Synthesis and Evaluation of Novel Erlotinib-NSAID Conjugates as More Comprehensive Anticancer Agents

Article abstract of DOI:10.1021/acsmedchemlett.5b00286

A series of novel anticancer agents were designed and synthesized based on coupling of different nonsteroidal anti-inflammatory drugs (NSAIDs) with the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib. Both the antiproliferative and pharmacokinetic activity of the target compounds were evaluated using HCC827 and A431 tumor cell lines. Among the derivatives made, compounds 10a, 10c, and 21g showed superb potency, comparable to that of erlotinib. Furthermore, preliminary SAR analysis showed that when the NSAIDs were conjugated via linkage to C-6 OH versus linkage to C-7 OH of the quinazoline nucleus, superior anticancer activity was achieved. Finally, the in vitro pharmacokinetic profile of several conjugates demonstrated the desired dissociation kinetics as the coupled molecules were effectively hydrolyzed, releasing both erlotinib and the specific NSAID in a time-dependent manner. The conjugation strategy represents a unique and simplified approach toward combination therapy, particularly for the treatment of cancers where both EGFR overexpression and inflammation play a direct role in disease progression.

Full text of DOI:10.1021/acsmedchemlett.5b00286

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Evaluation of Novel Erlotinib−NSAID Conjugates
as More Comprehensive Anticancer Agents
Yanmei Zhang,*,† Micky D. Tortorella,† Jinxi Liao,† Xiaochu Qin,† Tingting Chen,† Jinfeng Luo,†
Jiantong Guan,† John J. Talley,§ and Zhengchao Tu*,†
†Drug Discovery Pipeline, Guangzhou Institutes of Biomedicine and Health, Science City, Guangzhou 510530, P. R. China
§Euclises Pharmaceuticals, St. Louis, Missouri 63108, United States
S
* Supporting Information
ABSTRACT: A series of novel anticancer agents were designed
and synthesized based on coupling of different nonsteroidal anti-
inflammatory drugs (NSAIDs) with the epidermal growth-factor
receptor (EGFR) tyrosine kinase inhibitor, erlotinib. Both the
antiproliferative and pharmacokinetic activity of the target
compounds were evaluated using HCC827 and A431 tumor
cell lines. Among the derivatives made, compounds 10a, 10c,
and 21g showed superb potency, comparable to that of
erlotinib. Furthermore, preliminary SAR analysis showed that
when the NSAIDs were conjugated via linkage to C-6 OH
versus linkage to C-7 OH of the quinazoline nucleus, superior anticancer activity was achieved. Finally, the in vitro pharma-
cokinetic profile of several conjugates demonstrated the desired dissociation kinetics as the coupled molecules were effectively
hydrolyzed, releasing both erlotinib and the specific NSAID in a time-dependent manner. The conjugation strategy represents
a unique and simplified approach toward combination therapy, particularly for the treatment of cancers where both EGFR
overexpression and inflammation play a direct role in disease progression.
KEYWORDS: NSAIDs, EGFR, Erlotinib, cancer, COX, conjugate
ung cancer, in particular nonsmall cell lung carcinoma
(NSCLC), is a disease associated with a high mortality rate
familial adenomatous polyposis. Also, many studies have shown
the efficacy of COX-2 inhibitors in various animal models
of cancer. Mechanistically, COX-2 is highly expressed in many
tumor and stromal cells and PGE2, a major product of
COX-mediated arachidonic acid metabolism, is believed to be a
contributing component of angiogenesis in lung cancer.
Therefore, a combination drug that targets both EGFR and
cyclooxygenases may be a more comprehensive and effective
treatment modality for the treatment of lung cancer.
In this study we present a series of esterase hydrolyzable
NSAIDs conjugated with erlotinib, which may have potential as
a new class of chemotherapeutic agents that offers simplified
dosing regimen with improved efficacy.
The preparation of conjugated NSAIDs with erlotinib 10a−f,
11a−g, and 21a−g is outlined in Schemes 1 and 2. Compound 7
was obtained from commercially available 2-amino-4,5-dimethoxy-
benzoate (1) and elaborated according to the procedure pre-
viously described.20,21 Then the 6-OH of compound 7 was reacted
with 1-bromo-2-methoxyethane in the presence of K2CO3 to
generate 8. The desired conjugated derivatives 10a−f were
obtained by reacting 8 with 9a−f, which had been prepared by
esterification the corresponding acid with 2-bromoethanol in
L
due to its complexity and heterogeneity.1−3 Although there has
been significant progress made in its diagnosis, management,
and treatment, the five year survival rate for this disease is still
low.4 Chemotherapy is a primary option for patients with lung
cancers like NSCLC and the use of epidermal growth factor
receptor (EGFR) tyrosine kinase inhibitors has become a
frontline approach due to their demonstrable effectiveness in
slowing the progression of the disease as well as increasing the
overall survival rate of patients.5−7
However, in cancer treatment, using a single drug directed
toward just one target/mechanism is usually inadequate and
overtime disease management usually requires drug combina-
tions targeting multiple cancer pathways to sustain efficacy.8−10
Choosing the right combination of chemotherapeutic agents
is crucial for maximizing progression free survival while mini-
mizing side effects.
Inflammation has received considerable attention for its role
in the progression and aggressiveness of many cancers including
NSCLC. Many studies have shown the efficacy of NSAIDs in
animal models of cancer prevention, partly due their ability
to block cyclooxygenase (COX) activity.11−19 Mechanistically,
cyclooxygenases including COX-1 and especially COX-2 have
been shown to be important in many stages of oncogenesis. A
case in point is the USA FDA approval of the NSAID, celecoxib
(Celebrex, a selective COX-2 inhibitor), for use in patients with
Received: July 15, 2015
Accepted: September 3, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00286
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Compounds 10a−f and 11a−g
a
Reagents and conditions: (a) HCO2NH4, HCONH2, 140 °C, 24 h; (b) HBr, Ac2O; (c) Ac2O, py; (d) POCl3, CHCl3; (e) 3-ethynylaniline, i-PrOH,
reflux; (f) NH4OH; (g) 1-bromo-2-methoxyethane (1.0 equiv), K2CO3, DMF, 20 °C; (h) 9a−f, K2CO3, DMF; (i) 2-bromoethanol, SOCl2.
a
Scheme 2. Synthesis of Compounds 21a−g
a
Reagents and conditions: (a) 1-bromo-2-methoxyethane, NaH, DMF; (b) 2-bromoethyl acetate, K2CO3, DMF; (c) conc. HNO3, conc. H2SO4, HOAc; (d)
Fe, HOAc; (e) HCOONH4, HCONH2; (f) POCl3, CHCl3; (g) 3-ethynylaniline, i-PrOH, reflux; (h) NaOH, MeOH; (i) COCl2, DCM; (j) 9a−f, Et3N, DCM.
B
DOI: 10.1021/acsmedchemlett.5b00286
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
showed less activity in their coupled state compared to the
corresponding unconjugated molecules. The noncoupled com-
pounds effectively blocked their target enzymes with IC50
values consistent with previous reports.22−26 These data suggest
that the conjugated compounds are likely acting as pro-drugs
for their parent molecules. Release of the parent molecules
from these pro-drugs may be advantageous from a safety per-
spective as the plasma levels of fully active drugs may be more
precisely managed using this strategy. In addition, it has been
reported that esterases have elevated activity in tumors
suggesting that activation of the drug conjugates may be
enhanced at sites of disease relative to normal tissues.27,28
To assess the efficacy of the conjugated compounds as anti-
cancer agents, a series of coupled molecules were evaluated
for their antiproliferative activity against two tumor cell lines
HCC827 and A431. As hoped, multiple conjugated drugs
demonstrated excellent activity in blocking tumor cell prolifera-
tion (Table 2). Coupled compounds 10a, 10b, 10c, 10d, 10f,
21a, 21c, and 21g were particularly active at blocking prolifera-
tion of HCC827 cells. The efficacy potency of these com-
pounds was comparable to erlotinib, which was used as a
positive control. SAR analysis showed that NSAIDs conjugated
via linkage to C-6 OH versus linkage to C-7 OH of the quinazo-
line nucleus possessed superior in vitro anticancer activity. It is
important to point out that tumor cell-based assays are not
adequate to assess the synergistic activity between erlotinib and
the specific NSAID as cells grown under these conditions lack a
significant inflammatory component. Future studies will focus on
testing candidate conjugated drugs in animal models of cancer.
To make sure the drugs were acting on their respective tar-
gets, namely, EGFR tyrosine kinase and COX-2, as a repre-
sentative example, cells and media were collected from 10d
treated HCC827 cultures and analyzed for a reduction in both
phosphorylation of EGFR and PGE2 levels by Western blot
analysis and HPLC, respectively. As expected, a dose-dependent
reduction of phosphorylation of EGFR (Figure 1) and PGE2
(IC50 23 μM; data not shown) was observed indicating that 10d
is acting on its target/mechanism(s).
Table 1. Inhibition of EGFR, COX-1, and COX-2
enzyme inhibition IC50 value (μM)
compd
erlotinib
EGFR
COX-1
>10
COX-2
>10
0.0005 0.0004
>10
aspirin
ibuprofen
sulindac
naproxen
indomethacin
ketoprofen
10a
>10
>10
>10
>10
1.1 2.27
8.8 2.72
0.28 0.21
0.41 0.06
0.061 0.05
>10
>10
16 10.975
0.18 0.82
0.11 0.05
0.067 0.05
>10
>10
>10
>10
0.005 0.0006
0.009 0.0143
0.031 0.0034
0.072 0.0200
0.027 0.0015
0.032 0.0037
0.034 0.0068
>10
10b
44.67 20.61
>10
>10
10c
>10
10d
24.48 14.71
11.34 21.40
>10
>10
10e
>10
10f
>10
11a
39.57 12.97
>10
>10
11b
>10
11c
0.027 0.0019
0.39 0.0480
0.033 0.0028
0.88 0.2820
0.001 0.0003
>10
>10
11d
>10
>10
11e
31.38 0.31
>10
>10
11f
>10
GJT-0383
>10
>10
the presence of SOCl2. Analogues 11a−g were prepared by a
similar experimental procedure.
Ethyl 3,4-dihydroxybenzoate was selectively alkylated with
1-bromo-2-methoxyethane to afford 12, which was then
converted to compound 13 in the presence of 2-bromoethyl
acetate and K2CO3. Nitration of 13 with concentrated HNO3
and H2SO4 in acetic acid gave 14, which was converted into
aniline 15 by reduction with iron in acetic acid. Quinazolin-
4(3H)-one 16 was prepared by condensation of 15 and
HCOONH4 in HCONH2. Treatment of 16 with POCl3 in
CHCl3 gave 4-chloroquinazoline 17, which was treated with
3-ethynylaniline in isopropyl alcohol to afford 4-anilinoquinazo-
line derivative 18. Hydrolysis of the acetyl group of compound
18 was performed using sodium hydroxide in methanol to
provide the key intermediate 19. The desired conjugated com-
pounds 21a−g were prepared by reacting 19 with the cor-
responding NSAID acyl chloride using Et3N as the base in DCM.
To assess the potency of the conjugated compounds versus
the individual noncoupled molecules, selected compounds
were tested against EGFR tyrosine kinase, COX-1 and COX-2
enzymes (Table 1). As expected, the conjugated compounds
To assess the time-dependent hydrolysis of the conjugated
compounds and the corresponding dissociation of the parent
molecules, a pharmacokinetic analysis was performed using
compound 10d in cell culture. In the experiment, 10d was
added to culture media and then allowed to incubate with
HCC827 tumor cells for up to 24 h. During the incubation,
conditioned medium was collected at various time points
and assessed for the coupled compound 10d, and the parent
Table 2. Summary of the in Vitro Antiproliferative Activities of Conjugate Compounds against Two Human Cell Lines Including
Epidermoid and Lung Adenocarcinoma
IC50 (μM)
IC50 (μM)
compd
10a
HCC827
A431
compd
HCC827
A431
0.1 0.03
0.3 0.18
0.9 0.36
0.9 0.27
3.5 2.13
1.6 0.82
2.1 0.74
>10
1.8 0.49
5.4 1.83
>10
10b
10d
10f
0.4 0.23
0.1 0.04
0.2 0.05
16.5 18.35
2.3 0.32
>10
2.2 0.49
3.2 0.10
>10
10c
10e
11a
>10
11b
11d
11f
NA
11c
>10
NA
11e
>10
NA
11g
>10
21a
0.1 0.04
0.4 0.29
1.2 0.46
0.029 0.004
>10
21b
>10
21c
4.8 2.33
>10
21d
0.5 0.28
0.3 0.21
0.014 0.003
5.0 0.21
1.7 1.35
1.27 0.17
21e
21g
erlotinib
0.844 0.02
GJT-0383
C
DOI: 10.1021/acsmedchemlett.5b00286
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
naproxen overtime. Within 24 h approximately 50% of com-
pound 10d was hydrolyzed demonstrating that the conjugated
drug can be processed in a cell based system. With the loss of
compound 10d there is a corresponding increase in the
concentrations of GJT-0383 and naproxen. At time 0 a small
amount of GJT-0383 and naproxen was detected suggesting
some hydrolysis during storage of 10d.
In the cell based experiments, active serum was used bringing
up the possibility that esterases present in the serum are partly
or mostly responsible for the hydrolysis of 10d overtime. In
order to address this, 10d was incubated with both active versus
heat inactivated whole serum and measuring the hydrolysis
overtime. It was observed that 10d was effectively hydrolyzed
overtime in the active serum, but in contrast, little or no
hydrolysis of 10d occurred in the heat inactivated serum
presumably due to the denaturing of the esterases contained in
the serum (Figure 2). Based on these data it appears that a
major proportion of the activation of 10d observed in HCC827
and A431 cells is mediated by serum esterases.
Figure 1. Western blot analysis showing the effects of 10d at concen-
trations of 0 to 500 nM on the phosphorylation of EGFR in HCC827
cells.
Scheme 3. Hydrolysis of Compound 10d
In this study we describe a new series of conjugated com-
pounds that may be used as a potentially simplified approach
for treating cancers where both EGFR overexpression and
inflammation are involved. Future studies will focus on the
in vivo efficacy of the more potent coupled compounds in
animal models of colon and lung cancer where inflammation is
believed to be a significant contributing factor to disease
progression.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acsmedchemlett.5b00286.
■
Table 3. Hydrolysis of Compound 10d in HCC827
S
time (h)
10d (μg/L)
GJT-0383 (μg/L)
naproxen (μg/L)
0
3707 4.714
3567 26.24
3427 12.47
3233 12.47
3150 21.60
2923 33.00
2137 20.55
105.0 2.160
158.3 4.110
224.3 4.922
704.7 3.682
1277 18.86
2363 9.428
4397 123.9
91.20 0.8832
108.4 8.536
128.7 9.672
293.0 39.91
382.0 29.34
849.0 87.41
2017 65.49
Complete experimental details along with synthesis and
characterization of Erlotinib−NSAID conjugates (PDF)
0.25
0.5
2
4
AUTHOR INFORMATION
Corresponding Authors
*E-mail: zhang_yanmei@gibh.ac.cn.
*E-mail: tu_zhengchao@gibh.ac.cn.
Funding
This work was supported in part by the Guangzhou Science &
Technology Project (2011Y2-00026), Guangdong Science and
Technology International Cooperation projects (Grant No.
2013B050800009) (to Y.Z.), CAS Key Technology Talent Program
China (2013) (to Z.T.) and Guangdong Science and Technology
Plan projects, China (Grant No. 2013B040200031) (to Z.T.).
■
8
24
molecules naproxen and erlotinib. In the case of erlotinib,
following hydrolysis of compound 10d an additional hydrogen
atom is left behind giving the molecule a slightly higher
molecular mass and referred to as GJT-0383 in this study
(Scheme 3). To make sure GJT-0383 is as potent as erlotinib,
the compound was tested for activity against EGFR tyrosine
kinase. Both GJT-0383 and erlotinib showed comparable IC50
values (Table 1).
Notes
As can be seen in Table 3, there is a time-dependent hydro-
lysis of 10d resulting in the release of both GJT-0383 and
The authors declare no competing financial interest.
Figure 2. Hydrolysis of 10d in active versus heat inactivated serum.
D
DOI: 10.1021/acsmedchemlett.5b00286
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
assay for identification of natural products as inhibitors of
prostaglandin biosynthesis. J. Nat. Prod. 1998, 61, 2−7.
(23) Blobaum, A. L.; Uddin, J.; Felts, A. S.; Crews, B. C.; Rouzer, C.
A.; Marnett, L. J. The 2′-Trifluoromethyl Analogue of Indomethacin Is
a Potent and Selective COX-2 Inhibitor. ACS Med. Chem. Lett. 2013,
4, 486−490.
(24) Zarghi, A.; Arfaei, S. Selective COX-2 Inhibitors: A Review of
Their Structure-Activity Relationships. Iranian J. of Pharm. Res. 2011,
10, 655−683.
(25) Cicconetti, A.; Bartoli, A.; Ripari, F.; Ripari, A. COX-2 selective
inhibitors: a literature review of analgesic efficacy and safety in oral-
maxillofacial surgery. Oral Surgery, Oral Medicine, Oral Pathology, Oral
Radiology, and Endodontology. 2004, 97, 139−146.
(26) Patrono, C.; Rocca, B. Non-steroidal Anti-inflammatory Drugs:
Past, Present and Future. Pharmacol. Res. 2009, 59, 285−289.
(27) Giang, I.; Boland, E. L.; Poon, G. M. Prodrug applications for
targeted cancer therapy. AAPS J. 2014, 16, 899−913.
(28) Singh, Y.; Palombo, M.; Sinko, P. J. Recent Trends in Targeted
Anticancer Prodrug and Conjugate Design. Curr. Med. Chem. 2008, 15,
1802−1826.
REFERENCES
■
(1) Sakashita, S.; Sakashita, M.; Sound Tsao, M. Genes and
Pathology of non-small cell lung carcinoma. Semin. Oncol. 2014, 41,
28−39.
(2) Stella, G. M.; Luisetti, M.; Pozzi, E.; Comoglio, P. M. Oncogenes
in non-small-cell lung cancer: emerging connections and novel
therapeutic dynamics. Lancet Respir. Med. 2013, 1, 251−6.
(3) Hensing, T.; Chawla, A.; Batra, R.; Salgia, R. A personalized
treatment for lung cancer: molucular pathways, targeted therapies, and
genomic characterization. Adv. Exp. Med. Biol. 2014, 799, 85−117.
(4) Schiller, J. H.; Harrington, D.; Belani, C. P.; Langer, C.; Sandler,
A.; Krook, J.; Zhu, J.; Johnson, D. H. Comparison of four
chemotherapy regimens for advanced non−small-cell lung cancer. N.
Engl. J. Med. 2002, 346, 92−98.
(5) Goffin, J. R.; Zbuk, K. Epidermal growth factor receptor:
pathway, therapies, and pipeline. Clin. Ther. 2013, 35, 1282−1303.
(6) Zhang, J.; Gold, K. A.; Kim, E. Sorafenib in non-small cell lung
cancer. Expert Opin. Invest. Drugs 2012, 21, 1417−26.
(7) Di Maio, M.; Gridelli, C.; Normanno, N.; Perrone, F.; Ciardiello,
F. Trying to compose the puzzle with all the pieces: epidermal growth
factor tyrosine kinase inhibitors in non-small cell lung cancer. J. Cell.
Physiol. 2005, 205, 355−63.
(8) Genestreti, G.; Di Battista, M.; Cavalla, G.; Bartolotti, M.;
Brandes, A. A. Maintenance therapy in non-small cell lung cancer.
Expert Rev. Anticancer Ther. 2015, 31, 1−8.
(9) Queirolo, P.; Picasso, V.; Spagnolo, F. Combined BRAF and
MEK inhibition for the treatment of BRAF-mutated metastatic
melanoma. Cancer Treat. Rev. 2015, 41, S0305−7372.
(10) Diaby, V.; Tawk, R.; Sanogo, V.; Xiao, H.; Montero, A. J. A
review of systematic reviews of the cost-effectiveness of hormone
therapy, chemotherapy, and targeted therapy for breast cancer. Breast
Cancer Res. Treat. 2015, 151, 27−40.
(11) Chen, Z.; Li, M.; Wang, Z.; Wieand, H. S.; Grandis, J. R.; Shin,
D. M. Simultaneously targeting epidermal growth factor receptor
tyrosine kinase and cyclooxygenase-2, an efficient approach to
inhibition of squamous cell carcinoma of the head and neck. Clin.
Cancer Res. 2004, 10, 5930−5939.
(12) Wirth, L. J.; Haddad, R. I.; Lindeman, N. I.; Zhao, X.; Lee, J. C.;
Joshi, V. A.; Norris, C. M.; Posner, M. R. Phase I study of gefitinib plus
celecoxib in recurrent or metastatic squamous cell carcinoma of the
head and neck. J. Clin. Oncol. 2005, 23, 6976−6981.
(13) Vosooghi, M.; Amini, M. The discovery and development of
cyclooxygenase-2 inhibitors as potential anticancer therapies. Expert
Opin. Drug Discovery 2014, 9, 255−67.
(14) Misra, S.; Sharma, K. COX-2 signaling and cancer: new players
in old arena. Curr. Drug Targets 2014, 15, 347−59.
(15) Khan, Z.; Khan, N.; Tiwari, R. P.; Sah, N. K.; Prasad, G. B.;
Bisen, P. S. Biology of Cox-2: an application in cancer therapeutics.
Curr. Drug Targets 2011, 12, 1082−93.
(16) Kao, J.; Sikora, A. T.; Fu, S. Dual EGFR and COX-2 inhibition
as a novel approach to targeting head and neck aquamous cell
carcinoma. Curr. Cancer Drug Targets 2009, 9, 931−7.
(17) Ranger, G. S. Current Concepts in Colorectal Cancer
Prevention with Cyclooxygenase Inhibitors. Anticancer Res. 2014, 34,
6277−6282.
(18) Horn, L.; Backlund, M.; Johnson, D. H. Targeting the
eicosanoid pathway in non-small-cell lung cancer. Expert Opin. Ther.
Targets 2009, 13, 675−688.
(19) Spano, J. P.; Chouahnia, K.; Morere, J. F. Cyclooxygenase 2
̀
inhibitors and lung carcinoma. Bull. Cancer 2004, 91 (Suppl 2), S109−
112.
(20) Prasad, J.; Rao, N.; Chowdary, V. A novel process for the
preparation of erlotinib. PCT Patent Appl. WO 2007/060691A2,
2007.
(21) Chandregowda, V.; Rao, G. V.; Reddy, G. C. Convergent
Approach for Commercial Synthesis of Gefitinib and Erlotinib. Org.
Process Res. Dev. 2007, 11, 813−816.
(22) Noreen, Y.; Ringbom, T.; Perera, P.; Danielson, H.; Bohlin, L.
Development of a radiochemical cyclooxygenase-1 and −2 in vitro
E
DOI: 10.1021/acsmedchemlett.5b00286
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX