Structure-activity relationship investigation of methoxy substitution on anticancer pyrimido[4,5-c]quinolin-1(2H)-ones

Article abstract of DOI:10.1007/s00044-012-0428-9

Pyrimido[4,5-c]quinolin-1(2H)-one derivatives were shown to exert interesting biological activities including anticancer, antimicrobial, and cardiovascular. Substitution with methoxy groups played a crucial role in the anticancer activity of known antican

Full text of DOI:10.1007/s00044-012-0428-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0428-9
ORIGINAL RESEARCH
Structure–activity relationship investigation of methoxy
substitution on anticancer pyrimido[4,5-c]quinolin-1(2H)-ones
•
Kamel Metwally Ashraf Khalil Asmaa Sallam
•
•
•
Harris Pratsinis Dimitris Kletsas Khalid El Sayed
•
Received: 26 March 2012 / Accepted: 15 December 2012
Ó Springer Science+Business Media New York 2013
Abstract Pyrimido[4,5-c]quinolin-1(2H)-one derivatives
were shown to exert interesting biological activities
including anticancer, antimicrobial, and cardiovascular.
Substitution with methoxy groups played a crucial role in
the anticancer activity of known anticancer agents. This
study explores the contribution of diverse-positioned
methoxy substituents to the antimigratory and cytotoxic
activities of this class. Synthesized analogues were tested
in the MTT, cell cycle, and wound-healing assays. Previous
studies on this class reported weak to medium antimitotic
activity. Therefore, all compounds were subjected to
tubulin polymerization assay and in silico molecular
docking study at the colchicine binding site of tubulin. The
2-methoxy and 2,4-dimethoxy substitutions at the 2-aryl-
pyrimido functionality enhanced the antimigratory activity
in the 9-methoxy-substituted series like 6 and 9. The 3,4,5-
trimethoxy substitutions at the 2-arylpyrimido group also
significantly improved the antimigratory activity in the
presence or absence of the 9-methoxy substitution as
represented by 13 and 22, respectively. Docking experi-
ments showed two distinct orientations at the colchicine
binding site of tubulin. The first, achieved by 7 and 16–20,
coincides with that of colchicine and positions the meth-
oxy-substituted 2-aryl ring deep in the highly hydrophobic
narrow end of the funnel-shaped binding pocket. On the
other hand, 5, 6, 9–14 and 21, were oriented towards the
wider opening of the binding pocket. Pyrimido[4,5-
c]quinolin-1(2H)-ones are promising antimigratory hits
with potential for future use to control metastatic breast
cancers.
Keywords Anticancer Á Cytotoxicity Á
Methoxy substitution Á Pyrimido[4,5-c]quinolin-1(2H)-ones Á
Migration Á Tubulin Á Wound-healing
Introduction
The in vitro cytotoxic activity of pyrimido[4,5-c]quinolin-
1(2H)-one derivatives was previously reported by our group
(Metwally et al., 2007a, b, 2010). This class also exerted
several other interesting activities. Related phenylquinazoli-
none derivatives were patented for use as cardiovascular
agents (Hansen, 2010). Some of these derivatives demon-
strated activity by causing a C20 % increase in ApoA-I
mRNA at the lM dose level (Hansen, 2010). Related pyrim-
ido fused acridine derivatives also showed moderate anti-
bacterial and antifungal activities (Patel et al., 2006).
K. Metwally (&)
Department of Medicinal Chemistry, Faculty of Pharmacy,
Zagazig University, Sharkeya, Egypt
e-mail: kametwally@hotmail.com
A. Khalil
Pharmaceutical Sciences Section, College of Pharmacy,
Qatar University, Doha, Qatar
A. Sallam Á K. El Sayed
Department of Basic Pharmaceutical Sciences of College
of Pharmacy, University of Louisiana at Monroe,
Monroe, LA, USA
Structure–activity relationship study conducted on the 2,5-
diaryl series indicated that methoxy derivatives displayed the
highest cytotoxic activity (Metwally et al., 2010). The cyto-
toxicity of these compounds was attributed in-part to their weak
to medium antimitotic activity (Metwally et al., 2007a, b,
2010). Microtubule dynamics are implicated in cell adhesion,
H. Pratsinis Á D. Kletsas
Laboratory of Cell Proliferation and Ageing, Institute
of Biology, National Centre of Scientific
Research ‘‘Demokritos’’, Athens, Greece
123

Med Chem Res
migration, and morphology (Mane and Klobukowski, 2008;
Zhu et al., 1997; Rowinskyet al., 1992;Hayotet al., 2002). The
use of subtoxic doses of antimitotic agents was found to induce
significant antimigratory effects (Hayot et al., 2002). There-
fore, tubulin inhibitors with optimal potency are hypothesized
as potential antimetastatic entities with acceptable therapeutic
index and low toxicity profiles. This hypothesis, along with the
fact that most known antimitotic agents are rich in methoxy
groups (Chart 1), which markedly influence their biological
activities (Fitzgerald, 1976; Hu et al., 2006; Cushman et al.,
1991, 1992), have initiated the interest to explore the contri-
bution of diverse-positioned methoxy substituents to the
antimigratory and cytotoxic activities of pyrimido[4,5-c]quin-
olin-1(2H)-ones along with accurate in vitro and in silico
assessment of their antimitotic activity compared to the known
antimitotic colchicine.
(Metwally et al., 2007a, b). Lactonization of acids was
achieved by heating with acetic anhydride to give the
intermediate 1H-[1,3]oxazino[4,5-c]quinolin-1-ones (3 and
4) in 79 and 77 % yields, respectively. Treatment of lac-
tones with the appropriate anilines in the presence of
sodium acetate under reflux conditions afforded the target
compounds (5–22) in 34–63 % isolated yields.
4
R2
6
2
O
N
R1
N
N
Cl
Methods and materials
Compound R1
R2
Compound R1 R2
Chemistry
5
OCH3
H
14
H
H
H
H
H
H
H
H
H
H
6
OCH3 2-Methoxy
OCH3 3-Methoxy
OCH3 4-Methoxy
15
16
17
2-Methoxy
3-Methoxy
4-Methoxy
2,4-Dimethoxy
2,5-Dimethoxy
3,4-Dimethoxy
3,5-Dimethoxy
Melting points were determined on a Barnstead Electrother-
mal 9100 melting point apparatus, and are uncorrected. IR
spectra were recorded on a Shimadzu FTIR-8400S spectro-
photometer as KBr pellets. 1H NMR spectra were recorded in
DMSO-d6 on a Varian-Mercury 300BB at 300 MHz or on a
JEOL Eclipse NMR spectrometer at 400 MHz, while 13C
NMR spectra were recorded on a JEOL Eclipse NMR spec-
trometer at 100 MHz. Chemical shifts were reported as parts
per million (ppm) downfield from tetramethylsilane (TMS),
and coupling constants (J) are given in Hertz (Hz). Elemental
analyses (C, H, N) were performed at the Microanalytical
Unit, Cairo University, Cairo, Egypt. All compounds were
[95 %purityasroutinelyassessedby1H NMR and thin-layer
chromatography (TLC) on aluminum-backed silica gel plates
(E-Merck). All solvents were dried by standard methods.
Compounds 1–3 (Metwally et al., 2007a), 14, 15, and 17 were
previously reported (Metwally et al., 2007a).
7
8
9
OCH3 2,4-Dimethoxy 18
OCH3 2,5-Dimethoxy 19
OCH3 3,4-Dimethoxy 20
OCH3 3,5-Dimethoxy 21
10
11
12
13
OCH3 3,4,5-
Trimethoxy
22
3,4,5-
Trimethoxy
Preparation of 9-methoxy-3-methyl-5-(4-chlorophenyl)-
1H-[1,3]oxazino-[4,5-c]quinolin-1-one (4)
A suspension of 3-amino-2-(4-chlorophenyl)-6-methoxy-4-
quinolinecarboxylic acid (2; 10 mmol) in acetic anhydride
(10 mL) was heated at reflux for 5 h. The reaction mixture
was allowed to cool to room temperature and the precipi-
tated solid was filtered, washed with EtOH and dried.
Yield 77 %, mp 240–242 °C; 1H-NMR (DMSO-d6) d: 2.47
The synthetic route of pyrimido[4,5-c]quinolines (5–22)
is illustrated in Scheme 1. The starting 3-aminoquinoline-
4-carboxylic acids (1 and 2) were obtained following a
modified Pfitzinger procedure as previously reported
Chart 1 Structure of anti-
tubulin agents rich in methoxy
groups
OH
O
H3CO
H3CO
H3CO
O
O
N
O
H
H3CO
H3CO
O
H3CO
OH
OCH3
O
H3CO
OCH3
OCH3
Podophyllotoxin
OCH3
Colchicine
Combretastatin A-4
123

Med Chem Res
R2
O
O
COOH
NH2
R1
R1
Ac2O
N
O
N
Substituted aniline
AcONa
Reflux 5h
R1
N
N
N
AcOH
R1
H
R1
3 H
4 OCH3
Reflux 24h
Cl
1
2
Cl
N
OCH3
5-22
Cl
Scheme 1 Synthesis of 3-methyl-2,5-diarylpyrimido[4,5-c]quinolin-1(2H)-ones
(s, 3H, CH3), 3.96 (s, 3H, OCH3), 7.52–7.60 (m, 3H, Ar–
H), 8.01–8.09 (m, 3H, Ar–H), 8.58–8.59 (d, 1H,
J = 2.8 Hz, Ar–H). The compound was used without fur-
ther characterization in the next reactions.
9.05–9.06 (d, J = 2.9 Hz, 1H, Ar–H). 13C NMR (DMSO-
d6) d: 23.98, 55.96, 56.40, 105.41, 113.21, 118.49, 121.11,
121.71, 125.21, 126.05, 128.04, 129.73, 131.58, 131.70,
132.97, 133.96, 137.44, 140.66, 141.58, 153.33, 157.46,
160.07, 161.63, 162.76. Anal. calcd for C26H20ClN3O3: C,
68.20; H, 4.40; N, 9.18. Found: C, 68.40; H, 4.08; N, 9.10.
General procedure for 3-methyl-2,5-diary-
lpyrimido[4,5-c]quinolin-1(2H)-ones (5–22)
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-(3-methoxyphenyl)-
pyrimido[4,5-c]-quinolin-1(2H)-one (7) Yield 51 %, mp
A mixture of the appropriate 1H-[1,3]oxazino[4,5-c]quin-
olin-1-one (10 mmol), the appropriate substituted aniline
(11 mmol) and anhydrous sodium acetate (1.64 g,
20 mmol) in glacial acetic acid (20 mL) was heated under
reflux for 24 h. The reaction mixture was cooled, diluted
with water (10 mL) and the precipitated solid was filtered,
washed with saturated aqueous sodium bicarbonate then
water and dried. The crude product was subjected to silica
gel column chromatography (using petroleum ether/ethyl
acetate (9:1–7:3) as an eluent followed by recrystallization
from the appropriate solvent to afford the target compound.
1
216–218 °C (DMF/EtOH); H-NMR (DMSO-d6) d: 2.25
(s, 3H, CH3), 3.82 (s, 3H, OCH3), 3.91 (s, 3H, OCH3),
7.05–7.20 (m, 3H, Ar–H), 7.47–7.51 (dd, J = 2.9, 2.9 Hz,
1H, Ar–H), 7.53–7.56 (d, J = 8.0 Hz, 1H, Ar–H),
7.58–7.62 (m, 2H, Ar–H), 8.09–8.12 (d, J = 9.1 Hz, 1H,
Ar–H), 8.14–8.20 (m, 2H, Ar–H), 9.06–9.07 (d,
J = 2.9 Hz, 1H, Ar–H). 13C NMR (DMSO-d6) d: 24.65,
55.92, 55.97, 105.42, 114.34, 115.47, 118.71, 120.59,
120.97, 125.26, 128.05, 130.97, 131.63, 132.93, 133.96,
137.48, 139.16, 140.56, 141.51, 153.25, 156.94, 159.96,
160.80, 162.05. Anal. calcd for C26H20ClN3O3: C, 68.20;
H, 4.40; N, 9.18. Found: C, 68.22; H, 4.18; N, 9.16.
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-phenylpyrimido[4,5-
c]quinolin-1(2H)-one (5) Yield 62 %, mp 252–254 °C
(DMF/EtOH); H-NMR (DMSO-d6): 2.17 (s, 3H, CH3),
1
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-(4-methoxyphenyl)-
pyrimido[4,5-c]-quinolin-1(2H)-one (8) Yield 63 %, mp
3.88 (s, 3H, OCH3), 7.42–7.62 (m, 8H, Ar–H), 8.07–8.10
(d, J = 9.1 Hz, 1H, Ar–H), 8.13–8.15 (m, 2H, Ar–H),
9.05–9.06 (m, 1H, Ar–H). 13C NMR (DMSO-d6) d: 24.99,
55.97, 105.33, 118.75, 121.13, 125.34, 128.14, 128.67,
129.79, 130.32, 131.74, 133.01, 134.00, 137.49, 138.14,
140.60, 141.62, 153.43, 157.08, 159.99, 162.27. Anal.
calcd for C25H18ClN3O2: C, 70.18; H, 4.24; N, 9.82.
Found: C, 69.87; H, 4.38; N, 9.71.
1
209–211 °C (DMF/EtOH); H-NMR (DMSO-d6) d: 2.22
(s, 3H, CH3), 3.86 (s, 3H, OCH3), 3.91 (s, 3H, OCH3),
7.14–7.18 (m, 2H, Ar–H), 7.41–7.45 (m, 2H, Ar–H),
7.46–7.50 (dd, J = 2.9, 2.9 Hz, 1H, Ar–H), 7.58–7.62 (m,
2H, Ar–H), 8.09–8.12 (d, J = 9.1 Hz, 1H, Ar–H),
8.15–8.19 (m, 2H, Ar–H), 9.07–9.08 (d, J = 2.9 Hz, 1H,
Ar–H). 13C NMR (DMSO-d6) d: 25.02, 55.95, 56.01,
105.35, 115.41, 118.69, 121.04, 125.35, 128.12, 129.74,
130.57, 131.69, 133.01, 133.99, 137.50, 140.56, 141.55,
153.38, 157.60, 159.96, 160.04, 162.44. Anal. calcd for
C26H20ClN3O3: C, 68.20; H, 4.40; N, 9.18. Found: C,
68.06; H, 4.74; N, 9.07.
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-(2-methoxyphenyl)-
pyrimido[4,5-c]-quinolin-1(2H)-one (6) Yield 46 %, mp
1
237–240 °C (DMF); H-NMR (DMSO-d6) d: 2.19 (s, 3H,
CH3), 3.80 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 7.16–7.21
(m, 1H, Ar–H), 7.31–7.34 (d, J = 8.35 Hz, 1H, Ar–H),
7.47–7.51 (m, 2H, Ar–H), 7.54–7.55 (d, J = 1.6 Hz, 1H,
Ar–H), 7.58–7.61 (m, 2H, Ar–H), 8.09–8.13 (d,
J = 9.1 Hz, 1H, Ar–H), 8.13–8.18 (m, 2H, Ar–H),
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-(2,4-dimethoxy-
phenyl)-pyrimido [4,5-c]quinolin-1(2H)-one (9) Yield
37 %, mp 243–245 °C (DMF); 1H-NMR (DMSO-d6) d:
123

Med Chem Res
2.20 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 3.88 (s, 3H, OCH3),
3.92 (s, 3H, OCH3), 6.72–6.76 (dd, 1H, J = 2.5, 2.5 Hz,
Ar–H), 6.84–6.85 (d, J = 2.5 Hz, 1H, Ar–H), 7.33–7.39 (d,
J = 8.6 Hz, 1H, Ar–H), 7.45–7.51 (dd, J = 2.9, 2.9 Hz, 1H,
Ar–H), 7.57–7.61 (m, 2H, Ar–H), 8.09–8.12 (d, J = 9.1 Hz,
1H, Ar–H), 8.13–8.18 (m, 2H, Ar–H), 9.06–9.07 (d,
J = 2.8 Hz, 1H, Ar–H). Anal. calcd for C27H22ClN3O4: C,
66.46; H, 4.54; N, 8.61. Found: C, 66.80; H, 4.35; N, 8.50.
OCH3), 3.92 (s, 3H, OCH3), 6.93 (s, 2H, Ar–H), 7.47–7.51
(dd, J = 2.9, 2.9 Hz, 1H, Ar–H), 7.58–7.62 (m, 2H, Ar–H),
8.09–8.12 (d, J = 9.1 Hz, 1H, Ar–H), 8.14–8.18 (m, 2H,
Ar–H), 9.08–9.09 (d, J = 2.9 Hz, 1H, Ar–H). 13C NMR
(DMSO-d6) d: 24.68, 56.01, 56.67, 60.64, 105.29, 106.19,
118.73, 121.21, 125.40, 128.17, 128.22, 131.75, 132.96,
132.99, 133.78, 134.00, 137.51, 140.58, 141.56, 154.11,
157.50, 159.97, 162.29. Anal. calcd for C28H24ClN3O5:
C, 64.93; H, 4.67; N, 8.11. Found: C, 65.02; H, 4.38; N,
8.22.
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-(2,5-dimethoxy-
phenyl)-pyrimido[4,5-c] quinolin-1(2H)-one (10) Yield
41 %, mp 217–219 °C (DMF/EtOH); 1H-NMR (DMSO-d6)
d: 2.22 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 3.77 (s, 3H,
OCH3), 3.91 (s, 3H, OCH3), 7.12–7.13 (d, 1H, J = 3.1 Hz,
Ar–H), 7.14–7.15 (m, 1H, Ar–H), 7.23–7.26 (d, J = 8.8 Hz,
1H, Ar–H), 7.47–7.51 (dd, J = 2.9, 2.9 Hz, 1H, Ar–H),
7.57–7.61 (m, 2H, Ar–H), 8.09–8.12 (d, J = 9.1 Hz, 1H,
Ar–H), 8.14–8.17 (m, 2H, Ar–H), 9.05–9.06 (d, J = 2.8 Hz,
1H, Ar–H). 13C NMR (DMSO-d6) d: 24.01, 55.99, 56.23,
56.66, 105.30, 113.93, 115.51, 116.32, 118.52, 121.28,
125.24, 126.40, 128.13, 131.77, 133.02, 134.01, 137.43,
140.67, 141.58, 148.51, 153.42, 154.04, 157.50, 160.08,
161.65. Anal. calcd for C27H22ClN3O4: C, 66.46; H, 4.54; N,
8.61. Found: C, 66.33; H, 4.73; N, 8.59.
3-Methyl-5-(4-chlorophenyl)-2-(3-methoxyphenyl)-pyrim-
ido[4,5-c]quinolin-1(2H)-one (16) Yield 59 %, mp
1
229–231 °C (DMF/EtOH); H-NMR (DMSO-d6) d: 2.25
(s, 3H, CH3), 3.82 (s, 3H, OCH3), 7.08–7.18 (m, 3H, Ar–
H), 7.51–7.64 (m, 3H, Ar–H), 7.76–7.89 (m, 2H, Ar–H),
8.15–8.22 (m, 3H, Ar–H), 9.56–9.59 (m, 1H, Ar–H). 13C
NMR (DMSO-d6) d: 24.79, 55.98, 114.31, 115.50, 119.63,
120.58, 123.82, 126.17, 128.22, 129.52, 129.80, 130.28,
131.08, 133.13, 134.41, 137.35, 139.24, 141.43, 144.70,
156.58, 157.25, 160.79, 161.89. Anal. calcd for
C25H18ClN3O2: C, 70.18; H, 4.24; N, 9.82. Found: C,
70.33; H, 4.07; N, 9.96.
3-Methyl-5-(4-chlorophenyl)-2-(2,4-dimethoxyphenyl)-pyrimi-
do[4,5-c]quinolin-1(2H)-one (18) Yield 34 %, mp
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-(3,4-dimethoxy-
phenyl)-pyrimido[4,5-c]quinolin-1(2H)-one (11) Yield
52 %, mp 223–227 °C (DMF); 1H-NMR (DMSO-d6) d:
2.26 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
3.91 (s, 3H, OCH3), 7.02–7.06 (dd, 1H, J = 2.3, 2.4 Hz,
Ar–H), 7.14–7.18 (m, 2H, Ar–H), 7.46–7.50 (dd, J = 2.9,
2.9 Hz, 1H, Ar–H), 7.52–7.62 (m, 2H, Ar–H), 8.05–8.08
(d, J = 9.1 Hz, 1H, Ar–H), 8.14–8.19 (m, 2H, Ar–H),
9.08–9.09 (d, J = 2.9 Hz, 1H, Ar–H). Anal. calcd for
C27H22ClN3O4: C, 66.46; H, 4.54; N, 8.61. Found: C,
66.34; H, 4.17; N, 8.32.
1
267–270 °C (DMF); H-NMR (DMSO-d6) d: 2.20 (s, 3H,
CH3), 3.78 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.73–6.76
(dd, 1H, J = 2.5, 2.6 Hz, Ar–H), 6.84–6.85 (d,
J = 2.5 Hz, 1H, Ar–H), 7.36–7.39 (d, J = 8.6 Hz, 1H, Ar–
H), 7.60–7.64 (m, 2H, Ar–H), 7.76–7.89 (m, 2H, Ar–H),
8.15–8.22 (m, 3H, Ar–H), 9.54–9.58 (dd, J = 1.4, 1.1 Hz,
1H, Ar–H). 13C NMR (DMSO-d6) d: 24.18, 56.16, 56.53,
100.05, 106.32, 118.86, 119.31, 123.75, 126.16, 128.18,
129.56, 129.84, 130.18, 130.29, 133.18, 134.39, 137.32,
141.44, 144.76, 155.38, 156.64, 158.29, 161.60, 161.86.
Anal. calcd for C26H20ClN3O3: C, 68.20; H, 4.40; N, 9.18.
Found: C, 67.96; H, 4.72; N, 9.06.
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-(3,5-dimethoxy-
phenyl)-pyrimido[4,5-c]quinolin-1(2H)-one (12) Yield
55 %, mp 221–224 °C (DMF); 1H-NMR (DMSO-d6) d:
2.28 (s, 3H, CH3), 3.80 (s, 6H, 2 OCH3), 3.91 (s, 3H,
OCH3), 6.68–6.70 (m, 1H, Ar–H), 6.75–6.76 (d,
J = 2.0 Hz, 2H, Ar–H), 7.46–7.50 (dd, J = 2.7, 2.8 Hz, 1H,
Ar–H), 7.56–7.61 (m, 2H, Ar–H), 8.07–8.10 (d, J = 9.1 Hz,
1H, Ar–H), 8.14–8.17 (m, 2H, Ar–H), 9.05–9.06 (d,
J = 2.8 Hz, 1H, Ar–H). Anal. calcd for C27H22ClN3O4: C,
66.46; H, 4.54; N, 8.61. Found: C, 66.37; H, 4.59; N, 8.69.
3-Methyl-5-(4-chlorophenyl)-2-(2,5-dimethoxyphenyl)-pyrimi-
do[4,5-c]quinolin-1(2H)-one (19) Yield 38 %, mp
1
225–228 °C (DMF); H-NMR (DMSO-d6) d: 2.22 (s, 3H,
CH3), 3.74 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 7.12–7.13
(d, 1H, J = 3.1 Hz, Ar–H), 7.15–7.17 (m, 1H, Ar–H),
7.24–7.27 (d, J = 9.2 Hz, 1H, Ar–H), 7.59–7.64 (m, 2H,
Ar–H), 7.77–7.89 (m, 2H, Ar–H), 8.15–8.22 (m, 3H, Ar–
H), 9.54–9.57 (dd, J = 1.2, 1.7 Hz, 1H, Ar–H). 13C NMR
(DMSO-d6) d: 24.05, 56.22, 56.72, 114.01, 115.49, 116.32,
119.36, 123.70, 126.16, 126.44, 128.20, 129.60, 129.90,
130.31, 133.17, 134.41, 137.29, 141.44, 144.77, 148.50,
154.04, 156.62, 157.64, 161.32. Anal. calcd for
C26H20ClN3O3: C, 68.20; H, 4.40; N, 9.18. Found: C,
68.09; H, 4.32; N, 9.14.
3-Methyl-9-methoxy-5-(4-chlorophenyl)-2-(3,4,5-trimethoxy-
phenyl)-pyrimido[4,5-c]quinolin-1(2H)-one
(13) Yield
1
57 %, mp 253–256 °C (DMF/EtOH); H-NMR (DMSO-
d6) d: 2.30 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 3.80 (s, 6H, 2
123

Med Chem Res
3-Methyl-5-(4-chlorophenyl)-2-(3,4-dimethoxyphenyl)-py-
rimido[4,5-c]quinolin-1(2H)-one (20) Yield 56 %, mp
streptomycin (100 lg/mL), and 10 % fetal bovine serum
(media and antibiotics were from Biochrom KG, Berlin, Ger-
many, while the serum from Invitrogen Co., Carlsbad, CA,
USA) in an environment of 5 % CO2, 85 % humidity, and
37 °C, and they were subcultured using a trypsin 0.25 %-
EDTA 0.02 % solution. The cytotoxicity assay was performed
by a modification of the MTT method (Metwally et al., 2007a).
Briefly, the cells were plated at a density of approximately
5,000 cells/well in 96-well flat-bottomed microplates for 24 h
after which individual tested compound stocks were prepared
in DMSO and appropriate dilutions were prepared using the
assay media and added. After a 72-h incubation, the medium
was replaced with MTT (Sigma) dissolved at a final concen-
tration of 1 mg/mL in serum-free, phenol-red-free DMEM and
incubated for 4 h. Then, the MTT formazan was solubilized in
2-propanol, and the optical density was measured with a
microplate reader at a wavelength of 550 nm (reference
wavelength 690 nm). Doxorubicin hydrochloride was included
in the experiments as a positive control (Metwally et al.,
2007a). The results represent the mean of three independent
experiments and are expressed as IC50, the concentration that
reduced by 50 % the optical density of treated cells with respect
to untreated controls.
1
245–247 °C (DMF); H-NMR (DMSO-d6) d: 2.26 (s, 3H,
CH3), 3.77 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 7.02–7.06 (dd,
1H, J = 2.3, 2.3 Hz, Ar–H), 7.15 (s, 1H, Ar–H), 7.17–7.18
(d, J = 2.0 Hz, 1H, Ar–H), 7.58–7.64 (m, 2H, Ar–H),
7.76–7.89 (m, 2H, Ar–H), 8.16–8.21 (m, 3H, Ar–H),
9.57–9.60 (dd, J = 1.3, 1.1 Hz, 1H, Ar–H). 13C NMR
(DMSO-d6) d: 24.87, 56.22, 56.25, 112.16, 112.46, 119.59,
120.55, 123.85, 126.18, 128.21, 129.48, 129.74, 130.26,
130.67, 133.12, 134.40, 137.37, 141.38, 144.68, 149.60,
149.95, 156.56, 157.89, 162.11. Anal. calcd for
C26H20ClN3O3: C, 68.20; H, 4.40; N, 9.18. Found: C, 68.30;
H, 4.37; N, 9.20.
3-Methyl-5-(4-chlorophenyl)-2-(3,5-dimethoxyphenyl)-pyrimi-
do[4,5-c]quinolin-1(2H)-one (21) Yield 53 %, mp
1
219-221 °C (DMF); H-NMR (DMSO-d6) d: 2.33 (s, 3H,
CH3), 3.80 (s, 6H, 2 OCH3), 6.69–6.70 (m, 1H, Ar–H),
6.75–6.76 (d, J = 2.0 Hz, 2H, Ar–H), 7.61–7.63 (dd,
J = 2.0, 2.0 Hz, 2H, Ar–H), 7.78–7.85 (m, 2H, Ar–H),
8.16–8.20 (m, 3H, Ar–H), 9.56–9.57 (dd, J = 1.0, 1.0 Hz,
1H, Ar–H). 13C NMR (DMSO-d6) d: 24.45, 56.08, 101.53,
106.84, 119.55, 123.75, 126.10, 128.12, 129.38, 129.67,
130.19, 133.05, 134.36, 137.31, 139.75, 141.32, 144.67,
156.42, 157.11, 161.65, 161.69. Anal. calcd for
C26H20ClN3O3: C, 68.20; H, 4.40; N, 9.18. Found: C, 68.39;
H, 4.48; N, 9.31.
Cell cycle analysis
Cell–cycle analysis was performed following incubation of
exponentially growing HT-1080 cells with the test sub-
stances (2 lM) for 36 h. Treated cultures were then tryp-
sinized, washed in PBS, fixed in 50 % ethanol, and stained
with an RNAse-containing propidium iodide solution
(Metwally et al., 2007a). For apoptosis assessment through
subdiploid event analysis, a DNA extraction step with
phosphate-citrate buffer was included prior to flow
cytometry (Gong et al., 1994). DNA content was analyzed
on a FACS Calibur (Becton–Dickinson, San Jose, CA,
USA) flow cytometer using the ModFit software (Verity
Software House, Topsham, ME, USA).
3-Methyl-5-(4-chlorophenyl)-2-(3,4,5-trimethoxyphenyl)-py-
rimido[4,5-c]quinolin-1(2H)-one (22) Yield 54 %, mp
1
240–241 °C (DMF/EtOH); H-NMR (DMSO-d6) d: 2.31
(s, 3H, CH3), 3.77 (s, 3H, OCH3), 3.80 (s, 6H, 2 OCH3),
6.93 (s, 2H, Ar–H), 7.61–7.64 (d, J = 8.5 Hz, 2H, Ar–H),
7.77–7.89 (m, 2H, Ar–H), 8.16–8.22 (m, 3H, Ar–H),
9.58–9.61 (dd, J = 1.5, 1.5 Hz, 1H, Ar–H). 13C NMR
(DMSO-d6) d: 24.67, 56.67, 60.65, 106.18, 119.58, 123.80,
126.16, 128.23, 129.52, 129.79, 130.29, 133.11, 133.74,
134.41, 137.34, 138.11, 141.36, 144.70, 154.10, 156.54,
157.64, 161.92. Anal. calcd for C27H22ClN3O4: C, 66.46;
H, 4.54; N, 8.61. Found: C, 66.70; H, 4.80; N, 8.95.
Tubulin polymerization assay
The tubulin polymerization assay kit was purchased from
Cytoskeleton, Inc. (Denver, CO) and the assay performed
as per manufacturer’s recommendations (Cytoskeleton
Tubulin Polymerization Assay Booklet, Version 8.3).
Briefly, 10 lL of each of the controls and test compounds
at 100 lM (109 the desired final concentration) in general
tubulin buffer (provided with the kit) was added to separate
wells of a pre-warmed (37 °C) half area 96-well pla-
te. 100 lL of tubulin solution was then added and the plate
immediately transferred to a pre-warmed plate reader.
Absorbance was measured at 340 nm and 37 °C every
minute for 70 min. The tubulin solution contains 100 lL
Biological activity
MTT assay
Compounds 5–22 were tested for their cytotoxic activity on the
following human solid tumor cell lines: lung fibrosarcoma HT-
1080 (ATCC, Rockville, MD), mammary adenocarcinoma
MDA-MB-231 (ATCC), and colorectal adenocarcinoma HT-
29 (European Collection of Cell Cultures, Salisbury, U.K.). All
cells were routinely cultured in Dulbecco’s Minimal Essential
Medium (DMEM) supplemented with penicillin (100 IU/mL),
123

Med Chem Res
volume of 3 mg/mL tubulin in 80 mM pH 6.9, 0.5 mM
EGTA, 2 mM MgCl2, 1 mM GTP and 10 % glycerol.
et al., 1996; Ruppert et al., 1997). The 3D structure was
taken from the Brookhaven Protein Databank (PDB code:
3e22). To validate each docking experiment, the co-crys-
tallized ligand colchicine was extracted and re-docked.
SYBYL successfully and reliably produced the binding
mode observed in the crystal structure. The same docking
parameters were applied to all compounds and crystallo-
graphic colchicine was used as a reference.
Wound-healing assay
MDA-MB-231 human breast cancer cells were cultured
in RPMI 1640 medium containing 10 mM HEPES,
4 mM L-glutamine, 10 % fetal bovine serum, penicillin
(100 IU/mL), and streptomycin (50 lg/mL), and incubated
in a 5 % CO2 atmosphere at 37 °C (Sallam et al., 2010).
Cells were then plated onto sterile 24-well plates and
allowed to form a confluent monolayer ([95 % confluence)
overnight. Wounds were then inflicted in each cell mono-
layer using a sterile 200 lL pipette tip. Media was removed
and cells were washed twice with PBS and once with fresh
serum-free media. Test compounds prepared in serum-free
media, from DMSO stock solutions, at the desired con-
centrations (0.5, 1, 2, 5, 10, 20, 40, 50, and 100 lM) were
added to each well. Plates were then incubated for 24–48 h,
after which media was removed and cells were washed,
fixed and stained using Diff-QuickTM staining (Dade
Behring Diagnostics, Aguada, Puerto Rico). Cells which
migrated across the inflicted wound were counted under the
microscope in three or more randomly selected fields
(magnification: 9400). Final results were expressed as
percentage mean number of cell for tested compound/mean
number of cells for vehicle control SEM per 9400 field.
Results and discussion
The MTT assay allows for the measurement of cell via-
bility and proliferation of cell populations in a quantitative
colorimetric fashion by utilizing cellular ability to reduce
the MTT reagent to insoluble purple formazan dye (Sallam
et al., 2010). Compounds were tested for their cytotoxic
activity on the following human solid tumor cell lines: lung
fibrosarcoma HT-1080, mammary adenocarcinoma MDA-
MB-231 and colorectal adenocarcinoma HT-29. Each
compound was tested at five serial dilutions from 100 lM
down to 0.16 lM. The concentration of each compound
that resulted in 50 % cell growth inhibition, IC50, was
calculated (Table 1). With the exception of 13 and 22, all
compounds were not cytotoxic to HT-29 cell line. On the
Table 1 Cytotoxicity of 3-methyl-2,5-diarylpyrimido[4,5-c]quino-
lin-1(2H)-ones (IC50 in lM)
Molecular modeling
Compound
HT-1080
Mean
MDA-MB-231
HT-29
Mean
SEM
Mean
SEM
SEM
Three-dimensional structure building and all modeling were
performed using the SYBYL program package, version 8.1,
installed on DELL desktop workstations equipped with a dual
2.0 GHz IntelÒ XeonÒ processor running the Red Hat Enter-
prise Linux (version 5) operating system. Conformations of
each compound were generated using ConfortTM conforma-
tional analysis. Energy minimizations were performed using
the Tripos force field with a distance-dependent dielectric and
the Powell conjugate gradient algorithm with a convergence
criterion of 0.01 kcal/(mol A) (Clark et al., 1989). Partial
atomic charges were calculated using the semi-empirical AM1
method in MOPAC 6.0 program (Stewart, 1990).
b
b
a
a
a
a
a
a
a
5
27.67
9.47
0.18
6
10.55
a
15.35
a
0.36
7
a
a
8
9
5.54
0.29
1.63
0.94
10.68
0.83
1.78
10
17.13
51.74
a
11
97.35
a
a
12
13
12.71
13.02
32.34
38.82
33.28
14.13
19.53
52.27
76.18
3.16
2.14
0.30
0.77
7.10
9.92
1.09
2.40
2.72
0.94
0.12
0.04
16.92
b
1.17
53.46
b
6.72
14
a
a
a
a
a
a
a
15
44.33
55.39
56.8
1.02
3.05
16
Molecular docking
17
15.27
2.29
18
44.51
Surflex-Dock Program version 2.0 interfaced with SYBYL
8.1 was used to dock the compounds in the colchicine
binding site of the tubulin–colchicine–soblidotin: stathmin-
like domain complex (PDB 3e22) (Abdelbar et al., 2010).
Surflex-Dock employs an idealized active site ligand
(protomol) as a target to generate putative poses of mole-
cules or molecular fragments. These putative poses were
scored using the Hammerhead scoring function (Welch
19
47.07
a
0.05
20
21
46.47
4.45
0.03
1.15
0.62
0.02
22
68.07
0.35
7.27
0.12
Doxorubicin
0.02
SEM Standard error of the mean
a
b
IC50 [ 100 lM, Not tested
123

Med Chem Res
Tubulin polymerization assay
other hand, most compounds were moderately active
against MDA-MB-231, and HT-1080 cell lines. In general,
HT-1080 was the most susceptible among these two cell
lines, as previously observed (Metwally et al., 2010). The
C-9 methoxy group evidently reduces the cytotoxicity
compared to other compounds lacking this group in the
same position, except compounds 6 and 9 which showed
higher cytotoxicity compared to their respective 9-de-
methoxy structures 15 and 18, respectively. This could be
justified by reducing the tubulin binding affinity as
explained later. Finally, the most active compound against
MDA-MB-231 and HT-1080 was 22, with IC50 values 4.45
and 3.16 lM, respectively. Compounds 9, 6 and 13 were
the next most active with IC50 range from 5.54 to
16.92 lM (Table 1) against both cell lines.
The tubulin polymerization assay kit was used as the in vitro
model for the investigation of the effect of 5–22 on tubulin
polymerization (Fig. 1). In this assay, the extent of tubulin
polymerization is proportional to absorbance at 340 nm
(Cytoskeleton Tubulin Polymerization Assay Booklet, Ver-
sion 8.3). Three controls were used, namely DMSO (vehicle
control), paclitaxel and colchicine (Chen et al., 2005). All
compounds were tested at a single concentration of 10 lM.
Table 4 shows the AUC values for tested compounds relative
to the vehicle control as % inhibition. It reflects the total
amount of tubulin polymer formed per unit time relative to the
negative control. AUC values[100 % reflect an increase in
tubulin polymerization relative to the negative control,
whereas AUC values\100 % are consistent with inhibition of
tubulin polymerization. As expected, the AUC value is
enhanced by 1.6 fold in the presence of 10 lM paclitaxel,
which promotes and stabilizes microtubule polymerization,
thus resulting in an overall increase in AUC, and decreased by
4.7 fold in the presence of 10 lM colchicine (a tubulin
polymerization inhibitor, thus resulting in an overall decrease
in AUC). Generally, all compounds were much less active
compared to colchicine. This can be considered as an advan-
tage for future use as cancer migration inhibitors because they
may not be as toxic as colchicine. Compounds 6, 11, 13, 17,
18, 20 and 21 showed a relatively higher potency compared to
other compounds, inducing a decrease in the total amount of
polymerized tubulin as reflected by\80 % tubulin polymer-
ization relative to the vehicle control (Table 4). Compound
17, without C-9-methoxy substitution and with a 4-methoxy
substitution on the 2-arylpyrimido ring, shows a tubulin
polymerization inhibitory effect comparable to 13, which has
a C-9-methoxy substitution and 3,4,5-trimethoxy on the 2-a-
rylpyrimido substitution. Generally, C-9-methoxy substitu-
tion reduced the tubulin polymerization inhibition.
2-Methoxy, 4-methyoxy, 3,4-dimethoxy, 3,5-dimethoxy, or
3,4,5-trimethoxy-2-arylpyrimido substitution pattern was
consistent with enhancing the activity (Table 4).
Cell cycle analysis
Table 2 lists the results obtained with the most promising
compounds 6, 9, 13, and 22 administered at a concentration
of 20 lM for 48 h versus vehicle control. It is obvious that
the most cytotoxic compound 22 showed lowest S-phase,
highest G2/M, together with the lowest IC50 value in the
cytotoxicity assays. Notably, the microscopic appearance
of the cells treated with 22 and to a lesser extent with 13,
was suggesting apoptotic death (i.e. cell shrinkage and
detachment). This was supported by the existence of high
numbers of subdiploid events during cell cycle analysis,
especially when a phosphate-citrate buffer was used for
DNA extraction prior to flow cytometry (Gong et al.,
1994). In Table 3, a representative experiment showing
this induction in the number of subdiploid events during
DNA content-analysis by 22, which was comparable to that
produced by the well-known inducer of apoptotic cell death
staurosporine (500 lM) (Jarvis and Grant, 1999).
Table 2 Cell cycle phase distribution (%)
Compound
G0/G1
S
G2/M
Wound-healing assay
6
38.7 ( 2.6)
40.0 ( 3.3)
38.2 ( 3.1)
38.7 ( 2.4)
39.2 ( 1.2)
26.3 ( 0.4)
25.8 ( 0.3)
28.0 ( 0.6)
17.4 ( 0.7)
28.1 ( 0.5)
35.0 ( 2.9)
34.2 ( 3.1)
33.8 ( 2.5)
43.9 ( 3.2)
32.7 ( 1.8)
9
The wound-healing assay is a simple method for the study
of directional cell migration in vitro (Rodriguez et al.,
2005). Cell migration is relevant to many cellular processes
in morphogenesis, tissue repair, as well as cancer invasion
and metastasis (Ilina and Friedl, 2009). One of the key
steps of cell migration is cytoskeletal rearrangement. The
cytoskeleton is a complex, dynamic network consisting of
three types of polymers: actin, microtubules, and interme-
diate filaments (Millipore Product Selection Guide. www.
Millipore.com/techpublications/tech1/pb2932en00). There
is growing evidence suggesting that microtubules play a
13
22
Vehicle control
Average of two experiments
Table 3 Subdiploid events
during cell-cycle analysis (%)
Compound
%
22
66.4
61.5
13.5
Staurosporine
Vehicle control
123

Med Chem Res
Fig. 1 Tubulin polymerization
assay. Purified tubulin at a final
concentration of 3 mg/mL was
used to assess microtubule
formation in vitro in presence of
10 lM doses of test compounds.
Changes of absorbance at
340 nm were plotted versus
time (minutes)
Table 4 Tubulin polymerization assay and antimigratory activity
of 3-methyl-2,5-diarylpyrimido[4,5-c]quinolin-1(2H)-ones in WHA
using the human breast cancer cell line, MDA-MB-231
Figure 2 shows cell migration across a wound inflicted
in an MDA-MB-231 cell monolayer for the vehicle and
positive controls. The concentration of each analogue that
results in 50 % inhibition of cell migration, IC50, was
calculated relative to the vehicle (negative) control using
GraphPad Prism 5 (Table 4). The 2-methoxy and 2,4-
dimethoxy substitutions at the 2-arylpyrimido functionality
enhanced the antimigratory activity in the 9-methoxy-
substituted series like 6 and 9 with IC50 values of 5.00 and
5.86 lM, respectively. The 3,4,5-trimethoxy substitutions
at the 2-arylpyrimido group also significantly improved the
antimigratory activity in presence or absence of the
9-methoxy substitution as represented by 13 and 22 with
IC50 values of 2.40 and 1.52 lM, respectively. The most
active compounds in migration assay 6, 9, 13, and 22 were
also the most active in the MTT assay but at much lower
IC50 values, suggesting their activity is not due to direct
cytotoxicity. Compound 22, which is the most active in
MTT, cell cycle analysis, and migration assays was among
the least active in tubulin polymerization assay with
88.52 % inhibition. This clearly suggests different molec-
ular target for activity other than cytoskeleton.
Compound
% Inhibitiona
Antimigratory
IC50 (lM)
Vehicle control
100.00
157.12
21.81
107.98
79.79
87.87
83.10
84.49
86.32
74.72
108.74
78.43
90.43
98.73
85.44
77.62
79.90
88.36
75.09
72.91
88.52
–
Paclitaxel
–
Colchicine
7.07
5
20.84
5.00
6
7
23.12
15.58
5.86
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
a
8.61
41.51
26.81
2.40
17.98
21.62
20.56
29.42
20.79
14.60
10.17
9.30
Molecular modeling and docking studies
1.52
Compounds 5–22 were docked at the colchicine binding
site of the high-resolution crystal structure (resolu-
˚
tion = 3.80 A) of the tubulin–colchicine–soblidotin,
% Inhibition represents the area under the curve for each test
compound relative to the vehicle (negative) control
stathmin-like domain complex (PDB 3e22) using Surflex-
Dock interface implemented into SYBYL 8.1 (Abdelbar
et al., 2010; Hoshi et al., 1996; MacAulay and Woodgett,
2008; Liu and Shen, 2007). Surflex-Dock is a fully auto-
matic flexible molecular docking algorithm that combines
the scoring function from the Hammerhead docking system
key part in cell polarity and migration through interactions
with focal adhesion molecules. Agents affecting microtu-
bule dynamics, such as paclitaxel and nocodazole, also
affect cell migration via complex signaling mechanisms
(Nakano et al., 2010).
123

Med Chem Res
Fig. 2 Graphic Presentation for
the WHA using MDA-MB-231
cells after 24 h exposure to:
a Vehicle (DMSO) treatment as
a negative control. b A 2 lM
dose of the most active
compound 22
with a search engine that relies on a surface-based
molecular similarity method as a means to rapidly generate
suitable putative poses for molecular fragments (Hoshi
et al., 1996; MacAulay and Woodgett, 2008). Three
binding domains have been identified in tubulin to date.
They include; (a) the cholchicine site close to the a/b
interface, (b) the area where Vinca alkaloids binds, and
(c) the taxane binding pocket. The colchicine binding site
lies at the interface between the a- and b-subunits of
tubulin, mostly in the b-subunit (Tripathi et al., 2008). The
colchicine binding site is a funnel-shaped binding cavity
with its narrow part strongly hydrophobic whereas its wider
part is moderately polar and hydrophobic (Tripathi et al.,
2008). Colchicine is positioned such that its trimethoxy-
phenyl moiety fits into the narrow hydrophobic pocket and
is surrounded by the amino acids Cys241b, Leu242b,
Leu248b, Ala250b, and Leu252b (Fig. 3).
Docking of compounds 5–22 resulted in two general high-
scoring orientations (Figs. 4 and 5, Table 5). The first
achieved by 7 and 16–20 was very closely similar to the
binding mode of colchicine (Fig. 4). With the exception of 7,
all compounds showing this colchicine-like orientation lack
the C-9-methoxy substitution. The mono- or dimethoxy
substituted 2-arylpyrimido ring was deeply embedded in the
highly hydrophobic narrow end of the funnel-shaped binding
pocket, and was surrounded by Cys241b, Leu242b, Leu248b,
Ala250b, Leu255b, Ala354b, and Ileu378b amino acids
(Tripathi et al., 2008). Polar interactions also contributed to
binding as follows: the oxygen of the 3-methoxy group acts as
a hydrogen bond acceptor (HBA) and is located at
˚
(3.606–4.093)and(3.752–4.032) AawayfromtheamideHof
the amino acids Cys241b and Leu242b, respectively (Fig. 4
and Table 5). The quinoline nitrogen was also involved in
˚
hydrogen bonding and lies within (3.670–3.984) A of the NH2
group of Asn258b. Finally, N-4 of the pyrimidine ring was
Fig. 3 Detailed view of docked colchicine. Its binding site lies at the
interface between the a- and b-subunits of tubulin. Its trimethoxy
phenyl moiety fits into the narrow hydrophobic pocket and is
surrounded by the amino acids Cys241b, Leu242b, Leu248b,
Ala250b, and Leu252b
Fig. 4 Detailed view of docked structures 7 and 16–20 with a
colchicine-like binding mode
123

Med Chem Res
involved in hydrogen bonding with the NH2 group of
Asn101b (Fig. 4).
The second orientation was observed for compounds 5,
6, 9–14 and 21 with a C-9-methoxy substitution along with
14 and 21, which lack this C-9-methoxy substitution. The
oxygen of C-9-methoxy group forms a strong hydrogen
˚
bond (2.593–3.207 A) with the NH2 group of Lys254b,
thus anchoring each molecule in a way that prevented the
deep embedding of the methoxy substituted 2-arylpyrimido
ring in the narrow hydrophobic pocket (Fig. 5). Instead,
each molecule is oriented towards the wider opening of the
binding pocket. Compounds 8, 15 and 22 were exceptions
in that they do not fit any of the previously described
binding orientations and generally resulted in low docking
scores (Fig. 6 and Table 5).
In conclusion, methoxy substitutions on 2-arylpyrimido
moieties of Pyrimido[4,5-c]quinolin-1(2H)-ones play impor-
tant role in enhancing their antimigratory activity. The C-9
methoxy substitution significantly reduces the microtubule
polymerization inhibitory activity. Generally, all compounds
showed reduced tubulin binding affinity compared to
Fig. 5 Detailed view of docked structure for compounds 5, 6 and
9–13. Their 9-methoxy oxygens form strong hydrogen bond with
Lys254b NH2 group, anchoring each molecule in a way that
prevented the deep embedding of the methoxy-substituted 2-phen-
ylpyrimido ring in the narrow hydrophobic pocket
Table 5 Virtual binding affinity of tested compounds at the colchi-
cine binding site of tubulin (PDB 3e22) using SYBYL 8.1 Surflex-
Dock
Compound
Docking score
Crash
Polar
CScore
Colchicine
7.21
5.52
4.97
5.10
1.71
6.39
3.24
6.35
2.16
5.17
4.13
3.65
5.80
4.99
5.30
3.46
5.06
2.09
2.92
-1.35
-1.67
-2.39
-5.17
-4.74
-2.08
-4.10
-2.16
-5.76
-4.40
-1.62
-2.61
-4.08
-4.85
-4.17
-6.35
-5.13
-4.86
-10.44
0.02
1.48
1.00
0.00
0.00
1.22
0.80
1.42
0.08
1.58
0.66
0.00
0.00
0.00
0.00
0.00
0.00
0.76
2.68
3
4
2
4
4
4
2
4
4
2
1
1
4
2
4
4
2
4
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Total score was expressed in -log(Kd) units to represent binding
affinities. Crash is the degree of inappropriate penetration by the
ligand into the protein and of interpenetration between ligand atoms
that are separated by rotatable bonds. Polar score is the contribution
of the polar non-hydrogen bonding interactions to the total score. The
polar score may be useful for excluding docking results that make no
hydrogen bonds
Fig. 6 Docking mode for structures 8, 15 and 22 versus colchicine
123

Med Chem Res
Liu S, Shen Z (2007) A new target for diabetes therapy: advances in
the research of glycogen synthase kinase-3 inhibitors. Yaoxue
Xuebao 42:1227–1231
MacAulay K, Woodgett J (2008) Targeting glycogen synthase kinase-
3 (GSK-3) in the treatment of Type 2 diabetes. Expert Opin Ther
Targets 12:1265–1274
colchicine, which can aid their future use as cancer migra-
tion inhibitors because they will not be as toxic as
colchicine.
Mane J, Klobukowski M (2008) Free energy calculations on the
binding of colchicine and its derivatives with the a/b-tubulin
isoforms. J Chem Inf Model 48:1824–1832
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