Potential of substituted quinazolines to interact with multiple targets in the treatment of cancer

Article abstract of DOI:10.1016/j.bmc.2021.116061

The efficacy of quinazoline-based antiglioma agents has been attributed to their effects on microtubule dynamics.^1,2 The design, synthesis and biological evaluation of quinazolines as potent inhibitors of multiple intracellular targets, including microtubules and multiple RTKs, is described. In addition to the known ability of quinazolines 1 and 2 to cause microtubule depolymerization, they were found to be low nanomolar inhibitors of EGFR, VEGFR-2 and PDGFR-β. Low nanomolar inhibition of EGFR was observed for 1–3 and 9–10. Compounds 1 and 4 inhibited VEGFR-2 kinase with activity better than or equal to that of sunitinib. In addition, compounds 1 and 2 had similar potency to sunitinib in the CAM angiogenesis assay. Multitarget activities of compounds in the present study demonstrates that the quinazolines can affect multiple pathways and could lead to these agents having antitumor potential caused by their activity against multiple targets.

Full text of DOI:10.1016/j.bmc.2021.116061

Bioorg. Med. Chem. 35 (2021) 116061
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Potential of substituted quinazolines to interact with multiple targets in the
treatment of cancer
,
Shruti Choudharya 1, Arpit Doshia, Lerin Luckett-Chastainb, Michael Ihnatb, Ernest Hamelc,
,
*
Susan L. Mooberryd, Aleem Gangjeea
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, United States
b College of Pharmacy, University of Oklahoma Health Science Center, 1110 North Stonewall, Oklahoma City, OK 73117, United States
c Molecular Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer
Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, United States
d Department of Pharmacology, Mays Cancer Center, University of Texas Health Science Center, San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, United
States
A R T I C L E I N F O
A B S T R A C T
Keywords:
The efficacy of quinazoline-based antiglioma agents has been attributed to their effects on microtubule dy-
namics.1,2 The design, synthesis and biological evaluation of quinazolines as potent inhibitors of multiple
intracellular targets, including microtubules and multiple RTKs, is described. In addition to the known ability of
quinazolines 1 and 2 to cause microtubule depolymerization, they were found to be low nanomolar inhibitors of
EGFR, VEGFR-2 and PDGFR-β. Low nanomolar inhibition of EGFR was observed for 1–3 and 9–10. Compounds 1
and 4 inhibited VEGFR-2 kinase with activity better than or equal to that of sunitinib. In addition, compounds 1
and 2 had similar potency to sunitinib in the CAM angiogenesis assay. Multitarget activities of compounds in the
present study demonstrates that the quinazolines can affect multiple pathways and could lead to these agents
having antitumor potential caused by their activity against multiple targets.
Quinazolines
Microtubule targeting agents
Receptor tyrosine kinase
Multi-target inhibitors
Angiogenesis
1. Introduction
approval of several multi-RTK inhibitors such as axitinib10,11 (VEGFR,
PDGFR, c-kit), pazopanib12 (PDGFR, VEGFR, c-kit, FGFR), vandetanib13
Angiogenesis is the formation of new blood vessels from existing
vasculature and is an essential process for tumor growth and metas-
tasis.3 Proangiogenic growth factors such as VEGF, PDGF, and EGF are
released by tumors under hypoxic conditions. These growth factors bind
to and activate their respective RTKs: VEGFR, PDGFR-β and EGFR,
leading to a cascade of events that promote angiogenesis and tumor
growth, survival and metastasis.4–6 RTK inhibitors that prevent angio-
genesis established a new paradigm in cancer chemotherapy.7,8 The use
of multi-RTK inhibitors in cancer chemotherapy offers an advantage in
overcoming resistance mechanisms that limit single RTK inhibitors,
including point mutations in the ATP binding site and upregulation of
additional RTKs.9 Thus, the use of multi-RTK inhibitors has emerged as
an important approach in cancer chemotherapy and is validated by the
(VEGFR, EGFR, RET kinase), sunitinib14,15 (VEGFR, PDGFR and c-kit)
and sorafenib16,17 (VEGFR-2, PDGFR β and Raf kinase), among others.
The cytostatic activity of RTK inhibitors does not kill cancer cells, and
thus, they need to be combined with cytotoxic chemotherapeutic agents
and/or radiation to improve therapeutic outcomes.18–21
Microtubules are essential cytoskeletal components in eukaryotic
cells, and these cytoskeletal components are involved in cellular pro-
cesses during both interphase and mitosis.22 In interphase, microtubules
are involved in intracellular trafficking of proteins and organelles. In
dividing cells, they make up the mitotic spindle and are essential for the
separation of daughter chromosomes.22,23 Microtubule targeting agents
(MTAs) act by interfering with crucial processes involved in cell division
and cellular transport in interphase.22–24
Abbreviations: RTKs, Receptor tyrosine kinases; VEGF, vascular endothelial growth factor; VEGFR-2, vascular endothelial growth factor receptor-2; PDGFR-β,
platelet-derived growth factor receptor-β; EGFR, epidermal growth factor receptor; CA-1, combretastatin A-1; CA-4, combretastatin A-4; CAM, chorioallantoic
membrane; Pgp, P-glycoprotein, MTAs, microtubule targeting agents.
* Corresponding author.
E-mail address: gangjee@duq.edu (A. Gangjee).
1
Present address: The Scripps Research Institute, Jupiter, FL 33458, United States.
https://doi.org/10.1016/j.bmc.2021.116061
Received 11 November 2020; Received in revised form 28 January 2021; Accepted 1 February 2021
Available online 12 February 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
The strategy of using a combination of drugs, each acting at a
different biological target, has been successfully applied in the treatment
of many diseases.25,26 The basic premise for such an approach is to
overcome alternate pathways that may be activated when only one
pathway is inhibited. Similarly, combination chemotherapy with anti-
angiogenic and cytotoxic agents act by simultaneously inhibiting mul-
tiple modes, thereby inhibiting the growth of tumors dependent on
multiple signaling pathways.18–21
(EGFR) and vandetanib (VEGFR-2, VEGFR-3 and EGFR) (Fig. 2).
Structure-activity relationships with the quinazoline scaffold using N4-H
analogs as EGFR inhibitors suggest that the quinazoline scaffold has bulk
tolerance at the 6- and 7-positions.41,42 However, the corresponding N4-
CH3 analogs were not explored for their RTK inhibitory activities.
Separately, we have reported various scaffolds such as furo[2,3-d]py-
rimidines34 and pyrrolo[3,2-d]pyrimidines35,43 with N4-CH3 that are
dual acting antitubulin compounds with multi-RTK inhibitory activities.
Thus, on the basis of the structural similarities between 1 and the qui-
nazoline based RTK inhibitors (Fig. 2), it was of interest to determine the
RTK inhibitory properties (if any) of 1 and its antiangiogenic attributes
as well.
The vascular normalization theory proposes that the antiangiogenic
effects of RTK inhibitors causes pruning of abnormal blood vessels and
transiently normalizes the structural and functional integrity of the
vasculature.27 This normalization restores blood flow to the tumors,
improves delivery of co-administered cytotoxic agents and thus provides
an explanation, in part, of the improved efficacy observed with combi-
nation chemotherapy with antiangiogenic agents.27,28 Multi-target
directed agents are rationally designed analogs that incorporate essen-
tial pharmacophores for inhibiting two or more biological targets.29 In
cancer chemotherapy, such agents may help in preventing cancer cells
from developing resistance, lowering the risk of drug-drug interactions,
improving patient compliance and having a lower side effect profile.
Single agents with dual microtubule targeting and antiangiogenic
activities are highly desirable because these agents can exert their
cytotoxic effects throughout the tumor following normalization of tumor
via the antiangiogenic component.30 Clinical evaluation of the safety
and efficacy of metronomically dosed cytotoxic agents in conjunction
with antiangiogenic agents, such as sunitinib and sorafenib, has also
been explored.31–33 The dose-limiting cytotoxicity of conventional
combination chemotherapy could also potentially be avoided because
these agents work by complementary mechanisms.
Compound 1 was docked in the binding site of EGFR (Fig. 3) and had
a docked pose similar to erlotinib. The 2-CH3 group of 1 makes hydro-
phobic interactions with Ala719, the N1 and N3 make hinge region
binding interactions with Met769 and a water mediated H-bond with
Thr766, respectively. The 4-position aniline substitution is oriented in
the pocket in a manner similar to the 3-ethynyl substituted aniline in
erlotinib and lies in a pocket lined by residues Lys721 and Asp831. The
quinazoline scaffold of 1 is stabilized by hydrophobic interactions with
Leu820, Leu768 and Met769. The docked score of 1 was ꢀ 8.68 kcal/
mol, close to the ꢀ 9.29 kcal/mol score of erlotinib.45
The docked pose of 1 in the binding site of VEGFR-2 (Fig. 4)46 shows
that the quinazoline scaffold binds to the pocket where the thio-
benzamide of axitinib (a potent VEGFR inhibitor) binds and interacts
with the backbone of the hinge binding site residue Phe1047. In addi-
tion, the quinazoline scaffold makes a cation-pi interaction with the
sidechain of Lys868. The 4-position aniline substituent of 1 interacts
with Val848, Cys919, Leu1035 and Phe1047 and binds in the pocket
where the indazole scaffold of axitinib binds. The docked score of 1 in
VEGFR-2 was ꢀ 10.71 kcal/mol as compared to axitinib, which had a
docked score of ꢀ 13.25 kcal/mol.
Multi-target directed ligands with dual cytotoxic and antiangiogenic
activities have been shown to significantly reduce tumor growth,
metastasis and angiogenesis in tumor xenograft mouse models, without
any significant toxicity.34,35 Because of their crucial role in angiogen-
esis, VEGFR-2, PDGFR-β and EGFR were chosen as targets in the current
studies. Due to the clinical utility of cytotoxic tubulin inhibitors, such as
paclitaxel, in combination with antiangiogenic agents, tubulin was
chosen as the cytotoxic target.36–38
With the docked scores reported above, it was important to evaluate
1 as an RTK inhibitor. As expected, a preliminary evaluation of 1 in an in
vitro assay for inhibition of EGFR, VEGFR-2 and PDGFR-β resulted in
potent inhibitory activities against all three RTKs (Table 1). Compound
1 was more potent than the clinically used agent sunitinib (EGFR ~ 61-
fold, VEGFR-2 ~ 2-fold and PDGFR-β ~ 15-fold). Compared to erlotinib,
1 was 2-fold less potent in inhibiting EGFR but ~15-fold and ~2-fold
more potent in inhibiting VEGFR-2 and PDGFR-β, respectively. Thus, we
discovered that, in addition to tubulin inhibition, 1 was a multitargeted
RTK inhibitor as well.
Microtubule targeting agents, particularly those binding to the
colchicine site, have been shown to act on tumor vasculature and several
agents were tested in clinical trials for efficacy in glioblastoma multi-
forme (GBM),1,2,39 but none has yet advanced past phase II evaluation.
Compound 1 (verubulin, Fig. 1) was reported as a potent inducer of
apoptosis, binding at the colchicine site and inhibiting tubulin poly-
merization in cells (T47D EC50 = 2 ± 0.1 nM).2 The compound is not a
substrate for multidrug resistance transporters such as P-glycoprotein
(Pgp) and breast cancer resistance protein (BCRP1). In addition, com-
pound 1 is able to cross the BBB to attain 30-fold higher concentrations
in the brain compared to the plasma.2 These pharmacokinetic parame-
ters led to the evaluation of 1 in GBM and other cancers, but it failed to
achieve orphan drug status for GBM.40
1.1. Design of target compounds 2–11
The reported antitubulin activity of 1, along with its RTK inhibitory
activity (Table 1), provided the impetus for us to design quinazoline
based multi-targeted inhibitors with both tubulin and RTK inhibitory
properties in single agents. Compounds 2–4, Series I (Fig. 5), with a 2-
CH3 substitution, were designed to optimize the substitution at the 4-po-
sition for activity with multiple RTKs and at the colchicine site of
tubulin. The clinically used quinazoline RTK inhibitors (Fig. 2) all had a
2-H substitution and hence compounds in Series II (5–7) were designed
to mimic the 2-position substitution of the known RTK inhibitors and to
determine its effect, if any, on microtubule polymerization activity.
Compounds in Series III were expected to bind at the active sites of EGFR
and VEGFR-2 by maintaining the hinge region binding interactions seen
for erlotinib in EGFR (Fig. 3) and axitinib in VEGFR-2 (Fig. 4) while
retaining an interaction at the colchicine site of tubulin. In addition to
EGFR and VEGFR-2, the designed compounds 2–11 were anticipated to
inhibit PDGFR-β analogous to 1.
The quinazoline scaffold has also been widely explored as the basis
for RTK inhibitory activity as exemplified by gefitinib (EGFR), erlotinib
OCH3
H3C
N
4
5
3
6
7
N
The docked pose of 5 in the colchicine site of tubulin (Fig. 6) is very
similar to that of lead compound 1. The quinazoline scaffold in 1 and 5
undergoes hydrophobic interactions with Alaβ250, Leuβ248, Leuβ252,
Leuβ255, Ileβ318, Alaβ354, and Alaβ316. The N4-CH3 in 1 and 5 are
oriented towards and interact with Lysβ254. The N4-phenyl group
H3C
N
1
2
8
1
Fig. 1. Lead compound 1.
2

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
Br
F
F
NH
Cl
NH
N
O
NH
N
O
O
N
O
O
CH3
CH3
N
CH3
N
N
O
O
CH3
N
O
O
N
gefitinib
erlotinib
vandetanib
Fig. 2. Representative quinazoline based clinically used RTK inhibitors.
H3CO
CH3
N
N
5
N
H3C
1
NH
O
N
OCH3
OCH3
N
O
Erlotinib
Fig. 3. Docked pose of lead compound 1 (purple) superimposed on the crystallized ligand erlotinib (cyan) in EGFR (PDB: 1 M17).44
OCH3
H3C
N
N
H3C
N
1
N
N
N
H
O
S
N
H
Axitinib
Fig. 4. Docked pose of 1 (orange) superimposed on the crystallized ligand axitinib (cyan) in VEGFR-2 (PDB: 4AG8).46
3

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
and had docked scores of ꢀ 10.71 to ꢀ 12.15 kcal/mol. Compounds in
Series III docked in a manner similar to 5 and had docked scores of
ꢀ 10.66 to ꢀ 12.00 kcal/mol.
Table 1
EGFR, VEGFR-2 and PDGFR-β inhibitory activity of compound 1.
Compound
EGFR kinase
Flk-1(VEGFR-2) kinase
inhibition (nM)
PDGFR- β kinase
The 2-Cl substituted analog of 1 was reported to be an equipotent
inhibitor of cellular microtubule polymerization (T47D EC50 = 2 ± 0.1
nM) as compared with 12 and hence, by varying the bulk and electronics
at the 2-position, compounds 8–11 (Series III, Fig. 5) were designed to
assess the inhibitory effects on multiple RTKs. These compounds docked
at the colchicine site of tubulin (docked scores, ꢀ 8.06 to ꢀ 9.40 kcal/
mol), EGFR (docked scores, ꢀ 7.59 to ꢀ 8.46 kcal/mol) and VEGFR-2
(docked scores, ꢀ 10.72 to ꢀ 12.42 kcal/mol) in a mode similar to that
shown for lead compound 1 and were predicted be active at the
colchicine site and with EGFR and VEGFR-2.
inhibition (nM)
inhibition (nM)
1
2.8 ± 1.1
8.4 ± 2.2
5.6 ± 0.4
Sunitinib
Erlotinib
172.1 ± 19.4
1.2 ± 0.2
18.9 ± 2.7
124.7 ± 18.2
83.1 ± 10.1
12.2 ± 1.9
hydrophobically interacts with Lysβ352, Alaα180 and Valα181. The
docked score of 1 in the colchicine site of tubulin was ꢀ 8.68 kcal/mol
and that of 5 was ꢀ 8.43 kcal/mol. Compounds in Series I (2–4) had
docked scores of ꢀ 8.17 to ꢀ 9.83 kcal/mol and compounds in Series II
(5–7) ꢀ 8.06 to ꢀ 8.67 kcal/mol, suggesting that these compounds
should have similar or better activity than 1 as MTAs binding to the
colchicine site.
2. Materials and methods
In EGFR, compound 5 (Fig. 7) also undergoes a hinge region binding
interaction with Met769 and a water mediated H-bond with Thr766,
showing similar interactions at the binding site of EGFR as observed
with 1 (Fig. 3). Compounds in Series I docked in a manner similar to that
of 1 and had docked scores of ꢀ 7.38 to ꢀ 8.90 kcal/mol. Compounds in
Series II docked in a manner similar to that of 5 and had docked scores of
ꢀ 8.42 to ꢀ 9.09 kcal/mol.
Compounds 1–11 were synthesized by nucleophilic displacement of
4-chloroquinazolines (12a, 12b and 12c) by the appropriate anilines in
isopropyl alcohol/acetonitrile and a drop of conc. HCl at room tem-
perature for 12 h (see Schemes 1 and 2).
Yields of compounds in Series I were 58% (1, reported yield 71%),2
65% (2, reported yield 79%),48 70% (3, reported yield 41%),2 and 68%
(4). Yields of compounds in Series II were 76% (5, reported yield 79%),1
70% (6, reported yield 69%),49 and 72% (7). Yields of compounds in
Series III were 9% (8, reported yield 87%)1 and 25% (9, reported yield
87%).48 The synthesis of 10 was unsuccessful under the reaction con-
ditions described above, but instead dimer 18 was formed (1H NMR
indicated four methyl singlets, two for SCH3 at 2.48 and 2.49 ppm and
two for NCH3 at 3.29 and 3.57 ppm). Alternatively, a SNAr reaction in
acetonitrile at rt gave 10 in 22% yield. Compound 11 was synthesized
over two steps. The first step involved the nucleophilic displacement of
quinazoline 12c with 6-aminonaphthalen-1-ol (19) to yield 6-((2-
Compound 5 (Fig. 8), when docked in the X-ray crystal structure of
VEGFR-2,46 showed two low energy binding modes, which corroborate
similar findings reported for a series of furo[2,3-d]pyrimidine analogs.34
In Fig. 8A, 5 binds to VEGFR-2 in a binding mode similar to that seen for
compound 1 (Fig. 4) and has a docked score of ꢀ 10.66 kcal/mol. In
addition, due to the absence of bulk at the 2-position, 5 also binds to
VEGFR-2 in an alternate binding mode where the N1 of the quinazoline
scaffold in 5 undergoes H-bonding with Cys919 at the hinge region of
VEGFR-2 and has a similar docking score of ꢀ 9.32 kcal/mol. Com-
pounds in Series I docked in VEGFR-2 in a manner similar to that of 1
Series I
Series II
Series III
R
R
R
N
5
4
3
6
7
N
R
N
H
N
H3C
N
1
Cl
N
R
2
8
R
OCH3
OCH3
OCH3
SCH3
H3C
8
9
2
3
H3C
5
6
7
N
N
N
N
OCH3
SCH3
OCH3
SCH3
H3C
N
N
N
N
OCH3
H3C
4
H3C
10
H3C
N
OCH3
11
H3C
N
Fig. 5. Series I (2–4), Series II (5–7), Series III (8–11).
4

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
Fig. 6. Comparison of the docked poses of 1 (cyan) and 5 (peach) in the colchicine site of tubulin (PDB ID: 4O2B).47
OCH3
H3C
N
N
N
R
1
5
R = CH3
R = H
Fig. 7. Superimposition of the docked poses of 1 (magenta) and 5 (orange) in EGFR (PDB: 1 M17).44
chloroquinazolin-4-yl)amino)naphthalen-1-ol (20). N- and O-methyl-
ation of 20 using methyl iodide and sodium hydride yielded the desired
compound 11 in 47% yield.
N-formyl group using NaBH4 yielded aniline 16 (39%).50 To synthesize
the aniline 17 from 6-aminonaphthalen-1-ol (19), both N- and O-
methylation, were required, and a different strategy was necessary. 6-
Aminonaphthalen-1-ol (19) was first Boc protected to give the N-Boc
protected intermediate 22, which was subsequently dimethylated (both
N- and O-) with methyl iodide in the presence of sodium hydride51 and
To synthesize N-methylated aniline 16 (Scheme 3), commercially
available aniline 21 was formylated using paraformaldehyde in the
presence of sodium methoxide in methanol. Subsequent reduction of the
5

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
Fig. 8. Superimposition of the docked poses of 1 (orange) and 5 (magenta) in VEGFR-2 in two different binding modes (PDB: 4AG8).46
Cl
O
N
POCl3
(C2H5)3N
N
HN
H3C
toluene
H3C
N
13a
12a
(50%)
Cl
N
Cl
O
N
POCl3
(C2H5)3N
N
N
HN
Cl
toluene
N
12c
(Commercially available)
13b
12b
(80%)
Scheme 1. Synthesis of 4-chloro-2-methylquinazoline 12a and 4-chloroquinazoline 12b.
deprotected using trifluoroacetic acid to afford 5-methoxy-N-methyl-
naphthyl-2-amine (17) (55% over 3 steps).52
3. Results and discussion
3.1. Effect on cellular microtubules and cancer cell proliferation
2.1. Evidence of conformational restriction of 1 by 1H NMR
Compounds 2–11 were evaluated for their ability to depolymerize
microtubules in A-10 cells with a phenotypic assay to evaluate micro-
tubule loss. An EC50, the concentration that causes 50% cellular
microtubule loss, was determined. The antiproliferative effects were
evaluated in MDA-MB-435 melanoma cells using the sulforhodamine B
(SRB) assay, and the IC50, the concentration that causes 50% inhibition
of proliferation, was determined. The activities of the compounds were
compared with 1, paclitaxel and CA-4, a small molecule that binds the
Comparison of the 1H NMR spectra of compound 1 with the des-
methyl analog 1a2 in DMSO‑d6 suggested that the N-Me group imparts
conformational restriction along the C4-N bond. In 1, the 5-H proton
appears at δ 6.88. However in compound 1a, the 5-H proton is more
deshielded and appears at δ 8.06 (Fig. 9). The shielding effect of the 5-H
can be attributed to a diamagnetic anisotropic effect in 1 due to the
proximity of the phenyl ring on the 5-H proton in the more favorable
conformation. The same effect, however, does not occur in the less
favorable conformation since the phenyl ring lies anti to the 5-H and
leads to steric clashes between the N-Me and 5-H proton groups. The
diamagnetic anisotropy is also not observed for the conformationally
flexible analog 1a, and the 5-H proton appears more deshielded.
colchicine site of β-tubulin at its interface with α-tubulin (Table 2).
In the antiproliferative assay (Table 2), the potency of the com-
pounds followed the trend: 2-Cl (8–11) > 2-CH3 (1–4) > 2-H (5–7)
substitution, suggesting that small hydrophobic groups at the 2-position
could contribute to improved cellular permeability as indicated by the
CLogP values (Table 2). In addition, small hydrophobic groups at the 2-
position were tolerated for binding at the colchicine site. In the micro-
tubule depolymerization assay, compounds from Series I (1–4) and Se-
ries III (8–11) were equipotent and were 4- to 12-fold more potent than
Series II (5–7) compounds with 2-H substitution. For compounds 1–11,
6

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
OCH3
12a-c
OCH3
SCH3
2
6
9
(65%)
(70%)
(25%)
12a-c
1
5
8
(58%)
(76%)
(10%)
i-PrOH, 12 h
1 drop of conc. HCl
H3C
N
i-PrOH, 12 h
1 drop of conc. HCl
N
H
H
15
17
14
16
OCH3
12a-b
3
7
(70%)
(72%)
12a
H3C
i-PrOH, 12 h
1 drop of conc. HCl
N
H
4
(68%)
H3C
i-PrOH, 12 h
1 drop of conc. HCl
N
H
H3CS
CH3
N
N
H3CS
N
12c
i-PrOH, 12 h
N
SCH3
CH3
18
1 drop of conc. HCl
H3C
N
H
12c
16
10
(22%)
Acetonitrile,
1 drop of conc. HCl
rt, overnight
OCH3
OH
OH
H3C
N
N
N
HN
N
H2N
19
NaH, CH3I
DMF
12c
i-PrOH, 12 h
1 drop conc. HCl
Cl
Cl
N
20
11
(22%)
(47%)
Scheme 2. Synthesis of target compounds 1–11 (Series I, II and III).
the EC50/IC50 ratios (Table 2) are close to 1.0, suggesting a cytotoxic
mechanism of action that is primarily microtubule dependent.
Clinically relevant drug resistance mechanisms associated with most
clinically approved microtubule targeting agents, include expression of
either the β-III isotype of tubulin or Pgp, an ATP-dependent efflux
pump.53 Compounds 1–11 were tested for their efficacy in parental cells
and β-III and Pgp expressing cell lines (Table 3). In β-III overexpressing
HeLa cells, compounds 1–11 were able to all overcome β-III mediated
resistance compared with paclitaxel and were comparable to CA-4 in
their activity. The resistance ratio (Rr) values (Table 3) were 0.6–1.2
versus 1.0 for CA-4 and 8.6 for paclitaxel.
β assays suggested that these compounds were potent inhibitors of
multiple RTKs (Table 5). With respect to the structure–activity rela-
tionship, within Series I, the 4-methoxy-N-methylaniline substitution (1)
was equipotent with the tetrahydroquinoline substitution (2) and 3-fold
better than 3 and 4 with EGFR. For VEGFR-2, compounds 1–4 had
similar inhibitory IC50 values, suggesting the VEGFR-2 binding site is
more accommodating towards introduction of bulk at the 4-position of
the quinazoline scaffold. With PDGFR-β, 1 and 2 has similar activity, but
introduction of a thiomethyl group in 3 results in ~41-fold loss of po-
tency and ~5-fold loss in potency for compound 4 with PDGFR-β.
Comparing the tetrahydroquinoline compounds 2, 6 and 9 across Series
I-III, the 2-H substituted compound 6 resulted in ~7–10-fold loss in
potency for all the RTKs and also a similar loss of activity in the cyto-
toxicity assay, suggesting the presence of bulk at the 2-position is
important for activity in RTKs. 2-CH3 and 2-Cl substitution (2 and 9) did
not affect EGFR activity but results in ~6–10-fold loss of potency for
VEGFR-2 and PDGFR- β, respectively.
Paclitaxel is a known substrate for Pgp, and cells expressing Pgp are
resistant to its effects, resulting in a 240-fold loss in activity (Table 3).
Compounds 1–11 were evaluated for their ability to circumvent Pgp-
mediated drug resistance in the SK-OV-3 isogenic cell line pair. As ex-
pected, the compounds showed activity similar to that of CA-4 and were
essentially equipotent in the parental and Pgp-expressing cells (Rr
values 0.9–1.5). In contrast, paclitaxel had a Rr value of 240, indicating
its susceptibility to Pgp-mediated resistance (see Table 4).
In addition, comparing the 2-position quinazoline substitution across
Series I, II and III for EGFR, 2-CH3 and 2-Cl substitution were ~10-fold
better than the 2-H substitution. For VEGFR-2 binding, 2-CH3 was 6-fold
better than the 2-Cl substitution and ~12-fold better than the 2-H sub-
stitution. In contrast, for PDGFR-β, the 2-H substitution was 1.5-fold
better than the 2-Cl substitution, which in turn was 7-fold less potent
than the 2-CH3 substitution.
Selected quinazoline derivatives, on the basis of their activities in
Tables 2 and 3, were also tested for their activity as inhibitors of tubulin
assembly and their ability to inhibit the binding of [3H]colchicine to
tubulin. The compounds also had potencies similar to that of CA-4 for
inhibition of colchicine binding in both the assays.
Evaluation of compounds 1–4, 6, 8–9 in EGFR, VEGFR-2 and PDGFR-
Moreover, compounds 1–3 were 3- to 10-fold more potent than
7

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
i) paraformaldehyde
NaOMe, MeOH
SCH3
SCH3
H3C
N
H2N
ii) NaBH4
H
21
16
(39%)
OCH3
OH
OH
O
O
CH3I
(Boc)2O
O
N
NaH, rt 2 h
DMF
12 h, rt
O
N
H2N
H
CH3
19
23
(72%)
22
(85%)
OCH3
TFA
2 h, rt
H3C
N
H
17
(95%)
Scheme 3. Synthesis of anilines 16 and 17.
Fig. 9. Conformational restriction of C4-N observed for 1 due to presence of the N-Me group.
However, compounds in Series III were 1- to 4-fold less potent than
erlotinib in inhibiting angiogenesis.
Table 2
IC50 values for inhibition of proliferation of MDA-MB-435 cells and EC50 values
for microtubule depolymerization in A-10 cells.
4. Conclusion
Compound
IC50 ± SD in
MDA-435
EC50 (nM) for Microtubule
Depolymerization in A⋅10
Cells
EC50
IC50
/
CLogP
Substituted quinazolines have been explored as a scaffold for tar-
geting RTKs as well as microtubules. Here we describe the design, syn-
thesis and evaluation of 4-anilino quinazolines with multiple modes of
action contributing to their antiproliferative activities in cancer cells.
Compounds were synthesized by varying substitutions at the 2- and 4-
positions of the quinazoline scaffold. For the MTA effects in general, the
2-H substituted compounds (5–7) were 7- to 10-fold less potent than the
2-CH3 (1–4) and the 2-Cl (8–9) substituted compounds. This suggests
that bulk at the 2-position of the quinazoline scaffold is important for
binding to the colchicine site. In the RTK inhibitory assays, the 2-CH3
and 2-Cl substituted compounds were more potent than the 2-H
substituted compounds. For 2-substituted quinazolines (1–4), varying
the aniline substitution at the 4-position was well tolerated. The 2-Cl
substituted compound 8 was a potent inhibitor of EGFR but resulted
in a 10-fold and 15-fold loss of potency with VEGFR-2 and PDGFR-β,
respectively, when compared to the corresponding 2-CH3 compound 1.
Cells (nM)
Ratio
1
1.7 ± 0.1
1.1 ± 0.2
1.2 ± 0.2
2.0 ± 0.4
7.1 ± 0.8
12.4 ± 1.7
7.8 ± 0.8
0.6 ± 0.1
0.7 ± 0.2
2.7 ± 0.5
1.4 ± 0.1
4.4 ± 0.46
4.5 ± 0.52
2.1
3
1.2
2.7
1.7
1.2
1.2
1.9
1.6
3.3
2.9
1
4.2
4.8
4.8
5.3
3.7
4.3
4.3
4.4
5.1
5.1
5.6
2
3
2
4
2.4
8.2
23.4
12.3
2
5
6
7
8
9
2
10
2.7
1.9
9.8
–
11
1.4
2.2
–
CA-4
Paclitaxel
CLogP values were calculated using ChemDraw Professional 2018.
erlotinib in the CAM assay for angiogenesis inhibition (Table 5).
8

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
Contract No. HHSN261200800001E. The content of this publication
does not necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names, com-
mercial products, or organizations imply endorsement by the U.S.
Government.
Table 3
Compound activity in parental, β-III and Pgp expressing cell lines.
Compound
IC50
±
IC50
±
Rr
IC50
±
IC50 ± SD
in SK-OV-
3-MDR-
1–6/6
Rr Value
(MDR-
SD in
HeLa
(nM)
SD in
WT βIII
(nM)
Value
(WT/
HeLa)
SD in
SK-OV-
3 Cells
(nM)
6/6/SK-
OV-3)
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Cells
(nM)
1
2.0 ±
0.2
1.5 ±
0.2
0.8
1.0
0.8
0.7
0.9
0.8
0.6
1.0
1.2
0.6
0.6
1.0
8.6
2.2 ±
0.1
2.6 ± 0.1
2.2 ± 0.6
2.1 ± 1.1
3.5 ± 0.4
1.2
1.5
1.3
1.2
1.4
1.3
1.1
1.4
1.3
1.1
0.9
1.3
240
6. Experimental section
2
1.2 ±
0.5
1.2 ±
0.4
1.5 ±
0.3
3
1.3 ±
0.6
1.1 ±
0.4
1.6 ±
0.2
6.1. Chemistry
4
2.5 ±
0.3
1.7 ±
0.2
2.9 ±
0.3
All evaporations were carried out under vacuum with a rotary
evaporator. Analytical samples were dried in vacuo in a CHEM-DRY
drying apparatus over P2O5 at 50 ◦C. Melting points were determined
either using a MEL-TEMP II melting point apparatus with FLUKE 51 K/J
electronic thermometer or using an MPA100 OptiMelt automated
melting point system and are uncorrected. Thin-layer chromatography
(TLC) was performed on Whatman® PE SIL G/UV254 flexible silica gel
plates or Sorbetch silica gel TLC plates w/UV254, and the spots were
visualized under 254 and 365 nm ultraviolet illumination. Proportions
of solvents used for TLC are by volume. All analytical samples were
homogeneous on TLC in at least two different solvent systems. Flash
chromatography was carried out on the CombiFlash® Rf 200 (Teledyne
ISCO) automated flash chromatography system with prepacked
RediSep® Rf normal-phase flash columns (230–400 mesh) of various
sizes. The weight of silica gel for column chromatography was in the
range of 50–100 times the weight of the crude compounds being sepa-
rated. Nuclear magnetic resonance spectra for protons (1H NMR) were
recorded on the Bruker Avance II 400 (400 MHz) or Bruker Avance II
500 (500 MHz) NMR systems and were analyzed using MestReC NMR
data processing software. The chemical shift values (δ) are expressed in
ppm (parts per million) relative to tetramethylsilane as an internal
standard: s, singlet; d, doublet; dd, doublet of doublet; t, triplet; q,
quartet; m, multiplet; br, broad singlet; td, triplet of doublet; dt, doublet
of triplet; quin, quintet. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA. Element compositions are within ±0.4% of
the calculated values. Fractional moles of water or organic solvents
frequently found in some analytical samples could not be prevented
despite 24–48 h of drying in vacuo and were confirmed where possible
by their presence in the 1H NMR spectra. All solvents and chemicals
were purchased from Sigma-Aldrich or Fisher Scientific and were used
as received.
5
8.6 ±
0.3
7.4 ±
0.7
10.4 ±
1.1
14.3 ±
0.5
6
16.8 ±
2.2
13.1 ±
0.4
18.1 ±
1.8
23.4 ±
4.5
7
8.5 ±
0.8
7.3 ±
0.4
13.6 ±
0.9
15.6 ±
1.4
8
0.6 ±
0.2
0.6 ±
0.1
0.8 ±
0.1
1.1 ± 0.3
9
0.6 ±
0.2
0.7 ±
0.2
0.8 ±
0.2
1.0 ± 0.5
3.5 ± 0.6
2.6 ± 0.4
7.2 ± 1.1
10
5.0 ±
0.4
3.2 ±
0.2
3.3 ±
0.1
11
2.8 ±
0.5
1.6 ±
0.3
2.8 ±
0.4
CA-4
Paclitaxel
3.3 ±
0.4
3.3 ±
0.3
5.5 ±
0.5
2.8 ±
0.4
24.0 ±
0.2
5.0 ±
0.6
1200 ±
58
Table 4
Inhibition of [3H] colchicine binding and inhibition of tubulin assembly.
Compound
Inhibition of tubulin assembly IC50
Inhibition of colchicine
binding
(µM ± SD)
5 µM
1 µM
inhibitor
inhibitor
3
0.48 ± 0.07
0.64 ± 0.01
0.47 ± 0.02
0.73 ± 0.04
99 ± 0.3
98 ± 0.2
99 ± 0.8
98 ± 0.3
91 ± 0.5
88 ± 0.2
92 ± 0.2
87 ± 0.8
4
8
CA-4
Our studies establish that the quinazoline based analogs are indeed
single agents with multitargeted activities, and that they inhibit both
tubulin and RTKs and have potential as anticancer agents.
6.2. N-Methyl-4-(methylthio)aniline (16)
5. Disclaimer
4-(Methylthio)aniline (21) (1.13 g, 8.12 mmol) was added to a sus-
pension of sodium methoxide (2.19 g, 40.6 mmol) in MeOH (10 mL).
This mixture was poured into another suspension of paraformaldehyde
(0.340 g, 11.4 mmol) in MeOH (10 mL). The reaction mixture was then
This research was supported in part by the Developmental Thera-
peutics Program in the Division of Cancer Treatment and Diagnosis of
the National Cancer Institute, which includes federal funds under
Table 5
RTK inhibitory activities of quinazolines (1–4, 6, 8–9).
Compound
EGFR kinase inhibition
(nM)
Flk-1 (VEGFR-2) kinase
inhibition (nM)
PDGFR-β kinase
A431 Cytotoxicity
(nM)
U251 cytotoxicity
(nM)
CAM angiogenesis
inhibition (nM)
inhibition (µM)
1
2.8 ± 1.1
5.0 ± 0.4
8.7 ± 1.6
10.6 ± 1.8
48.9 ± 6.0
3.6 ± 0.6
5.4 ± 0.3
172.1 ± 19.4
1.2 ± 0.2
8.4 ± 2.2
5.6 ± 0.4
0.9 ± 0.2
2.5 ± 0.2
2.9 ± 0.5
5.3 ± 0.9
16.3 ± 2.0
1.2 ± 0.4
1.8 ± 0.1
–
2.8 ± 0.6
3.1 ± 0.3
4.9 ± 0.9
4.2 ± 2.0
27.8 ± 1.8
32.5 ± 4.1
14.8 ± 1.8
–
2.8 ± 1.1
3.1 ± 1.3
9.8 ± 0.7
ND
2
9.3 ± 3.9
7.8 ± 0.5
3
14.7 ± 2.8
16.8 ± 4.1
111.1 ± 19.2
97.5 ± 10.1
55.2 ± 11.1
18.9 ± 2.7
124.7 ± 18.2
231.5 ± 29.9
29.4 ± 5.1
54 ± 14.1
76.6 ± 16.2
80.6 ± 12.1
83.1 ± 10.1
12.2 ± 1.9
4
6
ND
8
130.0 ± 22.2
41.4 ± 6.1
–
29.1 ± 1.9
9
Sunitinib
Erlotinib
–
–
ND-Not Determined.
9

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
stirred at room temperature. After 5 h, sodium borohydride (0.31 g, 8.1
mmol) was added, and the mixture was kept at reflux for 2 h. After the
reaction was complete, the solvent was evaporated and 1 M KOH (10
mL) was added. The sample was then extracted 3 times using ether and
dried over MgSO4. The combined organic fractions were evaporated, a
silica gel plug was made, and the product was purified by column
chromatography using 10% EtOAc in hexanes to yield 16 as a brown
liquid (0.49 g, 39%). TLC Rf = 0.76 (Hex: EtOAc; 1:1); 1H NMR,
DMSO‑d6 (400 MHz): 2.33 (s, 3H, SCH3), 2.64–2.66 (d, 3H, J = 5.09,
NHCH3), 5.72–5.73 (m, 1H, exch, NH), 6.49–6.52 (d, 2H, J = 8.58, Ar),
7.11–7.13 (d, 2H, J = 8.57, Ar). 1H NMR agreed well with the litera-
ture54 values.
6.5. 4-Chloroquinazoline (12b)
To a 250 mL round-bottomed flask were added quinazolin-4(3H)-one
(13b) (2.00 g, 13.7 mmol), trimethylamine (3 mL), and toluene (30 mL).
The mixture was cooled to 0 ◦C, and phosphorus oxychloride (2 mL) was
added. The reaction mixture was stirred at room temperature for 1 h.
The reaction was then heated to 95 ◦C for 3 h. After monitoring the
reaction by TLC, the reaction mixture was cooled to room temperature
and diluted with 30 mL of ethyl acetate. The solution was then washed
with 10 mL of ice cold water, 10 mL of saturated NaHCO3, 10 mL of
water, 5 mL of 1 N HCl, 10 mL of water, 10 mL of saturated NaHCO3 and
10 mL of saturated NaCl. The organic layer was dried over Na2SO4,
filtered and concentrated to obtain 12b as a yellow solid (1.79 g, 80%).
TLC Rf
=
0.85 (MeOH:CHCl3; 1:5); mp, 144.2–146.1 ◦C (lit.56
6.3. 5-Methoxy-N-methylnaphthalen-2-amine (17)
145–147.5 ◦C); 1H NMR, DMSO‑d6 (400 MHz): 7.59–7.63 (dt, 1H, J1
=
1.09, J2 = 7.23, J3 = 8.01, Ar), 7.76–7.78 (d, 1H, J = 8.24, Ar),
7.89–7.92 (dt, 1H, J1 = 1.47, J2 = 7.24, J3 = 8.29, Ar), 8.15–8.17 (d, 1H,
J = 7.03, Ar), 8.58 (s, 1H, Ar).
Compound 17 was prepared over 3 steps. 6-Aminonaphthalen-1-ol
(19) (1.25 g, 7.85 mmol) was stirred with boc anhydride (5 mL) in
ethyl acetate (15 mL) at room temperature. After 12 h, boc anhydride
was removed by evaporation, and the residue was dissolved in ethyl
acetate and purified using flash chromatography in CHCl3/MeOH to
afford 22 as a brown liquid (1.74 g, 85%). TLC Rf = 0.67 (CH3OH:
CHCl3; 1:5); 1H NMR, DMSO‑d6 (400 MHz): 1.51 (s, 9H, (CH3)3),
6.71–6.73 (dd, 1H, J1 = 7.13, J2 = 1.21, Ar), 7.17–7.25 (m, 2H, Ar),
7.43–7.46 (dd, 1H, J1 = 1.97, J2 = 9.14, Ar), 8.00–8.01 (d, 2H, J = 8.87,
Ar), 9.54 (s, 1H, exch., OH), 10.00 (s, 1H, exch., NH). Compound 22
(1.70 g, 6.79 mmol) was dissolved in DMF (30 mL) under argon. Sodium
hydride (0.488 g, 20.4 mmol) was added, and the mixture was stirred at
0 ◦C for 15 min. Methyl iodide (2.12 g, 14.9 mmol) was added, and the
reaction mixture was stirred at room temperature for 2 h. At the end of
the reaction, solvent was evaporated, silica gel was added with methanol
to make a plug, and the mixture was purified using flash chromatog-
raphy with hexanes/ethyl acetate to afford 23 (1.36 g, 72%) as a yellow-
colored liquid. TLC Rf = 0.8 (Hex: EtOAc; 1:1); 1H NMR, DMSO‑d6 (400
MHz): 1.41 (s, 9H, (CH3)3), 3.28 (s, 3H, NCH3), 3.96 (s, 3H, OCH3),
6.92–6.94 (m, 1H, Ar), 7.42–7.43 (m, 2H, Ar), 7.45–7.46 (d, 1H, J =
2.19, Ar), 7.72–7.73 (d, 1H, J = 2.12, Ar), 8.07–8.09 (d, 1H, J = 9.03,
Ar). Boc deprotection of 23 (1.3 g, 4.5 mmol) was carried out in tri-
fluoroacetic acid (TFA, 15 mL) at room temperature for 2 h. At the end of
the reaction, TFA was removed by evaporation, and the mixture was
neutralized using an aq. solution of K2CO3. Extraction with ethyl acetate
(20 mL × 3), followed by concentration and drying over Na2SO4 yielded
17 (0.810 g, 95%) as a green-colored solid. TLC Rf = 0.77 (Hex: EtOAc;
1:1); 1H NMR, DMSO‑d6 (400 MHz): 2.75–2.76 (d, 3H, NCH3), 3.89 (s,
3H, OCH3), 5.98–5.99 (q, 1H, NHCH3, exch.), 6.57–6.59 (d, 1H, J =
7.43, Ar), 6.61–6.62 (d, 1H, J = 1.87, Ar), 6.88–6.90 (dd, 1H, J1 = 1.42,
J2 = 9.05, Ar), 7.15–7.23 (m, 2H, Ar), 7.83–7.86 (d, 1H, J = 9.05, Ar).
The compound was carried to the next step without further purification.
6.6. General method for the synthesis of compounds 1–10
To a 100 mL round-bottomed flask substituted-4-chloroquinazolines
(1 equivalent), appropriate anilines (1.2 equivalents) and isopropanol
(10 mL) were added and stirred at room temperature for 12 h. The
compounds precipitated as HCl salts, which were filtered and dried in
vacuo over P2O5 to afford the desired compounds.
6.6.1. N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine (1)
Using the general method described above, 12a (0.150 g, 0.840
mmol) and 4-methoxy-N-methylaniline (14) (0.138 g, 1.01 mmol) were
reacted for 12 h. At the end of the reaction, solvent was evaporated,
silica gel and methanol were added, and the methanol evaporated in
vacuo to give a dried plug. The reaction mixture was purified by flash
chromatography using chloroform/methanol to afford 1 as a brown
solid (0.136 g, 58%). TLC Rf
= 0.8 (MeOH:CHCl3; 1:5); mp,
99.1–101.2 ◦C; 1H NMR, DMSO‑d6 (400 MHz): 2.66 (s, 3H, 2-CH3), 3.60
(s, 3H, NCH3), 3.81 (s, 3H, OCH3), 6.88–6.90 (d, 1H, J = 8.35, Ar),
7.04–7.06 (d, 2H, J = 8.89, Ar), 7.14–7.18 (t, 1H, J1 = 7.49, J2 = 7.49,
Ar), 7.30–7.32 (d, 2H, J = 8.78, Ar), 7.68–7.75 (m, 2H, Ar). Anal. Calcd.
for C17H17N3O 0.52HCl: C, 68.46; H, 5.92; N, 14.08. Found C, 68.50; H,
6.24; N, 13.80; Cl, 4.54.
6.6.2. 4-(6-Methoxy-3,4-dihydroquinolin-1(2H)-yl)-2-methylquinazoline
(2)
Using the general method described above, 12a (0.150 g, 0.840
mmol) and 6-methoxy-1,2,3,4-tetrahydroquinoline (15) (0.164 g, 1.01
mmol) were reacted to afford 2 as a yellow precipitate (0.188 g, 65%).
TLC Rf = 0.85 (MeOH:CHCl3; 1:5); mp, 255.8–257.9 ◦C; 1H NMR,
DMSO‑d6 (400 MHz): 2.1 (quin, 2H, CH2), 2.75 (s, 3H, 2-CH3),
2.87–2.90 (t, 2H, CH2), 3.79 (s, 3H, OCH3), 4.20 (t, 2H, NCH2),
6.68–6.71 (dd, 1H, J1 = 2.88, J2 = 8.85, Ar), 7.02–7.03 (d, 1H, J = 2.81,
Ar), 7.05–7.08 (d, 1H, J = 8.87, Ar), 7.26–7.28 (d, 1H, J = 8.53, Ar),
7.36–7.40 (dt, 1H, J1 = 1.38 , J2 = 6.83, J3 = 8.28, Ar), 7.88–7.96 (m,
2H, Ar). Anal. Calcd. for C19H19N3O. 1.07HCl: C, 66.26; H, 5.87; N,
12.20. Found C, 66.28; H, 5.75; N, 12.11; Cl, 10.49.
6.4. 4-Chloro-2-methylquinazoline (12a)
To a 250 mL round-bottomed flask were added 2-methylquinazolin-4
(3H)-one (13a) (1.0 g, 6.2 mmol), trimethylamine (3 mL), and toluene
(30 mL). The mixture was cooled to 0 ◦C, and phosphorus oxychloride
(2 mL) was added. The reaction mixture was stirred at room temperature
for 1 h. The reaction was then heated to 95 ◦C for 3 h. After monitoring
the reaction by TLC, the reaction mixture was cooled to room temper-
ature and diluted with 30 mL of ethyl acetate. The solution was then
washed with 10 mL of ice cold water, 10 mL of saturated NaHCO3, 10 mL
of water, 5 mL of 1 N HCl, 10 mL of water, 10 mL of saturated NaHCO3
and 10 mL of saturated NaCl. The organic layer was dried over Na2SO4,
filtered and concentrated to obtain 12a as a yellow solid (0.600 g, 54%).
TLC Rf = 0.8 (MeOH:CHCl3; 1:5); mp, 83.9–85.3 ◦C (lit.55 85–86 ◦C); 1H
6.6.3. N,2-Dimethyl-N-(4-(methylthio)phenyl)quinazolin-4-amine (3)
Using the general method described above, 12a (0.135 g, 0.756
mmol) and N-methyl-4-(methylthio)aniline (16) (0.135 g, 0.907 mmol)
were reacted to afford 3 as a white precipitate (0.175 g, 70%). TLC Rf =
0.9 (MeOH:CHCl3; 1:5); mp, 235.2–236.4 ◦C; 1H NMR, DMSO‑d6 (400
MHz): 2.55 (s, 3H, 2-CH3), 2.77 (s, 3H, SCH3), 3.74 (s, 3H, NCH3),
6.87–6.89 (d, 1H, J = 8.39, Ar), 7.30–7.34 (dt, 1H, J1 = 1.33, J2 = 7.0,
J3 = 8.48, Ar), 7.43–7.50 (m, 4H, Ar), 7.85–7.89 (dt, 1H, J1 = 1.14, J2 =
7.05, J3 = 8.34, Ar), 7.93–7.95 (d, 1H, J = 8.32, Ar). Anal. Calcd. for
NMR, DMSO‑d6 (400 MHz): 2.58 (s, 3H, CH3), 7.59–7.63 (t, 1H, J1
=
7.58, J2 = 7.58, Ar), 7.78–7.80 (d, 1H, J = 7.98, Ar), 7.90–7.95 (t, 1H, J1
= 7.67, J2 = 7.67, Ar), 8.13–8.15 (d, 1H, J = 7.65, Ar).
C17H17N3S. 1.34HCl: C, 59.28; H, 5.37; N, 12.20; S, 9.31. Found C,
10

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
59.37; H, 5.62; N, 11.93; S, 9.03; Cl, 10.55.
1H, Ar), 7.32–7.35 (d, 2H, J = 8.95, Ar), 7.66–7.67 (m, 2H, Ar). Anal.
Calcd. for C16H14ClN3O: C, 64.11; H, 4.71; N, 14.02; Cl, 11.83. Found C,
64.08; H, 4.77; N, 13.93; Cl, 11.97.
6.6.4. N-(5-Methoxynaphthalen-2-yl)-N,2-dimethylquinazolin-4-amine
(4)
Using the general method described above, 12a (0.135 g, 0.840
mmol) and 5-methoxy-N-methylnaphthalen-2-amine (17) (0.156 g,
0.831 mmol) were reacted for 12 h. At the end of the reaction, solvent
was evaporated, and silica gel and methanol were added, and the
methanol evaporated in vacuo to give a dried plug. The reaction mixture
was purified using flash chromatography using chloroform/methanol to
afford 4 as a yellow solid (0.170 g, 68%). TLC Rf = 0.9 (MeOH:CHCl3;
1:10); mp, 203.0–204.4 ◦C; 1H NMR, DMSO‑d6 (400 MHz): 2.70 (s, 3H,
2-CH3), 3.72 (s, 3H, NCH3), 3.99 (s, 3H, OCH3), 6.91–6.93 (d, 1H, J =
8.26, Ar), 7.00–7.02 (d, 1H, J = 7.46, Ar), 7.05–7.09 (t, 1H, J1 = 7.70, J2
6.6.9. 2-Chloro-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)quinazoline
(9)
Using the general method described above, 12c (0.300 g, 1.51 mmol)
and 6-methoxy-1,2,3,4-tetrahydroquinoline (15) (0.295 g, 1.81 mmol)
were reacted for 12 h. At the end of the reaction, solvent was evapo-
rated, silica gel and methanol were added, and the methanol evaporated
in vacuo to give a dried plug. The reaction mixture was purified by flash
chromatography using chloroform/methanol to afford 9 as a yellow
solid (0.121 g, 25%). TLC Rf
= 0.97 (MeOH:CHCl3; 1:5); mp,
135.9–137.0 ◦C (lit.48 136–138 ◦C); 1H NMR, DMSO‑d6 (400 MHz):
2.13–2.18 (m, 2H, CH2), 2.88–2.91 (t, 2H, CH2), 3.85 (s, 3H, OCH3),
= 7.70, Ar), 7.40–7.49 (m, 3H, Ar), 7.66–7.69 (t, 1H, J1 = 7.59, J2
=
́
́
7.59, Ar), 7.74–7.76 (d, 1H, J = 8.33, Ar), 7.84 (s, 1H, Ar), 8.21–8.23 (d,
1H, J = 8.99, Ar). Anal. Calcd. for C21H19N3O. 0.41HCl: C, 73.22; H,
5.68; N, 12.20. Found C, 73.19; H, 5.82; N, 12.35; Cl, 2.22.
4.11–4.16 (m, 2H, CH2), 6.59–6.62 (dd, 1H, J1 = 2.89, J2 = 8.81, 7-Ar),
́
6.75–6.77 (d, 1H, J = 8.85, 8-Ar), 6.857–6.863 (d, 1H, J = 2.86, 5-Ar),
7.16–7.19 (m, 1H, J1 = 1.26, J2 = 6.96, J3 = 8.43, Ar), 7.31–7.33 (dd,
1H, J1 = 0.90, J2 = 8.53, Ar), 7.68–7.71 (m, 1H, J1 = 1.36, J2 = 6.97, J3
= 8.39, Ar), 8.05–8.07 (d, 1H, Ar). Anal. Calcd. for C18H16ClN3O . 0.24
(CH3)2CHOH: C, 66.08; H, 5.31; N, 12.34; Cl, 10.41. Found C, 66.30; H,
5.19; N, 12.35; Cl, 10.17.
6.6.5. N-(4-Methoxyphenyl)-N-methylquinazolin-4-amine (5)
Using the general method described above, 12b (0.150 g, 0.911
mmol) and N-methyl-4-methoxyaniline (14) (0.150 g, 1.09 mmol) were
reacted to afford 5 as a pale-yellow precipitate (0.210 g, 76%). TLC Rf =
0.8 (MeOH:CHCl3; 1:5); mp, 244.3–245.3 ◦C; 1H NMR, DMSO‑d6 (400
MHz): 3.75 (s, 3H, NCH3), 3.84 (s, 3H, OCH3), 6.84–6.86 (d, 1H, J =
8.43, Ar), 7.12–7.16 (d, 2H, J = 8.97, Ar), 7.32–7.36 (dt, 1H, J1 = 1.12,
J2 = 6.93, J3 = 8.45, Ar), 7.46–7.50 (d, 2H, J = 8.96, Ar), 7.87–7.91 (dt,
1H, J1 = 1.05, J2 = 6.94, J3 = 8.25, Ar), 7.95–7.97 (d, 1H, J = 8.15, Ar),
9.06 (s, 1H, Ar). Anal. Calcd. for C16H15N3O. 1.10 HCl: C, 62.89; H, 5.31;
N, 13.75. Found C, 62.89; H, 5.33; N, 13.75; Cl, 11.43.
6.6.10. 2-Chloro-N-methyl-N-(4-(methylthio)phenyl)quinazolin-4-amine
(10)
Using the general procedure described above, 12c (0.300 g, 1.51
mmol) and 16 (0.23 g, 1.5 mmol) were reacted in acetonitrile for 12 h.
At the end of reaction, solvent was evaporated, silica gel and methanol
were added, and the methanol evaporated in vacuo to give a dried plug.
The reaction mixture was purified by flash chromatography using
chloroform/methanol to afford 10 (0.1 g, 22%) as a dark yellow solid.
TLC Rf = 0.76 (MeOH:CHCl3; 1:5); mp, 123.6 ◦C, 1H NMR, CDCl3 (400
MHz): 2.55 (s, 3H, SCH3), 3.68 (s, 3H, NCH3), 6.97–7.02 (m, 1H, Ar),
7.10 (s, 1H, Ar), 7.17 (d, 2H, J = 8.6 Hz, Ar), 7.32 (d, 2H, J = 8.6 Hz, Ar),
7.64 (s, 1H, Ar), 7.95 (s, 1H, Ar). Anal. Calcd. for C16H14ClN3S: C, 60.85;
H, 4.47; N, 13.31; S, 10.15; Cl, 11.22. Found: C, 60.97; H, 4.55; N, 13.19;
S, 10.12; Cl, 11.07.
6.6.6. 4-(6-Methoxy-3,4-dihydroquinolin-1(2H)-yl)quinazoline (6)
Using the general method described above, 12b (0.150 g, 0.911
mmol) and 6-methoxy-1,2,3,4-tetrahydroquinoline (15) (0.179 g, 1.09
mmol) were reacted to afford 6 as a yellow precipitate (0.211 g, 70%).
TLC Rf = 0.8 (MeOH:CHCl3; 1:5); mp, 234.2–235.3 ◦C; 1H NMR,
DMSO‑d6 (400 MHz): 2.05–2.13 (quint, 2H, CH2), 2.87–2.91 (t, 2H,
CH2), 3.79 (s, 3H, OCH3), 4.20–4.21 (t, 2H, NCH2), 6.67–6.70 (dd, 1H,
J1 = 2.91, J2 = 8.85, Ar), 7.02–7.03 (d, 1H, J = 2.82, Ar), 7.08–7.10 (d,
1H J = 8.85, Ar), 7.33–7.35 (d, 1H, J = 8.50, Ar), 7.42–7.46 (dt, 1H, J1
= 1.55, J2 = 6.67, J3 = 8.32, Ar), 7.91–7.98 (m, 2H, Ar), 9.04 (s, 1H, Ar).
Anal. Calcd. for C18H17N3O. 1.22HCl: C, 64.38; H, 5.47; N, 12.51. Found
C, 64.39; H, 5.47; N, 12.45; Cl, 10.41.
6.6.11. N2,N4-Dimethyl-N2,N4-bis(4-(methylthio)phenyl)quinazoline-2,4-
diamine (18)
Compound 12c (0.200 g, 1.00 mmol) and N-methyl-4-(methylthio)
aniline (16) (0.185 g, 1.21 mmol) were stirred at room temperature for
12 h. At the end of the reaction, solvent was evaporated, silica gel and
methanol were added, and the methanol evaporated in vacuo to give a
dried plug. The reaction mixture was purified using flash chromatog-
raphy using chloroform/methanol which afforded dimer 18 as a yellow
semisolid (0.195 g, 45%). TLC Rf = 0.87 (Hex:EtOAc; 1:1); 1H NMR,
DMSO‑d6 (400 MHz): 2.48 (s, 3H, SCH3), 2.49 (s, 3H, SCH3), 3.29 (s, 3H,
NCH3), 3.57 (s, 3H, NCH3), 6.76–6.81 (m, 1H, Ar), 6.88–6.90 (dd, 1H, J
= 0.89, J = 8.43, Ar), 7.14–7.16 (d, 2H, J = 8.68, Ar) 7.25–7.29 (m, 4H,
Ar), 7.37–7.45 (m, 4H, Ar).
6.6.7. N-Methyl-N-(4-(methylthio)phenyl)quinazolin-4-amine (7)
Using the general method described above, 12b (0.150 g, 0.911
mmol) and N-methyl-4-(methylthio)aniline (16) (0.168 g, 1.09 mmol)
were reacted to afford 7 as a pale yellow precipitate (0.209 g, 72%). TLC
Rf = 0.8 (MeOH:CHCl3; 1:5); mp, 233.4–235.9 ◦C; 1H NMR, DMSO‑d6
(400 MHz): 2.54 (s, 3H, SCH3), 3.75 (s, 3H, NCH3), 6.94–6.96 (d, 1H, J
= 8.74, Ar), 7.35–7.39 (t, 1H, J1 = 7.69, J2 = 7.69, Ar), 7.43–7.45 (d,
2H, J = 8.70, Ar), 7.48–7.50 (d, 2H, J = 8.68, Ar), 7.88–7.92 (t, 1H, J1 =
7.55, J2 = 7.55, Ar), 7.95–7.97 (d, 1H, J = 7.68, Ar), 9.08 (s, 1H, Ar).
Anal. Calcd. for C16H15N3S. 1.13HCl: C, 59.59; H, 5.04; N, 13.03; S,
9.94. Found C, 59.56; H, 5.13; N, 12.97; S, 10.22; Cl, 10.22.
6.6.12. 6-((2-Chloroquinazolin-4-yl)amino)naphthalen-1-ol (20)
Using the general method described above, 12c (0.200 g, 1.00 mmol)
and 6-aminonaphthalen-1-ol 19 (0.144 g, 0.904 mmol) were reacted for
12 h. At the end of the reaction, the solvent was evaporated, silica gel
and methanol were added, and the methanol evaporated in vacuo to give
a dried plug. The reaction mixture was purified using flash chroma-
tography using chloroform/methanol to the afford 20 as white solid
(0.070 g, 22%). TLC Rf = 0.67 (MeOH:CHCl3; 1:5); 1H NMR, DMSO‑d6
(400 MHz): 6.83–6.87 (t, 1H, Ar), 7.32–7.33 (d, 2H, J = 4.55, Ar),
7.67–7.71 (dt, 1H, J1 = 1.13, J2 = 6.96, J3 = 8.09, Ar), 7.74–7.76 (dd,
1H, J1 = 0.79, J2 = 8.31, Ar), 7.81–7.84 (dd, 1H, J1 = 2.11, J2 = 9.07,
Ar), 7.90–7.94 (dt, 1H, J1 = 1.13, J2 = 7.08, J3 = 8.35, Ar), 8.16–8.18 (d,
1H, J = 9.02, Ar), 8.24–8.25 (d, 1H, J = 2.03, Ar), 8.63–8.65 (dd, 1H, J1
= 1.35, J2 = 8.66, Ar), 10.16 (s, 1H, exch., NH), 10.40 (s, br, 1H, exch.,
6.6.8. 2-Chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (8)
Using the general method described above, 12c (0.500 g, 2.51 mmol)
and 4-methoxy-N-methylaniline (14) (0.413 g, 3.01 mmol) were reacted
for 12 h. At the end of reaction, solvent was evaporated, silica gel and
methanol were added, and the methanol evaporated in vacuo to give a
dried plug. The reaction mixture was purified using flash chromatog-
raphy using chloroform/methanol to afford 8 as buff-colored solid
(0.070 g, 9%). TLC Rf = 0.95 (MeOH:CHCl3; 1:5); mp, 146.3–148.7 ◦C;
1H NMR, DMSO‑d6 (400 MHz): 3.52 (s, 3H, NCH3), 3.81 (s, 3H, OCH3),
6.86–6.88 (d, 1H, Ar), 7.03–7.06 (d, 2H, J = 8.96, Ar), 7.11–7.16 (m,
11

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
OH). The compound was used without further characterization.
8.4. Phosphotyrosine ELISA
6.6.13. 2-Chloro-N-(5-methoxynaphthalen-2-yl)-N-methylquinazolin-4-
amine (11)
A high-throughput phosphotyrosine ELISA was developed for eval-
uating the effect of compounds on RTKs.60 Cells used for these experi-
ments have been shown to overexpress particular RTKs; specifically
A431 for EGFR, U251 for VEGFR-2, and SH-SY5Y for PDGFR-β. Briefly,
cells at 60–75% confluence are placed in serum-free medium for 18 h to
reduce background phosphorylation. Cells were always > 98% viable by
trypan blue exclusion. Cells were then pretreated for 60 min with 100
nM-1.4 µM concentrations of the compound of interest followed by the
addition of 100 ng/mL of purified growth factor (EGF, VEGF, or PDGFR-
β) for 10 min. The reaction was stopped, and cells were permeabilized by
quickly removing the media and adding ice-cold Tris-buffered saline
(TBS) containing 0.05% Triton X-100, protease inhibitor cocktail, and
tyrosine phosphatase inhibitor cocktail (Sigma Chemical). The TBS so-
lution was then removed, and cells fixed to the plate for 30 min at 60 ◦C,
with a further incubation in 70% ethanol for an additional 30 min. Cells
were then exposed to a blocking solution (TBS with 1% BSA) for 1 h,
washed, and then a horseradish peroxidase (HRP)-conjugated phos-
photyrosine (PY) antibody was added overnight. The antibody was
removed, cells were washed again in TBS, exposed to an enhanced
luminol ELISA substrate (Pierce Chemical EMD, Rockford, IL), and light
emission was measured using a plate reader (BioTek, Winooski VT). The
known RTK-specific inhibitors (semaxanib, sunitinib, erlotinib) were
used as positive controls for kinase inhibition. Data were graphed as a
percent of cells receiving growth factor alone, and IC50 values were
calculated from two to three separate biological replicates (n = 8 tech-
nical replicates per biological replicate) using sigmoidal dose–response
relations in Prism 8.0 software (GraphPad).
Compound 20 (0.070 g, 0.22 mmol) was dissolved in anhydrous
DMF (10 mL) in a three necked round-bottomed flask, and the reaction
mixture was stirred at 0 ◦C under argon. Sodium hydride (0.016 g, 0.65
mmol) was added to the above mixture, which was stirred at 0 ◦C for an
additional 15 min. Methyl iodide (0.068 g, 0.40 mmol) was added while
maintaining the reaction at room temperature for 4 h. Methanol was
added at the end of the reaction, solvent was evaporated, and the re-
action mixture was then extracted with water and ethyl acetate (25 mL
× 3) and dried over Na2SO4. Silica gel was added to the combined
fractions, the solvent was removed by evaporation to yield a dry plug,
and a column was run using hexanes/ethyl acetate. The fractions con-
taining the product (TLC) were pooled and evaporated to obtain the
desired compound 11 as an off-white solid (0.036 g, 47%). TLC Rf = 0.82
(MeOH:CHCl3; 1:5); mp, 171.9–173.8 ◦C; 1H NMR, DMSO‑d6 (400
MHz): 3.64 (s, 3H, NCH3), 3.99 (s, 3H, OCH3), 6.86–6.88 (d, 1H, J =
8.54, Ar), 7.00–7.06 (m, 2H, Ar), 7.40–7.49 (m, 3H, Ar), 7.63–7.71 (m,
2H, Ar), 7.88–7.89 (d, 1H, J = 2.02, Ar), 8.21–8.23 (d, 1H, J = 8.92, Ar).
Anal. Calcd. for C20H16ClN3O . 0.11 CH3(CH2)4CH3: C, 69.07; H, 4.93; N,
11.68; Cl, 9.86. Found C, 68.84; H, 5.06; N, 11.69; Cl, 9.64.
7. Molecular modeling and computational studies
Molecular modeling was performed for all analogs with the tubulin
(PDB: 4O2B47), VEGFR-2 (PDB: 4AG846) and EGFR (PDB: 1M1757
)
crystal structures using the induced fit docking protocol of Maestro
11.9.45 The ligands were prepared using the Ligprep application of
Maestro. The docking protocol was validated by re-docking the co-
crystallized ligands colchicine, axitinib and erlotinib in tubulin,
VEGFR-2, and EGFR, respectively, with RMSD of 0.17 Å, 0.50 Å, and
1.19 Å, respectively. The centroids around the ligands were defined as
the ligand binding site. The OPLS3e force field was used, and amino acid
residues within 5 Å from the docked poses were allowed to be optimized
using prime refinement.
8.5. Chorioallantoic membrane (CAM) assay
The CAM assay is a standard assay for testing antiangiogenic agents.
The CAM assay used in these studies was performed as described pre-
viously.34 Briefly, fertile leghorn chicken eggs (Ideal Poultry, Cameron,
TX) were incubated for 10 days. The proangiogenic factors, human
VEGF-165 and bFGF (100 ng each), were then added at saturation to a 6
mm microbial testing disk (BBL, Cockeysville, MD) and placed onto the
CAM by breaking a small hole in the superior surface of the egg. Anti-
angiogenic compounds, at various doses, were added 8 h after the
VEGF/bFGF at saturation to the same microbial testing disk, and the
embryos were incubated for an additional 40 h. After 48 h, the CAMs
were perfused with 2% paraformaldehyde containing 0.025% Triton X-
100 for 20 s, excised around the area of treatment, fixed again in 4%
paraformaldehyde for 30 min, placed on Petri dishes, and a digitized
image was taken using a dissecting microscope (Leica Z16-APO; Wet-
zlar, Germany) at 7.5X magnification. A grid was then added to the
digital CAM images, and the average number of vessels within 5–7 grids
counted as a measure of vascularity. Sunitinib and semaxanib were used
as positive controls for antiangiogenic activity. Data were graphed as a
percent of CAMs receiving bFGF/VEGF only, and IC50 values calculated
from two to three separate experiments with 2–5 replicates per experi-
ment using non-linear regression dose–response relation analysis.
8. Biological studies
8.1. Effects of compounds on cellular microtubules
A-10 cells were used to evaluate the effects of the compounds on
cellular microtubules using indirect immunofluorescence techniques.
Cells were treated overnight with the compounds, fixed in cold MeOH,
and microtubules were visualized with a β-tubulin antibody (Sigma-
Aldrich, St. Louis, MO). EC50 values were calculated as previously
described58 and represent an average of at least three independent
experiments.
8.2. Sulforhodamine B (SRB) assay
The SRB assay was used, as previously described, to evaluate the
antiproliferative and cytotoxic effects of the compounds against cancer
cells.59 MDA-MB-435, SK-OV-3 and HeLa cells were purchased from the
American Type Culture Collection (Manassas, VA). Details about the
generation of the SK-OV-3-MDR1-6/6 and HeLa WT-βIII cells were
previously described.59 The IC50 values represent an average of at least 3
independent experiments using triplicate points in each experiment.
8.6. Quantitative tubulin studies
Bovine brain tubulin was purified as described previously.61 Briefly,
1.0 mg/mL of tubulin (10 µM) was incubated for 15 min with 0.8 M
monosodium glutamate (pH of 2 M stock solution adjusted to 6.6 with
HCl), varying compound concentrations and 4% (v/v) dimethyl sulf-
oxide as solvent. After a 15 min preincubation, 0.4 mM GTP was added.
The reaction mixtures were transferred to cuvettes held at 0 ◦C in a
cuvette holder equipped with an electronic temperature controller in a
recording spectrophotometer. After baselines were established, the
temperature was jumped over about 30 s to 30 ◦C, and changes in
turbidity were monitored for 20 min. The compound concentration that
8.3. Antibodies
The PY-HRP antibody was obtained from BD Transduction Labora-
tories (Franklin Lakes, NJ). Antibodies against EGFR, VEGFR-2, and
PDGFR-β were purchased from Cell Signaling Technology (Danvers,
MA).
12

S. Choudhary et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116061
20 El-Kenawi AE, El-Remessy AB. Angiogenesis inhibitors in cancer therapy:
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caused a 50% reduction in increase in turbidity, interpolated from the
values obtained with defined compound concentrations, was defined as
the IC50 value. The assay to measure inhibition of [3H]colchicine bind-
ing was described in detail previously.62 Briefly, 0.1 mg/mL (1.0 µM)
tubulin was incubated, at 37 ◦C, with 5.0 µM [3H]colchicine and po-
tential inhibitors at 1.0 or 5.0 µM, as indicated. Incubation was for 10
min, at which point 40–60% of the maximum colchicine that can be
bound without inhibitor is reached. The [3H]colchicine was a product of
Perkin-Elmer. CA-4 was a generous gift of Dr. G. R. Pettit, Arizona State
University.
21 Steinmetz MO, Prota AE. Microtubule-targeting agents: Strategies to hijack the
cytoskeleton. Trends Cell Biol. 2018;28:776–792.
22 Akhmanova A, Steinmetz MO. Control of microtubule organization and dynamics:
two ends in the limelight. Nat Rev Mol Cell Biol. 2015;16(12):711–726.
23 Field JJ, Kanakkanthara A, Miller JH. Microtubule-targeting agents are clinically
successful due to both mitotic and interphase impairment of microtubule function.
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Declaration of Competing Interest
27 Stylianopoulos T, Munn LL, Jain RK. Reengineering the tumor vasculature:
Improving drug delivery and efficacy. Trends Cancer. 2018;4:258–259.
28 Martin JD, Seano G, Jain RK. Normalizing function of tumor vessels: Progress,
opportunities, and challenges. Annu Rev Physiol. 2019;81:505–534.
29 Zhou J, Jiang X, He S, et al. Rational design of multitarget-directed ligands:
Strategies and emerging paradigms. J Med Chem. 2019;62:8881–8914.
30 Jain RK. Normalizing tumor microenvironment to treat cancer: bench to bedside to
biomarkers. J Clin Oncol. 2013;31(17):2205–2218.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
31 Murray A, Little SJ, Stanley P, Maraveyas A, Cawkwell L. Sorafenib enhances the in
vitro anti-endothelial effects of low dose (metronomic) chemotherapy. Oncol Rep.
2010;24:1049–1058.
This work was supported, in part, by a grant from the National In-
stitutes of Health, National Cancer Institute (RO1 CA142868 (AG,
SLM)); by the Duquesne University Adrian Van Kaam Chair in Scholarly
Excellence (AG), the Greehey Distinguished Chair (SLM); and by an NSF
equipment grant for NMR instrumentation (NMR: CHE 0614785).
32 Naganuma Y, Choijamts B, Shirota K, et al. Metronomic doxifluridine chemotherapy
combined with the anti-angiogenic agent TNP-470 inhibits the growth of human
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