Design and synthesis of potent 1,2,4-trisubstituted imidazolinone derivatives with dual p38αMAPK and ERK1/2 inhibitory activity

Article abstract of DOI:10.1016/j.ejmech.2015.03.008

The synthesis of new 1,2,4-trisubstituted imidazolinone derivatives was described. The new compounds were designed as dual p38αMAPK and ERK1/2 inhibitors through hybridization of pharmacophoric elements associated with inhibition of these kinases. The kin

Full text of DOI:10.1016/j.ejmech.2015.03.008

Accepted Manuscript
Design and synthesis of potent 1,2,4-trisubstituted imidazolinone derivatives with dual
p38αMAPK and ERK1/2 inhibitory activity
Fadi M. Awadallah, Sahar M. Abou-Seri, Mohamed M. Abdulla, Hanan H. Georgey
PII:
S0223-5234(15)00167-1
10.1016/j.ejmech.2015.03.008
EJMECH 7753
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 December 2014
Revised Date: 2 March 2015
Accepted Date: 3 March 2015
Please cite this article as: F.M. Awadallah, S.M. Abou-Seri, M.M. Abdulla, H.H. Georgey, Design and
synthesis of potent 1,2,4-trisubstituted imidazolinone derivatives with dual p38αMAPK and ERK1/2
inhibitory activity, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.03.008.
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ACCEPTED MANUSCRIPT
Graphical abstract:
N
H2N
O
HN
H
N
N
H
N
CF3
N
S
O
N
O
O
IV
F
I (LY2228820)
H
O
N
N
R1
O
p38 MAPK
inhibition
N
O
R
ERK1/2 inhibition
R= OCH3, R1= H, CH3, OCH3, Cl
6, 7
6d (R=3-OCH3, R1= 4-CH3)and 7h (R=4-OCH3, R1=2-Cl) exert inhibitory activity of 4.5 and 4.7 nM against p38αMAP, 25
and 24 nM against ERK1, and 3.2 and 3.5 nM against ERK2, respectively.

ACCEPTED MANUSCRIPT
Design and synthesis of potent 1,2,4-trisubstituted imidazolinone derivatives
with dual p38αMAPK and ERK1/2 inhibitory activity
Fadi M. Awadallah,1a Sahar M. Abou-Seri,*a Mohamed M. Abdulla,b Hanan H. Georgeya
a
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street 11562,
Cairo Egypt.
b Research Unit, Saco Pharm. Co., 6th October City, Egypt
Abstract.
The synthesis of new 1,2,4-trisubstituted imidazolinone derivatives was described. The
new compounds were designed as dual p38αMAPK and ERK1/2 inhibitors through
hybridization of pharmacophoric elements associated with inhibition of these kinases. The
kinase inhibition assay revealed excellent activity in the nanomolar range; especially
compounds 6d and 7h which seemed promising candidates for such dual activity with IC50
values of 4.5 and 4.7 nM against p38αMAP, 25.0 and 24.0 nM against ERK1, and 3.2 and 3.5
nM against ERK2, respectively. These compounds were further tested for their antiproliferative
activity against nine cancer cell lines, where they elicited high activity in the sub-micromolar
range against breast, prostate and melanoma cells.
Keywords: imidazolinones; breast cancer; melanoma; p38αMAPK; ERK1/2.
1 Corresponding author: fadi_mae@hotmail.com
Tel: 002-02-25353400, 002-012-23483941
Fax:002-02-23628426
* Corresponding author: saharshaarawy_69@yahoo.com
Tel: 002-02-25353400, 002-012-27059742
Fax:002-02-23628426
1

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1. Introduction.
The oncogenic process is a result of multiple dysfunctional pathways and pathway
genes. Therefore, targeting multiple pathways simultaneously is necessary for sustained tumor
remission. Signal transduction in cancer cells is a sophisticated process that involves receptor
tyrosine kinases (RTKs) that eventually trigger multiple cytoplasmic kinases, which are often
serine/threonine kinases. In clinical trials, highly selective or specific inhibitors of only one of
the kinases involved in these signaling pathways has been associated with limited or sporadic
responses. Therefore, simultaneous inhibition of several key kinases at the level of receptors
and/or downstream serine/threonine kinases may help to optimize the overall therapeutic
benefit associated with molecularly targeted anticancer agents [1-3].
Mamalian mitogen-activated protein kinases (MAPKs) are serine/threonine protein
kinases that participate in intracellular signaling during proliferation, differentiation, cellular
stress responses and apoptosis. Inhibition of MAPKs, including extracelluar signal-regulated
kinases 1 and 2 (ERK1/2), p38MAPK (having α, β, γ, δ isoforms) and the stress activated
protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), has been implicated in the activity of
numerous chemotherapy and genotoxic drugs [4,5]. p38MAPK regulates production of
cytokines by the tumor microenvironment and its activation enables cancer cells to survive in
the presence of oncogenic stress, radiation, chemotherapy and targeted therapies [6]. In
addition, p38MAPK is implicated in angiogenesis which is required for tumor growth and
metastasis, and many new cancer therapies are therefore directed against the tumor vasculature.
LY2228820 dimesylate I is a highly selective small molecule inhibitor of p38α and p38β
MAPKs that is currently under clinical investigation for human malignancies [7]. Another
example is the potent p38αMAP kinase inhibitor isoxazole isostere II [8] (Figure 1).
On the other hand, upon activation of ERK1/2 by growth factors, cytokines and
osmotic stresses, they can phosphorylate and regulate multiple substrates such as cytoskeletal
proteins, kinases and transcription factors within various cellular compartments. These events
in turn result in gene expression changes and alteration in cell proliferation, differentiation and
survival [9]. For this reason, inhibitors of the ERK1/2 are entering clinical trials as potential
anticancer agents [10]. The 3,4-diarylsubstituted pyrazole derivative III demonstrated potent
cytotoxic activity through ERK1/2 inhibition [11] (Figure 1).
Based on these observations, it seemed beneficial to co-target p38αMAPK and ERK1/2,
an approach that might lead to a synergistic effect on inhibiting cell proliferation and on
controlling metastasis. Moreover, it was reported that the inhibition of p38MAPK resulted in
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stimulation of ERK with subsequent induction of proliferation [12,13]. Therefore, dual
targeting of p38MAPK and ERK would prevent activation of ERK following p38MAPK
inhibition, and would potentiate the antiproliferative activity of the new compounds.
With the goal of producing new antitumor agents and based on the aforementioned
facts, the design of the new target compounds relied on a hybrid pharmacophoric approach in
which two molecular fragments were combined. The first was the vicinal diarylsubstituted 5-
membered heterocyclic nucleus, which is typical for p38MAPK inhibition, as could be featured
in compounds I and II. Accordingly, the vicinal diarylsubstituted imidazolinone moiety was
selected as a core for our target compounds 6 and 7. On the other hand, it could be observed
that the ERK1/2 inhibitor III was characterized by a structure extension at position 1 of the
pyrazole ring which was believed to contribute to ERK activity. Hence, a second molecular
fragment deemed essential to extend the imidazolinone core as to empower ERK inhibition
[11]. Searching for a bioactive moiety of interest in the field of chemotherapy, the literature
survey revealed the broad spectrum anticancer activity of compound IV against lung, prostate,
breast cancers and leukemia [14]. Appropriately, the benzylidene tail of the thiazolidinedione
derivative IV was selected to extend the imidazolinone scaffold with the hope to obtain new
compounds with potential activity against many types of cancer cells and acting via inhibition
of p38αMAPK and ERK1/2 (Figure 2).
Herein, we report the synthesis of novel series of diversely substituted imidazolinone
derivatives 6a-j and 7a-j together with the study of their inhibitory activity against p38αMAPK
and ERK1/2, and their antiproliferative activity toward selected cell lines.
2. Discussion.
2.1. Chemistry:
The synthetic strategy for the preparation of the target compounds 6a-j and 7a-j was
illustrated in Scheme 1. 4-(4-Hydroxybenzylidene)-2-phenyloxazol-5(4H)-one 2 was
synthesized according to the literature method [15] via the Erlenmyer reaction of hippuric acid
3

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with 4-hydroxybenzaldehyde and acetic anhydride. Subsequently, 4-(4-hydroxybenzylidene)-1-
(3/4-methoxyphenyl)-2-phenyl-1H-imidazol-5(4H)-ones 3 and 4 were obtained through the
reaction of 2 with 3/4-methoxyaniline in glacial acetic acid in the presence of anhydrous
sodium acetate. This reaction proceeded through open intermediates which were recyclized in
the presence of glacial acetic acid to afford the imidazolinones 3 and 4 [16]. IR spectra of
compounds 3 and 4 revealed the appearance of bands at 3327 and 3290 cm-1, respectively
assigned to the OH group. Also, 1H NMR showed the exchangeable singlet signal
corresponding to OH proton. Reaction of 3, 4 with 2-chloro-N-un/substituted phenylacetamide
5a-j; previously synthesized according to the literature method [17,18]; afforded the acetamide
derivatives 6a-j and 7a-j in good yields. IR spectra revealed the presence of an additional
1
carbonyl band assigned to the phenylacetamido function. H NMR revealed the presence of a
singlet signal integrated for 2 protons corresponding to the CH2 protons at 4.67-4.76 ppm and
an exchangeable singlet signal corresponding to NH proton mostly at δ 8.16-9.01 ppm. Also,
additional singlet signals corresponding to the 3 methyl or 3 methoxy protons resonating at
2.27-2.39 or 3.83-3.90 ppm in case of 6b-d, 7b-d or 6e-g, 7e-g, respectively, were observed.
Moreover, 13C NMR showed the characteristic signals of the CH2 signal at 67.4-67.6 ppm in
addition to signals at 17.4-21.4 ppm or 55.4-55.9 ppm for compounds bearing methyl or
methoxy groups, respectively.
2.2. Anticancer activity:
2.2.1 In vitro kinase inhibitory activity:
The target compounds were evaluated for their potential inhibitory effect on
p38αMAPK and ERK1/2 protein kinases using LanthaScreen Kinase activity assay kits
(Invitrogen). The activity was expressed by median growth inhibitory concentration (IC50) and
provided in Table 1. Sorafenib was used as reference drug. The results indicated that, the new
imidazolinones exhibited potent inhibitory activity against the three kinases at nanomolar level
(IC50 ranges: p38α = 3.7-354, ERK1= 21-95, ERK2= 2.3-89 nM). Moreover, the majority of
the tested compounds were more potent against p38αMAPK and/or ERK2 than ERK1. Several
derivatives showed potency higher than sorafenib against p38αMAPK and ERK2 but all of
them were less potent than the reference drug against ERK1. In particular, compounds 6d, 6f-
6j, 7h and 7i displayed excellent kinase suppressive effect at single digit nanomolar
concentrations against p38αMAPK and ERK2 (IC50 range = 3.7-6.5 and 3.4-7.0 nM,
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respectively). Compound 6e, 7g and 7j preferentially inhibited ERK2 with IC50= 4.7, 2.3 and
4.5 nM, respectively.
Analysis of p38αMAPK enzymatic assay results revealed that the inhibitory potency of
N1-3-methxoyphenyl imidazolinones 6a-j and their N1-4-methxoyphenyl analogs 7a-j was
primarily affected by the electronic nature and position of the substituent on the pendant phenyl
ring of the aryl acetamide moiety. The inhibitory potency of N1-3-methxoyphenyl
imidazolinones 6a-j was enhanced by grafting electron-withdrawing substituent like 3-methoxy
group 6f (IC50=6.5 nM) or chlorine atom 6h-i (IC50=3.7-5.6 nM) to the phenyl ring regardless
of its position. The potency was also favored by para-substitution on the phenyl with 4-methyl
6d (IC50=4.5 nM) or 4-methoxy group 6g (IC50= 4.3 nM), (c.f. 6a R= H, IC50= 35 nM).
However, ortho- and meta-methylphenyl 6b,c or ortho-methoxyphenyl 6e derivatives were the
least active in this series. Considering N1-4-methxoyphenyl imidazolinones 7a-j, compound 7a,
having unsubstituted phenyl ring, was the least active kinase inhibitor (IC50=354 nM). Similar
to the N1-3-methxoyphenyl imidazolinones 6a-j, compounds with electron-withdrawing 3-
methoxy group 7f (IC50=23 nM) or chloro 7h-i (IC50=4.5-44 nM) substituent on the pendant
phenyl were significantly more potent than those with electron-donating methyl group 7b-d or
2/4-methoxy group 7e and 7g (IC50=34-345 nM). However, substitution at the para position
(7d, 7g and 7j) was not well tolerated compared to ortho (7b, 7e and 7h) and meta (7c, 7f and
7i) substituted counterparts.
As per the activity against ERK2, it could be noticed that there was a high degree of
homogeneity between the activity of the compounds on p38αMAPK and ERK2; so that
compounds highly effective against p38αMAPK were similarly effective against ERK2 except
for derivatives 6e, 7g and 7j which were more active on ERK2 than p38αMAPK.
Finally, looking up for our target dual p38αMAP-ERK1/2 inhibitors, compounds 6d and
7h seemed promising candidates for such dual activity; exerting inhibitory activity of 4.5 and
4.7 nM against p38αMAP, 25 and 24 nM against ERK1, and 3.2 and 3.5 nM against ERK2,
respectively.
2.2.2. Antiproliferative activity of compounds 6d and 7h:
Constitutive activation of MAPK pathway was found in patient tumor samples in breast,
colon, melanoma, lung, blood and thyroid [19,20]. Therefore, the antiproliferative activity of
compounds 6d and 7h which demonstrated the best overall kinase inhibitory activity was
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evaluated against nine human tumor cell lines derived from different cancer types. The growth
inhibitory activity of the compounds was evaluated using MTT method [21] and sorafenib was
employed as a reference compound. The results were expressed as IC50 values and are
presented in Table 2.
The results revealed that both compounds exhibited potent
antiproliferative activity against most of the tested cell lines (IC50 range = 0.05-9.40 µM), but
were inactive against leukemia K562. Compounds 6d and 7h possessed superior activity at
sub-micromolar level, even higher than that of sorafenib, against breast cancer (MCF7 and
T47D), melanoma (LOX IMVI and MDA-MB-435) and prostate (PC3) cell lines with IC50
ranging from 0.05 to 0.99 µM. However, unlike sorafenib, the tested compounds were less
effective against colon cancer (Colo-205 and HT29) and lung cancer (A549) cell lines (IC50
range = 4.50-9.40 µM).
Comparing the activity of 6d and 7h against the sensitive cell lines, revealed that
compound 7h displayed higher potency than 6d toward breast (MCF7), colon (Colo-205 and
HT29), lung (A549) and melanoma (LOX IMVI and MDA-MB-435) cancer cell lines. On the
other hand, 6d was more effective against breast (T47D) and prostate (PC3) cell lines.
To examine the selectivity and hence the safety of compounds 6d and 7h, their
antiproliferative activity was assessed against normal mammalian cells (Vero cells). The data
indicated that the tested compounds had relatively low cytotoxic effect on normal cells (IC50=
17.34 and 13.20 ꢀM, respectively). The selectivity index calculated in relation to IC50 on Vero
cells revealed high selectivity toward most tested cancer cells (SI > 20) except colon (Colo-
205, HT-29) and lung (A-549) cells (Table2).
3. Conclusion.
The design and synthesis of new 1,2,4-trisubstituted imidazolinone derivatives of dual
p38αMAPK and ERK1/2 inhibitory activity was described. The structure of the new
compounds featured a vicinal diarylsubstituted imidazolinone core contributing to p38αMAPK
activity and an extended substitution at position 4 of the imidazolinone nucleus that favored
the ERK1/2 activity. In vitro kinase activity revealed preferential activity against p38αMAPK
and ERK2 with IC50 in the nanomolar level and the activity of some derivatives exceeded that
of sorafenib. Generally, the N1-3-methoxyphenyl-imidazolinones were more potent than their
N1-4-methoxyphenyl isomers. Compounds 6d and 7h exhibited the highest activity against the
three tested kinases. Therefore, their antiproliferative activity was assessed against several cell
lines belonging to breast, colon, lung, leukemia, melanoma and prostate cancers. These
compounds displayed activity in the sub-micromolar range and were found superior to
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sorafenib against breast cancer (MCF7 and T47D), melanoma (LOX IMVI and MDA-MB-435)
and prostate (PC3) cell lines with IC50 ranging from 0.05 to 0.99 µM. Also, the compounds
were highly selective to the same cancer cell lines as compared to normal Vero cells. Finally, it
could be claimed that the rationale behind the design of the target compounds succeeded in
achieving dual p38αMAPK and ERK1/2 inhibition, which in turn secured potent
antiproliferative activity against several cancer cell lines.
4. Experimental
4.1. Chemistry:
Melting Points were carried out by open capillary tube method using Stuart SMP3
Melting Point apparatus and they were uncorrected. Elemental Microanalysis was carried out at
the Regional Center for Mycology and Biotechnology, Al-Azhar University. Infrared spectra
were recorded on Shimadzu Infrared spectrometer IR Affinity-1 (FTIR- 8400S-Kyoto-Japan),
and expressed in wave number (cm-1), using potassium bromide discs. NMR spectra were
recorded on Bruker Avance III 400 MHz high performance digital FT-NMR spectrophotometer
(1H: 400, 13C: 100 MHz) in chloroform-d, using TMS as an internal standard. Chemical shifts
were expressed in δ units and were related to that of the solvents. All the reactions were
followed up by TLC using silica gel F254 plates (Merck), using chloroform: methanol 9:1 or
chloroform as eluting system and were visualized by UV-lamp. Compounds 2 [1] and 5a-j [3,4]
were prepared according to reported methods.
4.1. Chemistry
4.1.1. General procedure for the synthesis of compounds (3, 4).
A mixture of 2 (10 mmol, 2.65g), 3/4-methoxyaniline (12 mmol, 1.47g) and freshly
prepared anhydrous sodium acetate (0.3g) in glacial acetic acid (10 mL) was heated in a boiling
water bath for 3 h. The crystalline product separated on cooling was filtered off, washed with
water, dried, and recrystallized from ethanol.
4.1.1.1. 4-(4-Hydroxybenzylidene)-1-(3-methoxyphenyl)-2-phenyl-1H-imidazol-5(4H)-one (3).
o
Yield 87%; m.p. 252-253 C; IR (KBr, cm-1: 3327 (OH), 3061 (aromatic CH), 2954
(CH aliphatic), 1685 (C=O);1H NMR: δ 3.72 (s, 3H, OCH3), 6.79 (d, 2H, aromatic H, J= 7.72
Hz), 6.81 (s, 1H, aromatic H), 6.88 (d, 1H, aromatic H, J= 8.60 Hz), 6.91 (s, ex, 1H, OH), 6.99,
7.01 (dd, 1H, J= 2.24, 8.30 Hz), 7.20 (s, 1H, CH), 7.33-7.39 (m, 3H, aromatic H), 7.46 (t, 1H,
aromatic H, J= 7.36 Hz), 7.53 (d, 2H, aromatic H, J= 7.16 Hz), 8.24 (d, 2H, aromatic H, J=
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8.68 Hz); 13C NMR: δ 55.8, 114.2, 114.3, 116.6, 120.4, 125.6, 128.8, 129.1, 129.4, 130.4,
131.5, 135.2, 135.8, 136.3, 158.9, 160.1, 161.2, 169.9. Anal. Calcd. For C23H18N2O3 (370.12):
C, 74.58; H, 4.90; N, 12.96%. Found: C, 74.69; H, 4.93; N, 7.65%.
4.1.1.2. 4-(4-Hydroxybenzylidene)-1-(4-methoxyphenyl)-2-phenyl-1H-imidazol-5(4H)-one (4).
o
Yield 82%; m.p. 273-274 C; IR (KBr, cm-1): 3290 (OH), 3062 (aromatic CH), 2960
(CH aliphatic), 1689 (C=O); 1H NMR: δ 3.76 (s, 3H, OCH3), 6.89 (d, 2H, aromatic H, J= 8.80
Hz), 6.96 (d, 2H, aromatic H, J= 6.76 Hz), 7.16 (d, 2H, aromatic H, J= 6.96 Hz), 7.18 (s, 1H,
CH), 7.34-7.38 (m, 3H, aromatic H), 7.51 (d, 2H, aromatic H, J= 8.52 Hz), 8.24 (d, 2H,
aromatic H, J= 8.76 Hz), 10.49 (s, ex, 1H, OH); 13C NMR: δ 55.8, 114.9, 116.4, 127.8, 128.1,
128.8, 129.2, 129.4, 129.5, 131.5, 135.2, 136.1, 159.4, 160.7, 170.4; Anal. Calcd. For
C23H18N2O3 (370.12): C, 74.58; H, 4.90; N, 12.96%. Found: C, 74.72; H, 4.87; N, 7.68%.
4.1.2. General procedure for the synthesis of compounds (6a-j, 7a-j).
Potassium carbonate (10 mmol, 1.38g) was added to a solution of compound 3, 4 (10
mmol, 3.70g) in dry DMF (15 mL) and stirred at room temperature for 0.5h. Then, the
appropriate 2-chloro-N-un/substituted phenylacetamide 5a-j (10 mmol) was added and heated
at 100oC for 8h. The reaction mixture was poured onto ice/water and the precipitated solid was
filtered off, washed with water, dried and crystallized from ethanol.
4.1.2.1.
2-(4-((1-(3-Methoxyphenyl)-5-oxo-2-phenyl-1H-imidazol-4(5H)-ylidene)methyl)
phenoxy) -N-phenylacetamide (6a).
Yield: 82%; m.p. 220-221 oC; IR (KBr, cm-1): 3332 (NH), 3061 (aromatic CH), 2924
1
(CH aliphatic), 1706, 1685 (C=O); H NMR: δ 3.78 (s, 3H, OCH3), 4.71 (s, 2H, CH2), 6.74 (s,
1H, aromatic H), 6.76-6.78 (m, 1H, aromatic H), 6.94, 6.96 (dd, 1H, aromatic H, J= 2.16, 8.40
Hz), 7.09 (d, 2H, aromatic H, J= 8.80 Hz), 7.12 (t, 1H, aromatic H, J= 7.40 Hz), 7.28 (s, 1H,
CH), 7.31-7.41 (m, 5H, aromatic H), 7.44 (t, 1H, J= 7.44 Hz), 7.61 (d, 4H, aromatic H, J= 8.40
Hz), 8.25 (s, ex, 1H, NH), 8.34 (d, 2H, aromatic H, J= 8.40 Hz); 13C NMR: δ 55.4, 67.5, 113.0,
114.3, 115.1, 119.6, 120.1, 120.2, 125.0, 128.3, 128.4, 128.6, 129.0, 129.1, 130.0, 131.3, 134.7,
135.7, 136.6, 137.6, 158.5, 160.0, 160.2, 165.6, 170.4. Anal. Calcd. For C31H25N3O4 (503.55):
C, 73.94; H, 5.00; N, 8.34%. Found: C, 74.02; H, 5.04; N, 8.42%.
4.1.2.2.
2-(4-((1-(3-Methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-ylidene)
methyl)phenoxy)-N-(o-tolyl)acetamide (6b).
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Yield: 65%; m.p. 159-161oC; IR (KBr, cm-1): 3410 (NH), 3050 (aromatic CH), 2924
(CH aliphatic), 1728, 1693 (C=O); 1H NMR: δ 2.38 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 4.68 (s,
2H, CH2), 6.74 (s, 1H, aromatic H), 6.76-6-78 (m, 1H, aromatic H), 6.93, 6.95 (dd, 1H,
aromatic H, J= 2.24, 8.32 Hz), 6.99 (t, 1H, aromatic H, J= 7.50 Hz), 7.08 (d, 2H, aromatic H,
J= 8.80 Hz), 7.26 (s, 1H, CH), 7.24-7.48 (m, 7H, aromatic H), 7.61(d, 2H, aromatic H, J= 7.32
13
Hz), 8.23 (s, ex, 1H, NH), 8.33 (d, 2H, aromatic H, J= 8.76 Hz); C NMR: δ 21.4, 55.4, 67.5,
113.0,114.3, 115.1, 117.3, 120.7,120.8, 125.8, 128.3, 128.4, 128.8, 128.9, 129.0, 129.1, 130.0,
131.3, 134.7, 135.7, 136.6, 137.5, 139.1, 158.5,159.9,160.2,165.6, 170.4 . Anal. Calcd. For
C32H27N3O4 (517.58): C, 74.26; H, 5.26; N, 8.12%. Found: C, 74.34; H, 5.25; N, 8.22%.
4.1.2.3.
2-(4-((1-(3-Methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-ylidene)
methyl)phenoxy)-N-(m-tolyl)acetamide (6c).
Yield: 86%; m.p. 167-169 oC; IR (KBr, cm-1): 3414 (NH), 3066 (aromatic CH), 2924
1
(CH aliphatic), 1724, 1701(C=O); H NMR: δ 2.27 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 4.76 (s,
2H, CH2), 6.75 (s, 1H, aromatic H), 6.77-6-78 (m, 1H, aromatic H), 6.94, 6.96 (dd, 1H,
aromatic H, J= 1.96, 8.44 Hz), 7.10 (d, 2H, aromatic H, J= 8.80 Hz), 7.13 (d, 1H, aromatic H,
J= 7.72 Hz), 7.22 (t, 1H, aromatic H, J= 7.60 Hz), 7.28 (s, 1H, CH), 7.32 (s,1H, aromatic H),
7.33-7.37 (m, 3H, aromatic H), 7.44 (t, 1H, aromatic H, J= 7.44 Hz), 7.61(d, 2H, aromatic H,
J= 7.32 Hz), 7.98 (d, 1H, aromatic H, J= 7.92 Hz), 8.22 (s, ex, 1H, NH), 8.35 (d, 2H, aromatic
H, J= 8.80 Hz); 13C NMR: δ 17.4, 55.4, 67.5, 113.0, 114.3, 115.0, 119.6, 122.4, 125.5, 126.9,
128.3, 128.6, 128.8, 129.0, 129.1, 130.0, 130.5, 131.3, 134.7, 135.7, 137.5, 158.5, 160.0, 160.2,
165.6, 170.4. Anal. Calcd. For C32H27N3O4 (517.58): C, 74.26; H, 5.26; N, 8.12%. Found: C,
74.35; H, 5.29; N, 8.25%.
4.1.2.4.
2-(4-((1-(3-Methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-ylidene)
methyl)phenoxy)-N-(p-tolyl)acetamide (6d).
Yield: 84%; m.p. 151-153 oC; IR (KBr, cm-1): 3323 (NH), 3050 (aromatic CH), 2922
1
(CH aliphatic), 1710, 1683 (C=O); H NMR: δ 2.36 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 4.70 (s,
2H, CH2), 6.74 (s, 1H, aromatic H), 6.76-6-78 (m, 1H, aromatic H), 6.94, 6.96 (dd, 1H,
aromatic H, J= 2.08, 8.32 Hz), 7.09 (d, 2H, aromatic H, J= 8.80 Hz), 7.18 (d, 2H, aromatic H,
J= 8.24 Hz), 7.28 (s, 1H, CH), 7.33-7.38 (m, 3H, aromatic H), 7.44-7.50 (m, 3H, aromatic H),
7.61(d, 2H, aromatic H, J= 7.32 Hz), 8.19 (s, ex, 1H, NH), 8.34 (d, 2H, aromatic H, J= 8.76
Hz); 13C NMR: δ 20.9, 55.4, 67.5, 113.0, 114.3, 115.1, 119.6, 120.1, 128.3, 128.4, 128.8, 129.0,
129.1, 129.6, 130.0, 131.3, 134.1, 134.7, 135.7, 137.5, 158.5, 160.0, 160.2, 165.5, 170.4. Anal.
9

ACCEPTED MANUSCRIPT
Calcd. For C32H27N3O4 (517.58): C, 74.26; H, 5.26; N, 8.12%. Found: C, 74.39; H, 5.30; N,
8.25%.
4.1.2.5.
N-(2-Methoxyphenyl)-2-(4-((1-(3-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (6e).
Yield: 76%; m.p. 172-174 oC -; IR (KBr, cm-1): 3402 (NH), 3060 (aromatic CH), 2924
(CH aliphatic), 1710, 1683 (C=O); 1H NMR: δ 3.78 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.72 (s,
2H, CH2), 6.75 (s, 1H, aromatic H), 6.77-6-78 (m, 1H, aromatic H), 6.91-6.96 (m, 3H, aromatic
H), 6.99 (t, 1H, aromatic H, J= 7.52 Hz), 7.10 (d, 2H, aromatic H, J= 8.84 Hz), 7.28 (s, 1H,
CH), 7.31-7.37 (m, 3H, aromatic H), 7.44 (t, 1H, J= 7.44 Hz), 7.61 (d, 2H, aromatic H, J= 7.48
Hz), 8.34 (d, 2H, aromatic H, J= 8.76 Hz), 8.41 (d, 1H, aromatic H, J= 7.92 Hz), 8.96 (s, ex,
13
1H, NH); C NMR: δ 55.4, 55.9, 67.6, 110.1, 113.0, 114.3, 115.2, 119.6, 120.0, 121.1, 124.4,
126.6, 128.3, 128.5, 128.8, 129.1, 130.0, 131.3, 134.7, 135.7, 137.4, 148.3, 158.7, 159.9, 160.2,
165.4, 170.4. Anal. Calcd. For C32H27N3O5 (533.58): C, 72.03; H, 5.10; N, 7.88%. Found: C,
72.19; H, 5.07; N, 8.02%.
4.1.2.6.
N-(3-Methoxyphenyl)-2-(4-((1-(3-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (6f).
Yield: 87%; m.p. 137-140oC; IR (KBr, cm-1): 3383 (NH), 3062 (aromatic CH), 2924
(CH aliphatic), 1706, 1676 (C=O); 1H NMR: δ 3.78 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.70 (s,
2H, CH2), 6.73-6-77 (m, 3H, aromatic H), 6.94, 6.96 (dd, 1H, aromatic H, J= 2.20, 8.40 Hz),
7.08-7.11 (m, 3H, aromatic H), 7.25 (s, 1H, aromatic H), 7.28 (s, 1H, CH), 7.33-7.37 (m, 4H,
aromatic H), 7.44 (t, 1H, J= 7.40 Hz), 7.61 (d, 2H, aromatic H, J= 7.32 Hz), 8.24 (s, ex, 1H,
13
NH), 8.34 (d, 2H, aromatic H, J= 8.76 Hz); C NMR: δ 55.3, 55.4, 67.5, 105.9, 110.8, 112.3,
113.0, 114.3, 115.1, 119.6, 128.3, 128.4, 128.8, 129.0, 129.1, 129.8, 130.0, 131.3, 134.7, 135.7,
137.5, 137.8, 158.5, 160.0, 160.2, 160.3, 165.6, 170.4. Anal. Calcd. For C32H27N3O5 (533.58):
C, 72.03; H, 5.10; N, 7.88%. Found: C, 72.15; H, 5.09; N, 8.02%.
4.1.2.7.
N-(4-Methoxyphenyl)-2-(4-((1-(3-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (6g).
Yield: 72%; m.p. 204-206 oC; IR (KBr, cm-1): 3361 (NH), 3055 (aromatic CH), 2927
(CH aliphatic), 1714, 1683 (C=O); 1H NMR: δ 3.83 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.70 (s,
2H, CH2), 6.74 (s, 1H, aromatic H), 6.76-6-79 (m, 1H, aromatic H), 6.91-6-96 (m, 3H,
aromatic H), 7.09 (d, 2H, aromatic H, J= 8.48 Hz), 7.28 (s, 1H, CH), 7.31-7.37 (m, 3H,
aromatic H), 7.46 (d, 1H, aromatic H, J= 7.76 Hz), 7.50 (d, 2H, aromatic H, J= 8.60 Hz), 7.61
10

ACCEPTED MANUSCRIPT
(d, 2H, aromatic H, J= 7.60 Hz), 8.16 (s, ex, 1H, NH), 8.34 (d, 2H, aromatic H, J= 8.52 Hz);
13C NMR: δ 55.4, 55.5, 67.4, 113.0, 114.2, 115.1, 119.6, 122.0, 122.1, 128.3, 128.4, 128.8,
129.0, 129.1, 129.7, 130.0, 131.3, 134.7, 137.5, 156.9, 158.6, 160.0, 160.2, 160.3, 165.6, 170.4.
Anal. Calcd. For C32H27N3O5 (533.58): C, 72.03; H, 5.10; N, 7.88%. Found: C, 72.13; H, 5.13;
N, 7.96%.
4.1.2.8.
N-(2-Chlorophenyl)-2-(4-((1-(3-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (6h).
Yield: 78%; m.p. 162-164 oC; IR (KBr, cm-1): 3381 (NH), 3066 (aromatic CH), 2935
1
(CH aliphatic), 1712, 1685 (C=O), 837 (C-Cl) ; H NMR: δ 3.77 (s, 3H, OCH3), 4.71 (s, 2H,
CH2), 6.74 (s, 1H, aromatic H), 6.76-6-78 (m, 1H, aromatic H), 6.93, 6.95 (dd, 1H, aromatic H,
J= 2.08, 8.36 Hz), 7.07-7.10 (m, 3H, aromatic H), 7.28 (s, 1H, CH), 7.30-7.41 (m, 5H, aromatic
H), 7.43 (t, 1H, aromatic H, J= 7.40 Hz), 7.61 (d, 2H, aromatic H, J= 7.52 Hz), 8.33 (d, 2H,
aromatic H, J= 8.72 Hz), 8.46 (d, 1H, aromatic H, J= 8.24 Hz), 9.00 (s, ex, 1H, NH); 13C NMR:
δ 55.4, 67.4, 113.0, 114.2, 114.7, 119.6, 121.3, 123.1, 125.2, 127.8, 128.3, 128.4, 128.7, 129.0,
129.1, 130.0, 130.3, 131.0, 131.3, 134.7, 135.7, 137.5, 158.4, 159.9, 160.2, 165.7, 170.4. Anal.
Calcd. For C31H24ClN3O4 (538.00): C, 69.21; H, 4.50; N, 7.81%. Found: C, 69.30; H, 4.53; N,
7.92%.
4.1.2.9.
N-(3-Chlorophenyl)-2-(4-((1-(3-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (6i).
Yield: 74%; m.p. 157-159 oC; IR (KBr, cm-1): 3309 (NH), 3066 (aromatic CH), 2935
1
(CH aliphatic), 1710, 1685 (C=O), 839 (C-Cl); H NMR: δ 3.77 (s, 3H, OCH3), 4.68 (s, 2H,
CH2), 6.73 (s, 1H, aromatic H), 6.75-6-77 (m, 1H, aromatic H), 6.93, 6.95 (dd, 1H, aromatic H,
J= 2.12, 8.12 Hz), 7.07 (d, 2H, aromatic H, J= 8.76 Hz), 7.14 (d, 1H, aromatic H, J= 8.04 Hz),
7.27 (s, 1H, CH), 7.28-7.29 (m, 3H, aromatic H), 7.31-7.37 (m, 2H, aromatic H), 7.44 (t, 1H,
aromatic H, J= 7.66 Hz), 7.61 (d, 2H, aromatic H, J= 7.48 Hz), 7.74 (s, 1H, aromatic H), 8.32
(s, ex, 1H, NH), 8.34 (d, 2H, aromatic H, J= 8.56 Hz); 13C NMR: δ 55.4, 67.4, 113.0, 114.2,
114.7, 115.1, 118.1, 119.6, 120.2, 125.0, 128.3, 128.4, 128.8, 129.1, 130.0, 130.1, 131.3, 134.7,
135.7, 137.6, 137.8, 158.4, 160.0, 160.2, 165.8, 170.4. Anal. Calcd. For C31H24ClN3O4
(538.00): C, 69.21; H, 4.50; N, 7.81%. Found: C, 69.33; H, 4.52; N, 7.95%.
4.1.2.10.
N-(4-Chlorophenyl)-2-(4-((1-(3-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (6j).
11

ACCEPTED MANUSCRIPT
Yield: 75%; m.p. 215-217 oC; IR (KBr, cm-1): 3369 (NH), 3060 (aromatic CH), 2935
1
(CH aliphatic), 1707, 1674 (C=O), 831 (C-Cl); H NMR: δ 3.78 (s, 3H, OCH3), 4.69 (s, 2H,
CH2), 6.74 (s, 1H, aromatic H), 6.76-6.77 (m, 1H, aromatic H), 6.93, 6.95 (dd, 1H, aromatic H,
J= 2.16, 8.32 Hz), 7.07 (d, 2H, aromatic H, J= 8.80 Hz), 7.28 (s, 1H, CH), 7.30-7.37 (m, 5H,
aromatic H), 7.44 (t, 1H, aromatic H, J= 7.40 Hz), 7.57 (d, 2H, aromatic H, J= 8.76 Hz), 7.61
(d, 2H, aromatic H, J= 7.36 Hz), 8.29 (s, ex, 1H, NH), 8.33 (d, 2H, aromatic H, J= 8.56 Hz);
13C NMR: δ 55.4, 67.4, 113.0, 114.2, 114.7, 115.1, 119.6, 121.3, 121.4, 128.3, 128.4, 128.8,
129.1, 129.2, 130.0, 130.1, 131.3, 134.7, 135.3, 135.7, 137.6, 158.4, 160.0, 160.2, 165.7, 170.4.
Anal. Calcd. For C31H24ClN3O4 (538.00): C, 69.21; H, 4.50; N, 7.81%. Found: C, 69.32; H,
4.53; N, 7.91%.
4.1.2.11.
2-(4-((1-(4-Methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-ylidene)
methyl)phenoxy)-N-phenylacetamide (7a).
Yield: 70%; m.p. 229-230 oC -; IR (KBr, cm-1): 3338 (NH), 3052 (aromatic CH), 2923
(CH aliphatic), 1710, 1689 (C=O); 1H NMR: δ 3.86 (s, 3H, OCH3), 4.71 (s, 2H, CH2), 6.95 (d,
2H, aromatic H, J= 8.88 Hz), 7.09 (d, 2H, aromatic H, J= 6.84 Hz), 7.13 (d, 2H, aromatic H, J=
6.84 Hz), 7.19 (t, 1H, aromatic H, J= 7.44 Hz), 7.28 (s, 1H, CH), 7.31-7.48 (m, 5H, aromatic
H), 7.61 (d, 4H, aromatic H, J= 8.36 Hz), 8.25 (s, ex, 1H, NH), 8.34 (d, 2H, aromatic H, J=
8.76 Hz); 13C NMR: δ 55.5, 67.5, 114.7, 115.1, 120.2, 125.0, 127.3, 128.2, 128.3, 128.5, 128.9,
129.1, 129.2, 131.3, 134.7, 136.6, 137.6, 158.4, 159.4, 160.2, 165.6, 170.8. Anal. Calcd. For
C31H25N3O4 (503.55): C, 73.94; H, 5.00; N, 8.34%. Found: C, 73.98; H, 4.97; N, 8.51%.
4.1.2.12.
2-(4-((1-(4-Methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-ylidene)
methyl)phenoxy)-N-(o-tolyl)acetamide (7b).
Yield: 93%; m.p. 231-233 oC; IR (KBr, cm-1): 3309 (NH), 3062 (aromatic CH), 2960
1
(CH aliphatic), 1716, 1697 (C=O); H NMR: δ 2.27 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 4.75 (s,
2H, CH2), 6.95 (d, 2H, aromatic H, J= 8.88 Hz), 7.09-7.14 (m, 4H, aromatic H), 7.21 (t, 1H,
aromatic H, J= 7.28 Hz), 7.27-7.37 (m, 5H, aromatic H and CH), 7.44 (t, 1H, aromatic H, J=
7.40 Hz), 7.61 (d, 2H, aromatic H, J= 7.31 Hz), 7.98 (d, 1H, aromatic H, J= 7.91 Hz), 8.23 (s,
ex, 1H, NH), 8.35 (d, 2H, aromatic H, J= 8.80 Hz); 13C NMR: δ 17.4, 55.5, 67.5, 114.7, 115.0,
122.4, 125.5, 126.9, 127.3, 128.1, 128.3, 128.5, 128.6, 128.9, 129.1, 129.2, 130.5, 131.3, 134.7,
134.8, 137.6, 158.4, 159.4, 160.2, 165.6, 170.8. Anal. Calcd. For C32H27N3O4 (517.58): C,
74.26; H, 5.26; N, 8.12%. Found: C, 74.35; H, 5.24; N, 8.27%.
12

ACCEPTED MANUSCRIPT
4.1.2.13.
2-(4-((1-(4-Methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-ylidene)
methyl)phenoxy)-N-(m-tolyl)acetamide (7c).
Yield: 87%; m.p. 239-241 oC -; IR (KBr, cm-1): 3350 (NH), 3050 (aromatic CH), 2927
1
(CH aliphatic), 1710, 1678 (C=O); H NMR: δ 2.39 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 4.70 (s,
2H, CH2), 6.95 (d, 2H, aromatic H, J= 8.84 Hz), 7.00 (d, 1H, aromatic H, J= 7.96 Hz), 7.09-
7.13 (m, 4H, aromatic H), 7.28 (m, 1H, CH), 7.25-7.46 (m, 6H, aromatic H), 7.61 (d, 2H,
aromatic H, J= 7.40 Hz), 8.19 (s, ex, 1H, NH), 8.34 (d, 2H, aromatic H, J= 8.72 Hz); 13C NMR:
δ 21.4, 55.5, 67.5, 114.7, 115.1, 117.3, 120.8, 125.8, 127.3, 128.2, 128.3, 128.5, 128.9, 129.0,
129.1, 129.2, 131.3, 134.7, 136.5, 137.6, 139.1, 158.5, 159.4, 160.2, 165.6, 170.8. Anal. Calcd.
For C32H27N3O4 (517.58): C, 74.26; H, 5.26; N, 8.12%. Found: C, 74.33; H, 5.28; N, 8.25%.
4.1.2.14.
2-(4-((1-(4-Methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-ylidene)
methyl)phenoxy)-N-(p-tolyl)acetamide (7d).
Yield: 80%; m.p. 244-246 oC; IR (KBr, cm-1): 3361 (NH), 3051 (aromatic CH), 2918
1
(CH aliphatic), 1716, 1683 (C=O); H NMR: δ 2.36 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.69 (s,
2H, CH2), 6.94 (d, 2H, aromatic H, J= 8.84 Hz), 7.01 (d, 2H, aromatic H, J= 8.68 Hz), 7.11 (d,
2H, aromatic H, J= 7.68 Hz), 7.18 (d, 2H, aromatic H, J= 8.24 Hz), 7.28 (s, 1H, CH), 7.30-
7.35 (m, 3H, aromatic H), 7.48 (d, 2H, aromatic H, J= 8.36 Hz), 7.59 (d, 2H, aromatic H, J=
13
7.64 Hz), 8.21 (s, ex, 1H, NH), 8.33 (d, 2H, aromatic H, J= 8.76 Hz); C NMR: δ 20.9, 55.4,
67.5, 114.6, 115.1, 120.3, 127.3, 128.2, 128.3, 128.5, 128.9, 129.2, 129.3, 129.6, 130.3, 130.8,
131.3, 134.7, 138.0, 158.2, 159.2, 159.4, 165.5, 170.9. Anal. Calcd. For C32H27N3O4 (517.58):
C, 74.26; H, 5.26; N, 8.12%. Found: C, 74.41; H, 5.29; N, 8.19%.
4.1.2.15.
N-(2-Methoxyphenyl)-2-(4-((1-(4-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (7e).
Yield: 77%; m.p. 188-190 oC; IR (KBr, cm-1): 3367 (NH), 3055 (aromatic CH), 2927
(CH aliphatic), 1710, 1689 (C=O); 1H NMR: δ 3.85 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.72 (s,
2H, CH2), 6.91-6.99 (m, 4H, aromatic H), 7.01-7.13 (m, 5H, aromatic H), 7.28 (s, 1H, CH),
7.30-7.47 (m, 3H, aromatic H), 7.61 (d, 2H, aromatic H, J= 7.48 Hz), 8.34 (d, 2H, aromatic H,
J= 8.60 Hz), 8.41 (d, 1H, aromatic H, J= 7.52 Hz), 8.97 (s, ex, 1H, NH); 13C NMR: δ 55.5,
55.7, 67.6, 110.1, 114.7, 115.1, 119.9, 121.1, 127.3, 128.3, 128.4, 128.5, 128.9, 129.1, 129.2,
131.0, 131.2, 134.7, 134.8, 137.5, 148.3, 158.7, 159.4, 160.0, 165.4, 170.9. Anal. Calcd. For
C32H27N3O5 (533.58): C, 72.03; H, 5.10; N, 7.88%. Found: C, 72.09; H, 5.14; N, 7.96%.
13

ACCEPTED MANUSCRIPT
4.1.2.16.
N-(3-Methoxyphenyl)-2-(4-((1-(4-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (7f).
Yield: 74%; m.p. 196-197oC; IR (KBr, cm-1): 3438 (NH), 3062 (aromatic CH), 2938
(CH aliphatic), 1720, 1654 (C=O); 1H NMR: δ 3.82 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.67 (s,
2H, CH2), 6.09 (s, ex, 1H, NH), 6.59 (d, 1H, aromatic H, J= 7.76 Hz), 6.75-6.78 (m, 3H,
aromatic H), 6.94 (d, 2H, aromatic H, J= 8.48 Hz), 7.08 (d, 2H, aromatic H, J= 7.68 Hz), 7.25
(s, 1H, aromatic H), 7.28 (s, 1H, CH), 7.31-7.42 (m, 4H, aromatic H), 7.58 (d, 2H, aromatic H,
J= 7.16 Hz), 8.31 (d, 2H, aromatic H, J= 8.76 Hz); 13C NMR: δ 55.3, 55.5, 67.4, 105.9, 110.8,
112.3, 114.7, 115.9, 127.3, 128.2, 128.5, 128.6, 128.8, 129.1, 129.2, 130.2, 131.0, 134.7,
135.9, 137.8, 145.8, 158.5, 159.4, 160.2, 165.6, 170.9. Anal. Calcd. For C32H27N3O5 (533.58):
C, 72.03; H, 5.10; N, 7.88%. Found: C, 72.11; H, 5.12; N, 7.99%.
4.1.2.17.
N-(4-Methoxyphenyl)-2-(4-((1-(4-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (7g).
Yield: 80%; m.p. 239-240 oC; IR (KBr, cm-1): 3332 (NH), 3064 (aromatic CH), 2933
(CH aliphatic), 1712, 1699 (C=O); 1H NMR: δ 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.70 (s,
2H, CH2), 6.91 (d, 2H, aromatic H, J= 9.08 Hz), 6.95 (d, 2H, aromatic H, J= 8.48 Hz), 7.09 (d,
2H, aromatic H, J= 8.89 Hz), 7.17 (d, 2H, aromatic H, J= 8.84 Hz), 7.28 (s, 1H, aromatic H),
7.31-7.37 (m, 3H, aromatic H), 7.50 (d, 2H, aromatic H, J= 8.96 Hz), 7.61 (d, 2H, aromatic H,
J= 7.60 Hz), 8.15 (s, ex, 1H, NH), 8.34 (d, 2H, aromatic H, J= 8.72 Hz); 13C NMR: δ 55.5,
55.6, 67.4, 114.2, 114.7, 115.1, 122.1, 125.4, 127.3, 127.7, 128.2, 128.3,128.6, 129.0, 129.2,
130.0, 131.3, 134.7, 134.8, 137.6, 149.2, 158.5, 159.4, 160.2, 165.6, 170.8. Anal. Calcd. For
C32H27N3O5 (533.58): C, 72.03; H, 5.10; N, 7.88%. Found: C, 72.14; H, 5.15; N, 7.94%.
4.1.2.18.
N-(2-Chlorophenyl)-2-(4-((1-(4-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (7h).
Yield: 94%; m.p. 225-227 oC; IR (KBr, cm-1): 3383 (NH), 3053 (aromatic CH), 2928
1
(CH aliphatic), 1714, 1710 (C=O), 833 (C-Cl); H NMR: δ 3.85 (s, 3H, OCH3), 4.74 (s, 2H,
CH2), 6.95 (d, 2H, aromatic H, J= 8.84 Hz), 7.09-7.13 (m, 5H, aromatic H), 7.28 (s, 1H, CH),
7.30 (t, 1H, aromatic H, J= 7.80 Hz), 7.37-7.45 (m, 4H, aromatic H), 7.61 (d, 2H, aromatic H,
J= 7.40 Hz), 8.34 (d, 2H, aromatic H, J= 8.68 Hz), 8.47 (d, 1H, aromatic H, J= 8.20 Hz), 9.01
(s, ex, 1H, NH); 13C NMR: δ 55.5, 67.4, 114.7, 115.1, 117.3, 121.3, 125.2, 127.8, 128.2,
128.3, 128.5, 128.9, 129.1, 129.2, 131.0, 131.2, 133.7, 134.5, 134.7, 137.6, 158.3, 159.3, 160.1,
14

ACCEPTED MANUSCRIPT
165.7, 170.8. Anal. Calcd. For C31H24ClN3O4 (538.00): C, 69.21; H, 4.50; N, 7.81%. Found: C,
69.29; H, 4.51; N, 7.89%.
4.1.2.19.
N-(3-Chlorophenyl)-2-(4-((1-(4-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (7i).
o
Yield: 93%; m.p. 267-269 C; IR (KBr, cm-1): 3375 (NH), 3055 (aromatic CH), 2925
1
(CH aliphatic), 1708, 1681 (C=O), 832 (C-Cl); H NMR: δ 3.86 (s, 3H, OCH3), 4.71 (s, 2H,
CH2), 6.95 (d, 2H, aromatic H, J= 8.72 Hz), 7.08-7.09 (m, 4H, aromatic H), 7.16 (d, 1H,
aromatic H, J= 7.96 Hz), 7.28 (s, 1H, CH), 7.31-7.38 (m, 3H, aromatic H), 7.44-7.48 (m, 2H,
aromatic H), 7.61 (d, 2H, aromatic H, J= 7.48 Hz), 7.75 (s, 1H, aromatic H), 8.26 (s, ex, 1H,
NH) 8.34 (d, 2H, aromatic H, J= 8.72 Hz); 13C NMR: δ 55.5, 67.4, 114.7, 115.1, 118.1, 120.2,
125.1, 128.1, 128.3, 128.5, 129.2, 130.1, 131.3, 134.7, 134.8, 137.7, 137.8, 158.3, 159.4,
160.1, 165.7, 170.8. Anal. Calcd. For C31H24ClN3O4 (538.00): C, 69.21; H, 4.50; N, 7.81%.
Found: C, 69.31; H, 4.50; N, 7.92%.
4.1.2.20.
N-(4-Chlorophenyl)-2-(4-((1-(4-methoxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)phenoxy)acetamide (7j).
Yield: 91%; m.p. 276-278 oC; IR (KBr, cm-1): 3354 (NH), 3054 (aromatic CH), 2926
1
(CH aliphatic), 1714, 1699 (C=O), 831 (C-Cl) ; H NMR: δ 3.86 (s, 3H, OCH3), 4.70 (s, 2H,
CH2), 6.95 (d, 2H, aromatic H, J= 8.84 Hz), 7.08-7.13 (m, 4H, aromatic H), 7.28 (s, 1H, CH),
7.30-7.37 (m, 4H, aromatic H), 7.44 (t, 1H, aromatic H, J= 7.40 Hz), 7.57 (d, 2H, aromatic H,
J= 8.72 Hz), 7.61 (d, 2H, aromatic H, J= 7.48 Hz), 8.24 (s, ex, 1H, NH) 8.32 (d, 2H, aromatic
H, J= 8.72 Hz); 13C NMR: δ 55.5, 67.4, 114.7, 115.1, 121.4, 127.3, 128.1, 128.3, 128.5, 128.9,
129.1, 129.2, 130.0, 131.3, 134.7, 135.2, 137.7, 158.3, 159.4, 160.3, 165.7, 170.8. Anal. Calcd.
For C31H24ClN3O4 (538.00): C, 69.21; H, 4.50; N, 7.81%. Found: C, 69.34; H, 4.52; N, 7.94%.
4.2. Anticancer activity screening:
4.2.1. In vitro kinase inhibition:
All kinase reactions were performed using LanthaScreen Kinase activity assay kits
(Invitrogen), following the manufacturer’s instructions. In summary, kinase reactions were
performed in a 10 ꢀL volume in low-volume 384-well plates. Typically, Corning model 3676
(black) or 3673 (white) plates were used. The concentration of substrate [GFP-ATF2] in the
assay was 400 nM, and the kinase reaction buffer consisted of 50 mM HEPES pH 7.5, 0.01%
BRIJ-35, 10 mM MgCl2, and 1 mM EGTA, plus any additional additives that might be required
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for a specific kinase. Kinase reactions were allowed to proceed for 1 hour at room temperature
with substrate and ATP in the presence or absence of inhibitor before a 10 ꢀL preparation of
EDTA (20 mM) and Tb-labeled antibody [Tb-pAT F2(pThr71), 4 nM] in TR-FRET dilution
buffer were added. The final concentration of the antibody in the assay well was 2 nM, and the
final concentration of EDTA was 10 mM. The plate was allowed to incubate at room
temperature for at least 30 minutes. The data were generated using a BMG Pherastar plate
reader using the LanthaScreen™ filter block available from BMG. Inhibition studies were
performed against a dilution series of tested compounds and IC50 values determined from the
resulting inhibition curves.
4.2.2. MTT assay for antiproliferative activity:
The cancer cell lines and Vero cells were trypsinized and washed with Ca2+/Mg2+-free
PBS (pH 7.2). Cells were adjusted to 4×104 cells/ml with DMEM supplemented with 10% fetal
calf serum (Hyclone, Logan, UT,USA) and plated (50 ꢀl/well) in 96-well cell culture plate
(Corning, Corning, NY, USA) overnight at 37˚C with 5% CO2 and 95% humidity. Fifty
microliters of serial 10-fold diluted sterile tested compounds were added to final concentrations
of 0-100µM. Culture medium was used as negative control. Cultures were incubated for 72 h.
Supernatants were discarded, 20 ꢀl/well of methylthiazolyldiphenyl-tetrazolium bromide
(MTT) reagent (Promega, Madison, WI, USA) was added and incubated for 4 h at 37˚C with
5% CO2. Sterile sodium dodecyl sulfate (10% v/v in PBS) was added (25 ꢀl/well) and the plate
was kept at room temperature for 18 h before measuring optical density (OD) at 490 nm.
DMEM was used as a blank control. Results from three separate experiments were recorded
and the percentage of viable cells was calculated as [(O.D. of cell control–O.D. of treated
cells)/(O.D. of cell control–O.D. of initiated cells)] ×100.
References.
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[6] M. P. Goetz, A. W. Tolcher, P. Haluska, K. P. Papadopoulos, C. Erlichman, M. Beeram, J.
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[19] J. Rinehart, A. A. Adjei, P. M. LoRusso, D. Waterhouse, J. R. Hecht, R. B. Natale, O.
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Figure captions:
Figure 1. Structure of some reported inhibitors of p38αMAPK * and ERK1/2**.
Figure 2. Design of the target compounds 6 and 7.
Scheme1. Synthesis of the target compounds 6 and 7.
Table captions:
Table 1. IC50 values (nM) of tested compounds 6a-j and 7a-j against p38αMAPK and ERK1/2.
Table 2. Antiproliferative activity of compounds 6d and 7h against different cancer cell lines
and normal Vero cells.
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Table 1.
IC50 (nM) ± SEM*
Compound
R
R1
P38αMAPK
35.0±2.1
66.0±3.8
87.0±5.2
4.5±0.3
ERK1
ERK2
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
-
H
65.0±5.9
43.0±3.2
20.0±1.7
25.0±1.9
46.0±2.8
57.0±4.7
76.0±7.3
87.0±6.2
89.0±7.4
95.0±8.9
23.0±2.4
32.0±2.5
89.0±5.2
23.0±1.6
43.0±3.8
21.0±.1.2
23.0±2.0
24.0±2.1
35.0±2.3
47.0±3.1
18.1±1.6
54.0±4.3
44.0±3.6
43.0±2.7
3.2±0.32
4.7±0.45
5.4±0.27
4.3±0.34
5.3±0.41
3.4±0.30
4.6±0.3
6a
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2-Cl
6b
6c
6d
6e
54.0±3.7
6.5±0.5
6f
4.3±0.3
6g
5.6±0.3
6h
6i
3-Cl
3.7±0.3
4-Cl
4.6±0.3
6j
H
354.0±20.6
56.0±4.1
77.0±5.7
345.0±29.5
34.0±2.4
23.0±1.6
345.0±30.3
4.7±0.4
23.0±2.1
23.0±1.7
78.0±6.3
43.0±3.3
89.0±6.7
21.0±1.2
2.3±0.2
7a
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2-Cl
7b
7c
7d
7e
7f
7g
3.5±0.3
7h
7i
3-Cl
4.5±0.4
7.0±0.6
4-Cl
44.0±4.2
37.4±3.5
4.5±0.4
7j
-
109.5±9.8
Sorafenib
*IC50 values were calculated from the mean values of data from three separate experiments
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Table 2.
IC50 (µM) ± SEM*
Cancer
Type
Cell Line
6d
7h
Sorafenib SI**(6d)
SI**(7h)
24.90
26.40
2.93
Breast
MCF7
T47D
0.37±0.02 0.53±0.05 0.78±0.05
0.67±0.05 0.50±0.04 0.61±0.04
9.40±0.71 4.50±0.03 0.87±0.07
5.80±0.05 4.50±0.04 0.39±0.03
46.86
25.88
1.85
2.98
-
Colon
Colo-205
HT29
2.93
Leukemia
Lung
K562
>100
>100
1.22±0.01
-
A549
6.50±0.05 4.90±0.04 3.19±0.03
0.76±0.06 0.13±0.01 4.25±0.03
2.66
22.81
2.69
Melanoma LOX
IMVI
101.54
MDA-
MB-435
0.23±0.02 0.05±0.003 1.67±0.01
0.86±0.08 0.99±0.07 4.13±0.03
75.39
264.00
Prostate
PC3
-
20.16
-
13.33
-
Vero cells
17.34±1.42 13.20±1.20
-
* IC50 values were calculated from the mean values of data from three separate experiments.
**SI (selectivity index)= IC50 of Vero cells / IC50 of cancer cell. SI > 3 means that the
compound is selective to cancer cell.
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Cl
Cl
H
N
O
N
HN
O
N
CH3
CH3
N
N
F
N
N
N
N
H2N
N
NH
OH
Cl
F
CH3
H3C
I* (LY2228820)
Figure 1.
II*
III**
O
H
N
CF3
HN
S
O
O
O
IV
H
N
O
N
R1
O
p38 MAPK
inhibition
N
O
R
ERK1/2 inhibition
R= OCH3, R1= H, CH3, OCH3, Cl
6, 7
Figure 2.
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O
O
OH
OH
N
H
a
N
O
O
1
2
b
O
N
H
O
N
N
c
OH
O
N
N
R1
O
H
R
R
N
Cl
O
R1
6a-j
7a-j
5a-j
3, 4
3, 6 R= 3-OCH3
4, 7 R=4-OCH3
a R1= H
b R1= 2-CH3
d R1=4-CH3
f R1= 3-OCH3
g R1=4-OCH3 h R1=2-Cl
i R1=3- Cl j R1=4-Cl
c R1=3-CH3
e R1=2-OCH3
Scheme1.
Reagents and reaction conditions
CH3COONa; : 3/4-methoxyaniline, acetic acid, CH3COONa;
phenylacetamide 5a-j, DMF, K2CO3.
:
a
: 4-hydroxybenzaldehyde, acetic anhydride, anh.
b
c
: 2-chloro-N-un/substituted
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Highlights:
•
•
•
•
•
A series of 1,2,4-trisubstituted-imidazolinones was synthesized.
The new compounds proved active against p38αMAPK and ERK1/2.
IC50 of 6d and 7h on MDA-MB-435 is 0.23 and 0.05 µM, respectively.
IC50 of 6d against p38αMAPK and ERK2 = 4.5 and 3.2 nM, respectively.
Compound 7h has IC50 against p38αMAPK and ERK2 = 4.7 and 3.5 nM,
respectively.

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Compound 6a

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Compound 6a

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Compound 6b

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Compound 6b

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Compound 6d