Substituted benzoquinazolinones. Part 1: Synthesis of 6-aminobenzo[h]quinazolinones via Buchwald-Hartwig amination from 6-bromobenzo[h]quinazolinones

Article abstract of DOI:10.1016/j.tet.2014.05.117

6-(Morpholin-4-yl)benzo[h]quinazolin-4(3H)-one derivatives 18a,b were prepared under Buchwald-Hartwig conditions by reacting 6-bromobenzo[h]quinazolin-4(3H)-ones 13a,b with morpholine in the presence of a Pd(OAc)₂/XantPhos system in 1,4-dioxane as solvent. The starting 6-bromobenzo[h]quinazolin-4(3H)-ones 13 were synthesized via condensation of the ethyl 1-amino-4-bromonaphthalene-2-carboxylate (11) with formamide (Niementowski reaction), and then reaction of the obtained benzoquinazolinone 9 with appropriates benzyl bromides. Compound 11 was prepared using a three-step procedure involving (a) metalation of the N-Boc- or N-Piv-protected 1-aminonaphthalenes with t-BuLi, followed by reaction with ethyl chloroformate, (b) bromination of the naphthalene ring of ester 3 using NBS, and next (c) deprotecion of the amine group with TFA or HCl. Biological screening of the potential cytotoxicity of compounds 8, 9, 18b on A549 and HT29 cell lines, as well as on the lymphocytes showed that compound 18b has interesting anticancer activities. The detailed synthesis, spectroscopic data, and biological assays were reported.

Full text of DOI:10.1016/j.tet.2014.05.117

Tetrahedron xxx (2014) 1e8
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Substituted benzoquinazolinones. Part 1: Synthesis of
6-aminobenzo[h]quinazolinones via BuchwaldeHartwig
amination from 6-bromobenzo[h]quinazolinones
,
a
,
a
b
Monika Nowak a , Zbigniew Malinowski , Andrzej Jozwiak , Emilia Fornal ,
*
*
ꢀꢀ
Alina B1aszczyk c, Renata Kontek c
a University of Lodz, Faculty of Chemistry, Department of Organic Chemistry, Tamka 12 Street, 91-403 Lodz, Poland
b The John Paul II Catholic University of Lublin, Center for Interdisciplinary Research, Laboratory of Separation and Spectroscopic Method Applications,
Krasnicka Avenue 102, 20-718 Lublin, Poland
c University of Lodz, Faculty of Biology and Environmental Protection, Department of General Genetics, Molecular Biology, and Plant Biotechnology,
Laboratory of Cytogenetics, Banacha 12/16 Street, 90-237 Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
6-(Morpholin-4-yl)benzo[h]quinazolin-4(3H)-one derivatives 18a,b were prepared under Buch-
waldeHartwig conditions by reacting 6-bromobenzo[h]quinazolin-4(3H)-ones 13a,b with morpholine in
the presence of a Pd(OAc)2/XantPhos system in 1,4-dioxane as solvent. The starting 6-bromobenzo[h]
quinazolin-4(3H)-ones 13 were synthesized via condensation of the ethyl 1-amino-4-
bromonaphthalene-2-carboxylate (11) with formamide (Niementowski reaction), and then reaction of
the obtained benzoquinazolinone 9 with appropriates benzyl bromides. Compound 11 was prepared
Received 25 March 2014
Received in revised form 15 May 2014
Accepted 27 May 2014
Available online xxx
Keywords:
Metalation
Quinazolinones
Beznoquinazolinones
BuchwaldeHartwig reaction
Palladium coupling
Anticancer activity
using
a three-step procedure involving (a) metalation of the N-Boc- or N-Piv-protected 1-
aminonaphthalenes with t-BuLi, followed by reaction with ethyl chloroformate, (b) bromination of the
naphthalene ring of ester 3 using NBS, and next (c) deprotecion of the amine group with TFA or HCl.
Biological screening of the potential cytotoxicity of compounds 8, 9, 18b on A549 and HT29 cell lines, as
well as on the lymphocytes showed that compound 18b has interesting anticancer activities. The detailed
synthesis, spectroscopic data, and biological assays were reported.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
inspired us to synthesize a series of substituted benzoquinazoli-
nones as potential anticancer agents.
Quinazolinones and their various derivatives demonstrate
a broad spectrum of biological activity, such as antifungal, anti-
bacterial, and antiviral properties.1 Some aminoalkyl derivatives of
benzo[h]quinazolin-4(3H)-one are known to have potential thera-
peutic utility in the treatment of Alzheimer’s disease and also sleep
or pain disorders.2 Moreover, 6-amino derivatives of quinazolin-
4(3H)-one or their benzo analogs may be useful in the therapy of
metabolic and circulatory diseases also in nervous system
disorders.3
Literature surveys revealed that the construction of the benzo[h]
quinazolin-4(3H)-one skeleton can be achieved by the use of 1-
nitronaphthalene,8,9 3,4-dihydronaphthalene-1(2H)-one (
a-tetra-
lone),10 1-aminonaphthalene-2-carboxylic acid,11 naphthalene-2-
carbamate or their derivatives7,11,12 as starting materials.
To the best of our knowledge, one of the most common methods
for construction of the quinazolinone moiety via Niementowski
reaction has been used for the preparation of their benzo analogs
only once. The cyclization of aminonaphthalenecarboxylic acid
with formamide was described during the studies on the synthesis
and properties of Samoquasine A.9,10,13
In this paper we present the preliminary results of our studies
on the synthesis of novel benzo[h]quinazolin-4(3H)-ones A as well
as quinazolin-4(3H)-one B derivatives and their further trans-
formation into aminoquinazolinones C and D (Scheme 1) under
BuchwaldeHarwig palladium cross-coupling reaction. Selected
benzo[h]quinazolin-4(3H)-one derivatives were subjected to
There are some amino derivatives of quinazolinones with anti-
cancer activity that are able to act as a potential inhibitor of Pgp,
tubulin polymerization, and topoisomerases I, II.4e7 These facts
* Corresponding authors. Fax: þ48 42 6786583; e-mail addresses: monikus.7@
onet.eu (M. Nowak), zbigmal@uni.lodz.pl (Z. Malinowski).
http://dx.doi.org/10.1016/j.tet.2014.05.117
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Nowak, M.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.117

2
M. Nowak et al. / Tetrahedron xxx (2014) 1e8
In each case, a small amount of substrate 1a (8e10%) and N,N-
bis-(alkoxycarbonyl) derivative (4) (z6%) were also isolated from
reaction mixture, except with compound 3a.
When 2 equiv of ClCOOEt was used with bis-lithiated N-Boc-1-
naphthylamine, product 3a was not obtained. Instead, derivatives 4
(29%) and 5 (22%) were isolated from the crude material (Scheme
2).
Replacement of the tert-butoxycarbonylamino group by the tert-
butylcarbonylamino group in the naphthalene derivative did not
result in an increase in yield of the ethoxycarbonylation product. In
this case, in addition to derivative 3b (32%), the lactam 6 (8%) was
also isolated as the product of intramolecular cyclization of 3b
(Scheme 2).
B
Scheme 1. General route for the preparation of aminoquinazolinones.
With compounds 3a and 3b in hand, we decided to test two
approaches for the preparation of the benzo[h]quinazolinone
system (3a,b/9, Scheme 3). The first route involved deprotection
of amino esters 3a,b with TFA in DCM or HCl,17 then cyclization of
7 with formamide and finally, halogenation of benzo[h]quinazo-
linone 8. Compound 7 was obtained in 87% (from 3a) or 39% (from
3b), and 8 in 68% yield. The bromination process was carried out
under mild reaction conditions (rt) using NBS in acetonitrile.18
However, the relatively long time reaction (60 h) and low yield
(23%) were not satisfactory. The second approach that led to
bromobenzo[h]quinazolinone (9) was focused on using a three-
step procedure consisting of bromination of ester 3a,b (NBS, rt,
acetonitrile), deprotection of the amine group and then cyclization
of 11 under Niementowski reaction conditions. According to this
route the bromine atom was introduced into the 4-position of the
naphthalene ring of derivatives 10 before condensation with
formamide and formation of benzoquinazolinone skeleton
(Scheme 3). This path turned out to be more effective than first
one.
cytotoxicity tests against human non-small cell lung cancer cells
(A549) and human colon cancer cells (HT29) as well as against
lymphocytes.
2. Results and discussion
The first step of our studies involved the synthesis of 1-
aminonaphthalene-2-carboxylic acid ethyl ester as the key com-
pound in the preparation of benzoquinazolinone skeleton via Nie-
mentowski condensation.14
In a series of previous works we have presented15 our results of
the applications of regiospecific ortho-lithiation of aromatic com-
pounds, in particular N-phenylbenzamides, as an excellent pro-
cedure for the preparation of 1,2-disubstituted benzene derivatives.
In this project our aim was to extend the scope of this meth-
odology toward the synthesis of new 1,2-disubstituted naphthalene
derivatives, starting from easily available N-Boc- or N-Piv-protected
1-aminonaphthalenes (Scheme 2).
Scheme 2. Synthesis of compounds 3a and 3b. Reagents and conditions: (a) t-BuLi, Et2O, ꢀ20 ꢁC; (b) ClCOOEt, ꢀ20 ꢁC/rt.
Bis-(N- and C-ortho)-lithiated N-t-Boc protected 1-
naphthylamine was efficiently generated upon the treatment of
amides 1a,b with 2.1 equiv of t-BuLi in Et2O (ꢀ20 ꢁC). The treatment
of the lithiated species with ClCOOEt (1 equiv, Scheme 2) afforded
the corresponding ortho-ethoxycarbonylated ester 3a in 47% yield
as a single product.16
An increase in t-BuLi, used for the initial lithiation, from
2.1 equiv to 3 equiv resulted in a slight increase in yield of com-
pound 3a up to 52%. Unexpectedly, metalation of carbamate 1a
with t-BuLi, in the presence of TMEDA or HMPA, followed by re-
action with ethyl chloroformate led to ester 3a in a decrease in
yield.
The deprotection of compounds 10a,b using TFA or HCl afforded
the amino derivative 11 (99%, from 10a; 42%, from 10b), which after
cyclization with formamide, under microwave-assisted conditions,
formed 6-bromobenzo[h]quinazolin-4(3H)-one (9) in 84% yield
(Scheme 3). The 1H NMR spectrum of bromobenzoquinazolinone 9
shows two singlets at 8.41 and 8.33 ppm, which are specific for
protons H2 and H5, respectively.
In recent years, the Pd-catalyzed cross-coupling arylation of
amines has become an important and popular method for CeN
bond formation.19 The synthesis of amino quinazolin-4(3H)-one
and benzo[h]qinazoline derivatives in BuchwaldeHartwig type
amination reactions using, e.g., Pd(OAc)2 or Pd2(dba)3 and
Please cite this article in press as: Nowak, M.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.117

M. Nowak et al. / Tetrahedron xxx (2014) 1e8
3
in dry acetone under microwave-assisted conditions.26 Com-
pounds 13a,b and 14 were obtained in 55e70% yield. In one case,
when quinazolinone 12 was treated with benzyl bromide,
dibenzyl derivative 15 (10%) was isolated from the crude reaction
mixture besides the corresponding benzyl quinazolinone 14. The
1H NMR spectrum of compound 15 showed two characteristic
singlets at 5.85 and 5.39 ppm attributable to two groups of
benzylic protons.
The optimization of conditions for the palladium-catalyzed
amination of heteroaryl bromides 13a,b, 14 (Scheme 4) was per-
formed with 6-bromoquinazolin-4(3H)-one derivative 14 and
morpholine as the amine component. All experiments were carried
out by applying the Pd/XantPhos system in the presence of KOt-Bu
or Cs2CO3 as a base, in dry 1,4-dioxane or toluene as solvent
(90e100 ꢁC, 20 h). Detailed data are presented in the Table 1.
Table 1
Optimization of CeN bond forming reactions
Entry Starting [Pd]
material
Base
[Pd]/XantPhos Solvent
[mol %]
Yield
(%)
Scheme 3. Synthesis of benzo[h]quinazolinones 8, 9. Reagents and conditions: (a) TFA,
DCM, 0 ꢁC/rt or 6 M HCl, 90 ꢁC; (b) formamide, 190e200 ꢁC; (c) NBS, acetonitrile,
0
1
2
3
4
5
6
7
8
12
14
14
14
14
12
14
14
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
KOt-Bu
KOt-Bu
KOt-Bu 10:15
KOt-Bu 15:15
KOt-Bu 15:15
KOt-Bu 15:15
5:8
7:10
Toluene
Toluene
Toluene
Toluene
0b
ꢁC/rt; (d) formamide, 200 ꢁC, MW.
8b
15b
42a
frequently BINAP have been reported in the literature.7,20 There
have been studies in which it is shown that, e.g., DBBP,21 RuPhos,22
XantPhos, DavePhos, and Cy-JohnPhos,23 are good ligands in cross-
coupling reactions.
1,4-Dioxane 89a
1,4-Dioxane
0b
Pd(acac)2 KOt-Bu 15:15
Pd2(dba)3 Cs2CO3 15:15
1,4-Dioxane 62a
1,4-Dioxane 20b
Reaction conditions (entries 1e8): 1.5 equiv of a base, 3 equiv morpholine, time
reaction: 20 h, T¼90e100 ꢁC.
In our research we have concentrated on utilizing XantPhos, as it
has been suggested that the ligand is more effective than BINAP.24
Excellent properties of the XantPhos have been confirmed during
BuchwaldeHartwig amination reactions of quinazolin-4(3H)-one23
derivatives as well as various other compounds.25
As material for further modification, we chose benzyl de-
rivatives 13a,b and 14 (Scheme 4), which can be synthesized from
the easily available benzyl bromides and the appropriate lactams
9, 12. The N-benzylation of compounds 9 and 12 was carried out
a
Yield for isolated and purified products.
b
Yield from 1H NMR.
Initial attempts with various combinations of Pd(OAc)2/Xant-
Phos in ratio: 7 mol %/10 mol %, 10 mol %/15 mol %, 15 mol %/
15 mol % in toluene, in the presence of KOt-Bu afforded amine 16
with a maximum yield of 42% (Table 1, entries 2e4). The optimal
conditions for the arylation of morpholine were obtained by
B
Scheme 4. The amination reaction of 6-bromoquinazolin-4(3H)-ones 14 and 6-bromobenzo[h]quinazolin-4(3H)-ones 13a,b.
Please cite this article in press as: Nowak, M.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.117

4
M. Nowak et al. / Tetrahedron xxx (2014) 1e8
applying Pd(OAc)2/XantPhos system (15 mol %/15 mol %) in 1,4-
dioxane as a solvent (Table 1, entry 5, 89% of yield). A similar re-
sult was achieved under the same reaction conditions in the Pd-
amination reaction of quinazolinone 14 with 1-(2-fluorofhenyl)-
piperazine (88%). The use of Pd(acac)2 or Pd2(dba)3 as an alternative
palladium source with XantPhos in 1,4-dioxane, in the presence of
KOt-Bu or Cs2CO3 gave the corresponding amine 16 in low to
moderate yields (20e62%). We have also observed that Buch-
waldeHartwig amination reaction did not proceed without N-
protection of quinazolinone 12 (Table 1, entries 1,6).
We also attempted the amination of the benzyl derivative of 6-
bromobenzo[h]quinazolin-4(3H)-ones 13a,b with morpholine. The
amination products 18a and 18b were obtained in 40% and 45%
yields, respectively. As well as 18a and 18b compounds, there were
also some quantities of starting material (13a,b; 10e15%) isolated
from the post-reaction mixture.
4. Conclusion
In the present research, the application of o-metalation to ach-
ieve the benzo[h]quinazolin-4(3H)-one systems was shown, as
precursors in the synthesis of 6-aminoquinazolin-4(3H)-one de-
rivatives. Novel amino derivatives of benzo[h]quinazolin-4(3H)-
one and quinazolin-4(3H)-one were formed by BuchwaldeHartwig
amination using a combination of 15 mol % Pd(OAc)2, 15 mol %
XantPhos in 1,4-dioxane. To our knowledge, this report is the first,
which presents a results of modification the 6-position of the benzo
[h]quinazolin-4(3H)-one ring by applying amines in Buch-
waldeHartwig cross-coupling.
Based on the obtained results, it can be concluded that the level
of cytotoxicity of the new compounds depends on the type and
structure of the substituent that is connected to the base molecule.
Cytotoxic activity with regard to the substitution in the 6-position
of the quinazolinone ring increases in the order Br>H>N(R)2. The
amino group in 6-position of benzo[h]quinazolinone structure
seems to be necessary for the presence of antiproliferative activity.
Compound 18b proved to be highly cytotoxic to HT29 cancer cells
3. Antitumor activity
The choice of the compounds to be tested was determined by
the desire to check their influence on cell proliferation,
depending on the type of substituent in the 6-position of the
benzo[h]quinazolin-4(3H)-one system. Three quinazolin-4(3H)-
one derivatives obtained from the synthesis were chosen. Com-
pounds 8, 9, 18a were tested for their ability to inhibit the growth
of tumor cells. Cytotoxicity tests were performed on A549 and
HT29 cell lines (human non-small cell lung cancer cells and
human colon adenocarcinoma cells, respectively) as well as on
lymphocytes.
and weakly toxic to normal lymphocytes.
aminobenzoquinazolinone 18b showed the desired relationship:
low cytotoxicity to normal cellsdhigh toxicity to pathological cells.
A
new 6-
5. Experimental section
5.1. Chemistry
5.1.1. General. Melting points were determined on a Boetius hot
stage apparatus and were uncorrected. 1H, 13C, and 19F NMR spectra
were recorded on a Bruker Advance III spectrometer at 600 MHz,
150 MHz, and 565 MHz, respectively. The residual CDCl3 or DMSO-
d6 signal was used for reference (CDCl3 at 7.26 ppm or DMSO-d6 at
2.54 ppm for 1H NMR and CDCl3 at 77.0 ppm or DMSO-d6 at
39.0 ppm for 13C NMR). 19F NMR spectra were obtained without 1H-
decoupling. 2D Homonuclear 1H 1H COSY spectra and hetero-
nuclear 1H 13C COSY spectra (HSQC and HMBC) were used to assign
the proton and carbon signals. Coupling constants (J) are given in
hertz (Hz). IR spectra were recorded on a Nexus FT-IR spectrometer.
Microwave reactions were performed in a Synthos 3000 microwave
reactor from Anton Paar. LCeHRMS analyses were carried out using
a liquid chromatograph (Agilent Technologies series 1200) coupled
to a tandem mass spectrometer (Agilent Technologies 6538 UHD
Accurate Mass Q-TOF LC/MS) equipped with an HPLC-chip-cube
allowing nanoelectrospray ionization of analytes. The instrument
is housed in the Laboratory of Separation and Spectroscopic
Method Applications, Center for Interdisciplinary Research, KUL,
Lublin, Poland. Instrument control and data acquisition were per-
formed using an Agilent Technologies Mass Hunter Acquisition
module (version B.04). High-resolution mass spectra were acquired
in the positive ion scan mode at 100e1000 m/z. The capillary po-
tential was set at ꢀ1750 V and the fragmentor was set at 100 V.
Mobile phase consisted of water/acetonitrile/0.1% formic acid. A
Studies on the cytotoxic activity of samoquasine A (8) showed
a decrease in cell proliferation after cell treatment with increasing
concentration of the compound. The obtained results allowed us to
determine the IC50 values for cancer cells: 122.5
mM (A549) and
154.95 M (HT29). In the case of lymphocytes IC50 concentration
m
was not determined. These cells were less sensitivedabout 50%
reduction of cell proliferation was observed after the use of
160e180 mM concentrations.
Results of the studies on cytotoxicity of samoquasine A de-
rivative 9 did not allow the determination of IC50 values. A de-
crease in cell proliferation was shown after treatment of A549
cells with the compound even after using a concentration of
2 mM. Such a decrease remained at a similar level (between 50
and 60% as compared to the negative control), when a higher
concentration of the compound was used. 6-Bromobenzo[h]qui-
nazolin-4(3H)-one 9 caused also reduction of proliferation of
HT25 cells and lymphocytes, which was combined with in-
creasing concentrations of test compound. The highest inhibition
of cell proliferation was at the level of about 60e70% as compared
to the negative control.
Compound 18b was highly toxic against HT29 cells
(IC50¼4.12
lymphocytes was rather low (IC50¼134.05
respectively). Cytotoxicity of 18b against HT29 cancer cells
mM), while its cytotoxicity against A549 cells and
m
M and IC50¼175.11 M,
m
large capacity chip (160 nL) 150 mm C18, 5 mm was used. Internal
was higher than that of cisplatin (IC50 w8.47 mM), which is widely
mass calibration was enabled, using two reference mass ions
(121.0509 and 922.0098). The mass accuracy for MS scans was
<1 ppm. HRMS(EI) analyses were carried out in Mass Spectrometry
Laboratory of Polish Academy of Sciences in Lodz (CBMiM) using
mass spectrometer Finnigan MAT 95, Voyager-Elite, Automass III
and LKB 2091. The analytical thin layer chromatography tests (TLC)
were carried out on Merck silica gel plates (Kiselgel 60 F254, layer
thickness 0.2 mm) and the spots were visualized using UV lamp.
The flash column chromatography purifications were performed on
Fluka silica gel (Silica gel 60, 0.035e0.070 mm). tert-Butyllithium
solution in pentane (Aldrich) was each time titrated before use. All
reactions with organolithium and organopalladium compounds
used in cancer therapy.27 The summary of the results is shown in
Table 2.
Table 2
Cytotoxicity of compounds 8, 9, 18b against cancer cell lines
Testing compound
IC50 [mM]
Lymphocytes
HT29
154.95
>300
4.12
A549
122.50
>300
134.05
8
9
18b
>250
>300
175.11
Please cite this article in press as: Nowak, M.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.117

M. Nowak et al. / Tetrahedron xxx (2014) 1e8
5
were performed under an argon atmosphere using standard
Schlenk technique.
7.54e7.46 (m, 3H, 3,6,7AreH), 7.34e7.33 (m, 1H, 2AreH), 4.19e4.15
(m, 2H, CH2), 1.31 (s, 9H, BoceMe), 1.14e1.11 (m, 3H, Me); 13C NMR
Tetrahydrofuran, diethyl ether, and 1,4-dioxane were distilled
from sodium benzophenone ketyl prior to use. Commercially
available solvents and reagents: Boc2O, trimethylacetyl chloride, 1-
naphthylamine, ethyl chloroformate, DMF, NBS, anthranilic acid,
formamide, benzyl bromide, 4-methoxybenzyl bromide, 4,5-bis(-
diphenylphosphino)-9,9-dimethylxanthene (XantPhos) were pur-
chased from SigmaeAldrich and were used without further
purification.
(CDCl3): d 153.5, 151.7, 135.5, 134.4, 130.8, 128.6, 128.4, 127.0, 126.2,
125.9, 125.5, 122.1, 83.1, 63.0, 27.9, 14.2; HRMS (ESI-LC/MS) m/z
calcd for C18H21NO4: 315.1471; found [M]þ: 315.1470.
5.3.3. Ethyl 2-[(tert-butoxycarbonyl)(ethoxycarbonyl)amino]naph-
thalene-2-carboxylate (5). Beige solid, yield: 22%, Rf (DCM/PE/
AcOEt 5:1:1)¼0.66, mp 108e109 ꢁC; IR (KBr, cmꢀ1) 1768, 1750,
1710; 1H NMR (CDCl3):
d
8.05 (d, 1H, J¼8.6, 8AreH), 8.00e7.99 (m,
1H, 5AreH), 7.90e7.88 (m, 2H, 6,7AreH), 7.61e7.56 (m, 2H,
3,4AreH), 4.44e4.36 (m, 2H, CH2), 4.21e4.08 (m, 2H, CH2), 1.40 (t,
3H, J¼7.1, Me), 1.27 (s, 9H, Boc-Me), 1.10 (t, 3H, J¼7.1, Me); 13C NMR
5.2. Synthesis of N-t-Boc and N-Piv of 1-naphthylamine
derivatives
(CDCl3):
d 165.7, 152.3, 150.4, 136.2, 135.6, 130.8, 127.9, 127.8, 127.3,
N-Boc-1-naphthylamine (1a) and 1-naphthyl-N-pivalamide
(1b) were prepared by a procedure similar to that in the
literature.28e30
126.1,126.2,125.3,123.3, 82.5, 62.5, 61.3, 27.4,14.0,13.9; HRMS (ESI-
LC/MS) m/z calcd for C21H25NO6: 387.1682; found [M]þ: 387.1681.
5.3.4. Ethyl 1-[(2,2-dimethylpropanoyl)amino]naphthalene-2-
5.2.1. N-Boc-1-Naphthylamine (1a).28,30 Pastel pink needles, yield:
88%, mp 95e96 ꢁC (mp lit. 95e97 ꢁC30a); IR (KBr, cmꢀ1) 1684; 1H
carboxylate (3b). Yellow oil, yield: 32%, Rf (PE/AcOEt 4:1)¼0.68; IR
(KBr, cmꢀ1) 3355, 1694, 1629; 1H NMR (CDCl3):
d 9.91 (br s, 1H, NH),
NMR (CDCl3):
4AreH), 7.50e7.41 (m, 3H, 2,7,6AreH), 6.79 (br s, 1H, NH), 1.53 (s,
9H, BoceMe); 13C NMR (CDCl3):
153.6, 134.3, 133.1, 128.9, 126.7,
d
7.87e7.83 (m, 3H, 8,5,3AreH) 7.60 (d, 1H, J¼8.2,
7.97 (d, 1H, J¼8.7, 3AreH), 7.85e7.81 (m, 2H, 5,8AreH), 7.70 (d, 1H,
J¼8.7, 4AreH), 7.58e7.50 (m, 2H, 6,7AreH), 4.42 (q, 2H, J¼7.1, CH2),
d
1.46e1.42 (m, 12H, PiveMe; Me); 13C NMR (CDCl3):
d 178.6, 167.6,
126.2, 126.0, 125.5, 124.6, 120.6, 118.8, 80.8, 28.5.
138.7, 136.1, 128.9, 128.4, 127.9, 126.7, 126.2, 125.9, 125.4, 119.7, 61.5,
40.0, 27.8, 14.4; HRMS (ESI-LC-MS) m/z calcd for C18H21NO3:
299.1521; found [M]þ: 299.1524.
5.2.2. 1-Naphthyl-N-pivalamide (1b).29,31 Pale violet solid, yield:
66%, mp 149e151 ꢁC (mp lit. 150e151 ꢁC31); IR (KBr, cmꢀ1) 3259,
1648; 1H NMR (CDCl3):
d
7.96 (d, 1H, J¼7.5, 8AreH), 7.88e7.86 (m,
5.3.5. 1-(2,2-Dimethylpropanoyl)naphtho[1,2-b]azet-2(1H)-one
1H, 5AreH), 7.78e7.76 (m, 2H, 2AreH, NH), 7.69 (d, 1H, J¼8.2,
(6). White solid, yield: 8%, Rf (PE/AcOEt 4:1)¼0.84; mp 149e151 ꢁC;
4AreH), 7.54e7.46 (m, 3H, 3, 6, 7AreH), 1.44 (s, 9H, PiveMe); 13C
IR (KBr, cmꢀ1) 1752,1636; 1H NMR (CDCl3):
d 8.93e8.92 (m, 1H,
NMR (CDCl3):
120.8, 120.2, 39.8, 27.8.
d
176.9, 134.1, 132.4, 128.8, 127.3, 126.2, 125.8, 125.7,
8AreH), 8.09 (d, 1H, J¼8.6, 3AreH), 7.92e7.91 (m, 1H, 5AreH), 7.87
(d, 1H, J¼8.6, 4AreH), 7.75e7.69 (m, 2H, 6,7AreH), 1.51 (s, 9H,
PiveMe); 13C NMR (CDCl3):
d 169.8, 160.7, 145.8, 137.3, 130.2, 129.4,
5.3. General procedure for the preparation of 2-substituted
N-Boc- or N-Piv-1-naphthylamine derivatives 3a, 3b
128.3, 128.1, 127.4, 125.6, 122.4, 112.8, 38.6, 28.0; HRMS (ESI-LC/MS)
m/z calcd for C16H15NO2: 253.1103; found [M]þ: 253.1105.
Under argon, to a solution of N-Boc-1-naphthylamine (1a) or 1-
naphthyl-N-pivalamide (1b) (2.2 mmol) in dry Et2O (15 mL) at
ꢀ20 ꢁC, t-BuLi in pentane (4.6 mmol) was added dropwise. The
reaction mixture was stirred at this temperature for 2 h. Next, to the
solution of lithiated species was added dropwise at ꢀ20 ꢁC, a so-
lution of an appropriate electrophile (ethyl chloroformate,
2.2 mmol) in dry Et2O (10 mL). Then, the mixture was allowed to
warm to room temperature and was stirred under these conditions
for 2 h. After this time, to the reaction mixture a saturated solution
of NH4Cl (20 mL) was added and was stirred for 0.5 h. The water
layer was separated and extracted subsequently with Et2O
(3ꢂ20 mL). The organic phase was dried over MgSO4 and concen-
trated till dryness. The crude material was separated by flash
chromatography.
5.4. General procedure for the preparation of bromo de-
rivatives 10a and 10b
To a solution of the appropriate ester 3a or 3b (0.89 mmol) in
acetonitrile (12 mL) at 0 ꢁC, a solution of NBS (1.06 mmol) in ace-
tonitrile (4 mL) was added dropwise. The resulting mixture was
allowed to warm to ambient temperature and the reaction was
continued under these conditions until TLC analysis (DCM) of the
reaction mixture indicated the absence of starting material 3a or
3b. After the reaction was complete, acetonitrile was removed
under reduced pressure. The corresponding bromo derivative was
separated from the residue by flash chromatography.
5.4.1. Ethyl 4-bromo-1-[(tert-butoxycarbonyl)amino]naphthalene-2-
carboxylate (10a). Straw-yellow solid, yield: 95%, time of reaction
20 h, Rf (DCM)¼0.56, mp 135e136 ꢁC; IR (KBr, cmꢀ1) 3269, 1720,
5.3.1. Ethyl 1-[(tert-butoxycarbonyl)amino]naphthalene-2-
carboxylate (3a). Beige solid, yield: 47%, Rf (DCM)¼0.28, mp
1701; 1H NMR (CDCl3):
d 8.74 (br s, 1H, NH), 8.28 (s, 1H, 3AreH),
112e114 ꢁC; IR (KBr, cmꢀ1) 3421,1698,1687; 1H NMR (CDCl3):
d
8.80
8.23e8.22 (m, 1H, 8AreH), 8.10e8.09 (m, 1H, 5AreH), 7.70e7.68
(m, 1H, 7AreH), 7.61e7.59 (m, 1H, 6AreH), 4.44 (q, 2H, J¼7.1, CH2),
1.51 (s, 9H, BoceMe), 1.46 (t, 3H, J¼7.1, Me); 13C NMR (CDCl3):
(br s, 1H, NH), 8.08e8.06 (m, 1H, 8AreH), 7.96 (d, 1H, J¼8.7, 3AreH),
7.82e7.81 (m, 1H, 5AreH), 7.67 (d, 1H, J¼8.7, 4AreH), 7.58e7.53 (m,
2H, 6,7AreH), 4.43 (q, 2H, J¼7.1, CH2), 1.53 (s, 9H, Boc-Me), 1.46 (t,
d
166.5, 154.2, 138.2, 134.6, 130.3, 129.8, 129.3, 127.3, 127.1, 126.9,
3H, J¼7.1, Me); 13C NMR (CDCl3):
d
167.8, 154.4, 138.2, 136.3, 128.4,
120.1, 119.6, 81.3, 61.9, 28.4, 14.4; HRMS (EI) m/z calcd for
127.9, 126.5, 126.2, 125.6, 125.5, 125.4, 119.7, 80.9, 61.5, 28.4, 14.4;
C
18H20BrNO4, 393.0576; found [M]þ: 393.0588.
MS (EI) m/z: 316.2(37), 216.1(10), 215.1(76), 170(24), 169(100).
5.4.2. Ethyl
4-bromo-1-[(2,2-dimethylpropanoyl)amino]naphtha-
5.3.2. tert-Butyl ethyl naphthalen-1-yl-imidodicarbonate (4). Yellow
oil, yield: 6% (after reaction with 1 equiv of ClCOOEt) or yield: 29%
(after reaction with 2 equiv of ClCOOEt), Rf (DCM/PE/AcOEt 5:1:1)¼
lene-2-carboxylate (10b). Pale brown solid, yield: 54%, time of re-
action 20 h, Rf (PE/AcOEt 5:1)¼0.5, mp 122e124 ꢁC; IR (KBr, cmꢀ1
)
1694, 1620; 1H NMR (CDCl3):
d 9.87 (s, 1H, NH), 8.31 (s, 1H, 3AreH),
0.76; IR (thin film, cmꢀ1) 1778, 1751; 1H NMR (CDCl3):
d
7.89e7.87
8.26 (d, 1H, J¼8.5, 8AreH), 7.87 (d, 1H, J¼8.6, 5AreH), 7.72e7.69 (m,
(m, 1H, 8AreH), 7.84 (d, 1H, J¼8.3, 4AreH), 7.80 (m, 1H, 5AreH),
1H, 7AreH), 7.61e7.58 (m, 1H, 6AreH), 4.44 (q, 2H, J¼7.1, CH2),
Please cite this article in press as: Nowak, M.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.117

6
M. Nowak et al. / Tetrahedron xxx (2014) 1e8
1.48e1.45 (m, 12H, PiveMe; Me); 13C NMR (CDCl3):
138.8, 134.4, 130.1, 129.9, 129.1, 127.4, 127.2, 127.0, 120.1, 120.0, 62.0,
40.1, 27.8, 14.4; HRMS (ESI-LC/MS) m/z calcd for C18H20BrNO3:
377.0626; found [M]þ: 377.0629.
d
178.7, 166.6,
1.1 mmol) in formamide (8 mL) was heated at 190e200 ꢁC in an oil
bath for 10 h. After this time the reaction mixture was cooled to
ambient temperature and water was added. The precipitated solid
was filtered from the solution, washed with chloroform (10 mL),
and air-dried to give a pure product.
5.5. General procedure for the preparation of amino esters 7
and 11
5.6.2.1. Benzo[h]quinazolin-4(3H)-one (8).7,13a Beige solid, yield:
68%, mp 302e304 ꢁC (mp lit. >300 ꢁC13a); IR (KBr, cmꢀ1) 3424,
5.5.1. Synthesis of amino esters 7 and 11 prepared from N-Boc de-
rivatives. To a solution of N-Boc-amino ester 3a or 10a (0.83 mmol)
in DCM (5 mL) at 0 ꢁC, TFA (8.3 mmol) was added. The resulting
mixture was allowed to warm to ambient temperature and left in
these conditions until TLC analysis (DCM) of the reaction mixture
indicated the absence of starting material 3a or 10a. After the re-
action was complete all the volatile materials were removed under
reduced pressure and water (5 mL) was added to residue. The
mixture was adjusted to pH¼12 (10% KOHaq) and then extracted
with DCM (3ꢂ20 mL). The combined extracts were dried over
MgSO4 and concentrated under reduced pressure to give the ap-
propriate amine 7 or 11.
1666; 1H NMR (DMSO-d6):
d
12.58 (br s, 1H, NH), 8.92 (d, 1H, J¼8.1,
10AreH), 8.35 (s, 1H, 2AreH), 8.09e8.05 (m, 2H, 6,7AreH), 7.95 (d,
1H, J¼8.7, 5AreH), 7.79e7.72 (m, 1H, 8,9 AreH); 13C NMR (DMSO-
d6): d 161.2, 147.6, 145.5, 136.1, 129.9, 129.7, 128.5, 127.5, 127.2, 125.1,
121.6, 119.5.
5.7. Synthesis of 6-bromobenzo[h]quinazolin-4(3H)-one (9)
in an Anton Paar Synthos 3000 microwave reactor
A mixture of amino ester 11 (0.64 g, 2.2 mmol) and ammonium
carbonate (0.33 g, 3.5 mmol) in formamide (20 mL) was charged to
a PTFE tube, sealed in ceramic cases, and placed in the rotor. The
reaction mixture was stirred and heated to 200 ꢁC and held at this
temperature for 20 min. After this time reaction mixture was
cooled to 25 ꢁC and water was added (80 mL). The precipitated solid
was filtered from the solution, washed with chloroform (10 mL),
and air-dried to give product 9.
5.5.2. Synthesis of amino esters 7 and 11 prepared from N-Piv de-
rivatives. To a solution of N-Piv-amino ester 3b or 10b in 1,4-
dioxane (8 mL) 6 M HCl (8 mL) was added. The reaction mixture
was heated to 90 ꢁC for 8 h. The resulting mixture was basified with
a saturated aqueous solution of NaHCO3 to pH¼9e10 and then
extracted with DCM (3ꢂ20 mL). The combined extracts were dried
over MgSO4 and concentrated under reduce pressure to give ap-
propriate amine 7 or 11.
5.7.1. 6-Bromobenzo[h]quinazolin-4(3H)-one (9).2,8 Gray solid,
yield: 84%, mp 351e352 ꢁC (lit. only ESI/MS2,8); IR (KBr, cmꢀ1) 3442,
1660; 1H NMR (DMSO-d6):
d 12.76 (br s, 1H, NH), 9.01e8.99 (m, 1H,
10AreH), 8.41 (s, 1H, 5AreH), 8.33 (s, 1H, 2AreH), 8.26 (d,1H, J¼8.0,
5.5.3. Ethyl 1-aminonaphthalene-2-carboxylate (7).32,33 Claret-red
solid, yield: 87% (from 3a), 39% (from 3b), mp 109e110 ꢁC (lit.
gum32); IR (KBr, cmꢀ1) 3473, 3355, 1668; 1H NMR (CDCl3):
7AreH), 7.97e7.94 (m, 1H, 9AreH), 7.87e7.85 (m, 1H, 8AreH); 13C
NMR (DMSO-d6):
d 159.6, 146.8, 133.4, 130.8, 130.7, 128.1, 126.8,
125.3, 124.7, 120.0, 119.5; HRMS(EI) m/z calcd for C12H7BrN2O,
d
7.91e7.88 (m, 2H, 3,8AreH), 7.75 (d, 1H, J¼8.1, 5AreH), 7.56e7.53
273.9742; found [M]þ: 273.9748.
(m, 1H, 6 or 7AreH), 7.48e7.45 (m, 1H, 6 or 7AreH), 7.08 (d, 1H,
J¼8.7, 4AreH), 6.80 (br s, 2H, NH2),4.39 (q, 2H, J¼7.1, CH2), 1.43 (t,
5.8. General procedure for the benzylation in an Anton Paar
Synthos microwave reactor. Synthesis of compounds 13a, 13b,
14, 15
3H, J¼7.1, Me); 13C NMR (CDCl3):
d 169.1, 149.0, 136.6, 128.6, 128.5,
126.8, 125.3, 123.3, 121.6, 116.0, 104.6, 60.4, 14.6; HRMS(EI) m/z
calcd for C13H13NO2, 215.0946; found [M]þ: 215.0955.
A mixture of quinazolin-4(3H)-one 12 or 9 (4.44 mmol) and
potassium carbonate (4.44 mmol) in dry acetone (10 mL) was
charged to PTFE tubes, sealed in ceramic cases and placed in the
rotor. The reaction mixture was heated to 55 ꢁC, held for 30 min at
55 ꢁC and then cooled to 25 ꢁC. In the next step the corresponding
bromide (5.33 mmol BnBr or PMBBr) was added and the reaction
was continued at 55 ꢁC for 3.5 h. After cooling to room temperature
the separated solid was collected by filtration. The filtrate was
concentrated under reduced pressure and then the crude product
was purified by flash chromatography.
5.5.4. Ethyl 1-amino-4-bromonaphthalene-2-carboxylate
(11). Beige solid, yield: 99% (from 10a), 42% (from 10b), mp
94e95 ꢁC; IR (KBr, cmꢀ1) 3391, 3345, 1682; 1H NMR (CDCl3):
d
8.19e8.17 (m, 2H, 3,8AreH), 7.90 (d, 1H, J¼8.4, 5AreH), 7.68e7.66
(m, 1H, 7AreH), 7.55e7.52 (m, 1H, 6AreH), 6.85 (br s, 2H, NH2), 4.39
(q, 2H, J¼7.1, CH2), 1.43 (t, 3H, J¼7.1, Me); 13C NMR (CDCl3):
d 168.1,
148.8,134.6,130.2,129.7,128.1,126.2,124.5,122.0,108.4,105.3, 60.8,
14.6; HRMS (EI) m/z calcd for C13H12BrNO2, 293.0051; found [M]þ:
293.0049.
In the case of benzylation of quinazolinone 12 the precipitated
solid was washed with water (30 mL), next with
chloroform (20 mL) to provide salt 15.
5.6. Preparation of quinazolin-4(3H)-ones 8, 9, and 12 (the
Niementowski reaction)
5.6.1. Synthesis of 6-bromoquinazolin-4(3H)-one (12). 6-Bromo-
quinazolin-4(3H)-one (12) was prepared by a procedure similar to
that in the literature.14
5.8.1. 3-Benzyl-6-bromoquinazolin-4(3H)-one (14).33 White solid,
yield: 61%, Rf (Hex/AcOEt 1:1)¼0.48, mp 133e134 ꢁC (mp lit.
124e126 ꢁC33); IR (KBr, cmꢀ1) 3439, 1672; 1H NMR (CDCl3):
d 8.47
(d, 1H, J¼2.2, 5AreH), 8.10 (s, 1H, 2AreH), 7.84e7.83 (m, 1H,
5.6.1.1. 6-Bromoquinazolin-4(3H)-one (12).14b,c White solid,
7AreH), 7.59 (d, 1H, J¼8.7, 8AreH), 7.38e7.35 (m, 5H, BneH), 5.20
yield: 60%, mp 270e272 ꢁC (mp lit. 273e274 ꢁ
C
14c); IR (KBr, cmꢀ1
)
(s, 2H, CH2); 13C NMR (CDCl3):
d 160.1, 147.0, 146.7, 137.7, 135.6,
129.7, 129.5, 129.3, 128.6, 128.2, 123.8, 121.2, 49.9.
3431, 1691; 1H NMR (DMSO-d6):
d
12.39 (s, 1H, NH), 8.19 (d, 1H,
J¼2.4, 5AreH), 8.13 (s, 1H, 2AreH), 7.95e7.94 (m, 1H, 7AreH), 7.62
(d, 1H, J¼8.7, 8AreH); 13C NMR (DMSO-d6):
d
160.1, 148.2, 145.5,
5.8.2. 1,3-Dibenzyl-6-bromo-4-oxo-3,4-dihydroquinazolin-1-ium
137.6, 130.1, 128.4, 124.7, 119.6.
(15). White granules, yield: 10%, mp 243e245 ꢁC; IR (KBr, cmꢀ1
)
1725; 1H NMR (DMSO-d6):
d
10.44 (s, 1H, 2AreH), 8.44 (d, 1H, J¼2.3,
5.6.2. Synthesis of benzo[h]quinazolin-4(3H)-one (8). A mixture of
amino ester 7 (0.14 g, 0.67 mmol) and ammonium carbonate (0.1 g,
5AreH), 8.24e8.22 (m, 1H, 7AreH), 7.82 (d, 1H, J¼9.1, 8AreH),
7.60e7.54 (m, 4H, BneH), 7.46e7.36 (m, 6H, BneH), 5.86 (s, 2H,
Please cite this article in press as: Nowak, M.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.117

M. Nowak et al. / Tetrahedron xxx (2014) 1e8
7
CH2), 5.39 (s, 2H, CH2); 13C NMR (DMSO-d6):
136.4, 133.7, 132.8, 130.2, 128.8, 128.6, 128.5, 128.4, 128.3, 127.2,
d
156.7, 154.8, 139.2,
7.74e7.67 (m, 2H, 8,9AreH), 7.40e7.30 (m, 5H, BneH), 5.28 (s, 2H,
CH2), 4.01e4.00 (m, 4H, MOR), 3.19e3.18 (m, 4H, MOR); 13C NMR
122.8, 122.2, 121.2, 56.1, 52.4; HRMS (ESI-LC/MS) m/z calcd for
(CDCl3): d 160.9, 149.5, 144.8, 143.2, 135.8, 131.9, 129.0, 128.9, 128.8,
C
22H18BrN2O: 405.0597; found [MꢀBr]: 405.0598.
128.3, 128.1, 127.1, 125.6, 123.6, 119.0, 109.4, 67.3, 53.6, 49.8; HRMS
(ESI-LC/MS) m/z calcd for C23H21N3O2: 371.1634; found [M]þ:
371.1634.
5.8.3. 3-Benzyl-6-bromobenzo[h]quinazolin-4(3H)-one (13a). Beige
solid, yield: 55%, Rf (DCM/AcOEt/Hex 1:1:0.5)¼0.8, mp 161e162 ꢁC;
IR (KBr, cmꢀ1) 1668; 1H NMR (CDCl3):
d
9.00 (d, 1H, J¼8.3, 10AreH),
5.9.4. 3-(4-Methoxybenzyl)-6-(morpholin-4-yl)benzo[h]quinazolin-
4(3H)-one (18b). Beige solid, yield: 45%, Rf (PE/acetone 3:1)¼0.32,
mp 119e121 ꢁC; IR (KBr, cmꢀ1) 1676; 1H NMR (600 MHz, CDCl3):
8.58 (s, 1H, 5AreH), 8.33e8.31 (m, 2H, 2,7AreH), 7.83e7.81 (m, 1H,
9AreH), 7.76e7.73 (m, 1H, 8AreH), 7.40e7.33 (m, 5H, BneH), 5.28
(s, 2H, CH2); 13C NMR (CDCl3):
d
160.2, 146.8, 146.5, 135.6, 134.6,
d 9.01e8.99 (m, 1H, 10 AreH), 8.30e8.26 (m, 2H, 5,7 AreH), 7.82 (s,
131.2, 130.6, 129.3, 128.6, 128.3, 128.0, 127.6, 125.8, 125.5, 122.5,
50.1; HRMS (ESI-LC/MS) m/z calcd for C19H13BrN2O: 364.0211;
found [M]þ: 364.0213.
1H, 2AreH), 7.75e7.69 (m, 2H, 8,9 AreH), 7.38e7.36 (m, 2H,
PMBeH), 6.91e6.89 (m, 2H, PMBeH), 5.23 (s, 2H, CH2), 4.03e4.01
(m, 4H, MOR), 3.80 (s, 3H, Me), 3.21e3.20 (m, 4H, MOR); 13C NMR
(CDCl3): d 160.9, 149.5, 144.8, 143.2, 135.8, 131.9, 129.0, 128.9, 128.8,
5.8.4. 6-Bromo-3-(4-methoxybenzyl)benzo[h]quinazolin-4(3H)-one
128.3, 128.1, 125.1, 125.6, 123.6, 119.0, 109.4, 67.3, 53.6, 49.8; HRMS
(ESI-LC/MS) m/z calcd for C24H23N3O3: 401.1739; found [M]þ:
401.1740.
(13b). White solid, yield: 70%, Rf (Hex/AcOEt 3:1)¼0.44, mp
153e155 ꢁC; IR (KBr, cmꢀ1) 1673; 1H NMR (CDCl3):
d 9.00e8.98 (m,
1H, 10AreH), 8.57 (s, 1H, 5AreH), 8.34e8.31 (m, 2H, 2,7AreH),
7.83e7.80 (m, 1H, 9AreH), 7.75e7.72 (m, 1H, 8AreH), 7.36e7.34 (m,
2H, PMBeH), 6.90e6.88 (m, 2H, PMBeH), 5.21 (s, 2H, CH2), 3.79 (s,
6. Biology
3H, Me); 13C NMR (CDCl3):
d
160.2, 159.9, 146.7, 146.5, 134.6, 131.2,
6.1. Cells cultures
130.5, 129.9, 129.0, 128.0, 127.6, 125.8, 125.5, 122.4, 119.3, 114.7, 55.5,
49.7; HRMS (ESI-LC/MS) m/z calcd for C20H15BrN2O2: 394.0317;
found [M]þ: 394.0319.
The experiments were performed with A549 and HT29 cancer
cells (human non-small cell lung cancer cells and human colon
adenocarcinoma cells, respectively) from the American Type Cul-
ture Collection (ATCC) and human lymphocytes obtained from the
Blood Donation Centre (Lodz, Poland).
5.9. General procedure for Pd-catalyzed CeN bond-forming
reactions
A549 cells were cultured in DMEM (CytoGen) complemented
with FBS (10%; fetal bovine serum, Sigma) and penicillin/strepto-
mycin solution (1%). RPMI 1640 medium (CytoGen) used for
HT29cells contained FBS (10%), penicillin/streptomycin solution
(1%) and MEM non essential-amino acids solution (1%). Human
lymphocytes were cultured in RPMI 1640 medium complemented
with inactivated FBS (15%), penicillin/streptomycin (1%), and mi-
togen PHA (1%, phytohemaglutinin; CytoGen). Cells were cultured
at 37 ꢁC in a 5% CO2 humidified atmosphere. All solvents and re-
agents were obtained from SigmaeAldrich.
The reaction was carried out under an argon atmosphere in an
oven dried resealable Schlenk flask. A resealable Schlenk flask was
charged with Pd(OAc)2 (15 mol %), XantPhos (15 mol %), quinazo-
linone 14 or 13a or 13b (0.19 mmol), KOt-Bu (0.28 mmol), the ap-
propriate amine (0.57 mmol), and freshly distilled dioxane (3 mL).
The whole mixture was stirred and heated in an oil bath at
90e100 ꢁC for 10 h. After this time the reaction mixture was cooled
to an ambient temperature and diluted with chloroform (5 mL). The
product was purified by flash chromatography.
5.9.1. 3-Benzyl-6-(morpholin-4-yl)quinazolin-4(3H)-one (16). Beige
solid, yield: 89%, Rf (AcOEt/PE 10:1)¼0.32, mp 178e180 ꢁC; IR (KBr,
6.2. Inhibition growth assay
cmꢀ1) 1678; 1H NMR (CDCl3):
d
7.97 (s, 1H, 2AreH), 7.65 (d, 1H,
Cancer cells and human lymphocytes were grown for 24 h on
96-well plates at a density of 6e8ꢂ103 cells/well and 8ꢂ105 cells/
well, respectively. Then the cells were treated with the tested
compounds for 72 h. After the treatment MTT dye dissolved in PBS
J¼2.9, 8AreH), 7.63 (d, 1H, J¼9.0, 5AreH), 7.40e7.38 (m, 1H,
7AreH), 7.35e7.29 (m, 5H, BneH), 5.20 (s, 2H, CH2), 3.89e3.88 (m,
4H, MOR), 3.29e3.27 (m, 4H, MOR); 13C NMR (CDCl3):
d 161.1, 150.3,
143.8,141.6,136.0,129.0,128.5,128.2,128.0,123.3,123.0,109.6, 66.7,
49.6, 48.9; HRMS (ESI-LC/MS) m/z calcd for C19H19N3O2: 321.1477;
found [M]þ: 321.1479.
was added to each plate well (A549: 40
and HT29: 20 l, 5 mg/mL) for 4 h. Purple crystals formed in cancer
cells after reduction of MTT were dissolved by DMSO (100 L/well)
after removal of the medium. In the case of lymphocytes it was
done by adding 100 L of 20% DMF and 50% SDS mixture to each
mL, 3 mg/mL; lymphocytes
m
m
5.9.2. 3-Benzyl-6-[4-(2-fluorophenyl)piperazin-1-yl]quinazolin-
4(3H)-one (17). Pale yellow solid, yield: 88%, Rf (AcOEt/PE 10:1)¼
0.48, mp 164e165 ꢁC; IR (KBr, cmꢀ1) 1660, 1231; 1H NMR (CDCl3):
m
well for 24 h. Absorbance at 595 nm was measured with a spec-
trophotometer PowerWave XS (BioTek Instruments, Inc.).
d
7.97 (s, 1H, 2AreH), 7.73 (d, 1H, J¼2.9, 5AreH), 7.64 (d, 1H, J¼9.0,
The cell survival effect was expressed as the IC50 value, which is
the concentration of the compound required to reduce cell survival
to 50% as compared to the negative control. The experiments were
done in triplicate. All the results were presented as the meansꢃSD.
8AreH), 7.45e7.44 (m, 1H, 7AreH), 7.36e7.29 (m, 5H, BneH),
7.11e6.97 (m, 4H, 2-FPP), 5.20 (s, 2H, CH2), 3.51e3.49 (m, 4H, 2-
FPP), 3.28e3.27 (m, 4H, 2-FPP); 13C NMR (CDCl3):
d 161.1, 156.6,
155.0, 150.2, 143.7, 141.4, 139.8, 139.7, 135.9, 128.9, 128.4, 128.2,
127.9, 124.5 (d), 123.7, 122.9, 122.8 (d), 119.0 (d), 116.3, 116.1, 109.9,
6.3. MTT assay
50.4 (d), 49.6, 48.9; 19F NMR (CDCl3):
d
ꢀ122.74 to ꢀ122.78 (m);
HRMS (ESI-LC/MS) m/z calcd for C25H23FN4O: 414.1856; found
MTT assay is a quantitative colorimetric method to determine
cell proliferation after treatment with the tested compounds. It is
widely used to estimate the cytotoxic effect of chemicals on dif-
ferent types of cells. The assay is based on the reduction of the
yellow water-soluble tetrazolium MTT (3-(4,5-dimethylthiazolyl-
2)-2,5-diphenyltetrazolium bromide) by mitochondrial enzymes of
living (not dead) cells, which results in formation of an insoluble
[M]þ: 414.1860.
5.9.3. 3-Benzyl-6-(morpholin-4-yl)benzo[h]quinazolin-4(3H)-one
(18a). White solid, yield: 40%, Rf (Hex/acetone 3:1)¼0.4, mp
155e156 ꢁC; IR (KBr, cmꢀ1) 1679; 1H NMR (CDCl3):
d 8.99e8.98 (m,
1H, 10 AreH), 8.28e8.25 (m, 2H, 5,7 AreH), 7.80 (s, 1H, 2AreH),
Please cite this article in press as: Nowak, M.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.117

8
M. Nowak et al. / Tetrahedron xxx (2014) 1e8
14. (a) Mayer, J. F.; Wagner, E. C. J. Org. Chem. 1943, 8, 239e252; (b) Alexandre, F. R.;
Berecibar, A.; Wrigglesworth, R.; Besson, T. Tetrahedron 2003, 59, 1413e1419; (c)
Lehmann, H.; LaVecchia, L. Org. Process Res. Dev. 2010, 14, 650e656.
15. (a) Brzezinski, J. Z.; Epsztajn, J.; Bakalarz, A. D.; Lajszczak, A.; Malinowski, Z.
Synth. Commun. 1999, 29, 457e473; (b) Epsztajn, J.; Malinowski, Z.; Brzezinski,
J. Z.; Karzatka, M. Synthesis 2001, 14, 2085e2090; (c) Epsztajn, J.; Malinowski,
Z.; Urbaniak, P.; Andrijewski, G. Synth. Commun. 2005, 35, 179e192.
16. Clayden, J.; Frampton, C. S.; McCarthy, C.; Westlund, N. Tetrahedron 1999, 55,
14161e14184.
purple formazan product. Formazan crystals are solubilized with
organic solvent. The amount of formazan is measured spectro-
photometrically and it is directly proportional to the number of
living cells.
Acknowledgements
17. (a) Pflantz, R.; Christoffers, J. Chem. Eur. J. 2009, 15, 2200e2209; (b) Wang, Z.;
Wang, M.; Yao, X.; Li, Y.; Tan, J.; Wang, L.; Qiao, W.; Geng, Y.; Liu, Y.; Wang, Q.
Eur. J. Med. Chem. 2012, 53, 275e282; (c) Stokes, B. J.; Vogel, C. V.; Urnezis, L. K.;
Pan, M.; Driver, T. G. Org. Lett. 2010, 12, 2884e2887.
The authors are grateful to the University of Lodz for a partial
financial support.
18. Tercel, M.; Atwell, G. J.; Yang, S.; Stevenson, R. J.; Botting, K. J.; Boyd, M.; Smith,
E.; Anderson, R. F.; Denny, W. A.; Wilson, W. R.; Pruijn, F. B. J. Med. Chem. 2009,
52, 7258e7272.
19. (a) Muci, A. R.; Buchwald, S. L. Top. Curr. Chem. 2002, 219, 132e209; (b) Hartwig,
J. F. Angew. Chem., Int. Ed. 1998, 37, 2046e2067; (c) Hamann, B. C.; Hartwig, J. F.
J. Am. Chem. Soc. 1998, 120, 3694e3703.
Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2014.05.117.
20. (a) Astrazeneca A. B.; Aguila, B.; Lyne, P.; Pontz, T. US 2009/0170849 A1, Patent
Appl. No. 12/295,825, 2009; (b) Astrazeneca A. B.; Aguila, B.; Dakin, L.; Eshu-
thachan, J.; Lee, J.; Lyane, P.; Pontz, T. US 2008/0275022 A1, Patent Appl. No. 11/
569,918, 2008; (c) Letourneau, J.; Jokiel, P.; Napier, S. E.; Ho, K. K.; Ohlmeyer, M.;
McArthur, D. R.; Jeremiah, F.; Ratcliffe, P. D.; Schulz, J. US 2008/0090802 A1,
Patent Appl. No. 11/852,762, 2008; (d) Weber, C.; Demeter, A.; Szendrei, G. I.;
Greiner, I. Tetrahedron Lett. 2003, 44, 7533e7536.
References and notes
1. (a) Nanda, A. K.; Ganguli, S.; Chakraborty, R. Molecules 2007, 12, 2413e2426; (b)
Ilangovan, P.; Ganguly, S.; Pandi, V. J. Pharm. Res. 2010, 3, 703e706; (c)
Vijaychand, A.; Manjula, S. N.; Bharath, E. N.; Divya, B. Int J Pharm. Bio. Sci. 2011,
2, 780e809; (d) Kurogi, Y.; Inoue, Y.; Tsutsumi, K.; Nakamura, S.; Nagao, K.;
Yoshitsugu, H.; Tsuda, Y. J. Med. Chem. 1996, 39, 1433e1437; (e) Selvam, P.; Babu,
K.; Padamraj, R.; De Clercq, E.; Persoons, L. Afr. J. Pharm. Pharmacol. 2008, 2,
110e115.
2. Kuduk, S. D.; Chang, R. K.; DiMarco, Ch. Ng; Greshock, T. J. (Merck Sharp and
Dohme Corp.). US2012/0264761 A1, Patent Appl. No. 13/516,941, 2012.
3. Nagase, T.; Sato, N.; Kanatani, A.; Tokita, S. (Banyu Pharmaceutical Co. Ltd.).
US205/0182045 A1, Patent Appl. No. 11/058,444, 2005.
21. Dietrich, J.; Gokhale, V.; Wang, X.; Hurley, L. H.; Flynn, G. A. Bioorg. Med. Chem.
2010, 18, 292e304.
22. Ratcliffe, P. D.; Clarkson, T. R.; Jeremiah, F.; Kinnard, J.; MacLean, F. US 2012/
0208796 A1, Patent Appl. No. 13/501,587, 2012.
23. Garlapati, R.; Pottabathini, N.; Gurram, V.; Chaudhary, A. B.; Chunduri, V. R.;
Patro, B. Tetrahedron Lett. 2012, 53, 5162e5166.
24. Hesse, S.; Kirsch, G. Synthesis 2007, 10, 1571e1575.
25. (a) Surasani, R.; Kalita, D.; Rao, A. V. D.; Chandrasekhar, K. B. Beilstein J. Org.
Chem. 2012, 8, 2004e2018; (b) Yin, J.; Zhao, M. M.; Huffman, M. A.; McNamara,
J. M. Org. Lett. 2002, 4, 3481e3484; (c) Guari, Y.; Van Es, D. S.; Reek, J. N. H.;
Kamer, P. C. J.; Van Leeuwen, P. W. N. M. Tetrahedron Lett. 1999, 40, 3789e3790.
26. Guo, R. H.; Zhang, Q.; Ma, Y. B.; Luo, J.; Geng, C. A.; Wang, L. J.; Zhang, X. M.;
Zhou, J.; Jiang, Z. Y.; Chen, J. J. Eur. J. Med. Chem. 2011, 46, 307e319.
4. Muller, G. W. (Colgene Corporation, Summit, NJ 07901, US). US2007/020765,
Patent Appl. No. 07838876,6, 2007.
5. Wang, S.; Ryder, H.; Pretswell, I.; Depledge, P.; Milton, J.; Hancox, T. C.; Dale, I.;
Dangerfield, W.; Charlton, P.; Faint, R.; Dodd, R.; Hassan, S. Bioorg. Med. Chem.
Lett. 2002, 12, 571e574.
6. Hour, M. J.; Huang, L. J.; Kuo, S. C.; Xia, Y.; Bastow, K.; Nakanishi, Y.; Hamel, E.;
Lee, K. H. J. Med. Chem. 2000, 43, 4479e4487.
ꢀ
27. Kalinowska, U.; Mat1awska, K.; Che˛cinska, L.; Domaga1a, M.; Kontek, R.; Osiecka,
R.; Ochocki, J. J. Inorg. Biochem. 2005, 99, 2024e2031.
28. Vilaivan, T. Tetrahedron Lett. 2006, 47, 6739e6742.
29. Bruyneel, F.; Enaud, E.; Billottet, L.; Vanhulle, S.; Marchand-Brynaert, J. Eur. J.
Org. Chem. 2008, 72e79.
30. (a) Jahandi, F.; Tajbakhsh, M.; Golchoubian, H.; Khaksar, S. Tetrahedron Lett.
2011, 52, 1260e1264; (b) Manna, S.; Falck, J. R. J. Org. Chem. 1982, 47,
5021e5023.
31. Ikeda, M.; Ohno, K.; Takahasi, M.; Uno, T.; Tamura, Y. J. Chem. Soc., Perkin Trans. 1
1982, 741e748.
32. (a) Reddy, R. S.; Prasad, P. K.; Ahuja, B. B.; Sudalai, A. J. Org. Chem. 2013, 78,
5045e5050; (b) Reddy, S.R.; Pragati, K.P.; Ahuja, B.B.; Sudalai, A. (Council of
Scientific & Industrial Research). WO2013/105117 A1, Patent Inter. Appl. No.
PCT/IN2013/000019, 2013
7. Marzaro, G.; Dalla Via, L.; Toninello, A.; Guiotto, A.; Chilin, A. Bioorg. Med. Chem.
2011, 19, 1197e1204.
8. Kuduk, S. D.; Beshore, D. C.; DiMarco, Ch. Ng.; Greshock, T. J (Merck Sharp and
Dohme Corp.). US2011/0224198 A1, Patent Appl. No. 13/129,593, 2011.
9. Yang, Y. L.; Chang, F. R.; Wu, Y. C. Tetrahedron Lett. 2003, 44, 319e322.
10. Chakrabarty, M.; Sarkar, S.; Harigaya, Y. Synthesis 2003, 15, 2292e2294.
11. Reddy, M. S.; Ratnam, C. V. Bull. Chem. Soc. Jpn. 1985, 58, 2449e2450.
12. Marzaro, G.; Chilin, A.; Pastorini, G.; Guiotto, A. Org. Lett. 2006, 8, 255e256.
13. (a) Morita, H.; Sato, Y.; Chan, K. L.; Choo, C. Y.; Itokawa, H.; Takeya, K.; Ka-
bayashi, J. J. Nat. Prod. 2000, 63, 1707e1708; (b) Morita, H.; Sato, Y.; Chan, K. L.;
Choo, C. Y.; Itokawa, H.; Takeya, K.; Kabayashi, J. J. Nat. Prod. 2002, 65, 1748; (c)
ꢀ
Monsieurs, K.; Tapolcsanyi, P.; Loones, K. T. J.; Neumajer, G.; Dirk De Ridder, J.
ꢀ
ꢀ
A.; Goubitz, K.; Lemiere, G. L. F.; Dommisse, R. A.; Matyus, P.; Maes, B. U. W.
Tetrahedron 2007, 63, 3870e3881; (d) Timmons, C.; Wipf, P. J. Org. Chem. 2008,
73, 9168e9170.
33. Cabrera-Rivera, F. A.; Ortiz-Nava, C.; Roman-Bravo, P.; Leyva, M. A.; Escalante, J.
Heterocycles 2012, 85, 2173e2195.
Please cite this article in press as: Nowak, M.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.117