Design and development of novel thiazolidin-4-one-1,3,5-triazine derivatives as neuro-protective agent against cerebral ischemia–reperfusion injury in mice via attenuation of NF-?B

Article abstract of DOI:10.1111/cbdd.13744

The present study enumerates the discovery and development of novel thiazolidin-4-one-1,3,5-triazine as neuro-protective agent against cerebral ischemia–reperfusion injury in mice. These compounds showed significant inhibition of NF-?B transcriptional activity in LPS-stimulated RAW264.7 cells, displaying compound 8k as most potent inhibitor among the tested derivative. The compound 8k was further studied in in vivo middle cerebral artery occlusion (MCAO) mice model for neuro-protective action. Results suggest that compound 8k causes attenuation of inflammation (TNF-α, IL-β, and IL-6), oxidative stress (SOD, GSH, and MDA), and apoptosis (Bcl-2, Bax, and cleaved caspase-3) in MCAO mice in concentration-dependent manner. Collectively, our results documented that compound 8k pre-treatment protects cerebral I/R. This novel lead scaffold may be helpful for investigation of new neuro-protective agent by inactivation of NF-?B.

Full text of DOI:10.1111/cbdd.13744

PROFESSOR CHANGSONG XU (Orcid ID : 0000-0003-3843-5096)
Article type
: Research Article
Design and development of novel thiazolidin-4-one-1,3,5-triazine derivatives as
neuro-protective agent against cerebral ischemia reperfusion injury in mice via
attenuation of NF-ĸB
Min Lu1, Yujun Qi1, Yu Han1, Qiong Yi1, Lei Xu1, Wenlin Sun1, Guihua Ni2, Xiaoyu Ni1,
Changsong Xu2*
1Department of Rehabilitation Medicine, The Affiliated Huai'an No.1 People's Hospital of Nanjing
Medical University, Huai'an, Jiangsu 223300, P.R. China
2Department of Neurology, The Affiliated Huai'an No.1 People's Hospital of Nanjing Medical
University, Huai'an, Jiangsu 223300, P.R. China
Short running title: Thiazolidin-4-one-1,3,5-triazine as neuro-protective agent
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13744
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*Corresponding Author:
Changsong Xu,
Department of Neurology, The Affiliated Huai'an No.1 people's Hospital of Nanjing Medical
University,Huai'an, Jiangsu 223300, P.R. China
Tel/fax: +86 517 8495 9789
Email: coleridgerwqbvds@gmail.com
Abstract
The present study enumerates the discovery and development of novel thiazolidin-4-one-
1,3,5-triazine as neuro-protective agent against cerebral ischemia reperfusion injury in mice. These
compounds showed significant inhibition of NF-ĸB transcriptional activity in LPS-stimulated
RAW264.7 cells, displaying compound 8k as most potent inhibitor among the tested derivative.
The compound 8k was further studied in in-vivo middle cerebral artery occlusion (MCAO) mice
model for neuro-protective action. Results suggest that compound 8k causes attenuation of
inflammation (TNF-α, IL-β and IL-6), oxidative stress (SOD, GSH and MDA) and apoptosis (Bcl-
2, Bax and cleaved caspase-3) in MCAO mice in concentration dependent manner. Collectively,
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our results documented that compound 8k pre-treatment protects cerebral I/R. This novel lead
scaffold may be helpful for investigation of new neuro-protective agent by inactivation of NF-ĸB.
Keywords: Thiazolidin-4-one; 1,3,5-triazine; MCAO; NF-ĸB; inflammation; oxidative stress.
1. Introduction
The increased life expectancy has concomitantly raise chances of cerebrovascular disease
(CVD), where the blood flow to/in the brain is temporarily or permanently blocked. It is a serious
debilitating condition responsible for long-term disability and one of the three most prominent
cause of death worldwide (Avery et al., 2016; Truelsen et al., 2000). Cerebral ischemic stroke is
one of the major causes of CVD where, an artery to the brain is blocked. It reduces the blood flow
and oxygen to the brain, leading to damage or death of brain cells. However, after restoration of
blood flow it may induce cerebral ischemia and reperfusion (I/R) injury due to generation of
various reactive oxygen species (ROS), inflammatory cytokines and pro-inflammatory mediators
which worsen neuronal injury and induces neuronal apoptosis (Astrup et al., 1981; Broughton et
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al., 2009; Jin et al., 2010; Huang et al., 2006). Therefore, many researchers across the world are
currently involved in development of novel anti-inflammatory agents which can be able to inhibit
inflammatory cascade involved in cerebral I/R injury (Lakhan et al., 2009).
Nuclear factor κB (NF-κB), a member of transcription factors family recognized to transform
variety of metabolic events including cell survival, inflammation, apoptosis and even autophagic
cell death (Zhang et al., 2017; Hayden & Ghosh, 2004). The patients of cerebral I/R injury showed
aberrant activation of NF-ĸB which mediate cascade of inflammatory response upon activation by
any external stimuli, such as, cytokines (interleukins), bacterial products (LPS) and oxidative
stress (Ridder & Schwaninger 2009; Zhang et al., 2005). It was suggests being involved in gene
transcription after translocation to nucleus and induces expression of interleukin-1 (IL-1), IL-6,
tumor necrosis factor-α (TNF-α), and resulted in neuronal death (Karin & Ben-Neriah, 2000; Shih
et al., 2015). Thus, various studies showed NF-ĸB inhibitors have beneficial role against cerebral
I/R injury.
Thiazolidin-4-one is a heterocyclic molecule well-known for excellent pharmacological
activities, for instance, anti-bacterial (Vicini et al., 2006; Dwivedi et al., 2016), anti-cancer (Joseph
et al., 2013; Rashid et al., 2014), anti-viral (Murugesan et al., 2014) and anti-inflammatory
(Vazzana et al., 2004). On the contrary, 1,3,5-triazine is also a versatile lead molecule exhibiting
numerous biological activities, including antibacterial (Singh et al., 2014; Singh et al., 2012),
antifungal (Singh et al., 2012; Singh, et al., 2013; Vembu et al., 2016), anticancer ( Popowycz et
al., 2009; Hodous et al., 2007; Dao et al., 2015) and more importantly anti-inflammatory action
(Mogilski et al., 2017; Zacharie et al., 2018). Moreover, the pioneering study by Shrivastava et al.
reported a series of thiazolidin-4-one-1,3,5-triazine as potent NF-ĸB inhibitor for airway
inflammation in cystic fibrosis (Srivastava et al., 2015). Prompted by the above, in present study,
we wish to develop hybrid conjugates of thiazolidin-4-one-1,3,5-triazine in search of potent anti-
inflammatory agent against cerebral I/R injury.
2. Result and Discussion
2.1.
Synthesis of hybrid conjugates of thiazolidin-4-one-1,3,5-triazine
The compounds were synthesized using facile route as outlined in Scheme 1 in excellent
yields. Briefly, the synthesis was initiated by nucleophilic reaction between 2,4,6-tri chloro-1,3,5-
triazine (1) with substituted anilines 2(a-g) to furnish compounds 3(a-g). The morpholine was
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reacted with compounds 3(a-g) to afford compound 4(a-g). In the next step, aqueous ammonia
was used to liberate remaining chlorine atom to yield compounds 5(a-g). The last step corresponds
to the one-pot synthesis of targeted derivatives 8(a-n) by refluxing together compounds 5(a-g),
substituted aldehydes 6(a-b) and mercaptoacetic acid (7).
<Scheme 1>
2.2.
In vitro NF-ĸB transcription inhibitory activity
In the present study, we have developed hybrid molecules of thiazolidin-4-one and 1,3,5-
triazine in a search of better NF-ĸB inhibitor against cerebral I/R injury. The present strategy is
aimed to enhance the pharmacological spectrum of the hybrid derivatives by taking the advantage
of two different skeletons which might act in synergistic manner than individual component. The
multifactorial etiology of cerebral I/R injury is suitable towards this approach and might provide
additional benefit. The NF-ĸB is a dimeric protein corresponds to a family of transcription factors
(Rel/NF-ĸB) which bound to specific IĸB in cytoplasm in dormant state and upon activation it
translocates to nucleus where it bind to target genes and control transcription of numerous genes.
The experimental and clinical evidences have confirmed oxidative stress in the cerebral I/R injury
triggers the activation of NF-ĸB signaling pathway which induces neuronal inflammation (Conner
& Grisham, 1996; Lordan et al., 2019; Halliwell, 2006). Thus, inhibition of NF-ĸB activation
provides significant benefit against neuronal inflammation in cerebral I/R injury. As presented in
table 1, the synthesized hybrid conjugates of pyrazole-1,3,5-triazine (8a-n) were tested for
inhibitory effect on NF-ĸB transcriptional activity in LPS-stimulated RAW264.7 cells. The two
different set of molecules were developed by taking para-chloro (8a-g) and para-fluro (8h-n)
atoms on the phenyl of thiazolidin-4-one using different substitutions on the phenyl linked to
1,3,5-triazine core. The compound 8a containing un-substituted phenyl connected to 1,3,5-triazine
core along with para-chloro on the phenyl ring connected to thiazolidin-4-one scaffold showed
least effect on NF-ĸB transcriptional activity in LPS-stimulated RAW264.7 cells. However,
introduction of para-chloro at the un-substituted phenyl ring (8b) displayed drastic improvement
in inhibitory potency. The inhibitory potency was further improved significantly in the case of
compounds 8c (IC50 = 19.23 ± 2.11) and 8d (IC50 = 15.65 ± 1.92) containing para-fluro and para-
nitro atoms, respectively. To our surprise, the activity was significantly reduced on introduction of
para-hydroxy (IC50 = 31.36 ± 3.72) and para-methoxy (IC50 = 49.56 ± 4.73) in the case of
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compounds 8e and 8f, correspondingly. The last compound of this set (8g) showed least potent
activity among the developed compounds of both the series. On closely inspecting table 1, the
inhibitory activity was found increased in next set of compounds containing para-fluro (8h-n) on
the phenyl of thiazolidin-4-one as compared to para-chloro (8a-g) counterparts. The compound 8h
displayed moderate inhibitory activity on NF-ĸB transcriptional activity. The inhibitory activity
was significantly improved in the case of compound 8i with para-chloro substitution (IC50 = 3.33
± 0.23). On replacement of para-chloro with para-fluoro (8j), the activity was slightly reduced.
However, introduction of para-nitro (IC50 = 0.90 ± 0.12) render compound most potent among the
tested derivatives, even more potent that reference compound. The activity was found reduced in
the case of compounds 8l (IC50 = 5.89 ± 1.14), 8m (IC50 = 10.11 ± 2.33) and 8n (IC50 = 7.34 ±
1.50) against NF-ĸB transcriptional activity in LPS-stimulated RAW264.7 cells. In the present
study, the control group was deemed as RAW264.7 cells without stimulated by LPS, while LPS
group was considered as stimulated RAW264.7 cells by LPS without any treatment.
Dexamethasones serve as reference compound in the present study. Based on the comparative
inhibitory potency of compounds 8a-n against NF-ĸB transcriptional activity in LPS-stimulated
RAW264.7 cells, the structure-activity relationship (SAR) suggest that, the pattern of substitution
have strong influence on the pharmacological activity. The results corroborated that the
compounds containing para-fluro on the thiazolidin-4-one scaffold proved much efficient than
their para-chloro counterparts. The detailed analysis of activity chart further revealed that, un-
substitution is not favourable for inhibitory activity, while introduction of substitution render
compound more active. It was also marked to note that, more pronounced activity was revealed by
electron withdrawing group than their electron-donating counterpart, and the order of activity is as
follows NO2 > Cl > F >OH > OCH3 > CH3 > H.
<Table 1>
2.3.
In vivo pharmacological activity
Impressed by the excellent NF-ĸB transcription inhibitory activity, it is worthwhile to analyze
the effect of most potent NF-ĸB inhibitor compound 8k in the experimentally induced cerebral I/R
injury by middle cerebral artery embolism (MCAO) in mice (McCullough et al., 2011;Chiang et
al., 2011). Various clinical studies have shown impairment of neural function and cerebral edema
is the characteristic hallmark of cerebral injury (Werner & Engelhard, 2007; Unterberg et al.,
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2004). Therefore, initially after induction of cerebral I/R injury by MCAO, the effect of compound
8k was investigated on the neural function and cerebral edema in the brains of mice. As shown in
Fig.1A, the neurological deficit score of compound 8k treated group which signifies the neural
function of brain was found to be improved as compared to MCAO-treatment group in dose
dependent manner (P > 0.05). In the next instance, as shown in Fig. 1B, the MCAO treated mice
demonstrated increased cerebral edema as compared to sham. However, the cerebral edema was
found to be reduced significantly in compound 8k treated mice.
<Figure 1>
The effect of compound 8k was further studied visually by examining the histopathological
changes in MCAO-treated mice brains. It provides idea about clinical manifestation of disease and
the effect of drugs/treatment on the affected tissues via microscopic means. As shown in Fig. 2,
the MCAO treated mice showed reduced nerve cells, disordered arrangement of cells and
enhanced intracellular spaces, as compared to sham, which had not any such features. Whereas, in
8k treated mice, these features were found to be returned to near normal in concentration
dependent manner.
<Figure 2>
Infarction is the main consequence of ischemia of brain in cerebral injury, whereas,
restoration of normal brain function after reperfusion will be entirely dependent on the area of
non-infracted tissues (Chan, 2001). Therefore, many therapeutic modalities to treat cerebral injury
are majorly concentrated towards prompt reperfusion and drugs to prevent infarction. In this
regard, our next aim to determine the effect of compound 8k on the brain infarct size to assess its
protective effect against cerebral I/R injury. After 24h of MCAO surgery and behavioural
assessment, the mice were sacrificed and damage to the brain area due to ischemia has been
quantified using TTC staining method. As shown in Fig. 3, the sham treated mice showed no
infarct area, while MCAO treated mice showed significant increase in infarct size as compared to
sham. The infarct area was found to be significantly reduced in 8k treated mice in dose-dependent
manner as compared to MCAO treated mice.
<Figure 3>
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The neural cell death in the cerebral injury induces inflammation in the injured brain areas
and can exacerbate evolving tissue pathology. Therefore, in the next instance, we aimed to
determine the effect of compound 8k on the cytokines (TNF-α) and interleukins (IL-β and IL-6)
and results are presented in Fig. 4. It has been found that, MCAO treated mice showed significant
increase in concentration of examined cytokines and interleukins as compared to sham treated
mice. Moreover, the level of TNF-α, IL-β and IL-6 were found to be reduced considerably in 8k
treated mice.
<Figure 4>
Oxidative stress followed by cerebral I/R injury is the main determinant of to induce
neuro-inflammation due to the production of reactive oxygen species (ROS) (Mittal et al., 2014).
Therefore, effect of compound 8k was investigated on numerous oxidative stress markers, such as,
SOD, GSH and MDA, Fig. 5. It has been found that, the level of GSH and activity SOD was
reduced in MCAO treated mice together with increased MDA level. On the contrary, compound
8k treated mice showed reduction of oxidative stress as evident by restoration of these biomarkers
near to normal. This observation suggests that compound 8k has significant antioxidant effect
which might be responsible for protective action against neuro-inflammation and cerebral I/R
injury.
<Figure 5>
Apoptosis mediated cell death due to DNA damage is considered as the main pathological
feature of cerebral I/R injury. Various studies have shown that in response to oxidative stress, the
B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) translocated from the cytoplasm to the
mitochondria due to increased permeability of mitochondrial membrane. These Bcl-2 and Bax
genes will govern the apoptotic process by acting as antiapoptotic and proapoptotic genes,
respectively. Moreover, caspase-3 is the terminal protein of the cellular apoptosis cascade which
can promote the cellular apoptosis (Liu et al., 2013). Thus it is valuable to investigate the effect of
compound 8k on the Bcl-2, Bax, and cleaved caspase-3 using western blot analysis. As observed
in Fig. 6. The MCAO treated group showed increased expression of cleaved caspase-3 and Bax
along with reduced Bcl-2 level. It was significant to note that, compound 8k causes marked
improvement in the expression of these tested apoptotic/pro-apoptotic protein near to normal.
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<Figure 6>
Considering the significance of aberrant activation of NF-ĸB in cerebral I/R injury, the last
part of the study was aimed to investigate the effect of compound 8k on the mediators of NF-ĸB
signalling cascade by western blot analysis (Schneider et al., 1999). As shown in Fig. 7, MCAO
treated mice showed increased phosphorylation of IκBα after reperfusion, whereas treatment with
compound 8k showed significant reduction of phosphorylation in dose-dependent manner.
Moreover, compound 8k also causes attenuation of phosphorylation and nuclear translocation of
NF-κBp65, as compared to MCAO mice brains.
<Figure 7>
3. Material and methods
3.1.
Chemistry
1
The chemicals used in the present study were obtained from Sigma Aldrich (USA). H NMR
spectra were recorded in d6-DMSO on a Bruker Avance-400 NMR spectrometer with TMS as the
internal reference. 13C NMR spectra were recorded on a Bruker Avance-100 NMR spectrometer in
DMSO on the same spectrometers with TMS as the internal reference. The multiplicity of a signal
is indicated as: s – singlet, d – doublet, t – triplet, q – quartet, m – multiplet, br – broad, dd –
doublet of doublets, etc. Coupling constants (J) are quoted in Hz and reported to the nearest 0.1
Hz. Infrared spectra were recorded as a neat thin film on a Perkin-Elmer Spectrum One FT-IR
spectrometer using Universal ATR sampling accessories. Letters in the parentheses refer to the
relative absorbency compared to the most intense peak: w – weak, less than 40%; m – medium,
40-75%; s – strong, greater than 75%. Melting points were obtained using an OptiMelt automated
melting point system. MS spectra were recorded on an Agilent 1100 LC/MS.
Synthesis of compounds 3 (a-g), 4 (a-g) and 5 (a-g) were achieved by earlier reported
procedure and the authenticity of compounds were assessed by FT-IR, mass and elemental
analysis (Srivastava et al., 2015).
3.1.1. General procedure for the synthesis of title compounds 8 (a-g)
Corresponding substituted compound 5 (a–g) (0.1 mol), substituted benzaldehyde 6 (a–b)
(0.1 mol) and mercaptoacetic acid 7 (0.1 mol) were refluxed in the presence of toluene for 8–12 h.
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The product was filtered, washed with cold water and recrystallized with ethanol to afford the
corresponding pure product 8 (a–g).
3.1.1.1.
2-(4-Chlorophenyl)-3-(4-morpholino-6-(phenylamino)-1,3,5-triazin-2-
yl)thiazolidin-4-one (8a)
Yield: 83 %; M.p: 218-219 °C; MW: 468.96 ; Rf: 0.64; FTIR (νmax; cm-1 KBr): 3252 (N-H
stretching), 3086 (C–H broad), 2858 (CH2 stretching ), 1714 (C=O Stretching), 1684 (C=N aromatic),
1648 ( C=C ), 1078 (C-N stretching), 817 (C-S stretching), 794 (C-Cl stretching), 624; 1H NMR
(400MHz, DMSO-d6, TMS) δ ppm: 7.65 (d, 2H, J=1.49 Hz, Ar-H), 7.54 (d, 2H, J=1.57 Hz, Ar-
H), 7.51 (d, 2H, J=1.47 Hz, Ar-H), 7.27 (d, 2H, J=1.44 Hz, Ar-H), 7.06 (t, 1H, J=1.23 Hz, Ar-H),
6.23 (s, 1H, Thiazolidine-H), 3.94 (s, 2H, Thiazolidine-H), 3.97 (s, 1H, NH-), 3.74-3.64 (m, 8H,
CH2×4,Morpholine-H); 13C NMR (100MHz, DMSO) δ ppm:174.8, 171.3, 170.5, 169.2, 138.9,
137.4, 132.8, 130.1, 129.7, 128.7, 122.5, 117.9, 72.8, 66.3, 48.7, 33.5; Mass: 469.99 (M+H)+;
Elemental analysis for C22H21ClN6O2S: Calculated: C, 56.35; H, 4.51; N, 17.92. Found: C, 56.38;
H, 4.50; N, 17.94.
3.1.1.2.
2-(4-Chlorophenyl)-3-(4-((4-chlorophenyl)amino)-6-morpholino-1,3,5-triazin-2-
yl)thiazolidin-4-one (8b)
Yield: 71 %; M.p: 232-234 °C; MW: 503.40 ; Rf: 0.69; FTIR (νmax; cm-1 KBr): 3258 (N-H
stretching), 3081 (C–H broad), 2856 (CH2 stretching ), 1712 (C=O Stretching), 1687 (C=N aromatic),
1641 ( C=C ), 1072 (C-N stretching), 812 (C-S stretching), 791 (C-Cl stretching), 627; 1H NMR
(400MHz, DMSO-d6, TMS) δ ppm: 7.75 (d, 2H, J=1.54 Hz, Ar-H), 7.65 (d, 2H, J=1.49 Hz, Ar-
H), 7.54 (d, 2H, J=1.55 Hz, Ar-H), 7.42 (d, 2H, J=1.64 Hz, Ar-H), 6.25 (s, 1H, Thiazolidine-H),
3.95 (s, 2H, Thiazolidine-H), 3.99 (s, 1H, NH-), 3.76-3.65 (m, 8H, CH2×4,Morpholine-H); 13C
NMR (100MHz, DMSO) δ ppm:182.9, 171.3, 167.5, 164.4, 137.6, 137.1, 132.8, 130.2, 129.8,
128.8, 127.7, 122.1, 72.8, 66.4, 48.7, 33.6; Mass: 504.43 (M+H)+; Elemental analysis for
C22H20Cl2N6O2S: Calculated: C, 52.49; H, 4.00; N, 16.69. Found: C, 52.51; H, 4.04; N, 16.72.
3.1.1.3.
2-(4-Chlorophenyl)-3-(4-((4-fluorophenyl)amino)-6-morpholino-1,3,5-triazin-2-
yl)thiazolidin-4-one (8c)
Yield: 78 %; M.p: 225-226 °C; MW: 486.95 ; Rf: 0.79; FTIR (νmax; cm-1 KBr): 3254 (N-H
stretching), 3087 (C–H broad), 2852 (CH2 stretching ), 1709 (C=O Stretching), 1689 (C=N aromatic),
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1645 (C=C), 1156 (C-F stretching), 1076 (C-N stretching), 816 (C-S stretching), 793 (C-Cl
stretching), 637; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 7.72 (d, 2H, J=1.84 Hz, Ar-H), 7.66
(d, 2H, J=1.46 Hz, Ar-H), 7.56 (d, 2H, J=1.53 Hz, Ar-H), 7.06 (d, 2H, J=1.59 Hz, Ar-H), 6.24 (s,
1H, Thiazolidine-H), 3.96 (s, 2H, Thiazolidine-H), 3.98 (s, 1H, NH-), 3.75-3.63 (m, 8H,
CH2×4,Morpholine-H); 13C NMR (100MHz, DMSO) δ ppm:182.8, 171.3, 167.4, 164.3, 157.4,
137.5, 134.5, 132.8, 130.2, 128.9, 120.7, 116.4, 72.7, 66.4, 48.8, 33.7; Mass: 487.93 (M+H)+;
Elemental analysis for C22H20ClFN6O2S: Calculated: C, 54.26; H, 4.14; N, 17.26. Found: C,
54.27; H, 4.12; N, 17.29.
3.1.1.4.
2-(4-Chlorophenyl)-3-(4-morpholino-6-((4-nitrophenyl)amino)-1,3,5-triazin-2-
yl)thiazolidin-4-one (8d)
Yield: 84 %; M.p: 245-247 °C; MW: 513.96 ; Rf: 0.74; FTIR (νmax; cm-1 KBr): 3258 (N-H
stretching), 3083 (C–H broad), 2856 (CH2 stretching ), 1708 (C=O Stretching), 1685 (C=N aromatic),
1648 ( C=C ), 1538 (NO2 stretching), 1078 (C-N stretching), 817 (C-S stretching), 792 (C-Cl
stretching), 635; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 8.12 (d, 2H, J=1.82 Hz, Ar-H), 7.64
(d, 2H, J=1.48 Hz, Ar-H), 7.55 (d, 2H, J=1.56 Hz, Ar-H), 7.34 (d, 2H, J=2.24 Hz, Ar-H), 6.25 (s,
1H, Thiazolidine-H), 3.95 (s, 2H, Thiazolidine-H), 3.97 (s, 1H, NH-), 3.74-3.66 (m, 8H,
CH2×4,Morpholine-H); 13C NMR (100MHz, DMSO) δ ppm:174.7, 171.3, 170.8, 169.4, 145.1,
137.9, 137.4, 132.7, 130.2, 128.7, 124.7, 119.2, 72.8, 66.5, 48.7, 33.6 ; Mass: 514.98 (M+H)+;
Elemental analysis for C22H20ClN7O4S: Calculated: C, 51.41; H, 3.92; N, 19.08. Found: C, 51.40;
H, 3.95; N, 19.06.
3.1.1.5.
2-(4-Fluorophenyl)-3-(4-((4-hydroxyphenyl)amino)-6-morpholino-1,3,5-triazin-2-
yl)thiazolidin-4-one (8e)
Yield: 79 %; M.p: 238-239 °C; MW: 484.96 ; Rf: 0.70; FTIR (νmax; cm-1 KBr): 3262 (N-H
stretching), 3225 (O-H stretching), 3087 (C–H broad), 2859 (CH2 stretching ), 1719 (C=O
Stretching), 1685 (C=N aromatic), 1647 ( C=C ), 1342 (O-H bending), 1079 (C-N stretching), 813
(C-S stretching), 792 (C-Cl stretching), 631; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 7.67 (d,
2H, J=1.49 Hz, Ar-H), 7.54 (d, 2H, J=1.57 Hz, Ar-H), 7.27 (d, 2H, J=1.38 Hz, Ar-H), 6.67 (d, 2H,
J=2.63 Hz, Ar-H), 6.24 (s, 1H, Thiazolidine-H), 5.46 (s, 1H, Ar-OH), 3.97 (s, 2H, Thiazolidine-
13
H), 3.98 (s, 1H, NH-), 3.75-3.63 (m, 8H, CH2×4,Morpholine-H); C NMR (100MHz, DMSO) δ
ppm:182.9, 171.3, 167.5, 164.5, 148.6, 137.4, 132.8, 131.5, 130.1, 128.7, 122.1, 116.7, 72.8, 66.4,
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48.8, 33.6 ; Mass: 485.95 (M+H)+; Elemental analysis for C22H21ClN6O3S: Calculated: C, 54.49;
H, 4.36; N, 17.33. Found: C, 54.52; H, 4.34; N, 17.31.
3.1.1.6.
2-(4-Fluorophenyl)-3-(4-((4-methoxyphenyl)amino)-6-morpholino-1,3,5-triazin-2-
yl)thiazolidin-4-one (8f)
Yield: 67 %; M.p: 249-251 °C; MW: 498.99 ; Rf: 0.65; FTIR (νmax; cm-1 KBr): 3268 (N-H
stretching), 3084 (C–H broad), 2851 (CH2 stretching ), 2824 (OCH3 stretching), 1715 (C=O
Stretching), 1681 (C=N aromatic), 1649 ( C=C ), 1074 (C-N stretching), 812 (C-S stretching), 793
(C-Cl stretching), 638; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 7.65 (d, 2H, J=1.47 Hz, Ar-
H), 7.56 (d, 2H, J=1.59 Hz, Ar-H), 7.32 (d, 2H, J=1.68 Hz, Ar-H), 6.62 (d, 2H, J=2.72 Hz, Ar-H),
6.23 (s, 1H, Thiazolidine-H), 3.96 (s, 2H, Thiazolidine-H), 3.99 (s, 1H, NH-), 3.74-3.62 (m, 8H,
CH2×4,Morpholine-H), 3.71 (s, 3H, OCH3); 13C NMR (100MHz, DMSO) δ ppm:182.9, 171.3,
167.5, 164.2, 153.4, 137.5, 132.8, 131.2, 130.2, 128.9, 121.8, 115.2, 72.8, 66.5, 55.9, 48.9, 33.5;
Mass: 499.97 (M+H)+; Elemental analysis for C23H23ClN6O3S: Calculated: C, 55.36; H, 4.65; N,
16.84. Found: C, 55.34; H, 4.69; N, 16.85.
3.1.1.7.
2-(4-Fluorophenyl)-3-(4-morpholino-6-(p-tolylamino)-1,3,5-triazin-2-
yl)thiazolidin-4-one (8g)
Yield: 76 %; M.p: 241-242 °C; MW: 482.99 ; Rf: 0.78; FTIR (νmax; cm-1 KBr): 3262 (N-H
stretching), 3087 (C–H broad), 2952 (alkyl C-H stretching), 2852 (CH2 stretching ), 1718 (C=O
Stretching), 1685 (C=N aromatic), 1642 ( C=C ), 1467 (CH3 bending), 1078 (C-N stretching), 814
(C-S stretching), 792 (C-Cl stretching), 632; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 7.67 (d,
2H, J=1.48 Hz, Ar-H), 7.55 (d, 2H, J=1.56 Hz, Ar-H), 7.34 (d, 2H, J=1.34 Hz, Ar-H), 7.07 (d, 2H,
J=1.59 Hz, Ar-H), 6.24 (s, 1H, Thiazolidine-H), 3.96 (s, 2H, Thiazolidine-H), 3.98 (s, 1H, NH-),
3.76-3.64 (m, 8H, CH2×4,Morpholine-H), 2.24 (s, 3H, CH3); 13C NMR (100MHz, DMSO) δ
ppm:174.7, 171.3, 170.8, 169.4, 137.5, 135.9, 132.6, 131.2, 130.2, 129.8, 128.8, 120.3, 72.8, 66.4,
48.7, 33.6, 21.4; Mass: 483.94 (M+H)+; Elemental analysis for C23H23ClN6O2S: Calculated: C,
57.20; H, 4.80; N, 17.40. Found: C, 57.23; H, 4.78; N, 17.45.
3.1.1.8.
2-(4-Fluorophenyl)-3-(4-morpholino-6-(phenylamino)-1,3,5-triazin-2-
yl)thiazolidin-4-one (8h)
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Yield: 68 %; M.p: 196-197 °C; MW: 452.51 ; Rf: 0.69; FTIR (νmax; cm-1 KBr): 3259 (N-H
stretching), 3089 (C–H broad), 2851 (CH2 stretching ), 1709 (C=O Stretching), 1687 (C=N aromatic),
1646 ( C=C ), 1154 (C-F stretching), 1072 (C-N stretching), 814 (C-S stretching), 629; 1H NMR
(400MHz, DMSO-d6, TMS) δ ppm: 7.51 (d, 2H, J=1.47 Hz, Ar-H), 7.46 (d, 2H, J=1.33 Hz, Ar-
H), 7.27 (d, 2H, J=1.42 Hz, Ar-H), 7.09 (d, 2H, J=1.25 Hz, Ar-H), 7.05 (t, 1H, J=1.24 Hz, Ar-H),
6.20 (s, 1H, Thiazolidine-H), 3.95 (s, 2H, Thiazolidine-H), 3.98 (s, 1H, NH-), 3.75-3.63 (m, 8H,
CH2×4,Morpholine-H); 13C NMR (100MHz, DMSO) δ ppm:174.8, 171.3, 170.8, 169.5, 161.4,
138.9, 134.9, 130.4, 129.6, 122.6, 117.8, 115.4, 72.9, 66.4, 48.9, 33.7; Mass: 469.99 (M+H)+;
Elemental analysis for C22H21FN6O2S: Calculated: C, 58.39; H, 4.68; N, 18.57. Found: C, 58.43;
H, 4.67; N, 18.58.
3.1.1.9.
3-(4-((4-Chlorophenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)-2-(4-
fluorophenyl)thiazolidin-4-one (8i)
Yield: 73 %; M.p: 211-212 °C; MW: 486.95 ; Rf: 0.74; FTIR (νmax; cm-1 KBr): 3262 (N-H
stretching), 3088 (C–H broad), 2857 (CH2 stretching ), 1715 (C=O Stretching), 1689 (C=N aromatic),
1645 ( C=C ), 1156 (C-F stretching), 1078 (C-N stretching), 811 (C-S stretching), 798 (C-Cl
stretching), 621; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 7.78 (d, 2H, J=1.51 Hz, Ar-H), 7.41
(d, 2H, J=1.64 Hz, Ar-H), 7.46 (d, 2H, J=1.34 Hz, Ar-H), 7.09 (d, 2H, J=1.25 Hz, Ar-H), 6.21 (s,
1H, Thiazolidine-H), 3.94 (s, 2H, Thiazolidine-H), 3.97 (s, 1H, NH-), 3.73-3.61 (m, 8H,
CH2×4,Morpholine-H); 13C NMR (100MHz, DMSO) δ ppm:182.7, 171.4, 167.5, 164.5, 161.4,
137.2, 134.9, 130.4, 129.6, 127.8, 122.1, 115.5, 72.8, 66.4, 48.9, 33.6; Mass: 504.43 (M+H)+;
Elemental analysis for C22H20ClFN6O2S: Calculated: C, 54.26; H, 4.14; N, 17.26. Found: C,
54.29; H, 4.09; N, 17.28.
3.1.1.10. 2-(4-Fluorophenyl)-3-(4-((4-fluorophenyl)amino)-6-morpholino-1,3,5-triazin-2-
yl)thiazolidin-4-one (8j)
Yield: 84 %; M.p: 189-190 °C; MW: 470.50 ; Rf: 0.87; FTIR (νmax; cm-1 KBr): 3259 (N-H
stretching), 3083 (C–H broad), 2851 (CH2 stretching ), 1706 (C=O Stretching), 1687 (C=N aromatic),
1648 ( C=C ), 1159 (C-F stretching), 1073 (C-N stretching), 819 (C-S stretching), 645; 1H NMR
(400MHz, DMSO-d6, TMS) δ ppm: 7.74 (d, 2H, J=1.81 Hz, Ar-H), 7.46 (d, 2H, J=1.32 Hz, Ar-
H), 7.09 (d, 2H, J=1.24 Hz, Ar-H), 7.01 (d, 2H, J=1.56 Hz, Ar-H), 6.23 (s, 1H, Thiazolidine-H),
3.92 (s, 2H, Thiazolidine-H), 3.99 (s, 1H, NH-), 3.72-3.65 (m, 8H, CH2×4,Morpholine-H); 13C
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NMR (100MHz, DMSO) δ ppm:182.6, 171.4, 167.7, 164.4, 161.2, 157.5, 134.8, 134.5, 130.5,
120.7, 116.4, 115.6, 72.8, 66.4, 48.9, 33.5; Mass: 471.56 (M+H)+; Elemental analysis for
C22H20F2N6O2S: Calculated: C, 56.16; H, 4.28; N, 17.86. Found: C, 56.15; H, 4.24; N, 17.87.
3.1.1.11. 2-(4-Fluorophenyl)-3-(4-morpholino-6-((4-nitrophenyl)amino)-1,3,5-triazin-2-
yl)thiazolidin-4-one (8k)
Yield: 81 %; M.p: 252-253 °C; MW: 497.51 ; Rf: 0.77; FTIR (νmax; cm-1 KBr): 3254 (N-H
stretching), 3087 (C–H broad), 2858 (CH2 stretching ), 1713 (C=O Stretching), 1686 (C=N aromatic),
1642 ( C=C ), 1534 (NO2 stretching), 1156 (C-F stretching), 1075 (C-N stretching), 819 (C-S
stretching), 648; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 8.14 (d, 2H, J=1.84 Hz, Ar-H), 7.47
(d, 2H, J=1.35 Hz, Ar-H), 7.34 (d, 2H, J=2.24 Hz, Ar-H), 7.09 (d, 2H, J=2.21 Hz, Ar-H), 6.22 (s,
1H, Thiazolidine-H), 3.93 (s, 2H, Thiazolidine-H), 3.98 (s, 1H, NH-), 3.75-3.62 (m, 8H,
CH2×4,Morpholine-H); 13C NMR (100MHz, DMSO) δ ppm:176.7, 171.4, 170.8, 169.2, 161.4,
145.1, 137.9, 134.9, 130.4, 124.8, 119.2, 115.5, 72.8, 66.4, 48.8, 33.6; Mass: 498.12 (M+H)+;
Elemental analysis for C22H20FN7O4S: Calculated: C, 53.11; H, 4.05; N, 19.71. Found: C, 53.09;
H, 4.08; N, 19.75.
3.1.1.12. 2-(4-Fluorophenyl)-3-(4-((4-hydroxyphenyl)amino)-6-morpholino-1,3,5-triazin-2-
yl)thiazolidin-4-one (8l)
Yield: 86 %; M.p: 201-203 °C; MW: 468.51 ; Rf: 0.76; FTIR (νmax; cm-1 KBr): 3268 (N-H
stretching), 3223 (O-H stretching), 3081 (C–H broad), 2857 (CH2 stretching ), 1713 (C=O
Stretching), 1686 (C=N aromatic), 1649 ( C=C ), 1348 (O-H bending), 1159 (C-F stretching), 1072
(C-N stretching), 814 (C-S stretching), 648; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 7.48 (d,
2H, J=1.34 Hz, Ar-H), 7.27 (d, 2H, J=1.38 Hz, Ar-H), 7.08 (d, 2H, J=1.26 Hz, Ar-H), 6.68 (d, 2H,
J=2.65 Hz, Ar-H), 6.20 (s, 1H, Thiazolidine-H), 5.52 (s, 1H, Ar-OH), 3.92 (s, 2H, Thiazolidine-
13
H), 3.96 (s, 1H, NH-), 3.72-3.68 (m, 8H, CH2×4,Morpholine-H); C NMR (100MHz, DMSO) δ
ppm:182.7, 171.3, 167.6, 164.4, 161.2, 148.5, 134.9, 131.6, 130.3, 122.1, 116.8, 115.5, 72.8, 66.4,
48.9, 33.5; Mass: 469.52 (M+H)+; Elemental analysis for C22H21FN6O3S: Calculated: C, 56.40; H,
4.52; N, 17.94. Found: C, 56.42; H, 4.56; N, 17.93.
3.1.1.13. 2-(4-Fluorophenyl)-3-(4-((4-methoxyphenyl)amino)-6-morpholino-1,3,5-triazin-2-
yl)thiazolidin-4-one (8m)
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Yield: 85 %; M.p: 228-229 °C; MW: 482.53 ; Rf: 0.79; FTIR (νmax; cm-1 KBr): 3263 (N-H
stretching), 3085 (C–H broad), 2852 (CH2 stretching ), 2826 (OCH3 stretching), 1717 (C=O
Stretching), 1683 (C=N aromatic), 1645 ( C=C ), 1154 (C-F stretching), 1076 (C-N stretching), 815
(C-S stretching), 635; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 7.48 (d, 2H, J=1.35 Hz, Ar-
H), 7.32 (d, 2H, J=1.67 Hz, Ar-H), 7.09 (d, 2H, J=1.25 Hz, Ar-H), 6.64 (d, 2H, J=2.75 Hz, Ar-H),
6.21 (s, 1H, Thiazolidine-H), 3.94 (s, 2H, Thiazolidine-H), 3.96 (s, 1H, NH-), 3.77-3.63 (m, 8H,
CH2×4,Morpholine-H), 3.76 (s, 3H, OCH3); 13C NMR (100MHz, DMSO) δ ppm:182.5, 171.4,
167.5, 164.1, 161.2, 153.4, 134.9, 131.3, 130.2, 121.8, 115.6, 115.1, 72.8, 66.4, 55.8, 48.8, 33.5;
Mass: 483.57 (M+H)+; Elemental analysis for C23H23FN6O3S: Calculated: C, 57.25; H, 4.80; N,
17.42. Found: C, 57.27; H, 4.81; N, 17.40.
3.1.1.14. 2-(4-Fluorophenyl)-3-(4-morpholino-6-(p-tolylamino)-1,3,5-triazin-2-
yl)thiazolidin-4-one (8n)
Yield: 72 %; M.p: 213-214 °C; MW: 466.54 ; Rf: 0.71; FTIR (νmax; cm-1 KBr): 3269 (N-H
stretching), 3083 (C–H broad), 2954 (alkyl C-H stretching), 2856 (CH2 stretching ), 1714 (C=O
Stretching), 1682 (C=N aromatic), 1647 ( C=C ), 1469 (CH3 bending), 1157 (C-F stretching), 1072
(C-N stretching), 814 (C-S stretching), 639; 1H NMR (400MHz, DMSO-d6, TMS) δ ppm: 7.49 (d,
2H, J=1.32 Hz, Ar-H), 7.34 (d, 2H, J=1.29 Hz, Ar-H), 7.09 (d, 2H, J=1.25 Hz, Ar-H), 7.05 (d, 2H,
J=1.54 Hz, Ar-H), 6.23 (s, 1H, Thiazolidine-H), 3.92 (s, 2H, Thiazolidine-H), 3.97 (s, 1H, NH-),
3.74-3.62 (m, 8H, CH2×4,Morpholine-H), 2.21 (s, 3H, CH3); 13C NMR (100MHz, DMSO) δ
ppm:174.8, 171.2, 170.8, 169.4, 161.2, 135.8, 134.8, 131.3, 130.1, 129.8, 120.4, 115.5, 72.8, 66.4,
48.9, 33.6, 21.3; Mass: 467.56 (M+H)+; Elemental analysis for C23H23FN6O2S: Calculated: C,
59.21; H, 4.97; N, 18.01. Found: C, 59.25; H, 4.96; N, 18.02.
3.2.
In vitro NF-ĸB transcription inhibitory activity
RAW264.7 macrophages were purchased from American Type Culture Collection (ATCC,
USA). Cells were cultured in DMEM with 10% FBS, 100 mg mL-1 streptomycin and 100 U mL-1
penicillin in humidified 5% CO2 at 37 ̊C. The 80% confluency of RAW264.7 cells was achieved in
six well plates and transfected with 1 mg of the NF-ĸB reporter construct, along with 0.5 mg of
pSVGal plasmid using Lipofect-AMINE 2000 (Invitrogen) in Opti-MEM medium (Gibco). After
24 hours of transfection, cells were treated with LPS or target derivatives (100 µM) for an
additional 2 h, and then lysed using the reporter lysis buffer (Promega). Luciferase assays were
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performed using 20 µL of cell extract and 100 µL of luciferin substrate (Promega), and the
luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega)
according to the manufacturer's instructions. Luciferase and Renilla activities were measured by
an Orion microplate luminometer (Berthold detection systems).
3.3.
Pharmacological Activity
3.3.1. Animals
The C57/BL6 male mice were obtained from the Institutional animal house and housed in
controlled environment with ad libitum supply of food and water. The animals were kept under in
alternate light and dark cycles of 12 h. The study was approved reviewed and approved by the
Animal Ethical and Welfare Committee of People's Hospital of Nanjing Medical University,
China.
3.3.2. Animal model of cerebral ischemia
No food and water for 12 h before the MCAO surgery was given to animals. The MCAO
surgery was performed as per the earlier reported procedure. Towards this, the mice were
anesthetized with an intraperitoneal injection of 10% chloral hydrate (350 mg/kg) and fixed on the
surgery board in the supine position. The left carotid region was exposed through a midline
cervical incision; the external carotid artery and the common carotid artery were exposed. A non-
traumatic microvascular clamp was introduced from the carotid bifurcation into the internal
carotid artery, thereby occluding the origin of the middle cerebral artery. After a 2-hours
occlusion, reperfusion was initiated by withdrawal of the clamp. Finally, the incision was sutured
and the animal was allowed to recover from anesthesia. During the operation, the rectal
temperature of mice was maintained at 37 _C using a heating lamp. Sham operated group received
the same procedure but without MCA occlusion. The animals were allowed to recover from
anesthesia prior to being returned to their original housing.
3.3.3. Drug treatment
The lethal dose of compound 8k was found to be more than 2000mg/kg in mice as per the sub
acute toxicity assay according to OECD guidelines. Therefore, compound 8k was used in the dose
of 5, 10 and 15 mg/kg in mice. The compounds were suspended in 2% carboxy methyl cellulose
(CMC) for administration. The compounds were administered intraperitoneally immediately and
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after 12 h of MCAO surgery. The sham and MCAO treated mice administered with normal saline.
The animals were sacrificed after reperfusion for 24 h and brain infarct volume was determined
using TTC staining. On the other hand, ischemic area of brain containing the cortex and
hippocampus were separated and then lysed for the determination of inflammatory cytokine and
western blot analysis.
3.3.4. Assessment of neurological deficit
The effect of compound 8k was determined on the neurological dysfunction of mice after 24h
of MCAO surgery by an observer blinded to the treatment of experimental animals using a 5-point
neurological score. The score was assigned as follows, 0 for no deficit; 1 for flexion of
contralateral forelimb; 2 for decrease in resistance toward the contralateral plane; 3 for circling
monolaterally; and 4 for labored or absent ambulation. Higher scores represented more serious
dysfunction.
3.3.5. Evaluation of cerebral edema
For measurement of cerebral edema, the brains were rapidly excised onto ice after ischemia
reperfusion. Immediately, weigh the brain sample to get the wet weight (ww) and then the brain
tissue sample was dried at 60 °C for 48 h to get the dry weight (dw). After that, the brain water
content was calculated as follows: The cerebral edema=[(ww−dw)/(ww)]×100%.
3.3.6. Histological examination
The H and E staining of brain cornonal sections were performed after were fixed in 10%
neutral buffered formalin for 48 h then the samples were processed to get 4 µm paraffin embedded
sections. Then, the sections were stained with hematoxylin and eosin (H&E). The infarct areas
were determined, photographed and calculated in each histopathological section by using Lieca
Qwin 500 Image Analyzer (Leica, Cambridge, England).
3.3.7. Evaluation of mice brain infarct area
Briefly, the mice brains were excised, freezed and cutted into coronal sections of 2-mm. The
2% solution of TTC in saline phosphate was used to incubate the brain slices at 37 °C for 30 min.
The different colouring pattern of brain was recorded with the help of digital camera into two
distinct color zones, such as red and un-stained region which denotes normal tissue and infarct
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region, respectively. The volume of the whole brain and the infarct tissue was determined with
Image-Pro Plus software USA.
3.3.8. Detection of inflammatory cytokines in brain tissue
The effect of compound 8k on the TNF-α, IL-β and IL-6 using the ELISA kits as per the
manufacturer instruction (R&D Systems ELISA kit Minneapolis MN 55413, USA).
3.4.
Western blot analysis
Protein levels were determined using a BCA protein assay kit (Thermo Fisher Scientific).
Protein extract (50 μg) was resolved by 12% SDS-PAGE electrophoresis and electrotransferred
onto a nitrocellulose membrane. The membrane was blocked using TrisPBS (TPBS) with Tween
20 and 5% skimmed milk and probed with primary antibodies: Next, an anti-rabbit secondary
antibody conjugated to horseradish peroxidase (1: 10 000; Abcam/Cell Signaling Technology) in
TPBS was added and incubated at 37°C for 1 h. Bands were visualized using an enhanced
chemiluminescence image analyzer, and protein levels were quantified using Image-Pro Plus
software (Media Cybernetics, Inc., MD, USA).
3.5.
Statistical analysis
All data are presented as mean ± SD of three independent experiments. Data were statistically
analyzed by one-way analysis followed by the by a Tukey’s post hoc test using statistical software
GraphPad Prism 5.0 (California, USA). The P value < 0.05 was considered as statistically
significant.
4. Conclusion
As a concluding remark, the present study showed the development of thiazolidin-4-one-1,3,5-
triazine derivatives as protective agent against cerebral ischemia/reperfusion injury. The molecules
showed excellent in-vitro inhibition of NF-ĸB activation in RAW264.7 cells. The most potent NF-
ĸB inhibitor (8k) showed neuro-protective effect in mice mediated through attenuation of
inflammation, oxidative stress and apoptosis. Compound 8k also showed inhibition of activation
of NF-ĸB pathway in western blot analysis. In the SAR analysis, electron-withdrawing group
found more active than their electron donating counterparts which have encouraged us to
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investigate additional novel lead scaffolds exhibiting potent neuro-protective activity through
inactivation of NF-ĸB.
Competing interests
The authors declare that they have no competing interests.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon
reasonable request.
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Figure captions
Scheme 1: Synthesis of target compounds 8(a-n). Where a) various amines, stirring, aq. NaOH, 0–
5
̊
̊C; (b) NH3, reflux, 125–135 ̊C; (c) toluene, reflux,
̊
Figure 1: Effect of compound 8k on (a) neurological defict score and (b) cerebral edema
percentage. Values represent the mean ± SD and are representative of three independent
experiments. ##P < 0.05 vs sham; **P < 0.01 vs. MCAO, one-way analysis of variance followed by
a Tukey’s post hoc test.
Figure 2: Effect of 8k on the histopathology of brain coronal section.
Figure 3: Effect of compound 8k on the brain infarct area of MCAO mice. Values represent the
##
mean ± SD and are representative of three independent experiments. P < 0.05 vs sham; **P <
0.01 vs. MCAO, one-way analysis of variance followed by a Tukey’s post hoc test.
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Figure 4: Effect of compound 8k on pro-inflammatory mediators (a) TNF-α, (b) IL-β and (c) IL-6.
##
Values represent the mean ± SD and are representative of three independent experiments. P <
0.05 vs sham; **P < 0.01 vs. MCAO, one-way analysis of variance followed by a Tukey’s post
hoc test.
Figure 5: Effect of compound 8k on the oxidative stress in the MCAO mice (a) MDA, (b) SOD
and c) GSH. Values represent the mean ± SD and are representative of three independent
experiments. ##P < 0.05 vs sham; **P < 0.01 vs. MCAO, one-way analysis of variance followed by
a Tukey’s post hoc test.
Figure 6: Effect on the apoptosis related genes. (a) cleaved caspasse-3, (b) Bax and (c) Bcl-2.
##
Values represent the mean ± SD and are representative of three independent experiments. P <
0.05 vs sham; **P < 0.01 vs. MCAO, one-way analysis of variance followed by a Tukey’s post
hoc test.
Figure 7: Effect on Nf-kB signalling pathway. (a) p-IκBα and (b) NF-κB. Values represent the
##
mean ± SD and are representative of three independent experiments. P < 0.05 vs sham; **P <
0.01 vs. MCAO, one-way analysis of variance followed by a Tukey’s post hoc test.
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List of Tables
Table 1: Inhibitory activity of compounds (8a-n) on NF-ĸB transcriptional activity in LPS-
stimulated RAW264.7 cells.a
Entry
8a
8b
8c
8d
8e
8f
R1
R2
IC50 (in µM)a
> 100
H
4-Cl
22.12 ±5.67
19.23 ± 2.11
15.65 ± 1.92
31.36 ± 3.72
49.56 ± 4.73
> 100
4-F
4-NO2
4-OH
4-OCH3
4-CH3
H
4-Cl
8g
8h
8i
10.34 ± 0.86
3.33 ± 0.23
4.21 ± 0.67
0.90 ± 0.12
5.89 ± 1.14
10.11 ± 2.33
7.34 ± 1.50
4-Cl
8j
4-F
8k
8l
4-NO2
4-OH
4-OCH3
4-CH3
4-F
8m
8n
Dexamethasone (Reference) 0.95 ± 0.56
a
IC50 values expressed as mean ± SD of at least three independent assays, ** P < 0.01 vs.
Control, ## P < 0.01 vs. LPS
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cbdd_13744_f1.tif
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cbdd_13744_f2.tif
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cbdd_13744_f3.tif
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cbdd_13744_f4.tif
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