Inhibitory effect of novel tetrahydropyrimidine-2(1H)-thiones on melanogenesis

Article abstract of DOI:10.1016/j.bmc.2009.12.044

The series of imidazoldine-2-thiones 2 and tetrahydropyrimidine-2-thiones 3 were discovered as inhibitor of α-MSH-induced melanin production in melanoma B16 cells. The primary bioassay showed that 1-(4-ethylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3e (>1

Full text of DOI:10.1016/j.bmc.2009.12.044

Bioorganic & Medicinal Chemistry 18 (2010) 1135–1142
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitory effect of novel tetrahydropyrimidine-2(1H)-thiones
on melanogenesis
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, Cheongu 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:
The series of imidazoldine-2-thiones 2 and tetrahydropyrimidine-2-thiones 3 were discovered as inhib-
Received 10 November 2009
Revised 12 December 2009
Accepted 15 December 2009
Available online 22 December 2009
itor of
(4-ethylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3e (>100% inhibition at 10
(4-tert-butylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3f (>100% inhibition at 10
exhibited potent inhibitory effect against -MSH-induced melanin production. Compounds 3 inhibit
the biosynthesis of tyrosinase without affecting its catalytic activity in melanogenesis.
Ó 2010 Elsevier Ltd. All rights reserved.
a
-MSH-induced melanin production in melanoma B16 cells. The primary bioassay showed that 1-
M, IC50 = 1.2 M) and 1-
M, IC50 = 0.76 M)
l
l
l
l
a
Keywords:
Tetrahydropyrimidine-2(1H)-thione
a-MSH-induced melanin production
Hypopimenting
Melanogenesis
1. Introduction
phosphate (cAMP) dependent melanogenesis proteins other than
tyrosinase, has gain the attraction. Using the bioassay system for
measuring the amount of melanin formed from melanoma B16 cells
upon stimulation of a-melanocyte stimulating hormone (a-MSH),
we have screened many different compounds. As a result, highly po-
Melanin is the primary determinant of human skin color, and is
a heterogeneous biopolymer which plays an important role in the
absorption of free radicals formed in cytoplasm and in protecting
human skin from the harmful UV-radiation.1 Melanin is synthe-
sized in melanocytes through melanogenesis which is a complex
pathway involving series of enzymatic reactions.2–4 Among en-
zymes involved in the biosynthesis of melanin, tyrosinase is a most
tent hypopigmenting activity of 6-methyl-3-phenethyl-3,4-dihy-
dro-1H-quinazoline-2-thione (Fig. 1, 1, IC50 = 0.8 l
M) was found.18
This level of activity of 1 is approximately 70 and 180 times more po-
tent compared to those of kojic acid and arbutin in this bioassay sys-
tem, respectively. The compound 1 suppresses the biosynthesis of
tyrosinase, withoutaffectingtheactivityof thisenzyme. Preliminary
study on the structure–activity relationship of 1 revealed that qui-
nazolidine-2-thione motif is essential for their inhibition of melano-
genesis. In order to explore the role of fused benzene moiety of 1,
imidazolidine-2-thiones 2 and tetrahydropyrimidine-2-thiones 3
(Fig. 1) were designed, synthesized, and evaluated their inhibitory
activity for melanin production in melanoma B16 cell under the
critical enzyme that catalyzes the reaction of conversion of L-tyro-
sine to dopaquinone.2 Tyosinase, a copper bearing phenolase, is in-
volved in abnormal accumulation of melanin pigments
(hyperpigmentation).5,6 Tyrosinase was reported to be linked in
Parkinson diseases and some other neurodegenerative diseases.7,8
Therefore, tyrosinase inhibitors such as kojic acid,9 arbutin,10
ascorbic acid derivatives,11 hydroxylstilbine derivatives like resve-
ratol12–14 and methyl ester of genistic acid15,16 have been well
established as important constituents of cosmetic product and
depigmenting agents for the treatment of hyperpigmentation.
However, the recent study was reported that the most used kojic
acid has serious adverse effects such as skin cancer and dermatitis
and has been banned as cosmetic ingredient in many countries.17
Thus other type of molecules, which inhibit cyclic adenosine mono-
stimulus of
studied.
a-MSH. The mechanism of action of these analogs was
R
m
H3C
N
HN
N
n
N
S
S
H
Abbreviation: a-MSH, a-melanostimulating hormone.
* Corresponding author. Tel.: +82 42 821 5939; fax: +82 42 823 6566.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
2: m = 1, n = 1, 2 or 3
3: m = 2, n = 1, 2 or 3
1
Figure 1. cAMP dependent melanogenesis blocker 1 and cyclic thioureas 2 and 3.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.044

1136
P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1135–1142
on tetrahydropyrimidine-2-thione of 1 is not essential structural
2. Chemistry
unit for their activity and is obviously an enhancer of the activity
by increasing its lipophilicity. Thus tetrahydropyrimidine-2-thione
motif appears as novel scaffold for the inhibition of melanin
biosynthesis.
However, the cyclic urea compounds 11a–f and 12a–h showed
very weak activity, although their structures are very similar to
those of cyclic thioureas 2 and 3. Their IC50 values are more than
Imidazolidine-2-thiones 2 and tetrahydropyrimidine-2-thiones
3 were synthesized with the same synthetic procedures as shown
in Scheme 1. In the initial step, the commercially available ethy-
lenediamine (4) (or propan-1,3-diamine (5)) was mono-protected
with tert-butyloxycarbonyl (tBoc) group. Five equivalent of dia-
mine 4 (or 5) with 1 equiv of Boc2O in chloroform was stirred for
overnight at room temperature. The reaction mixture was filtered
and the filtrate was concentrated under vacuum. The resulting res-
idue was dissolved in ethyl acetate and the organic solution was
washed with brine and concentrated to give the corresponding
mono-protected 6 (or 7), which was pure enough for next step.19
The reductive amination20 of mono-protected diamine with
appropriate aldehyde (8) afforded amines 9 (or 10). The cyclization
of 9 (or 10) to the 11 (or 12) was performed by treatment with
potassium tert-butoxide (KOtBu)21 as summarized in Table 1.
According to literature22 the cyclization of these amines could be
facilitated either under basic catalysis or under Lewis acid cataly-
sis. Initial screen showed that strong base was most effective. In
the final step, the imidazoidinones 11 (or pyrimidinones 12) were
treated with Lawesson’s reagents23 under refluxing condition in
toluene for 12 h to produce corresponding thiones 2 (or 3) as sum-
marized in Table 1.
10 lM and less than 30% inhibitory activity at 10 lM. These facts
imply that thiourea group in the cyclic thioureas 2 and 3 has a piv-
otal role for the inhibition of melanin synthesis in the B16 cells.
This difference has been recognized in the activity of phenylthio-
urea (PTU, 58% inhibition at 100
tion at 100
M),24,25 although mechanism of action of PTU (IC50
value for tyrosinase inhibition: 1.8 M) is quite different from that
lM) and phenylurea (0% inhibi-
l
l
of the cyclic thioureas 2 and 3 as discussed later. The mechanism of
action of PTU has been known as inactivation of tyrosinase through
complex formation of copper ion in active site of tyrosinase.
Increase in lipophilicity of cyclic thioureas 2, 3 enhances the
activity of these compounds as indicated with C log P in Table 1.
The compound 3f shows the strongest activity (IC50: 0.76 lM)
and the highest C log P value (4.437). However, lipophilicity should
not be related to difference in activity of cyclic ureas and cyclic
thioureas since the activity of 12f (IC50: >10
of its higher C log P value (3.259) than those of 3b (C log P: 3.110,
IC50: 5.1 M) and 10 g (C log P: 2.940, IC50: 2.9 M). Thus cyclic
lM) is weak in spite
l
l
3. Pharmacology
thiourea motif obviously appears as essential group for inhibition
of melanin formation.
For all the synthesized compounds 11, 12, 2 and 3 the ability to
The lipophilicity of 2 and 3 is the important factor for the
enhancement of activity. The more enhanced activity of pyrimi-
dine-2-thiones 3 compared to those of imidazolidine-2-thiones 2
also can be explained with their increased lipophilicity of 3 as C log P
values of these compounds indicated. The role of phenylalkyl groups
on 1-position of 2 and 3 is the enhancer of their lipophilicity. Increas-
ing size of alkyl group on 4-position of phenyl increases the activity
of 2 and 3 as shown in comparison of activity of 2a and 2b as well as
3a, 3b, 3e, and 3d. Increasing the lipophilicity with chloro substitu-
inhibit formation of melanin from melanoma B-16 cells was deter-
mined under stimulus of
a-MSH (100 nM) for 3 day incubation.
Melanoma B16 cell (CRL6323) were obtained from ATCC (Manas-
sas, USA). Amounts of melanin released into the culture media
were determined by measuring absorbance values at 405 nm with
synthetic melanin as the standard.14 Data for % inhibition at 10
lM
and IC50 values are mean values from 3 to 5 separate experiments
as shown in Table 2.
ent as shown in compound 3c (IC50: 2.4
lM) also increases the activ-
4. Results and discussion
ity compared to that of 3a (IC50: >10 M). Increasing the number of
l
methylene groups between nitrogen and phenyl ring of 2 and 3 also
increased the activity. This also considered as enhancing effect of
lipophilicity. Introduction of 4-methoxy group on phenyl as shown
in 3d (IC50: 6.1 lM, C log P: 2.530) decreased the activity and lipo-
philicity compared to 3b, 3c, 3e, and 3f.
The cyclic thioureas 2b, 2c, 2f and 3b–h shows remarkably po-
tent inhibitory activity against melanin production in melanoma
B16 cells comparing the activity of kojic acid (IC50: 70
(IC50: 180 M) and almost similar to 1 (IC50 = 0.8 M). Their IC50
values range from 0.8 to 6.1 M, which are comparable to that of
1. Especially, compound 3g (IC50: 2.9 M) has the partial structure
of 1 with omission of methylbenzene unit. Thus benzene ring fused
lM), arbutin
l
l
l
The above discussion gave the more insight regarding the SAR
of 1 related to our current studies. Accordingly, the following struc-
l
H
H
o
N
O
+
(i)
H2N
H2N
O
NH2
m
m
R
O
R
4: m = 1
5: m = 2
8: o = 0, 1 or 2
(ii)
6: m = 1
7: m = 2
O
H
N
R
m
m
N
(iii)
(iv)
N
H
O
o
m
HN
N
HN
n
n
R
O
S
9: m = 1, o = 0, 1 or 2
10: m = 2, o = 0, 1 or 2
11: m = 1, n = 1, 2 or 3
12: m = 2, n = 1, 2 or 3
2: m = 1, n = 1, 2 or 3
3: m = 2, n = 1, 2 or 3
Scheme 1. Synthesis of compounds 2 and 3. Reagents and conditions: (i) Boc2O/CHCl3; (ii) NaBH4/TEA; (iii) KOtBu/THF; (iv) Lawesson’s reagent/toluene. Substituents for 9,
10, 11, 12, 2 and 3 are listed in Table 1.

P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1135–1142
1137
Table 1
The substituents of 11, 12, 2 and 3
Compound No.
m
n
R
C log P
Compound No.
m
n
R
C log P
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
1
1
1
2
3
1
1
1
1
1
1
2
3
H
CH3
Cl
OCH3
H
H
H
CH3
0.872
1.371
1.585
0.791
1.201
1.580
1.431
1.930
2.144
1.350
2.459
3.259
1.760
2.139
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
1
1
1
2
3
1
1
1
1
1
1
2
3
H
CH3
Cl
OCH3
H
H
H
CH3
2.052
2.551
2.765
1.971
2.381
2.760
2.611
3.110
3.324
2.530
3.369
4.437
2.940
3.319
Cl
Cl
OCH3
CH2CH3
C (CH3)3
H
OCH3
CH2CH3
C (CH3)3
H
12g
12h
3g
3h
H
H
Table 2
Inhibitory activity of 11, 12, 2, and 3 on melanogenesis of melanoma B16 cells
Compound No.
% Inhibition at 10
lM
IC50 value (
l
M)
Compound No.
% Inhibition at 10
lM
IC50 value (lM)
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
7
14
10
13
15
10
0
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2a
2b
2c
2d
2e
35
64
79
31
40
63
42
80
>100
60
>100
>100
93
>10
4.8
4.3
>10
>10
4.2
>10
5.1
2.4
6.1
1.3
0.8
2.9
2.1
0.8
180
2f
3a
3b
3c
3d
3e
5
26
20
29
30
10
10
3f
12g
12h
3g
3h
1
>100
Kojic acid
70
Arbutin
>100
tural modification on 1 was done successfully to get the compara-
ble activity as we observed in our previous studies18 (1) The bicy-
clic system as 1 can be replaced to cyclic thiourea derivative 3e–f,
(2) chain length also modified with the introduction of bulkiness at
p-position of phenyl ring 3e–f and (3) we also tried to replace thio-
urea group to urea one, unfortunately, without any activity. The
above points prove fruitful in the relationship of the compound 1
and the current synthesized compounds. Further, on comparing
oxidation velocity in the presence of 3f was measured as an indica-
tor of tyrosinase catalytic activity. Compound 3f did not signifi-
cantly inhibit the catalytic activity of cell-free tyrosinase
(Fig. 2B). Upon exposure to
the cells markedly increased from the baseline levels (Fig. 2C).
Compound 3f suppressed -MSH-induced levels of tyrosinase in
a-MSH alone, levels of tyrosinase in
a
a dose-dependent manner (Fig. 2C). However, at concentrations
effective for hypopigmentation activity, compound 3f did not af-
fect the viability of B16 cells (Fig. 2D), ruling out non-specific cyto-
toxicity. On the basis of these results, the compound 3f is an
the activity of 1 (IC50: 0.8
lM) with that of 3g–h (IC50: 2.9 lM
and 2.1 M), the level of activity is nearly the same. In fact, all
l
the derivatives 3b–f having bulky groups at p-position of phenyl
ring showed very comparable activity to 1. Thus benzene ring
fused on tetrahydropyrimidine-2-thione of 1 is obviously enhance-
ment the activity by increasing its lipophilicity. Thus tetrahydro-
pyrimidine-2-thione motif appears as novel scaffold for the
inhibition of melanin biosynthesis.
inhibitor of a-MSH-induced melanin production without affecting
the catalytic activity of tyrosinase. This result was unforeseen con-
sidering the well-known tyrosinase inhibitor NPT, because its thio-
urea moiety plays a key role in complexing the copper ion in the
active site of tyrosinase, thereby blocking enzyme activity.26
6. Conclusion
5. Mechanism of action
In summary, imidazolidine-2-thiones 2 and tetrahydropyrimi-
In defining the mechanism of action of 3, the effects on tyrosi-
dine-2-thiones 3 inhibited melanogenesis in a-MSH-induced and
nase activity, on tyrosinase synthesis upon stimulation with
MSH and on the depigmentation of melanin were evaluated with
3f. The effects of tyrosinase on melanin production were also stud-
a
-
melanin production. This implies that benzene ring fused on tetra-
hydropyrimidine-2-thione of 1 is not essential structural unit for
their activity. Thus tetrahydropyrimidine-2-thione motif appears
as novel scaffold for the inhibition of melanin biosynthesis. How-
ever, the urea analogs 11 and 12 did not show such effects. Among
all thiones 2 and 3, the compound 3f very effectively inhibits the
melanogenesis with an IC50 of an around 0.8 lM without affecting
the catalytic activity of tyrosinase. These results suggest that tetra-
hydropyrimidine-2-thiones 3 are quite promising for the treat-
ied during
showed an inhibitory effect on melanin synthesis in melanoma
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 exposure to -MSH for 72 h (Fig. 2A). The dopa
a-MSH stimulation with compound 3f. Compound 3f
a
6
l
4
l
a

1138
P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1135–1142
Figure 2. The effects of 3f 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 3f. 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
a-MSH-treated group. (B) Enzyme sources of tyrosinase were prepared from
cells stimulated with
conversion of 1 nmol dopa to dopachrome/min. The dopa oxidation velocity of cell-free tyrosinase (50
cells were stimulated with -MSH for 48 h in the presence of 3f. Cell extracts were subjected to Western blot analysis with an anti-tyrosinase antibody, and glyceraldehyde 3-
a-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
lg) was spectrophotometrically measured in the presence of 3f. (C) The
a
phosphate dehydrogenase (GAPDH) were used as an internal control. (D) The cells were treated with various concentrations of 3f for 72 h in the presence of a-MSH. After
reacting with MTT solution for 2 h, optical densities were measured at 590 nm.
ment of skin hyperpigmentation. Further exploration of the mech-
anism of activity of tetrahydropyrimidine-2-thiones 3 in depig-
mentation is in progress.
7.2.1. tert-Butyl 2-aminoethylcarbamate (6)
Yield 55%; colorless oil; 1H NMR (CDCl3, 400 MHz) d 1.49 (s, 9H,
tert-butyl), 2.79 (t, J = 6.4 Hz, 2H, NCH2), 3.18 (t, J = 6.0 Hz, 2H,
NCH2), 4.91 (br s, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 160 (M+), 161 (M+1).
7. Materials and methods
7.1. Chemistry
7.2.2. tert-Butyl 3-aminopropylcarbamate (7)
Yield 50%; Viscous oil; 1H NMR (CDCl3, 400 MHz) d 1.44 (s, 9H,
tert-butyl), 1.61 (m, 2H, NCH2CH2CH2N), 2.76 (t, J = 6.8 Hz, 2H,
NCH2C), 3.22 (t, J = 6.0 Hz, 2H, CH2NBoc). Mass spectra of com-
pound exhibited molecular ion peak at m/z 174 (M+), 175 (M+1).
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 tetra-
methylsilane by Varian Unity Inova 400 NMR (400 MHz) spectrom-
eters. The ESI-MS mass data were obtained on an 1100-LC-MSD
trap spectrometer (Agilent, Santa Clara, CA, USA).
7.3. General procedure for the preparation of compounds 9 and
10
To the solution of mono-protected diamine 6 or 7 (1 mmol) in
methanol (50 mL) and triethylamine (1 mmol) at 0 °C, the corre-
sponding aldehyde 8 (1 mmol) was added. The reaction mixture
was stirred at room temperature for 2 h and then cooled to 0 °C.
Sodium borohydride (2 mmol) was added portion wise over a per-
iod of 30 min, then the resulting solution was neutralized with 4 M
hydrochloric acid (5 mL) and extracted with Et2O (50 mL). The
ethereal phase was discarded and the aqueous phase was neutral-
ized with solid NaHCO3, extracted with ethyl acetate (200 mL),
dried over anhydrous Na2SO4, concentrated to produce the appro-
priate analogues 9 or 10.
7.2. General synthetic procedure for the preparation of mono-
protected amine 6 and 7
A 0.5 M solution of Boc2O (1 equiv) in CHCl3 was added over a
half an hour to a 0.25 M solution of diamine 4 (5 equiv) or 5 in
CHCl3 cooled with an ice-bath. The reaction mixture was stirred
overnight at room temperature and filtered. The filtrate was con-
centrated under vacuum and the resulting oil dissolved in ethyl
acetate (400 mL) than washed with half-saturated brine
(3 Â 150 mL), dried (Na2SO4) and concentrated reduced pressure
to afford the corresponding mono-BOC-protected diamine 6 or 7.
7.3.1. tert-Butyl 2-(benylamino)ethylcarbamate (9a)
Yield 80%; viscous oil; 1H NMR (CDCl3) d 1.49 (s, 9H, tert-butyl),
2.75 (t, J = 5.6 Hz, 2H, NCH2), 3.24 (t, J = 5.6 Hz, 2H, NCH2), 3.87 (s,
NCH2Ph) 5.02 (br s, 1H, NH), 7.23–7.34 (m, 5H, Ar-H). Mass spectra
of compound exhibited molecular ion peak at m/z 250 (M+), 251
(M+1), 252 (M+2).

P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1135–1142
1139
7.3.2. tert-Butyl 2-(4-methylbenzylamino)ethylcarbamate (9b)
Yield 75%; viscous oil; 1H NMR (CDCl3) d 1.49 (s, 9H, tert-butyl),
2.31 (s, 3H, CH3Ph) 2.72 (t, J = 6.0 Hz, 2H, CH2N), 3.22 (t, J = 6.0 Hz,
2H, CH2 NBoc), 3.74 (s, 2H, NCH2Ph), 4.95 (br s, 1H, NH) 7.21 (d,
J = 8.2 Hz, 2H, Ar-H),7.30 (d, J = 8.2 Hz, 2H, Ar-H). Mass spectra of
compound exhibited molecular ion peak at m/z 264 (M+), 265
(M+1), 266 (M+2).
J = 8.4 Hz, 2H, Ar-H). Mass spectra of compound exhibited molecu-
lar ion peak at m/z 294 (M+), 295 (M+1), 296 (M+2).
7.3.11. tert-Butyl 3-(4-ethylbenzylamino)propylcarbamate
(10e)
Yield 55%; viscous oil; 1H NMR (CDCl3) d 1.22 (t, J = 7.6 Hz, 3H,
CH3), 1.44 (s, 9H, tert-butyl), 1.63–1.69 (m, 2H, CCH2CN), 2.64 (q,
J = 5.6 Hz, 2H, PhCH2), 2.71 (t, J = 6.0 Hz, 2H, CH2N), 3.20 (t,
J = 6.0 Hz, 2H, CH2NBoc), 3.77 (s, 2H, Ar-CH2N), 7.14 (d, J = 7.6 Hz,
2H, Ar-H), 7.22 (d, J = 8.0 Hz, 2H, Ar-H). Mass spectra of compound
exhibited molecular ion peak at m/z 292 (M+), 293 (M+1), 294
(M+2).
7.3.3. tert-Butyl 2-(4-chlorobenzylamino)ethylcarbamate (9c)
Yield 70%; viscous oil; 1H NMR (CDCl3) d 1.49 (s, 9H, tert-butyl),
2.73 (t, J = 6.4 Hz, 2H, CH2N), 3.24 (t, J = 6.4 Hz, 2H, CH2NBoc), 4.90
(br s, 1H, NH), 7.22–7.30 (m, 4H, Ar-H). Mass spectra of compound
exhibited molecular ion peak at m/z 284 (M+), 286 (M+2).
7.3.12. tert-Butyl 3-(4-tert-butylbenzylamino)propylcarbamate
(10f)
7.3.4. tert-Butyl 2-(4-methoxybenylamino)ethylcarbamate (9d)
Yield 60%; viscous oil; 1H NMR (CDCl3) d 1.34 (s, 9H, tert-butyl),
2.49 (t, J = 6.4 Hz, 2H, CH2N), 3.01 (t, J = 6.0 Hz, 2H, CH2NBoc), 3.58
(s, 3H, OCH3), 3.70 (s, 2H, NCH2Ph) 4.31 (br s, 1H, NH) 6.88 (d,
J = 8.4 Hz, 2H, Ar-H), 7.19 (d, J = 8.4 Hz, 2H, Ar-H). Mass spectra of
compound exhibited molecular ion peak at m/z 280 (M+), 281
(M+1), 282 (M+2).
Yield 57%; viscous oil; 1H NMR (CDCl3) d 1.21 (s, 9H, tert-butyl),
1.41 (s, 9H, tert-butyl), 1.60 (m, 2H, CCH2CN), 2.71 (t, J = 6.4 Hz, 2H,
NCH2), 3.21 (t, J = 6.0 Hz, 2H, CH2N), 3.74 (s, 2H, PhCH2), 7.23 (d,
J = 7.6 Hz, 2H, Ar-H), 7.33 (d, J = 8.0 Hz, 2H, Ar-H). Mass spectra of
compound exhibited molecular ion peak at m/z 320 (M+), 321
(M+1), 322 (M+2).
7.3.5. tert-Butyl 2-(phenylethylamino)ethylcarbamate (9e)
Yield 55%; viscous oil; 1H NMR (CDCl3) d 1.43 (s, 9H, tert-butyl),
2.70 (t, J = 5.6 Hz, 2H, CH2Ph), 2.77–2.88 (m, 4H, CH2NCH2), 3.19 (t,
J = 5.6 Hz, 2H, CH2NBoc), 5.00 (br s, 1H, NH), 7.13–7.30 (m, 5H, Ar-
H). Mass spectra of compound exhibited molecular ion peak at m/z
264 (M+), 265 (M+1), 266 (M+2).
7.3.13. tert-Butyl 3-(phenethylamino)propylcarbamate (10g)
Yield 60%; viscous oil; 1H NMR (CDCl3); d 1.44 (s, 9H, tert-butyl),
1.62 (m, 2H, CCH2CN), 2.69 (t, J = 6.4 Hz, 2H, CH2Ph), 2.79 (t, J =
6.4 Hz, 2H, CH2N), 2.84 (t, J = 6.4 Hz, 2H, CH2N), 3.19 (t, J = 6.4 Hz,
2H, CH2NBoc), 5.14 (br s, 1H, NH), 7.11–7.26 (m, 5H, Ar-H). Mass
spectra of compound exhibited molecular ion peak at m/z 278
(M+), 279 (M+1), 280 (M+2).
7.3.6. tert-Butyl 2-(3-phenylpropylamino)ethylcarbamate (9f)
Yield 55%; viscous oil; 1H NMR (CDCl3) d 1.37 (s, 9H, tert-butyl),
1.69 (m, 2H, CCH2CPh), 2.54–2.61 (m, 6H, PhCH2CCH2NHCH2), 3.01
(t, J = 6.4 Hz, 2H, CH2NBoc), 7.13–7.30 (m, 5H, Ar-H). Mass spectra
of compound exhibited molecular ion peak at m/z 278 (M+), 279
(M+1), 280 (M+2).
7.3.14. tert-Butyl 3-(3-phenylpropylamino)propylcarbamate
(10h)
Yield 55%; viscous oil; 1H NMR (CDCl3) d 1.44 (s, 9H, tert-butyl),
1.52 (m, 2H, NCCH2CN), 1.62 (m, 2H, PhCCH2CN), 2.47 (m, 4H,
CH2NHCH2), 2.58 (t, J = 6.0 Hz, 2H, CH2Ph), 2.95 (t, J = 6.4 Hz, 2H,
CH2NBoc), 7.13–7.30 (m, 5H, Ar-H). Mass spectra of compound
exhibited molecular ion peak at m/z 292 (M+), 293 (M+1), 294
(M+2).
7.3.7. tert-Butyl 3-(benzylamino)propylcarbamate (10a)
Yield 80%; viscous oil; 1H NMR (CDCl3) d 1.44 (s, 9H, tert-butyl),
1.64–1.69 (m, 2H, NCCH2CN), 2.73 (t, J = 6.4 Hz, 2H, CH2N), 3.22 (t,
J = 6.4 Hz, 2H, CH2NBoc), 3.80 (s, 2H, CH2Ph), 5.19 (br s, 1H, NH),
7.27–7.32 (m, 5H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 264 (M+), 265 (M+1), 266 (M+2).
7.4. General procedure for the preparation of compounds 11
and 12
The appropriate amine 9 or 10 (16.90 mmol) charged with tet-
rahydrofuran (50 mL) in 100 mL round bottom flask. Solid potas-
sium tert-butoxide (51.0 mmol) was added and the resulting
yellow solution was heated to 60 °C for 3 h. The mixture was
cooled to ambient temperature and acidified with aqueous HCl
(35 mL, 1 M) and concentrated under reduced pressure. The aque-
ous residue was extracted with ethyl acetate, organic phase
washed twice with brine, dried over anhydrous Na2SO4 and con-
centrated under reduced pressure to give viscous oil which was
solidified on standing. The desired compound was purified by flash
column chromatography.
7.3.8. tert-Butyl 3-(4-methylbenzylamino)propylcarbamate
(10b)
Yield 90%; viscous oil; 1H NMR (CDCl3) d 1.42 (s, 9H, tert-butyl),
1.62–1.70 (m, 2H, CCH2CN), 2.30 (s, 3H, CH3Ph), 2.69 (t, J = 6.4 Hz,
2H, CH2N), 3.20 (t, J = 6.0 Hz, 2H, CH2NBoc), 3.73 (s, 2H, CH2Ph),
5.31 (br s, 1H, NH), 7.11–7.26 (m, 5H, Ar-H). Mass spectra of com-
pound exhibited molecular ion peak at m/z 278 (M+), 279 (M+1),
280 (M+2).
7.3.9. tert-Butyl 3-(4-chlorobenzylamino)propylcarbamate
(10c)
Yield 73%; viscous oil; 1H NMR (CDCl3) d 1.42 (s, 9H, tert-butyl),
1.67–1.70 (m, 2H, CCH2CN), 2.68 (t, J = 6.0 Hz, 2H, CH2N), 3.20 (t,
J = 6.0 Hz, 2H, CH2NBoc), 3.76 (s, 2H, CH2Ph), 5.30 (br s, 1H, NH),
7.27–7.33 (m, 4H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 298 (M+), 300 (M+2).
7.4.1. 1-Benzylimidazolidin-2-one (11a)
Yield 71%; yellow solid; mp 124 °C; Rf 0.27 (7:3 ethyl acetate/
hexane); IR (neat): 3236, 1677 cmÀ1 1H NMR (CDCl3) d 3.29 (m,
;
2H, NCH2CN), 3.42 (m, 2H, NCCH2N), 4.37 (s, 2H, CH2Ph), 4.53 (br
s, 1H, NH) 7.29 (m, 5H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 176 (M+), 177 (M+1).
7.3.10. tert-Butyl 3-(4-methoxybenzylamino)propylcarbamate
(10d)
7.4.2. 1-(4-Methylbenzyl)imidazolidin-2-one (11b)
Yield 67%; viscous oil; 1H NMR (CDCl3) d 1.37 (s, 9H, tert-butyl),
1.49–1.52 (m, 2H, CCH2CN), 2.43 (t, J = 6.4 Hz, 2H, CH2N), 2.94 (t,
J = 6.0 Hz, 2H, CH2NBoc), 3.56 (s, 3H, CH3OPh), 3.70 (s, 2H,
NCH2Ph), 5.74 (br s, 1H, NH), 6.82 (d, J = 8.4 Hz, 2H, Ar-H), (d,
Yield 67%; brown solid; mp 148 °C: Rf 0.34 (7:3 ethyl acetate/
hexane); IR (neat): 3228, 1681 cmÀ1 1H NMR (CDCl3) d 2.34 (s,
;
3H, CH3), 3.2 (t, J = 7.2 Hz, 2H, NCH2CN), 3.45 (t, J = 7.2 Hz, 2H,
NCCH2N), 4.32 (s, 2H, CH2Ph), 4.50 (br s, 1H, NH), 7.18–7.23 (m,

1140
P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1135–1142
4H, Ar-H). Mass spectra of compound exhibited molecular ion peak
CH2N), 3.82 (s, 3H, OCH3), 4.43 (s, 2H, NCH2Ph), 5.18 (br s, 1H,
NH), 6.82 (d, J = 8.2 Hz, 2H, Ar-H), 7.29 (d, J = 8.2 Hz, 2H, Ar-H).
Mass spectra of compound exhibited molecular ion peak at m/z
220 (M+), 221 (M+1).
at m/z 190 (M+), 191 (M+1).
7.4.3. 1-(4-Chlorobenzyl)imidazolidin-2-one (11c)
Yield 55%; white solid; mp 154 °C; Rf 0.14 (1:1 ethyl acetate/
hexane); IR (neat): 3235, 1678 cmÀ1
;
1H NMR (CDCl3) d 3.19 (m,
7.4.11. 1-(4-Ethylbenzyl)tetrahydropyrimidin-2(1H)-one (12e)
Yield 53%; colorless solid; mp 104 °C; Rf 0.10 (7:3 ethyl acetate/
hexane); IR (neat) 2926, 2856, 1791, 1651 cmÀ1; 1H NMR (CDCl3) d
1.22 (t, J = 7.6 Hz, 3H, CH3), 1.86–1.92 (m, 2H, CCH2C), 2.63 (q,
J = 7.6 Hz, 2H, PhCH2), 3.14 (t, J = 7.2 Hz, 2H, CH2N), 3.30 (t,
J = 6.8 Hz, 2H, CH2N), 4.85 (s, 2H, ArCH2N), 4.64 (br s, 1H, NH),
7.14 (d, J = 8.0 Hz, 2H, Ar-H), 7.18 (d, J = 8.0 Hz, 2H, Ar-H). Mass
spectra of compound exhibited molecular ion peak at m/z 218
(M+), 219 (M+1).
2H, NCH2CN), 3.41 (m, 2H, NCCH2N), 4.37 (s, 2H, CH2Ph), 4.41 (br
s, 1H, NH), 7.23 (d, J = 8.2 Hz, 2H, Ar-H), 7.32 (d, J = 8.2 Hz, 2H,
Ar-H). Mass spectra of compound exhibited molecular ion peak
at m/z 210 (M+), 212 (M+2).
7.4.4. 1-(4-Methoxybenzyl)imidazolidin-2-one (11d)
Yield 60%; yellow solid; mp 130 °C; Rf 0.23 (7:3 ethyl acetate/
hexane); IR (neat): 3236, 1677 cmÀ1 1H NMR (CDCl3) d 3.26 (t,
;
J = 6.4 Hz, 2H, NCH2CN), 3.40 (t, J = 6.4 Hz, 2H, NCCH2N), 3.8 (s,
3H, OCH3), 4.34 (s, 2H, CH2Ph), 4.28 (br s, 1H, NH), 6.82 (d,
J = 7.8 Hz, 2H, Ar-H), 7.22 (d, J = 7.8 Hz, 2H, Ar-H). Mass spectra of
compound exhibited molecular ion peak at m/z 206 (M+), 207
(M+1).
7.4.12. 1-(4-tert-Butylbenzyl)tetrahydropyrimidin-2(1H)-one
(12f)
Yield 59%; colorless solid; mp 127 °C; Rf 0.36 (ethyl acetate); IR
(neat) 2928, 2857, 1641 cmÀ1 1H NMR (CDCl3) d 1.39 (s, 9H, tert-
;
butyl), 1.87–1.93 (m, 2H, CCH2C), 3.18 (t, J = 6.0 Hz, 2H, CH2N), 3.30
(t, J = 6.4 Hz, 2H, CH2N), 4.51 (s, 1H, Ar-CH2N), 4.90 (br s, 1H, NH),
7.19 (d, J = 8.8 Hz, 2H, Ar-H), 7.19 (d, J = 8.4 Hz, 2H, Ar-H). Mass
spectra of compound exhibited molecular ion peak at m/z 246
(M+), 247 (M+1).
7.4.5. 1-Phenylehtylimidazolidin-2-one (11e)
Yield 62%; yellow solid; mp 132 °C; Rf 0.25 (7:3 ethyl acetate/
hexane); IR (neat): 3234, 1677 cmÀ1 1H NMR (CDCl3) d 2.87 (t,
;
J = 7.2 Hz, 2H, CH2Ph), 3.08–3.26 (m, 4H, NCH2CH2N), 3.50 (t,
J = 7.6 Hz, 2H, NCH2CPh), 5.01 (br s, 1H, NH), 7.26–7.31 (m, 5H,
Ar-H). Mass spectra of compound exhibited molecular ion peak
at m/z 190 (M+), 191 (M+1).
7.4.13. 3-Phenylethyltetrahydropyrimidin-2(1H)-one (12g)
Yield 61%; viscous oil; Rf 0.19 (7:3 ethyl acetate/hexane); IR
(neat) 3236, 1678 cmÀ1 1H NMR (CDCl3) d 1.98 (m. 2H, CCH2C),
;
7.4.6. 1-(3-Phenylpropyl)imidazolidin-2-one (11f)
2.72 (t, J = 7.6 Hz, 2H, CH2Ph), 3.09 (t, J = 6.0 Hz, 2H, CH2N), 3.25
(t, J = 6.4 Hz, 2H, CH2N), 3.53 (t, J = 7.6 Hz, 2H, CH2N), 5.28 (br s,
1H, NH), 7.38 (m, 5H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 204 (M+), 205 (M+1).
Yield 55%; yellow oil; Rf 0.20 (6:4 ethyl acetate/hexane); IR
(neat) 3227, 1683 cmÀ1 1H NMR (CDCl3) d 1.62–1.71 (m, 2H,
;
NCCH2CPh), 2.83 (t, J = 6.4 Hz, 2H, CH2Ph), 3.12–3.32 (m, 4H,
NCH2CH2N), 3.81 (t, J = 6.4 Hz, 2H, CH2N), 5.27 (br s, 1H, NH),
7.27–7.43 (m, 5H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 204 (M+), 205 (M+1).
7.4.14. 3-(3-Phenylpropyl)tetrahydropyrimidin-2(1H)-one
(12h)
Yield 55%; viscous oil; Rf 0.21 (7:3 ethyl acetate/hexane); IR
7.4.7. 1-Benzyltetrahydropyrimidin-2(1H)-one (12a)
(neat) 2926, 2859, 1650 cmÀ1 1H NMR (CDCl3) d 1.88–190 (m,
;
Yield 84%; orange solid; mp 146 °C; Rf 0.12 (7:3 ethyl acetate/
4H), 2.60 (t, J = 6.4 Hz, 2H, CH2Ph), 3.16 (t, J = 6.0 Hz, 2H, NCH2),
3.20–3.24 (m, 4H, CH2NCH2), 3.72 (t, J = 7.2 Hz, 2H, NCH2), 5.43
(s, 1H, NH), 7.15–7.27 (m, 5H, Ar-H). Mass spectra of compound
exhibited molecular ion peak at m/z 218 (M+), 219 (M+1).
hexane); IR (neat) 3236, 1677 cmÀ1 1H NMR (CDCl3) d 1.90 (m,
;
2H,CCH2C), 3.18 (t, J = 6.0 Hz, 2H, CH2N), 3.33 (t, J = 5.6 Hz, 2H,
CH2N), 4.56 (s, 2H, NCH2Ph), 4.96 (br s, 1H, NH), 7.31–7.37 (m,
5H, Ar-H). Mass spectra of compound exhibited molecular ion peak
at m/z 190 (M+), 191 (M+1).
7.5. General procedure for the preparation of compounds 2 and
3
7.4.8. 3-(4-Methylbenzyl)tetrahydropyrimidin-2(1H)-one (12b)
Yield 72%; brown solid; mp 108 °C; Rf 0.15 (7:3 ethyl acetate/
To the solution of urea compounds 11 or 12 in anhydrous tolu-
ene (75 mL) was added Lawesson’s reagent (1.2 equiv) under nitro-
gen atmosphere. The resulting solution was refluxed for overnight.
Toluene was removed and diluted with water. The aqueous mix-
ture was extracted with dichloromethane. The organic phase was
washed twice with brine, dried over anhydrous Na2SO4, and con-
centrated under reduced pressure to give viscous oil. The crude
material was purified by flash column chromatography
hexane); IR (neat) 3236, 1677 cmÀ1 1H NMR (CDCl3) d 1.88 (m,
;
2H, CCH2C), 2.33 (s, 3H, CH3Ph), 3.15 (t, J = 6.0 Hz, 2H, CH2N),
3.32 (t, J = 6.4 Hz, 2H, CH2N), 4.51 (s, 2H, NCH2Ph), 4.83 (br s, 1H,
NH), 7.18 (d, J = 8.0 Hz, 2H, Ar-H), 7.20 (d, J = 8.0 Hz, 2H, Ar-H).
Mass spectra of compound exhibited molecular ion peak at m/z
204 (M+), 205 (M+1).
7.4.9. 1-(4-Chlorobenzyl)tetrahydropyrimidin-2(1H)-one (12c)
Yield 67%; yellow solid; mp 102 °C; Rf 0.10 (7:3 ethyl acetate/
7.5.1. 1-Benzylimidazolidine-2-thione (2a)
hexane); IR (neat) 3229, 1677 cmÀ1
;
1H NMR (CDCl3) d 1.89 (m,
Yield 50%; white solid; mp 180 °C; Rf 0.29 (4:6 ethyl acetate/
2H, CCH2C), 3.16 (t, J = 5.6 Hz, 2H, CH2NH), 3.33 (t, J = 6.0 Hz, 2H,
CH2N), 4.50 (s, 2H, NCH2Ph), 5.08 (br s, 1H, NH), 7.21–7.28 (m,
4H, Ar-H). Mass spectra of compound exhibited molecular ion peak
at m/z 224 (M+), 226 (M+2).
hexane); IR (neat) 3205, 2360, 1494, 1452 cmÀ1 1H NMR (CDCl3)
;
d 3.56 (s, 4H, NCH2CH2N) 4.8 (s, 2H, CH2Ph), 5.72 (br s, 1H, NH),
7.31–7.37 (m, 5H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 192 (M+), 193 (M+1), 194 (M+2).
7.4.10. 3-(4-Methoxybenzyl)tetrahydropyrimidin-2(1H)-one
(12d)
7.5.2. 1-(4-Methylbenzyl)imidazolidine-2-thione (2b)
Yield 50%; white solid; mp 158 °C; Rf 0.41 (4:6 ethyl acetate/
Yield 50%; yellow solid; mp: 76 °C; Rf 0.11 (7:3 ethyl acetate/
hexane); IR (neat) 3215, 2360, 1500, 1477 cmÀ1 1H NMR (CDCl3)
;
hexane); IR (neat) 3233, 1674 cmÀ1
;
1H NMR (CDCl3) d 1.88 (m,
d 2.34 (s, 3H, CH3), 3.54 (s, 4H, NCH2CH2N), 4.76 (s, 2H, CH2Ph),
6.26 (br s, 1H, NH) 7.15 (d, J = 8.0 Hz, 2H, Ar-H), 7.24 (d,
2H, CCH2C), 3.13 (t, J = 6.0 Hz, 2H, CH2N), 3.28 (t, J = 4.8 Hz, 2H,

P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1135–1142
1141
J = 8.0 Hz, 2H, Ar-H). Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 206 (M+), 207 (M+1), 208 (M+2).
d 1.97–2.24 (m, 2H, CCH2C), 3.22 (t, J = 6.0 Hz, 2H, CH2N), 3.35 (t,
J = 6.4 Hz, 2H, CH2N), 3.38 (s, 3H, OCH3), 5.10 (s, 2H, NCH2Ph),
6.28 (br s, 1H, NH), 6.85 (d, J = 7.6 Hz, 2H, Ar-H), 7.33 (d,
J = 8.0 Hz, 2H, Ar-H). Mass spectra of compound exhibited molecu-
lar ion peak at m/z 236 (M+), 237 (M+1), 238 (M+2).
7.5.3. 1-(4-Chlorobenzyl)imidazolidine-2-thione (2c)
Yield 45%; white solid; mp 203 °C; Rf 0.22 (4:6 ethyl acetate/
hexane); IR (neat) 3258, 2359, 1543, 1464 cmÀ1 1H NMR (CDCl3)
;
d 3.56 (s, 4H, NCH2CH2N), 4.72 (s, 2H, NCH2Ph), 5.88 (br s, 1H,
NH), 7.23 (d, J = 8.2 Hz, 2H, Ar-H), 7.33 (d, J = 8.2 Hz, 2H, Ar-H).
Mass spectra of compound exhibited molecular ion peak at m/z
226 (M+), 228 (M+2).
7.5.11. 1-(4-Ethylbenzyl)tetrahydropyrimidine-2(1H)-thione
(3e)
Yield 45%; colorless solid; mp 155 °C; Rf 0.37 (7:3 ethyl acetate/
hexane); IR (neat) 3231, 2928, 2854, 1542, 1449 cmÀ1 1H NMR
;
(CDCl3) d 1.22 (t, J = 7.6 Hz, 3H, CH3), 1.92–1.98 (m, 2H, CCH2C),
2.60 (q, J = 7.6 Hz, 2H, Ar-CH2), 3.22 (t, J = 6.0 Hz, 2H, CH2N), 3.29
(t, J = 6.4 Hz, 2H, CH2N), 5.11 (s, 2H, Ar-CH2N), 6.31 (br s, 1H,
NH), 7.15 (d, J = 8.4 Hz, 2H, Ar-H), 7.28 (d, J = 8.4 Hz, 2H, Ar-H).
Mass spectra of compound exhibited molecular ion peak at m/z
234 (M+), 235 (M+1), 236 (M+2).
7.5.4. 1-(4-Methoxybenzyl)imidazolidine-2-thione (2d)
Yield 40%; brown solid; mp 162 °C; Rf 0.23 (4:6 ethyl acetate/
hexane); IR (neat) 3210, 2360, 1510, 1454 cmÀ1 1H NMR (CDCl3)
;
d 3.54 (s, 4H, NCH2CH2N), 3.80 (s, 3H, OCH3), 4.73 (s, 2H, CH2Ph),
6.25 (br s, 1H, NH), 6.82 (d, J = 8.4 Hz, 2H, Ar-H), 7.26 (d,
J = 8.8 Hz, 2H, Ar-H). Mass spectra of compound exhibited molecu-
lar ion peak at m/z 222 (M+), 223 (M+1), 224 (M+2).
7.5.12. 1-(4-tert-Butylbenzyl)tetrahydropyrimidine-2(1H)-
thione (3f)
7.5.5. 1-Phenylehtylimidazolidine-2-thione (2e)
Yield 45%; colorless solid; mp 187 °C; Rf 0.42 (7:3 ethyl acetate/
Yield 50%; brown solid; mp 93 °C; Rf 0.30 (4:6 ethyl acetate/
hexane); IR (neat): 2927, 2856, 1856, 1605, 1458 cmÀ1 1H NMR
;
hexane); IR (neat) 3297 2360, 1492, 1457 cmÀ1
;
1H NMR (CDCl3)
(CDCl3) d 1.31 (s, 9H, tert-butyl), 1.93–2.01 (m, 2H, CCH2C), 3.24
(t, J = 6.4 Hz, 2H, CH2N), 3.30 (t, J = 6.0 Hz, 2H, CH2N), 5.41 (s, 2H,
Ar-CH2), 6.31 (br s, 1H, NH), 7.27 (d, J = 8.4 Hz, 2H, Ar-H), 7.34 (d,
J = 8.0 Hz, 2H, Ar-H). Mass spectra of compound exhibited molecu-
lar ion peak at m/z 262 (M+), 263 (M+1), 264 (M+2).
d 2.82 (t, J = 7.2 Hz, 2H, CH2Ph), 3.35 (m, 4H, NCH2CH2N), 3.45 (t,
J = 7.2 Hz, 2H, NCH2CPh), 4.37 (br s, 1H, NH), 7.28 (m, 5H, Ar-H).
Mass spectra of compound exhibited molecular ion peak at m/z
206 (M+), 207 (M+1), 208 (M+2).
7.5.6. 1-(3-Phenylpropyl)imidazolidine-2-thione (2f)
7.5.13. 3-Phenylethyltetrahydropyrimidine-2(1H)-thione (3g)
Yield 50%; yellow solid; mp 111 °C; Rf 0.20 (4:6 ethyl acetate/
Yield 55%; brown solid; mp 61 °C Rf 0.30 (4:6 ethyl acetate/hex-
ane); IR (neat) 3224, 2360, 1496, 1456 cmÀ1
;
1H NMR (CDCl3) d
hexane); IR (neat) 3238, 2360, 1531, 1495 cmÀ1 1H NMR (CDCl3)
;
1.92–1.98 (m, 2H, CCH2CPh), 2.62 (t, J = 7.6 Hz, 2H, CH2Ph), 3.53
(t, J = 7.2 Hz, 2H, NCH2), 3.65–3.70 (each m, 4H, NCH2CH2N), 5.60
(br s, 1H,NH), 7.27 (m, 5H, Ar-H). Mass spectra of compound exhib-
ited molecular ion peak at m/z 220 (M+), 221 (M+1), 222 (M+2).
d 1.91- 1.96 (m, 2H), 3.02 (t, J = 6.4 Hz, 2H, PhCH2), 3.18 (t,
J = 5.6 Hz, 2H, CH2N), 3.26 (t, J = 6.0 Hz, 2H, CH2N), 4.74 (t,
J = 7.2 Hz, 2H, PhCCH2N), 6.23 (br s, 1H, NH), 7.29–7,32 (m, 5H,
Ar-H). Mass spectra of compound exhibited molecular ion peak
at m/z 220 (M+), 221 (M+1), 222 (M+2).
7.5.7. 1-Benzyltetrahydropyrimidine-2(1H)-thione (3a)
Yield 60%; brown solid; mp 166 °C; Rf 0.16 (4:6 ethyl acetate/
7.5.14. 3-(3-Phenylpropyl)tetrahydropyrimidine-2(1H)-thione
(3h)
hexane); IR (neat) 3194, 2360, 1536, 1516 cmÀ1 1H NMR (CDCl3)
;
d 1.95–2.05 (m, 2H, CCH2C), 3.23 (t, J = 6.0 Hz, 2H, CH2NH), 3.33
(t, J = 6.4 Hz, 2H, CH2N), 5.18 (s, 2H, NCH2Ph), 6.38 (br s, 1H, NH),
7.30–7.32 (m, 5H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 206 (M+), 207 (M+1), 208 (M+2).
Yield 68%; brownsolid; mp 131 °C; Rf 0.13 (4:6 ethyl acetate/hex-
ane); IR (neat) 3228, 2360, 1541, 1521 cmÀ1; 1H NMR (CDCl3) d 1.93–
2.03 (m, 4H), 2.67 (t, J = 7.6 Hz, 2H, PhCH2), 3.23–3.30 (m, 4H,
NCH2CCH2N), 3.90 (t, J = 7.6 Hz, 2H, NCH2CCPh), 6.16 (br s, 1H,
NH), 7.17–7.34 (m, 5H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 234 (M+), 235 (M+1), 236 (M+2).
7.5.8. 3-(4-Methylbenzyl)tetrahydropyrimidine-2(1H)-thione
(3b)
Yield 58%; white solid; mp 178 °C; Rf 0.28 (4:6 ethyl acetate/
7.6. Biological evaluation
hexane); IR (neat) 3216, 1531, 2360, 1501 cmÀ1 1H NMR (CDCl3)
;
d
1.91–1.97 (m, 2H, CCH2C), 2.33 (s, 3H, CH3Ph), 3.23 (t,
7.6.1. Chemicals and antisera
J = 5.6 Hz, 2H, CH2NH), 3.32 (t, J = 6.0 Hz, 2H, CH2N), 5.13 (s, 2H,
NCH2Ph), 6.53 (br s, 1H, NH), 7.23 (d, J = 7.6 Hz, 2H, Ar-H), 7.28
(d, J = 7.6 Hz, 2H, Ar-H). Mass spectra of compound exhibited
molecular ion peak at m/z 220 (M+), 221 (M+1), 222 (M+2).
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-
cals, including
a-MSH, were otherwise purchased from Sigma–
7.5.9. 1-(4-Chlorobenzyl)tetrahydropyrimidine-2(1H)-thione
(3c)
Aldrich (St. Louis, MO).
Yield 50%; Yellow solid; mp 208 °C; Rf 0.23 (4:6 ethyl acetate/
7.6.2. Cell culture
hexane); IR (neat) 3212, 2360, 1542, 1487 cmÀ1
;
1H NMR (CDCl3)
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 sul-
fate, 24 mM NaHCO3, pH 7.1) containing 10% FBS, and incubated at
d 1.98–2.23 (m, 2H, CCH2C), 3.22 (t, J = 6.0 Hz, 2H, CH2NH), 3.33
(t, J = 6.0 Hz, 2H, CH2N), 5.14 (s, 2H, NCH2Ph), 6.33 (br s, 1H, NH),
7.29 (m, 4H, Ar-H). Mass spectra of compound exhibited molecular
ion peak at m/z 240 (M+), 242 (M+2).
37 °C under 5% CO2 atmosphere.
7.5.10. 3-(4-Methoxybenzyl)tetrahydropyrimidin-2(1H)-thione
(3d)
7.6.3. Melanin quantification
Yield 55%; yellow solid; mp 161 °C; Rf 0.17 (4:6 ethyl acetate/
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
hexane); IR (neat) 3210, 1543, 1510, 1452 cmÀ1 1H NMR (CDCl3)
;

1142
P. Thanigaimalai et al. / Bioorg. Med. Chem. 18 (2010) 1135–1142
5% CO2 atmosphere for 24 h. The cells were then stimulated with
-MSH (10 nM) for 72 h, in the presence of sample. The cells were
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT,500 g/ml) for 2 h. The MTT-formazan complex was dissolved
a
l
harvested, and then disrupted in 1 N NaOH-10% dimethylsulfoxide
with heating at 80 °C. Melanin contents were measured by absor-
bance values at wavelength 405 nm with synthetic melanin as a
standard.
in 100% dimethyl sulfoxide and optical densities were then mea-
sured at wavelength 590 nm.
Acknowledgment
7.6.4. Measurement of tyrosinase activity
This research was financially supported by a grant (R01-2007-
000-20099-0) from the National Research Foundation of Korea.
B16 cells were treated with a-MSH (10 nM) alone for 72 h. After
washing, the cells were resuspended in sodium phosphate buffer
(50 mM, pH 6.8) containing 1% Triton X-100 and phenylmethylsul-
fonyl fluoride (1 mM), and then subjected to sonication on ice.
After centrifugation, supernatants were dialyzed against sodium
phosphate buffer and then used as the sources of cell-free tyrosi-
nase. Dopa oxidation activity of tyrosinase was determined as de-
scribed previously.14 Briefly, dopa (5 mM) and sample were mixed
in sodium phosphate buffer (50 mM, pH 6.8) and finally added
with enzyme sources. Initial velocity of dopachrome formation
from the reaction mixture was determined by the increase of
absorbance values at wavelength 475 nm per min. One unit of
tyrosinase activity was defined as the conversion of 1 nmol dopa
to dopachrome per min.
References and notes
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7.6.6. MTT assay
B16 cells were treated with various concentration of sample for
72 h, in the presence of a-MSH (10 nM). The cells were exposed to