Design, synthesis and anticancer activity of furochromone and benzofuran derivatives targeting VEGFR-2 tyrosine kinase

Article abstract of DOI:10.1039/c4ra16228e

In continuation of our work concerning the relation between the anticancer and anti-vascular endothelial growth factor receptor (anti-VEGFR-2) activity of some synthesized compounds, we hereby designed and prepared three new series of furochromone and benzofuran derivatives (carbonitriles, sulfonyl hydrazides and imides). The prepared compounds were evaluated for their in vitro VEGFR-2 inhibitory activity, their cytotoxicity on fifteen human cancer cell lines and their in vivo antiprostate cancer activity. The highest anti-VEGFR-2 activity was demonstrated by 6-acetyl-4-methoxy-7-methyl-5H-furo[3,2-g]chromen-5-one (3), which exhibited the same IC₅₀ value as the reference drug sorafenib (2.00 × 10^-3 μM). On the other hand, most of the synthesized compounds showed potent cytotoxicity against most of the tested cell lines, in particular, the carbonitrile series (4a,b and 5a,d) which exhibited the best activity with IC₅₀ values ranging from 3.56 × 10^-13 to 4.89 × 10^-7 μM. Moreover, the imide series (15-17) showed the most significant in vivo antiprostate cancer activity. An in silico GOLD molecular docking study has been done to explore the binding mode of interaction of the furochromone and benzofuran derivatives to VEGFR-2 kinase, and to reveal the correlation between IC₅₀ (μM) of the enzymatic inhibition of VEGFR-2 kinase and the GoldScore fitness for further therapeutic application. This journal is

Full text of DOI:10.1039/c4ra16228e

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Design, synthesis and anticancer activity of furochromone and benzofuran
derivatives targeting VEGFRꢀ2 tyrosine kinase
Omaima M. Abdelhafez,a Hamed I. Ali,b, c Kamelia M. Amin,d
Mohamed M. Abdalla, e Eman Y. Ahmed a
a Chemistry of Natural Products Dept., National Research Center, Dokki, Egypt
bPharmaceutical sciences Dept., Irma Lerma Rangel College of Pharmacy, Texas A&M
Health Science Center, TX, USA
cPharmaceutical Chemistry Dept., Faculty of Pharmacy, Helwan University, Egypt
dPharmaceutical Chemistry Dept., Faculty of Pharmacy, Cairo University, Egypt
e Research unit, Mapco Pharmaceutical industries Balteim, Egypt
Abstract:
In continuation of our work, concerning the relation between the anticancer and antiꢀ
vascular endothelial growth factor receptor (antiꢀVEGFRꢀ2) activity of some synthesized
compounds, we hereby, designed and prepared three new series of furochromone and
benzofuran derivatives (carbonitriles, sulfonyl hydrazides and imides). The prepared
compounds were evaluated for their in vitro VEGFRꢀ2 inhibitory activity, their
cytotoxicity on fifteen human cancer cell lines and their in vivo antiprostate cancer
activity. The highest antiꢀVEGFRꢀ2 activity was demonstrated by 6ꢀacetylꢀ4ꢀmethoxyꢀ7ꢀ
methylꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ5ꢀone (3), which exhibited the same IC50 value as the
reference drug sorafenib (2.00 × 10ꢀ3 ꢁM). On the other hand, most of the synthesized
compounds showed potent cytotoxicity against most of the tested cell lines, in particular,
the carbonitrile series (4a,b and 5a,d) which exhibited the best activity with IC50 values
ranging from (3.56 × 10ꢀ13 to 4.89 × 10ꢀ7 ꢁM). Moreover, the imide series (15, 16 and 17)
showed the most significant in vivo antiprostate cancer activity. In silico GOLD
molecular docking study has been done to explore the binding mode of interaction of the
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furochromone and benzofuran derivatives into VEGFRꢀ2 kinase, and to reveal the
correlation between IC50 (µM) of enzymatic inhibition of VEGFRꢀ2 kinase and the
Gold Score fitness for further therapeutic application.
Introduction
Angiogenesis of tumor mass is the process of forming new blood vessels from existing
ones of host, and is strictly controlled by the balance of a number of angiogenic factors.1
The imbalance is thought to affect angiogenesis, which is involved in the expansion and
metastasis of solid tumors such as prostate, colon, breast, and gastric cancers.2ꢀ6 The
blockade of the tyrosine kinase VEGFRꢀ2, (also known as Flkꢀ1 for fetal liver kinaseꢀ1 or
KDR for kinase insert domain–containing receptor),7ꢀ9 signaling pathway may disrupt the
angiogenesis process of solid tumor so that the developing tumor cells grow slowly or
even cease to grow.10,11 Antiangiogenic therapy is directed mainly at proliferating
capillary endothelial cells and does not cause bone marrow suppression, gastrointestinal
symptoms or hair loss.12
Some inhibitors of VEGFRꢀ2 such as sunitinib 13 and sorafenib,14 have been approved
by the Food and Drug Administration for treating different kinds of tumors, also in
literature chromone bioisosteres,15,16 benzofuran,17,18 carbonitrile,19,20 hydrazide,21,22 and
imide derivatives,23,24 were identified as antiꢀVEGFRꢀ2 and anticancer agents.
The previously mentioned studies encouraged us to design some furochromone and
benzofuran derivatives in order to evaluate their biological activities and molecular
docking.
Chemistry
The carbonitriles (4aꢀd and 5aꢀd), sulfonyl hydrazides (8a,b, 10a,b and 12aꢀc) and
imides (15, 16 and 17) were efficiently synthesized according to the protocols outlined in
(schemes 1ꢀ3). All the starting materials of the synthesized compounds were prepared
according to the reported methods, while the general reaction conditions, procedures and
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compounds characterization of the newly synthesized derivatives were described in the
experimental section.
Refluxing visnagin (1) in aqueous potassium hydroxide afforded the visnaginone
derivative (2),25 which reacted with acetic anhydride in the presence of anhydrous sodium
acetate under reflux to give the acetyl visnagin derivative (3).26 The reaction of compound
(3) with the respective aromatic aldehyde
namely, (benzaldehyde, 4ꢀ
bromobenzaldehyde, 5ꢀmethyl furfural and 3,4,5ꢀtrimethoxybenzaldehyde),in the
presence of malononitrile and/or ethylcyanoacetate and ammonium acetate afforded the
iminocarbonitrile (4aꢀd) and the oxocarbonitrile (5aꢀd) derivatives respectively.
Moreover, khellinone (7) and acetyl khellin (9) were prepared using the same
previously mentioned method of preparation of visnaginone (2) and acetyl visnagin (3)
respectively. The Mannich bases (11aꢀc)27 were prepared by the reaction of visnaginone
(2) with some secondary amines namely, (piperidine, Nꢀmethyl piperazine and
morpholine) in ethanol in the presence of formaline. The different aryl sulfonyl
hydrazides (8a,b, 10a,b and 12aꢀc) were synthesized by reacting the previously
mentioned acetyl containing compounds (2, 3, 7, 9 and 11aꢀc) with pꢀtoluene sulfonyl
hydrazide in methanol and few drops of conc. HCL.
Furthermore, chlorosulphonation of visnagin (1) at 0°C affored visnagin sulfonyl
chloride (13),28 which upon stirring at room temperature with hydrazine hydrate 98% in
20 ml absolute ethanol and few drops of triethylamine gave the visnagin sulfonyl
hydrazide (14),28 finally, the imide derivatives (15, 16 and 17) were obtained by refluxing
(14) with the appropriate anhydrides namely, (phthalic, succinic and maleic anhydrides)
in glacial acetic.
Results and Discussion
1) Biology
a) In vitro antiꢀVEGFRꢀ2 screening
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Studying the antiꢀVEGFRꢀ2 activity of the tested compounds (table 1) showed that 6ꢀ
acetylꢀ4ꢀmethoxyꢀ7ꢀmethylꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ5ꢀone (3), was the most active
compound and exhibited the same IC50 value as the reference drug sorafenib (2.00 × 10ꢀ3
ꢁM).
b) In vitro cytotoxicity screening
The cytotoxicity of the tested compounds (3, 4aꢀd, 5aꢀd, 8a,b, 10a,b, 11aꢀc, 12aꢀc,
14, 15, 16 and 17) was determined on fifteen different human cancer cell lines (tables 2aꢀ
d).
Screening the cytotoxicity of the tested compounds on cervical carcinoma (KB),
leukemia (K561), neuroblastoma (NBꢀ1), liver carcinoma (HepG2) cell lines, showed
that compound (4b) has a very remarkable activity, with IC50 values ranging from (4.56
× 10ꢀ9 to 3.67 × 10ꢀ7 ꢁM), compared to the reference drug used.
On ovarian carcinoma (SKOVꢀ3), colonoadenocarcinoma (RKOP27), melanoma (SKꢀ
MELꢀ28), neuroblastoma (GOTO), fibrosarcoma (HT1080) cell lines, compound (5d)
was significantly potent with IC50 values ranging from (3.56 × 10ꢀ13 to 4.89 × 10ꢀ7 ꢁM),
exhibiting more potency than the reference drugs.
Regarding the CNS cancer (SFꢀ268), nonꢀsmall lung cancer (NCI H460), leukemia
(HL60) cell lines, it was found that compound (5a) (IC50 ranging from 2.33 × 10−11 ꢁM
to 3.67 × 10−9 ꢁM) demonstrated a potent cytotoxicity among the other synthesized
derivatives.
Estimation of the cytotoxicity of the prepared compounds on leukemia (U937),
prostate cancer (PCꢀ3) cell lines revealed that compound (4a) showed a very pronounced
potency (IC50 = 4.39 × 10−7 ꢁM) and (IC50 = 4.78 × 10−8 ꢁM) respectively compared to
the reference drug and the other furochromone and benzofuran derivatives.
Evaluation of the cytotoxicity on melanoma (G361) cell lines showed that compound
(3) exhibited a significant IC50 (3.67 × 10−8 ꢁM), better than the reference drug
aldesleukin (IC50 = 6.66 × 10−3 ꢁM).
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The previous screening of the bioactivity of the tested compounds on the presented
panel of cell lines indicated that, the majority of the synthesized compounds were
significantly potent than the comparative reference drugs used. The carbonitrile series
(4a,b and 5a,d) showed the best potency against all the cell lines compared to the
reference drugs and to the rest of the newly synthesized furochromone and benzofuran
derivatives.
C) In vivo antiprostate cancer activity:
The results of the in vivo antiprostate cancer activity (table 3) proved that the most
significant ED50 values were observed for the imide derivatives (15, 16 and 17)
displaying (ED50 from 2.12 ꢁM to 2.82 ꢁM ) which was better than the reference drug
flutamide (ED50 = 11.60 ꢁM).
2) Docking Studies
The computational docking methods are intensively applied to screen a variety of
compounds, looking for new hits with specific binding affinities or by investigating a
range of structural manipulation for an existing compound.29 The proteinꢀligand docking
program GOLD facilitates this approach for prediction of the binding mode of the
compounds under investigation. GOLD is considered as one of the flexible docking
programs that were tested on large test sets to give a 68% success rates on the
CCDC/Astex validation set of 305 complexes taken from the Protein Data Bank (PDB).30
In this article, we have implemented the Goldscore as a search algorithmic function for
“drugꢀlike” which was reported to give superior results than the Chemscore function as a
scoring function for GOLD.31This Goldscore fitness means the higher the values of fitness
(Table 4) is the better binding interaction into the binding site.
GOLD Fitness = Shb_ext + Svdw_ext + Shb_int + Svdw_int
The fitness score is the negative sum of the component energy terms, such as proteinꢀ
ligand external hydrogen bond energy (Shb_ext), proteinꢀligand external van der Waals
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energy (Svdw_ext), proteinꢀligand intramolecular hydrogen bond energy (Shb_int), and the
ligand internal van der Waals energy (Svdw_int), so that larger fitness scores are better.
The implemented Goldscore algorithmic function was initially evaluated for its
accuracy into VEGFRꢀ2 kinase. As a function of RMSD (root mean square deviation) of
the runs done by flexible docking using GOLD 5.1, the docking poses were nearest to the
experimental binding mode of the coꢀcrystallized ligands of VEGFRꢀ232. Our docking
results are matched with the reported requirements for successful scoring function,33 being
of RMSD ≤ 2.0Å from the experimental one.
In this study, the newly synthesized compounds were docked into the target crystal structure
of human Protein tyrosine kinase of VEGFRꢀ2 kinase domain (pdb code: 3EWH) in complex
with a pyridylꢀpyrimidine benzimidazole inhibitor (K111). Many compounds revealed good
Gold fitness score (Gold Score) values namely, compounds (4a, 4b, 4d, 5a, 5b, 5c, 5d, 10b,
12a, 12b, 12c and 15) which revealed 72, 74, 79.85, 71.80, 70.86, 70.66, 81.17, 71.36, 74.71,
75.09, 78.28 and 72.88 respectively (Table 4). Almost all the docked compounds show the
common hydrogen bond interactions (1ꢀ4) with D1046 (NH, C=O), K868 (NH), E885 (C=O),
and R1027 (NH). Fitness of the best docked compounds takes place within RMSD of 2.39 –
6.27 Å as cited in (Table 4). Such binding mode was identically similar to the native coꢀ
crystallized ligand (K111) which binds with similar binding orientation involving similar
amino acids used by docked compounds. This reveals that the interactions between the K868
(NH) and the small molecules will be crucial to inhibit the VEGFRꢀ2 kinase activity.
Compound (4a) possess a high potential fitness (GoldScore: 72) into the binding site of
the 3D macromolecule (PDB: 3EWH). Its high affinity is presumably attributed to its
hydrogen bond formed between its 5ꢀC=O group and D1046 (NH) amino acid and RMSD
of 4.44 Å. In addition to the hydrophobic interaction of its 4`ꢀphenyl pyridine moiety
which occupies the hydrophobic pockets of the receptor site (I888, I889, I892, V898,
V899, and I1044), as illustrated in Figure 1. Compound (4b) has a better potential fitness
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(GoldScore: 74) into the binding site, forming one hydrogen bond between its Terminal
4``ꢀBr and R1027 (NH) amino acid. In addition, both compounds (4a and 4b) revealed
external vdw of 52.43 and 53.55, respectively.
As shown in (Figure 2) and cited in (Table 4), compound (12a) (GoldScore: 74.71)
has a promising antiproliferative activity through its higher kinase binding affinity.
Where it binds into VEGFR2 kinase through two hydrogen bonds between its 4ꢀCH3O
and 6ꢀO moieties and K868 (HN), and D1046 (HN) amino acids, respectively, where it
revealed excellent RMSD of 2.39 Å. Furthermore its 7ꢀpiperidinyl moiety occupies the
extended hydrophobic pocket similarly to the lipophilic trifluoromethylꢀarene portion of
the native cocrystallized (K111).
In addition to the role of the hydrogen bond and vdw interactions, the hydrophobic
interaction plays a significant role to occupy the 3(4`ꢀaryl pyridine) moiety of compounds
(4aꢀd and 5aꢀd), and the 7ꢀpiperidinyl, NꢀMe piprazinyl, and morpholino moieties of
compounds (11aꢀc and 12aꢀc) into the extended hydrophobic pocket similarly to the
lipophilic trifluoromethylꢀarene portion of the native coꢀcrystallized (K111).
In the analysis of GOLD docking results, a high correlation (R2=0.712) between IC50 of
(VEGFRꢀ2) enzymatic inhibition and the GoldScore fitness for compounds (4b, 4c, 5a,
5b, 5c, 8a, 10a, 10b, 11c, 12a, 16 and 17) into (VEGFRꢀ2) kinase is shown in (Figure
3A).
Also well correlated result (R2=0.687) is shown between IC50 (ꢁM) against prostate
cancer cell line (PCꢀ3) and gold score fitness of compounds (4a, 4d, 5d, 10a, 10b, 11b,
11c, 12a, 12c and 16) (Figure 3B).
On other hand the correlations between the biological results IC50 (ꢁM) against
different tumor cell lines namely, (G361, HL60, K561, KB, RKOP27, SKOVꢀ3, SKꢀ
MELꢀ28 and U937) were variably correlated and revealing correlation coefficients (R2)
of 0.67, 0.74, 0.73, 0.764, 0.66, 0.658, 0.79 and 0.686, respectively.
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Conclusion
The previously mentioned discussions indicated that the more lipophilic agents may
be antiangiogenic rather than cytotoxic agents, and suggested that VEGFRꢀ2 kinase
inhibition may not be critical for the cytotoxic responses of our compounds. For example,
the phenyl (5a) and trimethoxy phenyl (5d) derivatives have a potent cytotoxic effects
against different tumor cells such as HL60, U937, K561, G361, SKꢀMELꢀ28, PCꢀ3,
HT1080, HepG2, KB, SK OVꢀ3, SFꢀ268, NCI H460, and RKOP27 in IC50 of 10ꢀ6ꢀ10ꢀ13
µM range. Whereas, their 4ꢀbromophenyl derivative (5b) being more lipophilic inhibited
these tumor cells in much lower IC50 of 10ꢀ3ꢀ10ꢀ5 µM range, despite its higher VEGFRꢀ2
inhibition (IC50 of 2.2 x 10ꢀ3 µM). The VEGFRꢀ2 inhibition of compound (5b) is almost
of the same potency of the referenc drug sorafenib being of (IC50 of 2.00 x 10ꢀ3 µM).
On the other hand, the in vivo results of these compounds are correlated with this
outcome, where compound (5b) revealed better in vivo antiprostate cancer activity (ED50
= 13.11µM) than that of compounds (5a and 5d) (ED50 = 26.0 and 28.6 µM,
respectively). This suggests that their prostate cancer antiproliferative activities are
mediated through VEGFRꢀ2 pathway.
Likewise within the in vivo prostate cancer experiment, compounds (15ꢀ17) have
(ED50 of 3.73, 2.56, 2.82, and 2.12 µM, respectively). Similarly, they have equipotent
inhibitory activities against VEGFRꢀ2 of (3.49 x 10ꢀ2, 2.62 x 10ꢀ2, 4.22 x 10ꢀ2, and 2.17 x
10ꢀ2 µM, respectively).However, this correlation was deviated in the in vitro PC3 cell
assay, where compound (14 and 17) have equipotent activities of IC50: 1.25 x 10ꢀ5 and
5.0 x 10ꢀ5 µM, respectively, revealing highly more potent cytotoxic effect than
compounds (15 and 16) of IC50: 8.0 x 10ꢀ3 and 8.9 x 10ꢀ3 µM, respectively. This result
may suggest the resistance of PC3 cell lines against compounds (15 and 16), and
indicates that the in vitro data may not necessarily reflect the in vivo effect.
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Experimental
1) Chemistry
Melting points were determined on Electrothermal IA 9000 apparatus and were
uncorrected. The infrared (IR) spectra were recorded using Nexus 670 FTꢀIR FTꢀRaman
spectrometer as potassium bromide discs, at National Research Centre. The proton
nuclear resonance (1H NMR) spectra were determined on Varian mercury 500 MHz
spectrometer, using tetramethylsilane (TMS) as the internal standard, at National
Research Centre. The mass spectra were performed on JEOL JMSꢀAX500 mass
spectrometer at the National Research Centre. The reactions were followed by thin layer
chromatography (TLC) (Silica gel, aluminum sheets 60 F254, Merck) using benzene:
ethyl acetate (8:2 v/v) as eluent and sprayed with iodine–potassium iodide reagent. The
purity of the newly synthesized compounds was assessed by elemental analysis and was
found to be higher than 95%.
Preparation of (6ꢀhydroxyꢀ4ꢀmethoxybenzofuranꢀ5ꢀyl)ethanone (2), 6ꢀacetylꢀ4ꢀ
methoxyꢀ7ꢀmethylꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ5ꢀone (3).
Visnaginone (2)25 and acetyl visnagin (3)26 were prepared according to the reported
methods.
General procedure for the preparation of different iminocarbonitriles (4aꢀd).
Acetyl visnagin (2.5 mmol) together with the respective aromatic aldehyde (2.5 mmol)
namely, (benzaldehyde, 4ꢀbromobenzaldehyde, 5ꢀmethyl furfural and 3,4,5ꢀ
trimethoxybenzaldehyde), malononitrile (2.5mmol) and ammonium acetate (20 mmol)
were refluxed for 10ꢀ14 hours in ethanol (30ml). The precipitate formed was filtered,
washed with ethanol, dried and crystallized from DMFꢀethanol (1:10).
2ꢀAminoꢀ6ꢀ(4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ6ꢀyl)ꢀ4ꢀ
phenylpyridineꢀ3ꢀcarbonitrile (4a).
Yield: 90%; mp: 82ꢀ5 °C; Mol. wt.: 423.42. IR (cmꢀ1, KBr): 3343 (NH2), 2206 (C≡N),
1
1617 (C=O, γꢀpyrone), H NMR (DMSOꢀd6, δ, ppm): 2.40 (3H, s, CH3), 3.78 (3H, s,
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OCH3), 6.64 (1H, s, CH), 6.66 (1H, d, Hꢀ3 benzofuran), 7.20ꢀ7.70 (5H, m, ArꢀH), 7.30
(1H, s, CHꢀpyridine), 7.52 (1H, d, Hꢀ2 benzofuran), 11.70 (2H, s, NH2, D2O
exchangeable). EIMS; m/z (R.A. %): 424 (M++1) (65%), 393 (75%), 355 (100%), 213
(58%), 140 (57%), 91 (76%).
2ꢀAminoꢀ4ꢀ(4ꢀbromophenyl)ꢀ6ꢀ(4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ
6ꢀyl)pyridineꢀ3ꢀcarbonitrile (4b).
Yield: 71%; mp: 87ꢀ90°C; Mol. wt.: 502.32. IR (cmꢀ1, KBr): 3342 (NH2), 2199 (C≡N),
1
1617 (C=O, γꢀpyrone). H NMR (DMSOꢀd6, δ, ppm): 1.90 (3H, s, CH3), 3.73 (3H, s,
OCH3), 6.64 (1H, s, CH), 6.66 (1H, d, Hꢀ3 benzofuran), 7.30ꢀ7.80 (4H, m, ArꢀH), 7.38
(1H, s, CHꢀpyridine), 7.52 (1H, d, Hꢀ2 benzofuran), 10.38 (2H, s, NH2, D2O
exchangeable). EIMS; m/z (R.A. %): 502/504 (M+/M++2) (80%), 469 (100%), 355
(70%), 213 (90%), 140 (60%), 91 (99%).
2ꢀAminoꢀ6ꢀ(4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ6ꢀyl)ꢀ4ꢀ(5ꢀ
methylfuranꢀ2ꢀyl)pyridineꢀ3ꢀcarbonitrile (4c).
Yield: 70%; mp: 67ꢀ70°C; Mol. wt.: 427.41. IR (cmꢀ1, KBr): 3356 (NH2), 2214 (C≡N),
1609 (C=O, γꢀpyrone).1H NMR (DMSOꢀd6, δ, ppm): 2.41 (3H, s, CH3), 3.73 (3H, s,
OCH3), 5.1 (1H, d, Hꢀ4 furan), 5.9 (1H, d, Hꢀ3 furan), 6.60 (1H, s, CH), 6.62 (1H, d, Hꢀ3
benzofuran), 7.00 (1H, s, CHꢀpyridine), 7.52 (1H, d, Hꢀ2 benzofuran), 10.38 (2H, s, NH2,
D2O exchangeable). EIMS; m/z (R.A. %): 427 (M+) (70%), 330 (70%), 355 (100%), 214
(50%), 143 (65%), 83 (55%).
2ꢀAminoꢀ6ꢀ(4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ6ꢀyl)ꢀ4ꢀ(3,4,5ꢀ
trimethoxyphenyl)pyridineꢀ3ꢀcarbonitrile (4d).
Yield: 90%; mp: 255ꢀ8°C; Mol. wt.: 513.50. IR (cmꢀ1, KBr): 3394 (NH2), 2205 (C≡N),
1618 (C=O, γꢀpyrone). 1H NMR (DMSOꢀd6, δ, ppm): 1.75 (3H, s, CH3), 3.71ꢀ3.74 (12H,
m, OCH3), 6.00 (2H, m, ArꢀH), 6.54 (1H, s, CH), 6.60 (1H, d, Hꢀ3 benzofuran), 7.60 (1H,
s, CHꢀpyridine), 7.78 (1H, d, Hꢀ2 benzofuran), 9.38 (2H, s, NH2, D2O exchangeable).
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EIMS; m/z (R.A. %): 512 (M+ꢀ1) (65%), 496 (100%), 351 (20%), 204 (25%), 120 (15%),
100 (13%).
General procedure for the preparation of different oxocarbonitriles (5aꢀd).
Acetyl visnagin (2.5 mmol) together with the respective aromatic aldehyde (2.5 mmol)
namely (benzaldehyde, 4ꢀbromobenzaldehyde, 5ꢀmethyl furfural and 3,4,5ꢀ
trimethoxybenzaldehyde), ethylcyanoacetate (2.5mmol) and ammonium acetate (20
mmol) were refluxed for 10ꢀ14 hours in ethanol (30ml). The precipitate formed was
filtered, washed with ethanol, dried and crystallized from DMFꢀethanol (1:10).
1,2ꢀDihydroꢀ6ꢀ(4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ6ꢀyl)ꢀ2ꢀoxoꢀ4ꢀ
phenylpyridineꢀ3ꢀcarbonitrile (5a).
Yield: 87%; mp: 100ꢀ1°C; Mol. wt.: 424.40. IR (cmꢀ1, KBr): 3123 (CH aromatic), 2210
1
(C≡N), 1624 (C=O, γꢀpyrone). H NMR (DMSOꢀd6, δ, ppm): 2.00 (3H, s, CH3), 3.81
(3H, s, OCH3), 6.00 (1H, s, CHꢀpyridine), 6.46 (1H, s, CH), 6.64 (1H, d, Hꢀ3
benzofuran), 7.10ꢀ7.30 (5H, m, ArꢀH), 7.42 (1H, d, Hꢀ2 benzofuran), 8.70 (1H, s, NH,
D2O exchangeable). EIMS; m/z (R.A. %): 424 (M+) (60%), 364 (30%), 246 (38%), 169
(45%), 80 (100%).
4ꢀ(4ꢀBromophenyl)ꢀ1,2ꢀdihydroꢀ6ꢀ(4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀ
g]chromenꢀ6ꢀyl)ꢀ2ꢀoxopyridineꢀ3ꢀcarbonitrile (5b).
Yield: 75%; mp: 95ꢀ7°C; Mol. wt.: 503.30. IR (cmꢀ1, KBr): 3128 (CH aromatic), 2220
1
(C≡N), 1620 (C=O, γꢀpyrone). H NMR (DMSOꢀd6, δ, ppm): 1.70 (3H, s, CH3), 3.63
(3H, s, OCH3), 6.38 (1H, s, CHꢀpyridine), 6.44 (1H, s, CH), 6.66 (1H, d, Hꢀ3
benzofuran), 7.20ꢀ7.40 (4H, m, ArꢀH), 7.40 (1H, d, Hꢀ2 benzofuran), 9.33 (1H, s, NH,
D2O exchangeable). EIMS; m/z (R.A. %): 503/505 (M+/M++2) (67%), 359 (65%), 262
(85%), 114 (85%), 80 (100%).
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1,2ꢀDihydroꢀ6ꢀ(4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ6ꢀyl)ꢀ4ꢀ(5ꢀ
methylfuranꢀ2ꢀyl)ꢀ2ꢀoxopyridineꢀ3ꢀcarbonitrile (5c).
Yield: 70%; mp: 70ꢀ3°C; Mol. wt.: 428.39. IR (cmꢀ1, KBr): 3123 (CH aromatic), 2221
1
(C≡N), 1618 (C=O, γꢀpyrone), H NMR (DMSOꢀd6, δ, ppm): 2.00 (3H, s, CH3), 2.17
(3H, d, CH3ꢀfuran), 3.73 (3H, s, OCH3), 5.93 (1H, s, CHꢀpyridine), 6.10 (1H, d, Hꢀ4
furan), 6.42 (1H, s, CH), 6.62 (1H, d, Hꢀ3 benzofuran), 6.89 (1H, d, Hꢀ3 furan), 7.50 (1H,
d, Hꢀ2 benzofuran), 9.00 (1H, s, NH, D2O exchangeable). EIMS; m/z (R.A. %): 428 (M+)
(60%), 330 (100%), 200 (50%), 143 (65%).
1,2ꢀDihydroꢀ6ꢀ(4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ6ꢀyl)ꢀ4ꢀ(3,4,5ꢀ
trimethoxyphenyl)ꢀ2ꢀoxopyridineꢀ3ꢀcarbonitrile (5d).
Yield: 91%; mp: 104ꢀ7°C; Mol. wt.: 514.48. IR (cmꢀ1, KBr): 3122 (CH aromatic), 2216
1
(C≡N), 1627 (C=O, γꢀpyrone). H NMR (DMSOꢀd6, δ, ppm): 1.71 (3H, s, CH3), 3.71ꢀ
3.80 (12H, m, OCH3), 5.60 (1H, s, CHꢀpyridine), 6.20 (2H, m, ArꢀH), 6.54 (1H, s, CH),
6.61 (1H, d, Hꢀ3 benzofuran), 7.71 (1H, d, Hꢀ2 benzofuran), 8.88 (1H, s, NH, D2O
exchangeable). EIMS; m/z (R.A. %): 513 (M+ꢀ1) (60%), 410 (20%), 218 (55%), 105
(90%), 77 (100%).
Preparation of 1ꢀ(6ꢀhydroxyꢀ4,7ꢀdimethoxybenzofuranꢀ5ꢀyl)ethanone (7), 6ꢀacetylꢀ
4,9ꢀdimethoxyꢀ7ꢀmethylꢀ5Hꢀfuro[3,2ꢀg]chromenꢀ5ꢀone (9), 1ꢀ(6ꢀhydroxyꢀ4ꢀmethoxyꢀ
7ꢀ(piperidinꢀ1ꢀylmethyl)benzofuranꢀ5ꢀyl)ꢀ3ꢀ(5ꢀmethylfuranꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀone
(11a),
1ꢀ(6ꢀhydroxyꢀ4ꢀmethoxyꢀ7ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)methyl)benzofuranꢀ5ꢀ
yl)ethanone (11b), 1ꢀ(6ꢀhydroxyꢀ4ꢀmethoxyꢀ7ꢀ(morpholinomethyl)benzofuranꢀ5ꢀ
yl)ethanone (11c).
Khellinone(7)25, acetyl khellin(9)26 and the Mannich bases (11aꢀc)27were prepared
according to the reported methods.
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General procedure for the preparation of different aryl sulfonylhydrazides (8a,b,
10a,b, 12aꢀc).
To the appropriate ketone (2, 3, 7, 9 and 11aꢀc) (0.02 mol),dissolved in methanol (15
ml), pꢀtoluene sulfonyl hydrazide (0.025) dissolved in methanol (30 ml) was added, then
2ꢀ3 drops of conc. HCL were added to the reaction mixture. The reaction mixture was
kept at room temperature overnight, where crystals of the product were separated out,
filtered, dried and then crystallized from methanol.
5ꢀ(1ꢀTosylhydrazonoꢀethyl)ꢀ4ꢀmethoxyꢀbenzofuranꢀ6ꢀol (8a)
Yield: 87%; mp: 146ꢀ8°C; Mol. wt.: 374.41. IR (cmꢀ1, KBr): 3564 (OH aromatic), 3212
1
(NH), 1628 (C=N). H NMR (DMSOꢀd6, δ, ppm): 1.00 (3H, s, CH3), 2.35 (3H, s, CH3ꢀ
toluene), 3.87 (3H, s, OCH3), 5.00 (1H, s, OH, D2O exchangeable), 6.22 (1H, d, Hꢀ3
benzofuran), 6.70 (1H, s, CHꢀbenzofuran), 7.32ꢀ7.94 (4H, m, CHꢀtoluene), 7.50 (1H, d,
Hꢀ2 benzofuran), 9.00 (1H, s, NH, D2O exchangeable). EIMS; m/z (R.A. %): 374 (M+)
(65%), 250 (55%), 163 (90%), 79 (100%).
5ꢀ(1ꢀTosylhydrazonoꢀethyl)ꢀ4,7ꢀdimethoxyꢀbenzofuranꢀ6ꢀol (8b)
Yield: 85%; mp: 205ꢀ7°C; Mol. wt.: 404.44. IR (cmꢀ1, KBr): 3490 (OH aromatic), 3225
1
(NH), 1617 (C=N). H NMR (DMSOꢀd6, δ, ppm): 1.88 (3H, s, CH3), 2.35 (3H, s, CH3ꢀ
toluene), 3.87 (6H, s, OCH3), 5.00 (1H, s, OH, D2O exchangeable), 6.22 (1H, d, Hꢀ3
benzofuran), 7.32ꢀ7.94 (4H, m, CHꢀtoluene), 7.50 (1H, d, Hꢀ2 benzofuran), 8.00 (1H, s,
NH, D2O exchangeable). EIMS; m/z (R.A. %): 405 (M++1) (70 %), 266 (50%), 183
(80%), 79 (100%).
6ꢀ(1ꢀTosylhydrazonoꢀethyl)ꢀ4ꢀmethoxyꢀ7ꢀmethylꢀfuro[3,2ꢀg]chromenꢀ5ꢀone (10a)
Yield: 82%; mp: 150ꢀ2°C; Mol. wt.: 440.47. IR (cmꢀ1, KBr): 3211 (NH), 3008 (CH
1
aromatic), 2846 (CH aliphatic), 1628 (C=N). H NMR (DMSOꢀd6, δ, ppm): 1.88 (3H, s,
CH3), 2.00 (3H, s, CH3ꢀfurochromone), 2.35 (3H, s, CH3ꢀtoluene), 3.79 (6H, s, OCH3),
6.66 (1H, d, Hꢀ3 benzofuran), 6.74(1H, s, CHꢀbenzofuran), 7.23ꢀ7.66 (4H, m, CHꢀ
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toluene), 7.52 (1H, d, Hꢀ2 benzofuran), 10.47 (1H, s, NH, D2O exchangeable). EIMS;
m/z (R.A. %): 439 (M+ꢀ1) (60 %), 289 (70%), 186 (100%), 79 (80%).
6ꢀ(1ꢀTosylhydrazonoꢀethyl)ꢀ4,9ꢀdimethoxyꢀ7ꢀmethylꢀfuro[3,2ꢀg]chromenꢀ5ꢀone
(10b)
Yield: 80%; mp: 170ꢀ1°C; Mol. wt.: 470.49. IR (cmꢀ1, KBr): 3188 (NH), 3067 (CH
1
aromatic), 2915 (CH aliphatic), 1631 (C=N). H NMR (DMSOꢀd6, δ, ppm): 1.22 (3H, s,
CH3), 1.90 (3H, s, CH3ꢀfurochromone), 2.35 (3H, s, CH3ꢀtoluene), 3.73 (3H, s, OCH3),
6.69 (1H, d, Hꢀ3 benzofuran), 7.29ꢀ7.68 (4H, m, CHꢀtoluene), 7.77 (1H, d, Hꢀ2
benzofuran), 11.47 (1H, s, NH, D2O exchangeable). EIMS; m/z (R.A. %): 439 (M+ꢀ1) (60
%), 289 (70%), 183 (80%), 78 (100%).
5ꢀ(1ꢀTosylhydrazonoꢀethyl)ꢀ4ꢀmethoxyꢀ7ꢀpiperidinꢀ1ꢀylꢀmethylbenzofuranꢀ6ꢀol
(12a).
Yield: 70%; mp: 258ꢀ9°C; Mol. wt.: 473.59. IR (cmꢀ1, KBr): 3450 (OH aromatic), 3062
(NH), 1611 (C=N). 1H NMR (DMSOꢀd6, δ, ppm): 0.99 (3H, s, CH3), 2.39ꢀ2.49 (10H, m,
CH2ꢀpiperidine), 2.59 (3H, s, CH3ꢀtoluene), 3.40 (2H, s, CH2N), 3.48 (3H, s, OCH3),
6.00 (1H, s, OH, D2O exchangeable), 7.24 (1H, d, Hꢀ3 benzofuran), 7.27ꢀ7.46 (4H, m,
CHꢀtoluene), 7.70 (1H, d, Hꢀ2 benzofuran), 14.80 (1H, s, NH, D2O exchangeable).
EIMS; m/z (R.A. %): 473 (M+) (60%), 307 (65%), 226 (25%), 140 (40%), 66 (100%).
5ꢀ(1ꢀTosylhydrazonoꢀethyl)ꢀ4ꢀmethoxyꢀ7ꢀ(4ꢀmethylꢀpiperazinꢀ1ꢀylꢀmethyl)ꢀbenzoꢀ
furanꢀ6ꢀol (12b).
Yield: 71%; mp: 196ꢀ7°C; Mol. wt.: 488.60. IR (cmꢀ1, KBr): 3420 (OH aromatic), 3189
1
(NH), 1617 (C=N). H NMR (DMSOꢀd6, δ, ppm): 1.21 (3H, s, CH3), 2.00 (3H, s, CH3ꢀ
piperidine), 2.30ꢀ2.50 (8H, m, CH2ꢀpiperidine), 3.00 (3H, s, CH3ꢀtoluene), 3.40 (2H, s,
CH2N), 3.78 (3H, s, OCH3), 5.00 (1H, s, OH, D2O exchangeable), 7.00 (1H, d, Hꢀ3
benzofuran), 7.37ꢀ7.70 (4H, m, CHꢀtoluene), 7.90 (1H, d, Hꢀ2 benzofuran), 13.00 (1H, s,
NH, D2O exchangeable). EIMS; m/z (R.A. %): 488 (M+) (60%), 312 (25%), 229 (25%),
128 (32%), 65 (100%).
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5ꢀ(1ꢀTosylhydrazonoꢀethyl)ꢀ4ꢀmethoxyꢀ7ꢀmorpholinꢀ4ꢀylꢀmethylꢀbenzofuranꢀ6ꢀol
(12c)
Yield: 75%; mp: 120ꢀ1°C; Mol. wt.: 475.56.IR (cmꢀ1, KBr): 3410 (OH aromatic), 3201
(NH), 1640 (C=N).1H NMR (DMSOꢀd6, δ, ppm): 1.00 (3H, s, CH3), 2.22 (3H, s, CH3ꢀ
toluene), 2.47ꢀ3.49 (8H, m, CH2ꢀmorpholine), 3.70 (2H, s, CH2N), 3.98 (3H, s, OCH3),
5.00 (1H, s, OH, D2O exchangeable), 6.74 (1H, d, Hꢀ3 benzofuran), 7.37ꢀ7.66 (4H, m,
CHꢀtoluene), 7.90 (1H, d, Hꢀ2 benzofuran), 13.80 (1H, s, NH, D2O exchangeable).
EIMS; m/z (R.A. %): 476 (M++1) (60%), 345 (65%), 237 (45%), 159 (100%).
Preparation of 4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromeneꢀ9ꢀsulfonylchloꢀ
ride (13) and 4ꢀmethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromeneꢀ9ꢀsulfonylhydrꢀ
azide (14).
Visnagin sulfonyl chloride (13)28 and Visnagin sulfonyl hydrazide (14)28 were prepared
according to the reported methods.
General procedure for the preparation of different imino derivatives (15, 16 and 17).
To a solution of visnaginꢀ4ꢀsulphonyl hydrazide (0.01 mol) in glacial acetic acid, the
appropriate anhydrides namely, (phthalic anhydride, succinic anhydride and maleic
anhydride) (0.005 mol) were added. The mixture was refluxed for 6ꢀ8 hours, poured onto
ice cold water, the formed precipitate was filtered, washed with water and crystallized
from glacial acetic acid to give the product.
4ꢀMethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromeneꢀ9ꢀsulfonic acid(1,3ꢀdioxoꢀ1,3ꢀ
dihydroꢀisoindolꢀ2ꢀyl)amide (15).
Yield: 90%; mp: 160ꢀ1°C; Mol. wt.: 454.41. IR (cmꢀ1, KBr): 3160 (CH aromatic),
1
2925(CH aliphatic), 1740 (C=O, anhydride). H NMR (DMSOꢀd6, δ, ppm): 2.20 (3H, s,
CH3), 4.00 (3H, s, OCH3), 6.22 (1H, s, CH), 6.70 (1H, d, Hꢀ3 benzofuran), 7.78 (1H, d,
Hꢀ2 benzofuran), 7.70ꢀ8.20 (4H, m, ArꢀH), 13.00 (1H, s, NH, D2O exchangeable). EIMS;
m/z (R.A. %): 454 (M+) (80%), 382 (40%), 292 (76%), 185 (20%), 56 (100%).
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4ꢀMethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromeneꢀ9ꢀsulfonic acid(pyrrolidineꢀ2ꢀ5ꢀ
dioneꢀ1ꢀyl)amide (16).
Yield: 75%; mp: 190ꢀ2°C; Mol. wt.: 406.37. IR (cmꢀ1, KBr): 3166 (CH aromatic), 2927
(CH aliphatic), 1744 (C=O, anhydride). 1H NMR (DMSOꢀd6, δ, ppm): 1.20 (3H, s, CH3),
2.73 (4H, t , CH2ꢀpyrrolidineꢀdione), 3.50 (3H, s, OCH3), 6.11 (1H, s, CH), 6.60 (1H, d,
Hꢀ3 benzofuran), 7.58 (1H, d, Hꢀ2 benzofuran), 9.00 (1H, s, NH, D2O exchangeable).
EIMS; m/z (R.A. %): 406 (M+) (60%), 342 (45%), 210 (66%), 191 (20%), 61 (100%).
4ꢀMethoxyꢀ7ꢀmethylꢀ5ꢀoxoꢀ5Hꢀfuro[3,2ꢀg]chromeneꢀ9ꢀsulfonic
acid(pyrrolꢀ2ꢀ5ꢀ
dioneꢀ1ꢀyl)amide (17)
Yield: 70%; mp: 270ꢀ2°C; Mol. wt.: 404.35. IR (cmꢀ1, KBr): 3170 (CH aromatic), 2930
(CH aliphatic), 1750 (C=O, anhydride). 1H NMR (DMSOꢀd6, δ, ppm): 2.00 (3H, s, CH3),
4.50 (3H, s, OCH3), 6.31 (1H, s, CH), 6.60 (1H, d, Hꢀ3 benzofuran), 7.00 (2H, d, CH2ꢀ
pyrroleꢀdione), 8.18 (1H, d, Hꢀ2 benzofuran), 10.00 (1H, s, NH, D2O exchangeable).
EIMS; m/z (R.A. %): 404 (M+) (70%), 358 (49%), 217 (66%), 114 (40%), 66 (100%).
2) Biology
VEGFRꢀ2 kinase activity assays by ELISA.34
The assay was performed in 96ꢀwell plates preꢀcoated with 20 ꢁg mL−1 poly (Glu,
Tyr) 4:1 using sorafenib and DMSO as positive and negative controls respectively,
Experiments at varying concentrations (1nMꢀ1µM) were performed in triplicate, where
85ꢁL of an 8 ꢁM ATP solution and 10 ꢁL of the compound were added in each well.
The reaction was initiated by adding VEGFRꢀ2 kinase (5 ꢁL), followed by addition of
100 ꢁL of antiꢀphosphotyrosine antibody (PY99; 1:500 dilution) then Goat antiꢀmouse
IgG horseradish peroxidase (100 ꢁL; 1 : 2000 dilution) diluted in TꢀPBS containing 5 mg
mL−1 BSA was added. After each addition there was an incubation period of 1h at room
temperature followed by washing.
Finally, 100 ꢁL of (0.03% H2O2, 2 mg mL−1oꢀphenylenediamine in citrate buffer 0.1
M, pH 5.5) was added and incubated at room temperature until color developed. The
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reaction was terminated by the addition of 100 ꢁL of 2 M H2SO4. The inhibition rate (%)
was calculated using the following equation:
Inhibition rate (%) = [1ꢀ(A492/ A492Control)] x 100%, where, A492 was measured using a
multiwell spectrophotometer (VERSAmax™).
In vitro cytoxicity screening.35
Cells were plated in 96ꢀmultiwell plate (5000 to 40,000 cells /well) for 24 h before
treatment with the compounds to allow attachment of cell to the wall of plate, a solution
of each tested compound was prepared by solubilizing each in DMSO 400ꢀfold the
desired final maximum test concentration.
After being incubated for 24 h, cells were treated with different concentrations of each
compound under test, where an aliquot of frozen concentrate was diluted to twice the
desired final maximum test concentration and four 10 fold serial dilutions were made to
provide a total of five drug concentrations plus control. The cells were incubated with the
compounds for 48 h at 37 ºC and in atmosphere of 5% CO2. Control cells were treated
with vehicle alone.
After 48 h, cells were fixed, washed and stained with Sulforhodamine B (SRB) (0.4 %
w/v) dissolved in 1 % acetic acid for 10 min. Excess stain was washed with acetic acid
and attached stain was solubilized with 10 mM trizma base. The absorbance was
measured in an automated plate reader at wave length of 515 nm. The concentration
required for 50% inhibition of cell viability (IC50) was calculated and compared with the
reference drugs.
In Vivo evaluation of antiprostate cancer Activitiy.36
Testosterone propionate (TP, 0.5 mg/kg) dissolved in cotton seed oil containing 5%
ethanol was administered to castrated male Wistar rats treated with androgen, three days
after castration, once daily for 5days alone or in combination with the tested compounds
(10ꢀ30 mg/kg) suspended with 0.5% methylcellulose. The rats were sacrificed after final
dosing, and both ventral prostates and seminal vesiclesꢀcoagulate glands were removed
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and weighed. The antiprostate cancer activity was expressed as a percentage of inhibition
of the TP effect.
3) Molecular Docking
GOLD software package, version 5.1 (Cambridge Crystallographic Data Centre,
Cambridge, U.K.),37 was used, the receptors were prepared using Hermes visualizer in the
GOLD Suite, and the region of interest was selected as all the protein residues within the
10 Å of the reference ligands.
For pose selection and enrichment studies default values of speed settings and all other
parameters were used. GOLD Score fitness algorithmic function was used with a default
input and annealing parameters, which were managed within 4.0 and 2.5 Å for the van der
Waals and Hꢀbond interactions, respectively 38,39
All the docked ligands were energetically minimized by using MOPAC with 100
iterations and minimum RMS gradient of 0.10.
In this study, 10 genetic algorithm (GA) docking runs were used with internal energy
offset. For pose reproduction analysis, the radius of the binding pocket was set as the
maximal atomic distance from the geometrical center of the ligand plus 3Å. When the top
three solutions attained RMSD values within 1.5 Å, docking was terminated.
The top ranked docking pose was retained for the 3D cumulative success rate analysis.
Rescoring was conducted with the GOLD rescore option, in which poses would be
optimized by the program. The top 10 ranked docking poses were selected based on their
gold fitness scores and their reported mode of interactions, which were analyzed using
Accelrys Discovery studio to reveal the hydrogen bond interaction and binding mode
within the binding domain.
With respect to ligand flexibility, flipping of all planar RNR1R2 ring, NHꢀR ring,
carboxylic acids –COOH were included, and the torsion angle distribution and post
process rotatable bonds were set as default. All other Genetic Algorithm parameters were
set as default settings.
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The crystal structure of the (VEGFRꢀ2) kinase domain in complex with Nꢀ[4ꢀ({3ꢀ[2ꢀ
(methylamino)pyrimidinꢀ4ꢀyl]pyridinꢀ2ꢀyl}oxy)naphthenꢀ1ꢀyl]ꢀ6ꢀ(trifluoromethꢀyl)ꢀ1Hꢀ
benzimidazolꢀ2ꢀamine (K111) coꢀcrystalized ligand (pdb code: 3EWH) was used as target
protein structure, which was retrieved from the Protein Data Bank (PDB). PDB sum was
accessed for identification of the key amino acids and the flexible residues (E885, T916,
and D1046) in the binding site. All residues within 10 Å radius from the origin: 13.237, ꢀ
2.016 and 11.23 of the coꢀcrystallized ligand coordinates.
Author Information
Corresponding Author
Phone: +20 202 37608284; fax: +20 202 33370931. Eꢀmail address:
dromaima45@gmail.com (O.M. Abdelhafez).
Abbreviations Used
IC50, half maximal inhibitory concentration; ED50, median effective dose; TLC, thin layer
chromatography; DMSO, Dimethyl Sulfoxide; MOPAC, Molecular Orbital Package.
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DOI: 10.1039/C4RA16228E
List of Tables
Table 1: AntiꢀVEGFRꢀ2 activity of the synthesized compounds.
Enzymatic inhibition IC50ꢁM
Compound
VEGFRꢀ2
2.00 x 10ꢀ3
3
ꢀꢀꢀꢀꢀꢀꢀ
4a
4b
2.70 x 10ꢀ3
2.90 x 10ꢀ3
8.40 x 10ꢀ3
1.01 x 10ꢀ2
2.20 x 10ꢀ3
6.30 x 10ꢀ3
1.22 x 10ꢀ2
2.62 x 10ꢀ2
5.70 x 10ꢀ3
3.20 x 10ꢀ3
3.20 x 10ꢀ3
1.20 x 10ꢀ2
5.20 x 10ꢀ3
1.63 x 10ꢀ2
4.65 x 10ꢀ2
1.48 x 10ꢀ2
1.79 x 10ꢀ2
3.49 x 10ꢀ2
2.62 x 10ꢀ2
4.22 x 10ꢀ2
2.17 x 10ꢀ2
2.00 x 10ꢀ3
4c
4d
5a
5b
5c
5d
8a
8b
10a
10b
11a
11b
11c
12a
12b
12c
14
15
16
17
Sorafenib
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DOI: 10.1039/C4RA16228E
Table 2a: Cytotoxicity of the synthesized compounds.
IC50µM
Compound
KB
ꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀ
SK OVꢀ3
5.66 x10ꢀ7
4.35 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
SFꢀ268
2.56 x10ꢀ9
4.56 x10ꢀ7
3.45 x10ꢀ11
9.70 x 10ꢀ4
3.30 x 10ꢀ5
2.33 x10ꢀ11
8.86 x 10ꢀ4
5.30 x 10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
8.77 x 10ꢀ3
5.80 x 10ꢀ4
4.40 x 10ꢀ5
5.50 x 10ꢀ3
5.50 x 10ꢀ3
6.50 x 10ꢀ3
5.70 x 10ꢀ3
8.70 x 10ꢀ6
4.40 x 10ꢀ5
5.20 x 10ꢀ3
4.60 x 10ꢀ3
5.00 x 10ꢀ4
4.30 x 10ꢀ5
4.00 x 10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
NCI H 460 RKOP27
ꢀꢀꢀꢀꢀꢀꢀꢀ
2.67 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀꢀ
4.78 x10ꢀ1
3
4a
4b
4c
4d
5a
5b
5c
5d
8a
8b
10a
10b
11a
11b
11c
3.67 x10ꢀ7
3.33 x 10ꢀ6
2.36 x 10ꢀ4
2.55 x10ꢀ3
6.30 x 10ꢀ5
7.40 x 10ꢀ3
7.54 x10ꢀ7
6.40 x 10ꢀ4
2.10 x 10ꢀ4
6.00 x 10ꢀ4
4.70 x 10ꢀ4
4.30 x 10ꢀ4
7.74 x 10ꢀ3
6.80 x 10ꢀ3
7.74 x 10ꢀ3
2.27 x 10ꢀ3
7.98 x 10ꢀ5
6.98 x 10ꢀ3
3.00 x 10ꢀ5
4.50 x 10ꢀ4
7.00 x 10ꢀ3
4.46 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
2.39 x10ꢀ10 4.67 x10ꢀ11
8.80 x 10ꢀ3 9.32 x 10ꢀ3
6.40 x 10ꢀ3 8.80 x 10ꢀ5
4.62 x10ꢀ11 3.67 x10ꢀ13
8.56 x 10ꢀ6 8.00 x 10ꢀ4
8.70 x 10ꢀ4 8.97 x 10ꢀ5
1.45 x10ꢀ10 3.56 x10ꢀ13
2.27 x 10ꢀ3 7.27 x 10ꢀ3
8.56 x 10ꢀ4 7.70 x 10ꢀ6
6.60 x 10ꢀ5 7.80 x 10ꢀ5
4.40 x 10ꢀ3 3.30 x 10ꢀ5
3.30 x 10ꢀ3 4.80 x 10ꢀ3
5.50 x 10ꢀ3 9.40 x 10ꢀ3
9.47 x 10ꢀ3 5.50 x 10ꢀ3
6.60 x 10ꢀ5 9.40 x 10ꢀ3
4.79 x 10ꢀ3 9.00 x 10ꢀ3
9.00 x 10ꢀ4 9.59 x 10ꢀ4
4.20 x 10ꢀ3 1.25 x 10ꢀ5
6.00 x 10ꢀ4 8.00 x 10ꢀ3
7.60 x 10ꢀ3 8.90 x 10ꢀ3
3.00 x 10ꢀ3 5.00 x 10ꢀ5
8.36 x 10ꢀ3
4.40 x 10ꢀ4
ꢀꢀꢀꢀꢀꢀꢀ
3.42 x 10ꢀ3
6.34 x 10ꢀ3
2.67 x10ꢀ7
4.60 x 10ꢀ5
7.23 x 10ꢀ5
3.00 x 10ꢀ4
4.00 x 10ꢀ3
5.50 x 10ꢀ5
5.50 x 10ꢀ5
7.70 x 10ꢀ3
4.40 x 10ꢀ4
6.60 x 10ꢀ4
3.33 x 10ꢀ2
6.21 x 10ꢀ3
7.00 x 10ꢀ3
6.50 x 10ꢀ5
5.00 x 10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
12a
12b
12c
14
15
16
17
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
Fluorouracil
Doxorubicin
Cytarabine
Gemcitabine
Capecitabine
4.16 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
7.68 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
4.33x10ꢀ3
2.13 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
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DOI: 10.1039/C4RA16228E
Table 2b: Cytotoxicity of the synthesized compounds.
IC50 µM
Compound
Leukemia
Melanoma
HL60
U937
K561
ꢀꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀꢀ
G361
SKꢀMELꢀ28
ꢀꢀꢀꢀꢀꢀꢀꢀ
3.67 x10ꢀ8
4.67 x10ꢀ8
4.67 x10ꢀ9
8.00 x 10ꢀ4
4.40 x 10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀꢀ
3.76 x10ꢀ8
ꢀꢀꢀꢀꢀꢀ
3
4a
4b
4c
4d
5a
5b
5c
5d
8a
8b
10a
10b
11a
11b
11c
12a
12b
12c
14
15
16
17
4.39 x10ꢀ7
4.56 x10ꢀ7
9.86 x 10ꢀ3
6.60 x 10ꢀ3
8.55 x10ꢀ7
ꢀꢀꢀꢀꢀꢀꢀꢀ
4.56 x10ꢀ9
2.70 x 10ꢀ4
7.46 x 10ꢀ3
5.32 x10ꢀ7
8.60 x 10ꢀ3
4.80 x 10ꢀ4
7.88 x10ꢀ8
4.90 x 10ꢀ4
7.88 x10ꢀ8
9.60 x 10ꢀ5
5.60 x 10ꢀ3
8.63 x 10ꢀ3
6.60 x 10ꢀ5
5.50 x 10ꢀ5
5.50 x 10ꢀ4
6.77 x 10ꢀ3
8.50 x 10ꢀ3
4.30 x 10ꢀ3
7.64 x 10ꢀ4
6.00 x 10ꢀ6
7.70 x 10ꢀ3
6.66 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
5.33 x10ꢀ8
6.20 x 10ꢀ4
634 x 10ꢀ3
7.43 x10ꢀ8
7.00 x 10ꢀ3
4.80 x 10ꢀ5
3.34 x10ꢀ7
6.60 x 10ꢀ5
3.34 x10ꢀ7
7.70 x 10ꢀ3
8.89 x 10ꢀ3
7.50 x 10ꢀ3
9.90 x 10ꢀ5
7.70 x 10ꢀ3
8.80 x 10ꢀ4
8.90 x 10ꢀ5
5.50 x 10ꢀ3
7.35 x 10ꢀ3
8.00 x 10ꢀ5
8.00 x 10ꢀ4
8.60 x 10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
6.20 x 10ꢀ4
7.50 x 10ꢀ5
5.67 x10ꢀ7
6.00 x 10ꢀ5
4.80 x 10ꢀ3
4.89 x10ꢀ7
7.70 x 10ꢀ3
4.90 x10ꢀ7
8.80 x 10ꢀ3
9.90 x 10ꢀ3
3.00 x 10ꢀ3
8.00 x 10ꢀ5
2.00 x 10ꢀ5
5.87 x 10ꢀ7
3.00 x 10ꢀ5
6.60 x 10ꢀ5
3.40 x 10ꢀ5
8.50 x 10ꢀ3
8.00 x 10ꢀ4
5.70 x 10ꢀ5
ꢀꢀꢀꢀꢀꢀꢀ
3.67 x10ꢀ9 5.87 x10ꢀ7
4.80 x 10ꢀ3
4.80 x 10ꢀ5
4.55 x10ꢀ7
8.70 x 10ꢀ6
4.55 x10ꢀ7
4.00 x 10ꢀ4
3.00 x 10ꢀ5
2.48 x 10ꢀ4
9.70 x 10ꢀ4
9.60 x 10ꢀ4
6.30 x 10ꢀ4
9.00 x 10ꢀ4
9.86 x 10ꢀ3
8.00 x 10ꢀ3
2.60 x 10ꢀ5
7.53 x 10ꢀ4
7.00 x 10ꢀ3
1.13 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
8.00 x 10ꢀ3
4.80 x 10ꢀ3
5.44 x10ꢀ7
5.40 x 10ꢀ3
5.44 x10ꢀ7
6.00 x 10ꢀ6
9.00 x 10ꢀ3
9.60 x 10ꢀ5
4.00 x 10ꢀ4
9.70 x 10ꢀ4
5.00 x 10ꢀ5
6.00 x 10ꢀ3
4.40 x 10ꢀ3
7.50 x 10ꢀ4
5.00 x 10ꢀ3
5.96 x 10ꢀ5
7.70 x 10ꢀ3
4.45 x10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
Doxorubicin
Aldesleukin
6.66 x10ꢀ3
3.45 x10ꢀ3
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DOI: 10.1039/C4RA16228E
Table 2c: Cytotoxicity of the synthesized compounds.
IC50 µM (Neuroblastoma)
Compound
GOTO
NBꢀ1
4.89 x10ꢀ7
ꢀꢀꢀꢀꢀꢀꢀꢀ
5.32 x10ꢀ7
5.78 x10ꢀ7
3.44 x10ꢀ7
7.90 x 10ꢀ5
5.68 x 10ꢀ4
ꢀꢀꢀꢀꢀꢀꢀꢀ
3
4a
4b
4c
4d
5a
5b
5c
5d
6.55 x10ꢀ9
8.60 x 10ꢀ6
3.00 x 10ꢀ4
ꢀꢀꢀꢀꢀꢀꢀꢀ
8.60 x 10ꢀ6
8.00 x 10ꢀ4
4.66 x10ꢀ9
9.00 x 10ꢀ4
8.60 x 10ꢀ4
7.50 x 10ꢀ4
6.41 x 10ꢀ4
7.60 x 10ꢀ4
8.60 x 10ꢀ3
6.80 x 10ꢀ3
8.60 x 10ꢀ3
8.60 x 10ꢀ3
8.00 x 10ꢀ5
8.50 x 10ꢀ3
4.63 x 10ꢀ5
7.40 x 10ꢀ4
9.60 x 10ꢀ3
4.73 x10ꢀ3
9.70 x 10ꢀ5
3.64 x 10ꢀ4
ꢀꢀꢀꢀꢀꢀꢀꢀ
3.00 x 10ꢀ5
7.20 x 10ꢀ5
7.20 x 10ꢀ4
7.20 x 10ꢀ3
5.00 x 10ꢀ5
7.25 x 10ꢀ4
7.00 x 10ꢀ3
7.25 x 10ꢀ4
7.20 x 10ꢀ3
5.30 x 10ꢀ3
3.87 x 10ꢀ3
8.00 x 10ꢀ4
3.80 x 10ꢀ5
8.63 x 10ꢀ3
5.15 x10ꢀ3
8a
8b
10a
10b
11a
11b
11c
12a
12b
12c
14
15
16
17
Doxorubicin
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Table 2d: Cytotoxicity of the synthesized compounds.
IC50 µM
Compound
PCꢀ3
HT1080
HepG2
6.40 x10ꢀ3
4.78 x10ꢀ10
8.60 x10ꢀ3
8.00 x10ꢀ6
6.40 x10ꢀ4
9.70 x10ꢀ3
8.70 x10ꢀ5
6.88 x10ꢀ3
8.80 x10ꢀ6
8.00 x 10ꢀ4
4.74 x 10ꢀ4
5.70 x 10ꢀ4
5.00 x 10ꢀ4
7.25 x 10ꢀ5
6.50 x 10ꢀ3
8.65 x 10ꢀ3
4.60 x 10ꢀ3
7.00 x 10ꢀ3
9.70 x 10ꢀ5
1.25 x 10ꢀ5
8.00 x 10ꢀ3
8.90 x 10ꢀ3
5.00 x 10ꢀ5
6.70 x 10ꢀ6
ꢀꢀꢀꢀꢀꢀꢀ
5.43 x 10ꢀ7
8.90 x 10ꢀ7
5.65 x 10ꢀ7
4.64 x 10ꢀ3
8.08 x 10ꢀ5
7.68 x 10ꢀ7
7.00 x 10ꢀ3
8.70 x 10ꢀ3
2.34 x 10ꢀ7
9.70 x 10ꢀ3
9.70 x 10ꢀ4
9.76 x 10ꢀ5
9.70 x 10ꢀ3
8.00 x 10ꢀ3
3.46 x 10ꢀ4
3.57 x 10ꢀ3
4.97 x 10ꢀ3
8.60 x 10ꢀ3
7.90 x 10ꢀ3
6.00 x 10ꢀ3
7.80 x 10ꢀ4
8.00 x 10ꢀ5
4.60 x 10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
3.37 x10ꢀ7
5.64 x10ꢀ7
2.88 x10ꢀ7
8.00 x 10ꢀ3
8.00 x 10ꢀ3
5.55 x10ꢀ7
5.70 x 10ꢀ3
3.60 x 10ꢀ3
2.99 x10ꢀ7
7.50 x 10ꢀ3
8.60 x 10ꢀ4
9.70 x 10ꢀ5
9.00 x 10ꢀ4
1.46 x 10ꢀ3
6.35 x 10ꢀ4
3.60 x 10ꢀ3
6.34 x 10ꢀ3
6.30 x 10ꢀ3
1.34 x 10ꢀ3
7.60 x 10ꢀ3
7.00 x 10ꢀ5
7.00 x 10ꢀ3
9.86 x 10ꢀ3
ꢀꢀꢀꢀꢀꢀꢀ
3
4a
4b
4c
4d
5a
5b
5c
5d
8a
8b
10a
10b
11a
11b
11c
12a
12b
12c
14
15
16
17
Abiraterone acetate
Imatinib
Gemcitabine
13.24 x10ꢀ5
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀꢀꢀ
3.44 x10ꢀ3
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Table 3: In vivo antiprostate cancer activity of the synthesized compounds.
Compound
ED50 µM
13.11±0.004
21.49±0.006
23.64±0.005
10.64±0.003
8.00±0.004
26±0.004
3
4a
4b
4c
4d
5a
13.11±0.004
7.21±0.005
28.6±0.004
5.66±0.04
5b
5c
5d
8a
6.22±0.04
8b
6.85±0.05
7.53±0.06
10a
10b
11a
11b
11c
12a
12b
12c
14
3.40±0.006
16.14±0.006
19.53±0.006
14.68±0.005
17.76±0.007
5.14±0.005
3.73±0.004
2.56±0.005
2.82±0.006
2.12±0.007
11.60±0.09
15
16
17
Flutamide
Each ED50 value is the mean (three significant digits) ± SEM from five experiments. Level of statistical
significance: P < 0.01 with respect to ED50value of Flutamide as determined by ANOVA/Dunnett’s.
28

Page 29 of 37
RSC Advances
View Article Online
DOI: 10.1039/C4RA16228E
Table 4: The flexible docking results (GOLD 5.1), regarding the GoldScore Fitness and external VDW
of compounds docked intoVEGFRꢀ2 kinase (3EWH).
Hydrogen bonds between atoms of compounds
Com
p.
RMSDa
(Å)
and amino acids of VEGFR
Gold
Score
External
VDW
Distance
(Å)
Angle
(o)
Atoms of comp. Amino acids
4ꢀCH3O
6ꢀC=O
5ꢀC=O
HN of K868
HN of R1051
HN of D1046
1.97
1.95
2.32
166.6
164.8
164.3
44.02
25.40
6.96
3
72.00
74.00
69.03
52.43
53.55
49.37
4.44
4.16
4.73
4a
4b
4c
4``ꢀBr
HN of R1027
HN of D1046
2.45
2.07
152.0
164.8
5ꢀC=O
5ꢀC=O
4``ꢀ CH3O
5ꢀC=O
5ꢀC=O
5ꢀC=O
5ꢀC=O
6ꢀO
4ꢀO
6ꢀO
4ꢀO
6ꢀO
4ꢀO
6ꢀO
Furan O
6ꢀO
Furan O
6ꢀO
Furan O
6ꢀO
4ꢀCH3O
6ꢀO
HN of D1046
HN of R1027
HN of D1046
HN of D1046
HN of D1046
HN of D1046
HN of K868
HN of K868
HN of D1046
1HN of K868
2HN of K868
1HN of K868
2HN of K868
HN of K868
HN of D1046
HN of K868
HN of D1046
HN of K868
HN of D1046
HN of K868
HN of D1046
O=C of D1046
2.01
2.11
2.05
2.05
2.23
2.48
1.85
1.48
2.15
2.25
2.09
2.24
2.12
2.37
1.85
2.21
1.87
2.27
2.05
2.22
2.16
1.94
2.25
2.20
1.75
1.64
1.92
1.85
1.48
1.87
159.7
152.9
165.7
165.4
171.3
162.0
163.8
167.4
170.1
174.2
166.1
172.9
159.8
171.5
171.3
171.9
144.2
174.0
160.1
165.7
151.4
141.6
115.5
161.9
162.0
154.2
139.7
164.0
127.6
164.4
79.85
55.78
5.65
4d
71.80
70.86
70.66
81.17
61.23
52.78
51.09
53.09
58.32
35.49
4.68
4.63
4.77
5.48
3.34
5a
5b
5c
5d
8a
63.93
68.08
71.36
57.89
59.91
58.27
74.71
75.09
78.28
37.98
45.62
41.51
39.98
36.94
38.14
54.22
51.49
48.43
4.70
4.55
4.84
3.86
4.00
3.42
2.39
6.27
5.85
8b
10a
10b
11a
11b
11c
12a
12b
12c
6ꢀOH
5ꢀCHNHNH O=C of E885
Furan O
S=O
Furan O
5ꢀC=O
S=O
S=ONH
Furan O
HN of D1046
HN of K868
HN of K868
HO of T916
HN of K868
O=C of D1046
HN of K868
57.17
72.88
27.14
51.88
2.40
2.40
14
15
29