Design, synthesis and cytotoxicity of chimeric erlotinib-alkylphospholipid hybrids

Article abstract of DOI:10.1016/j.bioorg.2018.11.021

Two series of erlotinib-alkylphospholipid hybrids were prepared and evaluated for their antiproliferative activities against a panel of four cell lines representing lung, breast, liver and skin cancers using erlotinib and miltefosine as reference standards. Amide analogs elicited more enhanced cytotoxic activity than analogous esters. Amide derivatives 8d and 8e exhibited promising broad-spectrum antiproliferative activity and higher efficacy than reference erlotinib and miltefosine. Their cellular GI₅₀ values was in the ranges of 24.7–46.9 μM and 26.8–43.1 μM for 8e and 8d respectively. Assay results of the inhibitory activity of the prepared compounds on EGFR kinase reaction and Akt phosphorylation in conjugation with statistical correlation analysis indicated that other mechanisms might contribute to their elicited cytotoxicities. In addition, statistical correlation analysis revealed that mechanisms of elicited cytotoxicities for amide series might be different from ester series. In addition, correlation analysis indicated variations in the mechanisms according to the types of cell line.

Full text of DOI:10.1016/j.bioorg.2018.11.021

BioorganicChemistry84(2019)51–62
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and cytotoxicity of chimeric erlotinib-alkylphospholipid
hybrids
Md. Maqusood Alama,1, Ahmed H.E. Hassanb,c,1, Kun Won Leea, Min Chang Choa, Ji Seul Yanga,
⁎
Jiho Songd, Kyung Hoon Mind, Jongki Hongb, Dong-Hyun Kima, Yong Sup Leea,b,e,
a Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea
b Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
c Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
d Department of Pharmacy, College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea
e KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul 02447, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two series of erlotinib-alkylphospholipid hybrids were prepared and evaluated for their antiproliferative ac-
tivities against a panel of four cell lines representing lung, breast, liver and skin cancers using erlotinib and
miltefosine as reference standards. Amide analogs elicited more enhanced cytotoxic activity than analogous
esters. Amide derivatives 8d and 8e exhibited promising broad-spectrum antiproliferative activity and higher
efficacy than reference erlotinib and miltefosine. Their cellular GI50 values was in the ranges of 24.7–46.9 μM
and 26.8–43.1 μM for 8e and 8d respectively. Assay results of the inhibitory activity of the prepared compounds
on EGFR kinase reaction and Akt phosphorylation in conjugation with statistical correlation analysis indicated
that other mechanisms might contribute to their elicited cytotoxicities. In addition, statistical correlation ana-
lysis revealed that mechanisms of elicited cytotoxicities for amide series might be different from ester series. In
addition, correlation analysis indicated variations in the mechanisms according to the types of cell line.
Epidermal growth factor receptor (EGFR)
Akt phosphorylation
Erlotinib
Alkylphospholipids (APL)
1. Introduction
metastatic non-small cell lung cancer [13]. Unfortunately, tumors het-
erogeneity and evolving resistance limit its effectiveness [14–19].
Cancer is an arduous obstacle and a major cause for mortality in
both developed and developing countries [1,2]. Over the past decades,
considerable understanding and development of anticancer therapies
were achieved. Nevertheless, statistical analysis of the global burden of
disease (GBD) demonstrates that cancer is still the second leading cause
of death [3]. Alarmingly, the lifetime risk of cancer has increased to
53.5% for men and 47.5% for women in some populations [4]. Con-
sequently, there is an urgent need for development of more effective
antineoplastic therapies.
Combination and multitarget therapies are two approaches to overcome
resistant tumors via simultaneous inhibition of multiple pathological
pathways [20,21]. In addition, this would produce higher efficacy and
reduce side effects. A single multitarget molecule offers pharmacoki-
netic and pharmacodynamics advantages over combination therapy
[22]. The clinical agent CUDC-101 (2, Fig. 1) inhibits EGFR and histone
deacetylase (HDAC) simultaneously [23,24]. However, HDAC in-
hibitors are known to induce multidrug resistance (MDR), via up-reg-
ulation of adenosine triphosphate binding cassette (ABC) transporters
[25]. It seems that CUDC-101 designed to overcome resistance could
induce resistance [26]. An alternative design of a multitarget agent
might be needed.
Epidermal-growth factor receptor (known as EGFR, HER1, or
ErbB1) is a member of tyrosine kinase family involved in several signal
transduction pathways impacting cells’ proliferation and survival in-
cluding RAS/RAF/MAPK, and PI3k/PIP3/AKT pathways [5–7]. In fact,
many efforts to develop EGFR targeted anticancer therapies were re-
ported [7–12]. Erlotinib 1 (Tarceva®, OSI-744) is a FDA-approved EGFR
inhibitor for treatment and maintenance treatment of non-small cell
lung cancer, treatment of pancreatic cancer, and first-line treatment of
Alkylphospholipids (APLs) are known to possess antitumor activities
[27–31]. They inhibit Akt signaling pathway, which is a downstream of
EGFR signaling pathway. In addition, they integrate into cell membrane
changing its properties, trafficking and signaling. Furthermore, they
interfere with lipids metabolism and lipids-dependent signaling
⁎ Corresponding author at: Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea.
E-mail address: kyslee@khu.ac.kr (Y.S. Lee).
1 Both authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2018.11.021
Received 31 May 2018; Received in revised form 3 November 2018; Accepted 17 November 2018
Availableonline19November2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
Fig. 1. Structures of reported compounds.
cascades [30–33]. Miltefosine 3 (Miltex®) and edelfosine 4 (ET-18-
OCH3) are prominent antitumor lipids. Unfortunately, miltefosine 4
was approved only as a topical therapy [34] while edelfosine 4 is
limited to eradication of bone marrow in acute leukemia [32]. Recently,
we have reported compounds (5) in which the EGFR inhibitor gefitinib
was conjugated to the terminus of the alkyl chain of alkylpho-
sphocholines in attempt to develop multitarget anticancer agents [35].
However, the investigated molecules showed no improved anti-
proliferative activity. Hence, different design approach might be re-
quired to access more effective conjugates.
The literature report the antitumor 6b (Fig. 2) in which the anti-
tumor alkylphospholipids 6a were conjugated with chlorambucil at sn-
2 position of the glycerol moiety [36]. These conjugates were claimed
to undergo enzymatic hydrolysis releasing two anticancer agents. To
recruit the unique properties of EGFR inhibitors and APLs in a single
chemical entity, hybrid compounds 7 and 8, in which APLs 6a were
conjugated with the pharmacophoric moiety of erlotinib 1 via an ester
or amide functionality at sn-2 position were designed and synthesized.
The idea is that chimeric compounds 7 and 8 might combine EGFR
inhibitors and APLs mechanisms of actions. As a multitarget agent, if
one mechanism fails, other mechanisms would work. This might result
in a broader activity spectrum and a lower resistance liability. More-
over, if hydrolysis takes place at the sn-2 position, two mechanistically
distinct antitumor drugs would be released. The designed hybrid mo-
lecules would have APL moieties of variable alkyl chain length to in-
vestigate its impact on the elicited biological activity. Herein, we would
like to report our results.
Fig. 2. Design of Chimeric molecules 7 and 8 via linking the pharmacophoric
moiety of erlotinib 1 with antitumor phospholipids 6a.
pharmacophoric moiety of erlotinib (16) was prepared by synthetic
steps outlined in Scheme 1. First, alkylation of ethyl 4-hydroxy-3-
methoxybenzoate 9 with ethyl 6-bromohexanoate installed the spacer
moiety to the benzoate derivative 10 which was then converted to
quinazoline derivative 13 via sequential reactions of nitration to nitro-
derivative 11, reduction to amino-derivative 12, then condensation/
cyclization to quinazoline derivative 13. The cyclized quinazoline de-
rivative 13 was converted to chloro-derivative 14 then aromatic
2. Results and discussion
2.1. Chemistry
Hybrid molecules 7a–g, 8a, 8b and 8d–g were prepared con-
vergently as shown in Schemes 1 and 2. The unreported spacer-attached
52

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
Scheme 1. Reagents and conditions: (a) Ethyl 6-bromohexanoate, K2CO3, rt, 99%; (b) HNO3, AcOH, 20 °C, 99%; (c) Fe, HCl, EtOH, reflux, 85%; (d) HCONH2,
HCOONH4, 180 °C, 85%; (e) POCl3, reflux, 93%; (f) 3-Ethynylaniline, i-PrOH, reflux, 95%; (g) LiOH, THF/H2O, rt, 96%.
nucleophilic substitution afforded quinazoline derivative 15 having
erlotinib’s pharmacophore. Alkaline hydrolysis of ester derivative 15
afforded carboxylic acid derivative 16 ready for conjugation with sn-2-
hydroxy-alkylphospholipid derivatives 20a–g and sn-2-amino-alkyl-
phospholipid 22a, 22b and 22d–g.
alkyl bromides followed by acid-catalyzed ring cleavage to yield the
intermediate diol derivatives 18a–g. Similar to literature report [39],
trityl protection of the primary hydroxyl group followed by benzylation
then trityl deprotection afforded 2-(benzyloxy)-3-alkoxypropan-1-ol
derivatives 19a–g. Following reported procedure [38], esterification of
alcohol derivatives 19a–g with 2-chloro-1,3,2-dioxaphospholane-2-
oxide then ring opening with trimethylamine followed by palladium
catalyzed hydrogenation afforded sn-2-hydroxy-alkylphospholipid de-
rivatives 20a–g.
Synthesis of sn-2-hydroxy-alkylphospholipid derivatives 20a–g is
illustrated in Scheme 2A. Analogous to literature reports [37,38], DL-
1,2-isopropylideneglycerol 17 was converted to 3-alkoxypropane-1,2-
diol derivatives 18a–g via O-alkylation with different chain lengths
Scheme 2. Reagents and conditions: (a) (i) NaH, Bromoalkane, Toulene, reflux; (ii) 2 N HCl, MeOH, reflux, 88–95% over 2 steps; (b) (i) TrCl, Py, 50 °C; (ii) BnBr,
NaH, DMF, 0 °C to rt; (iii) p-Toluenesulfonic acid, CH2Cl2/MeOH, rt, 50–57% over 3 steps; (c) (i) 2-Chloro-1,3,2-dioxaphospholane-2-oxide, NEt3, benzene, rt; (ii)
TMA, CH3CN, 65 °C; (iii) H2, Pd/C, MeOH, rt, 70–88% over 3 steps; (d) (i) TrCl, Py, 50 °C; (ii) MsCl, NEt3, CH2Cl2, 0 °C to rt; (iii) NaN3, DMF, 120 °C; (iv) 1 N HCl,
Acetone, reflux, 42–50% over 4 steps; (e) DCC, DMAP, CHCl3, reflux, 60–80%.
53

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
Adopting the reported procedure [38], sn-2-amino-alkylpho-
spholipid derivatives 22a, 22b and 22d–g were synthesized as illu-
strated in Scheme 2B. Thus, 3-alkoxypropane-1,2-diol derivatives 18a,
18b and 18d–g prepared in Scheme 2A, were subjected to trityl pro-
tection of the primary hydroxyl group, activation of the sn-2-hydroxy
group as mesylate ester, displacement by azide followed by acid in-
duced trityl deprotection to afford 3-alkoxy-2-azidopropan-1-ol deri-
vatives 21a, 21b and 21d–g. Esterification of azido-alcohol derivatives
21a, 21b and 21d–g with 2-chloro-1,3,2-dioxaphospholane-2-oxide
then ring opening with trimethylamine followed by palladium cata-
lyzed hydrogenation afforded sn-2-amino-alkylphospholipid derivatives
22a, 22b and 22d–g.
erlotinib-APL hybrids. As shown in Table 1 and outlined in Fig. 3, at
100 μM concentration, erlotinib and miltefosine produced limited in-
hibition (39.4% and 36.6% respectively). Amide-linked erlotinib-APL
hybrids with long alkyl chain at the sn-3 position effectively inhibited
A-549 cells proliferation (analogs 8d, 8e, 8f, 8g with sn-3 alkyl chain of
16, 18, 20, and 22 carbons elicited 94.4%, 94.1%, 84.8% and 92.8%
growth inhibition respectively). The corresponding ester analogs, was
much less active, except for analog 7e having 18-carbon length alkyl
chain substitution at sn-3. The latter elicited remarkable, but inferior
inhibitory efficiency than the corresponding amide analog (77.1% and
94.1% inhibition for ester and amide analog respectively). Derivatives
with sn-3 alkyl chain equal to/or less than 13 carbons inhibited A-549
growth less effectively. Nevertheless, their efficacy was comparable to
reference standards. Concentrations producing 50% growth inhibition
Scheme 2C shows the final coupling step of hydroxy 20a–g and
amino 22a, 22b and 22d–g intermediates with carboxylic acid deri-
vative 16 obtained in Scheme 1 using N,N′-dicyclohexylcarbodiimide as
a coupling agent in presence of stoichiometric amounts of 4-dimethy-
laminopyridine to yield the coupled compounds 7a–g, 8a, 8b and 8d–g.
(GI50
) for the effective amide analogs were in the range of
43.1–55.8 μM.
Breast Cancer is the top cancer afflicting females and has the second
highest premature-mortality costs [40–42]. Incongruous with expres-
sion of EGFR in numerous breast cancers, erlotinib, as well as, other
EGFR inhibitors are not effective for breast cancers [45,46]. Interest-
ingly, elevated lipid rafts levels are found in breast malignancies [47].
Miltefosine has been approved topically for cutaneous metastatic breast
cancer [34]. Therefore, the prepared compounds and reference stan-
dards were evaluated at 100 μM concentrations against a breast cancer
cell line model. As shown in Table 1, erlotinib produced modest efficacy
against the employed breast cancer MCF-7 cell line (28.7% inhibition).
In contrast, miltefosine was more active producing average inhibition
of growth of MCF-7 cell line (69.4% inhibition). With exception of ester
derivatives 7a, 7f and 7g, all other tested compounds elicited activities
comparable to/or higher than miltefosine. As illustrated in Fig. 4, amide
analogs having long alkyl sn-3 substituent were highly effective (93.4%,
93.1%, 89.0%, and 90.7% inhibition for 8d, 8e, 8f, and 8g respec-
tively). Amide derivatives 8e and 8d were the most potent among
tested derivatives having GI50 values of 24.7 and 26.8 μM respectively.
Amide derivatives 8f and 8g having longer alkyl chains were less potent
eliciting GI50 values of 38.6 and 46.6 μM respectively. Among ester
analogs, only compound 7e showed significantly superior inhibition to
miltefosine (93.1% inhibition and GI50 = 53.19 μM).
2.2. Biological evaluation
2.2.1. Inhibition of cell line proliferation:
MTT cell proliferation assay (conducted in triplicates) was used to
evaluate the antiproliferative activities of the newly synthesized erlo-
tinib-APL hybrids against four human cancer cells (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). Erlotinib 1 and miltefosine 4
were used as reference standards. All of the employed cell lines elicited
limited response to erlotinib 1 (maximum percent growth inhibition at
100 μM concentration was not more than 42.3% for the best inhibited
cell line; Table 1).
Among all cancers, lung cancer is the leading cause of death and has
the highest premature cancer-related mortality cost [3,40–42]. Non-
small-cell lung cancer is the most frequent type of lung cancer (85% of
lung cancer cases). Erlotinib was approved for treatment of non-small
cell lung cancer and first-line treatment of metastatic non-small cell
lung cancer [13,43]. Nonetheless, erlotinib-resistance frequently en-
countered [43]. Additionally, insensitivity of non-small-cell lung ade-
nocarcinoma cell line A-549 to APL edelfosine was reported [44].
Therefore, cell line A-549 was used to assess efficacies of prepared
Hepatocellular carcinoma is the first primary liver cancer and the
third leading cause of death among cancer mortalities [48]. Patients
Table 1
In vitro cytotoxicity of erlotinib-APL hybrid compounds 7 and 8 against selected human cancer cells.
Comp
X
n
A-549a
MCF-7b
HepG2c
A-431d
% Inhibitione
GI50
% Inhibitione
GI50
% Inhibitione
GI50
% Inhibitione
GI50
ND
f
f
f
f
7a
7b
7c
7d
7e
7f
O
7
34.8
33.5
26.5
15.5
77.1
28.1
31.4
29.1
38.5
94.4
94.1
84.8
92.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
48.4
69.0
68.6
75.4
93.1
27.5
35.3
68.9
66.0
93.4
93.1
89.0
90.7
ND
ND
ND
ND
46.4
62.4
62.6
28.8
85.4
54.5
34.4
42.8
51.7
92.9
92.8
57.1
91.7
ND
ND
ND
ND
58.1
76.4
78.6
70.5
58.1
42.7
24.7
76.9
74.9
92.9
ND
O
11
12
15
17
19
21
7
71.9
65.5
55.5
ND
O
O
O
53.2
ND
58.3
ND
O
ND
7g
8a
8b
8d
8e
8f
O
ND
ND
ND
NH
NH
NH
NH
NH
NH
ND
ND
60.7
68.3
32.1
ND
11
15
17
19
21
ND
ND
43.1
46.9
55.8
53.6
26.8
24.7
38.6
46.6
35.4
31.3
ND
87.9
46.1
8g
93.2
44.0
Erlotinib
39.4
ND
28.7
ND
31.1
ND
ND
42.3
35.4
ND
ND
Miltefosine
36.6
ND
69.4
ND
31.3
a
Human lung cancer cells.
b
c
d
e
f
Human hepatocellular carcinoma cells.
Human breast carcinoma cells.
Human epidermoid carcinoma cells.
% inhibition at 100 μM.
GI50 was defined as the concentration resulting in 50% inhibition of cell proliferation.
54

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
Fig. 3. Percentage inhibition of A-549 cell line proliferation after treatment with erlotinib-APL hybrid compounds 7 and 8, erlotinib and miltefosine at 100 μM.
Dotted red-line represents inhibition percentage of erlotinib treated cell line while dashed green-line represents inhibition percentage of miltefosine treated cell line.
have average survival time of 6–20 months with less than 5% prob-
ability of 5-years survival. According to the clinical study of Thomas
et al., 71% of the patients showed high EGFR expression [49]. Para-
doxically, erlotinib treatment did not show significant difference be-
tween high and non-high EGFR expressing patients. Role of lipids in
hepatocellular carcinoma and up-regulation of Akt signals have been
confirmed [50]. At high concentrations, miltefosine elicited anti-
proliferative activity on hepatocellular carcinoma HepG2 cell line [51].
Therefore, the growth-inhibitory activity of the synthesized erlotinib-
APL hybrids was assessed using hepatic cancer HepG2 cell line model.
At 100 μM concentrations both of erlotinib and miltefosine produced
almost similar low efficacy level (31.1% and 31.3% inhibition respec-
tively). As shown in Fig. 5, four derivatives exhibited high efficacy
(85.4%, 92.9%, 92.8% and 91.7% inhibition for ester analog 7e and
amide analogs 8d, 8e and 8g respectively). These four derivatives were
also the most effective derivatives against A-549 and MCF-7 cell lines.
Measured GI50 values were 35.4, 31.3 and 58.3 μM for 8d, 8e and 7e
respectively. The remaining tested amide and ester derivatives were
less effective as antiproliferative agents, but elicited higher efficacy
than the reference standards. The only exception was derivative 7d
which elicited efficacy slightly lower than erlotinib and miltefosine.
Epidermal squamous cells carcinoma; subclass of non-melanoma
Fig. 4. Percentage inhibition of MCF-7 cell line proliferation after treatment with erlotinib-APL hybrid compounds 7 and 8, erlotinib and miltefosine at 100 μM.
Dotted red-line represents inhibition percentage of erlotinib treated cell line while dashed green-line represents inhibition percentage of miltefosine treated cell line.
55

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
Fig. 5. Percentage inhibition of HepG2 cell line proliferation after treatment with erlotinib-APL hybrid compounds 7 and 8, erlotinib and miltefosine at 100 μM.
Dotted red-line represents inhibition percentage of erlotinib treated cell line while dashed green-line represents inhibition percentage of miltefosine treated cell line.
Fig. 6. Percentage inhibition of A-431 cell line proliferation after treatment with erlotinib-APL hybrid compounds 7 and 8, erlotinib and miltefosine at 100 μM.
Dotted red-line represents inhibition percentage of erlotinib treated cell line while dashed green-line represents inhibition percentage of miltefosine treated cell line.
skin cancer (NMSC) is a widespread skin cancer among white people
[52]. Over the last fifty years, there was no achievement in decreasing
the mortality rate of skin cancer [53]. Targeted therapies hold promise
for increasing cure rates and diminishing excision of adjoined healthy
tissue [52]. Despite A-431 cells (a cell line model of epidermal squa-
mous cells carcinoma) overexpresses EGFR and responds to erlotinib,
exposure over 6 months results in erlotinib-resistant cells. This was at-
tributed to evolvement of constitutively active Akt pathway [54]. As
Akt signaling pathway is linked with lipid rafts, the efficacy of the
prepared derivatives was assessed using A-431 cell line. Interestingly,
the pattern of growth inhibition of A-431 cells differed from that of
other three employed cell lines. As illustrated in Fig. 6, at 100 μM
concentration, both erlotinib and miltefosine produced average low
inhibition responses of 42.3% and 35.4%. All tested derivatives other
than ester analogs 7g and 7f were more effective than erlotinib and
miltefosine. While the most effective ester analog among tested ester
analogs against A-549, MCF-7 and HepG2 cell lines was derivative 7e, it
was less active against A-431 cell line than ester analogs 7c, 7b, 7d and
7a which have shorter alkyl. Ester analog 7c elicited the highest effi-
ciency among ester derivatives producing (78.6% inhibition), while 7d
was the most potent among ester analogs (GI50 = 55.5 μM). Accord-
ingly, increasing alkyl chain length at sn-3 position of ester analogs
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Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
Table 2
analog 8a respectively). Although active as EGFR inhibitors, it was
surprisingly to note that analogs 7e, 8d, 8f and 8g which were char-
acterized by pronounced activities in performed cell line proliferation
assays, in comparison with other compounds, demonstrated lower in-
hibitory activities of EGFR kinase reaction. This might indicate a lim-
ited contribution of their EGFR inhibitory activities to their overall
elicited growth inhibitory activities.
The result of erlotinib-APL hybrid compounds 7 and 8 inhibitory assays using
enzymatic EGFR kinase assay and cellular Akt phosphorylation inhibition assay
in MCF-7 cell line.
Compound
X
n
% EGFR
IC50 (nM) % AKT
IC50 (µM)
inhibitiona
inhibitionb
7a
7b
7c
7d
7e
7f
O
O
O
O
O
O
O
N
N
N
N
N
N
7
91.3
48.0
ND
ND
ND
ND
ND
ND
21.0
ND
ND
ND
ND
ND
33.0
ND
ND
47.4
ND
ND
ND
ND
ND
ND
ND
36.2
ND
ND
11 78.1
12 56.9
15 77.5
17 61.8
19 89.0
21 40.9
9.4
2.2.3. Inhibition of Akt phosphorylation assay
41.8
16.2
11.6
14.6
12.4
13.3
27.8
35.5
56.8
26.1
20.2
Alkylphospholipids (APLs) were reported to inhibit Akt phosphor-
ylation [55,56]. Consequently, the Akt phosphorylation inhibitory ac-
tivities of the prepared erlotinib-APL hybrid derivatives 7a–g, 8a, 8b,
and 8d-g were evaluated in triplicates by cell based assay using mil-
tefosine and erlotinib as reference compounds. Thus, cells pretreated
with each compound be tested at 20 µM concentration were stimulated
for Akt phosphorylation followed by assay of phosphorylated Akt in cell
lysate. For compounds that demonstrated more than 40% inhibition,
IC50 was determined. As Table 2 indicates, standard reference milte-
fosine showed 81.4% inhibition while erlotinib elicited only 35.2%
downstream inhibition of Akt phosphorylation. Most of the tested
compounds showed inhibition of Akt phosphorylation lower than er-
lotinib and miltefosine. However, compounds 7c and 8e (IC50 values of
47.4, 36.2 µM, respectively) were more effective than erlotinib, but less
potent than miltefosine as Akt phosphorylation inhibitors. Generally,
amide derivatives elicited relatively higher efficiency than analogous
ester derivatives. Notably, derivatives 8d and 8e which have remark-
able activities in performed cell line proliferation assays, showed rela-
tively higher Akt phosphorylation inhibition, however, much lower
than that of miltefosine. In addition, derivative 7c which had Akt-
phosphorylation inhibitory activity relatively higher than derivative
8d, elicited antiproliferative activity lower than that of derivative 8d in
all tested four cell lines. This might indicate a limited contribution of
Akt phosphorylation inhibitory activity of these compounds to their
overall elicited antiproliferative activity.
7g
8a
8b
8d
8e
8f
7
93.5
11 87.2
15 63.6
17 ND
19 76.4
21 50.2
8g
Miltefosine
ND
ND
81.4
20.0
Erlotinib
97.6
3.0
35.2
ND
a
b
% EGFR at a single dose concentration of 1 µM.
% AKT phosphorylation inhibition at a single dose concentration of 20 µM.
decreases the efficacy in A-431 cell line. In contrast, the higher alkyl
chain length did not impair antiproliferative effectiveness of amide
analogs. In fact, the most effective amide analogs were derivatives 8g
and 8d where sn-3 was substituted with 22 and 16 carbon chains re-
spectively (92.9% and 93.2% inhibition and GI50 = 32.1 and 44.0 μM
for 8d and 8g respectively). Other tested amide analogs were also re-
markably more efficient (74.9–87.9% inhibition) than reference stan-
dards (42.3% and 35.4 for erlotinib and miltefosine respectively).
2.2.2. EGFR assay
The remaining EGFR kinase activity after treatment with tested
compounds and erlotinib was determined in triplicates using ADP-Glo™
kinase assay kit. The mean percentage inhibition was calculated re-
lative to the measured kinase activity of untreated EGFR and presented
in Table 2. For compounds elicited more than 90% inhibition of the
kinase reaction at 1 µM concentration, IC50 were determined and are
presented in Table 2. The standard reference erlotinib exhibited 97.6%
inhibition at 1 µM concentration and IC50 value of 3.0 nM. At 1 µM
concentration, all prepared compounds were less effective than erlo-
tinib. Generally, the amide analogs were more active. The inhibition
percentage decreased upon increasing length of the alkyl chain sub-
stituent at sn-3 position. Only two compounds, 7a and 8a, with the
smaller alkyl chain substituent of 8 carbons, exhibited inhibition per-
centage more than 90%. However, their determined IC50 was much
higher than erlotinib (48 and 21 nM for ester derivative 7a and amide
2.2.4. Statistical and computational study
For better understanding of relationship between inhibition of EGFR
and Akt-phosphorylation to the elicited antiproliferative activity, sta-
tistical analysis was performed. Thus, single and multiple correlation
coefficients, in addition to regression coefficients for multiple correla-
tion of EGFR kinase inhibition and Akt-phosphorylation inhibition as-
says as two independent variables, with elicited antiproliferative ac-
tivities as a dependent variable, were calculated. Correlations were
calculated overall the series of erlotinib-APL series, the ester derivatives
only, and the amide analogs only to detect differences between ester
and amide analogs (if any).
As presented in Table 3, the calculation results revealed profile
differences between cell lines, in addition to difference between ester
Table 3
Calculated multiple, single correlation coefficients and partial regression coefficients of EGFR kinase inhibition and/or Akt-phosphorylation inhibition assays with
elicited antiproliferative activities of erlotinib-APL hybrid compounds.
Cell line
Multiple Correlation
Regression Coefficient of
Regression Coefficient of
Single EGFR Correlation
Single Akt Correlation
Coefficient
EGFR
Akt
Coefficient
Coefficient
A-549
Overall Series
Ester analogs
0.39918
0.32351
−0.60502
−0.19635
−1.39974
0.459928
−0.40371
0.99983
−0.36091
−0.19665
−0.87365
0.18763
−0.26595
0.54747
Amide analogs 0.91196
MCF-7 Overall Series
0.27152
−0.28273
−0.16724
−0.62079
0.31924
−0.02466
0.17237
−0.22303
−0.13154
−0.76210
0.16534
−0.01933
0.35273
Ester analogs
0.13219
Amide analogs 0.76801
HepG2 Overall Series
0.41590
−0.51216
−0.03380
−1.45247
0.28996
−0.02246
0.07243
−0.39206
−0.03302
−0.88396
0.15738
−0.01611
0.32494
Ester analogs
0.03608
Amide analogs 0.88420
A-431
Overall Series
0.45200
0.15264
0.30527
−0.35882
0.85060
0.56365
0.11543
0.10308
0.30544
−0.94009
0.43465
0.37474
0.55974
Ester analogs
0.47258
Amide analogs 0.97280
57

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
and amide analogs. These might be a reflection of variation of the un-
derlying mechanisms responsible for activities of ester and amide
analogs, as well as, differences of the biological machinery of different
cell line. For A-549, MCF-7, and HepG2 cell lines; both of EGFR kinase
and Akt-phosphorylation inhibitory activities did not show a sig-
nificantly indicative correlation to the elicited antiproliferative activity.
In these three cell lines, ester analogs had small negative correlation
coefficient and small negative regression coefficients for both of their
EGFR and Akt-phosphorylation inhibitory activities. In contrast, for A-
431 cell line, ester analogs showed significant, however low, positive
correlation coefficient and positive regression coefficient for both of
their EGFR inhibitory and Akt-phosphorylation activities. Accordingly,
inhibition of EGFR and Akt-phosphorylation might contribute to the
elicited antiproliferative activity of ester analogs in A-431 cell line, but
ruled out in the other three cell lines. For amide analogs, statistical
analysis predicted negative correlation to EGFR inhibition. In contrast,
inhibition of Akt-phosphorylation showed significant, however average
to low positive correlation. Therefore, in the four cell lines, inhibition of
EGFR by amide analogs does not contribute to their antiproliferative
activity while inhibition of Akt-phosphorylation does. The calculated
average to low correlations for ester and amide analogs indicate that, in
addition to inhibition of EGFR and Akt phosphorylation, other me-
chanisms might also contribute to the elicited cytotoxicities.
4.1.1. Ethyl 4-(6-ethoxy-6-oxohexyloxy)-3-methoxybenzoate (10)
A
mixture of ethyl 4-hydroxy-3-methoxybenzoate 9 (5.0 g,
25.51 mmol), potassium carbonate (10.5 g, 76.5 mmol) and ethyl 6-
bromohexanoate (6.8 g, 30.6 mmol) in DMF (20 mL) was stirred over-
night at rt. The reaction mixture was filtered and concentrated in va-
cuum. The residue was dissolved in dichloromethane, and washed with
brine. The organic layer was dried over magnesium sulfate, filtered and
concentrated to yield derivative 10. Yield 8.6 g, 99%; 1H NMR
(400 MHz, DMSO-d6)
δ 1.18 (t, 3H, J = 7.0 Hz), 1.31 (t, 3H,
J = 7.0 Hz), 1.44–1.40 (m, 2H), 1.63–1.56 (m, 2H), 1.78–1.71 (m, 2H),
2.31 (t, 3H, J = 7.4 Hz), 3.82 (s, 3H), 4.08–4.00 (m, 4H), 4.28 (q, 2H,
J = 7.2 Hz), 7.04 (d, 1H, J = 8.4 Hz), 7.44 (d, 1H, J = 1.8), 7.56 (dd,
1H, J1 = 8.5 Hz, J2 = 2.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ 172.8,
165.4, 152.3, 148.5, 123.0, 121.9, 111.9 (2C), 68.1, 60.3, 59.6, 55.5,
33.4, 28.2, 25.0, 24.2, 14.2, 14.0; HR-MS m/z 339.1797 (calcd for
C
18H27O6 [M + H]+, 339.1802).
4.1.2. Ethyl 4-(6-Ethoxy-6-oxohexyloxy)-5-methoxy-2-nitrobenzoate (11)
To a stirred solution of compound 10 (8.5 g, 25.14 mmol) in acetic
acid (25 mL) at 20 °C, fuming nitric acid (15.8 g, 251.4 mmol) was
added dropwise. The reaction mixture was stirred at 20 °C for 1 h,
poured into ice-water, and extracted with dichloromethane. The com-
bined organic layer was washed with water, aqueous NaHCO3, brine;
dried over sodium sulfate; filtered and concentrated to give derivative
11. Yield 9.6 g, 99%; 1H NMR (400 MHz, DMSO-d6) δ 1.17 (t, 3H,
J = 7.2 Hz), 1.27 (t, 3H, J = 7.0 Hz), 1.44–1.39 (m, 2H), 1.61–1.59 (m,
2H), 1.79–1.72 (m, 2H), 2.31 (t, 3H, J = 7.2 Hz), 3.92 (s, 3H),
4.11–4.02 (m, 4H), 4.30 (q, 2H, J = 7.2 Hz), 7.29 (s, 1H), 7.62 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ 172.8, 164.9, 152.3, 149.5, 140.8,
120.5, 111.1, 108.0, 68.9, 61.8, 59.6, 33.4, 28.0, 24.9, 24.1, 14.1, 13.6;
HR-MS m/z 384.1696 (calcd for C18H26NO8 [M + H]+, 384.1653).
3. Conclusion
In this study, two series of erlotinib-APL hybrids have been prepared
and biologically evaluated as cytotoxic agents. In comparison with ester
derivatives, the amide series was more effective in inhibiting the
growth of the tested cell lines. Among them, analogs 8d, 8e, 8f and 8g
had the best efficacy in most of the employed cell lines. Particularly, 8d
and 8e were the most potent with cellular GI50 ranges of 24.7–46.9 μM
and 26.8–43.1 μM for 8e and 8d respectively. Compound 7e elicited the
best efficacy among ester analogs in three cell lines out of four em-
ployed cell lines. However, in A-431 cell line, 7b, 7c, and 7d were more
effective. Assay of the prepared compounds’ inhibitory activity for
EGFR kinase reaction and Akt-phosphorylation did not fully explain the
measured cytotoxicities. Statistical analysis of the results revealed low
contribution for inhibition of EGFR kinase reaction and Akt-phosphor-
ylation to the elicited activities, as well as, the possibility of the pre-
sence of other mechanisms mediating activities of ester and amide
analogs with variation of employed cell line.
4.1.3. Ethyl
2-amino-4-(6-ethoxy-6-oxohexyloxy)-5-methoxybenzoate
(12)
To a solution of compound 11 (6.0 g, 15.6 mmol) in a mixture of
ethanol (90 mL), water (30 mL) and conc. hydrochloric acid (4 mL);
powdered iron (8.5 g, 156.0 mmol) was added portionwise. The mixture
was stirred at reflux for 30 min, cooled to rt, then neutralized with 10%
sodium hydroxide till pH = 8. The formed solids were removed by fil-
tration and the filtrate was concentrated in vacuo. The residue was
extracted with dichloromethane, washed with brine, dried over sodium
sulfate, filtered and concentrated to give compound 12. Yield 4.6 g,
85%; 1H NMR (400 MHz, DMSO-d6) δ 1.17 (t, 3H, J = 7.2 Hz), 1.28 (t,
3H, J = 7.0 Hz), 1.43–1.38 (m, 2H), 1.64–1.57 (m, 2H), 1.79–1.72 (m,
2H), 2.31 (t, 3H, J = 7.2 Hz), 3.63 (s, 3H), 3.9 (t, 3H, J = 6.5 Hz), 4.05
(q, 2H, J = 7.0 Hz), 4.21 (q, 2H, J = 7.0 Hz), 6.35 (s, 1H), 6.41 (s, 2H),
7.13 (s, 1H), 7.63 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 173.3,
167.4, 154.8, 148,8, 139.8, 113.6, 100.3 (2C), 68.1, 60.1, 59.9, 56.8,
33.9, 28.6, 25.5, 24.6, 14.8, 14.6; HR-MS m/z 354.1952 (calcd for
4. Experimental
4.1. Chemistry
Reactions and manipulations were performed under nitrogen at-
mosphere using standard Schlenk techniques. The reactions solvents
were purchased from Aldrich Co., TCI and Alfa and were used without
any other purification. Column chromatography was performed on
Merck Kiesegel 60 Art 9385 (230–400 mesh). TLC was carried out using
glass sheets precoated with silica gel (E. Merck Kiesegel 60 F254, layer
thickness 0.25 mm). NMR spectra were obtained on Bruker AC 400 or
Agilent 500 spectrometer. High-resolution spectra were performed on
Jeol accuTOF (JMS-T100TD) equipped with a DART (direct analysis in
real time) ion source from Ionsense, Tokyo, Japan in the positive
modes. Samples purities were determined using HPLC Agilent 1100
series equipped with SHISEIDO CAPCELL PAK C18 column
(250 mm × 4.6 mm, 5 μm) at 25 °C. HPLC was performed in isocratic
elution mode using MeOH:ACN:DW (85:15: 5 = v:v) with flow a rate of
1.0 mL/min. Compounds 18a-g, 19a-g, 20a-g, 21a, 21b, 21d-g and
22a, 22b, 22d-g were prepared according to literature reports
[37–39,57–59].
C
18H28NO6 [M + H]+, 354.1917).
4.1.4. Ethyl 6-(6-methoxy-4-oxo-3,4-dihydroquinazolin-7-yloxy)hexanoate
(13)
A mixture of compound 12 (4.5 g, 12.7 mmol), ammonium formate
(1.2 g, 19.1 mmol) and formamide (30 mL) was heated with stirring at
180 °C for 4 h. The reaction mixture was cooled, diluted with di-
chloromethane, washed with brine, dried over sodium sulfate, filtered
and concentrated to give compound 13. Yield 3.6 g, 85%; 1H NMR
(400 MHz, DMSO-d6) δ 1.18 (t, 3H, J = 7.8 Hz), 1.46–1.42 (m, 2H),
1.63–1.57 (m, 2H), 1.80–1.76 (m, 2H), 2.32 (t, 3H, J = 7.3 Hz), 3.87 (s,
3H), 4.11–4.02 (m, 4H), 4.30 (q, 2H, J = 7.2 Hz), 7.12 (s, 1H), 7.44 (s,
1H). 7.99 (d, 1H, J = 2.9 Hz), 12.09 (s, 1H). 13C NMR (100 MHz,
DMSO-d6) δ 172.8, 160.0, 154.5, 147.8, 144.7, 143.7, 115.5, 108.0,
105.7, 68.2, 59.6, 55.9, 33.4, 28.2, 25.0, 24.2, 14.1; HR-MS m/z
335.1614 (calcd for C17H23N2O5 [M + H]+, 335.1607).
58

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
4.1.5. Ethyl 6-(4-chloro-6-methoxyquinazolin-7-yloxy)hexanoate (14)
A mixture of compound 13 (3.0 g, 8.9 mmol) and phosphoryl
trichloride (30 mL) was stirred at reflux for 4 h. Excess phosphoryl
trichloride was removed in vacuo. The residue was dissolved in di-
chloromethane, extracted with water and aqueous NaHCO3, dried over
sodium sulfate, filtered and concentrated to give compound 14. Yield
2.6 g, 93%; 1H NMR (400 MHz, DMSO-d6) δ 1.18 (t, 3H, J = 7.0 Hz),
1.50–1.44 (m, 2H), 1.64–1.59 (m, 2H), 1.84–1.80 (m, 2H), 2.33 (t, 2H,
J = 7.3 Hz), 3.99 (s, 3H), 4.05 (q, 2H, J = 7.0 Hz), 4.22 (t, 2H,
J = 6.4 Hz), 7.39 (s, 1H), 7.44 (s, 1H), 8.87 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 172.8, 157.8, 156.0, 152.1, 151.4, 148.5, 118.4, 107.3.
102.2, 69.0, 59.6, 56.1, 33.4, 27.9, 25.0, 24.1, 14.1; HR-MS m/z
353.1282 (calcd for C17H22ClN2O4 [M + H]+, 353.1263).
1H), 7.79 (d, 1H, J = 8.0 Hz), 7.94 (t, 1H, J = 1.4 Hz), 8.44 (s, 1H); 13
C
NMR (100 MHz, CD3OD) δ 173.6, 157.3, 155.0, 152.5, 150.3, 146.2,
139.5, 128.7, 127.6, 125.9, 123.2, 123.0, 109.2, 106.4, 101.4, 83.2,
78.3, 72.0, 71.5, 69.1, 66.3, 66.2, 64.3, 59.3, 55.8, 53.6, 53.4, 53.3,
33.9, 31.8, 29.6, 29.4, 29.3, 28.5, 26.0, 25.6, 24.6, 22.5, 13.3; 31P NMR
(202.5 MHz, CD3OD)
δ -0.56; HR-MS m/z 757.3956 (calcd for
C
39H58N4O9P [M + H]+, 757.3941).
4.1.8.2. 3-(Dodecyloxy)-2-(6-(4-(3-ethynylphenylamino)-6-
methoxyquinazolin-7-yloxy)hexanoyloxy)propyl
2-(trimethylammonio)
ethyl phosphate (7b). Compound 7b was obtained using compound
20b (42.6 mg, 0.10 mmol), Yield 54.5 mg, 67%; HPLC purity: 96.79%
(Rt = 4.67 min); 1H NMR (400 MHz, CD3OD) δ 0.88 (t, 3H, J = 6.8 Hz),
1.21 (s, 18H), 1.54–1.48 (m, 2H), 1.65–1.57 (m, 2H), 1.81–1.74 (m,
2H), 1.97–1.90 (m, 2H), 2.45 (t, 2H, J = 7.2 Hz), 3.23 (s, 9H),
3.50–3.37 (m, 2H), 3.67–3.61 (m, 4H), 4.04–3.99 (m, 1H), 4.05 (s,
3H), 4.18 (t, 2H, J = 6.2 Hz), 4.30–4.26 (m, 2H), 5.22–5.17 (m, 1H),
7.16 (s, 1H), 7.28 (d, 1H, J = 7.7 Hz), 7.39 (t, 1H, J = 8.0 Hz), 7.77 (s,
1H), 7.80 (d, 1H, J = 8.3 Hz), 7.94 (m, 1H), 8.45 (s, 1H); HR-MS m/z
813.4620 (calcd for C43H66N4O9P [M + H]+, 813.4567).
4.1.6. Ethyl 6-(4-(3-ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanoate (15)
A mixture of compound 14 (1.0 g, 2.8 mmol) and 3-ethynylaniline
(489 mg, 3.3 mmol) in isopropanol (20 mL) was stirred at reflux for 4 h
then allowed to cool to rt. The solid was filtered, washed with iso-
propanol and ether and dried to give compound 15. Yield 1.16 g, 95%;
1H NMR (400 MHz, DMSO-d6) δ 1.18 (t, 3H, J = 7.2 Hz), 1.50–1.44 (m,
2H), 1.64–1.58 (m, 2H), 1.84–1.81 (m, 2H), 2.32 (t, 3H, J = 7.2 Hz),
4.02–4.08 (m, 5H), 4.16 (t, 2H, J = 6.2 Hz), 4.31 (s,1H), 7.37 (s, 1H),
7.41 (dd, 1H, J1 = 6.7, Hz, J2 = 0.76 Hz), 7.50 (t, 1H, J = 9.0 Hz), 7.80
(dd, 1H, J1 = 7.2, Hz, J2 = 0.9 Hz), 7.89 (s, 1H), 8.39 (s, 1H), 8.86 (s,
1H), 11.55 (s, 1H).
4.1.8.3. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanoyloxy)-3-(tridecyloxy)propyl 2-(trimethylammonio)ethyl phosphate
(7c). Compound 7c was obtained using compound 20c (44.0 mg,
0.10 mmol), Yield 58.7 mg, 71%; HPLC purity: 97.76%
(Rt = 5.13 min); 1H NMR (400 MHz, CD3OD)
δ 0.88 (t, 3H,
J = 6.8 Hz), 1.20 (s, 20H), 1.52–1.47 (m, 2H), 1.63–1.57 (m, 2H),
1.80–1.72 (m, 2H), 1.95–1.88 (m, 2H), 2.45 (t, 2H, J = 7.2 Hz), 3.24 (s,
9H), 3.49–3.39 (m, 2H), 3.62–3.61 (m, 2H), 3.67–3.65 (m, 2H),
4.03–3.99 (m, 1H), 4.04 (s, 3H), 4.15 (t, 2H, J = 6.2 Hz), 4.30–4.26
(m, 2H), 5.22–5.17 (m, 1H), 7.12 (s, 1H), 7.27 (d, 1H, J = 7.7 Hz), 7.39
(t, 1H, J = 7.8 Hz), 7.73 (s, 1H), 7.82 (d, 1H, J = 8.3 Hz), 7.96 (t, 1H,
J = 1.4 Hz), 8.43 (s, 1H); 13C NMR (100 MHz, CD3OD) δ 174.8, 158.4,
156.1, 153.7, 151.1, 147.5, 140.7, 129.9, 128.7, 127.1, 124.3, 124.2,
110.5, 102.5, 90.2, 84.4, 78.8, 73.2, 73.1, 72.6, 70.3, 70.0, 67.5, 65.4
(2C), 60.5, 60.4, 57.0, 54.7 (3C), 35.1, 33.1, 30.8, (4C), 30.7, 30.5,
29.7, 27.2, 26.7, 25.9, 23.8, 18.2, 14.5; HR-MS m/z 827.4777 (calcd for
4.1.7. 6-(4-(3-ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanoic acid (16)
Lithium hydroxide monohydrate (869 mg, 20.5 mmol) was added to
a stirred solution of compound 15 (3.0 g, 6.9 mmol) in a mixture of
THF/water (3:1, 125 mL). Stirring was continued at rt for 24 h. The ice-
bath cooled reaction mixture was acidified to pH = 2 with HCl (1 N
aqueous solution). The precipitated solid was collected by filtration,
washed with water, and dried under vacuum to afford compound 16.
Yield 2.5 g, 96%; 1H NMR (400 MHz, DMSO-d6) δ 1.48–1.44 (m, 2H),
1.61–1.56 (m, 2H), 1.85–1.82 (m, 2H), 2.26 (t, 2H, J = 7.2 Hz), 4.03 (s,
3H), 4.17 (t, 2H, J = 6.4 Hz), 4.31 (s, 1H), 7.36 (s, 1H), 7.41 (d, 1H,
J = 8.0 Hz), 7.50 (t, 1H, J = 7.8 Hz), 7.79 (d, 1H, J = 8.4 Hz), 7.89 (s,
1H), 8.37 (s, 1H), 8.85 (s, 1H), 11.500 (s, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 174.9, 158.5, 156.1, 150.7, 149.4 (2C), 137.9, 129.7,
129.6, 127.9, 125.6, 122.5, 107.8, 104.3, 83.4, 81.8, 69.5, 57.4, 34.0,
C
44H68N4O9P [M + H]+, 827.4722).
4.1.8.4. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanoyloxy)-3-(hexadecyloxy)propyl 2-(trimethylammonio)ethyl phosphate
(7d). Compound 7d was obtained using compound 20d (48.2 mg,
0.10 mmol), Yield 60.0 mg, 69%; HPLC purity: 99.62% (Rt = 7.90 min);
1H NMR (400 MHz, CD3OD) δ 0.88 (t, 3H, J = 6.8 Hz), 1.20 (s, 20H),
1.52–1.47 (m, 2H), 1.63–1.57 (m, 2H), 1.78–1.72 (m, 2H), 1.94–1.87 (m,
2H), 2.43 (t, 2H, J = 7.2 Hz), 3.23 (s, 9H), 3.47–3.39 (m, 2H), 3.61–3.60
(m, 2H), 3.65–3.63 (m, 2H), 4.02–3.96 (m, 1H), 4.03 (s, 3H), 4.16 (t, 2H,
J = 6.2 Hz), 4.30–4.25 (m, 2H), 5.20–5.15 (m, 1H), 7.13 (s, 1H), 7.27 (d,
1H, J = 7.7 Hz), 7.38 (t, 1H, J = 7.8 Hz), 7.75 (s, 1H), 7.78 (d, 1H,
J = 8.2 Hz), 7.93 (t, 1H, J = 1.4 Hz), 8.46 (s, 1H); 13C NMR (100 MHz,
CD3OD) δ 173.3, 157.5, 155.4, 150.5, 138.5, 128.6, 128.1, 126.7, 126.1,
82.8, 77.8, 71.3, 68.5, 66.1, 64.1, 63.0, 59.5, 55.7, 53.4, 53.3 (2C), 33.7,
31.6, 29.4 (3C), 29.3, 29.2, 29.0, 25.8 (3C), 25.3, 25.2 (2C), 25.1, 24.4,
24.3 (2C), 22.3, 13.1; HR-MS m/z 869.5299 (calcd for C47H74N4O9P
[M + H]+, 869.5193).
28.4, 25.5, 24.6; HR-MS m/z 406.1815 (calcd for
C23H24N3O4
[M + H]+, 406.1767).
4.1.8. General procedure for the synthesis of compounds 7a–g, 8a, 8b, and
8d–g
To a solution of each of compounds 20a–g, 22a, 22b, or 22d–g (1
equivalent) in a mixture of dry chloroform (20 mL per mmol) and dry
DMF (2 mL per mmol), DMAP (3 equivalents), compound 16 (3
equivalents), and DCC (3 equivalents) were added. The reaction mix-
ture was refluxed for 24 h, cooled to rt, and evaporated in vacuo. The
residue was purified by silica gel chromatography, first eluting with
CHCl3/MeOH (9:1) to wash out the less polar compounds, followed by
CHCl3/MeOH/H2O (65:20:4).
4.1.8.1. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanoyloxy)-3-(octyloxy)propyl 2-(trimethylammonio)ethyl phosphate
(7a). Compound 7a was obtained using compound 20a (37.0 mg,
0.10 mmol). Yield 56.8 mg, 75%; HPLC purity: 99.15%
4.1.8.5. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanoyloxy)-3-(octadecyloxy)propyl 2-(trimethylammonio)ethyl phosphate
(7e). Compound 7e was obtained using compound 20e (51.0 mg,
0.10 mmol),
Yield
62.8 mg,
70%;
HPLC
purity:
98.94%
(Rt = 3.21 min); 1H NMR (400 MHz, CD3OD)
δ
0.86 (t, 3H,
(Rt = 11.08 min); 1H NMR (400 MHz, CD3OD)
δ 0.90 (t, 3H,
J = 6.8 Hz), 1.23 (s, 10H), 1.53–1.50 (m, 2H), 1.63–1.58 (m, 2H),
1.80–1.73 (m, 2H), 1.96–1.89 (m, 2H), 2.45 (t, 2H, J = 7.2 Hz), 3.23 (s,
9H), 3.48–3.40 (m, 2H), 3.66–3.60 (m, 4H), 4.02–3.99 (m, 1H), 4.05 (s,
3H), 4.16 (t, 2H, J = 6.2 Hz), 4.30–4.27 (m, 2H), 5.20–5.17 (m, 1H),
7.13 (s, 1H), 7.28 (d, 1H, J = 7.6 Hz), 7.39 (t, 1H, J = 7.8 Hz), 7.74 (s,
J = 6.8 Hz), 1.21 (s, 30H), 1.53–1.49 (m, 2H), 1.65–1.59 (m, 2H),
1.81–1.73 (m, 2H), 1.97–1.90 (m, 2H), 2.45 (t, 2H, J = 7.2 Hz), 3.24 (s,
9H), 3.48–3.40 (m, 2H), 3.62–3.61 (m, 2H), 3.67–3.65 (m, 2H), 4.03–3.99
(m, 1H), 4.05 (s, 3H), 4.18 (t, 2H, J = 6.2 Hz), 4.29–4.27 (m, 2H),
5.22–5.17 (m, 1H), 7.16 (s, 1H), 7.29 (d, 1H, J = 7.6 Hz), 7.40 (t, 1H,
59

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
J = 8.0 Hz), 7.78 (s, 1H), 7.80 (d, 1H, J = 8.4 Hz), 7.95 (m, 1H), 8.47 (s,
1H); 13C NMR (100 MHz, CD3OD) δ 173.3, 157.0, 154.6, 154.3, 150.0,
146.1, 139.3, 128.5, 127.2, 125.6, 122.8, 122.7, 109.1, 106.2, 101.1, 83.0,
77.4, 71.8, 68.9, 66.1, 66.0, 61.0, 59.1, 55.6, 53.3 (3C), 33.7, 31.7, 29.4
(4C), 29.3 (4C), 29.2 (3C), 29.1, 28.3 (2C), 25.4, 25.3, 24.5, 24.4, 22.4,
13.1; 31P NMR (202.5 MHz, CD3OD) δ -0.60; HR-MS m/z 897.5490 (calcd
for C49H78N4O9P [M + H]+, 897.5506).
(Rt = 4.57 min); 1H NMR (400 MHz, CD3OD) δ 0.86 (t, 3H, J = 6.8 Hz),
1.22 (s, 18H), 1.64–1.48 (m, 4H), 1.79–1.73 (m, 2H), 1.95–1.88 (m,
2H), 2.46–2.42 (m, 2H), 3.24 (s, 9H), 3.46–3.39 (m, 2H), 3.63–3.60 (m,
2H), 3.66–3.64 (m, 2H), 4.03–3.98 (m, 2H), 4.06 (s, 3H), 4.20 (t, 1H,
J = 5.5 Hz), 4.30–4.27 (m, 2H), 7.17 (s, 1H), 7.33–7.30 (m, 1H), 7.41
(t, 1H, J = 7.9 Hz), 7.78–7.77 (m, 1H), 7.80 (s, 1H), 7.94–7.93 (m, 1H),
8.49 (s, 1H); HR-MS m/z 812.4703 (calcd for C43H67N5O8P [M + H]+
,
812.4727).
4.1.8.6. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanoyloxy)-3-(icosyloxy) propyl 2-(trimethylammonio)ethyl phosphate
(7f). Compound 7f was obtained using compound 20f (53.8 mg,
0.10 mmol), Yield 61.1 mg, 66%; HPLC purity: 97.67%
4.1.8.10. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanamido)-3-(hexadecyloxy)propyl 2-(trimethylammonio)ethyl phosphate
(8d). Compound 8d was obtained using compound 22d (48.1 mg,
0.10 mmol), Yield 59.9 mg, 69%; HPLC purity: 98.11% (Rt = 7.50 min);
1H NMR (400 MHz, CD3OD) δ 0.88 (t, 3H, J = 6.8 Hz), 1.20 (s, 26H),
1.52–1.44 (m, 2H), 1.62–1.56 (m, 2H), 1.76–1.71 (m, 2H), 1.93–1.89 (m,
2H), 2.31–2.26 (m, 2H), 3.21 (s, 9H), 3.40 (td, 2H, J1 = 6.6 Hz,
J2 = 1.9 Hz), 3.52–3.49 (m, 2H), 3.64–3.61 (m, 2H), 3.95–3.90 (m, 2H),
4.02 (s, 3H), 4.20–4.14 (m, 3H), 4.28–4.25 (m, 2H), 7.13 (s, 1H), 7.25 (d,
1H, J = 7.6 Hz), 7.36 (t, 1H, J = 6.0 Hz), 7.74 (s, 1H), 7.78 (d, 1H,
J = 8.2 Hz), 7.92 (s, 1H), 8.42 (s, 1H); 13C NMR (100 MHz, CD3OD) δ
176.0, 158.3, 156.0, 153.8, 151.3, 147.6, 140.8, 129.9, 128.6, 126.9,
124.2, 124.1, 110.5, 107.7, 102.5, 84.5, 78.8, 72.5, 70.4, 69.9, 67.5 (2C),
67.4 (2C), 65.8, 65.7, 60.5, 60.4, 57.0, 54.7 (3C), 51.1, 51.0, 37.1, 33.1,
30.9, 30.8, 30.7, 30.5, 29.8, 27.3, 26.9, 26.8, 23.8, 14.5; HR-MS m/z
868.5393 (calcd for C47H75N5O8P [M + H]+, 868.5353).
(Rt = 15.89 min); 1H NMR (400 MHz, CD3OD)
δ 0.88 (t, 3H,
J = 6.8 Hz), 1.18 (s, 34H), 1.51–1.46 (m, 2H), 1.60–1.56 (m, 2H),
1.76–1.71 (m, 2H), 1.92–1.87 (m, 2H), 2.43 (t, 2H, J = 7.2 Hz), 3.21 (s,
9H), 3.46–3.38 (m, 2H), 3.60–3.58 (m, 2H), 3.64–3.62 (m, 2H),
4.00–3.95 (m, 1H), 4.02 (s, 3H), 4.13 (t, 2H, J = 6.2 Hz), 4.28–4.24
(m, 2H), 5.20–5.14 (m, 1H), 7.11 (s, 1H), 7.25 (d, 1H, J = 7.7 Hz), 7.36
(t, 1H, J = 7.8 Hz), 7.72 (s, 1H), 7.78 (d, 1H, J = 8.2 Hz), 7.93 (m, 1H),
8.42 (s, 1H); 13C NMR (100 MHz, CD3OD) δ 173.3, 157.0, 154.6, 154.3,
150.0, 146.1, 139.3, 128.5, 127.2, 125.6, 122.8, 122.7, 109.1, 106.2,
101.1, 83.0, 77.4, 71.8, 68.9, 66.1, 66.0, 61.0, 59.1, 55.6, 53.3 (3C),
33.7, 31.7, 29.4 (4C), 29.3 (4C), 29.2 (3C), 29.1, 28.3 (2C) 25.8, 25.4,
25.3, 24.5, 24.4, 22.4, 13.1; 31P NMR (202.5 MHz, CD3OD) δ -0.59; HR-
MS m/z 925.5820 (calcd for C51H82N4O9P [M + H]+, 925.5819).
4.1.8.7. 3-(Docosyloxy)-2-(6-(4-(3-ethynylphenylamino)-6-
4.1.8.11. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanamido)-3-(octadecyloxy)propyl 2-(trimethylammonio)ethyl phosphate
(8e). Compound 8e was obtained using compound 22e (50.9 mg,
methoxyquinazolin-7-yloxy)hexanoyloxy) propyl 2-(trimethylammonio)
ethyl phosphate (7g). Compound 7g was obtained using compound
20 g (56.6 mg, 0.10 mmol), Yield 67.7 mg, 71%; HPLC purity: 93.04%
0.10 mmol),
Yield
62.7 mg,
70%;
HPLC
purity:
99.86%
(Rt = 21.68 min); 1H NMR (400 MHz, CD3OD)
δ
0.88 (t, 3H,
(Rt = 23.03 min); 1H NMR (400 MHz, CD3OD)
δ 0.87 (t, 3H,
J = 6.8 Hz), 1.21 (s, 38H), 1.53–1.48 (m, 2H), 1.63–1.57 (m, 2H),
1.78–1.72 (m, 2H), 1.97–1.90 (m, 2H), 2.34–2.26 (m, 2H), 3.23 (s, 9H),
3.44–3.40 (m, 2H), 3.57–3.50 (m, 2H), 3.67–3.64 (m, 2H), 3.96–3.93
(m, 1H), 4.04 (s, 3H), 4.17 (t, 2H, J = 6.5 Hz), 4.30–4.26 (m, 2H),
5.20–5.14 (m, 1H), 7.14 (s, 1H), 7.27 (d, 1H, J = 7.7 Hz), 7.39 (t, 1H,
J = 7.8 Hz), 7.77 (s, 1H), 7.81 (d, 1H, J = 8.2 Hz), 7.96 (m, 1H), 8.45
(s, 1H); 13C NMR (100 MHz, CD3OD) δ 174.3, 158.0, 154.6, 153.3,
150.0, 146.1, 139.3, 128.5, 127.2, 125.6, 122.8, 122.7, 109.1, 106.2,
101.1, 83.0, 77.4, 71.8, 68.9, 66.1, 66.0, 61.0, 59.1, 55.6, 53.3 (3C),
33.7, 31.7, 29.4 (4C), 29.3 (4C), 29.2 (3C), 29.1, 28.3 (2C), 25.8, 25.4,
25.3, 24.5, 24.4, 22.4, 13.1; 31P NMR (202.5 MHz, CD3OD) δ -0.31; HR-
MS m/z 953.6090 (calcd for C51H82N4O9P [M + H]+, 953.6127).
J = 6.8 Hz), 1.17 (s, 30H), 1.51–1.44 (m, 2H), 1.60–1.52 (m, 2H),
1.75–1.69 (m, 2H), 1.91–1.85 (m, 2H), 2.41 (t, 2H, J = 7.2 Hz), 3.20 (s,
9H), 3.46–3.36 (m, 2H), 3.59–3.56 (m, 2H), 3.65–3.61 (m, 2H), 3.99–3.94
(m, 2H), 4.01 (s, 3H), 4.11 (t, 1H, J = 6.3 Hz), 4.29–4.23 (m, 2H), 5.16 (t,
1H, J = 4.7 Hz), 7.08 (s, 1H), 7.23 (d, 1H, J = 7.7 Hz), 7.34 (t, 1H,
J = 7.9 Hz), 7.70 (s, 1H), 7.78 (d, 1H, J = 8.2 Hz), 7.93 (s, 1H), 8.40 (s,
1H); 13C NMR (100 MHz, CD3OD) δ 174.7, 158.4, 156.1, 153.8, 151.4,
147.6, 140.8, 129.9, 128.7, 127.0, 124.2 (2C), 107.7, 102.5, 84.4, 78.8,
73.2, 73.1, 72.6, 70.4, 70.0, 67.5 (3C), 67.4, 65.4, (2C), 60.5, 60.4, 57.0,
54.8, 54.7 (3C), 35.2, 33.1, 30.9, 30.8. 30.7 (2C), 30.6, 30.5, 29.8, 27.3,
26.7, 25.9, 23.8, 14.5; 31P NMR (202.5 MHz, CD3OD) δ −0.54; HR-MS m/
z 896.5603 (calcd for C49H79N5O8P [M + H]+, 896.5666).
4.1.8.8. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanamido)-3-(octyloxy) propyl 2-(trimethylammonio)ethyl phosphate
(8a). Compound 8a was obtained using compound 22a (36.8 mg,
0.10 mmol), Yield 55.9 mg, 74%; HPLC purity: 98.14%
4.1.8.12. 2-(6-(4-(3-Ethynylphenylamino)-6-methoxyquinazolin-7-yloxy)
hexanamido)-3-(icosyloxy) propyl 2-(trimethylammonio)ethyl phosphate
(8f). Compound 8f was obtained using compound 22f (53.7 mg,
0.10 mmol), Yield 56.4 mg, 61%; HPLC purity: 97.16%
(Rt = 3.11 min); 1H NMR (400 MHz, CD3OD)
δ
0.86 (t, 3H,
(Rt = 14.82 min); 1H NMR (400 MHz, CD3OD)
δ 0.88 (t, 3H,
J = 6.8 Hz), 1.21 (s, 10H), 1.58–1.46 (m, 4H), 1.75–1.70 (m, 2H),
1.91–1.84 (m, 2H), 2.31–2.27 (m, 2H), 3.24 (s, 9H), 3.42–3.39 (m, 2H),
3.52–3.50 (m, 2H), 3.66–3.64 (m, 2H), 3.96–3.93 (t, 2H, J = 5.7 Hz),
4.00 (s, 3H), 4.07 (t, 2H, J = 6.2 Hz), 4.20 (t, 1H, J = 5.5 Hz),
4.30–4.27 (m, 2H), 7.02 (s, 1H), 7.25 (d, 1H, J = 7.6 Hz), 7.36 (t,
1H, J = 6.0 Hz), 7.64 (s, 1H), 7.80 (d, 1H, J = 8.1 Hz), 7.96 (s, 1H),
8.39 (s, 1H); 13C NMR (100 MHz, CD3OD) δ 176.0, 158.3, 155.9, 153.7,
151.3, 147.5, 140.8, 129.9, 128.6, 126.9, 124.2, 124.1, 110.4, 107.7,
102.5, 84.5, 78.9, 72.5, 70.4, 69.9, 67.5 (2C), 67.4 (2C), 65.7 (2C),
60.5, 60.4, 57.0, 54.8, 54.7 (2C), 51.0 (2C), 37.1, 33.0, 30.8, 30.6, 30.5,
29.8, 27.3, 26.8 (2C), 23.8, 14.5; HR-MS m/z 756.4062 (calcd for
J = 6.8 Hz), 1.19 (s, 34H), 1.59–1.46 (m, 4H), 1.75–1.70 (m, 2H),
1.92–1.85 (m, 2H), 2.31–2.25 (m, 2H), 3.22 (s, 9H), 3.40 (td, 2H,
J1 = 6.6 Hz, J2 = 1.8 Hz), 3.52–3.48 (m, 2H), 3.65–3.63 (m, 2H), 3.93
(t, 2H, , J = 5.9 Hz), 4.00 (s, 3H), 4.10 (t, 2H, J = 6.2 Hz), 4.23–4.17
(m, 1H), 4.27 (brs, 2H), 7.05 (s, 1H), 7.24 (d, 1H, J = 7.7 Hz), 7.35 (t,
1H, J = 7.8 Hz), 7.68 (s, 1H), 7.80 (d, 1H, J = 8.2 Hz), 7.96 (s, 1H),
8.40 (s, 1H); 13C NMR (100 MHz, CD3OD) δ 176.0, 158.3, 156.0, 153.7,
151.4, 147.6, 140.8, 129.9, 128.6, 126.9, 124.1 (2C), 110.5, 107.7,
102.5, 84.5, 78.8, 72.5, 70.4, 69.9, 67.5 (2C), 67.4, 65.8, 65.7, 60.5,
60.4, 57.0, 54.7 (3C), 51.1, 51.0, 37.1, 33.1, 30.9, 30.7, 30.6, 29.8,
27.3, 26.9, 26.8, 23.8, 14.6; HR-MS m/z 924.5958 (calcd for
C
39H59N5O8P [M + H]+, 756.4101).
C
51H83N5O8P [M + H]+, 924.5979).
4.1.8.9. 3-(Dodecyloxy)-2-(6-(4-(3-ethynylphenylamino)-6-
4.1.8.13. 3-(Docosyloxy)-2-(6-(4-(3-ethynylphenylamino)-6-
methoxyquinazolin-7-yloxy)hexanamido) propyl 2-(trimethylammonio)
ethyl phosphate (8b). Compound 8b was obtained using compound
22b (42.6 mg, 0.23 mmol), Yield 60.9 mg, 75%; HPLC purity: 92.68%
methoxyquinazolin-7-yloxy)hexanamido) propyl 2-(trimethylammonio)
ethyl phosphate (8g). Compound 8g was obtained using compound
22 g (56.5 mg, 0.10 mmol), Yield 64.7 mg, 68%; HPLC purity: 92.11%
60

Md. M. Alam et al.
BioorganicChemistry84(2019)51–62
(Rt = 10.54 min); 1H NMR (400 MHz, CD3OD)
δ
0.88 (t, 3H,
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J = 6.8 Hz), 1.29 (s, 38H), 1.59–1.46 (m, 4H), 1.75–1.70 (m, 2H),
1.92–1.85 (m, 2H), 2.31–2.25 (m, 2H), 3.21 (s, 9H), 3.46–3.36 (m, 2H),
3.52–3.48 (m, 2H), 3.65–3.63 (m, 2H), 3.93 (t, 2H, , J = 5.9 Hz), 4.05
(s, 3H), 4.10 (t, 2H, J = 6.2 Hz), 4.23–4.17 (m, 1H), 4.27 (brs, 2H), 7.13
(s, 1H), 7.24 (d, 1H, J = 7.7 Hz), 7.35 (t, 1H, J = 7.8 Hz), 7.68 (s, 1H),
7.80 (d, 1H, J = 8.2 Hz), 7.92 (s, 1H), 8.42 (s, 1H); 13C NMR (100 MHz,
CD3OD) δ 174.9, 157.3, 155.1, 152.3, 152.1, 150.4, 139.4, 128.8,
128.7, 127.7, 126.0, 123.2, 123.0 (2C), 106.2, 101.4, 83.2, 77.6, 71.3
(2C), 69.2, 68.9, 66.3, 64.6, 59.2, 55.3, 53.6, 53.4, 53.3, 35.8, 31.9,
29.4 (12C), 28.6, 28.5, 26.1 (2C), 25.6 (3C), 25.5, 22.6, 13.3; HR-MS
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Acknowledgments
This work was supported by the Basic Science Research Program
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