Design and synthesis of optically pure 3-aryl-6-methyl-2-thioxotetrahydropyrimidin-4(1H)-ones as anti-prostate cancer agents

Article abstract of DOI:10.1039/c4ra06391k

3-Aryl-6-methyl-2-thioxotetrahydropyrimidin-4(1H)-ones were proposed as a new class of anti-prostate cancer agents on the basis of molecular modeling studies. Stereoselective synthesis of 3-aryl-6-methyl-2-thioxotetrahydropyrimidin-4(1H)-one derivatives was achieved by chiral induction employing (R)/(S)-α-methyl benzylamine and subsequent debenzylation with HBr in AcOH afforded the desired enantiomers in good yields. The compounds were screened in vitro against prostate cancer cell lines, PC-3 and LNCaP and the most potent derivatives were identified. This journal is

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Table of contents
3-Aryl-6-methyl-2-thioxotetrahydropyrimidin-4(1H)-ones were designed and synthesized as new class of anti-
prostate cancer agents.

RSC Advances
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DOI: 10.1039/C4RA06391K
Design And Synthesis of Optically Pure 3-Aryl-6-methyl-2-
thioxotetrahydropyrimidin-4(1H)-ones as Anti-prostate
Cancer Agents
Journal: RSC Advances
Manuscript ID: RA-ART-06-2014-006391.R1
Article Type: Paper
Date Submitted by the Author: n/a
Complete List of Authors: Kumar, Varun; Latvian Institute of Organic synthesis, Chemistry; National
Institute of Pharmaceutical Education and Research, Medicinal Chemistry
Rachamalla, Mahesh; National Institute of Pharmaceutical Education and
Research, Pharmacology and Toxicology
Nandekar, Prajwal; National Institute of Pharmaceutical Education and
Research, Centre for Pharmacoinformatics
Khatik, Gopal; National Institute of Pharmaceutical Education and
Research, Medicinal Chemistry
Sangamwar, Abhay; National Institute of Pharmaceutical Education and
Research, Department of Pharmacoinformatics
Tikoo, Kulbhushan; National Institute of Pharmaceutical Education and
Research, Pharmacology and Toxicology
Nair, Vipin; National Institute of Pharmaceutical Education and Research,
Department of Medicinal Chemistry

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Journal Name
RSCPublishing
Paper
Design and Synthesis of Optically Pure 3-Aryl-6-
methyl-2-thioxotetrahydropyrimidin-4(1H)-ones as
Anti-prostate Cancer Agents
Cite this: DOI: 10.1039/x0xx00000x
Varun Kumar,a* Mahesh Rachamalla,b Prajwal Nandekar,c Gopal L. Khatik,a
Abhay T. Sangamwar,c Kulbhushan Tikoo,b and Vipin A. Naira*
Received 00th January 2014,
Accepted 00th January 2014
DOI: 10.1039/x0xx00000x
3ꢀArylꢀ6ꢀmethylꢀ2ꢀthioxotetrahydropyrimidinꢀ4(1
antiꢀprostate cancer agents on the basis of molecular modeling studies. Stereoselective
synthesis of 3ꢀarylꢀ6ꢀmethylꢀ2ꢀthioxotetrahydropyrimidinꢀ4(1 )ꢀone derivatives was achieved
by chiral induction employing ( )/( )ꢀ ꢀmethyl benzylamine and subsequent debenzylation
H)ꢀones were proposed as a new class of
www.rsc.org/
H
R
S α
with HBr in AcOH, afforded the desired enantiomers in good yields. The compounds were
screened in vitro against prostate cancer cell lines, PCꢀ3 and LNCaP and the most potent
derivatives were identified.
Introduction
Prostate cancer is a major concern because of high
prevalence, and is a leading cause of cancer related death in
men.1 The development and progression of the prostate cancer
is directly related to the nuclear steroidal androgen receptor.2 It
regulates the binding of androgens like testesterone and its
active metabolite dihydrotestesterone. Hence steroidal agents
(cypreterone acetate, spironolactone) that block the androgens
(antiandrogens) were employed for the treatment of prostate
cancer in hormone therapy (Figure 1).3 But the clinical
applications of steroidal antiandrogens were limited because of
cross reactivity, poor bioavailability, lack of tissue selectivity
and other side effects.3b Hence the focus shifted to nonꢀsteroidal
antiandrogens that not only overcome the side effects of the
steroidal agents, but also provide sufficient scope for structural
modifications to afford more potent scaffolds.3,4
Figure 1. Steroidal and nonꢀsteroidal antiandrogens
Flutamide, nilutamide and bicalutamide are the available
nonꢀsteroidal drugs for the treatment of prostate cancer,3,4 but
with elapse of time the therapy fails because of alterations in
cell signaling pathways and mutations leading to antiandrogen
withdrawal syndrome. Moreover, other adverse effects such as
hepatotoxicity, gynaecomastia, loss of libido etc. necessitate the
search for novel ligands for the treatment of prostate cancer.
Results and discussion
The Xꢀray crystal structure of bicalutmide in WL AR LBD
complex revealed a bent conformation for bicalutamide, due to
the hydrogen bonding interactions.5 Therefore we assumed that
a conformationally restricted model with similar interactions
will be of high relevance to investigate the antiꢀprostate
activity. With our current interests on heterocyclic scaffolds
and antiꢀprostate cancer agents,6 we designed conformationally
aDepartment of Medicinal Chemistry, National Institute of
Pharmaceutical Education and Research, Sector 67, Mohali, Punjab,
160062, India.
Eꢀmail: drvarun.niper@gmail.com, vnair@niper.ac.in
bDepartment of Pharmacology and Toxicology, National Institute of
Pharmaceutical Education and Research, Sector 67, Mohali, Punjab,
160062, India
cDepartment of Pharmacoinformatics, National Institute of Pharmaceutical
Education and Research, Sector 67, Mohali, Punjab 160062, India
restricted
2ꢀthioxotetrahydropyrimidinꢀ4(1
H
)ꢀones
as
pharmacophore of choice satisfying the criteria (Figure 2). The
rationale is supported by recent literature reports where
heterocycles with thioamide moiety, especially 3ꢀaryl
thiohydantoins, are found to be highly potent nonꢀsteroidal
antiandrogens.7 Further to understand the ligand binding
interactions, docking of the proposed compounds was
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PPaaggee42oof f191
VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C4RA06391K
performed on the active site of the receptor. The results formation of hydrogen bonds with Arg752 and Gln711, and
obtained from molecular docking of designed ligands are hence a stronger binding affinity. The higher binding affinity of
summarized in (Table 1), where the binding affinity of ligand compounds with meta substitution on
N3 aryl group 10(b-e)
towards mutant androgen receptor is represented in terms of and 10'(b-e) can be explained on the basis of hydrophobic
docking score.8
interactions with amino acid residues Met745, Val746, Met749,
Phe764, Met787 and Leu873 of the sub pocket B. A high
docking score was observed for compound 10'e due to the
preferred geometry that allows hydrogen bonding interactions
between the NH of the ligand and Asn705, in addition to the
hydrogen bonding of the cyano group with Arg752 and Gln711.
Figure 2. Design of proposed pharmacophore
Table 1. Docking scores for the proposed ligands
Entry
Compound
Stereo-
chemistry
Docking
scorea
ꢀ10.74
Docking
scoreb
ꢀ10.99
Glide
emodel
ꢀ94.62
1
Bicalutamide
10a
(
(
(
(
(
(
(
(
(
(
(
(
(
R
)
2
S
S
S
S
S
S
)
ꢀ7.41
ꢀ7.76
ꢀ8.28
ꢀ7.85
ꢀ8.07
ꢀ8.01
ꢀ7.46
ꢀ7.99
ꢀ8.52
ꢀ8.05
ꢀ8.35
ꢀ7.59
ꢀ5.19
ꢀ5.57
ꢀ6.67
ꢀ6.73
ꢀ7.15
ꢀ6.46
ꢀ5.63
ꢀ5.27
ꢀ7.07
ꢀ7.28
ꢀ6.94
ꢀ5.71
ꢀ55.65
ꢀ59.13
ꢀ62.36
ꢀ57.80
ꢀ63.20
ꢀ60.20
ꢀ56.76
ꢀ58.51
ꢀ65.23
ꢀ55.30
ꢀ60.78
ꢀ43.47
3
10b
)
4
10c
)
)
)
)
5
10d
Scheme 1. Diastereoselective synthesis of
(S)ꢀmethyl 3ꢀ[(S)ꢀ1ꢀ
6
10e
phenylethylamino]butanoate
7
10f
8
10'a
10'b
10'c
R
R
R
R
R
R
)
)
)
)
)
)
9
10
11
12
13
10'd
10'e
10'f
aDocking scores were calculated in standard precision mode
bDocking scores were calculated in extra precision mode
The results obtained from molecular docking of designed
and synthesized analogues are summarized in Table 1. The
molecular docking analysis showed that all ligands were docked in
same ligand binding site as that of bicalutamide. The results also
indicated the high binding affinity of the compounds towards the
androgen receptor.
To gain more insights of the binding site, an inꢀdepth analysis
was carried out, which showed that the active site is composed
of mainly five sub pockets: (i) hydrophilic subpocket A that
includes Arg752 and Gln711 and is responsible for hydrogen
bonding interactions with electron withdrawing substituent at
Scheme 2. Diastereoselective syntheses of 3ꢀarylꢀ6(S)/(R)ꢀmethylꢀ1ꢀ
[(S)/(R)ꢀ1ꢀphenylethyl)]ꢀ2ꢀthioxotetrahydropyrimidinꢀ4(1 )ꢀones
H
the para position of the
pocket B consists of Gly708, Met745, Val746, Met749,
Phe764, Met787, Leu873, and Met895 which stabilizes the R2
substituent as well as the thioamide moiety (iii) hydrophobic 4(1
sub pocket C comprising of of Met895, Thr877, Phe891,
Leu880 and Phe876, interacts with ꢀCH3 group of the
N3 aryl group (ii) hydrophobic sub
Further, our efforts were to synthesize the (
enantiomers of 3ꢀarylꢀ6ꢀmethylꢀ2ꢀthioxotetrahydropyrimidinꢀ
)ꢀones to validate the conclusions drawn from the
molecular modeling studies, and to identify the most active
enantiomer as a potential androgen receptor antagonist. The key
R) and (S)
H
R
ꢀ
stereoisomer (iv) hydrophobic sub pocket D composed of
Leu704, Leu701, Met873, Met742 and Thr877 is responsible
for interactions with the ꢀCH3 group of the Sꢀstereoisomer (v)
hydrophilic sub pocket E containing Asn705 is involved in
hydrogen bonding with the ꢀNH group of the ligands (Figure
intermediate
thioxotetrahydropyrimidinꢀ4(1
involved
in
H
the
)ꢀones are β
synthesis
ꢀaminoesters which
of
2ꢀ
upon condensation with aryl isothiocyanates can afford the
desired products. The diastereoselective synthesis of the chiral
β
ꢀaminoesters9 was attempted as depicted in Scheme 1. (
S
)ꢀαꢀ
with
3
). The compounds having cyano group at the para position of
Methyl
benzylamine
1
when
treated
the 3 aryl ring has higher docking scores in comparison with
N
other substrates. This may be attributed to the simultaneous
2 | J. Name., 2012, 00, 1-3
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Figure 3. Binding orientations of compound 10c (A), compound 10'c (B), compound 10e (C) and compound 10'e (D) in 1z95 binding pocket.
Hydrogen bonding interactions are highlighted in yellow color. Active site amino acid residues are in color lines model and ligands are in pink color
stick model.
benzaldehyde
catalyst under neat condition afforded the imine
subsequently reduced with NaBH4 to obtain the chiral (
benzylꢀ ꢀmethyl benzylamine in excellent yields. The chiral
2
in presence of magnesium perchlorate as
,which was
)ꢀ
Finally the oxidative cleavage of the
cericammonium nitrate in acetonitrileꢀwater mixture (5:1)
afforded the ( )ꢀmethyl 3ꢀ[( )ꢀ1ꢀphenylethylamino]butanoate 7
Nꢀbenzyl group using
3
S
Nꢀ
S
S
α
4
with good yields and high diastereoselectivity. A similar reaction
sequence with (R)ꢀαꢀmethyl benzylamine afforded the other
enantiomer (R)ꢀmethyl 3ꢀ[(R)ꢀ1ꢀphenylethylamino]butanoate (R,R)ꢀ
7'. Synthesis of 3ꢀarylꢀ6(S)ꢀmethylꢀ1ꢀ[(S)ꢀ1ꢀphenylethyl)]ꢀ2ꢀ
thioxotetrahydropyrimidinꢀ4(1H)ꢀones 9(a-f) were then achieved by
the condensation of (S)ꢀmethyl 3ꢀ[(S)ꢀ1ꢀphenylethylamino]butanoate
7 with aryl isothiocyanates 8(a-f) using lithium perchlorate as
catalyst and triethylamine as a base in acetonitrile medium under
reflux conditions (Scheme 2). The reaction afforded good yields
of the products in all the cases and recrystalization in MeOH
Table 2. Reactions of aryl isothiocyanates with (S)/(R)ꢀmethyl 3ꢀ
[(S)/(R)ꢀ1ꢀphenylethylamino]butanoates
Entry
R1
R2
Time (h) Product
Yield
(%)
78
de
1
2
Cl
F
H
2.5
2.5
9a
9b
>99
>99
Cl
80
3
4
5
6
7
8
CN
Cl
CN
NO2 CF3
Cl
F
Cl
3.0
3.0
4.0
4.0
2.5
2.5
9c
74
76
78
72
80
82
>99
>99
>99
>99
>99
>99
CF3
CF3
9d
9e
9f
9'a
9'b
afforded the (
(Table 2). The corresponding (
obtained from
phenylethylamino]butanoate (R,R
S
,
S
) diastereomers as the sole product (de>99%)
) enantiomers 9'(a-f) were
)ꢀmethyl 3ꢀ[( )ꢀ1ꢀ
)-7'. After achieving chiral
R,R
(
R
R
H
Cl
induction, our aim was to cleave the chiral handle in order to
obtain the desired enantiomers of 3ꢀarylꢀ6ꢀmethylꢀ2ꢀ
thioxotetrahydropyrimidinꢀ4(1H)ꢀones stereospecifically. The
cleavage of the handle, which involves the debenzylation at N1,
was attempted by conventional methods (Table 3). Accordingly
9
CN
Cl
Cl
3.0
9'c
72
>99
10
11
12
CF3
CF3
3.0
4.0
4.0
9'd
9'e
9'f
76
74
72
>99
>99
>99
CN
NO2 CF3
a
solution
3ꢀ(4ꢀchlorophenyl)ꢀ6(
S
)ꢀmethylꢀ1ꢀ[(
S)ꢀ1ꢀ
*Reactions were performed with 2.57 mmoles of aryl isothiocyante, 2.57mmoles
of ꢀaminoester and 0.257 mmoles of catalyst
phenylethyl)]ꢀ2ꢀthioxotetrahydropyrimidin ꢀ4(1
H)ꢀone 9a in
β
MeOH was treated with Pd catalyst under hydrogen atmosphere
for 12 h. The reaction was unsuccessful and the starting
material was recovered. A variation in the solvents or the
oxidation state of the catalyst did not alter the outcome of the
amine
lithium amide derivative which underwent Michael addition
diastereoselectively with methyl crotonate affording the
aminoesters, methyl 3ꢀ(benzyl[( )ꢀ1ꢀ
phenylethyl]amino)butanoate with ( ) isomer as the major.
4 when reacted with nꢀBuLi gave the corresponding
5
βꢀ
S
reaction. Oxidative
N
ꢀdebenzylation using CAN, DEAD and
6
S,S
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4
DDQ also failed. Further, acid catalyzed cleavage of the chiral
PCꢀ3 cell lines.10 The cell viability was determined by colorimetric
MTT assay.11 It was observed that all the compounds exhibited
antagonistic activity with IC50 values in mM to nM range. However
none of the compounds showed agonistic activity. Though most of
the compounds showed the antagonistic activity a total of four
compounds (compound 10a, 10e and their enantiomers 10'a, 10'e)
showed IC50 values below 50 ꢁM that were further examined for the
selectivity studies (Table 5). As expected on the basis of the docking
studies the most potent compound turned out to be 10'e that has the
aryl ring with the CN group at the para position and CF3 group at the
meta position. A comparison of 10'e with the standard drugs for
prostate cancer showed an IC50 value of 1.25 ꢁM, comparable to
doxorubicin (1.05 ꢁM) on PCꢀ3 cell lines (Figure 4) and a better
antagonistic activity of 782 nM on LNCap cell lines compared to
flutamide (1 ꢁM) and bicalutamide (1.7 ꢁM) as shown in Figure 5.
However the corresponding enantiomer 10e was found to be less
potent with IC50 value of 34 ꢁM and 13 ꢁM on PCꢀ3 and LNCaP cell
lines respectively which is indicative of the fact that chirality is also
an important factor governing the binding affinity. Thus the
cytotoxicity of the compounds not only depend on the substituent on
the aryl ring but also on the orientation of the methyl group, with the
S isomer being more active.
handle was investigated with HBr. Interestingly, the substrate in
33% HBrꢀAcOH at room temperature afforded the desired product 3ꢀ
(4ꢀchlorophenyl)ꢀ6(S)ꢀmethylꢀ2ꢀthioxotetrahydropyrimidinꢀ4(1H)ꢀ
one (S)-10a in good yield (Scheme 3). The reaction conditions were
then generalized (Table 4) with other substrates 9(a-f) and 9'(a-f)
affording the corresponding products (S)-10(a-f) and (R)-10'(a-f)
respectively in good yields.
Table 3. Reaction conditions employed for debenzylation at N1
Entry
Catalyst
Reagent
H2
Solvent
MeOH
AcOH
MeOH
AcOH
CH3CN
DCM
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
9
Pd
12
24
18
24
5
ꢀ
ꢀ
Pd
H2
Pd(OH)2
H2
ꢀ
Pd(OH)2
H2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
CAN
DDQ
DEAD
HBr
HBr
ꢀ
10
72
2
ꢀ
Toluene
AcOH
AcOH
ꢀ
55
72
Table 5.Cytotoxicity studies of most active compounds on PCꢀ3 and LNCaP
4
cellsa
*Reactions were performed with 1.2 mmole of substrate and 0.12 mmoles of catalyst
Entry
Compound
R1
R2
IC50 (µM) on
PC-3 cells
1.05
IC50 (µM) on
LNCaP cells
ndc
1
Doxorubicinb ꢀꢀꢀ
Flutamideb
Bicalutamide ꢀꢀꢀ
ꢀꢀꢀ
2
ꢀꢀꢀ
ꢀꢀꢀ
ndc
ndc
1.0
3
ꢀꢀꢀ
1.7
4
(S)-10a
(R)-10'a
(S)-10b
(R)-10'b
(S)-10c
(R)-10'c
(S)-10d
(R)-10'd
(S)-10e
(R)-10'e
(S)-10f
Cl
H
>100
>100
50
>100
>100
>100
>100
39
5
Cl
H
6
F
Cl
7
F
Cl
>100
100
8
CN
CN
Cl
Cl
Scheme 3. Debenzylation of 3ꢀarylꢀ6(S)/(R)ꢀmethylꢀ1ꢀ(S)/(R)ꢀ1ꢀphenyl
ethyl)ꢀ2ꢀthioxotetrahydro pyrimidinꢀ4(1H)ꢀones
9
Cl
90
14
10
11
12
13
14
15
CF3
CF3
CF3
CF3
CF3
CF3
>100
>100
34
>100
>100
13.0
0.8
Table 4. Syntheses of enantiopure 3ꢀarylꢀ6(S)/(R)ꢀmethylꢀ2ꢀthioxo
tetrahydropyrimidinꢀ4(1H)ꢀones
Cl
CN
CN
NO2
NO2
Entry
Substrate
R1
R2
Product
Time
(h)
3.0
3.5
3.5
4.0
4.0
4.0
3.0
3.5
3.5
4.0
4.0
4.0
Yield
(%)
72
1.25
>100
>100
>100
>100
1
9a
Cl
H
(S)-10a
(S)-10b
(S)-10c
(R)-10'f
2
9b
9c
F
Cl
68
aThe data represents the mean of three experiments in triplicate
bUsed as a positive control. cnd = not determined.
3
CN
Cl
Cl
75
4
9d
9e
CF3
CF3
CF3
H
(S)-10d
(S)-10e
65
5
CN
NO2
Cl
69
6
9f
(S)-10f
64
7
9'a
9'b
9'c
9'd
9'e
9'f
(R)- 10'a
(R)- 10'b
(R)- 10'b
(R)- 10'd
(R)- 10'e
(R)- 10'f
74
8
F
Cl
70
9
CN
Cl
Cl
67
10
11
12
CF3
CF3
CF3
62
CN
NO2
66
68
*Reactions were carried out with 1.2 mmole of substrate and 5ml of HBr at rt
To determine the cytotoxicity of the compounds, in vitro
experiments were carried out at varying concentrations (0.01 nM to
100 ꢁM) on prostate cancer cell lines, PCꢀ3 and LNCaP.
Bicalutamide was used as the positive control for the androgen
dependent LNCaP cells, whereas as doxorubicin was preferred for
Figure 4. Dose response curve on PCꢀ3cells
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crude product as a pale yellow oil. The product (S)ꢀmethyl 3ꢀ
(benzyl[(S)ꢀ1ꢀphenylethyl]amino) butanoate, 8 (7.70 mL, 62%) was
purified by column chromatography on silica gel (60–120 mesh)
using EtOAcꢀhexane mixture (1:9) as the eluent. To a solution of (S)ꢀ
methyl 3ꢀ(benzyl[(S)ꢀ1ꢀphenylethyl]amino)butanoate (6.2 g, 20.0
mmol) in CH3CN:H2O (5:1), at room temperature, was added ceric
ammonium nitrate (23.01 g, 42.0 mmol). The reaction mixture was
stirred for 2h, neutralized with aqueous NaHCO3, extracted with
Et2O, dried over Na2SO4, and concentrated in vacuum to afford a
crude oil which was purified by column chromatography on silica
gel (60ꢀ120 mesh) using hexaneꢀethyl acetate mixture (70:30) as the
eluent to obtain the desired βꢀamino ester 9, (3.3 g, 77%). (R)ꢀmethyl
3ꢀ(R)ꢀ1ꢀphenylethylamino butanoate was synthesized from (R)ꢀαꢀ
methylbenzylamine by an analogous procedure.
Figure 5. Dose response curve on LNCaP cells
(S)-N-benzyl-1-phenylethanamine (4): Colourless oil; Yield 85%;;
Rf 0.3 (1:10 EtOAcꢀhexane); 1H NMR (400 MHz, CDCl3): δ 1.40 (d,
3H, J = 6.80 Hz), 3.68 (dd, 2H, J = 13.20 and J = 14.40 Hz), 3.85 (q,
1H, J = 6.80 Hz), 7.24ꢀ7.39 (m, 10H); 13C NMR (100 MHz, CDCl3):
δ 24.5, 51.6, 57.6, 126.6, 126.8, 126.9, 128.1, 128.3, 128.4, 140.6,
145.6 ; MS (APCI): [M+1]+ = 212.40; [α]20D ꢀ39.6 (neat)
Further to study the selectivity of the compounds for prostate
cancer, cytotoxicity of the most potent compounds were examined
on non cancerous cell line MCF 10A and breast cancer cell line
MCFꢀ7. The results obtained were impressive as no compound
exhibited toxicity on the other cell lines at conc. of 1 ꢁM. Hence 3ꢀ
(4ꢀcyanoꢀ3ꢀ(trifluoromethyl)phenyl)ꢀ6(R)ꢀmethylꢀ2ꢀthioxotetrahyro
pyrimidnꢀ4(1H)ꢀone was identified as the lead compound for further
studies in this direction.
(S)-methyl 3-(benzyl[(S)-1-phenylethyl]amino) butanoate (6):
Light yellow oil; Yield 62%; Rf 0.6 (1:10 EtOAcꢀhexane); 1H NMR
(400 MHz, CDCl3): δ 1.17 (d, 3H, J = 6.9 Hz), 1.38 (d, 3H, J = 6.9
Hz), 2.15 (dd, 1H, J = 7.8 and 7.8 Hz), 2.40 (dd, 1H, J = 7.8 and 7.8
Hz), 3.48 (q, 1H, J = 6.6 Hz), 3.53 (s,3H), 3.69ꢀ3.74 (m, 2H), 3.92
(q, 1H, J = 6.9 Hz), 7.20–7.45 (m, 10H), 13C NMR (100 MHz,
CDCl3): δ 17.3, 18.3, 39.5, 49.5, 49.9, 51.3, 57.5, 126.6, 127.7,
128.0, 128.1, 128.2, 128.3, 141.6, 144.2, 172.6; MS (APCI): [M+1]+
= 312.48; [α]20D ꢀ1.1 (c=1.0, CHCl3).
Experimental Section
Chemical Synthesis
1
The H and 13C NMR spectra were recorded at 400 MHz and 100
MHz, respectively on a Bruker Avance 400 (400 MHz) spectrometer
in CDCl3 using TMS as an internal standard. The chemical shifts (δ)
for 1H and 13C are given in ppm relative to residual signals of the
solvent. Coupling constants are given in Hz. The following
abbreviations are used to indicate the multiplicity: s, singlet; d,
doublet; m, multiplet. Mass spectra were recorded on Finnigan Mat
LCQ LCMS. The reactions were monitored by TLC (Merck).
Evaporation of solvents was performed under reduced pressure using
a Buchi rotary evaporator. Commercial grade reagents and solvents
were used without further purification. (s)ꢀαꢀmethyl benzyl amine,
(S)-methyl 3-(S)-1-phenylethylamino butanoate (7): (S,3S)ꢀ5a:
Light brown oil; Rf 0.2 (1:10 EtOAcꢀhexane); 1H NMR (400 MHz,
CDCl3): δ 1.04 (d, J = 6.4 Hz, 3H), 1.31 (d, J = 6.4 Hz, 3H), 1.66 (br
s, 1H), 2.33ꢀ2.47 (m, 2H), 3.65 (s, 3H), 3.87 (q, J = 6.4 Hz, 1H),
7.19ꢀ7.36(m, 5H); 13C NMR (100 MHz, CDCl3): δ 21.4, 24.6, 40.6,
47.7, 51.3, 55.2, 126.5, 126.8, 128.4, 146.0, 172.7; MS (APCI):
[M+1]+ = 222.34; [α]20D ꢀ38.0 (c=1.0, CHCl3).
benzaldehyde,
diisopropylamide
sodium
(LDA),
borohydride
methyl
(NaBH4),
acrylate,
lithium
lithium
General procedure for the syntheses of 3-aryl-6(S)/(R)-methyl-1-
[(S)/(R)-1-phenylethyl)-2-thioxotetrahydro pyrimidin-4(1H)-ones
(9, 9')(a-f): To a solution of aryl isothiocyanate (2.57 mmol) in
hexamethyldisilazane (LHMDS), cerric ammonium nitrate, 4ꢀchloro
aniline,
4ꢀchloroꢀ3ꢀtrifluoromethylaniline,
4ꢀaminoꢀ2ꢀ
chlorobenzonitrile,
4ꢀcynoꢀ3ꢀtrifluoromethylaniline, 4ꢀnitroꢀ3ꢀ
acetonitrile
(30
mL)
was
added
ethyl
3ꢀ(1ꢀ
trifluoromethylaniline (Aldrich), thiophosgene, Methanol, THF
(Spectrochem); dichloromethane (DCM) 4ꢀchloroaniline (Merck)
and sodium hydrogen carbonate (CDH).
phenylethylamino)butanoate (0.6 g, 2.57 mmol) followed by
triethylamine (0.4 mL, 3.08 mmol) and LiClO4 (10 mol%, 0.03 g,
0.26 mmol). The reaction mixture was refluxed for 1.2h and then
concentrated under reduced pressure. The residue was diluted with
DCM, washed with water (2 x 25 mL), then with brine (1 x 25 mL)
and dried over anhydrous Na2SO4. From the concentrated reaction
mixture the purified product was obtained by column
chromatography on silica gel (60–120) using EtOAcꢀhexane mixture
(15:85) as the eluent. The products were recrystalized from MeOH.
Synthesis of (S)-methyl 3-[(S)-1-phenylethylamino] butanoate: In
a typical experiment, (S)ꢀαꢀmethylbenzylamine (6.36 mL, 50.0
mmol) was taken in MeOH and benzaldehyde (5.50 mL, 55 mmol)
followed by MgClO4 (0.24 g, 2.0 mmol) was added to the reaction
mixture. It was stirred at room temperature for 2h to obtain the
corresponding imine. After completion of the reaction as determined
from TLC, NaBH4 (2.22 g, 60.0 mmol) was slowly added to it and
stirred for 2.5h. The reaction mixture was then concentrated, diluted
with water and extracted into ethyl acetate. The organic layer was
dried and the solvent was evaporated to obtain the crude product. It
was purified by column chromatography on silica gel (60–120 mesh)
using EtOAcꢀhexane mixture (1:9) as the eluent to afford the (S)ꢀNꢀ
benzylꢀ1ꢀphenylethanamine (8.89 g, 85%). The amine (8.36 g, 40.0
3-(4-Chlorophenyl)-6(S)-methyl-1-[(S)-1-phenylethyl]-2-
thioxotetrahydropyrimidin-4(1H)-one 9(a): White solid; Yield
o
78%; mp 177ꢀ179 C; Rf 0.66 (3:7 EtOAcꢀhexane); 1H NMR (400
MHz, CDCl3): δ 1.45 (d, 3H, J = 6.80 Hz), 1.73 (d, 3H, J = 7.2 Hz),
2.41ꢀ2.58 (m, 2H), 3.67ꢀ3.70 (m, 1H), 6.97 (q, 1H, J = 7.2 Hz), 7.13
(s, 1H), 7.25ꢀ7.46 (m, 8H); 13C NMR (100 MHz, CDCl3): δ 15.3,
19.4, 38.7, 46.3, 59.7, 127.1, 128.3, 128.4, 129.0, 129.3, 134.2,
138.1, 139.3, 165.9, 180.7; MS (APCI): [M+1]+ = 359.03; HRMS
(ESI): m/z [M+Na]+ calcd for C19H19ClN2OS: 381.0805, found:
381.0808; [α]20D ꢀ173.62 (c=1.0, CHCl3)
o
mmol) dissolved in THF (100 mL) was cooled to 0 C and nꢀbutyl
lithium (27.47 mL, 1.6 M in hexanes, 44.0 mmol) was added to it.
The solution was stirred at 0 oC for 15 min and further cooled to ꢀ78
oC. Methyl crotonate (4.04 mL, 40.0 mmol) in THF (50 mL) was
added dropwise and the reaction mixture was stirred for another 15
3-(4-Chlorophenyl)-6(R)-methyl-1-[(R)-1-phenylethyl]-2-
o
min at ꢀ78 C before quenching with saturated ammonium chloride
thioxotetrahydropyrimidin-4(1H)-one 9'(a): White solid; Yield
o
80%; mp 177ꢀ179 C; Rf 0.66 (3:7 EtOAcꢀhexane); 1H NMR (400
(2 mL). The reaction was allowed to warm, poured into saturated
aqueous sodium chloride solution, extracted with ether (2 x 100 mL),
dried over anhydrous Na2SO4, filtered and concentrated to give the
MHz, CDCl3): δ 1.48 (d, 3H, J = 6.6 Hz), 1.75 (d, 3H, J = 7.0 Hz),
2.44ꢀ2.60 (m, 2H), 3.69ꢀ3.72 (m, 1H), 6.99 (q, 1H, J = 7.20 Hz), 7.15
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

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PPaaggee86oof f191
View Article Online
DOI: 10.1039/C4RA06391K
6
(s, 1H), 7.37ꢀ7.56 (m, 8H); 13C NMR (100 MHz, CDCl3): δ 15.4,
(q, J = 6.8 Hz, 1H), 7.27ꢀ7.48 (m, 7H), 7.60 (d, 1H, J = 8.2 Hz); 13
C
19.4, 38.7, 46.3, 59.7, 127.1, 128.3, 128.4, 129.0, 129.3, 134.2,
138.1, 139.3, 165.9, 180.6; MS (APCI): [M+1]+ = 359.07; HRMS
(ESI): m/z [M+Na]+ calcd for for C19H19ClN2OS: 381.0805, found:
381.0800; [α]20D +173.70 (c=1.000, CHCl3).
NMR (100 MHz, CDCl3): δ 15.3, 19.5, 38.7, 46.3, 59.8, 121.1,
123.8, 127.0, 128.5, 129.0, 132.0, 132.2, 138.2, 139.1, 165.8, 180.1;
MS (APCI): [M+1]+ = 426.95; HRMS(ESI):m/z [M+Na]+ calcd for
D
C20H18ClF3N2OS: 449.0678, found: 449.0685; [α]20 +146.91
(c=1.000, CHCl3).
3-(3-Chloro-4-fluorophenyl)-6(S)-methyl-1-[(S)-1-phenylethyl]-2-
thioxotetrahydropyrimidin-4(1H)-one 9(b): White solid; Yield
3-(4-Cyano-3-(trifluoromethyl)phenyl)-6(S)-methyl-1-[(S)-1-
phenylethyl]-2-thioxotetrahydropyrimidin-4(1H)-one 9(e): White
o
80%; mp 170ꢀ171 C; Rf 0.66 (3:7 EtOAcꢀhexane); 1H NMR; (400
o
1
MHz, CDCl3): δ 1.47 (d, 3H, J = 6.8 Hz), 1.75 (d, 3H, J = 7.2 Hz),
2.44ꢀ2.59 (m, 2H), 3.70ꢀ3.73 (m, 1H), 6.97 (q, 1H, J = 7.20 Hz),
7.21ꢀ7.48 (m, 8H); 13C NMR (100 MHz, CDCl3): δ 15.3, 19.4, 38.7,
46.3, 59.8, 116.6, 121.3, 127.0, 128.5, 129.0, 131.6, 135.9, 139.2,
156.5, 159.0, 165.8, 180.5; MS (APCI): [M+1]+ = 377.13; HRMS
(ESI): m/z [M+Na]+ calcd for C19H18ClFN2OS: 399.0710, found:
399.0714; [α]20D ꢀ168.20 (c=1.000, CHCl3).
solid; Yield 78%; mp 178ꢀ180 C; Rf 0.50 (3:7 EtOAcꢀhexane); H
NMR (400 MHz, CDCl3): δ 1.49 (d, 3H, J = 6.8 Hz), 1.75 (d, 3H, J =
7.2 Hz), 2.46ꢀ2.61 (m, 2H), 3.71ꢀ3.78 (m, 1H), 6.88 (q, J = 7.1 Hz,
1H), 7.25ꢀ7.52 (m, 6 H),7.62 (s, 1H), 7.91 (d, 1H, J = 8.2 Hz); 13C
NMR (100 MHz, CDCl3): δ 15.3, 19.6, 38.6, 46.4, 59.8, 109.7,
115.1, 117.9, 120.7, 123.4, 126.1, 127.0, 128.6, 129.1, 135.2, 138.9,
143.7, 165.6, 179.5; MS (APCI): [M+1]+ = 417.96; HRMS(ESI):m/z
[M+Na]+ calcd for C21H18F3N3OS: 440.1021, found: 440.1021; [α]D ꢀ
169.38 (c=1.000, CHCl3).
3-(3-Chloro-4-fluorophenyl)-6(R)-methyl-1-[(R)-1-phenylethyl]-
2-thioxotetrahydropyrimidin-4(1H)-one 9'(b): White solid; Yield
o
1
82%; mp 170ꢀ171 C; Rf 0.66 (3:7 EtOAcꢀhexane); H NMR; (400
MHz, CDCl3): δ 1.47 (d, 3H, J = 6.5 Hz), 1.75 (d, 3H, J = 6.9 Hz),
2.42ꢀ2.59 (m, 2H), 3.70ꢀ3.73 (m, 1H), 6.97 (q, 1H, J = 6.9 Hz),
7.11ꢀ7.60 (m, 8H); 13C NMR (100 MHz, CDCl3): δ 15.3, 19.4, 38.7,
46.3, 59.8, 116.6, 121.3, 127.1, 128.5, 129.0, 131.6, 135.8, 139.2,
156.5, 159.0, 165.9, 180.5; MS (APCI): [M+1]+ = 376.95; HRMS
(ESI): m/z [M+Na]+ calcd for C19H18ClFN2OS: 399.0710, found:
399.0704; [α]20D +168.35 (c=1.000, CHCl3).
3-(4-Cyano-3-(trifluoromethyl)phenyl)-6(R)-methyl-1-[(R)-1-
phenylethyl]-2-thioxotetrahydropyrimidin-4(1H)-one 9'(e): White
o
1
solid; Yield 74%; mp 178ꢀ180 C; Rf 0.50 (3:7 EtOAcꢀhexane); H
NMR (400 MHz, CDCl3): δ 1.49ꢀ1.51 (d, 3H, J = 6.28 Hz), 1.77 (d,
3H, J = 6.7 Hz), 2.48ꢀ2.63 (m, 2H), 3.76 (m, 1H), 6.87 (q, J = 7.1
Hz, 1H), 7.27ꢀ7.54 (m, 6H), 7.63 (s, 1H), 7.91 (d, 1H, J = 7.9 Hz);
13C NMR (100 MHz, CDCl3): δ 15.3, 19.6, 38.6, 46.4, 59.8, 109.7,
115.1, 117.9, 120.7, 123.4, 126.1, 127.0, 128.6, 129.1, 135.2, 138.9,
143.7, 165.6, 179.5; MS (APCI): [M+1]+ = 418.02; HRMS (ESI):
m/z [M+Na]+ calcd for C21H18F3N3OS: 440.1021, found: 440.1019;
[α]20D +169.42 (c=1.000, CHCl3).
3-(3-Chloro-4-cyanophenyl)-6(S)-methyl-1-[(S)-1-phenylethyl]-2-
thioxotetrahydropyrimidin-4(1H)-one 9(c): White solid; Yield
74%; mp 218ꢀ220 oC; Rf 0.50 (3:7 EtOAcꢀhexane); 1H NMR (400
MHz, CDCl3): δ 1.46 (d, 3H, J = 6.8 Hz), 1.74 (d, 3H, J = 7.2 Hz),
2.44ꢀ2.59 (m, 2H), 3.69ꢀ3.76 (m, 1H), 6.88 (q, 1H, J = 7.2 Hz), 7.22ꢀ
7.46 (m, 7H), 7.74 (d, 1H, J = 8.0 Hz); 13C NMR (100 MHz, CDCl3):
δ 15.4, 19.5, 38.6, 46.4, 59.7, 113.1, 115.6, 127.0, 128.6, 128.9,
129.1, 131.5, 134.0, 137.1, 138.9, 144.3, 165.5, 179.5; MS (APCI):
3-(4-Nitro-3-(trifluoromethyl)phenyl)-6(S)-methyl-1-[(S)-1-
phenylethyl]-2-thioxotetrahydropyrimidin-4(1H)-one
9(f):
Yellow solid; Yield 72%; mp 134ꢀ136 oC; Rf 0.50 (3:7 EtOAcꢀ
1
hexane); H NMR (400 MHz, CDCl3): δ 1.48ꢀ1.50 (d, 3H, J = 6.40
Hz), 1.75ꢀ1.77 (d, 3H, J = 7.20 Hz), 2.46ꢀ2.62 (m, 2H), 3.72ꢀ3.79
(m, 1H), 6.86ꢀ6.91 (q, 1H, J = 7.20 Hz), 7.25ꢀ7.64 (m, 7H), 7.99ꢀ
7.97 (d, 1H, J = 8.40 Hz); 13C NMR (100 MHz, CDCl3): δ 15.3,
19.6, 38.6, 46.4, 59.8, 120.3, 123.3, 124.8, 125.9, 127.1, 128.4,
129.0, 138.9, 140.6, 143.4, 147.0, 165.6, 179.5; MS (APCI): [M+1]+
= 437.93; HRMS (ESI): m/z [M+Na]+ calcd for C20H18F3N3O3S:
460.0919, found: 460.0921; [α]20D ꢀ119.81 (c=1.000, CHCl3).
[M+1]+
=
384.20; HRMS(ESI): m/z [M+Na]+ calcd for
C20H18ClN3OS: 406.0757, found: 406.0757; [α]20D ꢀ195.87 (c=1.000,
CHCl3).
3-(3-Chloro-4-cyanophenyl)-6(R)-methyl-1-[(R)-1-phenylethyl]-
2-thioxotetrahydropyrimidin-4(1H)-one 9'(c): White solid; Yield
= 72%; mp = 218ꢀ220 oC; Rf 0.50 (3:7 EtOAcꢀhexane); 1H NMR
(400 MHz, CDCl3): δ 1.48 (d, 3H, J = 6.7 Hz), 1.75 (d, 3H, J = 7.2
Hz), 2.45ꢀ2.60 (m, 2H), 3.71ꢀ3.77 (m, 1H), 6.92 (q, 1H, J = 7.2 Hz),
7.22ꢀ7.47 (m, 7H), 7.74 (d, 1H, J = 8.0 Hz); 13C NMR (100 MHz,
CDCl3): δ 15.3, 19.5, 38.7, 46.4, 59.7, 113.1, 115.5, 127.0, 128.6,
128.9, 129.0, 131.5, 133.9, 137.1, 139.0, 144.3, 165.4, 179.6; MS
3-(4-Nitro-3-(trifluoromethyl)phenyl)-6(R)-methyl-1-[(R)-1-
phenylethyl]-2-thioxotetrahydropyrimidin-4(1H)-one
9'(f):
Yellow solid; Yield 72%; mp 134ꢀ136 oC; Rf 0.50 (3:7 EtOAcꢀ
1
hexane); H NMR (400 MHz, CDCl3): δ 1.50 (d, 3H, J = 6.2 Hz),
1.76 (d, 3H, J = 6.8 Hz), 2.47ꢀ2.62 (m, 2H), 3.76 (m, 1H), 6.90 (q,
1H, J = 6.6 Hz), 7.25ꢀ7.64 (m, 7H), 7.99 (d, 1H, J = 8.4 Hz); 13C
NMR (100 MHz, CDCl3): δ 15.3, 19.6, 38.6, 46.5, 59.8, 120.3,
123.0, 124.8, 125.9, 127.0, 128.7, 129.1, 134.3, 138.9, 143.4, 147.0,
165.6, 179.5; MS (APCI): [M+1]+ = 437.97; HRMS (ESI): m/z
[M+Na]+ Calcd for C20H18F3N3O3S: 460.0919, found: 460.0919;
[α]20D +119.86 (c=1.000, CHCl3).
(APCI): [M+1]+ = 384.00; HRMS(ESI):m/z [M+Na]+ calcd for
D
C20H18ClN3OS: 406.0757, found: 406.0766; [α]20
+195.82
(c=1.000, CHCl3).
3-(4-Chloro-3-(trifluoromethyl)phenyl)-6(S)-methyl-1-[(S)-1-
phenylethyl]-2-thioxotetrahydropyrimidin-4(1H)-one 9(d): White
o
1
solid; Yield 76%; mp 126ꢀ127 C; Rf 0.50 (3:7 EtOAcꢀhexane); H
NMR (400 MHz, CDCl3): δ 1.49 (d, 3H, J = 6.8 Hz); 1.76 (d, 3H, J
= 7.2 Hz), 2.45ꢀ2.61 (m, 2H), 3.70ꢀ3.77 (m, 1H), 6.95 (q, J = 7.1 Hz,
1H), 7.27ꢀ7.50 (m, 7H), 7.60 (d, 1H, J = 8.4 Hz); 13C NMR (100
MHz, CDCl3): δ 15.3, 19.5, 38.7, 46.3, 59.8, 121.1, 123.8, 127.0,
128.5, 129.0, 132.0, 132.2, 138.2, 139.1, 165.8, 180.1; MS (APCI):
General procedure for the syntheses of 3-aryl-6-methyl-2-
thioxotetrahydropyrimidin-4(1H)-ones [10/10' (a-f): In a typical
experiment
3ꢀarylꢀ6(S)ꢀmethylꢀ1ꢀ[(S)ꢀ1ꢀphenylethyl]ꢀ2ꢀ
thioxotetrahydropyrimidinꢀ4(1H)ꢀone (1.2 mmol) was taken in a RB
flask and HBr in AcOH (5 mL) was added to it. The solution was
stirred at room temperature until the reaction was complete as
observed by TLC. It was concentrated and the residue was diluted
with DCM, washed with NaHCO3 (2 x 15 mL), then with brine (1 x
15 mL) and dried over anhydrous Na2SO4. The reaction mixture was
concentrated and purified by column chromatography on silica gel
(60–100) using EtOAcꢀhexane mixture (35:65) as the eluent to afford
3ꢀarylꢀ6(S)ꢀmethylꢀ2ꢀthioxotetrahydropyrimidinꢀ4(1H)ꢀone. By an
[M+1]+
=
426.93; HRMS(ESI):m/z [M+Na]+ calcd for
D
C20H18ClF3N2OS: 449.0678, found: 449.0677; [α]20 ꢀ146.99
(c=1.000, CHCl3).
3-(4-Chloro-3-(trifluoromethyl)phenyl)-6(R)-methyl-1-[(R)-1-
phenylethyl]-2-thioxotetra hydropyrimidin-4(1H)-one 9'(d):
White solid; Yield 76%; mp 126ꢀ127 oC; Rf 0.50 (3:7 EtOAcꢀ
1
hexane); H NMR (400 MHz, CDCl3): δ 1.49 (d, 3H, J = 6.4 Hz),
analogous
procedure
3ꢀarylꢀ6(R)ꢀmethylꢀ2ꢀ
1.76 (d, 3H, J = 6.8 Hz), 2.46ꢀ2.61 (m, 2H), 3.72ꢀ3.75 (m, 1H), 6.95
thioxotetrahydropyrimidinꢀ4(1H)ꢀone was obtained from 3ꢀarylꢀ
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

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Journal Name
RSC Advances
View Article Online
DOI: 10.1039/AC4RRTAIC06L3E91K
6(R)ꢀmethylꢀ1ꢀ[(R)ꢀ1ꢀphenylethyl]ꢀ2ꢀthioxotetrahydropyrimidinꢀ
4(1H)ꢀone.
MHz, DMSOꢀd6): δ 1.26 (d, 3H, J = 6.5 Hz), 2.67–2.74 (m, 1H),
2.84ꢀ2.89 (m, 1H), 3.88ꢀ3.93 (m, 1H), 7.52 (d, 1H, J= 8.5 Hz), 7.76
7.76 (s, 2H), 10.16 (bs, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ
19.3, 37.5, 45.0, 121.2, 123.9, 126.7, 129.6, 131.7, 135.7, 138.8,
167.2, 180.0; MS (APCI): [M+1]+ = 323.24; HRMS (ESI): m/z
[M+Na]+ calcd for C12H10ClF3N2OS 345.0052; found: 345.0044;
[α]20D –38.29 (c=1.00, acetone).
3-(4-Chlorophenyl)-6(S)-methyl-2-thioxotetrahydropyrimidin-
4(1H)-one, 10(a): White solid; Yield 72%; mp 249ꢀ251 °C; Rf 0.44
(5:5 EtOAcꢀhexane); 1H NMR (400 MHz, DMSOꢀd6): δ 1.25 (d, 3H,
J = 5.6 Hz), 2.66–2.72 (m, 1H), 2.84ꢀ2.88 (m, 1H), 3.87 (m, 1H),
7.16 (d, 2H, J = 7.5 Hz), 7.43 (d, 2H, J = 7.6 Hz), 10.06 (bs, 1H); 13
C
NMR (100 MHz, DMSOꢀd6): δ 19.3, 37.6, 44.9, 128.3, 131.6, 132.2,
138.2, 167.1, 180.4; MS (APCI): [M+1]+ = 255.20; HRMS (ESI): /z
[M+Na]+ calcd for C11H11ClN2OS: 277.0179; found: 277.0168;
[α]20D –20.18 (c=1.00, acetone).
3-(4-Chloro-3-(trifluoromethyl)phenyl)-6(R)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10'd:White solid; Yield
62%; mp 239ꢀ241 °C; Rf 0.51 (5:5 EtOAcꢀhexane); 1H NMR (400
MHz, DMSOꢀd6): δ 1.26 (d, 3H, J = 6.0 Hz), 2.70–2.74 (m, 1H),
2.84ꢀ2.89 (m, 1H), 3.91 (m, 1H), 7.52 (d, 1H, J= 8.4 Hz), 7.76 (s,
2H), 10.16 (bs, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 19.8, 38.0,
45.5, 121.7, 124.4, 126.9, 130.1, 132.2, 136.2, 139.2, 167.7, 180.5;
MS (APCI): [M+1]+ = 323.20; HRMS (ESI): m/z [M+Na]+ calcd for
C12H10ClF3N2OS 345.0052; found: 345.0048; [α]20D +38.37 (c=1.00,
acetone).
3-(4-Chlorophenyl)-6(R)-methyl-2-thioxotetrahydropyrimidin-
4(1H)-one, 10'a: White solid; Yield 74%; mp 249ꢀ251 °C; Rf 0.44
(5:5 EtOAcꢀhexane); 1H NMR (400 MHz, DMSOꢀd6): δ 1.26 (d, 3H,
J = 6.4 Hz), 2.66–2.73 (m, 1H), 2.84ꢀ2.89 (m, 1H), 3.87 (m, 1H),
7.16 (d, 2H, J = 7.6 Hz), 7.44 (d, 2H, J = 7.6 Hz), 10.06 (bs, 1H); 13
C
NMR (100 MHz, DMSOꢀd6): δ 19.8, 38.1, 45.4, 128.8, 132.1, 132.7,
138.7, 167.6, 180.9; MS (APCI): [M+1]+ = 255.27; HRMS (ESI):
m/z [M+Na]+ calcd for C11H11ClN2OS: 277.0179; found: 277.0169;
[α]20D +20.15 (c=1.00, acetone).
3-(4-Cyano-3-(trifluoromethyl)phenyl)-6(S)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10e: White solid; Yield
69%; mp 225ꢀ227 °C; Rf 0.35 (5:5 EtOAcꢀhexane); 1H NMR (400
MHz, DMSOꢀd6): δ 1.27 (d, 3H, J = 5.8 Hz), 2.69–2.76 (m, 1H),
2.86ꢀ2.91 (m, 1H), 3.93 (m, 1H), 7.77 (d, 1H, J = 7.9 Hz), 8.0 (s,
1H), 8.23 (d, 1H, J = 7.9 Hz), 10.24 (bs, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 19.2, 37.4, 45.1, 107.6, 115.2, 123.7, 129.2, 131.0,
135.1, 135.8, 144.3, 167.1, 179.5; MS (APCI): [M+1]+ = 314.27;
HRMS (ESI): m/z [M+Na]+ calcd for C13H10F3N3OS: 336.0395;
found: 336.0383; [α]20D –28.20 (c=1.00, acetone).
3-(3-Chloro-4-fluorophenyl)-6(S)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10b: White solid; Yield
1
68%; mp 230ꢀ233 °C; Rf 0.48 (5:5 EtOAcꢀhexane); H NMR (400
MHz, DMSOꢀd6): δ 1.25 (d, 3H, J = 5.4 Hz), 2.66–2.71 (m, 1H),
2.84ꢀ2.89 (m, 1H), 3.87 (m, 1H), 7.20 (s, 1H), 7.41ꢀ7.49 (m, 2H),
10.10 (bs, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 19.3, 37.6, 44.9,
116.3, 118.7, 118.9, 131.9, 136.2, 155.3, 157.7, 167.1, 180.3; MS
(APCI): [M+1]+ = 273.29; HRMS (ESI): m/z [M+H]+ calcd for
3-(4-Cyano-3-(trifluoromethyl)phenyl)-6(R)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10'e: White solid; Yield
1
D
66%; mp 225ꢀ227 °C; Rf 0.35 (5:5 EtOAcꢀhexane); H NMR (400
C11H10ClFN2OS: 273.0265; found: 273.0812; [α]20 –19.31 (c=1.00,
MHz, DMSOꢀd6): δ 1.27 (d, 3H, J = 5.8 Hz), 2.69–2.76 (m, 1H),
2.86ꢀ2.91 (m, 1H), 3.93 (m, 1H), 7.77 (d, 1H, J = 7.9 Hz), 8.0 (s,
1H), 8.23 (d, 1H, J = 7.9 Hz), 10.26 (bs, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 19.7, 37.9, 45.6, 108.1, 115.7, 124.2, 129.6, 131.2,
135.6, 136.3, 144.8, 167.6, 180.0; MS (APCI): [M+1]+ = 314.35;
HRMS (ESI): m/z [M+Na]+ calcd for C13H10F3N3OS: 336.0395;
found: 336.0388; [α]20D +28.26 (c=1.00, acetone).
acetone).
3-(3-Chloro-4-fluorophenyl)-6(R)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10'b: White solid; Yield
70%; mp 230ꢀ233 °C; Rf 0.48 (5:5 EtOAcꢀhexane); H NMR (400
1
MHz, DMSOꢀd6): δ 1.26 (d, 3H, J = 6.6 Hz), 2.65–2.72 (m, 1H),
2.84ꢀ2.89 (m, 1H), 3.85ꢀ3.90 (m, 1H), 7.18ꢀ7.21 (s, 1H), 7.41ꢀ7.50
(m, 2H), 10.10 (bs, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 19.3,
37.6, 44.9, 116.3, 118.7, 118.9, 131.9, 136.2, 155.3, 157.7, 167.1,
180.3; MS (APCI): [M+1]+ = 273.32; HRMS (ESI): m/z [M+Na]+
calcd for C11H10ClFN2OS: 295.0084; found: 295.0093;
3-(4-Nitro-3-(trifluoromethyl)phenyl)-6(S)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10f: Yellow solid;Yield
64%; mp 199ꢀ201 °C; Rf 0.52 (5:5 EtOAcꢀhexane); 1H NMR (400
MHz, DMSOꢀd6): δ 1.28 (d, 3H, J = 6.0 Hz), 2.70–2.76 (m, 1H),
2.86ꢀ2.91 (m, 1H), 3.94 (m, 1H), 7.82 (d, 1H, J = 8.4 Hz), 8.03 (s,
1H), 8.21 (d, 1H, J = 8.3 Hz), 10.26 (bs, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 19.2, 37.4, 45.1, 121.7, 123.2, 125.9, 130.0, 136.0,
143.6, 146.2, 167.2, 179.6; MS (APCI): [M+1]+ = 334.19; HRMS
(ESI): m/z [M+Na]+ calcd for C12H10F3N3O3S: 356.0293; found:
356.0286; [α]20D –36.97 (c=1.00, acetone).
[α]20D +19.37 (c=1.00, acetone).
3-(3-Chloro-4-cyanophenyl)-6(S)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10c: White solid; Yield
75%; mp 236ꢀ238 °C; Rf 0.35 (5:5 EtOAcꢀhexane); 1H NMR (400
MHz, DMSOꢀd6): δ 1.26 (m, 3H, J = 5.6 Hz), 2.67–2.73 (m, 1H),
2.85ꢀ2.90 (m, 1H), 3.89 (m, 1H), 7.42 (d, 1H, J= 8.0 Hz), 7.73( s,
1H), 8.02 (d, 1H, J = 8.0 Hz), 10.21 (bs, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 19.2, 37.4, 45.1, 111.2, 115.7, 130.1, 131.7, 134.3,
134.9, 144.8, 167.0, 179.5; MS (APCI): [M+1]+ = 280.28; HRMS
(ESI): m/z [M+Na]+ calcd for C12H10ClN3OS: 302.0131; found:
302.0136; [α]20D –30.72 (c=1.00, acetone).
3-(4-Nitro-3-(trifluoromethyl)phenyl)-6(R)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10'f: Yellow solid; Yield
68%; mp 199ꢀ201 °C; Rf 0.52 (5:5 EtOAcꢀhexane); 1H NMR (400
MHz, DMSOꢀd6): δ 1.29 (d, 3H, J = 6.6 Hz), 2.71–2.77 (m, 1H),
2.87ꢀ2.92 (m, 1H), 3.93 (m, 1H), 7.83 (d, 1H, J = 8.5 Hz), 8.04 (s,
1H), 8.22 (d, 1H, J = 8.5 Hz), 10.26 (bs, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 19.7, 37.9, 45.6, 122.2, 123.7, 126.4, 130.5, 136.5,
144.1, 146.7, 167.7, 180.1; MS (APCI): [M+1]+ = 334.27; HRMS
(ESI): m/z [M+Na]+ calcd for C12H10F3N3O3S: 356.0293; found:
356.0287; [α]20D +36.91 (c=1.00, acetone).
3-(3-Chloro-4-cyanophenyl)-6(R)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10'c: White solid; Yield
67%; mp 236ꢀ238 °C; Rf 0.35 (5:5 EtOAcꢀhexane); 1H NMR (400
MHz, DMSOꢀd6): δ 1.26 (m, 3H, J = 5.6 Hz), 2.67–2.73 (m, 1H),
2.85ꢀ2.90 (m, 1H), 3.89 (m, 1H), 7.42 (d, 1H, J= 8.0 Hz), 7.73 ( s,
1H), 8.02 (d, 1H, J = 8.0 Hz), 10.21 (bs, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 19.2, 37.4, 45.1, 111.2, 115.7, 130.1, 131.7, 134.3,
134.9, 144.8, 167.0, 179.5; [M+1]+ = 280.28; HRMS (ESI): m/z
[M+Na]+ calcd for C12H10ClN3OS: 302.0131; found: 302.0123;
Molecular Modeling Studies
The molecular docking studies were performed to predict the ligand
binding affinity as well as correct binding poses in the protein active
site.12 Molecular docking of the designed analogues was performed
using Glide software included in Schrodinger suite 9.0.211. The
crystal structure of the androgen receptor ligandꢀbinding domain,
W741L mutant complex with Rꢀbicalutamide (pdb id 1Z95) is used
D
[α]20 +30.78 (c=1.00, acetone).
3-(4-Chloro-3-(trifluoromethyl)phenyl)-6(S)-methyl-2-
thioxotetrahydropyrimidin-4(1H)-one, 10d: White solid; Yield
65%; mp 239ꢀ241 °C; Rf 0.51 (5:5 EtOAcꢀhexane); 1H NMR (400
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

RSC Advances
PaPgaeg1e08oof f191
View Article Online
DOI: 10.1039/C4RA06391K
8
for molecular docking.13 Protein structure was prepared using
2004, 10, 440; (c) A. Jemal, T. Murray, A. Samuels, A. Ghafoor, E.
Ward and M. J. Thun, CA Cancer J. Clin. 2003, 53, 5; (d) A. O.
Brinkmann and J. Trapman, Nat. Med., 2000, 6, 628; (e) M.ꢀE. Taplin,
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Klocker, G. Bartsch and A. Hobishc, Endocr. Relat. Cancer, 2002, 9,
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Res., 2001, 61, 5611; (b) W. Gao, C. E. Bohl and J. T. Dalton, Chem.
Rev., 2005, 105, 3352.
protein preparation wizard for molecular docking as follows;
hydrogen atoms were added, hydrogen bonding network was
optimized, and protein was minimized to RMSD (Root Mean Square
Deviation) 0.30Å using OPLS (Optimized Potential for Liquid
Simulations) 2005 force field. The designed ligands were prepared in
LigPrep and minimized using OPLS 2005 force field. The coꢀ
crystallized ligand bicalutamide was used to define binding site grid
in protein structure. The ligand docking calculations were done in the
standard precision mode of glide software. Ligands were passed
through a scaling factor of 0.80 and partial charge cutoff of 0.15.114
2
3
4
Pharmacological Studies
All inꢀvitro experiments for cell proliferation/inhibition were
performed in triplicates. PCꢀ3 cells were originally derived from
advanced androgen independent bone metastasized prostate cancer.
For the PCꢀ3 cell growth inhibition assay, cells were cultured in
DMEM supplement and trypsinized, further diluted to 2.0 x 104
cell/mL with DMEM supplemented with 10% fetal bovine serum.
This cell suspension was transferred to 96ꢀwell microtiter plates, and
incubated in the presence or absence of increasing concentration of
positive control (doxorubicin) or the test compounds (10ꢀ12M, 10ꢀ
10M, 10ꢀ8M, 10ꢀ6M and 10ꢀ4M) at 37oC and 5% CO2. After 24h
incubation, cells were treated with MTT solution for 4h in a cell
culture incubator at 37 oC and 5% CO2. Cell proliferation was
determined by the MTT method. MTT which is a tetrazolium salt is
converted into insoluble formazan by mitochondrial dehydrogenases
in live cells. Formazan is dissolved in DMSO (Merck) and
absorbance was measured at dual wavelength of 550 nm and 630 nm
on an ELISA plate spectrophotometer (Biotrek instruments). Similar
experiments were performed in LNCaP cells also. Since LNCaP cells
have demonstrated androgen dependent cell growth; promotion and
inhibition of cell growth was considered as agonistic and
antagonistic respectively. These cells are known to possess an
aberrant AR with a mutation of 877Thr to Ala. LNCaP cells were
cultured as described above in RPMI1640 media and were
transferred into a 96ꢀwell plate with a 2.0 x 104 cell/mL well density
supplemented with testosterone (final concentration in each well was
kept 10 nM). After 24h, the cells were treated with testosterone in the
presence or absence of each concentration of positive control
(Flutamide) or test compounds (10ꢀ10M, 10ꢀ8M, 10ꢀ6M and 10ꢀ4M) for
another 24h. The total number of viable cells relative to viable cells
in untreated control was plotted. For LNCaP cells, the number of
cells on wells with testosterone alone was defined as 100% viability.
(a) S. M. Singh, S. Gauthier and F. Labrie, Curr. Med. Chem., 2000, 7,
211; (b) J. C. Lanter, J. J. Fiordeliso, W. Jiang, G. F. Allan, M.ꢀT. Lai,
O. Linton, D. W. Hahn, S. G. Lundeen and Z. Sui, Bioorg. Med. Chem.
Lett., 2007, 17, 123; (c) H. Tucker, J. W. Crook and G. J. Chesterson,
J. Med. Chem., 1988, 31, 954.
5
6
C. E. Bohl, W. Gao, C. E. Bell and J. T. Dalton Proceed. Natl. Acd.
Sci., 2005, 102, 6201.
(a) V. Kumar and V. A. Nair, Tetrahedron Lett., 2010, 51, 966; (b) V.
Kumar, P. Raghavaiah, S. M. Mobin and V. A. Nair, Org. Biomol.
Chem., 2010, 8, 4960; (c) G. L. Khatik, A. Pal, S. M. Mobin and V. A.
Nair, Tetrahedron Lett., 2010, 51, 3654; (d) G. L. Khatik, J. Kaur, V.
Kumar, K. Tikoo, P. Venugopalan and V. A. Nair, Eur. J. Med. Chem.,
2011, 46, 3291ꢀ3301; (e) M. Chouhan, K. R. Senwar, R. Sharma, V.
Grover and V. A Nair, Green Chem., 2011, 13, 2553; (f) R. Sharma,
M. Chouhan, V. A. Nair, Tetrahedron Lett., 2010, 51, 2039ꢀ2043; (g)
M. Chouhan, R. Sharma and V. A. Nair, Appl. Organomet. Chem.,
2011, 25, 470; (h) R. Sharma, M. Chouhan, D. Sood and V. A. Nair,
Appl. Organomet. Chem., 2011, 25, 305ꢀ309 (i) V. Kumar, G. L.
Khatik and V. A. Nair Synlett, 2011, 2997; (j) V. Kumar, A. Pal, G. L.
Khati and V. A. Nair, Tetrahedron: Asymmetry,2012, 23, 434; (k) G.
L. Khatik, V. Kumar and V. A. Nair, Org. Lett., 2012, 14, 2442; (l) M.
Chauhan, R. Sharma and V.A. Nair, Org. Lett., 2012, 14, 5672; (m) V.
Kumar, K. Kumar, A. Pal, G. L. Khatik and V. A. Nair, Tetrahedron,
2013, 69, 1747; (n) V. Kumar, G. L. Khatik, A. Pal, S. Bhattarai, M. R.
Praneeth and V. A. Nair, Synlett, 2012, 2357.
7
(a) C. Tran, S. Ouk, N. J. Clegg, Y. Chen, P. A. Watson, V. Arora, J.
Wongvipat, P. M. SmithꢀJones, D. Yoo, A. Kwon, T. Wasielewska, D.
Welsbie, C. D. Chen, C. S. Higano, T. M. Beer, D. T. Hung, H. I.
Scher, M. E. Jung and C. L. Sawyers, Science, 2009, 324, 787; (b) M.
E. Jung, S. Ouk, D. Yoo, C. L. Sawyers, C. Chen, C. Tran and J.
Wongvipat, J. Med. Chem., 2010, 53, 2779; (c) H. I.; Scher, T. M.
Beer, C. S. Higano, A. Anand, M.ꢀE. Taplin, E. Efstathiou, D.
Rathkopf, J. Shelkey, E. Y. Yu, J. Alumkal, D. Hung, M. Hirmand, L.
Seely, M. J. Morris, D. C. Danila, J. Humm, S. Larson, M. Fleisher and
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R. MacVicar, J. Wang, D. MacLean and W. M. Stadler, J. Clin.
Oncol., 2010, 28, 3084.
Conclusions
In conclusion, optically pure enantiomers of 3ꢀarylꢀ6ꢀmethylꢀ2ꢀ
thioxotetrahydropyrimidinꢀ4(1
yields and high selectivities by asymmetric induction using
chiral ( )/( )ꢀαꢀmethyl benzylamine. In vitro cytotoxicity of
H)ꢀones were obtained in good
R
S
these derivatives was evaluated on PCꢀ3 and LNCaP cell lines
for the antiꢀprostate cancer activity. Compound 10'e was found
to be highly potent with cytotoxicity comparable with
doxorubicin on PCꢀ3 cell lines and better than the standard
drugs flutamide and bicalutamide on LNCap cell lines though
its enantiomer was 20 fold less active. Acknowledgements
8
9
Docking studies were carried out on Glide, version 5.7, Schrodinger,
Inc., New York, NY, 2011.
We thank the Department of Science and Technology,
Government of India for the research funding. We are also grateful
to Centre for Pharmacoinformatics, NIPER, S. A. S. Nagar for the
computational facilities.
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Davies and O. Ichihara, Tetrahedron Asymm., 1991, 2, 183; (c) S. G.
Davies; O. Ichihara and I. A. S. Walters, J. Chem. Soc. Perkin
Trans.1, 1994, 1, 1141.
.
10
11
H. Tamura, Y. Ishimoto, T. Fujikawa, H. Aoyama, H. Yoshikawac and
M. Akamatsu, Bioorg. Med. Chem., 2006, 14, 7160.
Notes and references
1
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7, abstr. 168); (b) T. J. Mosmann Immunol. Methods, 1983, 65, 55 (c)
8 | J. Name., 2012, 00, 1-3
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Journal Name
DOI: 10.1039/AC4RRTAIC06L3E91K
T. Ishioka, A. Kubo, Y. Koiso, K. Nagasawa, A. Itai and Y. Hashimoto
Bioorg. Med. Chem., 2002, 10, 1555.
12
13
14
D. A. Gschwend, A. C. Good and I. D. Kuntz, J. Mol. Rec., 1996, 9,
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Ligand preparation was performed by LigPrep, version 2.5,
Schrodinger, Inc., New York, NY, 2011.
R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J. Klicic,
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This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 9