A one-pot multicomponent facile synthesis of dihydropyrimidin-2(1: H)-thione derivatives using triphenylgermane as a catalyst and its binding pattern validation

Article abstract of DOI:10.1039/c6ra19162b

A series of substituted dihydropyrimidin-2(1H)-thione derivatives (1-8) have been synthesized using a facile and modified procedure with triphenylgermyl propionate as a catalyst. In comparison with the classical Biginelli reaction, this new protocol has the advantages of excellent yield and shorter reaction times. The synthesized compounds have been characterized by various spectroscopic techniques such as FT-IR, multinuclear (¹H/¹³C) NMR spectroscopy and single crystal XRD analysis. Molecular docking studies were performed to identify the probable binding modes of potent inhibitors in the active site of the enzymes human topoisomerase II alpha (4FM9) and Helicobacter pylori urease (1E9Y). Compound 3 was found to be the most potent inhibitor according to the molecular docking scores and molecular dynamic simulations which suggests it can be further processed as a lead molecule to interpret the pharmacological properties of these compounds.

Full text of DOI:10.1039/c6ra19162b

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2016, DOI: 10.1039/C6RA19162B.
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DOI: 10.1039/C6RA19162B
A One-pot Multicomponent Facile Synthesis of Dihydropyrimidin-
2(1H)-thione Derivatives using Triphenylgermane as a Catalyst
and its Binding Pattern Validation
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
c
Sohaila Andleeb, Imtiaz-ud-Din, * Muhammad Khawar Rauf, * Syed Sikander Azam, Amin
Badshah, a Haseeba Sadaf, a Ahmed Raheel, a Muhammad Nawaz Tahir, b Saad Raza, c
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of substituted dihydropyrimidin-2(1H)-thione derivatives (1-8) have been synthesized by
using a facile and modified procedure using triphenylgermyl propionate as a catalyst. On
comparison with classical Biginelli reaction, this new protocol has the advantages of excellent
yield and shorter reaction time. The synthesized compounds have been characterized by various
spectroscopic techniques such as FT-IR, multinuclear (1H/13C) NMR spectroscopy and single crystal
XRD analysis.The molecular docking studies was performed to identify the probable binding
modes of potent inhibitors in the active site of the enzymes Human topoisomerase II alpha (4fm9)
and Helicobacter pylori urease (1E9Y). Compound 3 was found to be the most potent inhibitor
according to the molecular docking scores and molecular dynamic simulation which suggests they
can further be processed as lead molecule to interpret the pharmacologic properties of these
compounds.
Introduction
till the 1980s. Synthesis of many other heterocyclic
Thiones, in particular dihydropyrimdin-2(1H)-thiones
have been reported to possess remarkable
pharmacological applications such as antiviral,
antibacterial, antihypertensive, anti-inflammatory,
anticancer activity [1, 2] and can act as calcium channel
modulators as well as multi- drug resistance reversal [3,
4] The ring system of pyrimidine, being present in many
biomolecules such as vitamins and nucleic acids, play
an important role in various biological processes [5].
Keeping in view these aspects, their synthesis has
become the focus of great interest for organic and
medicinal chemists [6]. Pietro Biginelli reported the
acid catalysed cyclocondensation reaction of ethyl
acetoacetate, benzaldehyde and urea [7], and the
novel product (3,4-dihydropyrimidin-2(1H)-thione) was
obtained by simply heating a mixture of the three
components dissolved in ethanol with a catalytic
amount of HCl at refluxing temperature. Unfortunately,
the synthetic potential of this new heterocyclic
compound remained unexplored for quite some time
scaffolds has also been attributed to these quite
flexible precursors [8, 9]. One of the major drawbacks
associated with Biginelli condensation is low products
yield (30–35%). In recent years, several methods to
improve the yield using triphenyl phosphine [10], ZrCl4
[11], SiO2/H2SO2 [12], granite [13], trichloroisocyanuric
acid [14], copper(II) sulfamate [15], nafion-H [16], p-
sulfonic acid calixarenes [17] and [Al(H2O)6](BF4)3] [18]
have been reported in the literature (Table 1). To
overcome the problems associated with the reported
methodologies there is a need for a simple and efficient
method to synthesize dihydropyrimdin-2(1H)-thiones
under mild conditions. Apart from the synthesis of
organogermanium compounds, their new applications
have also been explored [18-20]. A detailed literature
survey clearly suggested that there is no report yet on
the application of triphenylgermane as Lewis acid
catalyst for the classic Biginelli and Hantzsch
condensation reactions. Research work was further
extended to develop a most plausible mechanism for
the synthesis of dihydropyrimidines using Ph3GeH as
catalyst. We here are now able to propose a new
reaction mechanism whereby organogermane in
presence of catalytic amount of HCl ( produced in situ)
play an active role in the synthesis of dihydropyrimidin-
2(1H)-thiones in excellent yield.
aDepartment of Chemistry,Quaid-i-Azam University, Islamabad-45320, Pakistan,
E-mail: drimtiazuddin@yahoo.com; Fax: +92 51 90642241; Tel: +92 51 9064210;
mkhawarrauf@yahoo.co.uk; Fax: +92 51 90642241; Tel: +92 51 90642226
bDepartment of Physics, University of Sargodha, Sargodha, Pakistan.
cComputational Biology Lab, National Center for Bioinformatics, Quaid-i-Azam
University, Islamabad-45320, Pakistan
Electronic Supplementary Information (ESI) available: [CCDC nos. for
1, 2, 3, 4 and
7
are 1419339, 1063506, 1419338, 1063500, and 1419340. For crystallographic
data See DOI: 10.1039/x0xx00000x
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In the recent years, sulfur containing compounds as a catalyst facilitates imminium mechanism which
has shown remarkable medicinal applications due to involves the reaction of substituted anilines and HSCN
their pharmacological and biological significance. Prior to produce thiourea intermediates II(a-h) followed by
to the in vitro studies, we preferred to explore the their reaction with methylpent-3-en-2-one (I) whose
protein binding capacity of these compounds by using stereoselectivity has been tuned by hydrogermane
in silico techniques against Human topoisomerase II used as catalyst.
alpha (PDB ID=4fm9) and Helicobacter pylori urease Table 1. Comparative overview of the catalytic activity
(PDB ID=1E9Y). The targets used are considered to be of various catalysts for the synthesis of Biginelli’s type
representative for anticancer and urease inhibition 3,4-dihydropyrimidin-2(1H)-thiones/ -ones [1, 10-17].
studies.
Solvent/
Temp.b
Solvent
free/ 1000C
C2H5OH/
reflux
Time Yield
Catalyst/ Refs.a
Results and discussion
(h)
(%)
Triphenyl phosphine
[10]
Synthesis and Chemistry
10
70-80
The designed 3,4-dihydropyrimidin-2(1H)-thiones (1-8)
were synthesized in a single step. Catalytic amount of
triphenylgermyl propanoic acid was treated with SOCl2
and excess of SOCl2 was removed prior to dilution with
acetone. Calculated quantity of KSCN was added and
stirred for 30 minutes, and then a solution of
substituted aniline was added. Aqueous workup in
acidified chilled water affords dihydropyrimidin-2(1H)-
thione (1-8) in excellent yields (Scheme 1).
ZrCl4 [11]
6
6
80-88
85-90
60-65
50-56
85-90
C2H5OH/
reflux
Silica/H2SO4 [1]
Granite [12(a)]
InBr3 [12(b)]
C2H5OH/
reflux
3.5
10
12
C2H5OH/
reflux
Trichloroisocyanuric
acid [13]
C2H5OH/
reflux
Biginelli’s reaction is quite sensitive to the choice of
solvent, catalyst and the reactants, in particular,
carbonyl compounds being used. Parallel studies
evaluating the effect of Ph3GeH on product yield and its
isolation settled that use of catalytic amount of
triphenylgermane/HCl enhances the yield averaging to
be 92%. The advantages of this kinetically more facile
methodology are the lesser reaction time and ease of
isolation. The data as mentioned in Table-1 manifested
that our synthetic methodology is one of the best,
although there are reports showing about 98% yield
[13, 17(c)] but there are also some synthetic
approaches which yielded the product less than 65%
[12].
CH3COOH/
1000C
Copper(II) sulfamate
[14]
6
75-80
C2H5OH/
reflux
Nafion-H [15]
5
8
75-90
75-80
75-80
p-Sulfonic acid
calixarenes [16]
[Al(H2O)6](BF4)3]
[17(a)]
C2H5OH/
reflux
CH3CN/
reflux
20
Yttria-Zirconia base
Lewis acid [17(b)]
Metallo-
phthalocyanines
[17(c)]
CH3CN/
reflux
4-16 81-94
CH3CN/
reflux
5
82-98
C2H5OH/
reflux
LaCl3·7H2O [17(d)]
5
18
3
56-97
60-97
90-96
Phenylboronic acid
[17(e)]
CH3CN/
reflux
Earlier reports clearly depicts that Iminium, Enamine
and Knoevenagel mechanisms are the three main
mechanistic approaches that are well established for
the Biginelli’s reaction and a kinetic model also suggests
that a catalyst used in Biginelli reaction not only
improves the yield of products and reduces the
reaction time but also responsible for selection of
reaction pathway, thereby favouring the possible
mechanism involved [21]. Choice of triphenylgermane
Ph3GeH Present
acetone/
reflux
work
a References b Temperature
The overall reaction, involving the formation of 4-
methylpent-3-en-2-one (I) and thioureas II(a-h) with
the use of substituted anilines instead of aldehydes as
mentioned in Biginelli’s reaction, to produce
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dihydropyrimidin-2(1H)-thiones (1-8), has been shown
in Scheme 2. Triphenylgermylpropanoic acid produces
HCl on reaction with SOCl2 as shown in scheme 2 that
leads to a pathway study suggesting the formation of 4-
methylpent-3-en-2-one (I), produced by acid-catalyzed
aldol reaction between two ketonic molecules as
depicted in scheme 2. Formation methylpent-3-en-2-
one in step 1 has been reported [22].
Step-III: Formation of thiourea species II (a-h)
Step-IV: Organogermane catalyzed reaction between
(I) and (II).
Scheme 1. Synthesis of dihydropyrimidin-2(1H)-thiones
(1-8).
The overall reaction can be delineated as follows;
Scheme 2. Plausible mechanism for (1-8).
Step-I: Acid halide formation.
Formation of acid catalyzed acyl imine intermediate as
a key rate determining step in Biginelli reaction has
been proposed and established by Kappe [8].
Formation of methylpent-3-en-2-one (I) in scheme 2
Step-II: Catalyzed conversion of acetone to 4- (step I) proceeds with hyrometallation where
methylpent-3-en-2-one (I).
triphenylgemanium hydride adds to I following Anti
Markovnikov addition. Owing to expandable d-orbital
of germanium, an intermediate IIIb forms between IIIa
and II(a-h) through a coordinate bond following
associative mechanism which is not stable enough and
triphenylgemanium leaves as triphenylgemanium
hydride through dissociative mechanism forming IIIC. A
keto-enol tautomerisim establishes in the subsequent
step which under acidic conditions favors enol form. As
earlier reports suggest, the presence of HCl activates
the enol form whose quantity accounts for all the
facets of Lewis aid catalyzed Biginelli reaction [23]. This
enol form undergo cyclocondensation forming 3, 4-
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dihydropyrimidin-2(1H)-thiones (1-8) as shown in used in cross-coupling reactions after germanium
scheme 2 (step-IV). electronic tuning. Finally this tuning of the electron
It is important to mention that wider functional deficiency of the germanium atom makes cross
group tolerance is exhibited here by coupling of II(a-h) facile which further on losing water
hydrogermalylation as compared to that displayed in molecule produces the target compounds (1-8).
previously known Lewis acid catalyzed methodologies Catalytic role of organogermane, produced in situ, was
[24]. Indeed due to lower oxophilicity of germanium as further confirmed by repeating the same methodology
compare to silicon, chemoselectivity of the reaction is using acylation of trans-3-phenyl-2-propenoic acid
affected, giving much more clean and high yielding where no Ph3GeH moiety is present in the acid that
product without disturbing the alkoxy substituents results in a mixture of products containing low yield of
present on the aniline used, even if excess of the dihydropyrimidine thiones. When this study was
germane is used.
undertaken in the presence of organogermanium,
Triphenylgermylpropanoic acid readily produces there is a tremendous improvement in the yield as well
organogermane species on decomposition as evident as the product isolation becomes facile. This study was
from the mass spectral studies of our earlier reports further continued by decreasing the amount of
[25, 26]. The oxidation potential of triphenyl germane, triphenylgermyl propanoic acid to different mole ratios
as measured by cyclic voltammetry, is +0.94V, which and it was determined that the catalytic system works
conforms to its easy oxidation leading to efficiently up to 5 mole%.
triphenylgermyl species. Moreover, the intermediacy of Spectroscopic Studies
this oxidatively generated triphenylgermyl radical has The spectroscopic data, mentioned in the experimental
also been proven using spin trapping experiments with section, fully characterized the formation of target
phenyl t-butyl nitrone [27]. These organogermanium compounds. Tentative assignments of peaks in the FT-
hydrides have been used as transition-metal-catalyzed IR spectra of the compounds (1-8) have been made on
hydrogermylation of alkenes and alkynes since mid- the basis of earlier publications [35, 36]. The infrared
1950s [28]. Since the initial report of Gilman [29], spectra show five absorption bands of interest namely
limited reports have been published on radical ν(N-H), ν(C-H)Ar, ν(C-H)aliphatic., ν(C=C) and ν(C=S) at
hydrogermylation of π-systems [27]. Besides the radical 3231-3185, 3032-2944, 2883-2850, 1560-1515 which
mechanistic framework, transition-metal-catalyzed range from weak to strong intensities respectively.
hydrogermylation of terminal alkynes has been more Many other prominent peaks like C-F, C-Cl, SH and NO2
extensively studied with catalysts based on rhodium have also been observed in their respective regions.
1
[30], palladium [31], ruthenium [32] and iron [33]. Two
The H NMR data demonstrate that all the protons
last classes of hydrogermylation that support our show characteristic chemical shift values in their
concept of hydrogermylation of 4-methylpent-3-en-2- respective regions identified by their intensity and
one (I), as enunciated in Scheme 2, is the Gevorgyan multiplicity pattern and the total number of protons
Lewis acid catalysed hydrogermylation of terminal calculated from the integration curve is in agreement
alkynes and the Schweizer’s stereoselective with the presented molecular composition. The NH
hydrogermylation of α-trifluoromethylated alkynes protons in all the synthesized compounds appeared as
[34]. As an attempt to discover a plausible route to broad peak near 7 ppm while a quartet around 4.81-
meet the challenge of high yield synthesis of 5.04 ppm shows the splitting of methylene proton by
dihydropyrimidine thiones, we envisioned that
a
neighbouring methyl protons on vicinal carbon. Two
hydrogermylation reaction, whose stereoselectivity has methyl substituents at carbon next to thiourea NH
been tuned under the present set of catalytic acidic show two singlets at different chemical shifts due to
conditions, could potentially provide an efficient and magnetic non-equivalence. Three protons of methyl
divergent route to substrate (I) that could be further substituent at carbon next to the methylene carbon
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show doublet in range of 1.43-2.55 ppm while protons density over the thiourea moiety, which is evidenced
in the aromatic region are sensitive towards the by the shorter N–C bond lengths [1.317–1.479 Å] listed
substituent attached to phenyl ring and change their in Supplementary Table 2, and hence the shortest N-C
positions and multiplicities accordingly. In compound 1 bond lengths [1.317-1.326Å] confirm the partial double
proton attached to carbon in vicinity of nitro group bond character of N-C in (H)N–C=S moiety due to C=S/
shows a splitting into a doublet due to coupling with SH tautomerism.
three methyl protons in its close vicinity. Due to higher
electron withdrawing effect of NO2 groups this proton
(-C=CNO2) is highly deshielded and a higher downfield
shift at 7.58-7.59 ppm is observed. Methoxy protons
give a clear singlet at 3.92 ppm. In compound 2, 4 and 5
proton attached at carbon adjacent to F, Br and S
shown higher downfield shift and their chemical shift
values are slightly higher than other protons of phenyl
ring. In compound 3 a doublet is observed for proton at
carbon next to nitro group (=C-CNO2) around 8.28ppm
while the proton at carbon next to nitro group (-
C=CNO2) shows multiplet instead of singlet due to
coupling with methyl protons as for compound 1. A
singlet at 3.4ppm in compound 5 indicates the
presence of SH proton.
The 13C NMR data show that signals found
correspond with the presence of magnetically non-
equivalent carbon atoms, assigned by comparison of
the experimental chemical shift values with those
calculated from the incremental method [37]. Highly
deshielded carbon in 13C NMR spectra of target
compounds (1-8) at 177.7-176.6ppm indicate the
presence of C=S functionality. The phenyl ring carbons
containing strong electron withdrawing groups e.g.,
NO2, F, Cl, Br resonate at low field while carbon of
methyl substituent appear up field.
Crystallographic discussion (1-4, 7)
Crystallographically suitable single crystals of (1-4, 7)
for X-ray analysis were grown from dichloromethane
solution by slow evaporation at room temperature. The
ORTEP structures of dihydropyrimidin-2(1H)-thione (1-
4, 7) along with numbering schemes are illustrated in
Fig. 1 and the selected geometric parameters are
presented in Supplementary Table 2. The
dihydropyrimidin-2(1H)-thiones moiety is nearly planar
in all the molecules as evident from the associated
torsional angles. The reason for this planarity, however,
can be accredited to the delocalization of π-electron
Fig. 1 ORTEP view showing 35% probability ellipsoids
(a-e) of (1-4, 7) with atom numbering scheme.
The longer N-C bond lengths [1.452–1.472 Å] (1-4, 7)
and [1.470-1.479Å] (1, 3) observed for HN-Csp3 and
O2N-Csp2 respectively. These elongations in N-C bond
lengths can be associated with the lesser s character of
C
sp3and the electron withdrawing effect of –NO2group.
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The molecular structure of 1 {Figure 2(f)} shows
intermolecular N(3)-H(3A)···S(1) 2.500Å and C(12)-
H(12A)···O(2) 2.699Å hydrogen bonds stabilizing the
thiones geometry mainly due to the centrosymmetric
dimers {···H-N-C=S}2 connected by N–H···Si (2.500Å) ((i)
−x+1, −y+2, −z+1) as well as by comparatively weak C–
H···O (2.699Å) hydrogen bonds.
(f)
In the crystal packing of 2 {Figure 2(g)}S atom (C=S)
shows trifurcated intermolecular N(2)-H(2)···S(1)i
2.638Å, C(5)-H(5)···S(1) 2.920Åand C(6)-H(6)···S(1)ii
2.975Å ((ii) −x, −y+1, −z+1) hydrogen bonds stabilizing
the thiones geometry. These molecular structures also
fashioned the centrosymmetric dimers {···H-N-C=S}2
connected by N–H···Si (2.638Å) {(i) −x, −y, −z+1}. Similar
type of intermolecular interactions has also been
observed for 4 {Figure 2(i)}.
(g)
In the crystal packing of 3, the sulphur atom (C=S)
involved in the formation of synthones {···H-N-C=S}2 is
bifurcated {Figure 2(h)} forming N(3)-H(3A)···S(1)i
2.559Å {(i) −x+1, −y, −z)} hydrogen bonds and
additional C(7)-S(1)···Cl(1) 3.493Å interactions. The
weak intermolecular molecular interactions C(11)-
H(11C)···O(1) 2.392Å {(ii) x−1, y, z } and N(1)-O(1)···O(1)
2.970Å have also been observed to stabilizing the
crystal packing in 3. Similar to the crystal packing of
3,sulfur atoms (C=S) in 7also involved in the formation
of dimeric synthones {···H-N-C=S}2through N(2)-
H(2A)···S(1)i2.593Å {(i) −x+1, −y+1, −z+1} hydrogen
bonds is bifurcated {Figure 2(j)} forming additional
C(5)-H(5)···S(1) 2.873Å {(ii) −x+1, y−1/2, −z+1/2}
interactions {Supplementary Table 3}. Molecular
structures of 4 and 7 additionally show C-H···Br and C-
H···Cl hydrogen bonds stabilizing their geometry,
respectively. The dihedral angles between the aromatic
(h)
(i)
(J)
rings C(1)-C(6) and
dihydropyrimidin-2(1H)-thione
moieties for 1-4 and 7 are 86.06, 85.85, 88.66, 84.23
and 85.69˚, respectively. The narrow variation in
dihedral angles subtended by aromatic rings with
dihydropyrimidin-2(1H)-thione moieties through C-N
bond reveal that the substituents have minimal effect
on the conformational geometry of these molecules.
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compounds are involved in making conventional
hydrogen bonds, hydrophobic interactions with the
residues of active site region. The docking results
indicate that the inhibitor docked around His322
(residue of active site) showed acceptable binding
energy values (Table 2).
Fig. 2 View on the hydrogen bonded 1D structures (f-j)
for (1-4, 7).
Molecular Docking
Molecular Docking Studies of Human topoisomerase II
alpha
The docking results indicate that the inhibitor docked
around Met762 (residue of active site) showed
acceptable GoldScore (55.39) and acceptable binding
energy values. VMD [38] and ligplot [39] analysis was
performed in order to validate the in-depth interaction
patterns between inhibitor and active site of 4fm9
(Supplementary Fig. 1-3). Studied compounds are
involved in making conventional hydrogen bonds,
hydrophobic interactions with the residues of active
site region and salt bridge as well 8.
Binding mode analysis
The docked complex is showing appreciable
interactions with the active site residues as shown in
the Fig. 4. Vander-Waals and hydrogen bonding is also
involved in the stability of the compound used in the
active site pocket.
Binding mode analysis
The docked complex is showing appreciable
interactions of the active site residues as shown in the
Fig 3. Active site residues inside 10 Å regions are
involved in the stability of the compound inside the
active site pocket.
Fig. 4.Interaction diagram of 3 with 1e9y
Molecular Dynamics of complexes
Docked compound 3 with Human topoisomerase II
alpha and Helicobacter pylori urease were simulated in
a molecular environment to establish their significance
against these targets. Top scored compound 3 was
selected among the derivatives (1-8) to sum up their
behaviour, and we are looking for the best candidates
among these derivatives to be used as potential lead.
Initial docked conformation showed promising binding
with the active site for 1EY9 and 4FM9 but to further
establish their binding pattern and to give some
baseline for their mechanism against these enzymes,
molecular dynamics simulation was carried out on
these docked complexes. The residue numbers are
restarted from 1 by simulation package, if there is no
structural information of those residues in pdb file. So,
there is a difference in number of residues in pdb and
simulation trajectory of 4FM9 which is 432. During the
Fig.3.Interaction diagram of 3 with 4fm9
Molecular Docking Studies of Helicobacter pylori
Urease
The docking results indicate that the inhibitor docked
around active site (residue of active site) showed
acceptable Gold Score 55.39. VMD [38] and ligplot [39]
analysis was performed in order to validate the in-
depth interaction patterns between inhibitor and active
site of 1E9Y (Supplementary Fig.4-7). Studied
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simulation the average mean deviation in backbone of 1E9Y is observed as 2.02
Å
(Figure 5a) and
Fig. 5 Root mean square deviation (a) and (b) of backbone of complex of compound 3 with 1E9Y and 4FM9
respectively, and root mean square fluctuation (c) and (d) of residues for the trajectories of 1E9Y and 4FM9
respectively during the course simulation.
Table 2. Docking scores
No.
Human topoisomerase II alpha (4FM9)
Helicobacter pylori Urease (1E9Y)
Binding Energy Gold score
Binding Energy
Gold score
50.10
1
2
3
4
5
6
7
8
-6.0
-8.1
-7.3
-7.3
-6.9
-7.0
-7.3
-7.2
-6.8
-7.2
-7.6
-7.1
-6.9
-6.5
-6.0
-6.5
51.85
45.10
57.72
46.50
48.75
50.52
48.09
57.07
46.16
55.39
49.06
49.27
52.23
50.42
50.72
deviation (RMSD) and root mean square fluctuation
(RMSF) value you can see that the 1EY9 is relatively
We see a relatively larger deviation of 2.74 Å in 4FM9
(Figure 5b). According to the root mean square
8 | J. Name., 2012, 00, 1-3
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more stable than the 4FM9 (Figure 5c). The main cause After another 20 ns we can see it acquires new polar
of this deviation is the two large helices which make up residue around it (Figure 6c). For the last 10 ns we can
the unique folds of 4FM9. These helices are present see that these partners slight vary but the compound
from residue number 600 and onwards in the protein does not leave its initial position (Figure 6d). In case of
4FM9 which are clearly visible in RMSF graph of 4FM9 1E9Y the compound 3 drifts away from initial site but
showing higher rate of fluctuation then the rest of after 30 ns it chooses another site to interact and binds
protein (Figure 5d). Overall the 4FM9 is having higher slightly away from its initial position (Figure 7a). From
fluctuations than 1E9Y, button the other hand the the snapshots taken after 30 ns the compound 3
compound 3 is found to be in the vicinity of initial appears to have bonded to this site for the rest of 20 ns
docked site of 4FM9 throughout the simulation time of simulation. From the snapshot taken at 30 ns
frame. The snapshots taken at different intervals compound 3 has adjusted itself around polar residue
suggest that the compound 3 stays near the residue ASN 547, THR 545, ASN 796, LYS 797 and GLN 802
GLN 110 and ASP 111. Initially the compound is found (Figure 7b). These interacting partners of continue to
to be actively interacting via hydrogen bonds to polar exist around the compound 3 after 20 ns, which are
residue around it (Figure 6a). As the simulation proceed seen in a snapshot taken at 40 ns (Figure 7c) and 50 ns
for 20 nanoseconds (ns) we can see that some of the (Figure 7d). This behaviour of compound 3 in the
interacting partners are lost but it firmly attaches itself presence of enzymes 1E9Y and 4FM9 could suggest
to the backbone of GLN110 and ASP111 (Figure 6b).
that it can actively suppress the human topoisomerase
II alpha and it can indirectly or directly alter the activity
of Helicobacter pylori urease.
Experimental
Chemicals/instrumentation
All reagents and solvents used in this study were
obtained from the supplier and recrystallized/
redistilled as required. (E)-3-phenylprop-2-enoic acid
was purchased from Sigma Aldrich (Germany), while
procuration of germanium dioxide (99.9% purity) was
made from the People’s Republic of China and was
used as received. 3-triphenylgermyl-3-phenylpropanoic
acid was prepared according to reported methods [18,
20].Thin layer chromatography (TLC) was performed
using aluminium sheets coated with silica gel 60 F254
(Merck). Melting points of all the synthesized
compounds have been determined in open capillary
tubes by using a Gallenkamp apparatus (MP-D) and
were uncorrected. IR spectra in the range of 4000–400
cm-1 were obtained on a Thermo Nicolet-6700 FT-IR
Fig. 6 Snapshot taken at intervals of (a) 1 ns, (b) 20 ns
(c) 40 ns and (d) 50 ns from the simulation trajectory of
complex of compound 3 with 4FM9.
Spectrophotometer. The H and 13C NMR spectra were
1
recorded on a Bruker spectrometer at 300 MHz and 75
MHz in CDCl3, respectively using residual solvent signals
as a reference.
Fig. 7 Orientation of compound 3 around 1E9Y at
simulation time intervals of (a) 1ns, (b) 30 ns, (c) 40 ns
and (d) 50 ns.
Synthesis of dihydropyrimidin-2(1H)-thiones(1-8)
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The general procedure used for the synthesis of target 177.7 (C=S), 133.9, 131.9, 130.4, 128.5, 124.2, 124.1,
compounds (1-8) is as follows. About 1 ml SOCl2 was 116.1, 109.6, 52.5, 31.3, 31.2, 19.9. Anal. calc. for
added to 0.09 g (0.2 mmol) of 3-triphenylgermyl-3- C13H15FN2S (250.34): C, 62.37; H, 6.04; N, 11.19; S,
phenylpropanoic acid (5 mol %) and stirred for two 12.81. Found: C, 62.32; H, 6.00; N, 11.24; S, 12.79.
hours at 80˚C. Potassium thiocyanate 0.35 g (4.0 mmol) 1-(2-Chloro-5-nitrophenyl)-4,4,6-trimethyl-3,4-
in dry acetone (30 ml) was then added drop wise and dihydropyrimidine-2(1H)-thione (3)
the solution was refluxed for 5-10 minutes and cooled. The methodology used for the synthesis of 3 is the
Afterwards 4.0 mmol of the respective substituted same as used for 1 using 0.70 g (4.0 mmol) of 2-chloro-
aniline was added and reaction mixture was stirred for
5-nitroaniline to produce a pale yellow crystalline solid
an additional one hour. Progress of reaction was (yield; 90%) m.p.199-201oC; FTIR (cm-1): 3207 (N-H),
1
monitored using TLC. The reaction mixture was poured 3013 (C-Haromatic), 2873 (C-Haliphatic), 1547 (C=S); H NMR
in a beaker containing crushed ice and stirred well. The (300 MHz, CDCl3, ppm) δ: 8.28-8.20 (m, 2H, ArH), 7.68-
solidified product was isolated and dried. Pure product 7.64 (d, 1H, J = 9 Hz, ArH), 7.36 (s, 1H, -NH), 4.92-4.91
was obtained by solvent extraction technique using (m, 1H, -CH), 1.54 (s, 3H, -CH3), 1.43 (s, 3H, -CH3), 1.41
chloroform/water mixture (thrice). Organic layer was (s, 1H, -CH3); 13C NMR (75 MHz, CDCl3, ppm) δ: 176.5
collected and dried over MgSO4, and left for (C=S), 146.5, 141.6, 140.1, 139.5, 130.5, 127.6, 124.4,
crystallization to obtain the target compounds (1-8).
110.2,
52.9,
31.7, 31.2, 20.0. Anal. calc. for
Spectroscopic data for (1-8)
C13H14ClN3O2S (311.79): C, 50.08; H, 4.53; N, 13.48; S,
10.28. Found: C, 50.01; H, 4.51; N, 13.51; S, 10.30.
1-(2-Bromophenyl)-4,4,6-trimethyl-3,4-
1-(4-Methoxy-2-nitrophenyl)-4,4,6-trimethyl-3,4-
dihydropyrimidine-2(1H)-thione (1)
dihydropyrimidine-2(1H)-thione (4)
Generalized procedure described above was used for
synthesis of 1 using 0.65 g (4.0 mmol) of 2-nitro-5-
methoxyaniline to produce a pale yellow crystalline
The methodology used for the synthesis of 4 is the
same as used for 1 using 0.68 g (4.0 mmol) of 2-
bromoaniline to produce a pale yellow crystalline solid
solid (yield; 96%): m.p. 220-222 C; FTIR (cm-1): 3211
o
o
(yield; 90%) m.p. 170-172 C; FTIR (cm-1): 3190 (N-H),
(N-H), 3020 (C-Haromatic), 2876 (C-Haliphatic), 1552 (C=S). 1H
NMR (300 MHz, CDCl3, ppm) δ: 7.59-7.58 (d, 1H, J = 3
Hz, ArH), 7.33-7.27 (m, 1H, ArH), 7.20-7.19 (d, 1H, J = 3
Hz, ArH), 7.01 (s, 1H, -NH-), 4.88-4.89 (m, 1H, -CH), 3.92
(s, 3H, -OCH3), 1.57(s, 3H, -CH3), 1.43(s, 3H, -CH3),
1.37(s, 3H, -CH3). 13CNMR(75 MHz, CDCl3) δ: 177.0,
159.6, 147.5, 141.4, 133.7, 131.6, 127.6, 119.5, 109.8,
56.0, 52.8, 31.9, 31.5, 20.3. Anal. calc. for C14H17N3O3S
(307.37): C, 54.71; H, 5.57; N, 13.67; S, 10.43. Found: C,
54.65; H, 5.56; N, 13.75; S, 10.41.
1
2954 (C-Haromatic), 2851 (C-Haliphatic), 1517 (C=S), H NMR
(300 MHz, CDCl3) δ: 7.65 (d, 1H, J = 7.8 Hz, ArH), 7.46 (s,
1H, -NH), 7.39-7.37 (m, 1H, ArH), 7.29-7.19 (m, 2H,
ArH), 4.85-4.84 (m, 1H, -CH), 1.49 (d, 3H, -CH3), 1.44 (s,
3H, -CH3), 1.37 (s, 3H, -CH3); 13C NMR(75 MHz, CDCl3,
ppm) δ 176.6 (C=S), 140.0, 133.0, 132.3, 131.3, 129.8,
127.8, 124.9, 109.6, 52.7, 32.0, 31.3, 20.2. Anal. calc.
for C13H14ClN3O2S (311.24): C, 50.17; H, 4.86; N, 9.00; S,
10.30. Found: C, 50.16; H, 4.85; N, 9.06; S, 10.27.
1-(2-Mercaptophenyl)-4,4,6-trimethyl-3,4-
1-(2-Fluorophenyl)-4,4,6-trimethyl-3,4-
dihydropyrimidine-2(1H)-thione (5)
dihydropyrimidine-2(1H)-thione (2)
The methodology used for the synthesis of 5 is the
same as used for 1 using 0.5 g (4.0 mmol) of 2-
aminothiophenol to produce a pale yellow crystalline
solid (yield; 90%) m.p. 168-170 oC; FTIR (cm-1): 3185 (N-
The methodology used for the synthesis of 2 is the
same as used for 1 using 0.44 g (4.0 mmol) of 2-
fluoroaniline to produce pale yellow crystalline solid
(yield; 90%): m. p. 192-194 C; FTIR (cm-1): 3196 (N-H),
o
1
1
H), 2944 (C-Haromatic), 2850 (C-Haliphatic), 1515 (C=S); H
2963 (C-Haromatic), 2852 (C-Haliphatic), 1521(C=S); H NMR
NMR (300 MHz, CDCl3, ppm) δ: 7.67 (d, 1H, J = 6 Hz,
ArH), 7.54-7.52 (d, 1H, J = 6 Hz, ArH), 7.44 (s, 1H, -NH),
7.41-7.38 (m, 1H, ArH), 7.31-7.26 (t, 1H, J = 9 Hz), 7.07
(300 MHz, CDCl3, ppm) δ: 7.42-7.14 (m, 4H, ArH), 6.99
(s, 1H, -NH), 4.87-4.86 (m, 1H, -CH), 1.56 (s, 3H, -CH3),
1.40-1.37 (m, 6H, -CH3); 13C NMR (75 MHz, CDCl3) δ:
10 | J. Name., 2012, 00, 1-3
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(s, 1H), 5.0 (s, 1H, -SH), 4.84-4.81 (m, 1H, -CH), 3.30- = 6Hz, ArH), 7.68 (d, 1H, J = 6 Hz, ArH), 7.08 (s, 1H, -
3.28 (d, 1H, J = 6 Hz), 1.47 (d, 3H, J = 3 Hz, -CH3), 1.45 (s, NH), 4.93-4.92 (m, 1H, -CH), 1.55-1.54 (d, 3H, J = 3 Hz, -
3H, -CH3), 1.40 (s, 3H, -CH3); 13CNMR (75 MHz, CDCl3) δ: CH3), 1.45 (s, 3H, -CH3), 1.42 (s, 3H, -CH3); 13C NMR(75
177.1(C=S), 139.2, 134.0, 133.8, 129.1, 128.9, 128.5, MHz, CDCl3) δ: 176.5, 146.6, 141.9, 141.1, 139.5, 130.6,
126.5, 109.6, 49.6, 32.4, 31.1, 20.0. Anal. calc. for 128.0, 125.4, 110.2, 52.8, 31.6, 31.3, 20.0. Anal. calc.
C13H16N2S2 (264.41): C, 59.05; H, 6.10; N, 10.59; S, 24.25 for C13H14ClN3O2S (311.79): C, 50.08; H, 4.53; N, 13.48;
Found: C, 58.98; H, 6.08; N, 10.61; S, 24.22.
1-(2,3,5-Trichlorophenyl)-4,4,6-trimethyl-3,4-
dihydropyrimidine-2(1H)-thione (6)
S, 10.28. Found: C, 50.02; H, 4.50; N, 13.50; S, 10.46.
Crystallographic data collection and structural
refinement
The methodology used for the synthesis of 6 is the Single crystals of (1-4, 7) were mounted on a thin glass
same as used for 1 using 0.75 g (4.0 mmol) of 2,3,5- fiber at room temperature and the reflection data were
trichloroaniline to produce a pale yellow crystalline collected on a Bruker Kappa APE XII CCD diffractometer
solid (yield; 92%) m.p. 230-231oC; FTIR (cm-1): 3231 (N- equipped with graphite mono-chromated MoKα
1
H), 3032 (C-Haromatic), 2883 (C-Haliphatic), 1560 (C=S). H radiation (λ= 0.71073 Å). The data were also corrected
NMR (300 MHz, CDCl3, ppm) δ, 8.35 (s, 1H), 8.04- to Lorentz and polarization effect. The structure was
8.01(m, 1H), 7.01 (s,1H, -NH), 5.04-5.03 (m, 1H, -CH), solved using SHELXS-97. Final refinement on F2 was
2.55-2.54 (d, 3H, J = 3 Hz), 2.34 (s, 3H, -CH3), 2.31 (s, 3H, carried out by full-matrix least-squares techniques
-CH3); 13C NMR (75 MHz, CDCl3) δ: 179.0, 140.0, 139.3, using SHELXL-97 [40].The crystal data of (1-4, 7) and
135.6, 138.8, 131.0, 130.0, 129.8, 110.0, 54.1, 36.2, refinement values are summarized in Supplementary
35.3, 22.1. Anal. calc. for C13H13Cl3N2S (335.68): C, Table 1.
46.51; H, 3.90; N, 8.35; S, 9.55. Found: C, 46.46; H, 3.89; Docking protocols
N, 8.37; S, 9.56.
Gold was used to carry out molecular docking.
Parameters were used using default settings and
keeping receptor rigid. Active site residue His322 for
1-(2,4,6-Trichlorophenyl)-4,4,6-trimethyl-3,4-
dihydropyrimidine-2(1H)-thione (7)
The methodology used for the synthesis of 7 is the 1e9y and Met762 was selected for 4fm9 around which
same as used for 1 using 0.75 g (4.0 mmol) of 2, 4, 6- a grid of 10 Å was defined as binding site. Gold Score
trichloroaniline to produce a pale yellow crystalline fitness function, assigned to each docked ligand was
solid(yield; 92%) m.p. 237-238oC; FTIR (cm-1): 3231 (N- used to assess the results. Binding affinities of the used
1
H), 3032 (C-Haromatic), 2883 (C-Haliphatic), 1560 (C=S). H compounds were then calculated using AutoDock\Vina.
NMR (300 MHz, CDCl3, ppm) δ 8.35 (s, 2H, ArH), 7.08 (s, Grid spacing of 1.0 Å was used with a box size of
1H, -NH), 5.04-5.03 (m, 1H, -CH), 2.55-2.54 (d, 3H, J=3 10×10×10 with xyz coordinates of active site centre.
Hz, -CH3), 2.34 (s, 3H, -CH3), 2.31 (s, 3H, -CH3) ); 13C NMR Best ligand was selected on the bases of Gold Score and
(75 MHz, CDCl3) δ: 178.5, 141.2, 138.9, 137.8, 132.0, Binding energy values.
128.8, 111.0, 53.0, 36.1, 35.9, 23.6. Anal. calc. for Structure selection and preparation
C13H13Cl3N2S (335.68): C, 46.51; H, 3.90; N, 8.35; S, 9.55. For molecular docking studies, structures were
Found: C, 46.44; H, 3.88; N, 8.39; S, 9.53.
1-(2-Chloro-4-nitrophenyl)-4,4,6-trimethyl-3,4-
dihydropyrimidine-2(1H)-thione (8)
extracted from PDB data base having PDB:IDs (1e9y)
and (4fm9). These structures were then minimized
using Chimera 1.8.1.
The methodology used for the synthesis of 8 is the Docking studies
same as used for 1 using 0.70 g (4.0 mmol) of 2-chloro- GOLD (Genetic Optimization for Ligand Docking) was
4-nitroaniline to produce a pale yellow crystalline solid used for molecular docking studies [41]. This software
(yield; 90%) m.p. 201-203oC: FTIR (cm-1): 3219 (N-H), utilizes a genetic algorithm for docking flexible ligands
1
3022 (C-Haromatic), 2880 (C-Haliphatic), 1554 (C=S); H NMR into protein binding sites to explore the full range of
(300 MHz, CDCl3, ppm) δ 8.96 (s, 1H, ArH), 8.23 (d, 1H, J ligand conformational flexibility with partial protein
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flexibility [42]. The binding site definition should temperature scaling [44]. NPT ensemble was utilized
therefore be large enough to contain any possible with isotropic position scaling to maintain pressure.
binding mode of the ligand. In this work we specified Carbon alpha positions were restrained by 5 kcal/mol –
the approximate centre of the binding site and atoms Å2. Same set of parameters were used for another 50
that lie within a specified radius of 10 A°. The standard ps, with an exception of reducing the restraint on
default settings such as population size (100), selection carbon after every 10 ps by 1kcal/mol –Å2. The system
pressure (1.1), niche size(2), and operator weights for was then allowed to equilibrate itself under same set of
migrate (0), crossover (100), number of operations condition for 1 ns. For production NVT ensemble was
(10,000), number of islands (1) and number of dockings selected with Berendsen temperature coupling
10 were adapted for docking process. These algorithm [45]. SHAKE was applied on hydrogen bonds
calculations were performed with GOLD5.1 on Intel and cut off value for non-bonded interaction was set to
Xeon QuadTM Core processor 3.0 GHz Linux 8.0 Å. The production run was carried out for 50 ns on a
workstation running under open SUSE11.3.
time step of 2 fs. The simulation trajectories were
analysed using ccptraj program of AMBER package and
Molecular Dynamic Simulation
Docked complex of compound 3 with 1E9Y and 4FM9 structural parameters like RMSD and RMSF were
were simulated to obtained their detailed mechanistic calculated. The trajectory were visually analysed using
and dynamics with respect to their targets. AMBER 14 VMD [38] and Chimera [46]. There depictions were
simulation package was utilized [43]. Antechamber captured using these tools.
program was used to generate initial library and
parameters for compound 3 and a non-standard
residue KCX carbamylated lysine in 1E9Y. Leap program
helped in integration of docked complex into a TIP3P
water box of size 12 Å with ff14SB force field describing
the molecular interactions of the system. 10 Na+ and 5
Cl-were added to hydrated complexes of 1E9Y and
4FM9 respectively to neutralize the system. The system
energy were minimized gradually, first the Hydrogen of
the entire system were relax with 500 cycle of
minimization; followed by minimization of water box
with cycles of 1000 with a restraint of 200 kcal/mol –
Å2. After that the system was minimized with 1000
cycles and a restraint of 5 kcal/mol –Å2 on carbon alpha
atoms. Then all non-heavy atoms were minimized using
300 cycles and restraint of 100 kcal/mol –Å2. After
performing minimization, system was heated to 300 K
for 20 picoseconds (ps) with a time step of 2
femtoseconds (fs) and 5 kcal/mol –Å2 restraint on
carbon alpha atoms. Temperature was maintained
using langevin dynamics with gamma ln value set to
1.0. SHAKE was applied for constraining bonds with
hydrogen and NVT ensemble was selected for heating
the systems. The molecule was then equilibrated for
100 ps with 2 fs time step, with SHAKE applied on
hydrogen bonds and langevin dynamics for
Conclusions
In conclusion, we have developed an efficient and
convenient Ph3GeH-catalyzed one-pot reaction from 4-
methylpent-3-en-2-one (I) and thioureas II(a-h)
obtained from readily available substituted anilines for
the synthesis of
a
series of substituted
dihydropyrimidine derivatives. It is interesting that the
addition of thiourea to 4-methylpent-3-en-2-one (I) as
final step towards product formation successfully
explains the debate about addition of thiourea to
aldehyde or to ethyl acetoacetate as first step in
Biginelli dihydropyrimidine synthesis. Hence not only
simplicity of this Biginelli’s one-pot combination is
preserved here but also consistent production of
excellent yields of the dihydropyrimidin-2(1H)-thione is
also achieved along with higher functional groups
tolerance. By further elaboration and diversification of
the various functional groups, a wide range of N-
heterocycles can be produced. The Ph3GeH-catalyzed
one-pot process may find potential applications in the
synthesis of biologically and medicinally relevant
compounds in future research endeavours. In silico
studies were carried out to find the binding pattern of
urease inhibitors. Furthermore, the results suggest that
these derivatives may be used as preventive/
chemotherapeutic agents for cancer. Hence it is
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recommended to find their role as urease inhibitors 13 M.A. Bigdeli, S. Jafari, G. H. Mahdavinia and H.
and anticancer agents by applying in vitro and in vivo
studies to find some meaningful conclusions.
Acknowledgements
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The authors are thankful to Quaid-i-Azam University
and Higher Education Commission of Pakistan,
Islamabad, Pakistan for the grant of funds for this
project.
15 H. X. Lin, Q. J. Zhao, B. Xu and X. H. Wang, Chin.
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Graphical Abstract
A series of substituted dihydropyrimidin-2(1H)-thione derivatives (1-8) have been synthesized
and docked against enzymes Human topoisomerase II alpha (4fm9) and Helicobacter pylori
Urease (1E9Y) for binding modes validation.