Structure based medicinal chemistry-driven strategy to design substituted dihydropyrimidines as potential antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2016.03.022

In an attempt to explore novel and more potent antileishmanial compounds to diversify the current inhibitors, we pursued a medicinal chemistry-driven strategy to synthesize novel scaffolds with common pharmacophoric features of dihydropyrimidine and chalcone as current investigational antileishmanial compounds. Based on the reported X-ray structure of Pteridine reductase 1 (PTR1) from Leishmania major, we have designed a number of dihydropyrimidine-based derivatives to make specific interactions in PTR1 active site. Our lead compound 8i has shown potent in vitro antileishmanial activity against promastigotes of L. Major and Leishmania donovani with IC₅₀ value of 0.47 1/4g/ml and 1.5 1/4g/ml respectively. The excellent in vitro activity conclusively revealed that our lead compound is efficient enough to eradicate both visceral and topical leishmaniasis. In addition, docking analysis and in silico ADMET predictions were also carried out. Predicted molecular properties supported our experimental analysis that these compounds have potential to eradicate both visceral and topical leishmaniasis.

Full text of DOI:10.1016/j.ejmech.2016.03.022

Accepted Manuscript
Structure based medicinal chemistry-driven strategy to design substituted
dihydropyrimidines as potential antileishmanial agents
Umer Rashid, Riffat Sultana, Nargis Shaheen, Syed Fahad Hassan, Farhana Yaqoob,
Muhammad Jawad Ahmad, Fatima Iftikhar, Nighat Sultana, Saba Asghar, Masoom
Yasinzai, Farzana Latif Ansari, Naveeda Akhter Qureshi
PII:
S0223-5234(16)30196-9
10.1016/j.ejmech.2016.03.022
EJMECH 8447
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 May 2015
Revised Date: 8 March 2016
Accepted Date: 9 March 2016
Please cite this article as: U. Rashid, R. Sultana, N. Shaheen, S.F. Hassan, F. Yaqoob, M.J. Ahmad,
F. Iftikhar, N. Sultana, S. Asghar, M. Yasinzai, F.L. Ansari, N.A. Qureshi, Structure based medicinal
chemistry-driven strategy to design substituted dihydropyrimidines as potential antileishmanial agents,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.022.
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Structure based medicinal chemistry-driven strategy to design substituted
dihydropyrimidines as potential antileishmanial agents
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Umer Rashid a,b,*, Riffat Sultana a, Nargis Shaheen c, Syed Fahad Hassan d, Farhana Yaqoob a, Muhammad Jawad
Ahmad a, Fatima Iftikhar a, Nighat Sultana e, Saba Asghar f, Masoom Yasinzai f , Farzana Latif Ansari g,h, Naveeda
Akhter Qureshi c, **
a Department of Chemistry, Hazara University, Mansehra 21120, Pakistan
b Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
c Department of Animal Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
d Department of Pharmacy, University of Lahore, Defence Road Campus, Lahore 53700, Pakistan
e Department of Biochemistry, Hazara University, Mansehra 21120, Pakistan
f Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
g Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
h Pakistan Council for Science and Technology, G-5/2 Islamabad, Pakistan
Corresponding author. *E-mail: umerrashid@ciit.net.pk; umer_rashid39@hotmail.com (Umer Rashid);
Tel. Off.: +92 (992) 383591-6
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**E-mail: nqureshi@qau.edu.pk (NA Qureshi)
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Abstract
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In an attempt to explore novel and more potent antileishmanial compounds to diversify the current
inhibitors, we pursued a medicinal chemistry-driven strategy to synthesize novel scaffolds with common
pharmacophoric features of dihydropyrimidine and chalcone as current investigational antileishmanial
compounds. Based on the reported X-ray structure of Pteridine reductase 1 (PTR1) from L. major, we have
designed a number of dihydropyrimidine-based derivatives to make specific interactions in PTR1 active
site. Our lead compound 8i has shown potent in vitro antileishmanial activity against promastigotes of L.
Major and L. donovani with IC50 value of 0.47 µg/ml and 1.5 µg/ml respectively. The excellent in vitro
activity conclusively revealed that our lead compound is efficient enough to eradicate both visceral and topical
leishmaniasis. In addition, docking analysis and in silico ADMET predictions were also carried out.
Predicted molecular properties supported our experimental analysis that these compounds have potential to
eradicate both visceral and topical leishmaniasis.
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Key words: Leishmaniasis, Structure based drug design, Chalcones, Dihydropyrimidine, Docking,
Rigidification
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1. Introduction
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Leishmaniasis is an infectious disease caused by Leishmania parasites belonging to genus Leishmania in
the family Trypanosomatidae. It is transmitted into host blood by the bite of female sand fly [1]. These
parasites can attack on mammals and human beings. When sandfly bites an infected organism parasite pass
from the blood of infected organism into the gut of the sandfly. These parasites grow in the gut of sandfly
for 8-20 days. When an infected sandfly bites a healthy organism it delivers these parasites
(metacyclicpromastigote) in the blood of that organism. The infected stage, metacyclicpromastigotes
inoculated in the rupture skin where it is phagocytosed by macrophages. The promastigotes multiplies and
turn into amastigotes and infect many tissues which gradually express the disease. There are more than 20
Leishmanial protozoan species, that are responsible for a number of clinical forms of leishmaniasis such as
cutaneous, diffuse cutaneous, disseminated cutaneous, mucocutaneous, visceral and post-kala-azar dermal
leishmaniasis (PKDL). Leishmaniasis has many symptoms, such as destructive mucosal inflammation, skin
lesions, ulcer and visceral infection (effects internal organs such as liver and spleen), fever and sometime
anemia. This disease is prevalent in 88 countries, including Asia, Africa and Latin America. Visceral
leishmaniasis (VL) is the most devastating and fatal form of leishmaniasis. It is caused by L. donovani and
L. infantum. VL patients are prone to bacterial co-infection including tuberculosis and pneumonia etc. Post-
kala-azar dermal leishmaniasis (PKDL), a form of dermal leishmaniasis caused by L. donovani, is a sequel
of VL and develops months to years in VL cure patients [2].
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In recent years, a plethora of investigational compounds has been investigated for antileishmanial activity.
The major approved antileishmanial drugs are: pentavalent antimony drugs (meglumine antimoniate and
sodium stibogluconate), pentamidine, miltefosine and amphotericin B. All these available treatments are
not satisfactorily significant and have many draw backs such as renal dysfunction, nausea, anorexia, fever
etc. There are also some reports of cardiac deaths. Besides investigational drugs, natural products are
valuable sources in finding lead compounds. Flavonoids such as quercetin and luteolin emerged as potent
antileishmanial agents against L. donovani. Similarly, natural products like lichochalcone A, iridoids,
naphtoquinones, quinolone alkaloids, saponins, lignans and coumarins have shown promising
antileishmanial activities [3-5]. Immunotherapy is considered as best alternative for the treatment of VL.
Despite intense attempts to develop a prophylactic vaccine, there is no safe and efficacious vaccine against
leishmaniasis due to inadequate knowledge of early immune response and poor understanding of parasite
pathogenesis. However, Leish-111f+MPL-SE vaccine has been proved promising to control VL [6-7].
Apart from chemotherapy/vaccination, leishmaniasis can be controlled by taking some safety measures like
reservoir eradication, use of insect repellent, protective clothing and use of fine-mesh netting to prevent
exposure to female sand fly [8].
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Chalcones (1,3-diaryl-2-propen-1-ones), belonging to the flavonoid family, have been reported to possess
many pharmacological activities . Zhai et al reported oxygenated chalcones as potent inhibitors of L. major
with IC50 in the range of 4.0-10.5 mM [3]. Foroumadi et al investigated chromene-based chalcones namely,
1-(6-methoxy-2H-chromen-3-yl)3-phenylpropen-1-ones and 3-(6-methoxy-2H-chromen-3-yl)-1-phenyl-
propen-1-ones for their antileishmanial activity against promastigotes form of Leishmania major [9]. These
chalcones exhibited excellent activity at non-cytotoxic concentrations. Narender et al. reported promising
antileishmanial activity of naturally occurring chromenochalcones [10]. 3,4-Dihydropyrimidine (DHPM) is
the most attractive derivative of pyrimidines for a medicinal chemist. This structural motif may also be
described as a derivative of cyclic urea. These non-planar heterocyclic compounds have received
considerable attention of the pharmaceutical industry because of their interesting multifaceted
pharmacological profiles. In the exploration of new and more potent antileishmanial compounds to
diversify the current inhibitors, it is essential to design novel and potent inhibitors. Singh et al. identified
potent dihydropyrimidine (DHPM) based derivatives targeting Pteridine reductase (PTR1) [11-12].
Recently, Kaur et al. reported monastrol, a dihydropyrimidine based KSP inhibitor, as a potent
antileishmanial agent [13].
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In our group, a major part of our research is focused on computer-aided drug design with subsequent
synthesis and testing of new chemical entities as putative drugs for the treatment of various diseases [14-
16]. Our group recently identified a series of N-(1-methyl-1H-indol-3-yl)methyleneamines and eight new
3,3-diaryl-4-(1-methyl-1Hindol-3-yl)azetidin-2-ones against Leishmania major [17]. In another study, we
identified a variety of 2-aryl- and 5-nitro-2-arylbenzimidazoles as new antileishmanial agents with IC50
values ranging from 0.62-0.92 µg/ml [18]. In continuation of our endeavor and considering the
pharmacological importance of DHPM scaffold, it was planned to design and synthesize a variety of
DHPM-based potent antileishmanial compounds to diversify the current inhibitors.
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2. Results and discussion
2.1. The Design strategy
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Molecular docking has contributed a lot in the identification of novel small drug-like scaffolds exhibiting
high binding affinity and selectivity for the target. Hence, we extended our study to investigate in silico
binding orientation of the synthesized DHPMs. Pteridine reductase (PTR1) is an important enzyme
responsible for Pteridine salvage in leishmania and other trypanosomatid protozoans. PTR1 contributes to
antifolate resistance and is responsible for the failure of conventional therapies such as methotrexate
(MTX) against these protozoans [19]. In a study, Singh et al. reported DHPM analogues targeting PTR1,
therefore, we focused on molecular docking studies to investigate PTR1 (from L. major) as a possible
target for our newly synthesized DHPM derivatives. Crystal structure of PTR1 (PDB ID 1E7W, from L.
major) with MTX as the co-crystallized ligand was selected for these studies [20]. Docking experiments
were performed via Molecular Operating Environment (MOE) docking program [21].
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Table 1 enlists the 50% inhibitory concentration (IC50) values of dihydropyrimidines (1-13). Compounds
1–13 showed varying degrees of antileishmanial activities with IC50 values ranging between 0.61 and 0.99
µg/ml as compared to standard amphotericin B (IC50= 0.56 µg/ml). Compound 3 (IC50= 0.61±0.01), 4
(IC50= 0.67±0.04 µg/ml), 8 (IC50= 0.63±0.02 µg/ml), 12 (IC50= 0.63±0.01 µg/ml), 13 (IC50= 0.65±0.04
µg/ml) and were found to show good in vitro activity against the promastigote form of L. Major.
Compounds 6 (IC50= 0.71±0.01µg/ml), 7 (IC50= 0.69±0.01µg/ml) and 9 (IC50= 0.74±0.04µg/ml) exhibited
moderate activities. Compounds 1, 2, 5, 10 and 11 showed weak activities with IC50 values between 0.78
and 0.89 µg/ml.
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Active site of PTR1 is an elongated and rigid cleft. Arg17, Asn109, Ser111, Phe113, Asp181, Met183,
Gln186, Leu188, Tyr194, Lys198, His241 are catalytically important residues (Figure 1A-C). Docking
analysis of compound 8 with IC50= 0.63±0.02 µg/ml was performed and our analysis identified unoccupied
space, which may be available to attract further modifications. Superimposed model of compound 8 with
MTX into the binding site of PTR1 (as viewed in Chimera 1.8.1) [22] is shown in Figure 1A, B. It is
revealed from Figure 1C that pteridine head group of MTX is embedded between Met183, Gln186 and
Leu188 and formed interactions with Gln186 at the distance of 1.4 Å (Figure 1B). While the glutamate tail
accepted proton from Arg17 and Lys198. Phe113 forms π-stacking interactions with phenyl ring of para-
aminobenzoate (pABA)-glutamate tail (Figure 1C). The structure of compound 8 bound to PTR1 binding
site showed that carbonyl oxygen of the cyclic urea act as a strong hydrogen bond acceptor and formed
hydrogen bond with Lys198. Similarly, carbonyl oxygen of the C-5 acetyl group accepts hydrogen bond
from Arg17 (Figure 1D). The observed negative fitness value of binding interaction (-8.9001 Kcal/mol)
revealed that compound 8 was not tightly fitted into the active site. The orientation of MTX and its
hydrogen bonding and π-stacking interactions provide a framework for the novel scaffold design. On the
basis of these initial results, further molecular modeling, synthesis and in vitro PTR1 inhibition studies of
compound 8 and 12 were carried out.
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We realized that compound 8 can be chose as a benchmark compound for structural modification and both
lipophilic and polar groups can be incorporated to probe extra binding interactions with the drug target. For
this purpose, we turned our attention to further decorate DHPM scaffold by varying substituents around
DHPM 8 to generate additional interactions with the drug target (Series 1). More specifically, we focused
on a well-established chain extension strategy of medicinal chemistry which involves the incorporation of
another functional group in the lead compound to enhance extra binding interactions with the PTR1. Based
on the input from docking studies (See below), we designed analogues by varying substituent at C-5 and C-
6 positions. Starting from C-5 acetyl group modification, a set of 5-cinnamoyl derivatives was synthesized
in an effort to increase the potency (Table 2, Compounds 8a-c). Additionally, substitution at C-6 position
with 4-aminochalcone derivatives (8d-i) and the effect of placing 4-aminochalcone group at C-5 position of
thioxo-analogues (Compound 12a-d, IC50=0.63±0.01) were also explored.
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2.2. Chemistry
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Dihydropyrimidines (1-13) were synthesized by using three component Biginelli reaction. Aldehydes, 1,3-
dicarbonyl compounds and urea/thiourea were reacted in a test tube under ultrasonic irradiation by using
SnCl2 as catalyst and acetonitrile (ACN) as solvent (Scheme 1) [14]. DHPM 3 and 8, having acetyl group at
C-5 position, can undergo Claisen-Schmidt condensation with appropriate aldehydes to form chalcones.
Furthermore, these may serve as versatile synthons capable of undergoing facile allylic bromination at C-6,
which may undergo nucleophilic displacement with 4-amino chalcones or with any other nucleophile.
Kolsov et al. studied effect of tautomerism in these two reactions and concluded that by inserting an alkyl
substituent at N-1 position not only increased the solubility but also eliminated the possibility of amide-
imidol tautomerism [23-24]. Based on these results, we initially started our investigation at C-5 position of
N-methyl DHPM 8 and a set of three 5-cinnamoyl-6-methyl-4-aryl-3,4-dihydropyrimidin-2(1H)-one (8a-c)
derivatives were synthesized by using Claisen-Schmidt condensation conditions (Scheme 2). The structures
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of synthesized compounds were confirmed on the basis of their physical constants and spectral data. H
NMR data of 5-cinnamoyl derivatives revealed the absence of C-5 acetyl protons and showed two
deshielded doublets, one proton integration each, appeared at 7.79-7.89 ppm and 7.56-7.59 ppm which
could be assigned to olefinic protons of α, β-unsaturated ketone. Their coupling constant (J= 15.6-15.9 Hz)
indicated the trans-relationship of H-α and H-β and hence confirming E-configuration of the compounds.
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During one-pot Biginelli reaction, methyl group is generally introduced at C-6 position of the pyrimidine
ring by using a 1,3-dicarbonyl compound essentially having an acyl group. This methyl group can undergo
a facile bromination, described as allylic bromination, which may undergo nucleophilic substitution
reaction leading to C-6 modified DHPMs. Allylic bromination was carried out at low temperature (0°C) in
chloroform. The composition of crude reaction mixtures was monitored by LC/MS. In the chromatograms
of the initial reaction mixture, peaks of 6-methyl brominated product (19A), 5-bromo acetyl product (19B)
along with some di-brominated products was observed. The main peak (83%) corresponds to 19A. These
results were consistent with results reported in the literature [24]. The mixture of compounds was separated
through standard silica gel column chromatography using n-hexane/ethyl acetate mixture as eluent. A
mixture of 6-bromomethyl derivative of DHPM and 4′-aminochalcones (14-18, Scheme 3a) in THF were
stirred in ultrasonic bath at the 40-45oC to yield products (8d-h, Scheme 3b).
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Rigidification has been an important medicinal chemistry tactic used to increase the activity of drug.
Number of rotatable bonds measures molecular flexibility. Considering this medicinal chemistry approach,
we decided to rigidify the Compound 8e by converting chalcone (rotatable bonds=8) moiety to a five-
membered pyrazoline ring (8i, rotatable bonds=7) (Scheme 4). Synthesis of pyrazoline 8i involved [3+2]
annulation of enone functionality of chalcone 8e by reaction with hydrazine and NaOH [25]. A pale yellow
colored solid was obtained by ultrasonic assisted reaction of chalcone with hydrazine and NaOH in 51%
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yield. In H NMR spectrum, an AMX pattern was observed. HA, HM and HX protons of pyrazoline ring
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appeared as doublets of a doublet at 4.81 ppm (J=12.3 Hz, 7.8 Hz) 3.93 (J=17.1 Hz, 12.3 Hz) and 3.44
(J=7.8 Hz, 17.1 Hz) ppm. The appearance of a downfield broad signal at 9.10 ppm was noticed for the
phenolic-OH. In 13C-NMR spectra, C-4 and C-5 of pyrazoline ring appeared at 45.6 ppm and 56 ppm
respectively.
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We also synthesized a new series of thioxo-analogues by substituting C-5 ethyl ester group with 4-amino
chalcone. This nucleophillic displacement reaction was carried out with 4-amino chalcones (14-18, Scheme
3a) in THF in ultrasonic bath at 40-45oC to yield products (12a-d). The synthesized compounds were
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characterized on the basis of their physical constants and spectral data. H NMR data of new thioxo-
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analogues were recorded, and a few generalizations could be made. Absence of signals of C-5 ethyl ester (a
triplet at 1.09-1.14 ppm and a quartet at 4.01-4.10 ppm) and observation of a broad singlet, appeared at
9.23-9.27 ppm, was attributed to C-5 amide proton (NH-C=O) and was a clear indication of substitution
reaction. Similarly, two deshielded doublets, one proton integration each, appeared at 7.99-8.04 ppm and
7.61-7.63 ppm which could be assigned to olefinic protons of α, β-unsaturated ketone.
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Substitution at C-5 gave a comparable potency to lead compound DHPM 8. The presence of a nitro group
at 3-position of phenyl ring (8a) exhibited lower in vitro inhibition. However, compounds with hydroxyl
and chlorine group at 4-position of phenyl ring (8b & c respectively) have shown similar potency to DHPM
8. The results are summarized in Table 2. Furthermore, substitution at C-6 position with 4-aminochalcone
derivatives resulted in significant influence on antileishmanial activity with an IC50 values ranging from
0.51 to 0.71 µg/ml. As shown in Table 2, chalcone derivative 8f with nitro group at 3-position of phenyl
ring has shown reduced potency. Similarly, derivative 8h with 4-dimethtlamino group also exhibited
reduced potency with IC50 value 0.71±0.01ꢀg/mL. Compound 8e (IC50= 0.51 µg/ml) having 4-hydroxy
substituent at ring B of the chalcone moiety has emerged as the most promising compound of this series.
Compound 8g with chloro substituent at 4-position showed IC50 value of 0.53 µg/ml. This could suggest
that certain aromatic substituents at 4-position on ring B of the chalcone moiety are important for
antileishmanial activity. The significance of the 4-hydroxyl group was investigated further and compound
8e (IC50= 0.51 µg/ml) was selected as a representative derivative to explore the effect of cyclization. As can
be seen, our pyrazoline derivative 8i, with IC50= 0.47 µg/ml, is more potent than corresponding chalcone
analogue 8e.
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To determine whether the SAR study around thioxo-analogues has improved effect on the biological
activity, in vitro antileishmanial activity was evaluated. SAR data in Table 3 shows that the most potent
compound of this series 12c (IC50= 0.51+0.04) has hydroxyl group at meta position of the C-4 phenyl ring
on DHPM and at para position of chalcone ring B. Addition of nitro group at position R1 results in
decrease in activity compared to hydroxyl group.
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The synthesized compounds were also tested against the promastigotes of L. donovani. Most of compounds
have shown no inhibition. However, lead compound 8i have shown excellent activity with IC50= 1.5 µg/ml.
Compound 12c have shown good activity with IC50= 5.58µg/ml. It is concluded that our lead compound 8i
is efficient enough to eradicate both visceral and topical leishmaniasis.
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2.4. Molecular docking analysis of designed compounds
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In an attempt to gain insights and to explore the probable binding modes of synthesized compounds,
molecular docking of these compounds was performed into the active site of LmajPTR1 (LmPTR1, PDB
1e7w) via MOE docking program. We focused on the properties of pteridine reductase 1 (PTR1) as drug
target for antileishmanial drug discovery. From Protein Data Bank, the available solved X-ray structures for
LmajPTR1 are: 1E7W, 1W0C, 2BF7, 1E92 and 3H4V. These crystal structures are in ternary complexes
with methotrexate (MTX), 7,8-dihydrobiopterin and 2,4,6-triaminoquinazoline. Selection of MTX, a folate
antagonist, resistant Leshmania, has provided much information about drug resistance. Two step folate
reduction mechanisms is the similar approach followed in both leishmania as well as in resistant
trypanosome. Therefore, we opted 1E7W as a useful target against resistant strains of the L. major. Another
intracellular protozoan species, L. donovani, is responsible for the most severe form of leishmaniasis i.e.
visceral leishmaniasis. L. major and L. donovani enzymes share 91% sequence identity and the catalytic
residues Asp181, Tyr191, Tyr194, and Lys198 are conserved; therefore, the details of the catalytic
mechanism are expected to be identical between them [26-28].
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Present study encompasses the synthesis and in vitro evaluation of racemic DHPMs derivatives as
antileishmanial agents by exploiting the in silico predictive power. Hence, we were interested in exploring
the probable binding modes of enantiomerically pure (R) and (S) DHPMs into the active site of PTR1.
Molecular docking studies were carried out on both enantiomers of the synthesized DHPMs analogues.
Analysis of the 2D interactions of the compounds with better IC50 values showed a fair correlation between
the docking scores and their inhibitory activity against PTR1 (Figure 2). Docking analysis of the
compound 8a (IC50= 0.59 ꢀg/mL, binding score -11.7909 Kcal/mol) is shown in Figure 2A. Phenyl ring at
C-4 nucleus of DHPM ring formed strong π-π interactions with Phe113. Carbonyl oxygen of α,β-
unsaturated ketone pointed towards Arg17. Similarly, carbonyl oxygen of the cyclic urea moiety formed a
hydrogen bond with Lys198.
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In order to explore the potent inhibitory activity of chalcone (8e) and its pyrazoline analogue (8i), we
docked these compounds into the active site of PTR1. The binding model of compounds 8e and 8i is shown
in Figure 2B-C. Key interactions stabilized both compounds, and the important contact residues for the
docked ligands were Arg17, Ser111, Phe113, Met183, Gln186 and Leu188. The phenolic ring of compound
8i, similarly to 8e, stabilized through H-bond with Met183, Gln186 and Leu188. The distance between the
hydroxyl group and these residues is shown in Figure 2B and C. In addition, these compounds are also
involved in the π-π stacking interactions between the aromatic ring A of chalcone/pyrazoline and Phe113.
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The docking score, binding affinity and binding energy of ligand-protein complex were also compared.
Compound 8i has the docking score -14.0053, strong binding affinity -10.463 Kcal/mol and lower binding
energy -42.899 Kcal/mol. On the other hand, fitness values of interactions for compound 8e were -12.9871,
-9.236 Kcal/mol and -40.973 Kcal/mol respectively. Therefore, it is concluded that the difference of almost
2.0 Kcal/mol in the binding energy confirmed the stabilization of ligand-protein complex of 8i as compared
to 8e. This showed a fair correlation between the predicted binding free energy values of the compounds
and their inhibitory activity against PTR1 (1E7W). Superimposition of top-ranked docking conformations
revealed that phenolic ring of pyrazoline moiety of 8i undergoes a rotation of 180 degrees relative to 8e,
which alter the position of the hydroxyl group (Figure 3). Despite this difference in docking orientation,
phenolic group of both compounds share the same binding region and a trivial (from IC50= 0.51 to 0.47
ꢀg/mL) increase in the potency may be anticipated due to this conformational motion.
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Parallel studies on weakly active compounds, for example compound 8a and 8h, were also conducted. It
can be noticed from Table 2 that compounds with 5-cinnamoyl core (8a-c) has showed decreased activity
due to missing interactions. Visual inspection of docked binding pose of compound 8a indicates that it does
not share the complete space in the binding site of PTR1 and showed very few binding interactions with
target (Figure 4A). Similarly, compound 8h oriented in such a way that it shows only one hydrogen bond
with Lys198 (Figure 4B). This may be explained by its improper fitting into the active site due to bulky
dimethyl amino group. The observed negative fitness values of binding interactions (-8.763 and -9.106
Kcal/mol respectively) for these compounds revealed that these compounds were not tightly fitted into the
active sites. This clearly demonstrates the weak activity of these compounds.
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To explore how compounds of Series 2 interacts with the active site residues, molecular docking studies of
the most active compound 12 c was carried out. The hydroxyl group (R), pointed towards Met 183 and
Gln186 and acts as a hydrogen bond donor. Carbonyl oxygen of chalcone pointed towards the guanidinium
group of Arg17 as hydrogen bond acceptor. Docking poses of 12c are shown in Figure 5. In view of the
above, we were successful in occupying the empty space in the binding site of PTR1 and the observed
negative fitness values of interactions (i.e. docking score, binding affinity and binding energy) has shown a
rational correlation between the docking scores and their inhibitory activity toward PTR1.
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2.5. In silico ADMET predictions
It is well recognized that employing computational absorption, distribution, metabolism and excretion
(ADME) predictions in combination with in vitro prediction (Docking studies) as early as possible in the
drug discovery process helps to reduce the number of safety issues and help to improve prediction success.
This early in silico ADME profiling, has in fact, decreased the proportion of drug candidates that fail in
clinical trials for ADME reasons. Owing to the excellent in vitro activity, we initiated in silico calculations
for drug-like characteristics and ADME prediction of compounds. An inspection of the data given in Table
4 revealed that almost all compounds of Series 1 have octanol-water partition coefficient (i.e. LogP) values
ranging from 3.49 to 5.42. However, molecular hydrophobicity, calculated as LogP, is not the only
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indicator of drug absorption. Aqueous solubility of a compound is also an important factor and is the direct
measure of hydrophobicity of a compound [29]. Table 4 shows the predicted values of solubility of
compounds (Sw in mg/ml) and it is clear that due to the low predicted values of solubility, these compounds
have shown less LogPermeability. On the other hand, amphotericin B has shown the lowest
LogPermeability (-11.516) and decreased logD values (less than -2.3, Table 4) and these decreased values
of amphotericin B are key factors responsible for its availability as IV-infusions clinically. Lead compound
8i is also predicted to be administered via IV-infusion to attain 100% bioavailability due to its reduced
LogPermeability (-10.5468) values. However, compound 8b can be administered orally due to its improved
native solubility (0.0143 mg/mL), logP (3.49) and LogPermeability (-9.1511). Furthermore, compound 8h
and 8i are predicted to possess enhanced solubility in the gastric environment due to their reduced logD
values (-0.30 and -0.52) irrespective of curtailed native solubility of 0.0014 and 0.0038 mg/ml respectively
(Table 4). 12a-d (Series 2) can be utilized to be administered via oral administration due to improved logD
to molecular weight band distributions (>50% orally bioavailable) irrespective of their low solubilities.
Total polar surface area (TPSA), another biological membrane penetration indicator, was calculated for
different pH values. In the blood stream (pH=7.4), a trivial increase TPSA of our lead compound 8i (114.85
A2 to 116.46 A2) and amphotericin B (319.61 A2 to 324.06 A2) has been observed (Figure 6).
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It was seen that some compounds like 8f, 8h, 12b and 12c violate some of the Lipinski’s rule of 5 (Ro5) as
obvious from Table 4. But this Ro5 is not the only parameter to scrutinize the fate of the drug candidate to
enter the market. As referenced drug (amphotericin B) utilized in the present research also violate the MW
parameter with 924.09 Da. The allowed MW for amphotericin is <300 Da according to its log logD -2.31.
It is therefore, due its high molecular weight to logD distribution (less than 50% orally bioavailable), it is
administered as intravenous route to overcome the oral permeability barrier which is also obvious from its
low logPermeability (-11.516). Therefore, all the compounds were screened against their allowed molecular
weights to logD distributions at physiological pH 7.4.
It was found that allowed molecular weight range for these compounds depicts the accurate correlations
between the oral bioavailable >50% to that of calculated logPermeability of compounds. Where, compound
with no Ro5 violation (8i) but still pertain less logPermeability of -10.5468, as its allowed molecular
weight is between 350-400 Da. Henceforth, it pertains less oral bioavailability irrespective of obeying the
Ro5 and is suited to be administered intravenously.
With respect to tissue distribution, logD value of compounds was correlated with the plasma protein
binding (PPB). The logD values of all the compounds at various physiological pH of the alimentary canal is
tabulated and shown in Figure 7a-b. From the data presented in Figure 7, it is significant that almost all
the compounds pertaining logD values greater than 3 have more than 90% PPB, which is consistent with
results described in literature [30]. However, high protein binding of amphotericin B is eccentric to its high
molecular weight (i.e. greater than 500 Da) rather than logD [31]. Owing to the increased PPB capacity,
lead compounds 8i, 12a-12d pertain long duration of action and can be used to eradicate the visceral and
topical leishmaniasis (Table 4). Furthermore, increase in logD values of compound is responsible for
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increased metabolic clearance over renal clearance as renal clearance decreases with lipophilicity [32]. In
addition, renal clearance is also related with the molecular weight distribution and hydrogen bond donor
(HBD) capacity of compounds. Hence, small molecules with MW below 350 Da will eliminate via renal
and higher molecular weight compounds via partially fecal route [33]. Similarly, compounds with HBD >4
will have greater tendency to eliminate via phase-II metabolism (glucoronidation) [34]. The renal clearance
of the compounds were predicted by their unbound percentage in the blood and tabulated in ml/min/kg
(Table 4). These predicted renal clearances may vary due to molecular weight distribution, active secretion
(via ATP driven efflux transporter P-glycoprotein). Therefore active eliminations are prone at the distal
parts of nephron (Figure 8a-c). The literature cut off values for propensity of molecules to be the substrate
at P-glycoprotein is: > 400 Da for MW and > 90 A2 for TPSA [35]. These predictions are promising and
hence deserve to be investigated further to assess the complete drug-likeness.
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3. Conclusion
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In summary, we have carried out medicinal chemistry-driven structure based modifications of 3,4-
dihydropyrimidine core. It is clear from the SAR exploration around DHPM 8 analogues that certain
aromatic substituents at 4-position on ring B of the chalcone moiety are important for antileishmanial
activity. Attempt to decrease the number of rotatable bonds resulted in increased potency presumably due
to hydrogen bond donor pattern of 4-OH group. In terms of potency, SAR exploration in Series 2
compounds offered generally lower potency over Series 1 compounds. In addition, in silico ADME data
revealed that the results of both series encourage further investigation on structural optimization and on in
vivo models.
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4. Experimental
4.1 General
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All the reagents and solvents were purchased from standard commercial vendors and were used without
any further purification. Sonication was performed in Elma E 30 H (Germany) ultrasonic cleaner with a
frequency of 37 KHz and a nominal power of 250 W. 1H and 13C-NMR spectra were recorded in deutrated
solvents on a Bruker spectrometer at 300 and 75 MHz respectively using tetramethylsilane (TMS) as
internal reference. Chemical shifts are given in δ scale (ppm). Melting points were determined in open
capillaries using Gallenkamp melting point apparatus (MP-D). The progress of all the reactions was
monitored by TLC on 2.0 x 5.0 cm aluminum sheets pre-coated with silica gel 60F254 with a layer
thickness of 0.25 mm (Merck). LC-MS spectra were obtained using Agilent technologies 1200 series high
performance liquid chromatography comprising of G1315 DAD (diode array detector) and ion trap LCMS
G2445D SL.
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4.2. General method for the synthesis of 3,4-dihydropyrimidine-2-ones and 3,4-dihydropyrimidine-2-
thiones (1-13)
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Dihydropyrimidines were synthesized according to our previous report procedure using ultrasonication
[14].
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A mixture of an aldehyde (10 mmol), a diamino compound (12 mmol), a dicarbonyl compound (10 mmol,
mL), SnCl2.2H2O (10 mol %) and acetonitrile (10 mL) was mixed in a pyrex tube. The mixture was then
irradiated in ultrasonic bath at 70-75oC. The reaction was monitored by TLC. After the completion of the
reaction, the resulting precipitate was filtered and crude product was recrystallized from an appropriate
solvent or purified through column chromatography. Spectroscopic data was consistent with the previously
reported for these compounds.
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4.3. Synthesis of chalcones from dihydropyrimidines (Scheme 2, 8a-8c)
In a pyrex tube 25 mL ethanol and 30 mL of NaOH (4M) solution was added. The flask was cooled in an
ice-bath and then 2 mmol DHPM (0.488 gm) and substituted aldehydes (2 mmol) was finally added and
placed in ultrasonic bath at 40-50 ºC. After completion of the reaction (TLC), quenched in ice-cold water
and then acidified with aq. HCl (1N). The precipitate obtained was re-crystallized from mixture of
ethanol/water (1:0.5).
4.3.1. 5-cinnamoyl-1,6-dimethyl-4-(3-nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one (8a)
1
Yield 610 mg (81%). Brown solid; Mp. 219-221 ºC. Rf=0.45 (n-hexane/ethyl acetate 3:1). H NMR (300
MHz, DMSO-d6): 8.81 (br s, 1H, NH), 8.19 (m, 2H, Ar-H), 7.92 (d, 1H, J = 15.6 Hz, H-Cβ), 7.68 (m, 2H,
Ar-H), 7.41 (d, 1H, J = 15.6 Hz, H-Cα), 7.10 (m, 5H, Ar-H), 5.11 (d, 1H, J = 3.0 Hz, CH), 3.19 (s, 3H, N-
CH3), 2.15 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-): 190.1, 152.7, 151.0, 150.20, 149.0, 147.1, 144.3,
136.4, 134.9. 132.6, 130.3, 129.9, 129.1, 126.2, 125.3, 120.0, 54.1, 30.7, 18.1. MS (EI) m/z 377.0 [M]+.
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4.3.2. (E)-5-cinnamoyl-4-(4-hydroxyphenyl)-1,6-dimethyl-3,4-dihydropyrimidin-2(1H)-one (8b)
Yield 536 mg (77%). Yellow solid. Mp. 199-200ºC. Rf=0.41 (n-hexane/ethyl acetate 5:1);1H NMR (300
MHz, DMSO-d6): δ 9.3 (s, 1H, OH), 8.87 (br s, 1H, NH), 7.81 (d, J = 15.9 Hz, 1H, H-Cβ), 7.67 (d, J = 15.9
Hz, 1H, H-Cα), 7.14 (m, 7H, Ar-H), 6.83 (d, 2H, , J = 6.6 Hz, Ar-H), 5.07 (d, 1H, J = 3.3 Hz, CH), 3.17 (s,
3H, N-CH3), 2.18 (s, 3H, CH3); 13C-NMR (75 MHz, DMSO-): δ 190.0, 162.2, 157.9, 153.1, 152.0, 149.4,
145.1, 129.8, 128.4, 128.2, 122.0, 120.7, 118.0, 115.0, 54.9, 30.6, 18.0. MS (EI) m/z 348.0 [M]+.
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4.3.3. (E)-4-(4-chlorophenyl)-5-cinnamoyl-1,6-dimethyl-3,4-dihydropyrimidin-2(1H)-one (8c)
1
Yield 651 mg (89%). Yellow solid. Mp. 211-212 ºC. Rf: 0.47 (n-hexane/ethyl acetate 2:1). H NMR (300
MHz, DMSO-d6): δ 8.93 (br s, 1H, NH), 7.79 (d, 1H, J = 15.9 Hz, H-Cβ), 7.59 (d, 1H, J = 15.9 Hz, H-Cα),
7.43 (d, 1H, J = 8.4 Hz, Ar-H), 7.32 (d, 1H, J = 8.4 Hz, Ar-H), 7.14 (m, 5H, Ar-H), 5.13 (d, 1H, J = 3.3
Hz, CH), 3.21 (s, 3H, N-CH3), 2.23 (s, 3H, CH3); 13C-NMR (75 MHz, DMSO-): δ 192.6, 152.4, 150.9,
145.7, 135.3, 130.2, 128.32, 127.1, 125.2, 122.9, 121.1, 119.8, 55.1, 30.3, 17.8. MS (EI) m/z 366.0 [M]+.
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4.4. Synthesis of 4′-aminochalcones (Scheme 3a, 14-18)
4′-aminochalcones were synthesized according to literature procedure [36].
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To a stirred ice-cold solution of 4'-aminoacetophenone (1.352 gm, 10 mmol) and potassium hydroxide (1-2
pellets) in 20 mL ethanol was added drop-wise substituted aromatic benzaldehydes (10 mmol).
Temperature of the reaction mixture was kept at room temperature. After completion of reaction mixture
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was poured into ice-water with continuous stirring and kept at 0-4 C overnight. The precipitate obtained
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was filtered, washed and re-crystallized from mixture of ethanol/water (1:0.5).
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4.4.1. (E)-1-(4′-aminophenyl)-3-phenylprop-2-en-1-one (14)
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14 was synthesized by using 4'-aminoacetophenone (1.352 gm, 10 mmol) and benzaldehyde (1.06 gm, 10
mmol). Yellow solid; Yield 1.54 gm (69%). Mp = 146 ºC; Rf =0.51 (n-hexane:ethyl acetate 3:1);. 1H NMR
(300 MHz, CDCl3): δ (ppm) = 7.97 (d, J = 15.9 Hz, 1H, H-Cβ), 7.69 (m, 3H, H-Cα, 2 × Ar-H), 7.18-7.30
(m, 5H, Ar-H), 6.81 (m, 2H, Ar-H), 5.82 (bs, 2H, NH2),; EIMS: C15H13NO, 223.0.
4.4.2. (E)-1-(4′-aminophenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (15)
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15 was synthesized by using 4'-aminoacetophenone (1.352 gm, 10 mM) and 4-hydroxyphenyl
benzaldehyde (1.22 gm, 10 mM). Yellow solid. Yield. 1.67 gm (71%). Rf =0.55 (n-hexane:ethyl acetate
3:1). 1H NMR (300 MHz, CDCl3): δ 9.71 (s, 1H, OH), 7.93 (d, J = 15.6 Hz, 1H, H-Cβ), 7.65 (m, 3H, H-Cα,
2 × Ar-H), 7.27-7.29 (m, 2H, Ar-H), 6.79-6.83 (m, 4H, Ar-H), 5.87 (bs, 2H, NH2); MS (EI) m/z 239.0
[M]+.
4.4.3. (E)-1-(4′-aminophenyl)-3-(4-nitrophenyl)prop-2-en-1-one (16)
16 was synthesized by using 4'-aminoacetophenone (1.352 gm, 10 mM) and 4-nitrophenyl benzaldehyde
(1.51 gm, 10 mM). Dark yellow solid, Yield. 1.53 gm (57%). Rf =0.40 (n-hexane/ethyl acetate 3:1). Mp:
1
185 ºC.; H NMR (300 MHz, CDCl3) δ 8.21 (m, 2H, Ar-H), 7.98 (d, J = 15.6 Hz, 1H, H-Cβ), 7.71 (d, J =
15.6 Hz, 1H, H-Cα), 7.61 (m, 4H, Ar-H), 6.81 (m, 2H, Ar-H), 5.81 (bs, 2H, NH2); LC-MS m/z 269
[M+H].
4.4.4. (E)-1-(4′-aminophenyl)-3-(4-chlorophenyl)prop-2-en-1-one (17)
17 was synthesized by using 4'-aminoacetophenone (1.352 gm, 10 mM) and 4-chlorophenyl benzaldehyde
(1.40 gm, 10 mM). Yellow solid, Yield 1.31 gm (51%). Rf =0.53 (n-hexane/ethyl acetate 2:1). Mp: 171 ºC.;
1H NMR (300 MHz, CDCl3) δ 7.93 (d, J = 15.9 Hz, 1H, H-Cβ), 7.69 (m, 3H, H-Cα, 2 × Ar-H), 7.23-7.25
(m, 4H, Ar-H), 6.86-6.88 (m, 2H, Ar-H), 5.83 (bs, 2H, NH2); LC-MS m/z 258 [M+H].
4.4.5. (E)-1-(4′-aminophenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one (18)
18 was synthesized by using 4'-aminoacetophenone (1.352 gm, 10 mM) and 4-(dimethylamino)phenyl
benzaldehyde (1.492 gm, 10 mM). Light yellow solid, Yield. 81%. Rf =0.47 (n-hexane/ethyl acetate 3:1).
1
m.p =157 ºC; H NMR (300 MHz, CDCl3) δ 7.93 (d, J = 15.6 Hz, 1H, H-Cβ), 7.61 (m, 3H, H-Cα, 2 × Ar-
H), 7.23-7.27 (m, 2H, Ar-H), 6.73-6.77 (m, 4H, Ar-H), 5.86 (bs, 2H, NH2), 2.97 (s, 6H, N(CH3)2) ; LC-MS
m/z 267 [M+H].
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4.5. Synthesis of 6-bromo DHPM (Scheme 3b)
Dihydropyrimidine (8) (2.44 gm, 10 mmol) was suspended in 30 mL CHC13 and then bromine (0.52 ml, 10
mmol) was added drop-wise through dropping funnel and stirred at 4 ºC for 18 hrs. After completion of
reaction, the solvent was removed under reduced pressure and residue was purified by silica gel column
chromatography to afford the product.
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4.5.1. 5-acetyl-6-(bromomethyl)-1-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (19A)
Yield. 2.51 gm (78%). White solid. Mp: 135-137 ºC. Rf : 0.38 (n-hexane:ethyl acetate 3:1). UV absorption
at 297.00 nm, 1H NMR (300 MHz, DMSO-d6): δ 9.49 (s,1H, NH), 7.24 (m, 5H, Ar-H), 5.16 (d, 1H, J=3.0
Hz, CH), 4.64 (s, 2H, CH2Br), 3.16 (s, 3H, N-CH3), 2.50 (s, 3H, COCH3). 13C-NMR (75 MHz, DMSO-d6):
δ 194.7, 152.9, 145.9, 120.2-127.0, 101.18, 53.3, 39.78, 30.5, 29.4; LC-MS m/z 323 (M+H).
4.5.2. 5-(2-bromoacetyl)-1,6-dimethyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (19B)
Yield. 0.58 gm (18%). White solid. LC-MS m/z 323 (M+H)
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4.6. C-6 substitution reaction of brominated DHPM (Scheme 3b, 8d-h)
In a pyrex tube, a mixture of 4′-aminochalcones (14-18, 2 mmol) and 6-bromo DHPM (0.646 gm, 2 mmol)
in THF (5 mL) was heated in ultrasonic bath at 40 ºC. The reaction was monitored by TLC. The precipitate
obtained was filtered, washed with sodium bicarbonate (2 × 10 mL, 0.1M). The crude solid was
recrystallized from a mixture of petroleum ether/ethanol (1:1).
4.6.1.(E)-5-acetyl-6-((4-cinnamoylphenylamino)methyl)-1-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-
one (8d)
Yellow crystals. Yield 716 mg (77%). LC-MS purity 99.98%, tR= 5.3min. Mp.=211-213 ºC. Rf =0.53 (n-
hexane/ethyl acetate 3:1). 1H NMR (300 MHz, DMSO-d6) δ 9.53 (s,1H, NH pyrimidine), 7.83 (d, J = 15.9
Hz, 1H, H-β (Vinyl)), 7.71 (m, 3H, H-Cα 2 × Ar-H), 7.11 (m, 10H, Ar-H), 6..71 (d, 2H, Ar-H), 6.07 (t, 1H,
J=5.7Hz, 1H , CH2-NH), 5.17 (d, 1H, J=3.0 Hz, CH), 3.23 (s, 3H, N-CH3), 3.13 (d, 2H, J=7.2 Hz, CH2N),
2.31 (s, 3H, COCH3); 13C-NMR (75 MHz, DMSO-d6): δ 195.0, 190.0, 151.1, 147.3, 142.5, 133, 120.0-
129.0, 103.6, 59.33, 53.3, 30.1, 28.7. LC-MS m/z 466.2 [M+H].
4.6.2. (E)-5-acetyl-6-((4-(3-(4-hydroxyphenyl)acryloyl)phenylamino)methyl)-1-methyl-4-phenyl-3,4-
dihydro-pyrimidin-2(1H)-one (8e)
Yellow crystals. Yield. 548 mg (57%). Yield 716 mg (77%). LC-MS purity 100.00%, tR= 4.9 min. Mp:
223-224 ºC. Rf: 0.42 (n-hexane/ethyl acetate 5:1). 1H NMR (300 MHz, DMSO-d6) δ 9.71 (s, 1H, OH), 9.57
(br s, 1H, NH pyrimidine), 7.79 (d, 1H, J = 15.9 Hz, Hβ (Vinyl)), 7.63 (m, 3H, 1H-Cα, 2 × Ar-H), 7.13 (m,
7H, Ar-H), 6.79 (m, 4H, Ar-H), 6.13 (t, 1H, J=5.7 Hz, CH2-NH), 5.21 (d, 1H, J=3.0 Hz, CH), 3.25 (s, 3H,
N-CH3), 3.11 (d, 2H, J=7.2 Hz, CH2N), 2.37 (s, 3H, COCH3). 13C-NMR (75 MHz, DMSO-d6): δ 195.0,
190.5, 159.1, 150.2, 147.6, 142.7, 133.9, 131.0, 130.5, 129.5, 129.2, 128.1, 127.5, 118.9, 113.5, 103.3,
58.9, 52.1, 30.4, 28.1. LC-MS m/z 482.2 [M+H].
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4.6.3. (E)-5-acetyl-1-methyl-6-((4-(3-(3-nitrophenyl)acryloyl)phenylamino)methyl)-4-phenyl-3,4-
dihydropyrimidin-2(1H)-one (8f)
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Brown crystals. Yield 643mg (63%). LC-MS purity 100.00%, tR=6.0 min. Mp: 229-231 ºC. Rf: 0.42 (n-
1
4
hexane/ethyl acetate 3:1). H NMR (300 MHz, DMSO-d6) δ 9.61 (br s, 1H, NH pyrimidine), 8.37 (s, 1H,
5
Ar-H), 8.17 (m, 1H, Ar-H), 7.98 (d, 1H, J = 15.9 Hz, Hβ (Vinyl)), 7.71 (d, 1H, J = 15.9 Hz, H-Cα), 7.51 (m,
4H, Ar-H), 7.14 (m, 5H, Ar-H), 6.73 (d, 2H, Ar-H), 6.19 (t, 1H, J=5.7 Hz, CH2-NH), 5.17 (d, 1H, J=3.0 Hz,
CH), 3.29 (s, 3H, N-CH3), 3.13 (d, 2H, J=7.2 Hz, CH2N), 2.33 (s, 3H, COCH3). 13C-NMR (75 MHz,
DMSO-d6): δ 195.5, 189.1, 150.5, 149.0, 147.6, 142.3, 121.9, 113.8-132.0, 103.2, 58.7, 53.6, 30.9, 27.9.
LC-MS m/z 511.2 [M+H].
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4.6.4. (E)-5-acetyl-6-((4-(3-(4-chlorophenyl)acryloyl)phenylamino)methyl)-1-methyl-4-phenyl-3,4-
dihydro-pyrimidin-2(1H)-one (8g)
Yellow crystals. Yield. 509 mg (51%). LC-MS purity 99.53%, tR=8.9 min. Mp: 193-194 ºC. Rf: 0.50 (n-
hexane/ethyl acetate 2:1). 1H NMR (300 MHz, DMSO-d6) δ 9.58 (bs, 1H, NH pyrimidine), 7.91 (d, 1H, J =
15.9 Hz, Hβ (Vinyl)), 7.67 (m, 3H, H-Cα, 2 × Ar-H), 7.10 (m, 9H, Ar-H), 6.79 (m, 2H, Ar-H), 6.27 (t, 1H,
J=5.7Hz, 1NH , CH2-NH), 5.29 (d, 1H, J=3.0 Hz, CH), 3.37 (d, 2H, J=7.2 Hz, CH2N), 3.21 (s, 3H, N-CH3),
2.37 (s, 3H, COCH3); 13C-NMR (75 MHz, DMSO-d6): δ 195.5, 189.3, 159.6, 152.6, 151.1, 146.9, 144.3,
142.4, 135.2, 134.1, 130.0, 127.9, 126.6, 125.6, 123.0, 120.3, 119.9, 102.9, 58.3, 53.8, 30.3, 27.3. LC-MS
m/z 500.2 [M+H].
4.6.5. (E)-5-acetyl-6-((4-(3-(4-(dimethylamino)phenyl)acryloyl)phenylamino)methyl)-1-methyl-4-
phenyl-3,4-dihydropyrimidin-2(1H)-one (8h)
Brown solid. Yield 742 mg (73%). LC-MS purity 100.00%, tR=10.2 min. Mp: 237-239 ºC. Rf =0.45 (n-
hexane/ethyl acetate 3:1). 1H NMR (300 MHz, DMSO-d6) δ 9.60 (br s, 1H, NH pyrimidine), 7.99 (d, 1H, J
= 15.9 Hz, H-β), 7.65 (m, 3H, H-Cα, 2 × Ar-H), 7.06 (m, 7H, Ar-H), 6.68 (m, 4H, Ar-H), 6.26 (t, 1H,
J=5.7Hz, CH2-NH), 5.28 (d, 1H, J=3.0 Hz, CH), 3.37 (d, 2H, J=7.2 Hz, CH2N), 3.20 (s, 3H, N-CH3), 2.91
(s, 6H, N(CH3)2), 2.33 (s, 3H, COCH3). 13C-NMR (75 MHz, DMSO-d6): δ 195.0, 189.6, 151.4, 154.9,
151.4, 149.4, 146.5, 142.3, 132.0, 130.6, 129.6, 128.8, 128.5, 127.2, 126.5, 124.5, 122.2, 121.3, 120.2,
102.4, 58.0, 53.2, 41.4, 30.8, 27.0; LC-MS m/z 509.2 [M+H].
4.7. Synthesis of pyrazolines (Scheme 4, 8i)
In a pyrex tube containing 2.5 mmol of solid NaOH in ethanol (15 mL) was added chalcone (2 mmol) and
hydrazine (2 mmol). The reaction mixture was heated in ultrasonic bath at 40 ºC. Upon completion of the
reaction (TLC), the reaction mixture was cooled to room temperature and quenched with 5 mL of a solution
of dil. HCl. The reaction mixture concentrated to dryness in vacuum to afford the product as a solid. The
crude solid was recrystallized from a mixture of n-hexane and methanol.
4.7.1. 5-acetyl-6-((4-(5-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenylamino)methyl)-1-methyl-
4-phenyl-3,4-dihydropyrimidin-2(1H)-one (8i)
Pale yellow solid. Yield 502 mg (51%). LC-MS purity 100.00%, tR=3.3 min. Mp: 198-200 ºC. Rf =0.47 (n-
1
hexane/ethyl acetate 3:1). H NMR (300 MHz, DMSO-d6) δ 9.87 (s,1H, pyrimidine-NH), 9.10 (s, 1H, Ar-
15

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OH), 8.80 (s,1H, pyrazoline-NH), 7.79 (d, 2H, J=7.5 Hz, Ar-H), 6.98 (m, 7H, Ar-H), 6.64 (d, 2H, J=8.4
Hz, Ar-H), 6.42 (d, 2H, J=8.4 Hz, Ar-H), 5.99 (t, 1H, J=5.7 Hz, CH2-NH), 5.15 (d, 1H, J=3.0 Hz,
pyrimidine-CH), 4.81 (dd, J=12.3 Hz, 7.8 Hz, 1H, pyrazoline-CH), 3.93 (dd, J=17.1 Hz, 12.3 Hz, 1H,
pyrazoline-CH), 3.44 (dd, J=7.8 Hz, 17.1 Hz, 1H, pyrazoline-CH), 3.23 (s, 3H, N-CH3), 3.16 (d, 2H, J=7.2
Hz, CH2N), 2.39 (s, 3H, COCH3); 13C-NMR (75 MHz, DMSO-d6): δ 195.0, 159.95, 153.50, 151.1, 149.9,
148.9, 142.5, 139.0, 128.9, 128.8, 127. 9, 126.6, 124.9, 123.5, 121.1, 121.0, 120.0, 103.0, 76.0, 59.33, 56.0,
51.0, 30.5, 28.1; LC-MS m/z 493.2 [M+H].
4.8. Series 2: SAR exploration around thioxo-analogues 12 and 13 (12a-d).
9
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In a pyrex tube, a mixture of 4′-aminochalcones (2 mmol) and thioxo DHPM 12 (0.58 gm, 2 mmol) and 13
(0.585 gm, 2 mmol) in THF (5 mL) was heated in ultrasonic bath at 40 ºC. The reaction was monitored by
TLC. The precipitate obtained was filtered, washed with sodium bicarbonate (2 × 10 mL, 0.1M). The crude
solid was recrystallized from a mixture of petroleum ether/ethanol (1:1).
4.8.1. (E)-N-(4-(3-(4-hydroxyphenyl)acryloyl)phenyl)-6-methyl-2-thioxo-4-p-tolyl-,3,4-dihydropyrimidine
-5-carboxamide (12a)
Yellow crystals. Yield 589 mg (61%). LC-MS purity 99.98%, tR=17.6 min. Mp: 219-221 ºC. Rf =0.38 (n-
hexane/ethyl acetate 5:1). 1H NMR (300 MHz, DMSO-d6) δ 9.88 (s, 1H, OH), 9.57 (br s,1H, NH
pyrimidine), 9.23 (br s, 1H, NH-CO), 8.24 (br s, 1H, NH), 8.04 (d, 1H, J = 15.6 Hz, Hβ (Vinyl)), 7.71 (m,
4H, Ar-H), 7.59 (d, 1H, J = 15.6 Hz, H-Cα), 7.21 (m, 2H, Ar-H), 7.01 (m, 4H, Ar-H), 6.79 (m, 2H, Ar-H),
5.14 (d, 1H, J=3.3 Hz, CH), 2.21 (s, 6H, 2×CH3); 13C-NMR (75 MHz, DMSO-d6): δ 190.5, 182.8, 167.2,
159.6, 148.5, 144.7, 142.3, 139.9, 135.3, 133.5, 131.6, 129.8, 129.2, 129.0, 128.9, 128.8, 127.0, 125.6,
124.9, 113.0, 100.72, 54.6, 26.3, 18.2. LC-MS m/z 484.2 [M+H].
4.8.2. (E)-6-methyl-N-(4-(3-(3-nitrophenyl)acryloyl)phenyl)-2-thioxo-4-p-tolyl-3,4-dihydropyrimidine-5-
carboxamide (12b)
Dark yellow crystals. Yield 584 mg (57%). LC-MS purity 100.00%, tR=11.2 min. Mp =242-243 ºC. Rf
1
=0.38 (n-hexane/ethyl acetate 5:1). H NMR (300 MHz, DMSO-d6) δ 9.59 (bs,1H, NH pyrimidine), 9.27
(br s, 1H, NH-CO), 8.50 (s, 1H, Ar-H), 8.37 (br s, 1H, NH), 8.29 (m, 1H, Ar-H), 8.17 (d, 1H, J = 15.9 Hz,
Hβ), 7.99 (d, 1H, J = 15.9 Hz, Hα), 7.82 (d, 4H, Ar-H), 7.67 (d, 1H, Ar-H), 7.65 (d, 1H, Ar-H), 7.14 (s, 4H,
Ar-H), 5.17 (d, 1H, J=3.3 Hz, CH), 2.23 (s, 6H, 2×CH3); 13C-NMR (75 MHz, DMSO-d6): δ 190.5, 182.8,
167.2, 151.0, 148.5, 144.7, 124.9, 113.0-132.0, 54.6, 26.3, 18.2. LC-MS m/z 513.2 [M+H].
4.8.3. (E)-4-(3-hydroxyphenyl)-N-(4-(3-(4-hydroxyphenyl)acryloyl)phenyl)-6-methyl-2-thioxo-3,4-
dihydro-pyrimidine-5-carboxamide (12c)
Yellow crystals. Yield 650 mg (67%). LC-MS purity 100.00%, tR=10.3 min. Mp: 219-221 ºC. Rf =0.51
(Chloroform/Methanol 5:1). 1H NMR (300 MHz, DMSO-d6) δ 9.96 (br s, 1H, OH-pyrimidine ) 9.68 (s, 1H,
OH-chalcone), 9.47 (bs,1H, NH-pyrimidine), 9.21 (br s, 1H, NH-CO), 8.14 (br s, 1H, NH-pyrimidine), 7.99
(d, 1H, J = 15.9 Hz, Hβ), 7.61 (m, 5H, H-Cα, 4×Ar-H), 7.23 (d, 2H, J = 6.3 Hz, Ar-H), 6.96 (t, 1H, J = 8.1
Hz, Ar-H), 6.74 (m, 3H, J = 5.1 Hz, Ar-H), 6.71 (d, 2H, J = 5.1 Hz, Ar-H), 5.27 (d, 1H, J=3.3 Hz, CH),
2.26 (s, 3H, CH3); 13C-NMR (75 MHz, DMSO-d6): δ 191.8, 182.1, 167.6, 160.4, 155.9, 148.7, 145.1,
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144.9, 142.3, 131.1, 130.2, 129.6, 129.4, 128.7, 125.5, 124.1, 123.2, 120.1, 115.2, 112.0, 100.2, 54.9, 18.6.
LC-MS 486.1 [M+H].
3
4
4.8.4. (E)-4-(3-hydroxyphenyl)-6-methyl-N-(4-(3-(3-nitrophenyl)acryloyl)phenyl)-2-thioxo-3,4-dihydro
pyrimidine-5-carboxamide (12d)
5
6
Yellow crystals. Yield 648 mg (63%). LC-MS purity 100.00%, tR=11.9 min. Mp: 229-230 ºC. Rf =0.57
1
(Chloroform/Methanol 3:1). H NMR (300 MHz, DMSO-d6) δ 9.83 (br s, 1H, OH-pyrimidine ), 9.51 (bs,
7
1H, NH-pyrimidine), 9.23 (br s, 1H, NH-CO), 8.59 (s, 1H, Ar-H), 8.43 (br s, 1H, NH-pyrimidine), 8.29 (m,
1H, Ar-H), 8.18 (d, 1H, J = 15.9 Hz, Hβ), 7.94 (d, 1H, J = 15.9 Hz, H-Cα), 7.85 (d, 4H, J = 6.6 Hz, Ar-H),
7.63 (d, 1H, J = 6.3 Hz, Ar-H), 7.49 (m, 1H, Ar-H), 7.011 (m, 1H, Ar-H), 6.67 (m, 3H, Ar-H), 5.27 (d,
1H, J=3.3 Hz, CH), 2.26 (s, 3H, CH3); 13C-NMR (75 MHz, DMSO-d6): δ 191.2 (C=O), 181.3 (C=S), 167.1
(NHC=O), 161.9 (C-OH-pyrimidine), 151.8 (C-NO2), 148.3 (Cβ), 145.0 (CH3C=C), 124.1 (Cα), 113.3-
129.2 (C-aromatic), 54.7 (CH-Ar), 26.2 (C-aromatic-CH3), 18.5 (CH3); LC-MS m/z 515.1 [M+H].
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4.8. Computational studies
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3.8.1. Docking studies
Docking studies were performed, using the Molecular Operating Environment (MOE) version 2011.12. The
program operated under ‘Windows XP’ operating system. To initialize the in silico studies, high resolution
crystal structures of proteins were retrieved from the PDB (PDB ID 1E7W, from L. major). The 3D
protonation of the 1E7W was done and energy minimization of the retrieved protein molecule was carried
out by using default parameters of MOE energy minimization algorithm [gradient: 0.05, Force Field:
MMFF94X]. Root mean square deviation (RMSD) was used to compare the ligand between the predicted
and its corresponding crystal structure. The resulting docked poses with RMSD less than 1.5Å were
clustered together. The lowest energy minimized pose was used for further analysis.
4.8.2. ADMET predictions
Various descriptors like logP, solubility (Sw) calculated as logS by MOE software and converted to mg/ml
units. Hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), number of rotatable bonds (NOR)
and molecular weight (MW) were calculated by MOE software. While, logD7.4 and total polar surface area
(TPSA) was calculated via Marvin 6.0.0 software of Chemaxon [37]. Whereas, renal clearance (Cl), plasma
protein binding (PPB) and LogPermeability were calculated theoretically [38].
29
4.9. In vitro antileishmanial activity
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Antileishmanial activity of the compounds was assayed using a pre-established culture of clinical isolate of
L. major and L. donovani obtained from National Institute of Health (NIH), Islamabad, Pakistan [10, 18].
Promastigotes were cultured in medium 199 (Cassion Laboratories, Inc. USA) containing 10 % fetal bovine
serum (ICN Flow, UK), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium bicarbonate
(Sigma), penicillin (ICN Biochemicals, Inc. Germany), and streptomycin (Sigma). Incubation and growth
o
of the parasite were carried out at 24 C. Briefly after 6–7 days of the culture, the promastigotes were
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centrifuged at 3,000 rpm for 3 min, the supernatant was discarded and the pellet was washed three times
with phosphate buffer saline (MP-Biomedicals, Inc. France) and re-suspended in medium 199 at 2 9 10 6
cell/mL. Promastigotes were seeded in 96-well round bottom microtiter plates (TPP, Switzerland)
containing serial dilution of the compound and media 199. Amphotericin B (BioChemica, Germany) and
DMSO were used as positive and negative controls, respectively. The plates were incubated for 72 h at 24
oC in shaker incubator. All the compounds were assayed in triplicate and the number of alive parasites was
determined by counting in Neubauer chamber. The inhibitory concentrations for the 50 % of the inhibition
IC were calculated by GraphPad Prism (GraphPad Prism Software, Inc. USA) software and data reported as
the mean ± SD.
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Acknowledgments
The Higher Education Commission (HEC), Pakistan is thankfully acknowledged for providing financial
support to Umer Rashid for its startup grant under IPFP program (HEC No: PM-
IPFP/HRD/HEC/2011/346). The authors are also thankful to Chemistry Department, Quaid-i-Azam
University and National Institute of Health (NIH), Islamabad, Pakistan for providing facilities.
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Figure Legends
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7
Figure1: Computer generated molecular models of Compound 8 (gold) superimposed on MTX (purple)
docked into the binding site of PTR1 (A) Overall structure (B) Close-up view with important interactions
(C) & (D) 2D ligand interaction maps of MTX and compound 8 respectively.
Figure 2: (A-C) 2D ligand interaction maps of compounds of Series 1 docked into the binding site of
PTR1.
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Figure 3: a) Superposition of the best conformations of Compound 8e (Reddish brown ) and 8i (magenta)
into the binding site of PTR1.
Figure 4: 2D ligand interaction maps of weakly active compounds (A) compound 8a (B) compound 8h.
Figure 5: Docking of 12c in the active site of PTR1 a) 3D view b) 2D ligand interactions
Figure 6: Total polar surface area depicted as TPSA of the compounds calculated by Marvin 6.0.0 of
Chemaxon Software.
Figure 7: LogD values at various physiological pH. a) compounds of SAR series 1; 2) Compounds of
SAR series 2
Figure 8: (a) and (b) depicts the relation between logD and renal clearances Cl in mg/ml/min. (c) Renal
clearance in increasing trend of logD. (d) Increasing trend in renal clearance due to pharmacokinetic factors
as MW, TPSA, HBD count (outer ring) as percentile. Highest clearance (24%) shown by lead compound 8i
(inner ring).
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Table 1.
In vitro antileishmanial activity of dihydropyrimidines (1-13)
R2
NH
O
R3
R4
N
R1
X
1-13
Compound
R1
R2
R3
R4
X
IC50 (µg/mL)
±SEM
H
C6H5
OEt
Me
O
0.99+0.03
1
2
CH2.C6H5
C6H5
OEt
Me
Me
O
O
O
O
O
O
O
O
O
S
0.99+0.02
0.61±0.01
0.67+0.04
0.89±0.01
0.71±0.01
0.69±0.01
0.63±0.02
0.74±0.04
0.95±0.02
0.78±0.02
0.63±0.01
0.65+0.04
0.56±0.01
H
H
H
H
H
Me
H
H
H
H
H
C6H5
Me
3
3-NO2.C6H4
3-Me.C6H4
Me
Me
4
Me
Me
5
3-OMe,4-OH.C6H3
4-OMe.C6H4
C6H5
Me
Me
6
Me
Me
7
Me
Me
8
3-NO2.C6H4
3-NO2.C6H4
C6H5
OCHMe2
OMe
Me
Me
9
CH2OMe
Me
10
11
12
13
4-Me.C6H4
3-OH.C6H4
OEt
OEt
Me
S
Me
S
Amphoterecin B (Standard drug)

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Table 2. SAR exploration around DHPM analogues (8a-8i)
O
Y
X
NH
N
O
CH3
8a-i
IC50 (µg/mL) ±SEM
Compound
X
Y
L. major
L. donovani
CH3
CH3
0.73±0.04
0.59±0.05
NI
8a
NO2
NI
NI
8b
8c
HO
CH3
0.63+0.04
Cl
O
CH3
CH3
0.54±0.01
0.51±0.06
>100
7.5
8d
8e
N
H
O
HO
N
H
O
CH3
0.67±0.05
>100
8f
N
H
NO2
O
CH3
CH3
-
0.53+0.04
0.71±0.01
0.47±0.02
4.40+0.98
NI
8g
8h
Cl
N
H
O
N
N
H
NH
N
OH
8i
1.5+0.17
2.98
N
H
0.56±0.01
-
Amphoterecin B
SSG (sodium stibogluconate)
SEM=Standard error of the mean; NI=No inhibition

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Table 3. SAR exploration around thioxo-analogues (12a-d)
R
O
X
NH
H3C
N
S
H
12a-d
Compound
X
R
IC50 (µg/mL) ±SEM
L. major
L. donovani
O
4-CH3
0.53±0.03
NI
NI
12a
HO
N
H
O
4-CH3
0.65±0.01
12b
N
H
NO2
O
3-OH
3-OH
0.51+0.04
0.58±0.01
5.58+1.1
12c
12d
HO
N
H
O
NI
N
H
NO2
Amphoterecin B
0.56±0.01
-
SSG (sodium stibogluconate)
SEM=Standard error of the mean; NI=No inhibition
-
2.98

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Table 4:
Descriptors of synthesized compounds
Comp.
logP
logD7.4
Sw
mg/ml
LogPer
TPSA
A2
HBA
HBD
NOR
Cl
MW Da
logK
Allowed
MW
range
Oral
Bioavailability
MOE
ml/min/kg
3.77
3.49
4.39
4.78
4.48
4.76
5.38
4.7
3.36
3.1
0.0045
0.0143
0.0048
0.0015
0.0022
0.0007
0.0007
0.0016
0.0043
0.0003
0.0001
0.0002
0.0008
0.0149
-10.2294 187.879
7
5
1
2
5
4
4
8
8
9
8
9
7
7
8
8
7
3
0.10485
0.1848
0.0939
0.1052
0.1309
0.0957
0.0725
0.0897
0.4266
0.103
377.4
348.4
-1.124
-0.852
-1.175
-1.123
-1.019
-1.166
-1.294
-1.196
-0.401
-1.134
-1.245
-1.153
-1.195
-1.279
400-450
less 50%
S. less
>50%
8a
8b
-9.1511
-6.8452
-8.4391
-10.408
-11.675
-8.2351
-9.8329
157.403
107.21
350-400
450-500
450-500
450-500
450-500
450-500
450-500
350-400
>500
4.02
3.89
3.58
3.83
4.5
4
1
366.85
465.55
481.55
510.55
499.99
508.62
495.58
483.59
512.59
514.56
485.56
924.09
8c
162.639
211.401
246.272
164.301
203.068
6
2
>50%
8d
7
3
>50%
8e
9
2
less 50%
>50%
8f
6
2
8g
4
7
2
S. less
less 50%
>50%
8h
4.12
5.14
5.42
4.86
4.58
0.71
2.53
4.95
5.2
-10.5468 217.243
8
4
8i
-6.5709
-7.4393
-7.1931
-8.52543
-11.516
122.55
148.14
142.78
168.37
319.61
4
4
12a
12b
12c
12d
Ampho
3
3
0.081
>500
>50%
4.14
4.38
-2.31
4
4
0.098
450-500
450-500
<300
>50%
5
5
0.089
>50%
17
13
0.091
less 50%

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