Synthesis and structure-activity relationship study of 1-phenyl-1-(quinazolin-4-yl)ethanols as anticancer agents

Article abstract of DOI:10.1021/ml5004684

A quinazoline derivative PVHD121 (1a) was shown to have strong antiproliferative activity against various tumor-derived cell lines, including A549 (lung), NCI-H460 (lung), HCT116 (colon), MCF7 (breast), PC3 (prostate), and HeLa (cervical) cells with IC₅₀ values from 0.1 to 0.3 M. A structure-activity relationship (SAR) study at the 2- and 4-position of the quinazoline core lead to the discovery of more potent anticancer agents (14, 16, 17, 19, 24, and 31). The results of an in vitro tubulin polymerization assay and fluorescent-based colchicine site competition assay with purified tubulin indicated that 1a inhibits tubulin polymerization by binding to the colchicine site.

Full text of DOI:10.1021/ml5004684

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Letter
Synthesis and Structure-Activity Relationship Study of 1-
Phenyl-1-(quinazolin-4-yl)ethanols as Anticancer Agents
Kenta Kuroiwa, Hirosuke Ishii, Kenji Matsuno, Akira Asai, and Yumiko Suzuki
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml5004684 • Publication Date (Web): 10 Jan 2015
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Synthesis and Structure-Activity Relationship Study of 1-Phenyl-1-
(quinazolin-4-yl)ethanols as Anticancer Agents
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Kenta Kuroiwa,† Hirosuke Ishii,‡ Kenji Matsuno, ‡§ Akira Asai,*,‡ and Yumiko Suzuki*,†
†Department of Material and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho,
Chiyoda-ku, Tokyo 102-8554, Japan
‡Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Ja-
pan
ABSTRACT: A quinazoline derivative PVHD121 (1a) was shown to have strong anti-proliferative activity against various
tumor-derived cell lines, including A549 (lung), NCI-H460 (lung), HCT116 (colon), MCF7 (breast), PC3 (prostate), and
HeLa (cervical) cells with IC50 values
IC50 Value
OMe
OMe
from 0.1 to 0.3 µM. A structure-
activity relationship (SAR) study at
the 2- and 4-position of the quinazo-
line core lead to the discovery of
more potent anticancer agents (14,
16, 17, 19, 24 and 31). The results of an
in vitro tubulin polymerization assay
and fluorescent-based colchicine site
competition assay with purified tu-
R = Me (14)
0.053
0.038
0.027
0.058
0.067
Me
N
Me
N
HO
HO
16
17
CCl3(
)
)
Cl
(
SAR
OMe(19)
N
N
24
SMe(
)
R
(31)
0.035
PVHD Derivatives
PVHD121 (1a)
IC50 = 0.27
S
µ
M
bulin indicated that 1a inhibits tubulin polymerization by binding to the colchicine site. KEYWORDS: Quinazoline, Anti-
cancer, Tubulin inhibitor, Colchicine binding site, Structure-activity relationship study
Cancer is one of the most common causes of death
worldwide.1 According to the World Health Organization,
cancer killed 7.6 million people in 2008, representing ap-
proximately 13% of all deaths. The number of cancer
deaths is estimated to reach 13.1 million by 2030. Many
bioactive compounds have been shown to possess anti-
cancer activity.2 However, their use is limited due to side
effects or limited scope of activity. Thus, the identifica-
tion of novel, potent drugs with fewer side effects and a
broad spectrum of activity, is desirable to improve cancer
treatment. Among the available anticancer drugs, antimi-
totic agents, which interact with microtubules, are one of
the most successful therapeutics.3-8 Microtubules are a
component of the cytoskeleton in eukaryotic cells, and
consist of tubulin protein polymers. These proteins can
polymerize and depolymerize. Antimitotic agents interact
with binding sites on tubulin, such as the colchicine site
and the vinblastine site,9 and inhibit or promote microtu-
no groups at the 4-position of the core, rather than the 1-
hydroxyethyl group in 1a.11-13 In the present study, we de-
scribe the results of an SAR study for the development of
novel anticancer drugs, and demonstrate the biological
activity of 1a and its derivatives for the first time.
The hit compound 1a was originally prepared as a sub-
strate in the development of a new synthetic catalysis.14
According to the reported procedure, the synthesis of
derivatives 1a-n, which contain various aromatic rings on
the substituents at the 4-position of the quinazoline nu-
cleus, was initiated by NHC-catalyzed aroylation using an
aldehyde, followed by a Grignard reaction with methyl-
magnesium bromide. Derivatives 4o-q which have me-
thylamino, dimethylamino, and methylthio groups were
synthesized via nucleophilic aromatic substitution (SNAr)
of the fluoro group of 4b by N- or S- nucleophiles and a
subsequent Grignard reaction (Scheme 1).
The synthesis of compounds 5-8, which have ethyl, ben-
zyl, trifluoromethyl, and a hydrogen atom at the benzyl
position instead of a methyl moiety, is shown in Scheme 2.
The Grignard reaction between 4a and ethylmagnesium
iodide produced ethyl-substituted derivative 5. Benzyl-
substituted derivative 6 was similarly synthesized from 4a.
Trifluoromethylation of the keto group of 4a using TMS-
CF3 and TBAF15 yielded derivative 7. The reduction of the
keto group of 4a using NaBH4 afforded 8.
bule polymerization, thereby affecting their function.3-8
A
number of antimitotic compounds have been synthesized
as promising drug candidates in many laboratories.10
In the course of random screening for anticancer agents,
we discovered a quinazoline derivative, PVHD121 (1a),
which had strong antiproliferative activity against the
cervical cancer cell line, HeLa. Recently, there have been
reports of quinazoline-based anticancer agents with ami-
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aScheme 2
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2
Scheme 1a
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aReagent and Conditions : (i) RMgI, THF, rt ; (ii) TMS-CF3,
8
9
TBAF, THF, 0°C to rt ; (iii) NaBH4, ethanol, rt
aScheme 3
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aReagents and Conditions: (i) N,N´-
aReagents and Conditions: (i) N,N’-1,3-
dimethylimidazolium iodide (catalyst), p-anisaldehyde,
NaH, THF/DMF, reflux ; (ii) MeMgI, THF, rt
dimethylimidazolium iodide (catalyst), NaH, THF, reflux ;
(ii) MeMgBr, THF, rt ; (iii) MeNH2, 1,4-dioxane, reflux ;
(iv) Me2NH, 1,4-dioxane, reflux ; (v) NaSMe, 1,4-dioxane,
reflux
Synthesis of 18-34 from the 2-chloro derivative 17 was
performed by SNAr or Suzuki-Miyaura cross coupling
(Scheme 4).19-20
Derivative 9, which has a 1-(4-anisyl)-1-hydroxyethyl
group at position 2 of the quinazoline core, instead of the
4-position, was also prepared from 2,4-
dichloroquinazoline 13a via substitutions of the 2- and 4-
chloro groups and a Grignard reaction with methyl-
magnesium bromide. (See Scheme S-1, Supporting Infor-
mation).
Hit compound 1a induced G2/M accumulation in hu-
man lung carcinoma A549 cells (Figure 1A). In addition,
1a showed a broad spectrum of anti-proliferative activity
against various solid tumor-derived cell lines including
A549 (lung), NCI-H460 (lung), HCT116 (colon), MCF7
(breast), PC3 (prostate), and HeLa (cervical) cells, with
IC50 values ranging from 0.1 to 0.3 µM (See Figure S-1,
Supporting Information).
The compounds having methyl, phenyl, trichloromethyl,
and chloro groups at position 2 of the quinazoline (14-17)
were synthesized using the same procedure as 1a-n
(Scheme 3). Specifically, NHC-catalyzed aroylation of 2-
substituted 4-chloroquinazoline 10a-13a 12,16-18 was per-
formed, followed by Grignard reaction, yielding 14-17
(Scheme 3).
aScheme 4
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aReagent and Conditions: (i) NaOH, THF, reflux / R’ONa, MeOH, rt / MeSNa, 1,4-dioxane, reflux / amine, 1,4-dioxane,
reflux (See Supporting Information for details); (ii) ArB(OH)2, K2CO3, Pd(OAc)2, 1,4-dioxane, reflux
1
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We found that several 2-substituted compounds had
stronger anti-proliferative activity against A549 than 1a,
including 2-methyl (14), 2-trichloro (16), 2-chloro (17), 2-
both enantiomers of 1a separated by chiral column chro-
The obtained derivatives 1b-q, 5-9, and 14-34, along with
methoxy (19), 2-methythio (24), and 2-(3-thienyl) (31)
substituted derivatives. In particular, the 2-
chlorosubstituted analog 17 showed the highest activity
(IC50 = 0.027 µM), which is ten times more effective than
matography were tested for cytotoxicity against the hu-
man lung cancer cell line A549 (Table 1). Growth-
inhibition by the quinazoline derivatives was evaluated by
MTS assay.
9
the hit compound 1a. In constast, the introduction of 2-
Among the derivatives with various aromatic rings on
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substituents, such as hydroxyl (the analog with 2-
the substituent at the 4-position of quinazoline, 1f (con-
hydroxyl group mainly exists as its tautomer 18), propoxy
taining a 4-ethoxyphenyl group, IC50 = 0.30 µM) and 1q
(21), phenoxy (23), phenylamino (26), piperidin-1-yl (27),
(containing a 4-methylthiophenyl group, IC50 = 0.34 µM)
showed the same degree of anti-proliferative activity as 1a
(IC50 = 0.27 µM). However, none of the derivatives had a
higher antiproliferative activity than 1a in A549 cells. Alt-
hough their absolute configurations are not yet deter-
mined, (+)-1a showed stronger anti-proliferative activity
than (-)-1a (See Figure S-2, Supporting Information).
4-methylpiperadine-1-yl (28), 4-(4-fluorophenyl)piperadine-1-
yl (30), and 3-chlorophenyl (34) significantly decreased activi-
ty.
Table 1.
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(A) Flow cytometric analysis of cell cycle distribution in A549 cells (B) Immunomicroscopic analysis of tubulin destabi-
lization in A549 cells (C) In vitro tubulin polymerization assay (D) Competitive displacement binding assay on the colchi-
cine-binding site of tubulin (E) Concentration dependent binding of 1a to the colchicine-binding site
formation). Furthermore, such correlation was also seen
in the experiments using both enantiomers of 1a (Fig. S-2,
Supporting Information).
To gain further insight into the mechanism of microtu-
bule destabilization, we investigated the binding site of 1a
on the tubulin protein using a fluorescent-based colchi-
cine site competition assay with purified tubulin. Colchi-
To develop a novel anticancer drug, various substituents
cine is known to show clear fluorescent properties when
at the 2- and 4-positions of quinazoline were synthesized
bound to tubulin (excitation at 350 nm and emission at
via NHC-catalyzed aroylation. Based on the SAR studies,
the 2-chloroquinazoline derivatives 14, 16, 17, 19, 24, and
31 were shown to be the most potent analogs. In addition,
our results suggest that this type of quinazoline derivative
430 nm), although it is non-fluorescent by itself.21 The
fluorescence signal was robustly attenuated in the pres-
ence of nocodazole, which binds the colchicine-binding
site (Figure 1D). In contrast, vinblastine, which preferen-
exhibits anti-proliferative activity through binding to the
tially binds to a non-colchicine binding site, did not di-
minish the fluorescence (Figure 1E). In this competition
polymerization in cancer cells. Further derivatization to
assay, the fluorescence was clearly attenuated by 1a in a
concentration dependent manner (Figure 1D and 1E).
These results suggest that 1a has high affinity for the col-
colchicine-site of tubulin, thereby inhibiting microtubule
enhance activity and the elucidation of the detailed
mechanism of action are now under investigation.
chicine-binding site.
ꢀASSOCIATED CONTENT
One of the derivatives (1f) had comparable anti-
*S Supporting Information
proliferative activity to 1a, and showed similar inhibitory
activity in the tubulin polymerization assay. The more
potent inhibitor 15, inhibited tubulin polymerization
more robustly than 1a at 10 µM (Fig. S-3, Supporting In-
Scheme S-1, Figure S-1, S-2, S-3, experimental procedures,
1
analytical data, H- and 13C-NMR charts, and chiral HPLC-
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ACS Medicinal Chemistry Letters
(11) Wang, X.-F.; Guan, F.; Ohkoshi, E.; Guo, W.; Wang, L.;
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Li, L.; Qian, K.; Morris-Natschke, S. L.; Yuan, S.; Lee, K.-
H.; Xie, L. Optimization of 4-(N-
cycloamino)phenylquinazolines as a novel class of tubu-
lin-polymerization inhibitors targeting the colchicine site
J. Med. Chem. 2014, 57, 1390-1402.
the Internet at http://pubs.acs.org.
ꢀAUTHOR INFORMATION
Corresponding Author
*(A. A.) E-mail: aasai@u-shizuoka-ken.ac.jp
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A.; Anderson, M. B.; Mather, G. Discovery of N-(4-
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Miyashita, A. Carbon-carbon bond cleavage of a-
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*(Y. S.) E-mail: yumiko_suzuki@sophia.ac.jp
§Present Address: Department of Pharmaceutical Sci-
ences, Graduate School of Medicine, Dentistry and Phar-
maceutical Sciences, Okayama University, 1-1-1 Tsushima-
naka, Kita-ku, Okayama 700-8530
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