Design, synthesis and evaluation of alkylphosphocholine-gefitinib conjugates as multitarget anticancer agents

Article abstract of DOI:10.1007/s12272-017-0977-z

The evolving resistance to the currently used chemotherapeutic agents requires continuous efforts to develop new anticancer agents overcoming resistance and with lower side effects. Polypharmacology via designing a single molecule intercepting multiple signaling pathways is more effective than targeting a single one. Several alkylphosphocholines show anticancer activity via inhibition of Akt phosphorylation. On the other hand, several molecules having quinazoline scaffold elicit anticancer activity through inhibition of epidermal growth factor receptor (EGFR) tyrosine kinases. We report our efforts to develop alkylphosphocholines-gefitinib conjugates as multitarget anticancer agents. The antiproliferative activities of the newly synthesized compounds were evaluated against cell lines representing lung, breast, liver and skin cancers. In addition, the capability of the newly synthesized compounds to inhibit Akt phosphorylation and EGFR tyrosine kinases were determined. The results emphasized the influence of the linkers’ length on the elicited bioactivity. The long chain linkers possessing conjugates were more active regarding both of the elicited antiproliferative effect and inhibition of Akt phosphorylation, while maintained the ability to inhibit EGFR tyrosine kinases. Their cytotoxic activities were superior or comparable to erlotinib and miltefosine.

Full text of DOI:10.1007/s12272-017-0977-z

Arch. Pharm. Res.
Online ISSN 1976-3786
Print ISSN 0253-6269
DOI 10.1007/s12272-017-0977-z
RESEARCH ARTICLE
Design, synthesis and evaluation of alkylphosphocholine-gefitinib
conjugates as multitarget anticancer agents
Md. Maqusood Alam1 Ahmed H. E. Hassan2,3 Yeong Ho Kwon1
•
•
•
Hyo Jong Lee1 Nam Yong Kim1 Kyung Hoon Min4 Sang-Yoon Lee5
Dong-Hyun Kim1 Yong Sup Lee1,3
•
•
•
•
•
Received: 20 June 2017 / Accepted: 19 October 2017
Ó The Pharmaceutical Society of Korea 2017
Abstract The evolving resistance to the currently used
chemotherapeutic agents requires continuous efforts to
develop new anticancer agents overcoming resistance and
with lower side effects. Polypharmacology via designing a
single molecule intercepting multiple signaling pathways is
more effective than targeting a single one. Several
alkylphosphocholines show anticancer activity via inhibi-
tion of Akt phosphorylation. On the other hand, several
molecules having quinazoline scaffold elicit anticancer
activity through inhibition of epidermal growth factor
receptor (EGFR) tyrosine kinases. We report our efforts to
develop alkylphosphocholines-gefitinib conjugates as
multitarget anticancer agents. The antiproliferative activi-
ties of the newly synthesized compounds were evaluated
against cell lines representing lung, breast, liver and skin
cancers. In addition, the capability of the newly synthe-
sized compounds to inhibit Akt phosphorylation and EGFR
tyrosine kinases were determined. The results emphasized
the influence of the linkers’ length on the elicited bioac-
tivity. The long chain linkers possessing conjugates were
more active regarding both of the elicited antiproliferative
effect and inhibition of Akt phosphorylation, while main-
tained the ability to inhibit EGFR tyrosine kinases. Their
cytotoxic activities were superior or comparable to erloti-
nib and miltefosine.
Keywords Alkylphosphocholines Á Gefitinib Á Akt
phosphorylation inhibitors Á EGFR tyrosine kinase
inhibitors Á Antiproliferartive agents
Introduction
Cancer cells differs from normal cells in uptake and
metabolism of lipids (Beloribi-Djefaflia et al. 2016).
Increased lipids uptake by cancer cells enabled develop-
ment of visualizing agents for cancer cells via alkylphos-
phocholine-based hybrid molecules for delivering imaging
agents into cancer cells (Weichert et al. 2014). CLR1404
(1a, Fig. 1) is a radiolabeled molecule that has been pro-
posed for PET-imaging of tumors. Also, CLR1502 (1b,
Fig. 1) which incorporates the infrared dye IR-775 might
be suitable for intraoperative surgical detection of tumor’s
margin. In fact, some alkylphosphocholines (APCs) elicit
antitumor activities by themselves (Pachioni Jde et al.
2013; Faustino 2014; Kostadinova et al. 2015; Murray
et al. 2015). They may integrate into tumor cell’s mem-
brane changing its properties, trafficking and signaling. In
addition, they may interfere with lipids metabolism and
lipids-dependent signaling cascades (Danker et al. 2010;
van Blitterswijk and Verheij 2013; Faustino 2014). Milte-
fosine 2 (miltexÒ) is a prominent members of this class of
compounds. However, gastrointestinal toxicity restricts its
use to topical treatment of cutaneous metastasis of breast
cancer (Clive et al. 1999). Several studies indicate that
& Yong Sup Lee
kyslee@khu.ac.kr
1
Department of Life and Nanopharmaceutical Sciences,
Kyung Hee University, Seoul 02447, Republic of Korea
2
Department of Medicinal Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
3
Medicinal Chemistry Laboratory, Department of Pharmacy,
College of Pharmacy, Kyung Hee University, Seoul 02447,
Republic of Korea
4
Department of Pharmacy, College of Pharmacy, Chung-Ang
University, Seoul 06974, Republic of Korea
5
Neuroscience Research Institute, Gachon University,
Incheon 20565, Republic of Korea
123

Md. Maqusood Alam et al.
Fig. 1 Structures of reported alkylphosphocholines and gefitinib
APCs such as miltefosine 2 can form highly stable com-
plexes with the cholesterol-enriched rigid domains within
the cell membrane known as lipid rafts (Gomez-Serranillos
EGFR pathways might be a useful strategy for developing
novel anticancer agents. Investigation of the reported
crystals of gefitinib-EGFR complex (PDB codes 3UG2 and
4WKQ) shows that the aromatic rings of the quinazoline
core of gefitinib (3) interact with the hydrophobic pocket of
EGFR while the aliphatic alkoxy substituents extends out-
side of the binding site. Accordingly, modification of the
alkoxy substituents is anticipated to maintain the affinity of
the molecule to the binding pocket of EGFR. On the other
hand, analogues of miltefosine 2 such as CLR1404 and
CLR1502 which have moieties conjugated to the terminal
end of the alkyl chain maintained selectivity to cancerous
cells. Based on this, analogues of miltefosine (2) conjugated
with the core of gefitinib via hydroxyalkanoic acids linkers
might result in useful multitarget anticancer agent with
increased the efficacy (Fig. 2). In the designed conjugated
molecules, the impact of the length of the alkyl chain of the
APCs and moiety, as well as, the linker would be investi-
gated. Herein, we would like to report our results.
´
et al. 2004; Malta de Sa et al. 2015). Lipid rafts modulate
multiple signaling pathways (Simons and Toomre 2000;
Pike 2003; Fielding and Fielding 2004). PI3K/Akt signal-
ing pathway is one of these pathways impacted by lipid
rafts (Reis-Sobreiro et al. 2013). Cholesterol synthesis
inhibition or sequestration from lipid rafts down-regulates
Akt signaling (Zhuang et al. 2005; Fedida-Metula et al.
2008; Calay et al. 2010). The anticancer properties of APC
derivatives mediated via modulation of PI3K/Akt pathway
are known (Alam et al. 2012, 2013).
Gefitinib (3, Iressa) is a FDA approved kinase inhibitor
for the treatment of EGFR-mutated lung cancer in which
EGFR mutations abolish the activity of the wild EGFR
inhibitors such as erlotinib (Kazandjian et al. 2016).
Unfortunately, gefitinib resistance arise because of activa-
tion of PI3K/AKT pathway, at least in part (Li et al. 2013).
It is known that PI3K/AKT pathway communicates syn-
ergistically with the EGFR signaling (Block et al. 2012). A
combination of gefitinib and RAD001, an inhibitor of
PI3K/AKT pathway, was found to elicit synergistic inhi-
bition of cancer cell lines (Dragowska et al. 2011).
Importantly, the PI3K/AKT pathway inhibitor LY294002
was reported to restore the sensitivity to gefitinib in resis-
tant cell lines (Li et al. 2013).
Materials and methods
Chemistry
General
Polypharmacology via design of a single molecule
intercepting multiple signaling pathways could be more
effective than the simple combination therapy. A single
bifunctional molecule inhibiting both of PI3K/AKT and
All solvents were purified and used on scrupulously dry
condition. NMR spectra of all compounds were recorded
on Bruker AC-400 spectrometer (400 MHz for 1H and
100 MHz for 13C). Chemical shifts (d) are reported in ppm,
123

Design, synthesis and evaluation of alkylphosphocholine-gefitinib
Fig. 2 Design of alkylphosphocholine-gefitinib conjugates
downfield from internal TMS standard. High resolution
mass spectra (HRMS) were recorded on Jeol AccuTOF
(JMS-T100TD) equipped with a DART (direct analysis in
real time) ion source from ionsense, Tokyo, Japan in the
positive modes. Analytical thin layer chromatography
(TLC) was carried out using precoated silica gel (E. Merck
Kiesegel 60F254, layer thickness 0.25 mm), and chro-
matography was performed using Merck Kiesegel 60 Art
9385 (230–400 mesh).
was adjusted to 9–10 with ammonium hydroxide (25% aq.
solution). Concentration in vacuo, filtration and washing
with water followed by ether yielded the titled compound
1
7. Yield 98%. H-NMR (400 MHz, DMSO-d6) d: 4.00 (s,
3H), 7.28-7.04 (M, 2H), 7.48 (s, 1H), 7.84 (m, 1H), 8.56 (s,
1H), 8.80 (s, 1H).
General procedure for the synthesis of ethyl 7-(4-(3-
chloro-4-fluorophenylamino)-6-methoxyquinazolin-
7-yloxy)alkanoates (8)
7-(Benzyloxy)-N-(3-chloro-4-flurophenyl)-6-
methoxyquinazoline-4-amine (6)
A mixture of compound 7 (1.0 mmol), ethyl bromoalka-
noate (1.2 mmol) and potassium carbonate (3.0 mmol) in
DMF was stirred at 60 °C for 4 h. The mixture was cool to
rt and filtered. The filtrate was concentrated in vacuo. The
residue was dissolved in dichloromethane and washed with
brine, dried over magnesium sulfate and concentrated to
afford derivatives 8.
A mixture of 7-(benzyloxy)-4-chloro-6-methoxyquinazo-
line 5 (2.0 g, 6.6 mmol) and 3-chloro-4-fluoroaniline
(1.1 g, 7.9 mmol) in i-PrOH (20.0 mL) was refluxed for
4 h. Upon cooling to rt, the precipitated crystals were fil-
tered, washed with i-PrOH, ether and dried to give com-
1
pound 6. Yield 96%. H-NMR (400 MHz, DMSO-d6) d:
4.02 (s, 3H), 5.34 (s, 2H), 7.76-7.37 (m, 8H), 8.03 (d, 1H,
Ethyl 7-(4-(3-chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)heptanoate (8a)
J = 6.7 Hz), 8.31 (s, 1H), 8.84 (s, 1H).
4-(3-Chloro-4-flurophenylamino)-6-methoxyquinazolin-7-
ol (7)
Yield 89%. 1H-NMR (400 MHz, DMSO-d6) d: 1.18 (t, 3H,
J = 7.2 Hz), 1.57-1.32 (m, 6H), 1.82-1.74 (m, 2H), 2.32 (t,
3H, J = 7.2 Hz), 4.02-4.08 (m, 5H), 4.16 (t, 2H,
J = 6.2 Hz), 7.35 (s, 1H), 7.53 (t, 1H, J = 9.0 Hz), 7.79-
7.76 (m, 1H), 8.04 (d, 1H, J = 6.7 Hz), 8.38 (s, 1H), 8.85
(s, 1H), 11.6 (s, 1H).
A mixture of compound 6 (2.6 g, 6.3 mmol) and trifluo-
roacetic acid (20.0 mL) was refluxed for 1 h, cooled to rt,
filtered, dissolved in methanol (10.0 mL), and then the pH
123

Md. Maqusood Alam et al.
Ethyl 2-(4-(3-chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)acetate (8b)
chromatography on silica gel, first eluting with CHCl3-
MeOH (9:1) for separation of nonpolar product, and then
with CHCl3-MeOH-H2O (65:20:4). The obtained semi-
solids were precipitated from chloroformic solutions with
n-pentane to afford 13.
Yield 71%. 1H-NMR (400 MHz, DMSO-d6) d: 1.18 (t, 3H,
J = 7.2 Hz), 4.13-3.84 (m, 7H), 6.91 (s, 1H), 7.38 (t, 1H,
J = 9.1 Hz), 7.97-7.93 (m, 2H), 8.27 (d, 1H, J = 6.7 Hz),
8.34 (s, 1H), 9.78 (s, 1H).
9-(Benzyloxy)nonyl 2-(trimethylammonio)ethyl phosphate
(13a)
General procedure for the synthesis of 7-(4-(3-
chloro-4-fluorophenylamino)-6-methoxyquinazolin-
7-yloxy)alkanoic acids (9)
Yield 88%. 1H-NMR (400 MHz, methanol-d4) d: 1.46-1.32
(m, 10H), 1.83-1.57 (m, 4H), 3.23 (s, 9H), 3.49 (t, 1H,
J = 6.4 Hz), 3.65-3.63 (m, 2H), 3.72 (t, 1H, J = 5.1 Hz),
3.88 (q, 2H, J = 6.8 Hz), 3.98-3.94 (m, 2H), 4.34-4.24 (m,
2H), 7.37-7.26 (m, 3H), 7.51-7.46 (m, 1H), 8.04-8.01 (m,
1H).
Lithium hydroxide monohydrate (3.0 mmol) was added to
a solution of ethyl 7-(4-(3-chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)alkanoates 8 (1.0 mmol) in
THF (18.0 mL) and distilled water (6.0 mL). The solution
was stirred for 24 h. The reaction mixture cooled in an ice
bath was acidified to pH 2 with HCl (1 N aq. solution). The
solids were filtered, washed with distilled water and dried
to yield the titled derivatives 9.
10-(Benzyloxy)decyl 2-(trimethylammonio)ethyl phosphate
(13b)
Yield 83%. 1H-NMR (400 MHz, methanol-d4) d: 1.46-1.31
(m, 12H), 1.83-1.57 (m, 4H), 3.23 (s, 9H), 3.49 (t, 1H,
J = 6.4 Hz), 3.65-3.64 (m, 2H), 3.72 (t, 1H, J = 5.1 Hz),
3.88 (q, 2H, J = 6.8 Hz), 3.98-3.94 (m, 2H), 4.34-4.24 (m,
2H), 7.37-7.26 (m, 3H), 7.51-7.45 (m, 1H), 8.04-8.01 (m,
1H).
7-(4-(3-Chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)heptanoic acid (9a)
1
Yield 91%. H-NMR (400 MHz, DMSO-d6) d: 1.81-1.44
(m, 8H), 2.20-2.02 (m, 2H), 3.96 (s, 3H), 4.14-4.11 (t, 2H,
J = 6.4 Hz), 7.18 (s, 1H), 7.44 (t, 1H, J = 9.1 Hz), 7.82 (s,
1H), 8.14 (m, 1H), 8.48 (s, 1H) 9.58 (s, 1H).
12-(Benzyloxy)dodecyl 2-(trimethylammonio)ethyl
phosphate (13c)
2-(4-(3-Chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)acetic acid (9b)
Yield 53%. 1H-NMR (400 MHz, methanol-d4) d: 1.46-1.33
(m, 16H), 1.83-1.58 (m, 4H), 3.23 (s, 9H), 3.49 (t, 1H,
J = 6.4 Hz), 3.65-3.63 (m, 2H), 3.72 (t, 1H, J = 5.1 Hz),
3.88 (q, 2H, J = 6.8 Hz), 3.98-3.95 (m, 2H), 4.34-4.24 (m,
2H), 7.37-7.28 (m, 3H), 7.51-7.46 (m, 1H), 8.04-8.01 (m,
1H).
1
Yield 90%. H-NMR (400 MHz, DMSO-d6) d: 4.00 (s,
3H), 4.24 (s, 2H), 7.05 (s, 1H), 7.26 (t, 1H, J = 8.9 Hz),
7.57-7.48 (m, 1H), 7.86-7.82(m, 2H), 8.56 (s, 1H).
General procedure for the synthesis
of benzyloxyalkyl 2-(trimethylammonio)ethyl
phosphates (13)
General procedure for the synthesis of hydroxyalkyl
2-(trimethylammonio)ethyl phosphates (14)
To a solution of compound 13 (1.0 mmol) in methanol
(10.0 mL) 10% Pd/C was added (catalytic amount). The
reaction was stirred under hydrogen for 4 h at rt. The
mixture was filtered and evaporated under vacuum to
afford 14.
2-Chloro-1,3,2-dioxaphospholane 2-oxide (2.0 mmol) was
added to a stirred solution of compound 12 (1.0 mmol)
in benzene (5.0 mL) containing triethylamine (2.0 mmol)
at 0 °C. After warming up slowly to rt, the reaction
mixture was stirred for 4 h. The precipitated triethy-
lamine hydrochloride was filtered and washed with ben-
zene. The combined filtrate and washings was
concentrated, diluted with CH3CN (10.0 mL) and trans-
ferred into pressure tube. After cooling to -78 °C,
trimethylamine was added (ca. 1.5 mL). The tube was
closed, heated at 65 °C for 18 h, cooled back to rt and
evaporated. The residual solid was purified by column
9-Hydroxynonyl 2-(trimethylammonio)ethyl phosphate
(14a)
Yield 96%. 1H-NMR (400 MHz, methanol-d4) d: 1.54-1.29
(m, 12H), 1.65-1.60 (m, 2H), 3.22 (s, 9H), 3.64-3.51 (m,
4H), 3.92-3.85 (m, 2H), 4.32-4.18 (m, 2H).
123

Design, synthesis and evaluation of alkylphosphocholine-gefitinib
10-Hydroxydecyl 2-(trimethylammonio)ethyl phosphate
(14b)
10-(7-(4-(3-Chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)heptanoyloxy) decyl
2-(trimethylammonio)ethyl phosphate (4ab)
Yield 92%. 1H-NMR (400 MHz, methanol-d4) d: 1.40-1.22
(m, 12H), 1.54-1.50 (m, 2H), 1.66-1.61 (m, 2H), 3.23 (s,
9H), 3.54 (t, 2H, J = 5.3 Hz), 3.66-3.62 (m, 2H), 3.90-3.86
(m, 2H), 4.27 (m, 2H).
1
Yield 88%. H-NMR (400 MHz, methanol-d4) d:1.47-1.27
(m, 14H), 1.68-1.51 (m, 8H), 1.91-1.85 (m, 2H), 2.31 (t,
2H, J = 7.2 Hz), 3.20 (s, 9H), 3.62-3.59 (m, 2H), 3.83 (q,
2H, J = 6.5 Hz), 4.02 (s, 3H), 4.08 (t, 2H, J = 6.5 Hz),
4.25-4.10 (m, 4H), 7.10 (s, 1H), 7.24 (t, 1H, J = 7.9 Hz),
7.70-7.66 (m, 2H), 8.00 (d, 1H, J = 6.7 Hz), 8.42 (s, 1H).
13C-NMR (100 MHz, methanol-d4) d: 174.2, 156.9, 155.4,
154.7, 152.3, 150.1, 146.3, 136.2, 124.3, 122.3, 120.0,
116.0, 106.3, 101.0, 102.4, 70.0, 66.0, 65.4, 64.1, 58.9,
55.5, 53.3, 53.3, 53.2, 33.6, 30.5, 29.3, 29.0, 22.2, 29.0,
28.4, 28.4, 28.4, 25.7, 25.5, 25.4, 24.6. HRMS m/z
769.3506 (calcd for C37H56ClFN4O8P [M?H]? 769.3503).
12-Hydroxydodecyl 2-(trimethylammonio)ethyl phosphate
(14c)
Yield 93%. 1H-NMR (400 MHz, methanol-d4) d: 1.40-1.22
(m, 16H), 1.54-1.50 (m, 2H), 1.66-1.61 (m, 2H), 3.23 (s,
9H), 3.54 (t, 2H, J = 5.3 Hz), 3.66-3.62 (m, 2H), 3.90-3.86
(m, 2H), 4.27 (m, 2H).
General procedure for the synthesis of 9-(7-(4-(3-
chloro-4-fluorophenylamino)-6-methoxyquinazolin-
7-yloxy)alkanoyloxy) alky
12-(7-(4-(3-Chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)heptanoyloxy) dodecyl
2-(trimethylammonio)ethyl phosphate (4ac)
2-(trimethylammonio)ethyl phosphates (4)
Hydroxyalkyl 2-(trimethylammonio)ethyl phosphate 14
(1.0 mmol) was dissolved in anhydrous CHCl3 (20.0 mL)
and DMF (2.0 mL) and the mixture was heated to reflux
under an atmosphere of N2. DMAP (3.0 mmol), 7-(4-(3-
chloro-4-fluorophenylamino)-6-methoxyquinazolin-7-yloxy)
alkanoic acid 11 (3.0 mmol) and DCC (3.0 mmol) were
added. The reaction mixture was refluxed for 24 h. The
reaction mixture was cooled to rt. The residual semisolid
was purified by column chromatography on silica gel, first
eluting with CHCl3-MeOH (9:1) for separation of nonpolar
product, and then with CHCl3-MeOH-H2O (65:20:4).
Yield 63%. 1H-NMR (400 MHz, methanol-d4) d: 1.47-1.25
(m, 18H), 1.68-1.51 (m, 8H), 1.91-1.85 (m, 2H), 2.31 (t,
2H, J = 7.2 Hz), 3.20 (s, 9H), 3.62-3.59 (m, 2H), 3.83 (q,
2H, J = 6.5 Hz), 4.02 (s, 3H), 4.08 (t, 2H, J = 6.5 Hz),
4.15 (t, 2H, J = 6.1 Hz), 4.27-4.20 (m, 2H), 7.13 (s, 1H),
7.25 (t, 1H, J = 7.9 Hz), 7.70-7.66 (m, 2H), 8.00 (d, 1H,
J = 6.7 Hz), 8.42 (s, 1H). 13C-NMR (100 MHz, methanol-
d4) d: 174.4, 157.1, 155.6, 154.9, 153.7, 152.5, 150.3,
146.4, 136.5, 124.5, 122.5, 120.0, 116.3, 109.1, 106.5,
101.2, 68.8, 66.3, 65.8, 64.4, 59.1, 59.0, 53.5, 53.5, 53.5,
33.9, 30.7, 29.8, 29.6, 29.5, 29.5, 29.3, 29.2, 28.7, 28.6,
28.6, 28.6, 28.6, 25.9. HRMS m/z 797.3824 (calcd for
C39H60ClFN4O8P [M?H]? 797.3816).
9-(7-(4-(3-Chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)heptanoyloxy) nonyl
2-(trimethylammonio)ethyl phosphate (4aa)
9-(2-(4-(3-Chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)acetoxy)nonyl
2-(trimethylammonio)ethyl phosphate (4ba)
Yield 50%. 1H-NMR (400 MHz, methanol-d4) d: 1.38-1.19
(m, 12H), 1.81-1.42 (m, 10H), 2.31 (t, 2H, J = 7.2 Hz),
3.11 (s, 9H), 3.64-3.50 (m, 2H), 3.85 (q, 2H, J = 6.5 Hz),
3.93 (s, 3H), 4.08 (t, 2H, J = 6.5 Hz), 4.15 (t, 2H,
J = 6.1 Hz), 4.21-4.05 (m, 4H), 7.02 (s, 1H), 7.16 (t, 1H,
J = 7.9 Hz), 7.62-7.56 (m, 2H), 7.91 (d, 1H, J = 6.7 Hz),
8.33 (s, 1H). 13C-NMR (100 MHz, methanol-d4) d: 175.6,
158.3, 156.2, 153.7, 151.5, 147.6, 137.6, 130.4, 125.8,
123.9, 121.4, 117.5, 110.3, 107.7, 102.4, 70.0, 67.5, 66.9,
65.5, 60.3, 56.9, 54.7, 54.7, 54.7, 35.1, 31.9, 31.8, 30.6,
30.4, 30.3, 29.8, 29.8, 27.1, 26.9, 26.8, 26.0. HRMS m/z
755.3345 (calcd for C36H54ClFN4O8P [M?H]? 755.3346).
Yield 80%. 1H-NMR (400 MHz, methanol-d4) d: 1.58-1.05
(m, 14H), 3.11 (s, 9H), 3.56-3.49 (m, 2H), 3.77 (q, 2H,
J = 6.5 Hz), 3.91(s, 3H), 4.18-4.11 (m, 4H), 4.83 (s, 2H),
6.84 (s, 1H), 7.72-7.18 (m, 2H), 7.43-7.41 (m, 1H), 7.80 (s,
1H), 8.85 (s, 1H). 13C-NMR (100 MHz, methanol-d4) d:
168.8, 157.1, 155.7, 153.8, 152.7, 150.1, 146.1, 133.0,
129.2, 128.4, 122.6, 120.2, 116.3, 107.5, 101.9, 66.1, 65.5,
65.3, 65.2, 58.9, 55.7, 53.3, 53.3, 53.2, 30.5, 30.4, 29.2,
29.2, 29.0, 28.3, 25.6. HRMS m/z 685.2609 (calcd for
C31H44ClFN4O8P [M?H]? 685.2564).
123

Md. Maqusood Alam et al.
10-(2-(4-(3-Chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)acetoxy)decyl
2-(trimethylammonio)ethyl phosphate (4bb)
USA) and the data are presented as % inhibitions of the cell
growth.
Akt phosphorylation inhibitory activity assay
Yield 58%. 1H-NMR (400 MHz, methanol-d4) d: 1.35-1.17
(m, 12H), 1.63-1.54 (m, 4H), 3.12 (s, 9H), 3.63-3.60 (m,
2H), 3.83 (q, 2H, J = 6.5 Hz), 4.05 (s, 3H), 4.26-4.19 (m,
4H), 4.93 (s, 2H), 7.06 (s, 1H), 7.26 (t, 1H, J = 8.9 Hz),
7.72-7.68 (m, 2H), 7.78 (s, 1H), 8.43 (s, 1H). 13C-NMR
(100 MHz, methanol-d4) d: 168.6, 156.9, 155.7, 153.4,
153.3, 152.5, 149.9, 145.9, 136.1, 124.3, 122.4, 120.0,
116.1, 109.7, 107.3, 101.7, 66.1, 65.5, 65.3, 65.2, 58.9,
55.7, 53.3, 53.3, 53.2, 30.5, 30.4, 29.2, 29.0, 28.9, 28.3,
25.5. HRMS m/z 699.2715 (calcd for C32H46ClFN4O8P
[M?H]? 699.2720).
A549 human lung cancer cell line was grown to 70% con-
fluency and serum starved for 18 h. Cells were incubated for
2 h with 20 lM concentrations of each compound or mil-
tefosine after which Akt phosphorylation was stimulated by
adding insulin at 10 lg/mL and incubated for further 30 min.
As a negative control, insulin was not added and positive
control insulin was added for 30 min. For the control of an
active Akt pathway, the Akt phosphorylation inhibitors,
miltefosine, was added for 2 h prior to addition of insulin and
showed in all cases lack of Akt phosphorylation. After
30 min stimulation with insulin, the cells were washed in ice-
cold PBS and lysed using RIPA lysis buffer (Sigma-Aldrich,
MO, USA) before performing the ELISA-based phosphor-
Akt assay kit (R&D systems, MN, USA) according to the
manufacturer’s protocol. Data are presented as the means of
three independent experiments.
12-(2-(4-(3-Chloro-4-fluorophenylamino)-6-
methoxyquinazolin-7-yloxy)acetoxy)dodecyl
2-(trimethylammonio)ethyl phosphate (4bc)
Yield 73%. 1H-NMR (400 MHz, methanol-d4) d: 1.37-1.14
(m, 16H), 1.63-1.56 (m, 4H), 3.21 (s, 9H), 3.63-3.60 (m,
2H), 3.85 (q, 2H, J = 6.6 Hz), 4.05 (s, 3H), 4.26-4.19 (m,
4H), 4.94 (s, 1H), 7.06 (s, 1H), 7.26 (t, 1H, J = 8.9 Hz),
7.72-7.68 (m, 2H), 7.78 (s, 1H), 8.03 (d, 1H, J = 6.6 Hz),
8.44 (s, 1H). 13C-NMR (100 MHz, methanol-d4) d: 170.0,
158.4, 155.7, 154.8, 153.9, 151.3, 147.3, 137.6, 125.7,
123.8, 121.4, 117.5, 117.3 116.2, 108.7, 103.1, 67.5, 67.0,
66.7, 66.6, 60.3, 57.1, 54.7, 54.7, 54.7, 31.9, 31.9, 30.7,
30.7, 30.4, 30.3, 29.7, 27.0, 26.9. HRMS m/z 727.3068
(calcd for C34H50ClFN4O8P [M?H]? 727.3033).
EGFR phosphorylation inhibitory activity assay
The ability of the prepared derivatives to inhibit EGFR
kinase activity was assessed using ADP-GloTM Kinase
Assay kit (Promega) according to the manufacturer’s
instructions. Briefly, the assays were carried out in 96-well
plates in a final volume of 20 lL containing 1 9 reaction
buffer, 50 lM DTT, 2 mM MnCl2, 5 lg substrate, 10 lM
ATP, and 30 ng of wild-type human recombinant EGFR
expressed in sf9 insect cells. The enzyme incubated with
1 lM concentrations of each derivative as well as the
standard gefitinib for 10 min at room temperature prior to
the kinase reaction. Kinase reactions were initiated by
adding ATP and substrate and incubated for 25 min at
room temperature. Then, 20 lL ADP-GloTM Reagent
(promega) was added to stop the kinase reaction and the
reaction mixture was incubated for 50 min. After adding
the kinase detection reagent, the EGFR activity was
determined by measuring the luminescence of the reaction
mixture. Data are presented as the means of three inde-
pendent experiments.
Biological evaluation
Cell viability MTT assay
A549, MCF-7, HepG2 and A431 cell lines were purchased
from Korean cell line bank (KCLB). Cell lines were grown
under humidified atmosphere containing 5% CO2 at 37 °C
with Dulbecco’s Modified Eagle’s Medium (DMEM) and
10% (v/v) heat inactivated fetal bovine serum (FBS). Cells
were placed into 96-well plates at a density of 5 9 103
cells/mL per well, treated with compounds to be tested.
After incubation for 2 days under humidified atmosphere
containing 5% CO2 at 37 °C, the cells were harvested,
treated with 10 lL of Dye Solution (Cell Titer 96, Pro-
mega, Madison, WI) and incubated at 37 °C under 5% CO2
for 4 h. Then, 100 lL of solubilization solution/stop mix
was added and the plates were left to stand for overnight.
The absorbance at 570 nm was measured with a microplate
reader (VersaMax,Molecular Devices, and Sunnyvale, CA,
Results
Convergent synthesis was adopted for preparation of the
targeted alkylphosphocholine-gefitinib conjugates (4).
First, gefitinib core tethered with linkers were synthesized
as shown in Scheme 1. The easily prepared 4-chloro-
quinazoline derivative 5 (Schwan et al. 2014) upon aro-
matic nucleophilic substitution with 3-chloro-4-
123

Design, synthesis and evaluation of alkylphosphocholine-gefitinib
Scheme 1 Reagents and conditions: a 3-chloro-4-fluoroaniline, reflux, 96%; b TFA, reflux, 98%; c K2CO3, ethyl bromoalkanoate, 62 °C,
89 * 71%; d LiOH, rt, 91 * 92%
fluoroaniline afforded the core of gefitinib 6 as 7-benzy-
loxy protected derivative. Deprotection with trifluoroacetic
acid yielded the hydroxyl derivative 7 for tethering with
linkers. Tethers were attached via alkylation with corre-
sponding ethyl bromoalkanoates to afford derivatives 8.
Hydrolysis with lithium hydroxide provided carboxylic
acids 9 ready for coupling with alkylphosphocholine
derivatives.
combination treatment for breast cancer (Gutteridge et al.
2010; Girgert et al. 2017; Wu et al. 2017), liver cancer
¨
(Hopfner et al. 2004; Desbois-Mouthon et al. 2006; Shao
et al. 2016) and skin cancer (Godugu et al. 2016).
Accordingly, for the evaluation of the antiproliferative
activity of the prepared gefitinib-APC conjugates, four cell
lines were employed representing lung, breast, liver and
skin cancers (non-small-cell lung adenocarcinoma cells
line A-549; breast adenocarcinoma cell line MCF-7, liver
hepatocellular carcinoma cell line HepG2, and epidermoid
squamous carcinoma cell line A-431). As shown in
Table 1, gefitinib shows high micromolar IC50 values
against these cell lines. Three reference standard anticancer
agents were used. Two of them (gefitinib and erlotinib) are
EGFR kinase inhibitors while the third (miltefosine) is a
standard alkylphosphocholine derivative. With regard to
the length of the linker’s chain, two series of compounds
were prepared and evaluated. The results are presented in
Table 1.
As shown in Scheme 2A, the benzyloxy derivatives 10
(Raghavan and Krishnaiah 2010; Verma et al. 2016) were
converted to benzyloxyalkylphosphocholines 11 via reac-
tion with 2-chloro-1,3,2-dioxaphospholane-2-oxide fol-
lowed by opening of the dioxaphospholane ring with
anhydrous trimethylamine. Hydrogenation affords the
unprotected hydroxyl derivatives 12 ready for conjugation
with the prepared carboxylic acids 9. Finally, as illustrated
in Scheme 2B, DCC-promoted coupling of carboxylic
acids 9 and hydroxyl derivatives 12 yielded the desired
alkylphosphocholine-gefitinib conjugates (4).
Gefitinib is primarily used for lung cancer treatment.
However, it has been also considered as a single or in
As alkylphosphocholines are known to inhibit Akt
phosphorylation (Alam et al. 2012, 2013), the ability of the
123

Md. Maqusood Alam et al.
A
B
Scheme 2 Reagents and conditions: a (i) 2-Chloro-1,3,2-dioxaphospholane-2-oxide, TEA, rt; (ii) TMA, 65 °C, 88 * 53% over 2 steps; b H2,
Pd/C, MeOH, rt, 96 * 92%; c DCC, DMAP, CHCl3, reflux, 88 * 50%
prepared gefitinib-APCs conjugates 4 to inhibit Akt phos-
phorylation were measured by cell based assay using mil-
tefosine as a reference standard. Thus, A549 cells
pretreated with 20 lM concentration of each compound or
the standard miltefosine were stimulated to undergo Akt
phosphorylation for 30 min after which cells were lysed
and phosphorylated Akt was assayed in the lysate to cal-
culate the percent inhibition of Akt phosphorylation. The
results are shown in Table 1.
remaining EGFR activity after treatment with 1 lM
concentrations of the most active conjugates series (4aa,
4ab and 4ac) and gefitinib was determined. The results
are in Table 1.
Discussion
The results, as indicated in Table 1, revealed that the
longer linker’s alkyl chain series (m = 6) possesses higher
activity than the shorter linker’s alkyl chain series (m = 1).
Among the used four cell lines, breast cancer cell line
MCF-7 showed the highest sensitivity to the prepared
Next to the assay of the ability of the prepared con-
jugates 4 to inhibit phosphorylation of Akt, determina-
tion of the inhibitory activity EGFR kinase reaction was
addressed. Using ADP-GloTM kinase assay kit, the
123

Design, synthesis and evaluation of alkylphosphocholine-gefitinib
Table 1 Cytotoxicity against human cancer cell lines, inhibition of Akt Phosphorylation and EGFR kinase reaction of gefitinib-APC
conjugates 4
Compd.
MCF-7
% GIa
A431
% GI
HepG2
% GI
A549
% GI
% Akt Phos. Inhib.c
% EGFR Inhib.d
ICb50
IC50
IC50
IC50
Gefitinib
Erlotinib
Miltefosine
4aa
89.2
33.2
31.2
53.6
54.4
45.8
6.2
40.1
–
75.8
47.0
35.3
48.6
19.8
46.4
15.9
17.5
12.1
36.1
–
92.9
36.7
31.2
29.1
36.8
19.3
6.9
26.7
–
89.2
48.2
36.6
29.5
31.7
13.4
–
40.7
–
–
98.7 0.8
–
–
–
–
–
–
87.0 4.1
26.6 7.5
28.1 1.8
31.3 5.4
8.3 3.6
8.4 3.6
3.1 2.3
–
–
–
–
–
87.4 0.4
4ab
–
–
–
–
75.5 2.6
4ac
–
–
–
–
70.0 2.0
4ba
–
–
–
–
–
–
–
4bb
1.6
–
–
10.9
–
–
14.2
–
–
4bc
14.9
–
–
–
–
a% Growth inhibition at 100 lM
bMicromolar concentration that inhibits growth by 50%
cPercent inhibition of Akt phosphorylation in A549 cells at 20 lM
dPercent inhibition of EGFR kinase at 1 lM
conjugates. The measured growth inhibition percent for
derivatives 4aa and 4ab was around 54% while it was
lower (& 46%) for derivative 4ac. Despite these values for
inhibition percent are lower than that of gefitinib, they are
still better than those for erlotinib and miltefosine. The
response of skin cancer cell line A431 to derivatives 4aa
and 4ac (around 49 and 46% respectively) was not far from
that response for erlotinib (47%), but significantly higher
than that of miltefosine (35%) and lower than that of
gefitinib (& 76%). In contrast, the response for derivative
4ab was weak in inducing inhibition of the proliferation of
A431 cell line. However, the response of liver cancer cell
line HepG2 to compound 4ab was almost similar to erlo-
tinib (& 37%) and higher than that for miltefosine but
lower than gefitinib. While the response of HepG2 cell line
to derivative 4ac was lower than all of responses to gefi-
tinib, erlotinib and miltefosine, it showed response to 4aa
near to miltefosine. The response of the last used cell line
which represent lung cancer A549 to derivatives 4aa, 4ab
and 4ac was weaker than responses to gefitinib, erlotinib
and miltefosine. Nevertheless, responses to derivatives 4aa
and 4ab were significant and they were lower by 5 * 7
percent units relative to the response to miltefosine. Col-
lectively, these results show that the profiles of derivatives
4aa, 4ab and 4ac over the tested cell lines are discrete from
the profile of gefitinib. They are more skewed toward the
profile of miltefosine, although they are still distinct.
On the other side, the responses to derivatives 4ba, 4bb,
and 4bc with the short linker were very weak over the used
four cell lines. The best measured response among this
series was 17.5% growth inhibition by derivative 4bb
against A431 cell line. Responses to this derivative by
A549, HepG2 and MCF-7 were as low as 14.2, 10.9 and
1.6% growth inhibition respectively. In spite that derivative
4bc did not elicit inhibition of growth of two cell lines
(HepG2 and A549), it showed the best percent of growth
inhibition (& 15%) among the short linker series against
MCF-7 cell line. It was also the least active among this
series against A431 cell line (& 12%). Also, derivative
4ba did not show any inhibition of A549 cell line and
showed weak inhibitions against MCF-7, HepG2 and A431
(around 6, 7 and 16% respectively). These results show that
the longer linker chain length are in favor of more
antiproliferative effect.
The assay of inhibition of Akt phosphorylation, as
indicated in Table 1, revealed that the standard miltefosine
effectively induced 87% inhibition of Akt phosphorylation.
Paradoxically, the prepared conjugates 4 were not effective
inhibitors of Akt phosphorylation. The maximum inhibi-
tory activity for Akt phosphorylation was 31.3% for
derivative 4ac. Even with the measured low inhibitions, the
results revealed that conjugates 4aa, 4ab and 4ac pos-
sessing the longer linker induce higher Akt inhibition
values than the corresponding conjugates 4ba, 4bb and 4bc
with the shorter linker. This difference in Akt phosphory-
lation inhibition is proportional to the observed difference
in the antiproliferative activity of both series. This might
indicates that the length of the linker is crucial for the
elicited inhibition of Akt phosphorylation. Development of
conjugates with better inhibitory activity might results in
more effective antiproliferative activity.
As revealed from the obtained results (Table 1), the
standard gefitinib inhibited almost all of the EGFR activity
(98.7%). The tested conjugates at the used concentration
produced high inhibitions of EGFR kinase reaction, albeit,
incomplete inhibitions was achieved. The highest achieved
123

Md. Maqusood Alam et al.
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inhibition value induced by conjugates was for the 4aa
with the smallest alkyl chain (87.4%). The EGFR inhibi-
tory activity decreases with increasing the chain length.
This is clear from the measured values for 4ab with the
longer chain length (75.5%) and for 4ac with the longest
chain length (70%). This is in inverse relation to the
revealed behavior of inhibition of Akt phosphorylation
which increases with increasing chain length (26.6, 28.1
and 31.3% for 4aa, 4ab and 4ac respectively). These
results may indicate a needed careful balancing between
the inhibition of both of Akt phosphorylation and EGFR
kinase reaction.
In summary, two series of novel conjugates of
alkylphosphocholines and gefitinib were designed and
prepared as multitarget antiproliferative agents. While the
conjugates series with short chain length linker elicited
weak antiproliferative efficacy, the conjugates series with
the longer chain length linker possessed efficacy compa-
rable to erlotinib and miltefosine, however, weaker than
gefitinib. Despite that, the performed study provides
insights on the balance between the inhibitions of the Akt
phosphorylation and EGFR kinase reaction in the studied
conjugates. This strategy might offer opportunities for
preparation of more effective chemical entities by imple-
menting the gained lessons from this study to achieve a
better design of multitarget antiproliferative agents.
Acknowledgements This research was supported by the ‘‘Basic
Science Research Program’’ through the ‘‘National Research Foun-
dation of Korea (NRF)’’ funded by MEST, Republic of Korea (#2012-
006431).
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J, Gee J (2010) The effects of gefitinib in tamoxifen-resistant and
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
¨
Hopfner M, Sutter AP, Huether A, Schuppan D, Zeitz M, Scherubl H
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