Evaluation of 3,4-dihydroquinazoline-2(1H)-thiones as inhibitors of α-MSH-induced melanin production in melanoma B16 cells

Article abstract of DOI:10.1016/j.bmc.2010.01.005

Novel 3,4-dihydroquinazoline-2(1H)-thiones (QNTs) 1 were found to be potent inhibitors of α-MSH-induced melanin production. The effect of QNTs to inhibit melanin formation in B16 melanoma cells was screened in the presence of α-MSH. In defining the mechan

Full text of DOI:10.1016/j.bmc.2010.01.005

Bioorganic & Medicinal Chemistry 18 (2010) 1555–1562
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of 3,4-dihydroquinazoline-2(1H)-thiones as inhibitors
of a-MSH-induced melanin production in melanoma B16 cells
P. Thanigaimalai a, Ki-Cheul Lee a, Seong-Cheol Bang a, Jee-Hyun Lee a, Cheong-Yong Yun b, Eunmiri Roh b,
Bang-Yeon Hwang b, Youngsoo Kim b, Sang-Hun Jung a,
*
a College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 305-764, Republic of Korea
b College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 3,4-dihydroquinazoline-2(1H)-thiones (QNTs) 1 were found to be potent inhibitors of
a-MSH-
Received 22 October 2009
Revised 30 December 2009
Accepted 5 January 2010
Available online 11 January 2010
induced melanin production. The effect of QNTs to inhibit melanin formation in B16 melanoma cells
was screened in the presence of -MSH. In defining the mechanism of activity, the effects on tyrosinase
a
activity, on tyrosinase synthesis and on the depigmentation of melanin were evaluated. QNTs did not
affect the catalytic activity of tyrosinase, but rather acted as an inhibitor of tyrosinase synthesis.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
3,4-Dihydroquinazolines
a-Melanocyte stimulating hormone
Tyrosinase
Depigmentation
1. Introduction
mia-associated transcription factor (MITF), which, in turn, plays a
pivotal role in the expression of the tyrosinase gene.7,8 Tyrosinase
Melanin is a heterogeneous biopolymer and is important in pro-
tecting the skin from the harmful effects of sunlight, toxic drugs,
and chemicals.1 Melanin absorbs harmful UV radiation and trans-
forms the energy into heat through an ultrafast internal conversion
process.2 This process keeps the light-induced generation of free
radicals at a minimum. However, abnormal deposition of melanin
pigments can cause diverse hyperpigmentary disorders, such as
melasma, freckles, and age spots.3,4
catalyses the rate-limiting steps in the biosynthetic pathway of
melanin pigments in melanocytes.9,10 This key enzyme catalyses
the hydroxylation of tyrosine into 3,4-dihydroxyphenylalanine (L-
dopa) and the subsequent oxidation to form dopaquinone, a sub-
strate for the synthesis of pheomelanins or eumelanins. Thus, con-
trolling this enzyme has been useful in treating pigmentation
disorders and in the development of cosmetic whitening agents.
Many tyrosinase inhibitors have been reported, including kojic
acid,11 arbutin,12 ascorbic acid derivatives,13 hydroxylstilbene
derivatives, like resveratol,14,15 and genistic acid methyl ester,16,17
and the hypopigmenting effects of fatty acids.18 Because of several
recently reported adverse effects,19 such as cytotoxicity, skin can-
cer, and dermatitis, kojic acid, a frequently used skin whitening
agent, has been banned for cosmetic use in many countries. Addi-
tionally, compounds that inhibit other melanogenesis signaling
proteins have been discovered, including linoleic acid and terrain,
which inhibit melanogenesis by down-regulation of tyrosinase
expression without changing tyrosinase activity.20,21
The production of melanin in the melanocytes of hair and skin is
strictly controlled by the melanocortin 1 receptor (MC1R), the
activity of which is regulated positively by
ing hormone ( -MSH) and negatively by agouti signal protein
(ASP).5,6 Stimulation of MC1R by
-MSH results in the activation
a-melanocyte stimulat-
a
a
of adenylyl cyclase and the production of cAMP, which in turn af-
fects the pathways of cAMP-responsive element-binding protein
(CREB), MAP kinase, and P13 kinase, resulting in the formation of
melanin. Among the steps in this pathway, the CREB component
has attracted considerable attention. cAMP leads to the phosphor-
ylation of CREB transcriptional factors, which induces microphthal-
We attempted to identify, by a random screening process, com-
pounds that inhibited
a-MSH-stimulated melanogenesis in B16
melanoma cells. The potent hypopigmentation activity of 6-
Abbreviations: QNTs, 3,4-dihydroquinazolines-2(1H)-thiones;
nocyte stimulating hormone.
* Corresponding author at present address: College of Pharmacy and Institute of
Drug Research and Development, Chungnam National University, Daejeon 305-764,
Republic of Korea. Tel.: +82 42 821 5939; fax: +82 42 823 6566.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
a-MSH, a-mela-
methyl-3-phenethyl-3,4-dihydro-1H-quinazoline-2-thione (Fig. 1,
1a, IC50 = 0.8 l
M) was found as a result of these efforts.22 The po-
tency of 1a is approximately 80 and 200 times greater than that
of kojic acid and arbutin, respectively. Thus, the current study fo-
cused on the design and synthesis of its analogs for use as novel
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.005

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P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1555–1562
Table 1
The substituents of 7 and 8
N
Compd
R1
R2
n
Compd
R1
R2
N
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
6-CH3
7-CH3
5-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-Cl
Pha
Ph
Ph
4-CH3Ph
4-iPrPhb
4-ClPh
2,4-Cl2Ph
Ph
1
1
1
1
1
1
1
2
3
1
1
1
1
1
2
3
1
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
6-CH3
7-CH3
5-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-Cl
Pha
Ph
Ph
4-CH3Ph
4-iPrPhb
4-ClPh
2,4-Cl2Ph
Ph
1
1
1
1
1
1
1
2
3
1
1
1
1
1
2
3
1
N
H
S
Figure 1. 6-Methyl-3-phenethyl-3,4-dihydro-1H-quinazoline-2-thione (1a, IC50
0.8 M).
=
l
whitening agents and as a means of determining structure–activity
relationships and mechanism(s) of the observed activity.
Ph
Ph
cHx
cHx
Npc
Npc
2. Chemistry
Ph
Ph
6-Cl
6-Cl
6-Cl
6-Cl
4-CH3Ph
4-ClPh
Ph
6-Cl
6-Cl
6-Cl
6-Cl
4-CH3Ph
4-ClPh
Ph
Target compounds 1a–q were prepared using the synthetic
pathway in Scheme 1. The alkylation of amino groups in 3 was con-
ducted with two different reactions. Direct alkylation of 3a–i with
o-nitro benzyl chlorides 2a–d yielded key intermediates 5a–k.22
Intermediates 5l–q were obtained by reductive amination of 3a,
3b, 3e, 3f, 3g, or 3h with 2-nitro-5-chlorobenzaldehyde (4).23,24
The reduction of nitro compounds 5a–q with 10% Pd/C under a
30-psi hydrogen atmosphere at room temperature for 5 h yielded
intermediates 6a–q,22 which were used without further purifica-
tion. The subsequent cyclization of 6a–q was performed with car-
bonyldiimidazole in the presence of triethylamine to yield
quinazolones (QNO) 7a–q.22 Resulting data concerning the prepa-
ration of 7 are summarized in Table 1. Finally these were converted
to quinazoline-2-thiones (QNT) 1a–q, as shown in Table 1, by
refluxing overnight with Lawesson’s reagent in toluene.25 The
synthesized compounds were purified by flash column chromatog-
raphy. All QNOs and QNTs exhibited Clog [P] values of <5.8, as
calculated by ChemDraw (v. 9.0) and were fully characterized by
spectral analyses.
Ph
Ph
6-Cl
cHxd
6-Cl
cHxd
a
b
c
Ph = phenyl.
iPr = isopropyl.
Np = 1-naphthyl.
cHx = cyclohexyl.
d
Table 2. The amount of melanin released into the culture media
was determined by measuring sample absorbance at 405 nm
against a synthetic melanin standard15 (Fig. 2A and B).
The tyrosinase activity was defined as the rate of conversion of
1 nmol of dopa to dopachrome/min. The dopa oxidation of cell-free
tyrosinase was measured spectrophotometrically in the presence
of QNT 1g (Fig. 2C).
The cells extracted were subjected to Western blot analysis
with an anti-tyrosinase antibody and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were used as an internal control (Fig. 2D).
The optical densities were measured at 590 nm after reacting with
MTT solution (Fig. 2E).
3. Pharmacology
4. Results and discussion
For QNT 1a–q and QNO 7a–q, the ability to inhibit melanin
formation in B16 melanoma cells was determined in the presence
The activity of both QNOs (7) and QNTs (1) are summarized in
of
a
-MSH (100 nM) throughout three-day incubation as shown in
Table 2 with the mean value obtained from 3 to 5 independent
Cl
NO2
NH2
( )n
+
a
R2
( )n
R1
R2
N
H
NO2
5a-q
R1
3a, R2 = Ph, n =1
3b, R2 = 4-MePh, n =1
3c, R2= 4-iPrPh, n =1
3d, R2 = 4-ClPh, n =1
3e, R2 = 2,4-Cl2Ph, n =1
3f, R2 = Ph, n =2
3g, R2 = Ph, n =3
3h, R2 = cHx
2a, R1 = 5-CH3
2b, R1 = 6-CH3
2c, R1 = 4-CH3
2d, R1 = 4,5-O-CH2-O
b
3i, R2 = Np
O
Cl
H
3a, 3b, 3e, 3f, 3g or 3h
+
c
NO2
4
R2
( )n
R2
( )n
R2
( )n
N
H
NH2
N
N
R1
R1
d
R1
e
N
H
S
N
H
O
6a-q
7a-q
1a-q
Scheme 1. Synthesis of compounds 1. Reagents and conditions: TEA, CH2Cl2, reflux, overnight; (b) TEA, NaBH4, methanol, reflux, 4 h; (c) 10%Pd/C, 30 psi, rt, 5 h; (d) 1,1-
carbonyl diimidazole, THF, reflux, overnight; (e) Lawesson’s reagent, toluene, reflux, overnight. Substituents for 7a–q and 1a–q are listed in Table 1.

P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1555–1562
1557
Table 2
In vitro activity of compounds 7 and 1 against
a-MSH-induced melanin production in B16 cells
Compd
% Inhibition at 10
lM
IC50
(
lM)
Clog Pa
Compd
% Inhibition at 10
lM
IC50
(l
M)
Clog Pa
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
Kojic acid
16
24
18
10
6
15
11
22
38
17
5
18
6
7
16
17
3
>10
>10
9.8
3.629
3.629
3.629
4.128
5.056
4.342
5.055
4.008
4.537
4.711
4.803
4.145
4.644
4.858
4.522
5.053
5.227
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
Arbutin
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
96
0.8
0.8
1.1
2.7
0.5
4.3
0.5
3.9
0.9
0.8
2.4
2.8
5.5
1.6
0.8
5.5
4.3
180
4.289
4.289
4.289
4.788
5.716
5.002
5.715
4.668
5.197
5.371
5.463
4.503
5.002
5.216
4.882
5.411
5.585
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
70
>100
a
Clog P values were calculated by Chemdraw ver. 9.0.
experiments. The IC50 of QNTs 1 ranged from 0.5 to 5.5
eral, the activity of QNOs 7a–q (IC50: >10
l
M. In gen-
1j and 1q. Compound 1j (IC50: 0.8
of activity as 1a, and the activity of 1q (IC50: 4.3 l
l
M) exhibited the same level
M) was similar
l
M) was very weak. Thus,
the thiourea moiety of QNTs appeared to be key for the activity of
1a–p. This trend had been noticed in the activity of phenylthiourea
to that of 1l. Thus, planarity of the phenyl moiety of 1a is appar-
ently not required for activity and likely does not participate in
binding of receptor molecules. Thus, the N-phenyl alkyl groups of
QNT 1 may act as controllers of lipophilicity. Although Clog P val-
ues were much changed with introduction of substituents on 1a,
the variation in activity is not much recognized by increasing the
lipophilicity of these thioureas as indicated in Table. 2. For in-
(PTU, 58% inhibition at 100
100
M).26,27 Although the mechanism of PTU activity (IC50 value
for tyrosinase inhibition: 1.8 M) was quite different from that of
lM) and phenyl urea (0% inhibition at
l
l
QNTs 1a–p, as discussed later. Because the thiourea moiety of
PTU is important for complexing the copper ion in tyrosinase, it
was originally considered that the same mechanistic pathway ex-
isted for QNTs. However, this was not the case. The role of the thio-
urea moiety in QNT 1 is unclear.
stance, compounds 1e (IC50: 0.5
0.5 M, Clog P = 5.715) showed comparable activity to that of 1a
(IC50: 0.8 M, Clog P = 4.429). In addition to that, lipophilicity did
not differentiate between the activity of urea 7a–q and of thiourea
1a–q, considering the Clog P values and the activity of 7q (IC50
>10 M, Clog P = 5.227) and 1a (IC50 0.8 M, Clog P = 4.289).
lM, Clog P = 5.716) and 1g (IC50:
l
l
Positioning the methyl group at 5, 6, or 7 on quinazoline ring of
1 did not alter its activity, as demonstrated with 1a (IC50: 0.8
lM),
:
1b (IC50: 0.8 M), and 1c (IC50: 1.1 M), respectively. Replacement
l
l
l
:
l
of the 6-methyl group with a chlorine substituent on the quinazo-
line ring of QNT resulted in a slight variation in activity, as indi-
cated by a comparison of 1a–j and 1l–q. This demonstrates that
electronic effects on the quinazoline ring of 1 are detrimental to
QNT activity and may indicate that small substituents on the qui-
nazoline ring do not affect QNT activity.
Therefore lipophilicity is not critical factor for the activity of QNT 1.
In defining the mechanism of activity, the effects on tyrosinase
activity, on tyrosinase synthesis upon stimulation with a-MSH, and
on the depigmentation of melanin were evaluated. Additionally,
the effects of tyrosinase on melanin production were studied dur-
ing
inhibitory effect on melanin production in B16 cells stimulated
with -MSH, an elevator of intracellular cAMP concentration. The
amounts of melanin pigments were quite low in the resting cells
(2 g/mL) but markedly increased, to 44 g/mL upon expo-
sure to -MSH for 72 h (Fig. 2A). QNT 1g dose dependently inhib-
ited -MSH-induced melanin production, with an IC50 value of
1.2 M (Fig. 2A, B). Arbutin, a well-known skin whitener, also
inhibited the production of -MSH-induced melanin, with an IC50
value of 190 M (Fig. 2B).
a-MSH stimulation with compound 1g. QNT 1g showed an
Comparing the relative activities of 1a (IC50: 0.8
lM), 1d (IC50:
2.7
l
M), 1e (IC50: 2.7 M), and 1f (IC50: 4.3 M) reveals that substi-
l
l
a
tution at the 4-position of the phenyl group of 1a with methyl,
chloro, or isopropyl substituents decreased the activity slightly.
This trend was also apparent in the activity of the chloro-QNT ser-
6
l
4 l
a
ies 1l, 1m, and 1n. However, compound 1g (IC50: 0.5
l
M), with a
a
l
2,4-dichloro substitution, retained the same level of activity as
1a. To determine the optimum size of the aromatic group in 1a, a
1-naphthyl group was substituted for the phenyl group, as shown
a
l
in 1k (IC50: 2.4
lM) with a corresponding decrease in activity. The
To define the mode of hypopigmentation activity, the ability of
introduction of substituents or enlargement of the aromatic phenyl
group in 1a was thus not beneficial for QNT activity.
QNT 1g to influence the catalytic activity of cell-free tyrosinase,
prepared from B16 cells stimulated with only
a-MSH, was first
Elongation of the methylene unit connecting the phenyl and
quinazoline moieties from two to three units reduced the activity,
investigated. The dopa oxidation velocity in the presence of QNT
1g was measured as an indicator of tyrosinase catalytic activity.
Compound 1g did not significantly inhibit the catalytic activity of
cell-free tyrosinase (Fig. 2C). Western blot analysis was then per-
as indicated by comparison between compounds 1a and 1h (IC50
:
:
3.9
0.9
l
M). However, one additional methylene unit, as in 1i (IC50
l
M) resulted in the original level of activity. This trend was re-
M) was
M) was less
formed to determine the level of tyrosinase. Upon exposure to
MSH alone, levels of tyrosinase in the cells markedly increased
from the baseline levels (Fig. 2D). QNT 1g suppressed -MSH-in-
a-
versed in the chloro-QNT series. Compound 1o (IC50: 0.8
more potent than 1l (IC50: 2.8 M), but 1p (IC50: 5.5
l
l
l
a
active. Thus, elongation of methylene unit in 1a only marginally af-
fected activity.
To evaluate the necessity of phenyl group planarity in 1a, a
bulkier cyclohexyl group was introduced as shown in compounds
duced levels of tyrosinase in a dose-dependent manner (Fig. 2D).
However, at concentrations effective for hypopigmentation activ-
ity, compound 1g did not affect the viability of B16 cells (Fig. 2E),
ruling out non-specific cytotoxicity. Thus, QNT 1g is an efficient

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P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1555–1562
Figure 2. The effects of QNT 1g on
a-MSH-induced melanin production in melanoma B16 cells and its mode of action. (A) The cells were stimulated with a-MSH for 72 h in
the presence of QNT 1g. Melanin content was determined by measuring absorbance at 405 nm against a synthetic melanin standard. Data are expressed as the mean SD
from three independent experiments. #P <0.05, versus the media alone-treated group. *P <0.05, versus the
-MSH-treated group. (B) The effects of QNT 1g (solid circle) or
arbutin (open circle) on -MSH-induced melanin production are also shown in terms of percent inhibition. (C) Enzyme sources of tyrosinase were prepared from cells
stimulated with -MSH (10 nM) for 48 h and diluted to a specific activity of 80,000 U/mg of protein, in which one unit of tyrosinase activity was defined as the conversion of
1 nmol dopa to dopachrome/min. The dopa oxidation velocity of cell-free tyrosinase (50 g) was spectrophotometrically measured in the presence of QNT 1g. (D) The cells
were stimulated with -MSH for 48 h in the presence of QNT 1g. Cell extracts were subjected to Western blot analysis with an anti-tyrosinase antibody, and glyceraldehyde-
a
a
a
l
a
3-phosphate dehydrogenase (GAPDH) were used as an internal control. (E) The cells were treated with various concentrations of QNT 1g for 72 h in the presence of a-MSH.
After reacting with MTT solution for 2 h, optical densities were measured at 590 nm.
inhibitor of
a-MSH-induced melanin production and suppressed
unusual and potent mechanism suggests that QNTs may be prom-
tyrosinase levels without affecting the catalytic activity of the en-
zyme. This result was unexpected considering the well-known
tyrosinase inhibitor PTU, in which the thiourea moiety plays a
key role in complexing the copper ion in the active site of tyrosi-
nase, thereby blocking enzyme activity.28
ising whitening agents. Further exploration of the mechanism of
QNT activity in depigmentation is in progress.
6. Materials and methods
6.1. Chemistry
5. Conclusion
Melting points (mp) were determined on Electro thermal 1A
9100 MK2 apparatus and are uncorrected. All commercial chemi-
cals were used as obtained and all solvents were purified by the
standard procedures prior to use. Thin-layer chromatography
was performed on E Merck Silica Gel GF-254 precoated plates
and the identification was done with UV light and colorization
with spray 10% phosphomolybdic acid followed by heating. Flash
column chromatography was performed with E Merck Silica Gel
(230–400 mesh). A FT-IR spectrum was recorded with Nicolet-
380 models. NMR spectra were measured against the peak of
In summary, novel QNTs 1 were prepared and identified as po-
tent inhibitors of
a-MSH-induced melanin production. Although
QNOs 7 are structurally very similar to QNTs 1, these compounds
were nearly inactive. Thus, 6-methyl-3-phenylalkyl-3,4-dihydro-
quinazoline-2(1H)-thione was shown to be an essential scaffold
for inhibitory activity of melanin production from B16 melanoma
cells stimulated by a-MSH. Although the mechanism of QNT activ-
ity requires further exploration, QNTs were shown to block the for-
mation of tyrosinase, without affecting tyrosinase activity. This

P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1555–1562
1559
tetramethylsilane by Varian Unity Inova 400 NMR (400 MHz) spec-
trometers. High resolution mass spectrum (HRMS) was recorded
on API2000 mass spectrometer (PE Sciex, Toronto, Canada).
2H), 5.46 (br s, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.39 (s, 1H), 7.91 (d,
J = 8.4 Hz,1H).
6.1.1.11. N-(5-Methyl-2-nitrobenzyl)-2-(naphthalen-2-yl)eth-
anamine (5k). Yellow oil; yield 70%; 1H NMR (CDCl3): d 2.35 (s,
3H), 3.02 (s, 4H), 4.08 (s, 2H), 7.14 (d, J = 8.0 Hz, 1H), 7.31–7.47
(m, 4H), 7.62 (s, 1H), 7.71–7.81 (m, 3H), 7.86 (d, J = 8.4 Hz, 1H).
6.1.1. General procedure for the synthesis of compounds 5 (a–k)
To a corresponding benzyl chloride (1 equiv) in methylene chlo-
ride (50 mL) was added the corresponding amines (1.5 equiv) and
followed by triethylamine (3 equiv) Then the reaction mixture was
allowed to stir for overnight at 55 °C. After the reaction was com-
plete; the reaction mixture was cooled to room temperature and
washed with brine. The organic layer was dried over anhydrous
Na2SO4 and concentrated under vacuum. The product was isolated
by flash column chromatography using a mixture of 10–15% ethyl
acetate in hexane as an eluent.
6.1.2. General procedure for the synthesis of compounds 5 (l–q)
A mixture of corresponding amine (1 equiv), aldehyde (1 equiv)
and triethylamine (2 equiv) was taken in ethanol (50 mL), and
cooled to 0 °C. To the reaction mixture was added appropriate
amine and allowed to reflux for 4 h. The mixture was cooled to
0 °C and added NaBH4 portion wise over 10 min. The resulting
solution was neutralized with 4 M hydrochloric acid (5 mL) and ex-
tracted with diethyl ether (50 mL). The ethereal phase was dis-
carded and aqueous phase was neutralized with solid NaHCO3,
and extracted with ethyl acetate (200 mL), dried over Na2SO4, con-
centrated to produce an appropriate compound which was purified
by flash column chromatography.
6.1.1.1. N-(5-Methyl-2-nitrobenzyl)-2-phenylethanamine (5a).
Yellow oil; yield 80%; 1H NMR (CDCl3) d 2.35 (s, 3H), 2.81 (t,
J = 6.8 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H), 3.98 (s, 2H), 7.01–7.31 (m,
6H), 7.35 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H).
6.1.1.2. N-(4-Methyl-2-nitrobenzyl)-2-phenylethanamine (5b).
Yellow oil; yield 85%; 1H NMR (CDCl3) d 2.40 (s, 3H), 2.83 (t,
J = 6.4 Hz, 2H), 2.88 (t, J = 6.4 Hz, 2H), 4.00 (s, 2H), 7.19–7.44 (m,
7H), 7.75 (s, 1H).
6.1.2.1. N-(5-Chloro-2-nitrobenzyl)-2-phenylethanamine (5l).
Yellow oil; yield 65%; 1H NMR (CDCl3) d 2.81 (t, J = 6.8 Hz, 2H),
2.91 (t, J = 6.8 Hz, 2H), 4.06 (s, 2H), 7.20–7.36 (m, 6H), 6.56 (s, 1H),
7.91 (d, J = 8.0 Hz, 1H).
6.1.1.3. N-(2-Methyl-6-nitrobenzyl)-2-phenylethanamine (5c).
Yellow oil; yield 80%; 1H NMR (CDCl3) d 2.46 (s, 3H), 2.83 (t,
J = 7.2 Hz, 2H), 2.97 (t, J = 7.2 Hz, 2H), 3.78 (s, 2H), 7.18–7.31 (m,
6H), 7.38 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H).
6.1.2.2. N-(5-Chloro-2-nitrobenzyl)-2-p-tolylethanamine (5m).
Yellow oil; yield 68%; 1H NMR (CDCl3) d 2.32 (s, 3H), 2.77 (t,
J = 6.8 Hz, 2H), 2.94 (t, J = 6.4 Hz, 2H), 4.05 (s, 2H), 7.08–7.10 (s,
4H), 7.33 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H).
6.1.1.4. N-(5-Methyl-2-nitrobenzyl)-2-p-tolylethanamine (5d).
Yellow oil; yield 73%; 1H NMR (CDCl3) d 2.34 (s, 3H), 2.42 (s,
3H), 2.77 (t, J = 6.8 Hz, 2H), 2.90 (t, J = 6.4 Hz, 2H), 4.02 (s, 2H),
7.09 (s, 4H), 7.15 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.89 (d,
J = 8.4 Hz,1H).
6.1.2.3. N-(5-Chloro-2-nitrobenzyl)-2-(4-chlorophenyl)ethan-
amine (5n). Yellow oil; yield 61%; 1H NMR (CDCl3) d 2.77 (t,
J = 6.8 Hz, 2H), 2.88 (t, J = 6.8 Hz, 2H), 4.05 (s, 2H), 7.13 (d,
J = 8.4 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H),
7.61 (s, 1H), 7.91 (d, J = 8.8 Hz, 1H).
6.1.1.5. 2-(4-Isopropylphenyl)-N-(5-methyl-2-nitrobenzyl)eth-
anamine (5e). Yellow oil; yield 68%; 1H NMR (CDCl3) d 1.23 (d,
J = 4.0 Hz, 6H), 2.43 (s, 3H), 2.86 (t, J = 6.8 Hz, 2H), 2.84 -2.89 (m,
3H), 4.04 (s, 2H), 6.99- 7.17 (m, 5H), 7.34 (s, 1H), 7.90 (d,
J = 8.4 Hz, 1H).
6.1.2.4. N-(5-Chloro-2-nitrobenzyl)-3-phenylpropan-1-amine
(5o). Yellow oil; yield 67%; 1H NMR (CDCl3) d 1.81–1.88 (m, 2H),
2.65–2.70 (m, 4H), 4.00 (s, 2H), 7.16 (m, 3H), 7.26 (t, J = 8.4 Hz,
2H), 7.35 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 7.92 (d, J = 8.4 Hz, 1H).
6.1.1.6. 2-(4-Chlorophenyl)-N-(5-methyl-2-nitrobenzyl)ethan-
amine (5f). Yellow oil; yield 79%; 1H NMR (CDCl3) d 2.43 (s, 3H),
2.80 (t, J = 6.8 Hz 2H), 2.91 (t, J = 6.4 Hz, 2H), 4.02 (s, 2H), 7.04 (d,
J = 8.0 Hz, 1H), 7.10–7.32 (m, 5H), 7.35 (s, 1H), 7.90 (d, J = 8.0 Hz,
1H).
6.1.2.5. N-(5-Chloro-2-nitrobenzyl)-4-phenylbutan-1-amine
(5p). Yellow oil; yield 58%; 1H NMR (CDCl3) d 1.51–1.71 (m, 4H),
2.61 (m, 4H), 4.02 (s, 2H), 7.16–7.48 (m, 6H), 7.68 (s, 1H), 7.92
(d, J = 8.4 Hz, 1H).
6.1.1.7. 2-(2,4-Dichlorophenyl)-N-(5-methyl-2-nitrobenzyl)eth-
anamine (5g). Yellow oil; yield 73%; 1H NMR (CDCl3) d 2.41 (s,
3H), 2.86–2.94 (m, 4H), 4.08 (s, 1H), 7.07 (s, 2H), 7.08 (s, 1H),
7.34 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 1H).
6.1.2.6. N-(5-Chloro-2-nitrobenzyl)-2-cyclohexylethanamine (5q).
Yellow oil; yield 74%; 1H NMR (CDCl3) d 1.54–1.90 (m, 11H),
2.14 (t, J = 6.8 Hz, 2H), 2.71 (t, J = 6.8 Hz, 2H), 4.05 (s, 2H), 5.47 (s,
1H), 7.36 (d, J = 8.4 Hz, 1H), 7.71 (s, 1H), 7.95 (d, J = 8.8 Hz, 1H).
6.1.1.8. N-(5-Methyl-2-nitrobenzyl)-3-phenylpropan-1-amine
(5h). Yellow oil; yield 75%; 1H NMR (CDCl3) d 1.81–1.87 (m, 2H),
2.42 (s, 3H), 2.67–2.75 (m, 4H), 3.99 (s, 2H), 7.09–7.29 (m, 6H),
7.36 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H).
6.1.3. General procedure for the synthesis of compounds 7 (a–
q)
To the amine compound (1 equiv) in tetrahydrofuran (50 mL)
was added CDI (1.2 equiv) followed triethylamine (2 equiv) under
nitrogen atmosphere and this mixture was allowed to reflux for
overnight. After disappearance of starting material, the mixture
was neutralized with 2 N HCl, extracted with methylene chloride
(100 mL) and dried over anhydrous sodium sulfate. The pure prod-
uct was isolated by flash column chromatography using 30–40%
ethyl acetate in hexane. In order to determine the purity, all com-
pounds were tested HPLC analysis using C18 reverse phase column
by the mobile phase of methanol and acetonitrile (1:1).
6.1.1.9. N-(5-Methyl-2-nitrobenzyl)-4-phenylbutan-1-amine (5i).
Yellow oil; yield 79%; 1H NMR (CDCl3) d 1.52–1.70 (m, 4H), 2.41
(s, 3H), 2.57–2.67 (m, 4H), 3.98 (s, 1H), 7.16 (d, J = 6.8 Hz, 4H), 7.26
(t, J = 7.6 Hz, 2H), 7.37 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H).
6.1.1.10. N-(5-Methyl-2-nitrobenzyl)-2-cyclohexylethanamine
(5j). Yellow oil; yield 83%; 1H NMR (CDCl3) d 1.5–1.99 (m, 11H),
2.13 (t, J = 6.8 Hz, 2H), 2.41 (s, 3H), 2.72 (t, J = 6.8 Hz, 2H), 4.00 (s,

1560
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6.1.3.1. 6-Methyl-3-phenethyl-3,4-dihydroquinazolin-2(1H)-one
(7a). Brown solid; yield 64%; purity 97.55%; mp 165–167 °C; IR
1.65–1.71 (m, 4H), 2.24 (s, 3H), 2.65 (t, J = 7.2 Hz, 2H), 3.44 (t,
J = 7.2 Hz, 2H), 4.36 (s, 2H), 6.52 (d, J = 8.4 Hz, 1H), 6.65 (br s,
1H), 6.82 (s, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.96–7.28 (m, 5H); HRMS
calcd for C19H22N2O m/z 294.1732, found 294.1721.
(neat) 3021, 3084, 1650, 1506 cmÀ1 1H NMR (CDCl3) d 2.25 (s,
;
3H), 2.94 (t, J = 7.2 Hz, 2H), 3.62 (t, J = 6.8 Hz, 2H), 4.30 (s, 2H),
6.53 (d, J = 8.0 Hz, 1H), 6.71 (br s, 1H), 6.76 (s, 1H), 6.94 (d,
J = 8.4 Hz, 1H), 7.19–7.31 (m, 5H); HRMS calcd for C17H18N2O m/z
266.1419, found 266.1408.
6.1.3.10. 6-Methyl-3-(2-cyclohexylethyl)-3,4-dihydroquinazo-
lin-2(1H)-one (7j). White solid; yield 69%; purity 97.5%; mp 149–
151 °C; IR (neat) 3048, 2915, 2847, 1665, 1501 cmÀ1 1H NMR
;
6.1.3.2. 7-Methyl-3-phenethyl-3,4-dihydroquinazolin-2(1H)-one
(7b). Brown solid; yield 68%: purity 96.93%; mp 161–164 °C; IR
(CDCl3) d 0.92–1.77 (m, 13H), 2.27 (s, 3H), 3.46 (t, J = 7.6 Hz, 2H),
4.38 (s, 2H), 6.53 (d, J = 8.0 Hz, 1H), 6.65 (br s, 1H), 6.85 (s, 1H),
6.9 (d, J = 8.0 Hz, 1H); HRMS calcd for C17H24N2O m/z 272.1889,
found 272.1876.
(neat) 3200, 3024, 1662, 1598, 1525 cmÀ1 1H NMR (CDCl3) d
;
2.29 (s, 3H), 2.95 (t, J = 7.6 Hz, 2H), 3.65 (t, J = 7.6 Hz, 2H), 4.31 (s,
2H), 6.48 (s, 1H), 6.73 (d, J = 7.6 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H),
7.00 (br s, 1H), 7.22–7.31 (m, 5H); HRMS calcd for C17H18N2O m/z
266.1419, found 266.1409.
6.1.3.11. 6-Methyl-3-(2-(naphthalen-2-yl) ethyl)-3,4-dihydroqui-
nazolin-2(1H)-one (7k). Yellow solid; yield 57%; purity 95.08%;
mp 179–181 °C; IR (neat) 2959, 2851, 1663, 1514 cmÀ1 1H NMR
;
6.1.3.3. 5-Methyl-3-phenethyl-3,4-dihydroquinazolin-2(1H)-one
(7c). Brown solid; yield 68%; purity 95.12%; mp 158–160 °C; IR
(CDCl3) d 2.40 (s, 3H, CH3), 3.14 (t, J = 7.2 Hz, 2H), 3.73 (t,
J = 6.8 Hz, 2H), 4.99 (s, 2H), 7.034 (d, J = 8.4 Hz, 1H), 7.40–7.52
(m, 3H), 7.60 (br s, 1H), 7.67–7.84 (m, 5H), 8.05 (d, J = 8.4 Hz,
1H); HRMS calcd for C21H20N2O m/z 316.1576, found 316.1552.
(neat) 3203, 3056, 1655, 1611, 1455 cmÀ1 1H NMR (CDCl3) d
;
2.08 (s, 3H), 2.96 (t, J = 7.6 Hz, 2H), 3.68 (t, J = 7.6 Hz, 2H), 4.26 (s,
2H), 6.49 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 7.6 Hz, 1H), 6.98 (br s,
1H), 7.04 (t, J = 8.0 Hz, 1H), 7.24–7.29 (m, 5H); HRMS calcd for
C17H18N2O m/z 266.1419, found 266.1408.
6.1.3.12. 6-Chloro-3-phenethyl-3,4-dihydroquinazolin-2(1H)-
one (7l). Yellow solid; yield 56%; purity 95.01%; mp 141–142 °C;
IR (neat) 3290, 2924, 2856, 1665, 1604, 1493 cmÀ1 1H NMR
;
6.1.3.4. 6-Methyl-3-(4-methylphenethyl)-3,4-dihydroquinazolin-
(CDCl3) d 2.95 (t, J = 7.6 Hz, 2H), 3.65 (t, J = 7.6 Hz, 2H), 4.35 (s,
2H), 6.59 (d, J = 8.4 Hz, 1H), 6.89 (br s, 1H), 6.91 (m, 2H), 7.10–
7.30 (m, 5H); HRMS calcd for C16H15ClN2O m/z 286.0873, found
286.0856.
2(1H)-one (7d). Yellow solid; yield 73%; purity 97.07%; mp 132–
134 °C; IR (neat) 2924, 2857, 1659, 1503 cmÀ1 1H NMR (CDCl3) d
;
2.33 (s, 3H), 2.63 (s, 3H), 2.89 (t, J = 7.2 Hz, 2H), 3.62 (t, J = 7.2 Hz,
2H), 4.31 (s, 2H), 6.53 (d, J = 8.0 Hz, 1H), 6.67 (br s, 1H, NH), 6.73
(s, 1H), 6.94 (d, J = 8.0 Hz, 1H), 7.13 (dd, J = 8.0, 8.0 Hz, 4H); HRMS
calcd for C18H20N2O m/z 280.1576, found 280.1569.
6.1.3.13. 6-Chloro-3-(4-methylphenethyl)-3,4-dihydroquinazo-
lin-2(1H)-one (7m). Yellow solid; yield 72%; purity 97.62%; mp
156–158 °C; IR (neat) 3059, 2925, 2856, 1660, 1605 cmÀ1 1H
;
6.1.3.5. 3-(4-Isopropylphenethyl)-6-methyl-3,4-dihydroquinaz-
NMR (CDCl3) d 2.31 (s, 3H), 2.89 (t, J = 7.6 Hz, 2H), 3.63 (t,
J = 7.6 Hz, 2H), 4.32 (s, 2H), 6.62 (d, J = 8.0 Hz, 1H), 6.80 (br s,
1H), 6.86–6.97 (m, 2H), 7.08 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.0 Hz,
2H); HRMS calcd for C17H17ClN2O m/z 300.1029, found 300.1013.
olin-2(1H)-one (7e). Yellow solid; yield 79%; purity 97.65%; mp
143–145 °C; IR (neat) 3171, 2926, 2856, 1655, 1456 cmÀ1 1H
;
NMR (CDCl3) d 1.24 (d, J = 6.8 Hz, 6H), 2.25 (s, 3H), 2.88 (m, 3H),
3.61 (t, J = 7.6 Hz, 2H), 4.30 (s, 2H), 6.56 (d, J = 8.0 Hz, 1H), 6.74
(s, 1H), 6.96 (d, J = 8.4 Hz, 1H), 7.26 (m, 4H), 7.32 (br s, 1H); HRMS
calcd for C20H24N2O m/z 308.1889, found 308.1878.
6.1.3.14. 6-Chloro-3-(4-chlorophenethyl)-3,4-dihydroquinazo-
lin-2(1H)-one (7n). Yellow solid; yield 56%; purity; 95.02%; mp
139–140 °C; IR (neat) 3190, 2926, 1663, 1595 cmÀ1 1H NMR
;
6.1.3.6. 3-(4-Chlorophenethyl)-6-methyl-3,4-dihydroquinazolin-
(CDCl3) d 2.83 (t, J = 7.2 Hz, 2H), 3.84 (t, J = 7.2 Hz, 2H), 4.35 (s,
2H), 6.63 (d, J = 8.0 Hz, 1H), 6.74 (br s, 1H), 6.89–6.96 (m, 2H),
7.10–7.29 (m, 5H); HRMS calcd for C16H14Cl2N2O m/z 320.0483,
found 320.0466.
2(1H)-one (7f). Yellow solid; yield 63%; purity 96.0%; mp 140–
142 °C; IR (neat) 3193, 2923, 2855, 1660, 1506 cmÀ1 1H NMR
;
(CDCl3) d 2.23 (s, 3H), 2.93 (t, J = 7.2 Hz, 2H), 3.63 (t, J = 7.2 Hz,
2H), 3.43 (s, 2H), 6.59 (d, J = 8.4 Hz, 1H), 6.79 (s, 1H), 7.09 (d,
J = 8.4 Hz, 1H), 7.22–7.32 (m, 4H), 7.44 (br s, 1H); HRMS calcd for
C17H17ClN2O m/z 300.1029, found 300.1017.
6.1.3.15. 6-Chloro-3-(3-phenylpropyl)-3,4-dihydroquinazolin-
2(1H)-one (7o). Yellow solid; yield 66%; purity 96.87%; mp 128–
130 °C; IR (neat) 3198, 3057, 2925, 2857, 1666, 1490 cmÀ1 1H
;
6.1.3.7. 3-(2,4-Dichlorophenethyl)-6-methyl-3,4-dihydroquinaz-
NMR (CDCl3) d 1.92–2.00 (m, 2H), 2.56 (t, J = 7.6 Hz, 2H), 3.47 (t,
J = 7.6 Hz, 2H), 4.42 (s, 2H), 6.64 (d, J = 8.0 Hz, 1H), 6.70 (br s,
1H), 6.91 (t, J = 7.6 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 7.14–7.27 (m,
5H); HRMS calcd for C17H17ClN2O m/z 300.1029, found 300.1017.
olin-2(1H)-one (7g). White solid; yield 69%; purity 95.07%; mp
151–153 °C; IR (neat) 2926, 1855, 1661, 1461 cmÀ1 1H NMR
;
(CDCl3) d 2.43 (s, 1H), 3.14 (t, J = 7.2 Hz, 2H), 3.73 (t, J = 6.8 Hz,
2H), 4.99 (s, 2H), 7.03 (d, J = 8.4 Hz, 1H), 7.46 (m, 2H), 7.60 (br s,
1H), 7.67 (s, 1H), 7.77 (m, 2H), 8.05 (d, J = 8.4 Hz, 1H); HRMS calcd
for C17H16Cl2N2O m/z 334.0640, found 334.0622.
6.1.3.16. 6-Chloro-3-(4-phenylbutyl)-3,4-dihydroquinazolin-
2(1H)-one (7p). Yellow solid; yield 62%; purity 98.24%; mp 147–
148 °C; IR (neat) 3345, 2924, 2856, 1671, 1495 cmÀ1 1H NMR
;
6.1.3.8. 6-Methyl-3-(3-phenylpropyl)-3,4-dihydroquinazolin-
(CDCl3) d 1.66 (t, J = 7. 6 Hz, 4H), 2.51–2.68 (m, 4H), 4.40 (s, 2H),
6.63 (d, J = 7.6 Hz, 1H), 6.78 (br s, 1H), 6.90 (t, J = 7.6 Hz, 1H),
7.00 (d, J = 7.6 Hz, 1H), 7.13–7.18 (m, 5H); HRMS calcd for
C18H19ClN2O m/z 314.1186, found 314.1168.
2(1H)-one (7h). White solid; yield 59%; purity 98.30%; mp 137–
139 °C; IR (neat) 3193, 2925, 1662, 1492 cmÀ1 1H NMR (CDCl3) d
;
1.91–1.99 (m, 2H), 2.27 (s, 3H), 2.67 (t, J = 7.6 Hz, 2H), 3.48 (t,
J = 7.6 Hz, 2H), 4.37 (s, 2H), 6.53 (d, J = 8.0 Hz, 1H), 6.66 (br s,
1H), 6.83 (s, 1H), 6.95 (d, J = 6.8 Hz, 1H), 7.16–7.29 (m, 5H); HRMS
calcd for C18H20N2O m/z 280.1576, found 280.1559.
6.1.3.17. 6-Chloro-3-(2-cyclohexylethyl)-3,4-dihydroquinazolin-
2(1H)-one (7q). Yellow solid; yield 78%; purity 96.5%; mp 113–
115 °C; IR (neat) 3202, 2921, 1667, 1601, 1481 cmÀ1 1H NMR
;
6.1.3.9. 6-Methyl-3-(4-phenylbutyl)-3,4-dihydroquinazolin-2(1H)-
one (7i). White solid; yield 57%; purity 99.20%; mp 129–131 °C;
(CDCl3) d 0.90–1.93 (m, 13H), 3.45 (t, J = 7.6 Hz, 2H), 4.29 (s, 2H),
6.65 (d, J = 7.6 Hz, 1H), 6.98 (br s, 1H), 7.02 (d, J = 7.6 Hz, 1H),
IR (neat) 3193, 2926, 2856, 1665, 1509 cmÀ1 1H NMR (CDCl3) d
;

P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1555–1562
1561
7.15 (d, J = 7.6 Hz, 1H); HRMS calcd for C16H21ClN2O m/z, 292.1342,
6.1.4.7. 3-(2,4-Dichlorophenethyl)-6-methyl-3,4-dihydroquinaz-
found 292.1334.
oline-2(1H)-thione (1g). White solid; yield 39%; purity 96.23%;
mp 160–161 °C; IR (neat) 3195, 2926, 2856, 1595, 1509 cmÀ1 1H
;
NMR (CDCl3) d 2.28 (s, 3H), 3.09 (t, J = 7.6 Hz, 2H), 4.09 (t,
J = 7.6 Hz, 2H), 4.35 (s, 2H), 6.54 (d, J = 8.0 Hz, 1H), 6.72–6.84
(m, 2H), 6.97 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 1H), 7.82
(br s, 1H); HRMS calcd for C17H16Cl2N2S m/z 350.0411, found
350.0402.
6.1.4. General procedure for the synthesis of compounds 1 (a–q)
To the carbonyl compounds 7a–q in anhydrous toluene
(75 mL) was added Lawesson’s reagent (1.2 equiv) under nitrogen
atmosphere. The resulting solution was refluxed for overnight.
The toluene was removed and the aqueous mixture was extracted
with dichloromethane. The organic phase was washed twice with
brine, dried over anhydrous sodium sulfate, and concentrated un-
der reduced pressure to give the crude product, which was then
purified by flash column chromatography. In order to determine
the purity, all compounds have tested HPLC analysis using C18 re-
verse phase column by the mobile phase of methanol and aceto-
nitrile (1:1).
6.1.4.8. 6-Methyl-3-(3-phenylpropyl)-3,4-dihydroquinazoline-
2(1H)-thione (1h). White solid; yield 49%; purity 99.26%; mp
131–133 °C; IR (neat) 3189, 2925, 2856, 1496 cmÀ1 1H NMR
;
(CDCl3) d 2.04–2.13 (m, 2H), 2.26 (s, 3H), 2.73 (t, J = 7.6 Hz, 2H),
3.89 (t, J = 7.6 Hz, 2H), 4.43 (s, 2H), 6.60 (d, J = 8.0 Hz, 1H), 6.79
(s, 1H), 6.96 (d, J = 8.0 Hz, 1H), 7.09–7.28 (m, 5H), 8.24 (br s, 1H);
HRMS calcd for C18H20N2S m/z 296.1347, found 296.1328.
6.1.4.1. 6-Methyl-3-phenethyl-3,4-dihydroquinazoline-2(1H)-
thione (1a)22. Light yellow solid; yield 47%; purity 96.72%; mp
6.1.4.9. 6-Methyl-3-(4-phenylbutyl)-3,4-dihydroquinazoline-
175–177 °C; IR (neat) 3200, 3020, 2920, 1620, 1550–1480, cmÀ1
;
2(1H)-thione (1i). White solid; yield 57%, purity 97.04%; mp 135–
1H NMR (CDCl3) d 2.27 (s, 3H), 3.10 (t, J = 7.7 Hz, 2H), 4.12 (t,
J = 7.7 Hz, 2H), 4.36 (s, 2H), 6.60–7.02 (m, 3H), 7.28 (m, 5H),
8.48 (br, NH); HRMS calcd for C17H18N2S m/z 282.1191, found
282.1174.
136 °C; IR (neat) 3191, 2927, 2857, 1534 cmÀ1 1H NMR (CDCl3) d
;
1.63–1.87 (m), 2.27 (s, 3H), 2.69 (t, J = 7.2 Hz, 2H), 3.93 (t,
J = 7.2 Hz, 2H), 4.44 (s, 2H), 6.54 (d, J = 8.0 Hz, 1H), 6.81 (s, 1H),
6.98 (d, J = 9.2 Hz, 1H), 7.16–7.32 (m, 5H), 7.79 (br s, 1H); HRMS
calcd for C19H22N2S m/z 310.1504, found 310.1500.
6.1.4.2. 7-Methyl-3-phenethyl-3,4-dihydroquinazoline-2(1H)-
thione (1b). White solid; yield 44%; purity 96.12%; mp 181–
6.1.4.10. 6-Methyl-3-(2-cyclohexylethyl)-3,4-dihydroquinazoline-
183 °C; IR (neat) 3200, 3025, 1599, 1525, 1479 cmÀ1
;
1H NMR
2(1H)-thione (1j). White solid; yield 60%; purity 98.0%; mp 169–
(CDCl3) d 2.30 (s, 3H), 3.10 (t, J = 7.6 Hz, 2H), 4.12 (t, J = 7.6 Hz,
2H), 4.36 (s, 2H), 6.49 (s, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.89 (d,
J = 8.0 Hz, 1H), 7.23–7.30 (m, 5H), 7.90 (br s, 1H); HRMS calcd for
C17H18N2S m/z 282.1191, found 282.1176.
171 °C; IR (neat) 3733, 2922, 2852, 1538 cmÀ1 1H NMR (CDCl3) d
;
0.97–1.80 (m, 13H), 2.28 (s, 3H), 3.94 (t, J = 7.6 Hz, 2H), 4.47 (s,
2H), 6.54 (d, J = 8.4 Hz, 1H), 6.83 (s, 1H), 6.99 (d, J = 8.0 Hz, 1H),
7.79 (br s,1H); HRMS calcd for C17H24N2S m/z 288.1660, found
288.1646.
6.1.4.3. 5-Methyl-3-phenethyl-3,4-dihydroquinazoline-2(1H)-
thione (1c). White solid; yield 45%; purity 99.20%; mp 128–
6.1.4.11. 6-Methyl-3-(2-(naphthalen-2-yl) ethyl)-3,4-dihydro-
130 °C; IR (neat) 3199, 3134, 2922, 1616, 1598, 1523 cmÀ1 1H
;
quinazoline-2(1H)-thione (1k). White solid; yield 45%; purity
98.08%; mp 199–201 °C; IR (neat) 2922, 1551, 1463 cmÀ1 1H
;
NMR (CDCl3) d 2.03 (s, 3H, Ar-CH3), 3.12 (t, J = 7.6 Hz, 2H), 4.14
(t, J = 7.6 Hz, 2H), 4.30 (s, 2H), 6.49 (d, J = 8.0 Hz, 1H), 6.80 (d,
J = 7.6 Hz, 1H), 7.07 (t, J = 8.0 Hz, 1H), 7.23–7.30 (m, 5H), 7.87 (br
s, 1H); HRMS calcd for C17H18N2S m/z 282.1191, found 282.1174.
NMR (CDCl3) d 2.23 (s, 3H), 3.25 (t, J = 7.6 Hz, 2H), 4.20 (t,
J = 7.6 Hz, 2H), 4.34 (s, 2H), 6.54 (s, 1H), 6.56 (d, J = 8.0 Hz, 1H),
6.96 (d, J = 8.0 Hz, 1H), 7.41–7.46 (m, 3H), 7.68 (m, 2H), 7.78 (m,
2H), 7.91 (br s, 1H); HRMS calcd for C21H20N2S m/z 332.1347, found
332.1334.
6.1.4.4. 6-Methyl-3-(4-methylphenethyl)-3,4-dihydroquinazo-
line-2(1H)-thione (1d). White solid; yield 44%; purity 95.95%; mp
135–138 °C; IR (neat) 3198, 2925, 2855, 1711, 1533 cmÀ1; 1H NMR
(CDCl3) d 2.26 (s, 3H), 2.32 (s, 3H), 3.05 (t, J = 7.6 Hz, 2H), 4.08 (t,
J = 7.2 Hz, 2H), 4.36 (s, 2H), 6.54 (d, J = 8.0 Hz, 1H), 6.72 (s, 1H),
6.99 (d, J = 8.4 Hz, 1H), 7.08 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz,
2H), 7.83 (br s, 1H); HRMS calcd for C18H20N2S m/z 296.1347, found
296.1332.
6.1.4.12. 6-Chloro-3-phenethyl-3,4-dihydroquinazoline-2(1H)-
thione (1l). White solid; yield 33%; purity 98.43%; mp 175–
177 °C; IR (neat) 2926, 1473 cmÀ1 1H NMR (CDCl3) d 3.10 (t,
;
J = 7.6 Hz, 2H), 4.13 (t, J = 7.6 Hz, 2H), 4.38 (s, 2H), 6.65 (d,
J = 8.0 Hz, 1H), 6. 91 (s, 1H), 6.97 (d, J = 8.0 Hz, 1H), 7.17–7.29 (m,
5H), 7.87 (br s, 1H); HRMS calcd for C16H15ClN2S m/z 302.0644,
found 302.0627.
6.1.4.5. 3-(4-Isopropylphenethyl)-6-methyl-3,4-dihydroquinaz-
oline-2(1H)-thione (1e). White solid; yield 54%; purity 95.46%;
mp 156–158 °C; IR (neat) 2926, 2855, 1493 cmÀ1; 1H NMR (CDCl3)
d 1.22 (d, J = 7.2 Hz, 6H), 2.26 (s, 3H), 2.29 (m, 1H), 3.03 (t,
J = 7.6 Hz, 2H), 4.09 (t, J = 7.6 Hz, 2H), 4.54 (s, 2H), 6.56 (d,
J = 8.0 Hz, 1H), 6.69 (s, 1H), 6.96 (d, J = 8.0 Hz, 1H), 7.13–7.21 (m,
4H), 7.90 (br s, 1H); HRMS calcd for C20H24N2S m/z 324.1660, found
324.1646.
6.1.4.13. 6-Chloro-3-(4-methylphenethyl)-3,4-dihydroquinazo-
line-2(1H)-thione (1m). White solid; yield 27%; purity 95.03%;
mp 153–154 °C; IR (neat) 3191, 2927, 1499 cmÀ1; 1H NMR (CDCl3)
d 2.35 (s, 3H), 3.06 (t, J = 7.6 Hz, 2H), 4.08 (t, J = 6.4 Hz, 2H), 4.39 (s,
2H), 6.65 (d, J = 7.6 Hz, 1H), 6.95 (t, J = 8.0 Hz, 2H), 7.08 (d,
J = 8.0 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.90 (br s, 1H); HRMS calcd
for C16H15ClN2S m/z 302.0644, found 302.0627.
6.1.4.6. 3-(4-Chlorophenethyl)-6-methyl-3,4-dihydroquinazo-
line-2(1H)-thione (1f). White solid; yield 54%; purity 95.15%; mp
157–159 °C; IR (neat) 3193, 2924, 2855, 1708, 1508 cmÀ1; 1H NMR
(CDCl3) d 2.23 (s, 3H), 3.09 (t, J = 7.6 Hz, 2H), 4.11 (t, J = 7.2 Hz, 2H),
4.36 (s, 2H), 6.55 (d, J = 8.0 Hz, 1H), 6.72 (s, 1H), 6.97 (d, J = 8.4 Hz,
1H), 7.21–7.32 (m, 4H), 7.84 (br s, 1H); HRMS calcd for C17H17ClN2S
m/z 316.0801, found 316.0789.
6.1.4.14. 6-Chloro-3-(4-chlorophenethyl)-3,4-dihydroquinazo-
line-2(1H)-thione (1n). White solid; yield 42%, purity 98.03%; mp
157–159 °C; IR (neat) 3190, 2926, 2853, 1598 cmÀ1 1H NMR
;
(CDCl3) d 3.08 (t, J = 7.6 Hz, 2H), 4.10 (t, J = 7.6 Hz, 2H), 4.38 (s,
2H), 6.60 (d, J = 8.0 Hz, 1H), 6.89 (s, 1H), 6.98 (d, J = 8.0 Hz, 1H),
7.17–7.29 (m, 4H), 7.91 (br s, 1H); HRMS calcd for C16H14Cl2N2S
m/z 336.0255, found 336.0244.

1562
P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1555–1562
6.1.4.15. 6-Chloro-3-(3-phenylpropyl)-3,4-dihydroquinazoline-
6.2.5. Western blot analysis
2(1H)-thione (1o). White solid; yield 56%; purity 95.04%; mp
B16 cells were stimulated with a-MSH (10 nM) for 48 h, in the
presence of sample, and then disrupted in a lysis buffer (50 mM
166–167 °C; IR (neat) 3171, 2928, 2857, 1723, 1594 cmÀ1
;
1H
NMR (CDCl3) d 1.61–1.80 (m, 2H), 2.69 (t, J = 7.2 Hz, 2H), 3.94 (t,
J = 7.2 Hz, 2H), 4.48 (s, 2H), 6.65 (d, J = 8.0 Hz, 1H), 6.80 (s, 1H),
6.85 (d, J = 8.0 Hz, 1H), 6.92–7.29 (m, 5H), 7. 96 (br s, 1H); HRMS
calcd for C17H17ClN2S m/z 316.0801, found 316.0792.
Tris, 50 mM NaCl, 0.1% SDS, 1% NP-40, 1 mM phenylmethanesulfo-
nyl fluoride, 10 lg/mL aprotinin, 10 lg/mL leupeptin, pH 7.4).
Equal amounts of the proteins were resolved on SDS–acrylamide
gels by electrophoresis and transferred to a polyvinylidene difluo-
ride membrane. Either 5% non-fat milk in PBS containing Tween 20
or 5% BSA in Tris-buffered saline containing Tween 20 was used as
the blocking buffer. The blots were usually incubated at 4 °C over-
night with primary antisera (dilution); anti-tyrosinase (1:1000),
and anti-GAPDH (1:5000). The blots were then incubated with
appropriate horseradish peroxidase-conjugated secondary antisera
at room temperature for 2–5 h. Immune complexes on the blots
were finally visualized by exposure to X-ray film after reacting
with an enhanced chemiluminescence’s reagent (GE Healthcare,
Chalfont St. Giles, UK).
6.1.4.16. 6-Chloro-3-(4-phenylbutyl)-3,4-dihydroquinazoline-
2(1H)-thione (1p). White solid; yield 46%; purity; 97.0%; mp
172–174 °C; IR (neat) 3171, 2928, 2857, 1594 cmÀ1 1H NMR
;
(CDCl3) d 1.60–1.80 (m, 4H), 2.67 (t, J = 7.2 Hz, 2H), 3.98 (t,
J = 7.2 Hz, 2H), 4.48 (s, 2H), 6.64 (d, J = 8.0 Hz, 1H), 6.85 (s, 1H),
6.91–7.33 (m, 6H), 7.90 (br s, 1H); HRMS calcd for C18H19ClN2S
m/z 330.0957, found 330.0943.
6.1.4.17. 6-Chloro-3-(cyclohexylethyl)-3,4-dihydroquinazoline-
2(1H)-thione (1q). White solid; yield 63%; purity 97.8%; mp 148–
150 °C; IR (neat) 3191, 2920, 1605, 1493 cmÀ1 1H NMR (CDCl3) d
;
0.95–1.77 (m, 13H), 3.97 (t, J = 7.6 Hz, 2H), 4.52 (s, 2H), 6.67 (d,
J = 7.6 Hz, 1H), 7.01 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 7.99 (br s,
1H); HRMS calcd for C16H21ClN2S m/z 308.1114, found 308.11012.
6.2.6. MTT assay
B16 cells were treated with various concentration of sample for
72 h, in the presence of
to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT, 500 g/mL) for 2 h. The MTT–formazan complex was dis-
a-MSH (10 nM). The cells were exposed
l
6.2. Biological evaluation
solved in 100% dimethyl sulfoxide and optical densities were then
measured at wavelength 590 nm.
6.2.1. Chemicals and antisera
Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s media
and other culture supplements were purchased from Invitrogen
(Carlsbad, CA). Antisera against tyrosinase or GAPDH were pur-
chased from Santa Cruz Biotech (Santa Cruz, CA). All other chemi-
Acknowledgment
This research was financially supported by a grant (R01-2007-
000-20099-0) from the National Research Foundation of Korea.
cals, including
a-MSH, were otherwise purchased from Sigma–
Aldrich (St. Louis, MO).
References and notes
6.2.2. Cell culture
B16 melanoma cells were obtained from American Type Culture
Collection (Manassas, USA). The cells were cultured in DMEM
(13.4 mg/mL Dulbecco’s modified Eagle’s medium, 10 mM HEPES,
143 U/mL benzylpenicillin potassium, 100 lg/mL streptomycin
sulfate, 24 mM NaHCO3, pH 7.1) containing 10% FBS, and incubated
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B16 cells were seeded in culture plates, a density of 2.5 Â 103
cells per well of 96-well microplates, and incubated at 37 °C under
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6.2.4. Measurement of tyrosinase activity
B16 cells were treated with a-MSH (10 nM) alone for 72 h. After
washing, the cells were resuspended in sodium phosphate buffer
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