Synthesis, molecular modeling and NAD(P)H:quinone oxidoreductase 1 inducer activity of novel 2-phenylquinazolin-4-amine derivatives

Article abstract of DOI:10.3109/14756366.2016.1158714

Reactive oxygen species (ROS) play an integral role in the pathogenesis of most diseases. This work presents the design and synthesis of novel 2-phenylquinazolin-4-amine derivatives (2–12) and evaluation of their NAD(P)H:quinone oxidoreductase 1 (NQO1) inducer activity in murine cells. Also, molecular docking of all the new compounds was performed to assess their ability to inhibit Keap1–Nrf2 protein–protein interaction through occupying the Keap1–Nrf2-binding domain which biologically leads to a consequent Nrf2 accumulation and enhanced gene expression of NQO1. Docking results showed that all compounds have the ability to interact with Keap1; however compound 7, the most active compound in this study, showed more interactions with key amino acids.

Full text of DOI:10.3109/14756366.2016.1158714

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, molecular modeling and
NAD(P)H:quinone oxidoreductase 1 inducer
activity of novel 2-phenylquinazolin-4-amine
derivatives
Mostafa M. Ghorab, Mansour S. Alsaid, Maureen Higgins, Albena T. Dinkova-
Kostova, Abdelaaty A. Shahat, Nehal H. Elghazawy & Reem K. Arafa
To cite this article: Mostafa M. Ghorab, Mansour S. Alsaid, Maureen Higgins, Albena T.
Dinkova-Kostova, Abdelaaty A. Shahat, Nehal H. Elghazawy & Reem K. Arafa (2016): Synthesis,
molecular modeling and NAD(P)H:quinone oxidoreductase 1 inducer activity of novel 2-
phenylquinazolin-4-amine derivatives, Journal of Enzyme Inhibition and Medicinal Chemistry
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1158714
Published online: 07 Apr 2016.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [McMaster University]
Date: 08 April 2016, At: 07:10

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1158714
!
RESEARCH ARTICLE
Synthesis, molecular modeling and NAD(P)H:quinone oxidoreductase 1
inducer activity of novel 2-phenylquinazolin-4-amine derivatives
Mostafa M. Ghorab1,2, Mansour S. Alsaid1, Maureen Higgins3, Albena T. Dinkova-Kostova3,4, Abdelaaty A. Shahat1,5
Nehal H. Elghazawy6, and Reem K. Arafa6,7
,
2
1Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia, Department of Drug Radiation
Research, National Center for Radiation Research & Technology, Atomic Energy Authority, Nasr City, Egypt, 3Jacqui Wood Cancer Centre, Division of
4
Cancer Research, Medical Research Institute, University of Dundee, Dundee, UK, Departments of Medicine and Pharmacology and Molecular
5
Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA, Phytochemistry Department, National Research Center, Cairo, Egypt,
6Zewail City of Science and Technology, Cairo, Egypt, and 7Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo,
Egypt
Abstract
Keywords
Reactive oxygen species (ROS) play an integral role in the pathogenesis of most diseases. This
work presents the design and synthesis of novel 2-phenylquinazolin-4-amine derivatives (2–12)
and evaluation of their NAD(P)H:quinone oxidoreductase 1 (NQO1) inducer activity in murine
cells. Also, molecular docking of all the new compounds was performed to assess their ability to
inhibit Keap1–Nrf2 protein–protein interaction through occupying the Keap1–Nrf2-binding
domain which biologically leads to a consequent Nrf2 accumulation and enhanced gene
expression of NQO1. Docking results showed that all compounds have the ability to interact
with Keap1; however compound 7, the most active compound in this study, showed more
interactions with key amino acids.
Cytoprotection, Keap1/Nrf2, molecular
modeling, NQO1 induction,
2-phenylquinazolin-4-amine derivatives
History
Received 1 January 2016
Revised 19 February 2016
Accepted 21 February 2016
Published online 5 April 2016
Introduction
However, upon increased oxidative load, reactive cysteine resi-
dues within Keap1 serve as sensors for oxidants, leading to loss of
its ability to target Nrf2 for degradation, subsequent accumulation
of the transcription factor, activation of the pathway and increased
Excessive unbalanced production of reactive oxygen species
(ROS), namely superoxide anion radical (Oꢀ2 ꢁ), singlet oxygen
(1O2), hydroxyl radical (ꢁOH) and hydrogen peroxide (H2O2), has
been well established to be associated with many pathological
conditions including atherosclerosis, stroke, diabetes, Alzheimer’s
disease, cancer and inflammation1. In a counter action and to
maintain homeostasis, living cells combat the deleterious effects
of ROS by an arsenal of biochemical antioxidants2,3. Under
oxidative stress conditions; where the balance between ROS and
biochemical antioxidants is lost due to over production of the
former or under availability of the latter; ROS can attack and
damage cellular membrane lipids, DNA nucleotides4, proteins’
sulphydryl groups5 and ribonucleoproteins6. Among the most
important antioxidant mechanisms activated under conditions of
oxidative stress is the Kelch-like ECH-associated protein 1
(Keap1)–nuclear factor erythroid 2-related factor 2 (Nrf2)-
antioxidant response elements (AREs) pathway7. This pathway
is associated with the expression of more than 100 cytoprotective
genes and their respective enzymes, one of which is
NAD(P)H:quinone oxidoreductase 1 (NQO1, EC 1.6.99.2).
Under homeostatic conditions, Nrf2 is continuously targeted
for ubiquitination and proteasomal degradation by Keap1.
transcription of cytoprotective antioxidant proteins8–10
.
Medicinal chemists view the Keap1–Nrf2 protein–protein
interaction as a critical point of the pathway that can be targeted
for intervention and for potential management of a variety of
oxidative stress-related ailments like cancer, Parkinson’s,
Alzheimer’s and diabetes11–14. Design of non-covalent small
molecule modulators of the Keap–Nrf2 interaction has been
widely explored15,16. Quinazoline-based nitrogenous heteroaryls
are an excellent reservoir of bioactive substances where a number
of biological activities have been associated with antioxidant
activity17–19
In continuation of our quest for new cytoprotective agents20,21
.
,
we herein report the synthesis, NQO1 inducer activity and Keap1
inhibitory in silico screening results for a novel class of N-aryl
substituted 4-amino-2-phenylquinazolines.
Materials and methods
Chemistry
Melting points (uncorrected) were determined in open capillary
tube on a Gallen Kamp melting point apparatus (Sanyo Gallen
Kamp, Leicestershire, UK). Precoated silica gel plates (Kieselgel
0.25 mm, 60 F254, Merck, Darmstadt, Germany) were used for
thin layer chromatography. A developing solvent system of
Address for correspondence: Mostafa M. Ghorab, Department of
Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh, 11451, Kingdom of Saudi Arabia. E-mail:
mmsghorab@gmail.com

2
M. M. Ghorab et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
chloroform/methanol (8:2) was used and the spots were detected with D2O]. 13C-NMR (DMSO-d6): ppm 22.6 (2), 25.0, 29.6,
by ultraviolet light. IR spectra (KBr disc) were recorded using an 113.0 (2), 125.1, 125.3, 125.8, 128.0, 128.7 (2), 129.1 (2),
FT-IR spectrophotometer (Perkin Elmer, Waltham, MA). NMR 129.3 (2), 133.1, 134.1, 135.7, 135.8, 138.4, 157.7, 160.1, 163.4.
spectra were scanned on a NMR spectrophotometer (Bruker AXS MS m/z (%): 351 (M+) (7.13), 294 (100). Anal. Calcd. for
Inc., Geneva, Switzerland), operating at 500 MHz for 1H and C24H21N3 (351): C, 82.02; H, 6.02; N, 11.96. Found: C, 82.34;
125.76 MHz for 13C. Chemical shifts are expressed in ꢀ-values H, 5.81; N, 12.21.
(ppm) relative to TMS as an internal standard, using DMSO-d6 as
a solvent. Elemental analyses were done on a model 2400
CHNSO analyzer (Perkin Elmer, Waltham, MA). All the values
were within 0.4% of the theoretical values. All reagents used
were of AR grades. The starting material 4-chloro-2-phenylqui-
nazoline (1) was purchased from Sigma (St. Louis, MO) and was
directly used for the preparation of target compounds.
N-(naphthalen-1-yl)-phenylquinazolin-4-amine (6). White solid;
yield, 79%; m.p. 143–145 ꢂC. IR (KBr, cmꢀ1): 3401 (NH), 3100
(CH arom.), 2981, 2780 (CH aliph.), 1629 (C¼N). 1H-NMR
(DMSO-d6): ppm 7.1–8.9 [m, 16H, Ar–H], 11.2 [s, 1H, NH,
exchangeable with D2O]. 13C-NMR (DMSO-d6): ppm 113.7,
119.4, 122.4, 122.8, 124.1, 124.7, 125.2, 126.0, 126.2, 126.5 (2),
127.3, 127.4, 128.2, 128.7, 129.0, 130.0, 131.8, 133.1, 134.1,
135.0, 158.6, 160.8, 163.4. MS m/z (%): 347 (M+) (11.23), 269
(100). Anal. Calcd. for C24H17N3 (347): C, 82.97; H, 4.93; N,
12.10. Found: C, 82.64; H, 5.21; N, 11.86.
Synthesis of 2-phenyl-4-substituted quinazoline
derivatives 2–12
General procedure
2-Phenyl-N-(quinolin-2-yl)quinazolin-4-amine (7). White solid;
yield, 69%; m.p. 191–192 ꢂC. IR (KBr, cmꢀ1): 3411 (NH), 3024
(CH arom.), 2977, 2839, 2780 (CH aliph.), 1635 (C¼N). 1H-
NMR (DMSO-d6): ppm 7.1–8.9 [m, 15H, Ar–H], 9.1 [s, 1H, NH,
exchangeable with D2O]. 13C-NMR (DMSO-d6): ppm 112.6,
116.7, 117.2, 123.1, 126.6 (2), 127.7, 127.8 (2), 128.0 (2), 129.2
(2), 129.3, 129.5, 129.8, 133.1, 135.4, 138.4 (2), 157.1, 161.7,
164.8. MS m/z (%): 348 (M+) (24.07), 209 (100). Anal. Calcd. for
C23H16N4 (348): C, 79.29; H, 4.63; N, 16.08. Found: C, 79.55; H,
4.38; N, 16.36.
A mixture of 2-phenyl 4-chloroquinazoline 1 (2.40 g, 0.01 mol)
and different amines (0.01 mol) in dimethylformamide (10 mL)
containing a catalytic amount of trimethylamine (three drops) was
refluxed for 24 h and then left to cool. The solid product formed
was collected by filtration and recrystallized from acetic acid to
give the desired quinazoline derivatives 2–12.
N-(2,3-dihydro-1H-inden-5-yl)-2-phenylquinazolin-4-amine (2).
White solid; yield, 76%; m.p. 118–119 ꢂC. IR (KBr, cmꢀ1):
3401 (NH), 3080 (CH arom.), 2976, 2778 (CH aliph.), 1631
(C¼N). 1H-NMR (DMSO-d6): ppm 1.9–2.8 [m, 6H, 3CH2,
Cyclo], 7.1–9.0 [m, 12H, Ar–H], 11.8 [s, 1H, NH, exchangeable
with D2O]. 13C-NMR (DMSO-d6): ppm 25.6, 32.9 (2), 113.1,
121.6, 123.0, 124.4, 125.4, 128.3 (2), 129.3, 129.4, 129.7, 129.8,
133.7 (2), 135.3, 136.1, 142.3, 144.5 (2), 157.3, 159.1. MS m/z
(%): 337 (M+) (16.73), 219 (100). Anal. Calcd. for C23H19N3
(337): C, 81.87; H, 5.68; N, 12.45. Found: C, 81.53; H, 5.94; N,
12.14.
2-Phenyl-N-(quinolin-3-yl)quinazolin-4-amine (8). White solid;
yield, 91%; m.p. 281–282 ꢂC. IR (KBr, cmꢀ1): 3321 (NH), 3059
(CH arom.), 1624 (C¼N). 1H-NMR (DMSO-d6): ppm 7.4–9.3 [m,
15H, Ar–H], 10.3 [s, 1H, NH exchangeable with D2O]. 13C-NMR
(DMSO-d6): ppm 114.4, 121.4, 124.8, 125.6, 126.3, 126.5, 127.0
(2), 127.9, 128.1, 128.4, 128.9 (2), 129.1, 130.2, 131.8, 132.2,
133.9, 138.2, 144.7, 149.2, 162.7, 163.2. MS m/z (%): 348 (M+)
(39.41), 270 (100). Anal. Calcd. for C23H16N4 (348): C, 79.29; H,
4.63; N, 16.08. Found: C, 79.56; H, 4.32; N, 16.29.
N-(benzo[d][1,3]dioxol-5-yl)-2-phenylquinazolin-4-amine (3).
Pale yellow solid; yield, 89%; m.p. 107–108 ꢂC. IR (KBr,
cmꢀ1): 3441 (NH), 3081 (CH arom.), 2984, 2874 (CH aliph.),
N-(2-methylquinolin-4-yl)-2-phenylquinazolin-4-amine
(9).
White solid; yield, 88%; m.p. 236–238 ꢂC. IR (KBr, cmꢀ1):
3433 (NH), 3023 (CH arom.), 2976, 2839, 2779 (CH aliph.), 1609
(C¼N). 1H-NMR (DMSO-d6): ppm 2.5 [s, 3H, CH3], 7.5–8.0 [m,
14H, Ar–H], 12.5 [s, 1H, NH exchangeable with D2O]. 13C-NMR
(DMSO-d6): ppm 19.8, 102.0, 115.5, 119.7, 121.4, 124.2, 126.1,
126.4 (2), 127.0, 127.9, 128.2, 128.9 (2), 129.7, 131.8, 133.1,
133.9, 149.1, 152.8, 153.2, 158.1, 162.7 (2). MS m/z (%): 362
(M+) (24.09), 347 (100). Anal. Calcd. for C24H18N4 (362): C,
79.54; H, 5.01; N, 15.46. Found: C, 79.22; H, 4.87; N, 15.18.
1
1617 (C¼N). H-NMR (DMSO-d6): ppm 6.0 [s, 2H, O–CH2–O],
7.0–8.5 [m, 12H, Ar–H], 9.7 [s, 1H, NH, exchangeable with D2O].
13C-NMR (DMSO-d6): ppm 101.4, 104.8, 108.6, 114.4, 115.8,
123.3, 126.2 (2), 128.3, 128.5, 128.8 (2), 130.6, 133.5, 133.9,
138.8 (2), 147.4, 150.8, 159.6, 162.7. MS m/z (%): 341 (M+)
(3.65), 294 (100). Anal. Calcd. for C21H15N3O2 (341): C, 73.89;
H, 4.43; N, 12.31. Found: C, 74.11; H, 4.75; N, 12.65.
N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-phenylquinazolin-4-amine
(4). Tan white solid; yield, 80%; m.p. 180–181 ꢂC. IR (KBr,
cmꢀ1): 3238 (NH), 3069 (CH arom.), 2944, 2791 (CH aliph.),
1618 (C¼N). 1H-NMR (DMSO-d6): ppm 4.8 [s, 2H, CH2], 5.9 [s,
2H, O–CH2–O], 6.8–8.6 [m, 12H, Ar–H], 10.2 [s, 1H, NH,
exchangeable with D2O]. 13C-NMR (DMSO-d6): ppm 44.5,
101.3, 108.4, 108.5, 113.4, 120.9, 124.2, 127.2 (2), 129.1 (2),
132.4 (2), 132.9, 133.3, 134.8 (2), 146.5, 146.7, 147.7, 158.4,
163.4. MS m/z (%): 355 (M+) (22.15), 233 (100). Anal. Calcd. for
C22H17N3O2 (355): C, 74.35; H, 4.82; N, 11.82. Found: C, 74.08;
H, 4.59; N, 12.10.
4-Methyl-7-(2-phenylquinazolin-4-ylamino)-2H-chromen-2-one
(10). Orange solid; yield, 78%; m.p. 279–280 ꢂC. IR (KBr,
cmꢀ1): 3356 (NH), 3062 (CH arom.), 2946, 2869, 2765 (CH
1
aliph.), 1699 (C¼O), 1618 (C¼N). H-NMR (DMSO-d6): ppm
2.4 [s, 3H, CH3], 6.2 [s, 1H, CH Chromene], 7.5–8.6 [m, 12H,
Ar–H], 10.1 [s, 1H, NH exchangeable with D2O]. 13C-NMR
(DMSO-d6): ppm 18.5, 108.1 (2), 112.7, 114.5, 115.3, 123.5,
125.9, 126.7 (2), 128.3, 128.7, 129.0 (2), 130.9, 134.0, 138.6,
143.3, 151.0, 153.6, 154.0, 158.1, 159.3, 160.6. MS m/z (%): 379
(M+) (9.64), 294 (100). Anal. Calcd. for C24H17N3O2 (379): C,
75.97; H, 4.52; N, 11.08. Found: C, 75.61; H, 4.87; N, 10.78.
2-Phenyl-N-(5,6,7,8-tetrahydronaphthalen-1-yl)quinazolin-4-
amine (5). White solid; yield, 77%; m.p. 192–193 ꢂC. IR (KBr,
cmꢀ1): 3412 (NH), 3068 (CH arom.), 2922, 2782 (CH aliph.), 7-(2-Phenylquinazolin-4-ylamino)-4-(trifluoromethyl)-2H-chro-
1
1609 (C¼N). H-NMR (DMSO-d6): ppm 1.6–2.8 [m, 8H, 4CH2 men-2-one (11). Yellow solid; yield, 72%; m.p. 255–257 ꢂC. IR
Cyclo], 7.0–9.0 [m, 12H, Ar–H], 11.4 [s, 1H, NH, exchangeable (KBr, cmꢀ1): 3355 (NH), 3058 (CH arom.), 1697 (C¼O), 1617

DOI: 10.3109/14756366.2016.1158714
2-Phenylquinazolin-4-amine derivatives
3
F
O
F
F
N
HN
N
HN
N
O
O
HN
N
O
O
N
N
N
12
10
O
11
F
N
F
F
H2N
O
O
H2N
O
O
NH2
N
HN
N
NH2
N
HN
Cl
H2N
N
N
N
N
N
2
9
1
O
O
O
O
HN
NH2
N
H2N
HN
N
N
N
N
N
3
8
O
HN
O
N
NH2
HN
N
O
N
H2N
O
N
N
N
4
7
NH2
H2N
HN
HN
N
N
N
N
5
6
Scheme 1. Synthesis of quinazoline derivatives 2–12.

4
M. M. Ghorab et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
1
(C¼N). H-NMR (DMSO-d6): ppm 6.2 [s, 1H, CH Chromene], concentrations of inducers. All compounds were dissolved in
7.2–8.6 [m, 12H, Ar–H], 10.1 [s, 1H, NH exchangeable with DMSO and diluted in the cell culture medium at a ratio of 1:1000
D2O]. 13C-NMR (DMSO-d6): ppm 106.3, 108.1, 112.6, 112.9, prior to screening experiments. The final solvent concentration in
114.5, 121.4, 123.5, 124.7, 125.9 (2), 126.5, 127.0, 128.3 (2), the medium was 0.1% (v/v) in all wells. At the end of the
129.0, 130.9, 133.1, 143.5, 149.2, 154.0, 158.1, 160.6, 160.7, treatment period, cell lysates were prepared in digitonin and the
162.6. MS m/z (%): 433 (M+) (1.98), 355 (100). Anal. Calcd. for specific activity of NQO1 was determined using menadione as a
C24H14F3N3O2 (433): C, 66.51; H, 3.26; N, 9.70. Found: C, 66.21; substrate as previously described22. The Concentration which
H, 3.54; N, 9.98.
Doubles the specific activity of NQO1 (CD value) was used as a
measure of inducer potency. Mean values for the eight replicate
wells are shown for each data point. The standard deviation for
each data point was within 5% of the value.
N-(4-morpholinophenyl)-2-phenylquinazolin-4-amine
(12).
White solid; yield, 86%; m.p. 228–229 ꢂC. IR (KBr, cmꢀ1):
3334 (NH), 3059 (CH arom.), 2977, 2891, 2860 (CH aliph.), 1617
(C¼N). 1H-NMR (DMSO-d6): ppm 2.8–3.7 [m, 8H, 4CH2 Cyclo],
7.0–8.5 [m, 13H, Ar–H], 9.9 [s, 1H, NH exchangeable with D2O].
13C-NMR (DMSO-d6): ppm 49.2 (2), 66.6 (2), 114.5 (2), 115.4,
123.4 (2), 126.1, 128.3 (2), 128.5 (2), 128.8 (2), 130.6, 131.6,
133.4 (2), 138.9, 148.0, 159.6, 162.7. MS m/z (%): 382 (M+)
(12.23), 295 (100). Anal. Calcd. for C24H22N4O (382): C, 75.37;
H, 5.80; N, 14.65. Found: C, 75.04; H, 5.44; N, 14.31.
Molecular modeling study
The 3D molecular models of the newly synthesized phenylqui-
nazolinamine derivatives were built according to the default
parameters, with the MOE software suite version 10.2009.
Geometry optimization as well as a systematic conformational
˚
search was carried out to an RMS gradient of 0.01 A employing
the ConfSearch module implemented in MOE. All computations
were performed with the Merck Force Field (MMFF94s). To
evaluate our compounds’ ability to access and block the Nrf2-
binding site of Keap1, a molecular docking study was performed
using the crystallographic structure of the Kelch domain of
Keap1 obtained from the Protein Data Bank (PDB ID: 4IQK).
The protein target was prepared for docking by addition of the
missing hydrogens and calculating the partial charges. Internal
validation was performed to the native ligand followed by
docking of the compounds where the target protein was kept
rigid, while ligands were allowed to rotate to accommodate
freely inside the protein cavity. Following multiple separate
docking simulations using default parameters, the best conform-
ations were chosen based on the combination of S score data, E
conformation and appropriate fitting with the relevant amino
acids in the binding pocket.
Biological evaluation
Hepa1c1c7 murine hepatoma cells were maintained in a
humidified atmosphere at 37 ꢂC, 5% CO2 in a-MEM supple-
mented with 10% (v/v) fetal bovine serum. Cells (104 per well)
were grown for 24 h in 96-well plates. For evaluation of the
potential NQO1 inducer activity of the new 2-phenylquiazolin-4-
amines 2–12, the medium was replaced with inducer-containing
fresh medium, and the cells were further grown for 48 h. There
were eight replicates of each treatment of eight different
Results and discussion
Chemistry
The aim of this work was to design and synthesize a novel series
of quinazolines bearing biologically active heterocyclic moieties
to evaluate and explore their NQO1 inducer activity. Therefore,
4-aminoquinazoline derivatives 2–12 were synthesized starting
from 4-chloro-2-phenylquinazoline (1) as depicted in Scheme 1.
Compounds 2–12 were obtained in good yield via reaction of 1
Figure 1. Concentration dependance of NQO1 inducer activity of
quinazoline derivatives.
Table 1. NQO1 inducer activity and CD values of the test compounds.
Compound#
Concentration (mM)
2
3
4
5
6
7
8
9
10
11
12
0.09765
0.1953
0.3906
0.781
1.563
3.125
6.25
12.5
25
50
100
NR*
NR
NR
NR
NR
NR
2.31
2.42
2.44
2.41
2.2
1.88
1.7
1.5
0.5
NR
NR
NR
NR
NR
NR
NR
NR
1.34
1.49
1.81
2.43
3.33
4.3
5.14
5.5
NR
NR
NR
NR
NR
1.93
2.36
2.39
2.2
2.12
1.76
NR
NR
NR
NR
1.34
1.5
NR
NR
NR
1.50
1.77
2.09
2.21
2.37
2.36
NR
NR
3
NR
NR
NR
1.83
2.03
2.08
2.04
2.02
NR
NR
NR
NR
1.64
1.66
1.45
1.53
1.47
NR
1.27
1.41
1.53
1.66
1.71
1.78
1.77
1.56
NR
1.16
1.02
0.93
0.88
0.87
0.81
0.83
0.8
1.19
1.33
1.44
1.5
1.55
1.71
1.82
1.87
NR
1.82
2.24
2.98
3.71
4.34
4.69
4.69
3.65
1
1.81
2.3
2.92
3.57
4.61
4.82
4
NR
NR
1.6
NR
NR
NR
NR
0.4
NR
0.9
CD**
NR
NR
*NR ¼ not recorded.
**CD data presented are the averages of three independent experiments, each with eight replicate wells of cells and SD (standard deviation) for each
data point was within 5% of the value.

DOI: 10.3109/14756366.2016.1158714
2-Phenylquinazolin-4-amine derivatives
5
Figure 2. Interactions of the native ligand
with the Kelch domain of Keap1 (PDB ID:
41QK).
Figure 3. Interactions of compound 11 with
the Kelch domain of Keap1 (PDB ID: 41QK).
with the appropriate amine in dry dimethyl formamide Biological evaluation
(Scheme 1). All the synthesized compounds were established on
basis of their elemental analyses, IR, 1H-NMR, 13C-NMR and
mass spectral data. IR spectra of compounds 2–12 revealed the
presence of the characteristic bands for the NH group ranging
from 3441 to 3238 cmꢀ1, the aromatic CH around 3100–
NQO1 inducer activity assessment represented in Figure 1 and
Table 1 revealed that the dihydroindenyl derivative 2, the dioxo
bioisostere N-benzo[d][1,3]dioxol-5-yl derivative 3, its methylene
spacer-containing derivative 4, the tetrahydronaphthyl derivative
5 and the morpholinophenyl compound 12 were all essentially
inactive. In contrast, the naphth-1-yl derivative 6 showed a
moderate dose-dependent inducer activity (CD ¼ 1 mM). The 2-
1
3023 cmꢀ1, the C¼N group from 1635 to 1609 cmꢀ1. H-NMR
spectra of compounds 2–12 exhibited signals at ꢀ 12.5–19.1 ppm
attributed to the NH group. Further details of the spectroscopic
analysis findings of all the synthesized compounds are thoroughly methyl-quinolin-4-yl analog 9 displayed a four times weaker
described in the chemistry experimental section.
inducer potency (CD ¼ 4 mM), indicating that these structural

6
M. M. Ghorab et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
small molecule compounds were reported to bind to the Keap1
Kelch domain and antagonize its activity23–25. For assessing the
newly synthesized compounds’ ability to access and block the
Kelch domain of Keap1, a molecular docking study was
performed using the Kelch domain of Keap1 crystal coordinates
obtained from the PDB ID: 4IQK. The main interactions observed
between the validated native ligand and the protein target were
found to be arene–cation interaction with Arg415, arene–arene
interaction with Tyr525 and three hydrogen bonds with Ser602,
Ser508 and Ser555 with S¼ ꢀ13.306 kcal/mol and rmsd
modifications were not in favor of activity as compared to the
naphthyl bioisostere 6. The bioisosteres 7 and 8 demonstrated a
large difference in activity. Whereas, the quinolin-2-yl derivative
7 had the highest potency among the compounds in this study
(CD ¼ 0.4 mM), the activity of the three positional isomer 8 was
lower (CD ¼ 0.9 mM); furthermore, the absence of dose-depend-
ence for compound 8 is indicative of low specificity and potential
off-target effects. Finally, with regards to the bioisosteric pair 10
and 11 (CD ¼ 3 and 1.6 mM, respectively) it was evident that
replacement of the methyl group with a triflouromethyl almost
doubled the potency and improved the dose responsiveness.
Overall, compound 7 was the most potent inducer (CD ¼ 0.4 mM),
and importantly, had the most favorable inducer properties within
this series.
˚
0.6635 kcal/mol/A (Figure 2). Upon docking of the synthesized
compounds, they all showed an arene–cation binding interaction
with Arg415 via their aromatic rings. Compound 11
(S ¼ ꢀ10.913 kcal/mol) has adopted a conformation allowing
the presence of three arene–cation interactions with Arg415 and
multiple rings of the ligand (Figures 3 and 4).
Molecular modeling study
Finally, compound 7 that showed the most potent NQO1
inducer activity (CD ¼ 0.4 mM) was the only compound that
showed three arene–cation interactions with Arg415 and two
arene–arene interactions with Tyr525 indicating a possible
correlation between those multiple interactions and the noted
high potency (S¼ ꢀ10.7652 kcal/mol) (Figures 5 and 6).
One limitation of our study is the lack of a well-established
biological assay for assessing the ability of small molecules to
disrupt the Keap1–Nrf2 protein–protein interactions. Therefore,
although the molecular docking study suggests that these
compounds can potentially bind to Keap1 and disrupt its
interaction with Nrf2, further experimental evidence is required
to confirm this possibility. Regardless of the precise mode of
action in activating Nrf2, the inducer potency of compound 7 in
upregulating NQO1 strongly suggests that this compound could
be used as a lead for further optimization of the quinazoline
scaffold.
Keap1 is known to bind to Nrf2 promoting its degradation,
resulting in low levels of cytoprotective gene products. Various
Conclusion
In conclusion, this study presents the synthesis of N-substituted 2-
phenylquinazolin-4-amine based derivatives 2–12 as cytoprotec-
tive NQO1 inducers. The most potent inducer in this study was
the analogue 7 which caused a dose-dependent induction of
NQO1 with a CD value of 0.4 mM. In silico evaluation of these
derivatives as Keap1–Nrf2 protein–protein interaction inhibitors
Figure 4. Overlap of compound 11 (maroon) over native ligand (yellow)
(PDB ID: 41QK).
Figure 5. Interaction of compound 7 with the
Kelch domain of Keap1 (PDB ID: 41QK).

DOI: 10.3109/14756366.2016.1158714
2-Phenylquinazolin-4-amine derivatives
7
8. Kobayashi M, Li L, Iwamoto N, et al. The antioxidant defense
system Keap1–Nrf2 comprises a multiple sensing mechanism for
responding to a wide range of chemical compounds. Mol Cell Biol
2009;29:493–502.
9. McMahon M, Lamont DJ, Beattie KA, Hayes JD. Keap1 perceives
stress via three sensors for the endogenous signaling molecules nitric
oxide, zinc, and alkenals. Proc Natl Acad Sci USA 2010;107:
18838–43.
10. Dinkova-Kostova AT, Holtzclaw WD, Cole RN, et al. Direct
evidence that sulfhydryl groups of Keap1 are the sensors regulating
induction of phase 2 enzymes that protect against carcinogens and
oxidants. Proc Natl Acad Sci USA 2002;99:11908–13.
11. Li J, Ichikawa T, Janicki JS, Cui T. Targeting the Nrf2 pathway
against cardiovascular disease. Expert Opin Ther Targets 2009;13:
785–94.
12. Zhao J, Redell JB, Moore AN, Dash PK. A novel strategy to activate
cytoprotective genes in the injured brain. Biochem Biophys Res
Commun 2011;407:501–6.
13. Steel R, Cowan J, Payerne E, et al. Anti-inflammatory effect of a
cell-penetrating peptide targeting the Nrf2/Keap1 interaction. ACS
Med Chem Lett 2012;3:407–10.
14. Williamson TP, Amirahmadi S, Joshi G, et al. Discovery of potent,
novel Nrf2 inducers via quantum modeling, virtual screening, and in
vitro experimental validation. Chem Biol Drug Des 2012;80:
810–20.
15. Hu L, Magesh S, Chen L, et al. Discovery of a small-molecule
inhibitor and cellular probe of Keap1–Nrf2 protein–protein inter-
action. Bioorg Med Chem Lett 2013;23:3039–43.
16. Zhuang C, Miao Z, Sheng C, Zhang W. Updated research and
applications of small molecule inhibitors of Keap1–Nrf2 protein–
protein interaction: a review. Curr Med Chem 2014;21:1861–70.
17. Nesterova NA, Kovalenko SI, Belenichev IF, et al. Formation of
combinational library of quinazoline-4-yl-hydrazones with antioxi-
dant activity. Ukraine Med Khim 2004;6:14–21.
18. Nesterova NA, Kovalenko SI, Karpenkos OV, Belenichev IF.
Synthesis and antioxidant activity of 4-arylidenehydrazinoquinazo-
lines. Ukr Farmatsevtichnii Zhurnal (Kiev) 2004;22:5–10.
19. Al-Omar MA, Al-Rashood ST, El-Subbagh HI, Abdel Hamide SG.
Interaction of 2-thio-4-oxo-quinazoline derivatives with guinea pig
liver molybdenum hydroxylases, xanthine oxidase and aldehyde
oxidase. J Biol Sci 2005;5:370–8.
Figure 6. Overlap of compound 7 (magenta) over native ligand (cyan)
(PDB ID: 41QK).
was performed using molecular docking techniques employing
MOE software version 10.2009. Derivatives were found to bind to
key amino acids in the binding site with a network or arene–arene
and arene–cationic interactions. These molecules represent
promising quinazoline scaffold-based leads for further optimiza-
tion as cytoprotective agents and for future investigations into
other plausible modes of action.
Declaration of interest
The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at King Saud University for
funding of this research through the Research Group Project no.
RGP-VPP-302. Maureen Higgins and Albena T. Dinkova-Kostova
are also grateful to Cancer Research UK (C20953/A10270 and
C20953/A18644) for their financial support.
20. Ghorab MM, Higgins M, Alsaid MS, et al. Synthesis, molecular
modeling and NAD(P)H:quinone oxidoreductase 1 inducer activity
of novel cyanoenone and enone benzenesulfonamides. J Enzyme
Inhib Med Chem 2014;29:840–5.
21. Ghorab MM, Higgins M, Dinkova-Kostova AT, et al. Novel
thioureido derivatives carrying thione and sulfonamide moieties
induce the cytoprotective enzyme NAD(P)H:quinone oxidoreduc-
tase 1. Asian J Chem 2014;26:8501–4.
22. Prochaska HJ, Santamaria AB. Direct measurement of
NAD(P)H:quinone reductase from cells cultured in microtiter
wells: a screening assay for anticarcinogenic enzyme inducers.
Anal Biochem 1988;169:328–36.
23. Marcotte D, Zeng W, Hus JC, et al. Small molecules inhibit the
interaction of Nrf2 and the Keap1 Kelch domain through a
noncovalent mechanism. Bioorg Med Chem 2013;21:4011–19.
24. Magesh S, Chen Y, Hu L. Small molecule modulators of Keap1–
Nrf2–ARE pathway as potential preventive and therapeutic agents.
Med Res Rev 2012;32:687–726.
25. Bertrand HC, Schaap M, Baird L, et al. Design, synthesis, and
evaluation of triazole derivatives that induce Nrf2 dependent gene
products and inhibit the Keap1–Nrf2 protein–protein interaction.
J Med Chem 2015;58:7186–94.
References
1. Waris G, Ahsan H. Reactive oxygen species: role in the development
of cancer and various chronic conditions. J Carcinog 2006;5:14.
2. Scandalios JG. The rise of ROS. Trends Biochem Sci 2002;27:
483–6.
3. Klein JA, Ackerman SL. Oxidative stress, cell cycle, and
neurodegeneration. J Clin Invest 2003;111:785–93.
4. Ahsan H, Ali A, Ali R. Oxygen free radicals and systemic
autoimmunity. Clin Exp Immunol 2003;131:398–404.
5. Knight JA. Diseases related to oxygen-derived free radicals. Ann
Clin Lab Sci 1995;25:111–21.
6. Waris G, Alam K. Attenuated antigenicity of ribonucleoproteins
modified by reactive oxygen species. Biochem Mol Biol Int 1998;
45:33–45.
7. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to
environmental stresses via the Keap1–Nrf2–ARE pathway. Annu
Rev Pharmacol Toxicol 2007;47:89–116.