100%).
The histoscore was created by multiplying the stain proportion
score (1, <5%; 2, 5%e25%; 3, 26%e50%; 4, 51%e75%; 5, >75%) with
the intensity score (0e3) to obtain values between 0 and 15.
4.2.6. Cell scratch assay
MiaPaCa2 cells were seeded in a 6-well plate. The next day
media was discarded, scratched with 200
ml pipette tips, washed
with 1x PBS. Scratched cells were treated with 5
mM of analog 84.
The gap generated by the pipette tip was examined and captured by
a bright-field microscope (Nikon, Japan) at 0, 24 and 48 h. The
result was analyzed using ImageJ (NIH, USA) software. The per-
centage of gap closure was calculated for quantitative comparisons.
4.2.7. Growth inhibition assay
We followed the method previously reported by us [61]. Con-
4.5. Statistical analysis
centrations (20, 10, 5, 2.5, 1.25 and 0,625 mM) of analog 84 used in
the study to derive the reported IC50 values.
Data are presented as the average SEM. Live/dead cell assay
was analyzed by 2-way ANOVA with Bonferroni's multiple com-
parisons test. Colony formation, gap closure, tumor weight, tumor
4.2.8. Pharmacokinetic studies
CD1 mice were orally administered 20 mg/kg of 13e197 or 84 in
DMSO:PEG400:H2O ¼ 1:4:5 or intravenously administered 10 mg/
kg of 84 in DMSO:PEG400:H2O ¼ 1:5:4. At following time points
volume, Ki-67 and Histoscore of p-IKKb was analyzed by unpaired
student's t-test. The p value less than 0.05 was considered statis-
tically significant.
13

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
[17] N. Gautam, S.P. Bathena, Q. Chen, A. Natarajan, Y. Alnouti, Pharmacokinetics,
protein binding and metabolism of a quinoxaline urea analog as an NF-kappaB
inhibitor in mice and rats by LC-MS/MS, Biomed. Chromatogr. 27 (2013)
900e909.
[18] N.K. Chaturvedi, R.N. Rajule, A. Shukla, P. Radhakrishnan, G.L. Todd,
A. Natarajan, J.M. Vose, S.S. Joshi, Novel treatment for mantle cell lymphoma
including therapy-resistant tumor by NF-kappaB and mTOR dual-targeting
approach, Mol. Canc. Therapeut. 12 (2013) 2006e2017.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[19] R. Rajule, V.C. Bryant, H. Lopez, X. Luo, A. Natarajan, Perturbing pro-survival
Acknowledgement
proteins using quinoxaline derivatives:
a structure-activity relationship
study, Bioorg. Med. Chem. 20 (2012) 2227e2234.
This work was supported in part by NIH grants CA182820,
CA197999, CA251151, CA208108 and CA036727. Support from the
NRI-POC is also gratefully acknowledged. JIC was supported by
UNMC fellowship and thank Dr. Divya Thomas for technical
assistance.
[20] Q. Chen, V.C. Bryant, H. Lopez, D.L. Kelly, X. Luo, A. Natarajan, 2,3-Substituted
quinoxalin-6-amine analogs as antiproliferatives: a structure-activity rela-
tionship study, Bioorg. Med. Chem. Lett 21 (2011) 1929e1932.
[21] J.V. Napoleon, S. Singh, S. Rana, M. Bendjennat, V. Kumar, S. Kizhake,
N.Y. Palermo, M.M. Ouellette, T. Huxford, A. Natarajan, Small molecule binding
to inhibitor of nuclear factor kappa-B kinase subunit beta in an ATP non-
competitive manner, Chem. Commun. 57 (2021) 4678e4681.
[22] F. Liu, Y. Xia, A.S. Parker, I.M. Verma, IKK biology, Immunol. Rev. 246 (2012)
239e253.
[23] J.A. DiDonato, F. Mercurio, M. Karin, NF-kappaB and the link between
inflammation and cancer, Immunol. Rev. 246 (2012) 379e400.
[24] A. Israel, The IKK complex, a central regulator of NF-kappaB activation, Cold
Spring Harb Perspect Biol 2 (2010) a000158.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113579.
[25] D.F. Lee, H.P. Kuo, C.T. Chen, J.M. Hsu, C.K. Chou, Y. Wei, H.L. Sun, L.Y. Li,
B. Ping, W.C. Huang, X. He, J.Y. Hung, C.C. Lai, Q. Ding, J.L. Su, J.Y. Yang,
A.A. Sahin, G.N. Hortobagyi, F.J. Tsai, C.H. Tsai, M.C. Hung, IKK beta suppression
of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway,
Cell 130 (2007) 440e455.
[26] E. Maniati, M. Bossard, N. Cook, J.B. Candido, N. Emami-Shahri,
S.A. Nedospasov, F.R. Balkwill, D.A. Tuveson, T. Hagemann, Crosstalk between
the canonical NF-kappaB and Notch signaling pathways inhibits Ppargamma
expression and promotes pancreatic cancer progression in mice, J. Clin. Invest.
121 (2011) 4685e4699.
[27] J. Ling, Y. Kang, R. Zhao, Q. Xia, D.F. Lee, Z. Chang, J. Li, B. Peng, J.B. Fleming,
H. Wang, J. Liu, I.R. Lemischka, M.C. Hung, P.J. Chiao, KrasG12D-induced IKK2/
beta/NF-kappaB activation by IL-1alpha and p62 feedforward loops is
required for development of pancreatic ductal adenocarcinoma, Canc. Cell 21
(2012) 105e120.
References
[1] J.A. Pereira, A.M. Pessoa, M.N. Cordeiro, R. Fernandes, C. Prudencio,
J.P. Noronha, M. Vieira, Quinoxaline, its derivatives and applications: a State of
the Art review, Eur. J. Med. Chem. 97 (2015) 664e672.
[2] J.W. Coe, P.R. Brooks, M.G. Vetelino, M.C. Wirtz, E.P. Arnold, J. Huang,
S.B. Sands, T.I. Davis, L.A. Lebel, C.B. Fox, A. Shrikhande, J.H. Heym, E. Schaeffer,
H. Rollema, Y. Lu, R.S. Mansbach, L.K. Chambers, C.C. Rovetti, D.W. Schulz,
F.D. Tingley 3rd, B.T. O'Neill, Varenicline: an alpha4beta2 nicotinic receptor
partial agonist for smoking cessation, J. Med. Chem. 48 (2005) 3474e3477.
[3] D. Cambridge, UK-14,304,
a potent and selective alpha2-agonist for the
characterisation of alpha-adrenoceptor subtypes, Eur. J. Pharmacol. 72 (1981)
413e415.
[28] Y. Xia, N. Yeddula, M. Leblanc, E. Ke, Y. Zhang, E. Oldfield, R.J. Shaw,
I.M. Verma, Reduced cell proliferation by IKK2 depletion in a mouse lung-
cancer model, Nat. Cell Biol. 14 (2012) 257e265.
[4] M.F. Sugrue, New approaches to antiglaucoma therapy, J. Med. Chem. 40
(1997) 2793e2809.
[5] H.C. Richards, J.R. Housley, D.F. Spooner, Quinacillin: a new penicillin with
unusual properties, Nature 199 (1963) 354e356.
[6] T. Pillaiyar, V. Namasivayam, M. Manickam, Macrocyclic hepatitis C virus NS3/
4A protease inhibitors: an overview of medicinal chemistry, Curr. Med. Chem.
23 (2016) 3404e3447.
[7] J.A. McCauley, M.T. Rudd, Hepatitis C virus NS3/4a protease inhibitors, Curr.
Opin. Pharmacol. 30 (2016) 84e92.
[8] T.P.S. Perera, E. Jovcheva, L. Mevellec, J. Vialard, D. De Lange, T. Verhulst,
C. Paulussen, K. Van De Ven, P. King, E. Freyne, D.C. Rees, M. Squires, G. Saxty,
M. Page, C.W. Murray, R. Gilissen, G. Ward, N.T. Thompson, D.R. Newell,
N. Cheng, L. Xie, J. Yang, S.J. Platero, J.D. Karkera, C. Moy, P. Angibaud,
S. Laquerre, M.V. Lorenzi, Discovery and pharmacological characterization of
[29] K. Vlantis, A. Wullaert, Y. Sasaki, M. Schmidt-Supprian, K. Rajewsky,
T. Roskams, M. Pasparakis, Constitutive IKK2 activation in intestinal epithelial
cells induces intestinal tumors in mice, J. Clin. Invest. 121 (2011) 2781e2793.
[30] H. Shaked, L.J. Hofseth, A. Chumanevich, A.A. Chumanevich, J. Wang, Y. Wang,
K. Taniguchi, M. Guma, S. Shenouda, H. Clevers, C.C. Harris, M. Karin, Chronic
epithelial NF-kappaB activation accelerates APC loss and intestinal tumor
initiation through iNOS up-regulation, Proc. Natl. Acad. Sci. U. S. A. 109 (2012)
14007e14012.
[31] M. Naramura, A. Natarajan, Mouse pancreatic tumor model independent of
tumor suppressor gene inactivation, Pancreas 47 (2018) e27ee29.
[32] H. Suzuki, T. Chiba, M. Kobayashi, M. Takeuchi, T. Suzuki, A. Ichiyama,
T. Ikenoue, M. Omata, K. Furuichi, K. Tanaka, IkappaBalpha ubiquitination is
catalyzed by an SCF-like complex containing Skp1, cullin-1, and two F-box/
WD40-repeat proteins, betaTrCP1 and betaTrCP2, Biochem. Biophys. Res.
Commun. 256 (1999) 127e132.
JNJ-42756493 (Erdafitinib),
a functionally selective small-molecule FGFR
family inhibitor, Mol. Canc. Therapeut. 16 (2017) 1010e1020.
[9] J.D. Karkera, G.M. Cardona, K. Bell, D. Gaffney, J.C. Portale, A. Santiago-Walker,
C.H. Moy, P. King, M. Sharp, R. Bahleda, F.R. Luo, J.D. Alvarez, M.V. Lorenzi,
S.J. Platero, Oncogenic characterization and pharmacologic sensitivity of
activating fibroblast growth factor receptor (FGFR) genetic alterations to the
selective FGFR inhibitor Erdafitinib, Mol. Canc. Therapeut. 16 (2017)
1717e1726.
[33] M. Kroll, F. Margottin, A. Kohl, P. Renard, H. Durand, J.P. Concordet,
F. Bachelerie, F. Arenzana-Seisdedos, R. Benarous, Inducible degradation of
IkappaBalpha by the proteasome requires interaction with the F-box protein
h-betaTrCP, J. Biol. Chem. 274 (1999) 7941e7945.
[34] B. Tian, D.E. Nowak, M. Jamaluddin, S. Wang, A.R. Brasier, Identification of
direct genomic targets downstream of the nuclear factor-kappaB transcription
factor mediating tumor necrosis factor signaling, J. Biol. Chem. 280 (2005)
17435e17448.
[10] K.J. Addess, J. Feigon, NMR investigation of Hoogsteen base pairing in qui-
noxaline antibiotic–DNA complexes: comparison of 2:1 echinomycin, triostin
A and [N-MeCys3,N-MeCys7] TANDEM complexes with DNA oligonucleotides,
Nucleic Acids Res. 22 (1994) 5484e5491.
[35] B. Tian, D.E. Nowak, A.R. Brasier, A TNF-induced gene expression program
under oscillatory NF-kappaB control, BMC Genom. 6 (2005) 137.
[36] T.D. Gilmore, M. Herscovitch, Inhibitors of NF-kappaB signaling: 785 and
counting, Oncogene 25 (2006) 6887e6899.
[37] M.L. de Castro Barbosa, R.A. da Conceicao, A.G.M. Fraga, B.D. Camarinha,
G.C. de Carvalho Silva, A.G.F. Lima, E.A. Cardoso, V. de Oliveira Freitas Lione,
NF-kappaB signaling pathway inhibitors as anticancer drug candidates,
Anticancer Agents Med Chem 17 (2017) 483e490.
[38] V. Ramadass, T. Vaiyapuri, V. Tergaonkar, Small molecule NF-kappaB pathway
inhibitors in clinic, Int. J. Mol. Sci. 21 (2020).
[39] B. Gatto, Biologics targeted at TNF: design, production and challenges, Reu-
matismo 58 (2006) 94e103.
[40] S. Rana, E.C. Blowers, C. Tebbe, J.I. Contreras, P. Radhakrishnan, S. Kizhake,
T. Zhou, R.N. Rajule, J.L. Arnst, A.R. Munkarah, R. Rattan, A. Natarajan, Isatin
derived spirocyclic analogues with alpha-Methylene-gamma-butyrolactone
as anticancer agents: a structure-activity relationship study, J. Med. Chem.
59 (2016) 5121e5127.
[41] V.C. Bryant, G.D. Kishore Kumar, A.M. Nyong, A. Natarajan, Synthesis and
evaluation of macrocyclic diarylether heptanoid natural products and their
analogs, Bioorg. Med. Chem. Lett 22 (2012) 245e248.
[11] G. Ughetto, A.H. Wang, G.J. Quigley, G.A. van der Marel, J.H. van Boom, A. Rich,
A comparison of the structure of echinomycin and triostin A complexed to a
DNA fragment, Nucleic Acids Res. 13 (1985) 2305e2323.
[12] T. Kaushal, G. Srivastava, A. Sharma, A. Singh Negi, An insight into medicinal
chemistry of anticancer quinoxalines, Bioorg. Med. Chem. 27 (2019) 16e35.
[13] A. Simeonov, A. Yasgar, A. Jadhav, G.L. Lokesh, C. Klumpp, S. Michael,
C.P. Austin, A. Natarajan, J. Inglese, Dual-fluorophore quantitative high-
throughput screen for inhibitors of BRCT-phosphoprotein interaction, Anal.
Biochem. 375 (2008) 60e70.
[14] G.L. Lokesh, A. Rachamallu, G.D. Kumar, A. Natarajan, High-throughput fluo-
rescence polarization assay to identify small molecule inhibitors of BRCT
domains of breast cancer gene 1, Anal. Biochem. 352 (2006) 135e141.
[15] D. Maroni, S. Rana, C. Mukhopadhyay, A. Natarajan, M. Naramura,
A quinoxaline urea analog uncouples inflammatory and pro-survival func-
tions of IKKbeta, Immunol. Lett. 168 (2015) 319e324.
[16] P. Radhakrishnan, V.C. Bryant, E.C. Blowers, R.N. Rajule, N. Gautam,
M.M. Anwar, A.M. Mohr, P.M. Grandgenett, S.K. Bunt, J.L. Arnst, S.M. Lele,
Y. Alnouti, M.A. Hollingsworth, A. Natarajan, Targeting the NF-kappaB and
mTOR pathways with a quinoxaline urea analog that inhibits IKKbeta for
pancreas cancer therapy, Clin. Canc. Res. 19 (2013) 2025e2035.
14

S. Sagar, S. Singh, J.R. Mallareddy et al.
European Journal of Medicinal Chemistry 222 (2021) 113579
Chem. 54 (2011) 4508e4522.
[51] D. Ray, A.M. Nyong, A. Natarajan, Synthesis of unnatural amino acid de-
rivatives via palladium catalyzed 1,4 addition of boronic acids, Tetrahedron
Lett. 51 (2010) 2655e2656.
[52] C.M. Robb, S. Kour, J.I. Contreras, E. Agarwal, C.J. Barger, S. Rana, Y. Sonawane,
B.K. Neilsen, M. Taylor, S. Kizhake, R.N. Thakare, S. Chowdhury, J. Wang,
J.D. Black, M.A. Hollingsworth, M.G. Brattain, A. Natarajan, Characterization of
CDK(5) inhibitor, 20-223 (aka CP668863) for colorectal cancer therapy,
Oncotarget 9 (2018) 5216e5232.
[42] D. Wen, Y. Nong, J.G. Morgan, P. Gangurde, A. Bielecki, J. Dasilva, M. Keaveney,
H. Cheng, C. Fraser, L. Schopf, M. Hepperle, G. Harriman, B.D. Jaffee, T.D. Ocain,
Y. Xu, A selective small molecule IkappaB Kinase beta inhibitor blocks nuclear
factor kappaB-mediated inflammatory responses in human fibroblast-like
synoviocytes, chondrocytes, and mast cells, J. Pharmacol. Exp. Therapeut.
317 (2006) 989e1001.
[43] P.L. Podolin, J.F. Callahan, B.J. Bolognese, Y.H. Li, K. Carlson, T.G. Davis,
G.W. Mellor, C. Evans, A.K. Roshak, Attenuation of murine collagen-induced
arthritis by a novel, potent, selective small molecule inhibitor of IkappaB Ki-
nase
2,
TPCA-1
(2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-
[53] J.I. Contreras, C.M. Robb, H.M. King, J. Baxter, A.J. Crawford, S. Kour, S. Kizhake,
Y.A. Sonawane, S. Rana, M.A. Hollingsworth, X. Luo, A. Natarajan, Chemical
genetic screens identify kinase inhibitor combinations that target anti-
apoptotic proteins for cancer therapy, ACS Chem. Biol. 13 (2018) 1148e1152.
[54] S. Kour, S. Rana, J.I. Contreras, H.M. King, C.M. Robb, Y.A. Sonawane,
M. Bendjennat, A.J. Crawford, C.J. Barger, S. Kizhake, X. Luo,
M.A. Hollingsworth, A. Natarajan, CDK5 inhibitor downregulates mcl-1 and
sensitizes pancreatic cancer cell lines to navitoclax, Mol. Pharmacol. 96 (2019)
419e429.
thiophenecarboxamide), occurs via reduction of proinflammatory cytokines
and antigen-induced T cell Proliferation, J. Pharmacol. Exp. Therapeut. 312
(2005) 373e381.
[44] J.R. Burke, M.A. Pattoli, K.R. Gregor, P.J. Brassil, J.F. MacMaster, K.W. McIntyre,
X. Yang, V.S. Iotzova, W. Clarke, J. Strnad, Y. Qiu, F.C. Zusi, BMS-345541 is a
highly selective inhibitor of I kappa B kinase that binds at an allosteric site of
the enzyme and blocks NF-kappa B-dependent transcription in mice, J. Biol.
Chem. 278 (2003) 1450e1456.
[45] K. Nagashima, V.G. Sasseville, D. Wen, A. Bielecki, H. Yang, C. Simpson,
E. Grant, M. Hepperle, G. Harriman, B. Jaffee, T. Ocain, Y. Xu, C.C. Fraser, Rapid
TNFR1-dependent lymphocyte depletion in vivo with a selective chemical
inhibitor of IKKbeta, Blood 107 (2006) 4266e4273.
[55] P. Smolewski, J. Grabarek, H.D. Halicka, Z. Darzynkiewicz, Assay of caspase
activation in situ combined with probing plasma membrane integrity to
detect three distinct stages of apoptosis, J. Immunol. Methods 265 (2002)
111e121.
[46] F.R. Greten, M.C. Arkan, J. Bollrath, L.C. Hsu, J. Goode, C. Miething, S.I. Goktuna,
M. Neuenhahn, J. Fierer, S. Paxian, N. Van Rooijen, Y. Xu, T. O'Cain, B.B. Jaffee,
D.H. Busch, J. Duyster, R.M. Schmid, L. Eckmann, M. Karin, NF-kappaB is a
negative regulator of IL-1beta secretion as revealed by genetic and pharma-
cological inhibition of IKKbeta, Cell 130 (2007) 918e931.
[47] S. Rana, A. Natarajan, Face selective reduction of the exocyclic double bond in
isatin derived spirocyclic lactones, Org. Biomol. Chem. 11 (2013) 244e247.
[48] E.A. Kumar, Q. Chen, S. Kizhake, C. Kolar, M. Kang, C.E. Chang, G.E. Borgstahl,
A. Natarajan, The paradox of conformational constraint in the design of
Cbl(TKB)-binding peptides, Sci. Rep. 3 (2013) 1639.
[49] A. Natarajan, Y. Guo, H. Arthanari, G. Wagner, J.A. Halperin, M. Chorev, Syn-
thetic studies toward aryl-(4-aryl-4H-[1,2,4]triazole-3-yl)-amine from 1,3-
diarylthiourea as urea mimetics, J. Org. Chem. 70 (2005) 6362e6368.
[50] J. Deng, E. Feng, S. Ma, Y. Zhang, X. Liu, H. Li, H. Huang, J. Zhu, W. Zhu, X. Shen,
L. Miao, H. Liu, H. Jiang, J. Li, Design and synthesis of small molecule RhoA
inhibitors: a new promising therapy for cardiovascular diseases? J. Med.
[56] N.A. Franken, H.M. Rodermond, J. Stap, J. Haveman, C. van Bree, Clonogenic
assay of cells in vitro, Nat. Protoc. 1 (2006) 2315e2319.
[57] D. Thomas, P. Radhakrishnan, Tumor-stromal crosstalk in pancreatic cancer
and tissue fibrosis, Mol. Canc. 18 (2019) 14.
[58] L.A. Peterson, Reactive metabolites in the biotransformation of molecules
containing a furan ring, Chem. Res. Toxicol. 26 (2013) 6e25.
[59] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings, Adv. Drug Deliv. Rev. 46 (2001) 3e26.
[60] S. Rana, Y.A. Sonawane, M.A. Taylor, S. Kizhake, M. Zahid, A. Natarajan, Syn-
thesis of aminopyrazole analogs and their evaluation as CDK inhibitors for
cancer therapy, Bioorg. Med. Chem. Lett 28 (2018) 3736e3740.
[61] S. Rana, S. Kour, Y.A. Sonawane, C.M. Robb, J.I. Contreras, S. Kizhake, M. Zahid,
A.R. Karpf, A. Natarajan, Symbiotic prodrugs (SymProDs) dual targeting of
NFkappaB and CDK, Chem. Biol. Drug Des. 96 (2020) 773e784.
15