Design, synthesis, and in vitro biological evaluation of novel thiazolopyrimidine derivatives as antileishmanial compounds

Article abstract of DOI:10.1002/ardp.201900325

A series of thiazolopyrimidine derivatives was designed and synthesized as a Leishmania major pteridine reductase 1 (LmPTR1) enzyme inhibitor. Their LmPTR1 inhibitor activities were evaluated using the enzyme produced by Escherichia coli in a recombinant way. The antileishmanial activity of the selected compounds was tested in vitro against Leishmania sp. Additionally, the compounds were evaluated for cytotoxic activity against the murine macrophage cell line RAW 264.7. According to the results, four compounds displayed not only a potent in vitro antileishmanial activity against promastigote forms but also low cytotoxicity. Among them, compound L16 exhibited an antileishmanial activity for both the promastigote and amastigote forms of L. tropica, with IC₅₀ values of 7.5 and 2.69 μM, respectively. In addition, molecular docking studies and molecular dynamics simulations were also carried out in this study. In light of these findings, the compounds provide a new potential scaffold for antileishmanial drug discovery.

Full text of DOI:10.1002/ardp.201900325

Received: 1 November 2019
Revised: 21 April 2020
Accepted: 11 May 2020
|
|
DOI: 10.1002/ardp.201900325
F U L L P A P E R
Design, synthesis, and in vitro biological evaluation of novel
thiazolopyrimidine derivatives as antileishmanial compounds
Huseyin Istanbullu1
| Gulsah Bayraktar2
| Hasan Akbaba3
| Ali Ahmet Kilimcioglu4
| Ercin Erciyas2
| Ibrahim Cavus4
|
Gunes Coban2
| Bilge Debelec Butuner3
| Vildan Alptuzun2
|
Ahmet Ozbilgin4
1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Izmir Katip Celebi
University, Cigli, Izmir, Turkey
Abstract
A series of thiazolopyrimidine derivatives was designed and synthesized as a
Leishmania major pteridine reductase 1 (LmPTR1) enzyme inhibitor. Their LmPTR1
inhibitor activities were evaluated using the enzyme produced by Escherichia coli in a
recombinant way. The antileishmanial activity of the selected compounds was tested
in vitro against Leishmania sp. Additionally, the compounds were evaluated for cy-
totoxic activity against the murine macrophage cell line RAW 264.7. According to
the results, four compounds displayed not only a potent in vitro antileishmanial
activity against promastigote forms but also low cytotoxicity. Among them, com-
pound L16 exhibited an antileishmanial activity for both the promastigote and
amastigote forms of L. tropica, with IC50 values of 7.5 and 2.69 µM, respectively. In
addition, molecular docking studies and molecular dynamics simulations were also
carried out in this study. In light of these findings, the compounds provide a new
potential scaffold for antileishmanial drug discovery.
2Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ege University, Bornova,
Izmir, Turkey
3Department of Pharmaceutical
Biotechnology, Faculty of Pharmacy, Ege
University, Bornova, Izmir, Turkey
4Department of Parasitology, Manisa Celal
Bayar University, Manisa, Turkey
Correspondence
Huseyin Istanbullu, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Izmir Katip Celebi University,
35620 Cigli, Izmir, Turkey.
Email: huseyin.istanbullu@ikc.edu.tr
Funding information
Türkiye Bilimsel ve Teknolojik Arastirma
Kurumu, Grant/Award Number: SBAG‐213‐S‐
026; Ege Üniversitesi, Grant/Award Number:
2014BIL003
K E Y W O R D S
antileishmanial, Leishmania major, Leishmania tropica, neglected disease, PTR1 enzyme
inhibition, thiazolo[5,4‐d]pyrimidine
agent for VL.[3] These parasites are also seen in Turkey, which is the
bridge between Asia and Europe.[4]
|
1
INTRODUCTION
Leishmaniasis, one of the 20 neglected diseases classified by the
WHO, is caused by a protozoa parasite from Leishmania species,
which is transmitted to humans by the bite of the infected female
phlebotomine sandflies. There are three main forms of the disease:
visceral leishmaniasis (VL, Kala‐azar), cutaneous leishmaniasis (CL),
and mucocutaneous leishmaniasis (MCL).[1] Approximately one billion
people are at the risk of this disease across the globe, and an esti-
mated 0.7–1 million new cases of this disease and 26,000–65,000
deaths occur annually.[1,2]
The first‐line drugs for the treatment of leishmaniasis are
pentavalent antimonial compounds such as sodium stibogluco-
nate (Pentostam™) and meglumine antimoniate (Glucantime™).
Although, these drugs have been used since 1940s, their chemical
structures were elucidated in the last two decades, but their
mechanism of action has not been clarified yet.[5,6] However, it is
known that these drugs are converted into an active form with
the reduction from pentavalentent to a trivalent form. The
second‐line drugs are amphotericin B (AmpB) and its liposomal
form (AmBisome®), pentamidine, miltefosine, paromomycin, and
some azoles (ketoconazole, itraconazole, and fluconazole).[7]
Among these, miltefosine and azoles are the only options for oral
treatment.
More than 20 species of the unicellular protozoan parasites of
the Leishmania genus can cause leishmaniasis in mammalian hosts.
Among these, Leishmania tropica and Leishmania major are common
causative agents for CL, whereas Leishmania infantum is a causative
|
Arch Pharm. 2020;e1900325.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
1 of 14
https://doi.org/10.1002/ardp.201900325

2 of 14
ISTANBULLU ET AL.
|
Regrettably, there are some limiting factors for using these drugs
in the treatment of leishmaniasis: increasing drug resistance, high
costs for AmBisome®, and severe adverse effects. For instance, it has
been reported that resistance to miltefosine and antimonial drugs is
up to 60% in India.[8‐10] There are no data about resistance to these
drugs in Turkey, but it is a fact that the war conditions in Syria and
Iraq, and the advent of refugees in Turkey have increased the num-
ber of cases in Turkey.[11,12] Therefore, there is an urgent need for
effective and safe chemotherapeutics against leishmaniasis.
Targeting the intracellular pathway is an important method for
rational drug design and discovery. PTR1 is an NADPH‐dependent,
short‐chained reductase enzyme family member, which can reduce a
variety of unconjugated pteridines, as well as folates.[13,14] Folic acid
and the related pteridines are essential cofactors for all life forms,
especially for kinetoplastids. The active forms of both folate and
biopterine are the reduced tetrahydro derivatives, which increase in
Leishmania through the action of dihydrofolate reductase (DHFR) or
the alternative pteridine reductase PTR1.[15] Unlike humans, kine-
toplastids have a PTR1 enzyme. Therefore, the inhibition of the PTR1
enzyme appears to be a rational strategy for selective antileishmanial
drug development.
antileishmanial activity.[22‐24] On the contrary, bioisosteric replace-
ment of pteridine and quinoxaline ring gave nonfolate inhibitors
having benzothiazole, aminobenzimidazole, and pyrrolopyrimidine
scaffolds. These nonfolate inhibitors showed good inhibition against
the PTR1 enzyme at the micromolar level.[25‐27]
The aims of this study are (a) to design and synthesize com-
pounds bearing a thiazolopyrimidine ring, which is a bioisostere of
the pteridine ring, (b) to test their PTR1 enzyme inhibitor activity,
and (c) to investigate their antileishmanial and cytotoxic activities. In
this study, thiazolopyrimidine scaffold was evaluated for antil-
eishmanial activity for the first time.
|
2
RESULTS AND DISCUSSION
|
2.1
Chemistry
We designed novel compound derivatives from a 2‐aminothiazolo
[5,4‐d]pyrimidine scaffold, which is an isostere of the pteridine ring
(Figure 1). The compounds were derivatized by amidation of 2‐amino
function group with aliphatic and/or aromatic acyl groups.
The first information about the PTR1 enzyme (EC 1.5.1.33) was
reported in 1992.[16,17] Later it was reported that this enzyme was an
alternate source for the reduction of folate compounds and ex-
pressed PTR1 in transformed Escherichia coli.[13] Then, it was found
that PTR1 was able to reduce folates to a minimal extent; its over-
expression could compensate for the inhibition of DHFR by metho-
trexate (MTX).[18] The enzyme structure was reported by Hunter
et al., and proteomic analyses for drug targets and resistance me-
chanisms were studied.[19,20]
All compounds were synthesized according to formerly published
procedures in four steps (Scheme 1).[28‐30] In the first step,
5‐nitrouracil was converted to 2,4‐dichloro‐5‐nitropyrimidine (1) by
using phosphoryl chloride and N,N‐dimethylaniline. Compound 1 was
reacted with potassium thiocyanate to yield 2‐chloro‐4‐thiocyanato‐
5‐nitropyrimidine (2) in glacial acetic acid. Later, 2‐amino‐
5‐chlorothiazolo[5,4‐d]pyrimidine (3) ring was obtained with the
reduction of the nitro group using iron powder, followed by in situ
cyclizations of compound 2. In the last step, final amide derivatives,
According to a literature survey, the first reported PTR1 enzyme
inhibitors are folate‐like compounds bearing pteridine and quinoxa-
line ring as heterocyclic cores.[21] Among the six‐membered fused
rings, the structure–activity relationship of the substituted quinazo-
line core was examined extensively as PTR1 enzyme inhibitors with
L1–L23, were obtained from compound 3 in the presence of
4‐dimethylaminopyridine (DMAP) with various acyl chlorides in
pyridine (Scheme 1).
The structures of the final compounds were determined by
spectral analyses and the spectroscopic properties were in
FIGURE 1 The chemical structures of
biopterin (with pteridine ring structure), a
substrate of PTR1 enzyme (i); antifolate
compound aminopterin (ii); reported
compounds with a PTR1 inhibitor activity
(iii),[15] and synthesized compounds with
thiazolopyrimidine ring, designed as
bioisostere of the pteridine ring (iv)

3 of 14
ISTANBULLU ET AL.
|
SCHEME 1 The synthesis pathway of
compounds L1–L23
accordance with the proposed structures. According to the Fourier‐
transform infrared spectroscopy (FTIR) data, amide I and II bands
were observed between 1,724–1,686 cm−1 and 1,572–1,585 cm−1
fields for aliphatic amides, whereas they were observed between
1,660–1,698 cm−1 and 1,575–1,584 cm−1 fields for aromatic amides.
In the 1H‐NMR (nuclear magnetic resonance) spectral data, com-
pound 3 (2‐amino‐5‐chlorothiazolo[5,4‐d]pyrimidine) was identified
with two proton signals: 8.49 ppm (singlet belonging to hydrogen on
the aromatic ring) and 8.29 ppm (broad singlet of amino hydrogens).
Unlike L3, the proton signal due to the amide NH group of L1–L23
was recorded between 13.57 and 10.35 ppm as a broad singlet, and
also the single proton signal of the heterocyclic ring was determined
between 9.15 and 8.60 ppm as a singlet. In the spectrum, the shifts of
these protons to the lower field supported the amide formation. On
the contrary, in the case of compound L21, two doublets were ob-
served at 6.93 and 7.81 ppm, with a 16‐Hz coupling constant, which
were assigned to the ethylenic protons that indicated E configuration.
Some 1H‐NMR spectra indicated the organic solvent impurities in the
aliphatic region of the spectra, which were confirmed by the ele-
mental analysis data as well.[31,32] The obtained elemental analysis
data of the compounds were within 0.4% impurity of the predicted
structures. In addition, observed carbon signals in the 13C‐NMR of
the compounds were in accordance with the molecular structures;
however, some compounds were not subjected to 13C‐NMR analysis
due to solubility issues. According to electrospray ionization–mass
spectroscopy (ESI–MS) data, molecular ion peaks, isotope peaks, and
fragmentation patterns were determined as expected.
MTX was used as the reference compound. IC50 values of the title
compounds are listed in Table 1.
According to activity results, most of the compounds bearing
amide group displayed a better enzyme inhibitor activity than com-
pound 3, with an amine group at 2‐position of 5‐chlorothiazolo[5,4‐d]
pyrimidine. Among the studied compounds, the compound bearing
acetyl moiety (L1) displayed the best inhibitor potency with the IC50
value of 43.8 1.9 µM. In general, the replacement of the acetyl
group with the other aliphatic acyl groups such as propanoyl (L2, IC50
75.5 1.7 µM), butanoyl (L3, IC50 52.6 1.4 µM), and isobutanoyl
(L4, IC50 81.1 2.5 µM) did not improve the inhibitory activity
against LmPTR1.
When benzoyl derivatives were evaluated within themselves, it
was observed that the introduction of a substituent to the phenyl
ring resulted in an increased inhibitor activity, except for fluorine
substituent.
Generally, the results showed that the conversion of the amine
groups on 5‐chlorothiazolo[5,4‐d]pyrimidine ring to amide function
by attaching an aliphatic or an aromatic moiety affected the enzyme
inhibitory activity positively.
|
2.2.2
In vitro antileishmanial activity
Targeting the folate pathway by inhibiting the PTR1 enzyme, which is
a bypass of DHFR inhibition in the parasites' folate pathway, is a
feasible strategy for selective antileishmanial chemotherapy.[33]
As LmPTR1 enzyme inhibition is one of the possible targets to
combat leishmaniasis, the compounds were tested for their in vitro
antileishmanial activity. For antileishmanial activity, the percentage
viability of parasites was studied against L. tropica, L. infantum, and
L. major at 24, 48, and 72 hr, respectively. As the observed percen-
tage viabilities were similar in terms of time and parasite species,
IC50 values were calculated for 24 hr in L. tropica promastigote (Ta-
ble 1). L19 was found to be the most active one with the IC50 value of
0.5 0.4 µM, followed by L16 with the IC50 value of 7.5 4.6 µM.
Among the phenyl derivatives, generally, the introduction of a sub-
stituent to para position resulted in a remarkable improvement in the
antileishmanial activity, whereas ortho substitution led to a lack of
activity. This result was noticeably observed in chlorine substitution.
The characterization data of all compounds are reported in the
experimental section in detail, and all compounds, except L1, are
reported for the first time in this study. The spectral data of L1
were in accordance with the formerly reported literature.[28]
|
2.2
Biological activities
|
2.2.1
LmPTR1 enzyme expression and in vitro
inhibition assay
In vitro enzyme inhibition studies were performed according to the
literature with slight modifications, as explained in Section 4.[21]

4 of 14
ISTANBULLU ET AL.
|
TABLE 1 Synthesized compounds and
their LmPTR1 enzyme inhibitor activity and
in vitro antileishmanial activity against
Leishmania tropica (L. tropica) promastigote
LmPTR1 IC50
(µM SD)
L. tropica
R (Ar)
IC50 (µM SD)
L1
methyl
43.8 1.9
75.5 1.7
52.6 1.4
81.1 2.5
62.8 2.4
86.5 2.0
52.4 2.1
72.1 2.3
69.1 2.6
>100
>100
L2
ethyl
>100
L3
propyl
67.7 7.9
>100
L4
isopropyl
L5
cyclohexane
phenyl
27.3 2.8
>100
L6
L7
2‐methylphenyl
2‐methoxyphenyl
2‐chlorophenyl
2‐fluorophenyl
2‐bromophenyl
2‐iodophenyl
2‐trifluoromethyl phenyl
4‐methylphenyl
4‐methoxyphenyl
4‐chlorophenyl
4‐fluorophenyl
4‐nitrophenyl
2,4‐dichlorophenyl
2‐phenylethyl
styryl
>100
L8
>100
L9
>100
L10
L11
L12
L13
L14
L15
L16
L17
L18
L19
L20
L21
L22
L23
3
>100
73.8 2.4
nda
>100
>100
nda
>100
76.5 1.8
nda
>100
37.1 21.0
7.5 4.6
61.0 13.1
10.1 6.2
0.5 0.4
22.5 3.3
8.4 2.8
>100
74.8 1.2
>100
77.2 2.8
nda
>100
nda
2‐furyl
>100
2‐thienyl
nda
>100
–
97.1 2.6
6.3 0.5
>100
Methotrexate
Amphotericin B®
Glucantime®
–
–
0.2 0.02
5.8 1.4
Abbreviation: SD, standard deviation.
aThe activity of the compound was non‐detectable due to solubility problems.
Interestingly, the addition of 2‐chlorine substitution to 4‐chlorine
derivative improved the activity significantly. On the contrary, the
derivative bearing phenylethyl group showed up to a four‐fold better
activity, compared with ethyl‐chained aliphatic amide; therefore, the
substitution of ethyl with phenyl group improved the activity.
Additionally, styryl derivative was found to be nearly three‐fold more
active than its saturated analog, phenylethyl derivative.
assay. The results were expressed in the means of percent decrease as
compared with the positive control. Infection percentages were calcu-
lated by the multiplication of infected macrophage percent with an
average number of amastigotes per infected macrophage.[34] IC50 values
of L16 and L19 were found to be 2.69 µM (0.07–9.17) and 185.4 µM
(97.22–418.2), respectively (Table 2 and Figure S15). Negative control
amphotericin B gave an IC50 value of 0.065 µM (0.04–0.1).
On the basis of antileishmanial activity results against L. tropica
promastigote, L16 and L19 were selected for the further amastigote
Taken together, L16 has an antileishmanial potential for both
promastigotes and amastigotes.

5 of 14
ISTANBULLU ET AL.
|
TABLE 2 The antileishmanial activity of the selected compounds
against L. tropica amastigotes
TABLE 3 Selected compounds' in vitro cytotoxic activity against
RAW 264.7 murine macrophage cell line
Compound
L16
IC50 (µM)
Compound
L16
In vitro cytotoxicity IC50 (µM SD) RAW 264.7
2.69 (0.07–9.17)
185.4 (97.22–418.2)
0.065 (00.4–0.1)
>100
L19
L18
77.1 0.14
82.9 1.02
>100
Amphotericin B
L19
Note: The bottom and top values are given in brackets.
L21
Pentostam®
>100
Abbreviation: SD, standard deviation.
|
2.2.3
Cytotoxicity assay
The cytotoxicity assay was performed against RAW 264.7 murine
macrophage cell line for the compounds which showed the best in
vitro antileishmanial activity.[35,36] The compounds showed low (L18,
L19) or no (L16, L21) cytotoxicity (Table 3). Therefore, these results
indicate that the title compounds exhibit selective toxicity.
thiazolopyrimidine ring of these compounds was shifting toward
Ser111 and the phosphate and ribose components of the cofactor,
the titled ring was sandwiched between Phe113 and nicotinamide
component of NADPH as the quinazoline ring of CB3717 in the
crystal form of the ternary complex.
In addition, the occupancy of the substituted benzamide groups
of L9 and L18 in the catalytic site was similar to p‐aminobenzamide
group of CB3717 in the catalytic site of LmPTR1 (Figures S1, S5, and
S11). L16, the other selected compound, occupied the catalytic site of
LmPTR1 by an opposite orientation as compared with orientations of
L9 and L18 inside LmPTR1. It was detected that, when substituted
benzamide group of L16 settled between Phe113 and nicotinamide
component of the cofactor, thiazolopyrimidine ring shifted to be
placed between Leu188, Tyr191, Tyr194, and Lys198 (Figure S8).
The comparison of the first‐ranked solutions of the studied com-
pounds in LmPTR1 is given in Figure 2.
|
2.3
Molecular modeling studies
In the current study, molecular docking was carried out to detect
binding orientations and affinity of thiazolopyrimidine derivatives in
the active site of LmPTR1 (PDB id: 2BFA resolved at 2.7 Å) using
Gold 5.2.1 program. Docking scores of the first‐ranked docking so-
lutions of the studied compounds are reported in Table 4. Before the
docking study, 10‐propargyl‐5,8‐dideazafolic acid (CB3717) taken
from the crystal structure of LmPTR1 (PDB id: 2BFA) was docked in
LmPTR1 to validate the docking method.
The first‐ranked docking pose and the scoring value of CB3717
inside the LmPTR1 are figured in Figure S1 and reported in Table 4.
It was observed that the quinazoline ring of CB3717 obtained from
the docking study was settled into the pteridine‐binding pocket of
LmPTR1, similar to the ternary complex of LmPTR1 (PDB id: 2BFA;
Figure S1). The minor difference between the docking pose and the
crystal form of CB3717 is the orientation of the glutamate tail of
the inhibitor compound. Although the glutamate tail obtained from
the docking pose extended Gln186 and Leu189, the tail of the crystal
form oriented toward the opposite side. The comparison of the
first‐ranked solutions and crystal form of CB3717 inside LmPTR1 is
given in Figure S1.
TABLE 4 Docking scores for thiazolopyrimidine derivatives in the
active site of LmPTR1 (PDB id: 2BFA) using Goldscore
LmPTR1 (2BFA)
Compounds Goldscore
LmPTR1 (2BFA)
Compounds
L14
Goldscore
L1
51.5110 (1)
53.3057 (1)
56.9219 (1)
56.0127 (1)
62.5664 (1)
61.8942 (1)
64.1272 (1)
64.8221 (1)
64.2589 (1)
61.0443 (1)
65.3434 (1)
66.8230 (1)
64.0401 (1)
64.3328 (1)
65.9658 (1)
64.7663 (1)
62.5242 (1)
64.7168 (1)
66.4583 (1)
62.5019 (1)
70.2491 (1)
58.7457 (1)
63.0418 (1)
45.7886 (1)
L2
L15
L3
L16
L4
L17
L5
L18
In consideration of enzyme inhibition assay, L1 displayed the
best inhibitory activity against LmPTR1 among the studied com-
pounds. According to docking study results, the first‐ranked docking
pose of L1 partially occupied the catalytic center of LmPTR1 (Figure
S2). It was observed that the acetamide group of the L1 was placed
between Ser111, Ser112, Phe113, and the phosphate and ribose
fragment of the cofactor; while the thiazolopyrimidine ring of the L1
was sandwiched between Phe113 and nicotinamide group of the
cofactor. The other selected compounds, L9 and L18, were placed in
the catalytic site of LmPTR1, formed by amino acid residues Ser111,
Phe113, Asp181, Tyr194, and Lys198, and nicotinamide and ribose
component of the cofactor. It was observed that while the
L6
L19
L7
L20
L8
L21
L9
L22
L10
L11
L12
L13
L23
3
Methotrexate 76.8028 (1)
CB3717 75.1539 (1)
Note: The absolute ranking positions for the suggested binding poses are
given inside brackets.

6 of 14
ISTANBULLU ET AL.
|
FIGURE 2 The comparison of the first‐ranked solutions of the studied compounds in LmPTR1 (PDB id: 2BFA). Cyan, light purple, orange, and
army green sticks represent compounds L1, L9, L16, and L18, respectively. The active site residues are named and represented as gray sticks in
LmPTR1. For a clear image, all hydrogen atoms were hidden
In our study, the molecular dynamics (MD) simulations and
MM‐GBSA binding free energy calculations were executed to analyze
noncovalent interactions of thiazolopyrimidine derivatives, namely
compounds L1, L9, L16, and L18, in the active site of LmPTR1. For
this purpose, the first‐ranked solutions of compounds L1, L9, L16, and
L18, yielded by using the GOLD 5.2.1 program, were chosen for MD
simulations. The protein–ligand–cofactor ternary complexes of the
selected compounds were then subjected to 100‐ns simulations after
the equilibrium step.
LmPTR1 (Figure S7). About the average RMSD value of the L18,
periodical changes were viewed between ∼0.5 and ∼1.0 Å and this
was observed when the substituted benzamide group of L18 was
rotating, ligand kept its orientation and location (Figures S12 and
S13). There was a gradual increase in the average RMSD value of the
ternary complex of L16, ∼1.5 to ∼3.5 Å, throughout 75 ns of MD
simulations, and then it decreased to ∼2.5 Å during the rest of MD
simulation. As the RMSD value of L16 was evaluated, it was observed
that there were periodically changing fluctuations between ∼0.5 and
∼1.0 Å in the entire simulation (Figure S9). It was monitored that L16
returned starting binding orientations, however, it was located dif-
ferent regions of the active site of LmPTR1 during the MD simula-
tions (Figure S10).
The analysis of the stability of ternary complexes of the selected
compounds revealed that the MD simulations for the stability of
these complexes were observed to be stable during the whole MD
simulation. The average root mean square deviation (RMSD) value of
the ternary complex of L1 was detected, which fluctuated between
~1.5 Å and ~2.5 Å throughout the whole simulation. Moreover, the
average RMSD value of L1 was determined, revealing that it pre-
served its stability, ~0.5 Å, from beginning to end of MD simulation
(Figure S3). It was displayed that L1 kept its position in the active site
of LmPTR1, preserving key interactions with protein and cofactor
(Figure S4). The RMSD value of the ternary complex of L9 was found
to be increased, fluctuating from ∼1.5 Å to ∼2.0 Å at the first 20 ns of
MD simulations. Later, its fluctuations increased to ∼2.5 Å, and it
fluctuated between ∼2.0 and ∼3.0 Å at rest of the MD simulations.
A similar fluctuation behavior was observed for the RMSD plot of the
ternary complex of L18 in its MD simulations. The RMSD value of the
ternary complex was found to be increased, fluctuating from ∼1.5 to
∼2.5 Å at the first 20 ns of MD simulations, and after this increase, it
fluctuated between ∼2.0 and ∼3.0 Å at rest of the MD simulations.
The average RMSD value of L9 was stable, ∼0.5 Å, in the entire MD
simulation (Figure S6), similar to the RMSD value of L1. Therefore, no
change was observed at the position of L9 in the active site of
In the MD plots of the binary complex formed by NADPH and
LmPTR1, the average RMSD value fluctuated between ∼1.5 and
∼2.2 Å throughout the whole MD simulation (Figure S14).
The binding mode analysis revealed that several noncovalent
interactions were observed between the studied compounds and
NADPH–LmPTR1 complexes such as hydrogen bonding, cation–π,
and π−π interactions. It was observed that major efficiencies of the
titled interactions were different for the binding mode of the
studied compounds. According to analysis results, hydrogen
bonding formed by a direct bonding with Tyr194, cofactor, and
water molecules, and water‐mediated bonding with backbone re-
sidues and cofactor is the major factor for binding of L1 inside the
active site of LmPTR1 (Tables S1–S3). Besides hydrogen‐bonding
network, π−π interactions formed by the nicotinamide group of the
cofactor, Phe113, and thiazolopyrimidine ring of L1 as sandwich‐
type π−π stacking contributed to the bonding of the title com-
pound (Figure S4). The main interaction contributing to the binding
mode of L9 and L18 is a sandwich‐type π−π interaction formed by

7 of 14
ISTANBULLU ET AL.
|
Phe113, the nicotinamide group of the cofactor, and the thiazo-
lopyrimidine ring of the studied compounds (Figures S7 and S13).
Hydrogen bonding, formed by water molecules and water‐
mediated bonding with backbone residues and cofactor, is the
secondary interaction that contributes to the formation of the
binding mode of L9 and L18 inside LmPTR1. For these two mole-
cules, direct hydrogen bonding with backbone residues and co-
factor was observed with low frequencies during the whole MD
simulation (Tables S4–S6 and S10–S12). Lastly, in the binding
mode analysis of L16, two main interactions, which contribute to
the binding process inside LmPTR1, were observed as hydrogen
bonding and cation–π interactions between thiazolopyrimidine
ring and the positively charged guanidine group of Arg284
(Figure S10 and Tables S7–S9). For all studied compounds,
hydrogen bond formed between related heteroatoms of the com-
pounds and the hydroxyl group of ribose ring of cofactor with
several hydrogen‐bonding frequencies was detected (Tables S1,
S4, S7, and S10).
simulations of the title compounds, confirm that the hydrogen
bond was formed by water molecules. The electrostatic energies
(in vacuum) only contribute to the binding free energy of L1 and
L16. The energy decomposition analysis of L1 revealed that
Phe113, Met183, Tyr19,4, and NADPH provide important energy
contributions in a ternary complex system (Table S13). The van
der Waals and nonpolar solvation energies are dominant com-
ponents for the binding of the ligand, which are supplied by
Phe113 and NADPH; therefore, this shows the contribution of
Phe113 and NADPH to form a π–π interaction with ligand in the
active site of LmPTR1. For L9, the van der Waals and nonpolar
solvation energies are the main factors for the contribution of the
binding free energy provided by Phe113, Leu188, Tyr194, and
cofactor. In addition to this, polar solvation energy provided by
NADPH contributes to binding free energy as well as the van der
Waals and nonpolar solvation energies (Table S14). The van der
Waals and nonpolar solvation energies provided by Phe113 and
NADPH support the sandwich π–π interaction between thiazo-
lopyrimidine ring and titled residues. The energy decomposition
analysis of L16 revealed that the van der Waals and nonpolar
solvation energies play a crucial role in the binding process in the
ligand–NADPH–LmPTR1 ternary complex. The calculated van der
Waals and nonpolar solvation energies display the contribution of
hydrophobic interactions for the binding process (Table S15).
Also, the contribution of electrostatic energy supplied by Arg284
supports the formation of cation–π interaction between the po-
sitively charged guanidine group of Arg284 and thiazolopyr-
imidine ring of the L16 (Table S15). The energy decomposition
analysis of L18 revealed that the van der Waals and nonpolar
solvation energies are the largest contributions to binding free
energy (Table S16). Especially, the favorable contributions for
binding free energy are the van der Waals and nonpolar solvation
energies provided by NADPH and Phe113, interacting with L18
for the binding process. These contributions support the binding
mode of L18, similar to the binding mode of compound L9 that is
sandwiched between Phe113, cofactor, and ligand.
The MM‐GBSA binding free energy calculations and energy de-
composition analysis were performed for compounds L1, L9, L16, and
L18 from free MD simulations. The average binding free energies of
the title compounds inside the active site of LmPTR1 are tabulated in
Table 5, and the results of energy decomposition analysis are tabu-
lated in Tables S13–16.
The average binding free energies of the studied compounds
were calculated around −20 kcal/mol, and no significant differ-
ence was observed among the compounds. At the same time, the
experimental binding energies of the studied compounds were
calculated between −5.5 and −6.0 kcal/mol (Table 5). According
to binding free energy calculations, the unfavorable contribution
is provided from the electrostatic energies (in vacuum and sol-
vent) for both systems. Besides, the van der Waals energies and
nonpolar solvation energies are the important factors in contrast
to the electrostatic (in vacuum) and polar solvation energies (in
solvent) for the binding of the title compounds to the active site
of LmPTR1 (Table 5). Polar solvation energies, obtained from MD
TABLE 5 The calculated MM‐GBSA (Molecular Mechanics ‐ Generalized Born Surface Area) binding free energies (delta total energy) and
their components for compounds L1, L9, L16, and L18 inside LmPTR1 with standard errors of the mean
L1
L9
L16
L18
van der Waals energy (kcal/mol)
Electrostatic energy (kcal/mol)
Polar solvation energy (kcal/mol)
Nonpolar solvation energy (kcal/mol)
Delta gas‐phase free energy (kcal/mol)
Delta solvation free energy (kcal/mol)
Delta total energy (kcal/mol)
−27.0527 0.0396
−14.4281 0.0923
25.4838 0.049
−3.2060 0.0051
−41.4808 0.0979
22.2778 0.0624
−19.2031 0.0489
43.8 1.9
−35.6598 0.0406
2.2817 0.0682
16.4761 0.0681
−3.3994 0.0049
−33.3781 0.0947
13.0767 0.0650
−20.3014 0.0417
69.1 2.6
−32.4403 0.0498
−15.2893 0.0821
29.7441 0.0656
−3.9665 0.0043
−47.7296 0.0877
25.7776 0.0633
−21.9519 0.0425
74.8 1.2
−37.6610 0.0584
0.3036 0.0604
18.1820 0.0572
−3.8218 0.0066
−37.3574 0.0935
14.3602 0.0545
−22.9972 0.0590
77.2 2.8
IC50 (μM)
Experimental binding energy (kcal/mol)
−5.9824a
−5.7106a
−5.6634a
−5.6445a
aExperimental binding free energy was calculated from IC50 values according to ΔG ≈ RT ln IC50
.

8 of 14
ISTANBULLU ET AL.
|
|
3
CONCLUSION
Mercury Plus NMR Varian (Varian Inc., Palo Alto, CA). Chemical
shifts were measured in parts per million (δ). Coupling constants (J)
were reported in hertz (Hz). Liquid chromatography–mass spectro-
metry (MS) was recorded on a Thermo MSQ Plus/DSQ II (San Jose,
CA) mass spectrometer using the ESI+ method. Elemental analyses
were performed on Leco TruSpec Micro CHNS (Leco, St. Joseph, MI),
and the values were found to be within 0.4% of the theoretical
values.
In this study, a series of thiazolopyrimidine derivatives was designed
and synthesized as antileishmanial compounds with LmPTR1 in-
hibitor activity. Regarding the activity results, the conversion of the
amine group to the amide function on the main ring affects the en-
zyme inhibitory activity. Among them, the 2,4‐dichlorobenzoyl deri-
vative (L19) was found to be the most active compound. First, the
antileishmanial activity of the compounds against promastigote form
was evaluated, and then the most promising ones were tested against
amastigote forms of the parasite. It was found out that L16 was quite
active for both promastigote and amastigote forms of the parasite,
whereas L19 showed remarkable activity against the promastigote
form of the parasite. Moreover, selected derivatives displayed low
cytotoxicity. Interestingly, there is no correlation between enzyme
inhibitory activity and antileishmanial activity. Taken together, it
might be speculated that the antileishmanial activity occurs via dif-
ferent mechanisms.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
|
4.1.2
Synthesis of the title compounds
2‐Amino‐5‐chlorothiazolo[5,4‐d]pyrimidine derivatives were pre-
pared in four steps according to the procedure in the literature
(Scheme 1).[28–30] In the first step, 7.3 ml of freshly distilled POCl3
(0.08 mol) was carefully added to the mixture of 5‐nitrouracil (3.14 g,
0.028 mol) and N,N‐dimethylaniline (3.54 ml, 0.028 mol) in an ice bath
for 30 min. The reaction mixture was stirred at 60°C for 5.5 hr. After
cooling to room temperature, the mixture was poured into 100 ml of
ice water. The solution was extracted with diethyl ether (3 × 100 ml).
The organic layer was washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo to obtain 2,4‐dichloro‐5‐
nitrouracil (compound 1). Compound 1 was used without further
purification.
MD simulations, guided by docking studies, of compounds L1, L9,
L16, and L18 inside LmPTR1 showed that hydrogen‐bonding net-
works played a key role in the stability of the title compounds in the
active site of LmPTR1. Especially, the formation of hydrogen bonds
with backbone residues for L1 was observed more frequently, com-
pared with the other studied compounds. It was determined that
hydrogen bonding formed by water molecules and water‐mediated
bridges contributed to the binding process of all studied compounds
inside LmPTR1. In addition to hydrogen bonding, it was detected that
the cation–π interaction was the secondary dominant factor for the
binding process of the L16, and π–π interaction was the secondary
dominant factor for the binding process of L9 and L18 inside
LmPTR1. According to enzyme inhibition assay, L1 has a slightly
higher inhibitory activity against LmPTR1, compared with the other
studied compounds, and considering the molecular modeling studies,
it might be concluded that hydrogen bonds formed especially with
Tyr194 caused this difference.
Compound 1 (1.91 g, 0.01 mol) was added to 5‐ml glacial acetic
acid and cooled down to 0°C using an ice bath. Potassium thio-
cyanate (1.07 g, 0.0111 mol) was added in portions over 30 min
and the mixture was stirred at 0°C for 2 hr. The reaction mixture
was poured into crushed ice, filtered, and washed with cold
diethyl ether to give 2‐chloro‐5‐nitro‐4‐thiocyanatopyrimidine
(compound 2). The crude product was used without further pur-
ification for the next step.
In the third step, the mixture of compound 2 (2 g, 9.23 mmol) and
iron powder (1.55 g, 27.7 mmol) in glacial acetic acid (18.5 ml) was
refluxed for 2 hr. The reaction mixture was cooled to room tem-
perature and filtrated. The filtrate was evaporated under reduced
pressure. The residue was diluted with water and then extracted with
ethyl acetate (3 × 50 ml). The combined organic layer was washed
with saturated NaHCO3 solution and saturated NH4Cl solution, dried
over anhydrous Na2SO4, and evaporated under vacuum. The crude
product was crystallized from ethanol to yield 2‐amino‐5‐
chlorothiazolo[5,4‐d]pyrimidine ring (compound 3).
|
4
EXPERIMENTAL
|
4.1
Chemistry
|
4.1.1
General
Most of the reagents and solvents were commercially available ma-
terials of reagent grade and were purchased from Alfa Aesar, TCI
Chemicals, Sigma‐Aldrich, or Merck. Melting points were determined
with the capillary melting point apparatus (Barnstead Electrothermal
IA 900). Analytical thin‐layer chromatography was run on Merck
silica gel plates (Kieselgel 60 F254) with detection by ultraviolet light
(254 nm). The FTIR spectra of the compounds were monitored by
attenuated total reflectance (PerkinElmer Spectrum 100 FT‐IR,
Shelton). The NMR spectra (400 MHz for 1H‐NMR and 100 MHz
for 13C‐NMR) were recorded in the deuterated solvent on AS400
The final compounds, L1–L23, were obtained from compound 3
with various acyl chlorides. Compound 3 (0.3 g, 1.6 mmol) in pyridine
was treated with acyl chlorides (3.2 mmol) in an ice bath. DMAP was
added to the reaction mixture in a catalytic amount, and it was
stirred at room temperature overnight. The pH of the reaction
medium was adjusted to ~2 with 2‐N HCl solution, and then it was
extracted with ethyl acetate (3 × 50 ml). After the combined organic
extracts were washed with saturated NaHCO3 solution, dried over
anhydrous Na2SO4, and evaporated under reduced pressure, the final

9 of 14
ISTANBULLU ET AL.
|
compounds were crystallized from ethanol, methanol, ethyl acetate,
or tetrahydrofuran.
J = 8.0 Hz), 8.01 (d, 2H, J = 8.0 Hz), 8.60 (s, 1H), and 10.69 (br, 1H).
13C‐NMR (CDCl3, 100 MHz) δ: 127.8 (2C), 129.4 (2C), 130.9, 134.0,
139.3, 149.2, 154.8, 159.5, 165.5, and 165.9. MS (ESI+): m/z: 290.9
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)acetamide (L1)[28]
Yield 71% (yellow solid). mp. 205°C. FTIR νmax cm−1: 1,724 and 1,584.
1H‐NMR (dimethyl sulfoxide DMSO‐d6, 400 MHz) δ: 2.21 (s, 3H), 9.05
(s, 1H), and 12.78 (br, 1H). 13C‐NMR (DMSO‐d6, 100 MHz) δ: 23.1,
140.4, 149.7, 153.4, 158.9, 166.0, and 170.7. MS (ESI+): m/z: 229.1 [M
+H]+ and 231.1 [M+2+H]+. Anal. (C7H5ClN4OS.0.5H2O.0.33CH3OH)
calcd: C 35.47, H 2.97, N 22.57, S 12.92; found: C 35.85, H 3.36, N
22.55, S 12.59.
[M+H]+ and 292.9 [M+2+H]+, Anal. (C12H7ClN4OS.0.5H2O.0.1C6H14
)
calcd: C 49.08, H 3.07, N 18.17, S 10.40; found: 48.93, H 2.97,
N 17.94, S 10.33.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐2‐methyl benzamide (L7)
Yield 77% (yellow solid). mp. 172°C. FTIR νmax cm–1: 1,676 and 1,575.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 2.41 (s, 3H), 7.32–7.36 (m, 2H), 7.48
(d, 1H, J = 7.6 Hz), 7.64 (d, 1H, J = 7.6 Hz), 9.13 (s, 1H), and 13.21 (br,
1H). 13C‐NMR (DMSO‐d6, 100 MHz) δ: 20.1, 126.2, 128.9, 131.5,
131.9, 133.2, 137.3, 140.3, 150.1, 153.5, 159.2, 165.9, and 169.3. MS
(ESI+): m/z: 305.0 [M+H]+ and 307.0 [M+2+H]+. Anal. (C13H9ClN4
OS.0.2C2H5OH) calcd: C 51.26, H 3.27, N 17.84, S 10.21; found:
50.95, H 3.51, N 17.49, S 10.35.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)propionamide (L2)
Yield 73% (yellow solid). mp. 241°C. FTIR νmax cm−1: 1,692 and 1,572.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 1.10 (t, 3H, t, J = 7.6 Hz), 2.54
(q, 2H, J = 7.6 Hz), 9.07 (s, 1H), and 12.75 (br, 1H). 13C‐NMR (DMSO‐
d6, 100 MHz) δ: 9.1, 28.9, 140.3, 149.7, 153.2, 158.8, 165.9, and
174.4. MS (ESI+): m/z: 243.2 [M+H]+ and 245.2 [M+2+H]+, Anal.
(C8H7ClN4OS) calcd: C 39.59, H 2.91, N 23.09, S 13.21; found: C
39.77, H 3.10, N 23.47, S 13.45.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐2‐methoxybenzamide (L8)
Yield 52% (yellow solid). mp. 220°C (decomposition). FTIR νmax
cm–1: 1,673 and 1,575. 1H‐NMR (DMSO‐d6, 400 MHz) δ: 3.90
(s, 3H), 7.02–7.22 (m, 2H), 7.56–7.73 (m, 2H), 9.11 (s, 1H), and
11.33 (br, 1H). 13C‐NMR: n.d. MS (ESI+): m/z: 321.0 [M+H]+ and
323.1 [M+2+H]+. Anal. (C13H9ClN4O2S.0.1H2O.0.2C4H8O2) calcd:
C 48.73, H 3.20, N 16.47, S 9.42; found: C 48.63, H 3.51, N 16.12,
S 9.13.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin ‐2‐yl)butyramide (L3)
Yield 72% (beige solid). mp. 194°C. FTIR νmax cm−1: 1,709 and 1,585.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 0.91 (t, 3H, J = 7.6 Hz), 1.63 (m, 2H,
J = 7.2 Hz), 2.48 (br, 2H), 9.04 (s, 1H), and 12.77 (br, 1H). 13C‐NMR
(DMSO‐d6, 100 MHz) δ: 13.9, 18.2, 37.4, 140.3, 149.8, 153.2, 158.8,
165.8, and 173.6. MS (ESI+): m/z: 257.0 [M+H]+ and 259.1 [M+2+H]+.
2‐Chloro‐N‐(5‐chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)benzamide (L9)
Yield 77% (yellow solid). mp. 188°C (decomposition). FTIR νmax cm–1
:
Anal. (C9H9ClN4OS.0.2H2O.0.2CH3OH) calcd:
C
41.43,
H
3.85,
N 21.01, S 12.02; found: C 41.33, H 3.43, N 20.83, S 11.92.
1,678 and 1,579. 1H‐NMR (CDCl3, 400 MHz) δ: 7.45–7.50 (m, 2H),
7.53–7.57 (m, 1H), 7.95 (dd, 1H, J1 = 2.0 Hz, J2 = 7.4 Hz), 8.65 (s, 1H),
and 10.56 (br, 1H). 13C‐NMR (CDCl3, 100 MHz) δ: 127.8, 131.0,
131.4, 131.4, 133.8, 139.2, 145.7, 149.2, 154.8, 158.8, 164.5, and
165.8. MS (ESI+): m/z: 325.00 [M+H]+, 327.0 [M+2+H]+, and 329.0
[M+4+H]+. Anal. (C12H6Cl2N4OS.0.5C4H8O2) calcd: C 45.54, H 2.73,
N 15.17, S 8.68; found: C 45.73, H 2.37, N 15.52, S 9.01.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)isobutyramide (L4)
Yield 45% (light green solid). mp. 179°C. FTIR νmax cm−1: 1,686 and
1,583. 1H‐NMR (DMSO‐d6, 400 MHz) δ: 1.14 (d, 6H, J = 6.8 Hz), 2.81
(m, 1H, J = 6.8 Hz), 9.05 (s, 1 H), and 12.80 (br, 1H). 13C‐NMR (DMSO‐
d6, 100 MHz) δ: 19.2, 34.6, 140.3, 149.7, 153.2, 159.0, 165.9, and
177.5. MS (ESI+): m/z: 257.0 [M+H]+ and 259.2 [M+2+H]+. Anal.
(C9H9ClN4OS.0.33H2O.0.25CH3OH) calcd: C 41.05, H 3.97, N 20.70,
S 11.84; found: C 41.10, H 3.95, N 20.83, S 11.42.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐2‐fluorobenzamide (L10)
Yield 47% (yellow solid). mp. 218°C. FTIR νmax cm–1: 1,667 and 1,576.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 7.36–7.42 (m, 2H), 7.65–7.71
(m, 1H), 7.80 (d, 1H, J = 7.2 Hz), 9.13 (s, 1H), and 10.35 (br, 1H). 13C‐
NMR (DMSO‐d6, 100 MHz) δ: 117.1, 121.7, 125.2, 130.9, 135.0,
140.3, 150.3, 153.6, 158.9, 161.2, 164.8, and 165.8. MS (ESI+): m/z:
309.1 [M+H]+ and 311.1 [M+2+H]+. Anal. (C12H6ClFN4OS.0.33H2-
O.0.33CH3OH) calcd: C 45.54, H 2.47, N 17.23, S 9.81; found:
C 45.85, H 2.09, N 17.68, S 9.86.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)cyclohexane
carboxamide (L5)
Yield 62% (gray solid). mp. 192°C. FTIR νmax cm–1: 1,694 and 1,579.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 1.16–1.87 (m, 10H), 2.56 (tt, 1H,
J1 = 3.6 Hz, J2 = 11.2 Hz), 9.05 (s, 1H), and 12.72 (br, 1H). 13C‐NMR
(DMSO‐d6, 100 MHz) δ: 25.4 (2C), 25.6, 29.0 (2C), 44.0, 140.3, 149.8,
153.2, 159.0, 165.9, and 176.5. MS (ESI+): m/z: 297.0 [M+H]+ and
299.1 [M+2+H]+. Anal. (C12H13ClN4OS.0.5H2O.0.33CH3OH) calcd: C
46.66, H 5.01, N 17.41, S 9.96; found: C 46.43, H 4.63, N 17.22,
S 9.90.
2‐Bromo‐N‐(5‐chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)benzamide (L11)
Yield 65% (dark beige solid). mp. 216 °C. FTIR νmax cm–1: 1,698 and
1,581. 1H‐NMR (DMSO‐d6, 400 MHz) δ: 7.47–7.56 (m, 2H), 7.69 (dd,
1H, J1 = 2.0 Hz, J2 = 7.2 Hz), 7.77 (dd, 1H, J1 = 1.2 Hz, J2 = 7.4 Hz), 9.11
(s, 1H), and 13.46 (br, 1H). 13C‐NMR (DMSO‐d6, 100 MHz) δ: 119.6,
128.3, 130.0, 133.0, 133.5, 136.0, 140.3, 150.4, 153.6, 158.7, 165.8,
and 167.7. MS (ESI+): m/z: 368.8 [M+H]+, 370.9 [M+2+H]+, and 372.9
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)benzamide (L6)
Yield 75% (yellow solid). mp. 229°C. FTIR νmax cm–1: 1,671 and 1,577.
1H‐NMR (CDCl3, 400 MHz) δ: 7.55 (t, 2H, J = 8.0 Hz), 7.68 (t, 1H,

10 of 14
ISTANBULLU ET AL.
|
[M+4+H]+. Anal. (C12H6BrClN4OS) calcd: C 38.99, H 1.64, N 15.16, S
8.68; found: C 39.14, H 1.99, N 15.45, S 8.46.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐4‐fluorobenzamide (L17)
Yield 64% (dark yellow solid). mp. 272°C. FTIR νmax cm–1: 1,669 and
1,577. 1H‐NMR (DMSO‐d6, 400 MHz) δ: 7.41 (t, 2H, J = 8.8 Hz), 8.21
(dd, 2H, J1 = 3.6 Hz, J2 = 5.2 Hz), 9.12 (s, 1H), 13.37 (br, 1H). 13C‐NMR
(DMSO‐d6, 100 MHz) δ: 116.2, 116.4, 128.0, 128.1, 132.9, 132.0,
140.3, 150.0, 153.5, 160.0, 165.9, and 166.0. MS (ESI+): m/z: 309.1 [M
+H]+ and 311.1 [M+2+H]+. Anal. (C12H6ClFN4O2S.0.5H2O.0.2-
CH3OH) calcd: C 45.21, H 2.43, N 17.29, S 9.89; found: C 45.48,
H 2.80, N 17.42, S 10.29.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐2‐iodobenzamide (L12)
Yield 80% (yellow solid). mp. 216°C. FTIR νmax cm–1: 1,690 and 1,579.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 7.30 (td, 1H, J1 = 2.0 Hz,
J2 = 7.6 Hz), 7.54 (td, 1H, J1 = 1.2 Hz, J2 = 7.6 Hz), 7.62 (dd, 1H,
J1 = 1.6 Hz, J2 = 7.6 Hz), 7.98 (dd, 1H, J1 = 0.8 Hz, J2 = 8.0 Hz), 9.15
(s, 1H), and 13.40 (br, 1H). 13C‐NMR (DMSO‐d6, 100 MHz) δ: 94.0,
128.6, 129.3, 132.63, 139.8, 139.9, 140.4, 150.4, 153.6, 158.8, 165.8,
169.3. MS (ESI+): m/z: 416.8 [M+H]+ and 418.9 [M+2+H]+. Anal.
(C12H6ClIN4OS.0.25H2O.0.2CH3OH) calcd: C 34.27, H 1.72, N 13.10,
S 7.49; found: C 34.64, H 2.16, N 12.69, S 7.93.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐4‐nitrobenzamide (L18)
Yield 53% (yellow solid). mp. 214°C. FTIR νmax cm–1: 1,672 and 1,580.
1H‐NMR (CDCl3, 400 MHz) δ: 8.20 (d, 2H, J = 8.0 Hz), 8.39 (d, 2H,
J = 8.0 Hz), and 8.97 (s, 1H). 13C‐NMR (DMSO‐d6, 100 MHz) δ: 123.8
(2C), 130.0 (2C), 136.9, 139.9, 150.0, 150.2, 151.0, 153.7, 159.6, and
166.09. MS (ESI+): m/z: 335.9 [M+H]+ and 337.9 [M+2+H]+. Anal.
(C12H6ClN5O3S.0.25C2H5OH) calcd: C 43.24, H 2.18, N 20.17, S 9.23;
found: C 43.10, H 2.36, N 19.93, S 9.64.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐2‐trifluoromethyl
benzamide (L13)
Yield 59% (yellow solid). mp. 195°C. FTIR νmax cm–1: 1,693 and 1,579.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 7.78–7.82 (m, 1H), 7.84–7.87
(m, 2H), 7.91 (d, 1H, J = 7.6 Hz), 9.15 (s, 1H), 13.57 (br, 1H). 13C‐NMR
(DMSO‐d6, 100 MHz) δ: 127.1, 127.1, 129.6 (2C), 131.9, 133.2, 140.3,
150.5 (2C), 153.7, 158.7, 165.8, 167.6. MS (ESI+): m/z: 359.00 [M+H]+
and 361.00 [M+2+H]+. Anal. (C13H6ClF3N4OS.1H2O.0.25C2H5OH)
calcd: C 41.76, H 2.47, N 14.43, S 8.26; found: C 41.54, H 2.52,
N 14.22, S 8.30.
2,4‐Dichloro‐N‐(5‐chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐
benzamide (L19)
Yield 54% (light brown solid). mp. 217°C. FTIR νmax cm–1: 1,696 and
1,582. 1H‐NMR (DMSO‐d6, 400 MHz) δ: 7.60 (dt, 1H, J1 = 2.0 Hz,
J2 = 8.4 Hz), 7.74–7.81 (m, 2H), 9.11 (s, 1H), and 13.57 (br, 1H);. 13C‐
NMR (DMSO‐d6, 100 MHz) δ: 128.0, 130.0, 131.6, 132.2, 132.6,
137.0, 140.3, 150.4, 153.8, 158.7, 165.9, and 166.0. MS (ESI+): m/z:
358.9 [M+H]+, 360.9 [M+2+H]+, and 362.9 [M+4+H]+. Anal.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐4‐methyl benzamide (L14)
Yield 75% (yellow solid). mp. 158°C. FTIR νmax cm–1: 1,673 and 1,576.
1H‐NMR (CDCl3, 400 MHz) δ: 2.46 (s, 3H), 7.35 (d, 2H, J = 8.0 Hz),
7.93 (d, 2H, J = 8.0 Hz), and 8.69 (s, 1H). 13C‐NMR (DMSO‐d6,
100 MHz) δ: 21.6, 129.0 (3C), 129.7 (2C), 140.3, 144.4, 149.7, 153.6,
160.2, 164.7, 165.8. MS (ESI+): m/z: 304.9 [M+H]+ and 306.9
[M+2+H]+. Anal. (C13H9ClN4OS.0.1H2O.0.33CH3OH) calcd: C 50,49,
H 3,34, N 17,67, S 10,11; found: C 50.84, H 3.45, N 17.56, S 10.32.
(C12H5Cl3N4OS.0.33H2O.0.66CH3OH) calcd:
C 39.32, H 2.16,
N 14.49, S 8.29; found: C 39.72, H 2.23, N 14.46, S 8.03.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐3‐phenyl
propionamide (L20)
Yield 38% (green solid). mp. 202°C. FTIR νmax cm–1: 1,693 and
1,583. 1H‐NMR (DMSO‐d6, 400 MHz) δ: 2.85 (t, 2H, J = 7.8 Hz),
2.94 (d, 2H, J = 8 Hz), 7.17 (td, 1H, J1 = 2.0 Hz, J2 = 6.8 Hz),
7.23–7.29 (m, 4H), 9.03 (s, 1H), and 12.78 (br, 1H). 13C‐NMR
(DMSO‐d6, 100 MHz) δ: 30.6, 37.2, 126.1, 128.7 (2C), 128.84 (2C),
140.5, 140.8, 150.0, 153.3, 158.5, 166.1, and 173.4. MS (ESI+): m/z:
319.1 [M+H]+ and 321.0 [M+2+H]+. Anal. (C14H11ClN4O-
S.0.5C4H8O) calcd: C 54.16, H 4.26, N 15.79, S 9.04; found:
C 54.55, H 4.02, N 16.01, S 8.89.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)‐4‐
methoxybenzamide (L15)
Yield 69% (yellow solid). mp. 245°C. FTIR νmax cm–1 1,660 and 1,583.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 3.83 (s, 3H), 7.09 (d, 2 H, J = 8.0 Hz),
8.13 (d, 2H, J = 8.0 Hz), and 9.09 (s, 1H). 13C‐NMR (DMSO‐d6,
100 MHz) δ: 56.5, 114.6 (2C), 123.7, 131.2 (2C), 140.4, 149.5, 153.5,
160.0, 164.0, 166.9, and 166.3. MS (ESI+): m/z: 320.9 [M+H]+ and
322.9 [M+2+H]+. Anal. (C13H9ClN4O2S.0.25H2O.0.33CH3OH) calcd:
C 47.68, H 3.25, N 16.68, S 9.55; found: C 47.53, H 3.68, N 16.58,
S 9.52.
(E)‐N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)cinnamamide (L21)
Yield 47% (yellow solid). mp. 204°C. FTIR νmax cm–1: 1,694 and
1,584. 1H‐NMR (DMSO‐d6, 400 MHz) δ: 6.93 (d, 1H, J = 16 Hz),
7.45–7.46 (m, 3H), 7.65 (dd, 1H, J1 = 3.0 Hz, J2 = 7.0 Hz), 7.79 (d,
1H, J = 16 Hz), and 9.06 (s, 1H). 13C‐NMR (DMSO‐d6, 100 MHz) δ:
119.8, 128.6 (2C), 129.5 (2C), 131.0, 134.7, 140.6, 144.5, 149.5,
153.3, 159.4, 165.6, 166.2. MS (ESI+): m/z: 317.1 [M+H]+ and
319.1 [M+2+H]+. Anal. (C14H9ClN4OS.1H2O.0.1C4H8O) calcd: C
50.57, H 3.48, N 16.38, S 9.37; found: C 50.20, H 3.50, N 16.75,
S 9.70.
4‐Chloro‐N‐(5‐chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)benzamide (L16)
Yield 64% (yellow solid). mp. 293°C. FTIR νmax cm–1: 1,668 and 1,577.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 7.61 (d, 2H, J = 8.0 Hz), 8.10
(d, 2H, J = 8.0 Hz), and 9.07 (s, 1H). 13C‐NMR: n.d. MS (ESI+): m/z:
324.9 [M+H]+, 326.9 [M+2+H]+, and 328.9 [M+4+H]+. Anal.
(C12H6Cl2N4OS.0.5H2O.0.1C4H8O2) calcd: C 43.42, H 2.29, N 16.34,
S 9.35; found: C 43.16, H 2.37, N 16.69, S 9.04.

11 of 14
ISTANBULLU ET AL.
|
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)furan‐2‐carboxamide (L22)
N‐terminus of the LmPTR1 enzyme and allows the use of metal
chelate affinity chromatography for the purification. The unbound
proteins were washed from the column with a washing buffer. The
His‐tagged protein was eluted using elution buffer. The removal of
the histidine tag was not found necessary for activity due to the
disadvantages of the second purification step. The sodium dodecyl
sulfate polyacrylamide gel electrophoresis analysis was performed to
analyze the purity and molecular weight of the isolated enzyme.
Plasmid DNA isolation was carried out using the Invitrogen
MiniPrep pDNA isolation kit. HindIII and EcoRV restriction enzymes
were applied to confirm the transformation of pET15b–LmPTR1.
Yield 64% (orange solid). mp. 225°C (decomposition). FTIR νmax cm–1
1,675 and 1,583. 1H‐NMR (DMSO‐d6, 400 MHz) δ: 6.77 (q, 1H,
J = 1.6 Hz), 7.77 (dd, 1H, J1 = 0.8 Hz, J2 = 3.6 Hz), 8.07 (dd, 1H,
J1 = 0.8 Hz, J2 = 1.8 Hz), 9.10 (s, 1H), and 13.36 (br, 1H). 13C‐NMR
(DMSO‐d6, 100 MHz) δ: 112.6, 118.9. 140.2, 145.4, 148.5, 149.7,
153.8, 157.3, 159.2, and 166.4. MS (ESI+): m/z: 281.0 [M+H]+ and
:
283.1 [M+2+H]+. Anal. (C10H5ClN4O2S.0.5H2O) calcd:
C 41.46,
H 2.09, N 19.34, S 11.07; found: C 41.80, H 2.35, N 19.49, S 11.33.
N‐(5‐Chlorothiazolo[5,4‐d]pyrimidin‐2‐yl)thiophene‐2‐
carboxamide (L23)
Yield 49% (yellow solid). mp. 253°C. FTIR νmax cm–1: 1,694 and 1,584.
1H‐NMR (DMSO‐d6, 400 MHz) δ: 7.27 (t, 1H, J1 = 4.8 Hz, J2 = 4.0 Hz),
8.04 (d, 1H, J = 4.8 Hz), 8.32 (d, 1H, J = 4.0 Hz), 9.09 (s, 1H), and 13.36
(br, 1H). 13C‐NMR (DMSO‐d6, 100 MHz) δ: 128.7, 133.3, 135.2,
136.9, 140.2, 150.0, 153.7, 159.9, 161.2, and 165.5. MS (ESI+): m/z:
296.9 [M+H]+ and 298.9 [M+2+H]+. Anal. (C10H7ClN4O2S2.0.4H2-
O.0.1C2H5OH) calcd: C 39.70, H 2.09, N 18.16, S 20.78; found:
C 39.62, H 2.52, N 18.49, S 20.36.
|
4.2.2
In vitro evaluation of LmPTR1 enzyme
inhibition studies
The inhibitory activity of the recombinant LmPTR1 enzyme was de-
termined by measuring the absorbance at 340 nm in a 96‐well plate
with a Varioskan Flash multimode reader (Thermo Fisher Scientific).
Before the reaction, the enzyme concentration was determined by a
bicinchoninic assay (QuantiPro BCA Assay Kit, Sigma‐Aldrich). Then,
the reaction was carried out at 30°C in the presence of recombinant
LmPTR1 (5 μM), NADPH (100 μM), folic acid (30 μM), and dithio-
treitol (5 mM) in a sodium phosphate buffer (pH 6.0).[21,37] Folic acid
was added to the reaction as the substrate of the LmPTR1 enzyme,
and the reaction was initiated by the addition of freshly prepared
NADPH. The total volume of each reaction was 300 μl. The IC50
values were calculated by the addition of testing compounds in in-
creasing concentrations (6.25, 12.5, 25, 50, and 100 μM) in DMSO.
The final concentration of DMSO was kept below 1% to avoid an
impact on enzyme activity. MTX (Cayman Chemicals) was used at the
same concentration range as a positive control. Isolation and pur-
ification of the LmPTR1 enzyme were carried out from a single stock
at the beginning of each experiment. The purified enzyme was kept at
4°C and it was not used for longer than 3 days. Experiments were
carried out at least in triplicate. The IC50 concentration of each
compound was calculated by using GraphPad Prism 6.0.
|
4.2
Biological activities
|
4.2.1
Preparation and purification of recombinant
PTR1 enzyme
The E. coli expression plasmid carrying the LmPTR1 gene cloned with
the T7 promoter—pET15b–LmPTR1—was kindly provided by Prof.
W. N. Hunter's (University of Dundee, UK) laboratory. This plasmid
was transformed into the chemically competent E. coli BL21 strain by
heat shock. After transformation, a single colony was selected in
Luria‐Bertani (LB) agar plates containing 100 μg/ml ampicillin. The
selected colony was amplified by incubation at 37°C in 5 ml LB liquid
medium containing 50 μg/ml ampicillin for 4 hr, and it was then
transferred to 50 ml LB liquid (50 μg/ml ampicillin) medium to in-
crease the volume and incubated until the optical density value
reached 0.5–0.6. To obtain a cell pellet, the medium was centrifuged
at 10,000 g for 2 min at 4°C. The supernatant was discarded and the
pellet was transferred to 250 ml LB liquid medium containing 1 mM
of isopropyl β‐D‐1‐thiogalactopyranoside. Incubation was done at
27°C for 16 hr with shaking at 150 rpm.
|
4.2.3
In vitro antileishmanial activity
In vitro antileishmanial activity of the compounds was tested against
L. tropica (MHOM/AZ/1974/SAF‐K27), L. major (MHOM/SU/1973/
5ASKH), and L. infantum (MHOM/TN/1980/IPT1) promastigotes.
Promastigotes were grown in Rosewell Park Memorial Institute
(RPMI)‐1640 medium (Biochrom AG), supplemented with 10% fetal
calf serum. The parasites were incubated at 26°C and passaged until
log phase (108 promastigotes/ml). After centrifuging the parasites for
10 min at 1,500 rpm, they were washed with saline twice. Promas-
tigotes were cultured in 96‐well plates (106 parasites/well) with or
without the compounds. For in vitro experiments, 300, 150, 75, 37.5,
18.8, 9.4, 4.7, and 2.3‐μM concentrations of the compounds were
prepared in DMSO. As reference drugs, glucantime and AmpB were
Cells were harvested after incubation by centrifugation (2,500g)
at 4°C. The cell lysate was prepared using following procedures: in-
cubation with 1 mg/ml lysozyme for 30 min at 4°C with shaking;
sonication 10 times for 15‐s burst–15‐s cooling cycles; incubation
with 5 μg/ml DNase for 15 min at 4°C; and centrifugation of the
lysate at 10.000 g for 30 min at 4°C. Then, the supernatant was
transferred to a new Falcon tube. The supernatant was filtered
through a 0.45‐μm filter to eliminate the particles. Five microliters of
supernatant were then applied to an equilibrated affinity chroma-
tography purification column (GE‐Protino Ni‐TED 2000). The
pET15b–LmPTR1 plasmid codes for a hexa‐histidine tag on the

12 of 14
ISTANBULLU ET AL.
|
prepared in DMSO at the same concentrations of the compounds.
The highest concentration of DMSO and RPMI medium was also used
for control groups. Microplates were incubated at 25°C. The number
of parasites was counted with a hemocytometer under a light mi-
croscope after 24, 48, and 72 hr. All the in vitro experiments were
run in triplicate and the results were expressed as the percent in-
hibition in the parasite number. The percentage of viable parasites
(viability %) was calculated using the following equation: viability
% = (mean of treated live parasites − blank/mean of untreated (con-
trol) parasites − blank) × 100.[38–42]
were added to each well and incubated for 2.5 hr at 37°C. Viable cells
enzymatically convert the colorless tetrazolium salt to an orange‐
colored formazan compound. The intensity of the formed color was
measured spectrophotometrically at 475 nm in a Varioskan Flash
multimode reader (Thermo Fisher Scientific). Blank samples were
used to subtract background absorption. To eliminate nonspecific
absorbance occurrences from the medium, the absorbance at 660 nm
was also measured. Specific absorbance values were expressed
mathematically with the following equation: Specific absorbance =
A475 nm (test) – A475 nm (blank) − A660 nm (test).
J774.2 mouse macrophages were obtained from Parasite Bank
located in Manisa Celal Bayar University Faculty of Medicine, Turkey,
for amastigote assay. Macrophages were grown in RPMI‐1640
medium, supplemented with 15% inactivated fetal bovine serum.
Cells were seeded at a density of 1 × 104 cells/well in eight‐well
microplates with rounded coverslips on the bottom, and they were
cultured in a humidified 95% air and 5% CO2 atmosphere at 37°C for
24 hr. Afterward, the cells were infected with in vitro promastigote
forms of L. tropica in the stationary phase, at a ratio of 10:1, for 24 hr
(Figure S15). The nonphagocytosed parasites were removed by
washing, and then L16 and L19 (300, 150, 75, 37.5, 18.75, 9.37, 4.68,
and 2.34 µM) were added. AmpB was used as positive control in 1,
0.5, 0.25, 0.13, 0.06, 0.03, 0.02, and 0.01‐µM concentrations. Mac-
rophages were incubated with the compounds for 24 hr at 37°C in
5% CO2. After incubation, plates were washed and treated with
methanol. Determined preparations were Giemsa‐stained and mi-
croscopically examined. The number of cellular amastigotes was de-
termined by analyzing 200 host cells distributed in randomly chosen
microscopic fields.[43]
Cell viability was calculated on the basis of the specific absor-
bance of the cells, compared with the absorbance of the control
group consisting of untreated RAW264.7 cells. IC50 values were
calculated according to the cell viability values using the Prism 6.0
(GraphPad) software. Experiments were carried out at least in
triplicate.
|
4.3
Molecular modeling studies
The studied compounds were generated with a builder panel of
MOE2016.08, protonated using the protonate three‐dimensional
protocol, and subjected to energy minimization with MOE2016.08
using the MMFF94x force field.[44,45] The crystal structure of pter-
idine reductase 1 (PDB id: 2BFA resolved at 2.7 Å) was taken from
the RCSB Protein Data Bank (http://www.rcsb.org/pdb). Chain A and
NADPH of the crystal structure were selected to prepare docking
structure and chain B; heteroatoms and water molecules in the PDB
file were deleted. Antechamber and parmchk modules of AmberTools
16 were used to calculate the partial atomic charges for NADPH with
the AM1‐BCC charge model and generate the parameter files of
NADPH, respectively.[46,47] The complex of chain A and NADPH was
prepared using the xleap module of AmberTools 16 with the General
Amber force field (GAFF) for NADPH and AMBER99SB force field
for protein, and it was solvated in an octahedral box with TIP3P
water molecules with 10 Å distance between the protein surface and
the box boundary.[48] The solvated system was neutralized with an
appropriate number of chloride counter ions and subjected to energy
minimization with Sander.MPI module of Amber 12 suite. Before the
docking study, created chloride counterions and water molecules
(excluded the water molecules included in the binding site) were
removed from the system.
|
4.2.4
Cytotoxicity
The cytotoxicity evaluation of selected compounds was performed on
RAW264.7 murine macrophage cell line (ATCC® TIB‐71™). Cells
were cultured in a Dulbecco's modified Eagle's medium, supple-
mented with 2‐mM L‐glutamine and 10% fetal bovine serum.
Bacterial contamination was prevented by the addition of
penicillin–streptomycin (100 UI/ml penicillin, 100 μg/ml streptomy-
cin). For cytotoxicity assay, the cells were seeded in 96‐well culture
plates at a density of 105 cells/ml. After overnight incubation, the
medium was removed and cells were washed with Dulbecco's
phosphate‐buffered saline (DPBS). One hundred microliters of fresh
growth medium including increasing concentrations of synthesized
compounds were immediately added after the washing step. The
following concentrations of samples were added into the wells: 6.25,
12.5, 25, 50, and 100 μM/well. Control groups and blind groups for
spectroscopic analysis were also applied.
A docking study was performed using the GOLD 5.2.1 program
with default generic algorithm parameters. The studied compounds
were docked within a radius of 20 Å around the oxygen atom (O) of
the carbonyl group of Gly225 residue. In total, 100 conformations
were allowed per structure. The GoldScore fitness function was
used as scoring functions.[49,50] Figures were created with the
MOE2016.08 program.
The viability of cells was determined by the colorimetric WST‐1
cell proliferation assay kit as per the manufacturer's instructions
(Roche). Briefly, after overnight incubation, the growth medium
containing the samples was removed and cells were washed with
DPBS. Then, 100‐µl fresh growth medium and 10‐µl WST‐1 reagent
Molecular dynamics simulations were carried out for
NADPH–protein (LmPTR1) and ligand–NADPH–protein complexes
using AMBER 12 suite.[46] The initial ligand–NADPH–protein systems
were generated using the first solution of compounds L1, L9, L16, and

13 of 14
ISTANBULLU ET AL.
|
L18 in LmPTR1 yielded from docking studies. The partial atomic
charges for the title compounds and NADP were calculated with
antechamber module of AmberTools 16 using the AM1‐BCC charge
model.[46,47] The Xleap module of AmberTools 16 was used to
parameterize the complexes using general AMBER force field (gaff)
for ligands and NADP and AMBERff99SB force field for proteins, to
solvate the complexes in an octahedral box with TIP3P water mo-
lecules with 10 Å distance between the protein surface and the box
boundary, and to neutralize the solvated systems with an appropriate
number of chlorine counter ions.[46,48,51,52] The energy minimizations
and MD simulations of the systems were executed with SAN-
DER.MPI and PMEMD.CUDA modules of AMBER 12, respectively.[47]
The initial systems were exposed to energy minimization in two
steps: restraining initial structures for 1,000 iterations with the
steepest descent algorithm and for 1,000 iterations with conjugate
gradient methods, and then minimizing entire systems for 2,500
iterations using steepest descent algorithm and for 2,500 iterations
using conjugate gradient methods. The systems were heated from 0
to 300 K, with 10‐kcal·mol−1·Å−1 restraint force permitting water
molecules and ions to move freely for 0.1 ns, and the temperature
was equilibrated at 300 K with a collision frequency of 1.0 ps−1 in
constant volume periodic boundary and pressure at 1 bar. The posi-
tional restraints were kept for the solute using constant pressure
periodic boundary conditions with isotropic position scaling method
using Langevin dynamics for 2 ns in the MD simulations. The posi-
tional constraints were gradually removed, keeping the temperature
at 300 K and pressure at 1 bar, and the systems were subjected to
free MD simulation for 100 ns. The SHAKE algorithm was performed
to constrain band vibrations involving hydrogen atoms in the equi-
libration part of MD and the free MD simulations.[53] The Particle
Mesh Ewald method was executed for long‐range electrostatic in-
teractions using 10‐Å cutoff for the short‐range nonbonded inter-
actions and 2 fs for a time step.[54] Xmgrace program was used for
visualization of the trajectories.[55] The hydrogen bonding was de-
termined with Cpptraj module of AmberTools 16 using default
parameters.[56] Binding free energies of compounds were calculated
with MMPBSA.py.MPI module of AmberTools 16 using the Gen-
eralized Born (GB) model from 100 spaced snapshots of the last
40 ns of the unrestrained MD simulations.[57] MD snapshots were
taken out from free MD simulations using the UCSF Chimera pack-
age.[58] Figures were set up with the MOE2016.08 program.
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
ORCID
Huseyin Istanbullu
Gulsah Bayraktar
Hasan Akbaba
http://orcid.org/0000-0002-0102-4181
http://orcid.org/0000-0001-7271-1619
http://orcid.org/0000-0001-9273-6346
Gunes Coban
http://orcid.org/0000-0003-4512-4392
http://orcid.org/0000-0001-8112-9241
Bilge Debelec Butuner
Ahmet Ozbilgin
Vildan Alptuzun
http://orcid.org/0000-0003-3613-8741
http://orcid.org/0000-0002-1477-4440
REFERENCES
[1] WHO Leishmaniasis Fact Sheet, Updated March 2019. https://www.
who.int/en/news‐room/fact‐sheets/detail/leishmaniasis
October 2019)
(accessed:
[2] DNDi Leishmaniasis Fact Sheet, https://www.dndi.org/wp‐content/
uploads/2019/09/Factsheet2019_Leishmaniasis.pdf
(accessed:
October 2019)
[3] P. Pottinger, C. R. Sterling Sherris Medical Microbiology, 6th ed. (Eds:
K. C. Ryan, C. G. Ray), McGraw‐Hill Education, New York 2014,
pp. 823–845.
[4] A. Özbilgin, G. Çulha, S. Uzun, M. Harman, S. G. Topal, F. Okudan, F.
Zeyrek, C. Gündüz, İ. Östan, M. Karakuş, S. Töz, Ö. Kurt, I. Akyar, A.
Erat, D. Güngör, Ç. Kayabaşı, İ. Çavuş, P. Bastien, F. Pratlong, T.
Kocagöz, Y. Özbel, Trop. Med. Int. Health 2016, 21, 783.
[5] W. L. Roberts, W. J. McMurray, P. M. Rainey, Antimicrob. Agents
Chemother. 1998, 42, 1076.
[6] H. R. Hansen, C. Hansen, K. P. Jensen, S. H. Hansen, S. Sturup,
B. Gammelgaard, Anal. Chem. 2008, 80, 5993.
[7] WHO Technical Report, Series 949, Control of the Leishmaniasis:
Report of a meeting of the WHO Expert Committee on the Control
of Leishmaniases, Geneva, Switzerland 2010.
[8] S. L. Croft, S. Sundar, A. H. Fairlamb, Clin. Microbiol. Rev. 2006, 19,
111.
[9] S. Sundar, A. Singh, M. Rai, V. K. Prajapati, A. K. Singh, B. Ostyn, M.
Boelaert, J. C. Dujardin, J. Chakravarty, Clin. Infect. Dis. 2012, 55, 543.
[10] O. P. Singh, B. Singh, J. Chakravarty, S. Sundar, Infect. Dis. Poverty
2016, 5, 19.
[11] A. Alawieh, U. Musharrafieh, A. Jaber, A. Berry, N. Ghosn, A. R. Bizri,
Int. J. Infect. Dis. 2014, 29, 115.
[12] N. Salam, W. M. Al‐Shaqha, A. Azzi, PLOS Negl. Trop. Dis. 2014, 8,
e3208.
[13] A. R. Bello, B. Nare, D. Freedman, L. Hardy, S. Beverley, Proc. Natl.
Acad. Sci. U. S. A. 1994, 91, 11442.
[14] B. Nare, L. W. Hardy, S. M. Beverley, J. Biol. Chem. 1997, 272, 13883.
[15] T. J. Vickers, S. M. Beverley, Essays Biochem. 2011, 51, 63.
[16] H. L. Callahan, S. M. Beverley, J. Biol. Chem. 1992, 267, 24165.
[17] B. Papadopoulou, G. Roy, M. Ouellette, EMBO J. 1992, 11, 3601.
[18] D. G. Gourley, A. W. Schüttelkopf, G. A. Leonard, J. Luba, L. W. Hardy,
S. M. Beverley, W. N. Hunter, Nat. Struct. Biol. 2001, 8, 521.
[19] K. McLuskey, F. Gibellini, P. Carvalho, M. A. Averyc, W. N. Hunter,
Acta Crystallogr. Sect. D: Biol. 2004, D60, 1780.
ACKNOWLEDGMENTS
We would like to thank Prof. W. N. Hunter (University of Dundee,
UK) for the LmPTR1 construct and Pharmaceutical Sciences Research
Centre (FABAL) at Ege University Faculty of Pharmacy for spectral
analyses of the compounds and Molecular Modeling Software sup-
port, and Parasite Bank of Medical School of Manisa Celal Bayar
University for all support. The authors extend their appreciation to
the Scientific and Technological Research Council of Turkey (TUBI-
TAK), project no. SBAG‐213‐S‐026 and Ege University Science and
Technology Centre (EBILTEM), grant no. 2014BIL003.
[20] J. Drummelsmith, V. Brochu, I. Girard, N. Messier, M. Ouellette, Mol.
Cell. Proteomics 2003, 2, 146.
[21] A. Cavazzuti, G. Paglietti, W. N. Hunter, F. Gamarro, S. Piras, M.
Loriga, S. Allecca, P. Corona, K. McLuskey, L. Tulloch, F. Gibellini, S.
Ferrari, M. P. Costi, Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 1448.
[22] K. S. van Horn, X. Zhu, T. Pandharkar, S. Yang, B. Vesely, M.
Vanaerschot, J. C. Dujardin, S. Rijal, D. E. Kyle, M. Z. Wang, K. A.
Werbovetz, R. Manetsch, J. Med. Chem. 2014, 57, 5141.

14 of 14
ISTANBULLU ET AL.
|
[23] C. Mendoza‐Martínez, J. Correa‐Basurto, R. Nieto‐Meneses, A.
Márquez‐Navarro, R. Aguilar‐Suárez, M. D. Montero‐Cortes, B.
Nogueda‐Torres, E. Suárez‐Contreras, N. Galindo‐Sevilla, Á. Rojas‐
Rojas, A. Rodriguez‐Lezama, F. Hernández‐Luis, Eur. J. Med. Chem.
2015, 96, 296.
[45] T. A. Halgren, J. Comput. Chem. 1996, 17, 490.
[46] D. A. Case, T. A. Darden, T. E. Cheatham, C. L. Simmerling, J. Wang, R.
E. Duke, R. Luo, R. C. Walker, W. Zhang, K. M. Merz, B. P. Roberts, S.
Hayik, A. Roitberg, G. Seabra, J. Swails, A. W. Götz, I. Kolossváry, K. F.
Wong, F. Paesani, J. Vanicek, R. M. Wolf, J. Liu, X. Wu, S. R. Brozell, T.
Steinbrecher, H. Gohlke, Q. Cai, X. Ye, J. Wang, M.‐J. Hsieh, D. R. Roe,
D. H. Mathews, M. G. Seetin, R. Solomon‐Ferrer, C. Sagui, V. Babin, T.
Luchko, S. Gusarov, A. Kovalenko, P. A. Kollman, AMBER 12, Uni-
versity of California, San Francisco, CA 2012.
[24] A. H. Romero, N. Rodriguez, H. Oviedo, Bioorg. Chem. 2019, 83, 145.
[25] S. Ferrari, F. Morandi, D. Motiejunas, E. Nerini, S. Henrich, R. Luciani,
̀
A. Venturelli, S. Lazzari, S. Calo, S. Gupta, V. Hannaert, P. Michels, R.
C. Wade, M. P. Costi, J. Med. Chem. 2011, 54, 211.
[26] C. P. Mpamhanga, D. Spinks, L. B. Tulloch, E. J. Shanks, D. A.
Robinson, I. T. Collie, A. H. Fairlamb, P. G. Wyatt, J. A. Frearson, W. N.
Hunter, I. H. Gilbert, R. Brenk, J. Med. Chem. 2009, 52, 4454.
[27] A. I. Khalaf, J. K. Huggan, C. J. Suckling, C. L. Gibson, K. Stewart, F.
Giordani, M. P. Barrett, P. E. Wong, K. L. Barrack, W. N. Hunter,
J. Med. Chem. 2014, 57, 6479.
[47] A. Jakalian, B. L. Bush, D. B. Jack, J. Comput. Chem. 2000, 21, 132.
[48] W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, J. Chem. Phys. 1983,
79, 926.
[49] G. Jones, P. Willett, R. C. Glen, J. Mol. Biol. 1995, 245, 43.
[50] G. Jones, P. Willett, R. C. Glen, A. R. Leach, R. Taylor, J. Mol. Biol.
1997, 267, 727.
[28] Q. Dong, X. Gong, X., M. Hirose, B. Jin, F. Zhou, WO 2010/008847
A2, 2010.
[51] J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman, D. A. Case,
J. Comput. Chem. 2004, 25, 1157.
[29] N. Whittaker, J. Chem. Soc. 1951, 1565.
[52] V. Hornak, R. Abel, A. Okur, B. Strockbine, A. Roitberg, C. Simmerling,
Proteins: Struct., Funct., Bioinf. 2006, 65, 712.
[30] A. C. Schuemacher, R. W. Hoffmann, Synthesis 2001, 2, 243.
[31] H. E. Gottlieb, V. Kotlyar, A. Nudelman, J. Org. Chem. 1997, 62, 7512.
[32] G. R. Fulmer, A. J. M. Miller, N. H. Sherden, H. E. Gottlieb, A.
Nudelman, B. M. Stoltz, J. E. Bercaw, K. I. Goldberg, Organometallics
2010, 29, 2176.
[53] J. P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comput. Phys. 1977,
23, 327.
[54] U. Essmann, L. Perera, M. L. Berkowitz, T. Darden, H. Lee, L. G.
Pedersen, J. Chem. Phys. 1995, 103, 8577.
[33] B. Nare, J. Luba, L. W. Hardy, S. Beverley, Parasitology 1997, 114,
S101.
[55] Grace Development Team, http://plasma‐gate.weizmann.ac.il/Grace/.
Accessed October 5, 2019.
[34] L. Monzote, A. Lackova, K. Staniek, S. Steinbauer, G. Pichler, W. Jäger,
L. Gille, Parasitology 2017, 144, 747.
[56] D. R. Roe, T. E. Cheatham III, J. Chem. Theory Comput. 2013, 9, 3084.
[57] B. R. Miller III, T. D. McGee Jr., J. M. Swails, N. Homeyer, H. Gohlke,
A. E. Roitberg, J. Chem. Theory Comput. 2012, 8, 3314.
[58] E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M.
Greenblatt, E. C. Meng, T. E. Ferrin, J. Comput. Chem. 2004, 13,
1605.
[35] A. A. Dar, M. Shadab, S. Khan, N. Ali, A. T. Khan, J. Org. Chem. 2016,
81, 3149.
[36] M. Machado, P. Pires, A. M. Dinis, M. Santos‐Rosa, V. Alves, L. Sal-
gueiro, C. Cavaleiro, M. C. Sousa, Exp. Parasitol. 2012, 130, 223.
[37] L. W. Hardy, W. Matthews, B. Nare, S. M. Beverley, Exp. Parasitol.
1997, 87, 157.
[38] I. Ostan, H. Saglam, M. E. Limoncu, H. Ertabaklar, S. O. Toz, Y. Ozbel,
A. Ozbilgin, New Microbiol. 2007, 30, 439.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
[39] A. Ozbilgin, C. Durmuskahya, H. Kayalar, H. Ertabaklar, C. Gunduz, I.
Ural, F. Zeyrek, O. Kurt, I. Cavus, S. O. Toz, Y. Ozbel, Trop. J. Pharm.
Res. 2014, 13, 2047.
[40] E. Polat, H. Cakan, M. Aslan, S. Sirekbasan, Z. Kutlubay, T. Ipek, A.
Ozbilgin, Exp. Parasitol. 2012, 132, 129.
How to cite this article: Istanbullu H, Bayraktar G, Akbaba H,
et al. Design, synthesis, and in vitro biological evaluation of
novel thiazolopyrimidine derivatives as antileishmanial
compounds. Arch Pharm. 2020;e1900325.
[41] M. E. Limoncu, B. Erac, T. Gurpinar, A. Ozbilgin, C. Balcioglu, M.
Hosgor‐Limoncu, Turkiye. Parazitol. Derg. 2013, 37, 1.
[42] C. Williams, O. A. Espinosa, H. Montenegro, L. Cubilla, T. L. Capson, E.
Ortega‐Barria, L. I. Romero, J. Microbiol. Methods 2003, 55, 813.
[43] C. Marín, I. Ramírez‐Macías, M. J. Rosales, B. Muro, F. Reviriego, P.
Navarro, V. J. Arán, M. Sánchez‐Moreno, Acta Trop. 2015, 148, 170.
[44] Molecular Operating Environment (MOE) Chemical Computing
Group Inc., Montreal, Canada 2014.
https://doi.org/10.1002/ardp.201900325