Design, synthesis and biological evaluation of novel heptamethine cyanine dye-erlotinib conjugates as antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2020.127557

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) has been proved as a target for the treatment of non-small cell lung cancer (NSCLC) with specific gene mutations. However, EGFR-TK inhibitors (EGFR-TKIs) need to enter cancer cells and then competitively interact with the active site of tyrosine kinase receptors to suppress the downstream signaling pathway to inhibit tumor proliferation. In this study, in order to improve the tumor cell targeting ability of EGFR-TKI, EGFR-TKI erlotinib was conjugated with the cancer cell-targeting heptamethine cyanine dyes to form seventeen novel erlotinib-dye conjugates. The efficiency of tumor targeting properties of conjugates against cancer cell growth and EGFR-TK inhibition was evaluated in vitro. The result revealed that most erlotinib-dye conjugates exhibited stronger inhibitory effect on A549, H460, H1299 and MDA-MB-231 cell lines than the parent drug erlotinib. Meanwhile, representative compounds exhibited weak cytotoxicity on human normal mammary epithelial MCF-10A cells. Moreover, the conjugate CE17 also showed ~14-fold higher EGFR-TK inhibition activity (IC₅₀ = 0.124 μM) than erlotinib (IC₅₀ = 5.182 μM) in A549 cell line. Finally, molecular docking analysis verified that the erlotinib moiety of compound CE17 could form hydrogen bond with Met-769 and occupy active cavity of EGFR-TK. Therefore, we believed the integration strategy between heptamethine cyanine dyes and EGFR-TKI will contribute to enhancing the therapeutic effect of EGFR-TKI for NSCLC treatment.

Full text of DOI:10.1016/j.bmcl.2020.127557

Bioorganic&MedicinalChemistryLetters30(2020)127557
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of novel heptamethine cyanine
dye-erlotinib conjugates as antitumor agents
⁎
⁎
Xiaoguang Yanga,1, Zhuang Houa,1, Dun Wanga, Yanhua Moub, , Chun Guoa,
a School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
b School of Life Sciences and Biological Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Epidermal growth factor receptor tyrosine kinase (EGFR-TK) has been proved as a target for the treatment of
non-small cell lung cancer (NSCLC) with specific gene mutations. However, EGFR-TK inhibitors (EGFR-TKIs)
need to enter cancer cells and then competitively interact with the active site of tyrosine kinase receptors to
suppress the downstream signaling pathway to inhibit tumor proliferation. In this study, in order to improve the
tumor cell targeting ability of EGFR-TKI, EGFR-TKI erlotinib was conjugated with the cancer cell-targeting
heptamethine cyanine dyes to form seventeen novel erlotinib-dye conjugates. The efficiency of tumor targeting
properties of conjugates against cancer cell growth and EGFR-TK inhibition was evaluated in vitro. The result
revealed that most erlotinib-dye conjugates exhibited stronger inhibitory effect on A549, H460, H1299 and
MDA-MB-231 cell lines than the parent drug erlotinib. Meanwhile, representative compounds exhibited weak
cytotoxicity on human normal mammary epithelial MCF-10A cells. Moreover, the conjugate CE17 also showed
~14-fold higher EGFR-TK inhibition activity (IC50 = 0.124 μM) than erlotinib (IC50 = 5.182 μM) in A549 cell
line. Finally, molecular docking analysis verified that the erlotinib moiety of compound CE17 could form hy-
drogen bond with Met-769 and occupy active cavity of EGFR-TK. Therefore, we believed the integration strategy
between heptamethine cyanine dyes and EGFR-TKI will contribute to enhancing the therapeutic effect of EGFR-
TKI for NSCLC treatment.
EGFR-TK inhibitor
Non-small cell lung cancer
Heptamethine cyanine dye
Non-small cell lung cancer (NSCLC) is considered as a highly het-
erogeneous disease that is still difficult to treat.1 The traditional treat-
ment of NSCLC at advanced stage is mainly combination chemotherapy
or radiotherapy. However, the advent of targeted drugs provides hope
for the treatment of NSCLC.2 Epidermal growth factor receptor tyrosine
kinase (EGFR-TK) is a crucial target for NSCLC with EGFR gene muta-
tions treatment and is also one of the key factors for adult embryo
generation, tissue regeneration and organ maturation. EGFR-related
proteins are involved in three important tumorigenic signaling path-
ways, including RAS/RAF/MAPK, PI3K/AKT/mTOR and JAK/STAT
signaling.3,4 EGFR-TK inhibitors (EGFR-TKIs) have been used for the
clinical treatment of EGFR-mutated NSCLC patients.5,6 However, EGFR-
TKIs need to enter the cancer cell to block the transduction of mutated
EGFR signals through competitively inhibiting the binding between
adenosine triphosphate (ATP) and tyrosine kinase receptors, thus in-
hibiting cancer cell proliferation.7 Hence, the improvement of tumor
cell targeting and the transmembrane ability of EGFR-TKIs might en-
hance their therapeutic effect and even reduce side effects.
Heptamethine carbocyanine dye is a kind of near infrared fluor-
escent (NIFR) dye, which has been widely applied in tumor imaging in
vivo.8–13 Recent evidence suggests that several heptamethine cyanine
dyes including IR-780, IR-783 and IR-808 have preferential abilities to
target cancer cells and have the ability to accumulate in the tumors.14
Relevant mechanism research shows that the preferential uptake of
heptamethine carbocyanine dyes in cancer cells is related to hypoxia-
inducible factor 1α/organic anion transporting polypeptides (HIF1α/
OATPs) signaling pathway.15,16 In fact, the tumor tissue hypoxic en-
vironment increases the expression of HIF1α leading to the upregula-
tion of OATPs expression, which may mediate the transport of hepta-
methine cyanine dyes into tumor cells. In addition, the negative
mitochondrial membrane potential of tumor cells is one of mechanism
that these dyes can accumulate in tumor cells.17,18 Therefore, hepta-
methine cyanine dyes have been identified as excellent cancer-targeting
small molecules. In this study, some heptamethine cyanine dyes were
applied to couple with EGFR-TKI erlotinib to help achieve synergistic
targeting effect. As exhibited in Fig. 1, seventeen novel erlotinib-dye
⁎ Corresponding authors.
E-mail addresses: mu_hua_jj@sina.com (Y. Mou), chunguo@syphu.edu.cn (C. Guo).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2020.127557
Received 6 July 2020; Received in revised form 10 September 2020; Accepted 11 September 2020
Availableonline16September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

X. Yang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127557
Fig. 1. Schematic diagram of the novel compounds design.
O
O
O
COOEt
O
COOEt
HO
O
COOEt
HO
HO
c
b
a
AcO
Ph
AcO
O
HO
Ph
4
1
8
2
3
d
O
N
O
O
O
COOEt
NH2
O
O
COOEt
NO2
O
COOEt
g
e
AcO
O
NH
f
AcO
O
AcO
O
AcO
O
O
O
7
6
5
h
Cl
N
HN
HN
N
O
O
O
O
j
O
i
AcO
O
N
AcO
O
N
HO
N
O
N
O
9
10
E
Scheme 1. Synthetic route of compound E. Reagents and reaction conditions: a) benzyl alcohol, PPh3, DIAD, THF, −5℃ to r.t.; b) 2-bromoethyl acetate, K2CO3,
DMF, r.t.; c) Pd/C, H2, AcOEt, r.t.; d) 1-bromo-2-methoxyethane, K2CO3, DMF, r.t.; e) HNO3, H2SO4, AcOH, 0℃ to r.t.; f) Pd/C, H2, AcOEt, r.t.; g) HCONH2,
HCOONH4, 180℃; h) POCl3 ; i) 3-aminophenylacetylene, isopropyl alcohol, 80℃; j) KOH, methanol, r.t.
R1
R1
R1
R1
Cl
Cl
R1
b
a
N
N
N
N
Br
Br
O
Cl
N
Br
H
OH
HOOC
O
12
I
HOOC
CE1-CE4
11
COOH
COOH
O
O
O
O
N
H
N
N
Reagents and reaction conditions: a) AcONa, Ac2O, 70 ; b) E, EDCI, DMAP, CH2Cl2, r.t.
Scheme 2. Synthetic route of target compounds CE1-CE4. Reagents and reaction conditions: a) AcONa, Ac2O, 70℃; b) E, EDCI, DMAP, CH2Cl2, r.t.
conjugates were designed and synthesized through pH-sensitive ester
linkage.
available ethyl 3,4-dihydroxybenzoate (1), which was converted into
ethyl 4-(benzyloxy)-3-hydroxybenzoate (2) through Mitsunobu reac-
tion. What is noteworthy is that the selective etherification reaction of
catechol was achieved by a SN2 reaction using the reagents of PPh3 and
diisopropyl azodiformate (DIAD) in the first step.20 Etherification of the
Firstly, as illustrated in Schemes 1 and 2-(4-(3-ethynylaniline)-7-(2-
methoxyethoxy) quinazoline-6-oxy) ethanol (E) was synthesized
through 10-step reactions.19 The synthesis started with commercially
2

X. Yang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127557
R1
R1
R1
R1
R1
a
b
N
N
N
N
N
H
Br
Br
N
N
Br
·
HCl
13
O
11
COOH
HOOC
CE5-CE7
II
HOOC
COOH
O
O
O
O
N
H
N
N
Reagents and reaction conditions: a) Et3N, Ac2O, 70 ; b) E, EDCI, DMAP, CH2Cl2, r.t.
Scheme 3. Synthetic route of target compounds CE5-CE7. Reagents and reaction conditions: a) Et3N, Ac2O, 70℃; b) E, EDCI, DMAP, CH2Cl2, r.t.
R2
R2
a
b
H
N
N
N
N
N
Br
Br
O
·
HCl
13
14
15
R2
R1
R2
R1
c
N
N
N
N
Br
Br
III
CE8-CE17
Py
COOH
·
O
O
O
O
O
N
H
N
N
Reagents and reaction conditions: a) Ac2O, 70 ; b) 11, pyridine, 60 ; c) E, EDCI, DMAP, CH2Cl2, r.t.
Scheme 4. Synthetic route of target compounds CE8-CE17. Reagents and reaction conditions: a) Ac2O, 70℃; b) 11, pyridine, 60℃; c) E, EDCI, DMAP, CH2Cl2, r.t.
phenol in 2 was carried out with 2-bromoethyl acetate, producing the
intermediate 3. The benzyl group was removed under Pd/C and H2
conditions. The product 4 was alkylated with 1-bromo-2-methox-
yethane to obtain 5. After the nitration of 5, the nitro group in 6 was
reduced by Pd/C and H2. The quinazolin heterocycle in 8 was formed
by the intermediate 7, formamide and ammonium formate. The halo-
genation of phenolic hydroxyl group in 8 produced intermediate 9. The
nucleophilic substitution of 9 was conducted in the presence of 3-
aminophenylacetylene. Finally, the product 10 was hydrolyzed with
KOH, resulting in the formation of compound E.
The structural modification of heptamethine cyanine dyes includes
the substitution of different groups on the C-5 or N-1 position of the
indole structure and the introduction of rigid cyclohexenyl ring in the
middle of the methine chain. The quaternary ammonium compound 11
was synthesized according to our previous study (see in Scheme S2).21
Heptamethine cyanine dyes I (Scheme 2) were obtained by an one-step
Aldol reaction between compound 11 and 2-chloro-3-(hydro-
xymethylene)cyclohex-1-enecarbaldehyde (12) with the addition of
base (AcONa).22 And the intermediate 12 was synthesized by Vilsmeier-
Haack reaction using POCl3, DMF and cyclohexanone as reacting
3

X. Yang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127557
Table 1
Anti-proliferation on tumor cells (μM) of test compounds in A549, H460, H1299 and MDA-MB-231 cells.
R1
R2
R1
R1
R1
R1
Cl
N
N
N
N
N
N
Br
Br
Br
O
O
O
HOOC
CE1-CE4
HOOC
CE5-CE7
CE8-CE17
O
O
O
O
O
O
O
O
O
O
O
N
O
N
H
N
H
H
N
N
N
N
N
N
Compd.
R1
H
R2
Cytotoxicity IC50 (μM)
A549
H460
H1299
MDA-MB-231
CE1
–
9.04
15.20
0.816
2.70
6.96
4.31
CE2
CH3
OCH3
Br
–
2.38
1.55
3.36
CE3
–
1.96
2.21
4.23
CE4
–
7.33
13.66
7.25
17.58
5.14
0.609
1.62
CE5
H
–
1.13
CE6
CH3
OCH3
H
–
2.10
5.65
4.15
6.21
CE7
–
2.59
5.12
14.94
2.00
3.30
CE8
H
H
H
H
H
0.631
2.07
0.57
< 0.125
0.313
< 0.125
< 0.125
0.356
0.306
0.531
< 0.125
0.833
0.311
15.51
< 0.025
CE9
CH3
OCH3
Br
2.12
1.74
CE10
CE11
CE12
CE13
CE14
CE15
CE16
CE17
Erlotinib
Taxol
3.94
2.47
0.68
0.579
0.23
0.655
0.813
0.549
0.827
0.217
0.855
0.301
25.32
< 0.001
0.693
0.541
0.378
1.348
0.11
CF3
H
OCH3
OCH3
OCH3
OCH3
OCH3
–
0.388
0.678
0.455
2.08
CH3
OCH3
Br
0.246
0.079
28.92
0.005
CF3
–
0.177
2.63
–
–
< 0.001
reagents. It is noted that there is no need to purify intermediate I after
the reaction was finished. The dyes (I) can be acquired just through
diluting the acetic anhydride solvent with water and filtration. Com-
pound CE1-CE4 were prepared by the reaction of the intermediates I
and E with the addition of N-(3-dimethylaminopropyl)-N'-ethylcarbo-
diimide hydrochloride (EDCI) and DMAP.
reaction of dye III and E. The structure of the above target compounds
was confirmed by NMR spectra and high resolution mass spectrum.
Subsequently, the anti-proliferation effect of compounds CE1-CE17
on tumor cells by MTT assay (erlotinib and taxol as the control drugs).
EGFR is highly expressed in lung cancer and breast cancer. Therefore,
human lung adenocarcinoma cells A549, human large cell lung cancer
cells H460, human lung cancer cells H1299 and human breast cancer
cells MDA-MB-231 were used to investigate the antiproliferative ac-
tivity of compounds CE1-CE17. The results are shown in Table 1.
The following SAR can be gathered from the data shown in Table 1.
Firstly, all compounds exhibited superior antiproliferative activity
against H460, H1299 and MDA-MB-231 cells, and most compounds
(except CE1, CE4 and CE10) displayed better antiproliferative activity
over A549 cells in contrast with erlotinib. Secondly, the anti-
proliferative activity of compounds CE8-CE17 is better than com-
pounds CE1-CE7, indicating that heptamethine cyanine dye might
possess superior tumor targeting ability when N-1 position is butyl.
Thirdly, the antiproliferative activity of the conjugates could be
Different from the synthetic route of I, the Aldol reaction was car-
ried out at the presence of 11, N-(5-anilino-2,4-pentadienylidene)ani-
line hydrochloride (13) and EtN3 as the base when producing hepta-
methine cyanine dyes II. Similarly, the reaction between compound II
and E could provide conjugates CE5-CE7 (Scheme 3).
The general synthetic strategy for the formation of the target com-
pounds CE8-CE17 is shown in Scheme 4. Asymmetric heptamethine
cyanine dyes III were obtained by two-step Aldol reaction of 14, N-(5-
anilino-2,4-pentadienylidene)aniline hydrochloride (13) and 11. The
synthetic route of quaternary ammonium compounds 14 is similar to
11. However, there is no need to add base in the first step Aldol reac-
tion, and the pyridine serves as the solvent directly in second step Aldol
reaction. Finally, compounds CE8-CE17 were synthesized by the
Table 3
Table 2
The inhibition effect of test compounds on
The cytotoxicity of CE8 and CE17 on MCF-
EGFR activity in A549 cells.
Compd.
10A cells.
Compd.
IC50 (μM)
IC50 (μM)
CE12
0.205
0.124
5.182
CE8
33.2
38.6
CE17
CE17
Erlotinib
4

X. Yang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127557
Fig. 2. Stereoview of the binding site and nearby residues from compound CE17 in EGFR-TK (PDB: 1M17).
significantly improved when the central methine chain is lack of the
rigid cyclohexene ring. Finally, surprisingly, the IC50 of compound
CE17 reduced approximately 14-fold (IC50 = 0.177 μM) compared with
the parent drug erlotinib (IC50 = 2.628 μM) in A549 cells, indicating
that the trifluoromethyl might enhance the antiproliferative activity of
above conjugates.
addition, the erlotinib moiety of compound CE17 successfully enters
into the active hydrophobic cavity (a hydrophobic pocket with the
surrounding residues like Leu-694, Met-742, Leu-768 and Leu820, and
the N-1 of the quinazoline accepts an H-bond from the Met-769 amide
nitrogen),23 while the heptamethine cyanine dye moiety locates at the
outside of the cavity. Thus, above results might interpret the possible
binding mode between compound CE17 and EGFR in tumor cells.
In conclusion, we designed and synthesized a series of novel hep-
tamethine carbocyanine dyes-erlotinib conjugates. Most compounds
exhibited superior antiproliferative activity against A549, H460, H1299
and MDA-MB-231 cell lines compared with the parent drug erlotinib.
Meanwhile, representative compounds (CE8 and CE17) exhibited weak
cytotoxicity on human normal mammary epithelial MCF-10A cells.
Moreover, the conjugate CE17 also showed higher EGFR-TK inhibition
activity (IC50 = 0.124 μM) than erlotinib (IC50 = 5.182 μM) in A549
cell line. Together with the result of the antiproliferative activity assay
and EGFR-TK inhibition assay, erlotinib conjugates probably enhanced
their antitumor activity by synergistic targeting effect. Moreover, the
molecular docking results were consistent with the suppression of
tumor cells. Therefore, the conjugation of tumor cell targeting hepta-
methine dyes and EGFR-TKI will be an attractive approach for the an-
titumor drug design.
To verify whether the combination of EGFR-TKIs with the hepta-
methine cyanine dye will improve their tumor cell targeting and the
transmembrane ability, we investigated the cytotoxicity effect of re-
presentative compounds (CE8 and CE17) on MCF-10A cells (human
normal mammary epithelial cells). As shown in Table 2, the compounds
CE8 and CE17 demonstrated relatively weak cytotoxicity on MCF-10A
cells, suggesting the in vitro tumor cells selectivity of the representative
compounds could be enhanced.
As a result, the activities of most compounds are more excellent,
which demonstrates that integrating heptamethine cyanine dye with
EGFR-TK inhibitor perhaps enhance the antiproliferative effect by the
synergistic targeting to tumor cells.
Since the direct test of the inhibitory effect of above compounds on
EGFR-TK makes little sense, we chose to investigate the cancer cells
EGFR-TK inhibiting effect of above compounds in vitro, so as to better
reflect the transmembrane effect of the target compounds. A549 cells
were selected to determine the EGFR-TK inhibiting activity of com-
pounds CE12 and CE17 with the best antiproliferation activity in A549
cell. Therefore, the inhibition of intracellular EGFR-TK activity was
assessed by human EGFR ELISA Kit (Boster Biological Technology,
China, EK0327). According to the instructions of the kit, after treating
with the concentration of 0.1 μM, 5 μM, 50 μM, 200 μM, and 1000 μM
of compounds CE12, CE17 and erlotinib 30 h, A549 cells were lysed.
The protein level was determined by the bicinchoninic acid (BCA)
method, and the EGFR activity of the cells was measured by the ELISA
method. The absorbance (OD value) at 450 nm was measured using a
microplate reader (US Synergy-HT). The results are shown in Table 3.
Compounds CE12 and CE17 can both inhibit EGFR-TK activity, and
compound CE17 exhibited more excellent EGFR-TK inhibition than
compound CE12 and erlotinib, which is consistent with the MTT re-
sults. Therefore, the antiproliferative activity and EGFR-TK inhibitory
activity of compound CE17 are superior to erlotinib, implicating that
the antitumor effect is probably associated with the increased in-
tracellular concentrations of target conjugates due to their enhanced
targeting and transmembrane ability to tumor cells.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors appreciate the financial supports from the National
Natural Science Foundation of China (Grant No. 81903463), the
Natural Science Foundation of Liaoning Province (Grant No.
20180540032), the Career Development Support Plan for Young and
Middle-aged Teachers in Shenyang Pharmaceutical University (Grant
No. ZQN2018018) and China Postdoctoral Science Foundation (Grant
No. 2020M673042).
Finally, in order to better understand and verify the potential action
mode of compounds with EGFR-TK, the molecular docking was con-
ducted. Compound CE17 was chosen to dock with EGFR-TK (PDB:
1 M17). As exhibited in Fig. 2, there is an H-bond interaction between
the N-1 of the quinazoline moiety in CE17 and Met-769 of 1 M17. In
Appendix A. Supplementary data
Supplementary data (synthetic procedures and NMR spectra of
compounds CE1-CE17) to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127557.
5

X. Yang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127557
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