Refinement of the pharmacophore of 3,4-dihydroquinazoline-2(1H)-thiones for their anti-melanogenesis activity

Article abstract of DOI:10.1016/j.bmcl.2010.06.123

In order to define the structural requirements of quinazoline-2(1H)-thiones 1 for their inhibitory activity on melanogenesis, a novel series of 3,4-dihydroquinazoline-2(1H)-thiones (3a-h) were prepared and screened for their melanogenesis inhibition on melanoma B16 cell line under the stimulant of α-MSH. The anti-melanogenesis activity of 3 is mainly mediated by the hydrogen bonding ability of thioamide unit in addition to complexation ability of thione and the hydrophobic binding power of side chain substitutions at 3-position. Thus, the pharmacophore of 3,4-dihydroquinazoline-2(1H)-thiones for their anti-melanogenesis activity could be refined as 3-hydrophobic substituted quinazolinethione.

Full text of DOI:10.1016/j.bmcl.2010.06.123

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4771–4773
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Refinement of the pharmacophore of 3,4-dihydroquinazoline-2(1H)-thiones
for their anti-melanogenesis activity
Pillaiyar Thanigaimalai a, Vinay K. Sharma a, Ki-Cheul Lee a, Cheong-Yong Yun 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:
In order to define the structural requirements of quinazoline-2(1H)-thiones 1 for their inhibitory activity
on melanogenesis, a novel series of 3,4-dihydroquinazoline-2(1H)-thiones (3a–h) were prepared and
Received 21 May 2010
Revised 19 June 2010
Accepted 23 June 2010
Available online 1 July 2010
screened for their melanogenesis inhibition on melanoma B16 cell line under the stimulant of
a-MSH.
The anti-melanogenesis activity of 3 is mainly mediated by the hydrogen bonding ability of thioamide
unit in addition to complexation ability of thione and the hydrophobic binding power of side chain sub-
stitutions at 3-position. Thus, the pharmacophore of 3,4-dihydroquinazoline-2(1H)-thiones for their anti-
melanogenesis activity could be refined as 3-hydrophobic substituted quinazolinethione.
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Keywords:
3,4-Quinazoline-2(1H)-thiones
Inhibitors
Melanogenesis
In human, skin is the main barrier from external environment,
and relies on melanocytes to provide photoprotection by produc-
ing melanin through the process known as melanogenesis. Chem-
ically, melanin is heterogeneous biopolymers yielded from the
combination of enzymatic and chemical reaction in melanocytes.1,2
Melanin synthesized from the oxidation of tyrosine to dopaqui-
none by tyrosinase, which occurs in two steps: hydroxylation of
duced melanogenesis in melanoma B16 cell line.18 This is one of
the important approaches to inhibit melanogenesis by down
regulation of tyrosinase expression other than changing the tyros-
inase catalytic activity. These results encourage us to go for more
detailed structure–activity relationship (SAR) of the compound 1
to get more potent and safer whitening agents. Thus, the current
research focused on the design and synthesis of number of deriva-
tives of compound 1. In our previous studies,19,20 we discovered
the following structural requirements for our lead 1 regarding
(i) role of ring A and their substitutions, (ii) optimum size of the
ring C (thiourea unit) and (iii) nature of the substituents on ring
C. However, the exact requirements of ring C (thiourea unit) and
the substituents on ring C in 1 have not been fully explored. Thus,
a novel series of quinazolines 2, 3 and 4 (Chart 1) were synthesized
and their inhibitory activity were measured against melanogenesis
L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and then oxi-
dation of the latter to an o-quinone (dopaquinone).3 This first step
is the key step in melanogenesis because the reminder of the reac-
tion sequence can proceed spontaneously at physiological pH.4
Tyrosinase, a copper bearing phenolase, not only involves in abnor-
mal production of melanin (hyperpigmentation)5,6 but also has
been reported to be linked with Parkinson disease and related neu-
rodegenerative diseases.7,8 In addition, the well known whitening
agents such as hydroquinone-O-b-glucopyranoside (arbutin)9–11
and kojic acid12 are considered as harmful agents due to their
undesirable side effects.13 Taken together the current therapies
are considered to be inadequate for the skin treatment despite
many compounds have been reported as tyrosinase inhibitor.14–
17 These reports induced researchers to seek new potent tyrosinase
inhibitors. Therefore, to meet the current demand for more safer
whitening agents, we discovered compound 1, (6-methyl-3-phen-
in melanoma B 16 cells under the stimulant of a-MSH.
B
R
R1
N
N
A
C
C
A
ethyl-3,4-dihydro-1H-quinozoline-2-thione, IC50 = 0.8
1) that inhibited -melanocyte stimulated hormone (
l
M, Chart
N
R2
X
N
H
S
a
a-MSH) in-
1
2, 3 and 4
* Corresponding author. Tel.: +82 42 821 5939; fax: +82 42 823 6566.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
Chart 1. Melanogenesis inhibitors 1, 2, 3 and 4.
0960-894X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.123

4772
P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4771–4773
R1
R1
N
H
NH2
a
Cl
NO2
c
N
H
NO2
R
+
R
R
NH2
8
10
6
11
d
b
R1
O
N
R
Cl
H
N
H
O
NH2
+
2
NO2
7
9
e
R1
S
R1
S
N
f
N
R
R
N
H
N
R2
3
4
Scheme 1. Preparation of 2, 3 and 4. Reagents and conditions: (a) TEA, CH2Cl2, reflux, overnight; (b) TEA, NaBH4, methanol, reflux, 4 h; (c) 10% Pd/C, 30-psi, rt, 5 h; (d) 1,1-
carbonyldiimidazole, THF, reflux, overnight; (e) Lawesson’s reagent, toluene, reflux, overnight; (f) KOH, tetra-(n-butyl)ammonium bromide.
Table 1
The substituents of 2, 3 and 4
sium hydroxide.27 Various substituents and the physiochemical
properties of 2, 3 and 4 are indicated in Table 1.
R
R1
X
N
All the above synthesized derivatives were evaluated for their
inhibitory activity on melanogenesis19,20 using
a
-melanocyte stim-
-MSH) induced melanogenesis in melanoma
B16 cell line as shown in Table 2. Quinazolinethiones 3a–g (100%
inhibition at 10 M and their IC50 values: ranged from 1.2 to
3.0
N
ulated hormone (
a
R2
l
Compd
R
R1
R2
X
Yieldb (%)
Mp (°C)
l
M) containing thiourea motif showed strong inhibition
2a
2b
2c
2d
2e
2f
2g
2ha
3a
3b
3c
3d
3e
3f
CH3
CH3
CH3
CH3
CH3
CH3
Cl
C(CH3)2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
S
S
S
S
S
80
83
85
75
82
90
85
95
60
55
63
68
70
55
52
65
73
70
166–167
160–161
143–144
150–152
158–160
162–163
166–167
231–233
157–158
168–169
169–170
179–181
175–177
185–186
169–161
199–201
129–131
137–139
against melanogenesis.20 However, quinazolinones 2a–h contain-
ing urea unit do not exhibit the activity. This trend has also been
noticed in earlier reports,28,29 as indicated that phenylthiourea
CH2C(CH3)2
CH2CH2C(CH3)2
CH2)5CH3
(CH2)6CH3
CH2C6H11
CH2C6H11
H
(PTU, 58% inhibition at 100
phenylurea (0% inhibition at 100
l
M) showed better inhibition than
M), though the mechanism of
l
PTU activity (IC50 value for tyrosinase inhibition: 1.8 lM) was quite
different from quinazolinethiones 3 and 4.
In the first set of experiment, we studied the role of ring B in
the SAR of lead 1. The resulting synthesized compounds 3a and
3b by replacing ring B with aliphatic groups as the isopropyl ana-
H
CH3
CH3
CH3
CH3
CH3
CH3
Cl
H
CH3
CH3
C(CH3)2
CH2C(CH3)2
CH2CH2C(CH3)2
CH2)5CH3
CH2)6CH3
CH2C6H11
CH2C6H11
H
log 3a (>100% inhibition at 10
lM, IC50 = 1.9
lM, C log P = 3.030)
S
S
S
S
and isobutyl 3b (>100% inhibition at 10
lM, IC50 = 1.9 M, C
l
3g
3ha
4a
4b
log P = 3.649) exhibited similar activity to 1. This study revealed
that the phenyl group (ring B) can be replaced by an aliphatic
chain to maintain the activity. These results motivated us to
investigate the effects of volume or lipophilicity on the activity
by varying the sizes of alkyl groups as side chain of 3. Thus, more
bulky aliphatic groups were introduced at this position such as
C (CH3)2
CH2C(CH3)2
CH3
CH3
S
a
Compounds 2h and 3h are reported with following Refs. 23,25.
The yield of 4(a–b) was obtained from the corresponding cyclic thiourea 3(a–b),
3(a–h) form 2(a–h) and 2(a–h) from an intermediate 11.
b
isopentyl 3c (>100% inhibition at 10
4.178), n-hexyl 3d, (>100% inhibition at 10
log P = 4.837), n-heptyl 3e (>100% inhibition at 10
1.4 M, C log P = 5.366) and cyclohexyl methyl 3f (>100% inhibi-
tion at 10 M, IC50 = 1.6 M, C log P = 4.842) as a replacement of
ring B. From these results, we observed that there was not much
change in activity along with the variation of alkyl chain. These
outcomes not only prove that the ring B can be replaced by ali-
phatic groups but also the chain length of among these groups
dose not have influence on the activity of quinazolinethiones.
Thus, the side chain of 1 and 3 may have a panhandle effect
for proper arrangement of the molecules by their binding to
hydrophobic pocket in the binding site rather than controller of
lipophilicity (C log P values in Table 2).
l
M, IC50 = 3.0
M, IC50 = 1.2
M, IC50
l
M, C log P =
l
lM, C
The detailed chemistry20 involved in synthesis of 2, 3 and 4 as
shown in Scheme 1. The alkylation of amine 8 with o-nitrobenzyl
chloride 6 yielded key intermediate 10. On the other hand, the
cyclohexylmethylamine 9 treated with aldehyde 7 to produce 10
by reductive amination.21,22 This nitro compound 10 was
underwent reduction with 10% Pd/C under 30 psi hydrogen atmo-
sphere at room temperature for 5 h to get the intermediate 1120
which was used without further purification. Compound 2 ob-
tained by the cyclization of 11 using 1,1-carbonyldiimidazole,18,23
which was further treated with Lawesson’s reagent24–26 in toluene
to get corresponding thio analogs 3. Finally, N-methylated analog 4
was obtained from 3 using methyl iodide in the presence of potas-
l
=
l
l
l

P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4771–4773
4773
Table 2
The inhibitory activity of 2, 3 and 4 on melanogenesis using
a
-melanocyte stimulated hormone (
a-MSH) induced melanogenesis in melanoma B16 cell line
a
a
Compd
Inhibition at 10
l
M (%)
IC50
(lM)
C log Pb
Compd
Inhibition at 10
lM (%)
IC50
(
lM)
C log Pb
2a
2b
2c
2d
2e
2f
2g
2h
1
13
11
15
12
19
18
10
11
>10
>10
>10
>10
>10
>10
>10
>10
0.8
2.370
2.989
3.518
4.177
4.706
4.182
4.6984
0.877
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
>100
>100
>100
>100
>100
>100
>100
36
1.9
1.9
3.0
1.2
1.4
1.6
1.2
15.8
19.0
17.8
3.030
3.649
4.178
4.837
5.366
4.842
5.056
0.657
3.561
4.180
>100
10
18
Arbutin
180
a
IC50 values are taken as mean from three independent experiments.
C log P values were calculated by Chem Draw 9.0 v.
b
In our previous study20 we observed the importance of ring B of
1 as well as substitution on ring A for their activity. In order to con-
firm this fact, we synthesized an analog 3h (36% inhibition at
the National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology.
10 lM, IC50 = 15.8 lM, C log P = 0.657) with omission of side chain
Supplementary data
and substitution on ring A of 1. As expected, the analog 3h showed
very low inhibition compared to 1. This result strongly implies that
its low melanogenesis inhibition may be due to lack of binding to
the hydrophobic pocket of putative receptor. This also proves that
the panhandle effect of the side chain of 1 and 3 is important for
their activity.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.123.
References and notes
1. Prota, G. Med. Res. Rev. 1988, 8, 525.
2. Kuzumaki, T.; Matsuda, A.; Wakamatsu, K.; Ito, S.; Ishikawa, K. Exp. Cell Res.
1993, 207, 33.
3. Hearing, V. J.; Jimenez, M. Int. J. Biochem. 1987, 19, 1141.
4. Halaban, R.; Patton, R. S.; Cheng, E.; Svedine, S.; Trombetta, E. S.; Wahl, M. L.;
Ariyan, S.; Hebert, D. N. J. Biol. Chem. 2002, 277, 14821.
5. Mishima, Y. Pigment Cell Res. 1994, 7, 376.
6. Virador, V.; Matsunaga, N.; Matsunaga, J.; Valencia, J.; Oldham, R. J.;
Kameyama, K.; Peck, G. L.; Ferrans, V. J.; Vieira, W. D.; Abdel-Malek, Z. A.;
Hearing, V. J. Pigment Cell Res. 2001, 14, 289.
7. Xu, Y.; Stokes, A. H.; Roskosi, R. J.; Vrana, K. E. J. Neurosci. Res. 1998, 54, 691.
8. Asanuma, M.; Miyazaki, I.; Ogawa, N. Neurotox. Res. 2003, 5, 165.
9. Maeda, K.; Fukuda, M. J. Pharm. Exp. Ther. 1996, 276, 765.
10. Nihei, K. I.; Kubo, I. Bioorg. Med. Chem. Lett. 2003, 13, 2409.
11. Saruno, R.; Kato, F.; Ikeno, T. Agric. Biol. Chem. 1979, 43, 1337.
12. Battaini, G.; Monzani, E.; Casella, L.; Santagostini, L.; Pagliarin, R. J. Biol. Inorg.
Chem. 2000, 5, 262.
Finally, we introduced the methyl group at 1-position of quinaz-
olinethiones. The resulting analogs 4a (10% inhibition at 10
l
l
M,
M,
IC50 = 19.0
IC50 = 17.8
l
l
M, C log P = 3.561) and 4b (18% inhibition at 10
M, C log P = 4.180) showed poor activity compared to
their corresponding analogs 3a and 3b. This is also one of the impor-
tant finding for SAR of quinazolinethiones. The loss of activities in
analogs 4a and 4b might be originated from steric congestion at this
position for their binding in appropriate region or the loss of hydro-
gen bonding property of –NH group. Thus, the free 1-NH group of 3
also has an important role for the inhibition of melanogenesis activ-
ity in melanoma B16 cells under the stimulant of a-MSH.
We also studied the electronic effect of the substituent at ring A
by replacing the methyl group in 3f with chloro as shown in 3g
13. Maeda, K.; Fukuda, M. J. Soc. Cosmet. Chem. 1991, 42, 361.
14. Kojima, S.; Yamaguchi, H.; Morita, K.; Ueno, Y. Biol. Pharm. Bull. 1995, 18, 1076.
15. Verallo-Rowell, V. M.; Verallo, V.; Graupe, K.; Lopez-Villafuerte, L.; Garcia-
Lopez, M. Acta Derm. Venereol. 1989, 143, 58.
16. Takiwake, H.; Shirai, S.; Kohono, H.; Soh, H.; Arase, S. J. Invest. Dermatol. 1994,
103, 642.
17. Kimbrough-Green, C. K. Arch. Dermatol. 1994, 130, 727.
18. Kim, Y.; Jung, S. H.; Min, K.; Park, J.; Lee, J. Korean Patent KR747042, 2007.
19. Thanigaimalai, P.; Lee, K. C.; Bang, S. C.; Lee, J. H.; Yun, C. Y.; Roh, E.; Hwang, B.
Y.; Kim, Y.; Jung, S. H. Bioorg. Med. Chem. 2010, 18, 1135.
20. Thanigaimalai, P.; Lee, K. C.; Bang, S. C.; Lee, J. H.; Yun, C. Y.; Roh, E.; Hwang, B.
Y.; Kim, Y.; Jung, S. H. Bioorg. Med. Chem. 2010, 18, 1555.
21. Ishikawa, F.; Watanabe, Y.; Saegusa, J. Chem. Pharm. Bull. 1980, 25, 1357.
22. Jurczak, J.; Gryko, D.; Kobrzycka, E.; Gruza, H.; Prokopowicz, P. Tetrahedron
1998, 54, 6051.
(>100% inhibition at 10 lM, IC50 = 1.2 lM) dose not alter the activ-
ity. This result implies that the 3,4-dihydroquinazoline-2(1H)-thi-
ones are only the sole pharmacophore for the melanogenesis
inhibition.
In summary, a novel series of 3,4-dihydroquinazoline-2(1H)-
thiones were prepared, screened for their melanogenesis inhibition
on melanoma B16 cell line under the stimulant of
a-MSH and
structure features responsible have been identified. The SAR stud-
ies revealed that the anti-melanogesis activity of 3 is mainly med-
iated by the hydrogen bonding ability of thioamide unit in addition
to complexation ability of thione and the hydrophobic binding
power of side chain substitutions at 3-position. Thus, the pharma-
cophore of 3,4-dihydroquinazoline-2(1H)-thiones 1 and 3 for their
anti-melanogenesis activity could be refined as 3-hydrophbic
substituted quinazolinethione.
23. Leung, M. K.; Lai, J. L.; Lau, K. H.; Yu, H. H.; Hsiao, H. J. J. Org. Chem. 1996, 61,
4175.
24. Cava, M. P.; Levinson, M. I. Tetrahedron 1985, 41, 5061.
25. Delcros, J. H.; Tomasi, S.; Duhieu, S.; Foucault, M.; Martin, B.; Roch, M. L.; Lima,
V. E.; Renault, J.; Uriac, P. J. Med. Chem. 2006, 49, 232.
26. John, A. G.; David, E. N.; Maxine, B. N.; George, K. W. Y. J. Med. Chem. 1980, 23,
1261.
27. Takahata, H.; Tomiguchi, A.; Hagiwara, A.; Yamazaki, T. Chem. Pharm. Bull.
1982, 30, 3959.
Acknowledgments
28. Criton, M.; Mellay-Hamon, V. Bioorg. Med. Chem. Lett. 2008, 18, 3607.
29. Poma, A.; Bianchini, S.; Mirranda, M. Mutat. Res. 1999, 446, 143.
This work was supported by a grant (R01-2007-000-20099-0)
and Priority Research Centers Program (2009-0093815) through