Design, synthesis, neuroprotective, antibacterial activities and docking studies of novel thieno[2,3-d]pyrimidine-alkyne Mannich base and oxadiazole hybrids

Article abstract of DOI:10.1016/j.bmcl.2018.03.030

A series of thieno[2,3-d]pyrimidine alkyne Mannich base derivatives (7a-e, 8a-e) and thieno[2,3-d]pyrimidine 1,3,4-oxadiazole derivatives (9a-e, 10a-e) have been synthesized and evaluated for their neuroprotective and neurotoxicity activities where 9a, 10d displayed good neuroprotection 10.6 and 11.88 μg/mL respectively against the H₂O₂ induced cell death at the EC₅₀ values and 9b, 9d showed respective toxic effects on PC12 cells at CC₅₀ 86.12 and 94.16 μg/mL. Compounds 9a, 9e, 10a and 10b showed strong antibacterial activity against two gram positive (S. aureus, B. subtilis) and two gram-negative strains (E. coli, P. aeruginosa) and showed good binding affinities with C(30) carotenoid dehydrosqualene synthase, Gyrase A and LpxC. This is the first report for the demonstration of thieno[2,3-d] pyrimidine derivatives as promising neuroprotective agents against H₂O₂ induced neurotoxicity on PC12 cells.

Full text of DOI:10.1016/j.bmcl.2018.03.030

Accepted Manuscript
Design, synthesis, neuroprotective, antibacterial activities and docking studies
of novel thieno[2,3-d]pyrimidine-alkyne Mannich base and oxadiazole hybrids
Settypalli Triloknadh, Chunduri Venkata Rao, Kerru Nagaraju, Nallapaneni
Hari Krishna, Chintha Venkata Ramaiah, Wudayagiri Rajendra, Daggupati
Trinath, Yeguvapalli Suneetha
PII:
S0960-894X(18)30219-1
https://doi.org/10.1016/j.bmcl.2018.03.030
BMCL 25684
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 September 2017
5 March 2018
12 March 2018
Please cite this article as: Triloknadh, S., Rao, C.V., Nagaraju, K., Hari Krishna, N., Ramaiah, C.V., Rajendra, W.,
Trinath, D., Suneetha, Y., Design, synthesis, neuroprotective, antibacterial activities and docking studies of novel
thieno[2,3-d]pyrimidine-alkyne Mannich base and oxadiazole hybrids, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.030
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Design, synthesis, neuroprotective, antibacterial activities and docking studies
of novel thieno[2,3-d]pyrimidine-alkyne Mannich base and oxadiazole hybrids
Settypalli Triloknadha, Chunduri Venkata Rao*a, Kerru Nagarajua, Nallapaneni Hari Krishnaa,
Chintha Venkata Ramaiahb, Wudayagiri Rajendrab, Daggupati Trinathb, Yeguvapalli Suneethab
aDepartment of Chemistry, bDepartment of Zoology, Sri Venkateswara University, Tirupati-
517502, Andhra Pradesh, India.
* To whom correspondence should be addressed.
Email : cvrsvu@gmail.com
Tel: +91 9849605140
Fax: +918772289555
1

Abstract
A series of thieno[2,3-d]pyrimidine alkyne Mannich base derivatives (7a-e, 8a-e) and thieno[2,3-
d]pyrimidine 1,3,4-oxadiazole derivatives (9a-e, 10a-e) have been synthesized and evaluated for
their neuroprotective and neurotoxicity activities where 9a, 10d displayed good neuroprotection
10.6 and 11.88 µg/mL respectively against the H2O2 induced cell death at the EC50 values and
9b, 9d showed respective toxic effects on PC12 cells at CC50 86.12 and 94.16 µg/mL.
Compounds 9a, 9e, 10a and 10b showed strong antibacterial activity against two gram positive
(S.aureus, B.subtilis) and two gram-negative strains (E.coli, P.aeruginosa) and showed good
binding affinities with C(30) carotenoid dehydrosqualene synthase, Gyrase A and LpxC. This is
the first report for the demonstration of thieno[2,3-d] pyrimidine derivatives as promising
neuroprotective agents against H2O2 induced neurotoxicity on PC12 cells.
Keywords: Thieno[2,3-d]pyrimidine; Alkyne Mannich base; 1,3,4-oxadiazole; Neuroprotective,
Neurotoxicity; Antibacterial activity; Molecular Docking.
Neuroprotection refers to the relative preservation of neuronal structure or function.
Neuroprotection aims to prevent or slow the disease progression and secondary injuries by
halting or dawdling the loss of neurons.1 It is the widely explored treatment option for various
central nervous system (CNS) disorders, including neurodegenerative diseases i.e., Alzheimer's
disease (AD), Parkinson's disease (PD), Huntington’s disease (HD), Amyotrophic lateral
sclerosis (ALS) stroke and trauma.2 Neurons include the brain and spinal cord which are the
building blocks of the nervous system. Neurons usually don’t reproduce or restore themselves, so
when they become injured or die they can’t be replaced by the body. Further, neurodegenerative
diseases result from the gradual and progressive loss of neural cells. These affect millions of
people worldwide, Alzheimer’s disease and Parkinson’s disease are most common types.
Therefore neurodegenerative diseases are not curable at debilitating conditions that result in
progressive degeneration or death of nerve cells. Meanwhile these neurodegenerative disorders
involve various unknown pathogenic mechanisms i.e. apoptosis, inflammation, oxidative stress,
mitochondrial dysfunction and oxidative stress induced by overproduction of reactive oxygen
species (ROS) leading to injury of neural cells.3 Therefore there is an urgent need for
development of potent neuroprotective compounds for the treatment of stroke as well as
neurodegenerative disorders.
2

Further for the past few decades the prevalence of MDR microbial infections have been
rising dramatically. It has become a big task for present chemists to develop potent
antimicrobials. However continuous use of antibiotics has unfortunately helped the emergence of
bacterial resistance and also mostly all the bacteria have developed resistance mechanism against
most antibiotics. Hence there is an urgent need for development of new antimicrobial agents with
potent activity against MDR bacterial pathogens4-6 and that is why neuroprotective and
antimicrobial agents are very important in present situation.
Based on the above significance, the potent neuroprotective, anti bacterial compounds are
searched and identified a few efficient compounds among all chemical classes (Fig. 1), such as
LOXblock-1 (I) is a fused thiophene inhibitor of lipoxygenase protects both HT22 cells from
oxidative glutamate toxicity, oligodendroglial cells from oxidative stress induced cell death7 and
tirilazad mesylate (U-74006F) (II) a steroid compound which exhibits neuroprotective activity in
several models of central nervous system (CNS).8 Further 2,4-diaminopyrrolo[2,3-d]pyrimidines
(U-87663) showed similarly promising pharmacological profile9 and lipoxygenase inhibitors
exhibited potent neuroprotective activities.10,11 G. Rai & V. Kenyon et al have screened 74,000
small molecules in search of human reticulocyte 15-hLO-1 inhibitors through a quantitative high
throughput (qHTS) and selected 1,3,4-oxadiazole-2-thiol chemotype (III) for optimization and
synthesized superior molecules than III.12 M. Kitabatake et al have synthesized 1-alkyl and 1-
alkyl–N-propargyl-1,2,3,4-tetrahydroisoquinoline (TIQ) (IV)13 derivatives and estimated
cytotoxicity and protective effects towards MPP+ mediated PC12 cells death in which N-
propargyl functional group played a crucial role for the effective reduction of cytotoxicity. Some
aryloxy acetamide derivatives (V)14 have also showed potential protection from glutamate-
induced neurotoxicity in PC12 cells. Furthermore pyrimidines are the basic building scaffolds of
DNA and RNA-uracil, thymine, cytosine is probably a reason for diversity of their biological
activities and thienopyrimidines are important class of heterocyclic compounds which show
similarity to the biogenic pyrimidines and show broad spectrum of biological activities like anti-
inflammatory,15,16
antimalarial,17,18
antimicrobial
(VI),19,20-22
antiproliferative,23,24
antiprotozoal,25,26 antitumor,27 adenosine A2A receptor antagonists,28 antituberculosis/
leishmanicidal/cytotoxic etc.29
In addition 1,3,4-oxadiazoles attracted interest in medicinal chemistry, pesticide
chemistry, polymer science. In the recent past considerable evidences have been gathered to
demonstrate the efficacy of 1,3,4 oxadiazoles as anticonvulsant,30,31 antimalarial/
3

antitubercular,32,33 antimicrobial,34-37 antiproliferative,38 antiviral,39 anti-inflammatory and
analgesic,40,41 anti-HIV-142 and ulcerogenic43 and also the oxadiazole derivative furamizole (VII)
has strong antibacterial activity.44
1,3,4 Oxadiazole-2-thiol chemo
LOXblock-1 (I)
Tirilazad Mesylate U-74006F (II)
type (III)
1-alkyl TIQ (IV)
Aryloxy acetamide derivative (V)
4-aminothienopyrimidines (VI)
Furamizole (VII)
Our target compounds
Fig. 1. Reported neuroprotective and antibacterial compounds and our target compounds.
Inspired by the literature survey, twenty novel targets were designed (Fig. 2) and synthesized by
incorporating the core units which were present in the potent neuroprotective and antibacterial
active compounds and evaluated their activities.
4

Fig.2. Designing strategy for target compounds.
The general synthetic route for the preparation of target compounds was depicted in
Scheme 1, in which ethyl 5-amino-4-cyano-3-methylthiophene-2-carboxylate 1 was obtained by
reaction of ethyl acetoacetate and sulphur with malononitrile, in the presence of triethylamine
according to well known Gewald reaction.45 Cyclization of 1 with formic acid under reflux
conditions at 120 ºC yielded ethyl 5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxylate 2. 2 on treatment with POCl3 formed ethyl 4-chloro-5-methylthieno[2,3-d]
pyrimidine-6-carboxylate 3 which on further reacted with morpholine/piperidine in the presence
of triethylamine under reflux condition resulted 4-morpholine/piperidine substituted ethyl
thieno[2,3-d]pyrimidine-6-carboxylate 4a-b in excellent yields. Next the ester groups were
hydrolyzed in 4a-b by NaOH/MeOH(1:1) to form the corresponding thieno[2,3-d]pyrimidine-6-
carboxylic acids 5a-b which on treatment with propargyl bromide in DMF using K2CO3 for 12 h
at rt to yield 4-morpholine/piperidine substituted prop-2-yn-1-yl thieno[2,3-d]pyrimidine-6-
carboxylates 6a-b. Final target compounds 7a-e and 8a-e were obtained by refluxing 6a-b with
formaldehyde and various phenyl substituted piperazines at 110 ºC in 1,4 dioxane in the presence
of Cu(I)Cl (cat). Thieno[2,3-d] pyrimidine-1,3,4 oxadiazole hybrids 9a-e & 10a-e were obtained
in good yields from 5a-b on treatment with substituted aryl acid hydrazides in POCl3. The
13
structures of synthesized compounds were established on the basis of 1H-NMR, C-NMR, LC-
MS and IR spectroscopy analysis (see the Supporting Information Pg. No. 22−102).
5

Scheme 1. Synthesis of thieno[2,3-d]pyrimidine-alkyne Mannich base and oxadiazole hybrids
7a-e, 8a-e, 9a-e & 10a-e.
Spectral studies of compound 1 showed -ve molecular ion peak at m/z = 209 (M-H)− in
its mass and a broad singlet at δ 5.19 ppm; a singlet at δ 2.51 ppm corresponding to -NH2, -CH3
groups respectively and ethyl group quartet and triplet at δ 4.26 ppm, δ 1.33 ppm from NMR
spectra. IR of 2 showed two strong bands at 3386 cm−1, 1685 cm−1 corresponding to −NH, −C=O
groups stretching frequencies respectively and the two singlets at δ 12.60 ppm & δ 8.19 ppm
1
corresponding to −CONH-, −C2-H protons and the absence −CONH-, signal in H-NMR
evidences the formation of 3. 4a-b 1H-NMR signals at δ 3.87 ppm & δ 3.54 ppm 4a / δ 3.51 ppm
13
& δ 1.68 ppm 4b and C-NMR peaks at δ 66.51 ppm & 50.93 ppm 4a / δ 50.80 ppm, δ 25.10
ppm, & δ 23.58 ppm 4b corroborated to the morpholine & piperidine rings. IR absorptions at
2922−2857 cm−1, 1697–1703 cm−1and 1360–1362 cm−1 respectively indicate the presence of
−CH, −C=O & −C−N groups in 4a-b. The 4a (M+H)+ peak at m/z = 308 found to be in
6

agreement with the formula C14H17N3O3S. IR absorption bands at 3488 & 3482 for −OH and
1
absence of −OCH2CH3 group signals in the H & 13C-NMR spectrum confirms 5a & 5b
structure. Propargyl group in 6a was established by the appearance of 1H & 13C -NMR signals at
δ 4.96, δ 2.56 ppm and δ 52.59, δ 75.40, δ 77.23 ppm respectively for −OCH2- and −C≡CH
groups. 6a showed stretching frequencies for −C≡CH, −C≡C- at 3174 cm−1 and 2308 cm−1
respectively. Further positive molecular ion peak at m/z = 318 (M+H)+ confirmed structure of
1
6a. In H & 13C-NMR spectra the appearance of signals for −NCH2-, N-phenyl substituted
piperazine, phenyl group and lack of alkyne proton signals in the Mannich reaction based
compounds supported 7a-e & 8a-e. The 7d & 8d cyclopropyl ring protons and carbon signals
appeared at δ 0.73–1.88 ppm and δ 10.29–16.13 ppm respectively. [(M+2)+H]+, (M+H)+ 1:1
intensity ratio peaks at 597 & 595 and 595 & 593 respectively in the LCMS spectra showed the
presence of one bromine atom in 7e & 8e and for 7d at m/z = 557 (M+H)+. 7c, 7d, 7e, 8c, 8d, 8e
IR bands were in the range of 2216–2220 cm−1 characteristic for −C≡N group and 9b-e, 10b-e
had A2B2 pattern 1H NMR signals at δ 7.04-8.15 ppm, evidenced for para disubstituted nature of
phenyl group. The oxadiazole ring carbons’ signals of 9a-e, 10a-e indicated the formation of
various 2,5-disubstituted oxadiazole ring. 9e, 10e IR spectra showed absorption band at 731–733
cm−1 indicating the presence of C-Cl group and further supported by [(M+2)+H]+, (M+H)+ peaks
at 416, 414 for 9e and 414, 412 for 10e, 380 for 9a and 378 for 10a which were in good
agreement with the molecular formula of the respective compounds.
Prior to neuroprotective studies, we have evaluated the cytotoxic nature of the
synthesized compounds by determining the maximum non-toxic concentrations (MNTC) and
cytotoxic concentrations (CC50) using PC12 cell lines in a dose dependent manner (Table 1). At
different concentrations, all of the compounds were showed cytotoxic effects on cultured PC12
cells.
7

Table 1 Neuroprotection (EC50), Cytotoxicity (CC50) activities of the synthesized compounds (7a-e, 8a-e, 9a-e & 10a-e).
b
c
Compou
nds
Substituents
R1 R2
MNTCa
% CV ± SD Conc.
µg/mL
CC50
EC50
X
R
R3
R4
Conc.
µg/mL
% CV ± SD
Conc.
µg/mL
% CV ± SD
-O-
-O-
-O-
-O-
-O-
-CH2- -H
-CH2- -H
-CH2- -Me
-H
-H
-OMe -H
-H
-
-
-
-
-
-
-
-
60
98.14 ± 0.04 144.16 49.11 ± 0.17
98.11 ± 0.01 160.88 49.98 ± 0.02
98.46 ± 0.01 146.18 49.11 ± 0.02
99.10 ± 0.07 198.18 49.88 ± 0.02
99.64 ± 0.03 122.14 48.98 ± 0.02
99.44 ± 0.04 140.02 49.36 ± 0.21
98.18 ± 0.12 144.48 51.02 ± 0.02
98.18 ± 0.12 114.44 48.97 ± 0.07
99.64 ± 0.02 148.14 50.06 ± 0.02
99.11 ± 0.07 136.36 51.17 ± 0.21
99.50 ± 0.04 200.01 50.40 ± 0.01
36.16
38.38
41.32
12.44
32.28
35.28
40.44
38.16
16.18
40.86
10.60
26.60
28.18
16.18
28.16
22.12
28.18
20.14
11.88
21.82
74.89 ± 2.2
76.78 ± 3.1
74.98 ± 2.7
75.15 ± 1.2
75.44 ± 2.7
76.16 ± 2.3
75.08 ± 3.2
76.04 ± 2.1
75.14 ± 2.1
76.18 ± 2.7
76.18 ± 1.5
74.20 ± 1.2
75.15 ± 2.1
75.11 ± 2.1
77.18 ± 1.4
76.88 ± 3.1
74.98 ± 2.7
75.12 ± 3.1
74.78 ± 2.1
75.04 ± 2.4
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
-H
-H
-OMe -H
114
118
150
100
80
96
96
110
96
160
68
-Me
-C3H5 -H
-H -Br
-H
-H
-H
-CN
-CN
-CN
-H
-OMe -H
-H
-H
-OMe -H
-H
-H
-H
-
-
-
-
-
-
-
-CN
-CN
-CN
-CH2- -C3H5 -H
-CH2- -H
-
-
-Br
-O-
-O-
-O-
-O-
-O-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-H
-CH3
-OMe 120
-NO2
-Cl
-H
-CH3
-OMe 100
-NO2
-Cl
97.60 ± 0.03
98.28 ± 0.01 140.14 51.86 ± 0.08
99.18 ± 0.06 49.18 ± 0.07
49.18 ± 0.06
86.12
80
9d
9e
94.16
120
100
110
98.28 ± 0.01 168.16 51.08 ± 0.04
99.18 ± 0.07 146.18 54.14 ± 0.06
99.50 ± 0.06 160.86 49.18 ± 0.06
98.98 ± 0.03 124.14 49.88 ± 0.12
99.01 ± 0.06 144.86 49.88 ± 0.05
98.18 ± 0.11 150.88 48.88 ± 0.14
-CH2- -
10a
10b
10c
10d
10e
-CH2-
-CH2-
-CH2-
-CH2-
-
-
-
-
-
-
-
125
136
aMaximum NonToxic Concentration-it is a maximum concentration of compound required to show up to 100% of PC12 cell viability.
bCytotoxic concentration: It is a concentration of compound required to show 50% cell death on PC12 cells.
cEffective concentration: It is a concentration of compound which protects 75% of PC12 cells from H2O2-induced cell death.
8

In the target compounds by change of the substituents on the C4-position of the
thienopyrimidine ring and terminal phenyl rings showed notable effects on the CC50 values. The
compounds 9b, 9d (X = -O-, Morpholino) have shown effective CC50 value 86.12, 94.16 µg/mL
respectively. This could be due to the presence of R4 = -Me, -NO2 groups on the phenyl ring of
1,3,4-oxadiazole moiety. Whereas 7d (X = -O-, R3 = -CN, R = -C3H5), 9a (X = -O-, R4 = -H)
exhibited highest cytotoxic concentrations 198.18, 200 µg/mL. Subsequently, 7b (R2 = -OMe), 9e
(R4 = -Cl), 10b (R4 = -CH3) exhibited lower CC50 values compared with 7d, 9a. The left over
compounds 7a, 7c, 8a, 8b, 8d, 9c, 10a, 10d & 7e, 8c, 10c were displayed cytotoxic concentrations
(CC50) at the range of 140-148.14 µg/mL & 114.14-124.14 µg/mL respectively, this result indicated
that substitution effect is very less on the cytotoxic concentrations of those compounds.
To examine the neuroprotective effects of synthesized compounds against H2O2 induced cell
death, different concentrations of synthesized compounds were incubated with PC12 cells before
H2O2 treatment. Cell viability was assessed by MTT assay and the obtained EC50 values were
summarized in Table 1. As shown in Fig. 3, compared with normal control group, the viability of
the cells exposed to H2O2 treatment was reduced to 50 %. Meanwhile, pretreatment with
synthesized compounds significantly reduced the H2O2 induced cell apoptosis in a dose dependent
manner and the viability of these cells was considerably increased to 74.20-77.18 % at the dose
range of 10.60-41.32 µg/mL. Among the twenty novel compounds, 9a (X = -O-, Morpholino, R4 = -
H), 10d (X = -CH2, Piperidinyl, R4 = -NO2) showed good neuroprotection from the H2O2 induced
cell death at the lowest EC50 of 10.60, 11.88 µg/mL respectively. 7d (X = -O-, Morpholino) with 2-
cyano-5-isopropyl substitutions (R3= -CN, R= -C3H5) on the terminal phenyl ring, showed good
neuroprotection with EC50 value 12.44 µg/mL. While the introduction of R1 = -OMe, R2 = -OMe,
(R3 = -CN, R = -Me) and (R3 = -CN, R1 = -Br) groups in 7a-c & 7e led to a 3-fold increment in the
EC50 values appeared at 36.16, 38.38, 41.32 and 32.28 µg/mL respectively, which indicated that
instead of cyano, isopropyl groups any other substitutions were not tolerated in this region for good
activity. On the other hand, due to the presence of R3 = -CN, R = -C3H5 substitutions on the phenyl
ring of piperazine compound 8d (X = -CH2-) showed lower EC50 value of 16.18 µg/mL. But
compounds 8a (R1 = -OMe), 8b (R2 = -OMe), 8c (R3 = -CN, R = -Me) & 8e (R3 = -CN, R1 = -Br)
displayed respective higher effective concentrations 35.28, 40.44, 38.16 & 40.86 µg/mL than 8d. 9d
having R4 = -NO2 group showed EC50 value at 16.18 µg/mL. R4 = -Me, -OMe, -Cl substituents were
exhibited negligible effects on the EC50 of 9b (26.6 µg/mL), 9c (28.18 µg/mL), 9e (28.16 µg/mL),
9

10b (28.18 µg/mL). Moreover, 10a (22.12 µg/mL), 10c (20.14 µg/mL), 10e (21.82 µg/mL) have
lower EC50 values than 9b, 9c, 9e, 10b.
The overall observations indicated that 9a, 10d displayed good neuroprotective activity at
the lowest EC50 values of 10.60, 11.88 µg/mL. However 9b, 9d showed highest cytotoxic effects on
PC12 cell lines at the lowest CC50 of 86.12, 94.16µg/mL. 9a, 7d exhibited uppermost MNTC values
at 160, 150 µg/mL. But 9b, 9d showed lower MNTC values 68, 80 µg/mL with low CC50 values at
86.12, 94.16 µg/mL. Concerning to neuroprotective activity, the representative PC12 cell images
obtained by the presence or absence of active compounds (9a, 10d) were shown in Fig. 3.
Fig. 3. Representative images of PC12 cells after treatment with 200µM H2O2 in the absence or
presence of compound 9a & 10d. A) Control cells without 200µM H2O2 treatment. B) 200µM H2O2
alone induced neurotoxicity. C) Co-incubation of compound 9a (10.60 µg/mL) and 200µM H2O2.
D) Co-incubation of compound 10d (11.88 µg/mL) and 200 µM H2O2.
Further, all of the synthesized compounds were screened for their antibacterial activity
against the four bacterial strains such as S.aureus, B.subtilis, E.coli and P.aeruginosa on par with
reference drug gentamicin using agar well diffusion method as well as 96-well plate dilution
method. The zone of inhibition (ZOI) & minimum inhibitory concentration (MIC) values of these
compounds against the above four bacterial strains were reported in Table 2.
10

Most of the compounds have exhibited potent antibacterial activity against the above
bacterial strains. Among the target compounds, the most potent antibacterial activity was achieved
for compounds 9a, 9e, 10a and 10b with MIC values ranging from 0.0125 µg/mL to 0.0412 µg/mL
against all bacterial strains, which were better than that of gentamicin (MIC = 0.0212 to 0.0422
µg/mL). 7b (X = -O-, Morpholino) showed potent antibacterial activity against the three bacterial
strains of S.aureus, B.subtilis, P.aeruginosa with the MIC values of 0.0311, 0.0120, 0.0124 µg/mL
and 0.5010 µg/mL with E.coli may be due to the presence of R2 = -OMe substitution on the phenyl
ring of piperizine. However, moving the –OMe group from 3-position to 4-position (7a) led to
decrease in the bacterial activity against all strains. Moreover (R3 = -CN, R= -Me) and (R3 = -CN,
R1 = -Br) analogues (7c, 7e) exhibited specific activity against the B.subtilis (MIC = 0.0156
µg/mL), P.aeruginosa (MIC = 0.0128 µg/mL) than compared with reference drug gentamicin and
7d (R3 = -CN, R = -C3H5) had the low activity against four bacterial strains. Compounds 8a (R1 = -
OMe), 8b (R2 = -OMe) & 8d (R3 = -CN, R = -C3H5) exhibited good activity against the B.subtilis
(8a), S.aureus (8b, 8d) with the MIC 0.0158, 0.0244, 0.0422 µg/mL. 8c (B.subtilis: MIC = 0.0312
µg/mL, E.coli: MIC = 0.0144 µg/mL) and 8e (S.aureus: 0.0124 µg/mL, B.subtilis: 0.0186 µg/mL,
P.aeruginosa: 0.0112 µg/mL) showed potent antibacterial activity against the two and three
bacterial strains respectively. This result may be due to the presence of (R3 = -CN, R= -Me) and (R3
= -CN, R1 = -Br) groups on the phenyl ring. Electron donating group such as (R4 = -Me) analogue
9b exhibited good activity against E.coli (MIC = 0.0132 µg/mL), but it is less active compared to
no substituent on the phenyl ring 9a (R4 = -H) and cyano, amino substituent on the phenyl ring 9c
(R3 = -CN, R= -Me). 9d having R4 = -NO2 substituent on the phenyl ring of oxadiazole exhibited
good activity against B.subtilis. Electron releasing group R4 = -OMe in 10c (active with S.aureus at
0.0366 µg/mL) and electron withdrawing group -NO2 group in 10d (active with S.aureus at 0.0440
µg/mL) did not showed noticeable influence on the bacterial activity. 4-Cl substituted 10e showed
potent antibacterial activity against S.aureus, B.subtilis at 0.0224, 0.0284 µg/mL, but showed very
less activity with E.coli, P.aeruginosa. and 10c was active against the one strain. On the whole
thienopyrimidine-1,3,4-oxadiazole hybrids showed potent antibacterial activity than
thienopyrimidine-alkyne Mannich base hybrids.
11

Table 2 Antibacterial activity of synthesized compounds (7a-e, 8a-e, 9a-e & 10a-e)
Zone of Inhibition (mm)
Minimum inhibitory concentration (MIC)a in
µg/mL
Bacterial strains
Bacterial strains
Compounds
Gram-positive
S.aureus B.subtilis
Gram-negative
Gram-positive
Gram-negative
E.coli P.aeruginosa S.aureus B.subtilis E.coli P.aeruginosa
8 ± 0.060 12 ± 0.120 12 ± 0.010 14 ± 0.030
12 ± 0.030 16 ± 0.012 14 ± 0.012 14 ± 0.001
7 ± 0.051 13 ± 0.125 13 ± 0.011 14 ± 0.021
13 ± 0.011 10 ± 0.012 12 ± 0.015 13 ± 0.020
10 ± 0.060 12 ± 0.015 13 ± 0.062 12 ± 0.012
13 ± 0.031 11 ± 0.031 11 ± 0.012 10 ± 0.021
10 ± 0.012 13 ± 0.012 13 ± 0.030 14 ± 0.012
13 ± 0.031 13 ± 0.020 15 ± 0.041 14 ± 0.024
14 ± 0.036 16 ± 0.001 17 ± 0.001 17 ± 0.024
13 ± 0.016 16 ± 0.026 14 ± 0.031 15 ± 0.004
16 ± 0.034 12 ± 0.067 16 ± 0.026 11 ± 0.261
18 ± 0.211 15 ± 0.321 18 ± 0.021 14 ± 0.011
12 ± 0.101 13 ± 0.124 12 ± 0.122 16 ± 0.020
16 ± 0.012 13 ± 0.041 16 ± 0.061 16 ± 0.020
13 ± 0.050 18 ± 0.020 13 ± 0.061 18 ± 0.012
11 ± 0.062 14 ± 0.013 10 ± 0.011 13 ± 0.012
14 ± 0.031 17 ± 0.021 11 ± 0.051 12 ± 0.004
10 ± 0.026 14 ± 0.026 17 ± 0.028 12 ± 0.032
8 ± 0.0281 18 ± 0.032 14 ± 0.034 17 ± 0.036
16 ± 0.011 13 ± 0.144 15 ± 0.012 14 ± 0.008
0.0620
0.0311
0.0620
0.3120
0.5260
0.3126
0.0244
0.1221
0.0422
0.0124
0.0412
0.0546
0.0132
0.0460
0.0136
0.0125
0.0404
0.0366
0.0440
0.0224
0.0422
0.0620
0.0120
0.0156
0.1240
0.0628
0.0158
0.5210
0.0312
0.0581
0.0186
0.0366
0.0464
0.0112
0.0381
0.0144
0.0128
0.0142
0.0642
0.0641
0.0284
0.0386
0.1200
0.5010
0.0324
0.1860
0.0324
0.6224
0.0384
0.0144
0.5211
0.0244
0.0211
0.0132
0.0120
0.0382
0.0172
0.0132
0.0138
0.0486
0.0361
0.1861
0.0212
0.0620
0.0124
0.0624
0.0634
0.0128
0.0614
0.0842
0.0624
0.0686
0.0112
0.0186
0.0442
0.0440
0.0266
0.0144
0.0144
0.0201
0.0448
0.0382
0.1441
0.0242
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
10a
10b
10c
10d
10e
17
16
11
13
Gentamicin
aMIC-it is lowest concentration of compounds required to inhibit the visible growth of a microorganism.
12

To understanding the SAR deeply, we examined the effect of C4-substituent on the cytotoxic,
neuroprotective and antibacterial activities by changing of morpholino, piperidinyl groups. As
shown in Table 1, the results indicated that the introduction of six-membered cyclic secondary
amino groups had a good influence on activity. For example, 9b (X = -O-, Morpholino) exhibited
higher cytotoxic effects on PC12 cells than 10b (X = -CH2-, Piperidinyl) but both are electron
donating group such as R4 = -Me analogues. In no substituent analogues (9a, 10a R4 = -H),
compound 9a (X = -O-, Morpholino) showed good neuroprotection when compared to 10a (X = -
CH2-, Piperidinyl). Subsequently, 10b is good antibacterial than 9b.
In the SARs studies, it was observed that thienopyrimidine-oxadiazole molecular hybrids
showed good neuroprotection and antibacterial activities than the thienopyrimidine-alkyne Mannich
bases due to the amino groups on the C4-position of the thienopyrimidine ring and substituents on
terminal phenyl ring of oxadiazole. Either morpholine or piperidine rings on the C4 position of
thienopyrimidine and phenyl and p-NO2 phenyl on the oxadiazole ring showed good
neuroprotection. The substituents like -CH3, -NO2 groups and morpholine increased the toxicity of
the molecules towards the PC12 cell lines which were only responsible for low MNTC values for
9b & 9d. SARs shown in Fig. 4(B), states that morpholine, piperidine rings and -H, -CH3, -OCH3, -
Cl groups in side phenyl ring enhance the antibacterial activities. These detailed data was
summarized in Fig. 4 (see the Supporting Information).
In order to strengthen the in vitro antibacterial experimental results docking predictions
were performed on the compounds 7a-e, 8a-e, 9a-e and 10a-e with target proteins of C(30)
carotenoid dehydrosqualene synthase of S.aureus (PDB Id: 3ACX), Gyrase A of E.coli (PDB Id:
1AB4), LpxC of P.aeruginosa (PDB Id: 5U39). The binding energies (∆Gb) and inhibition constant
(Ki) values due to the interaction of ligands with the active site of proteins are shown in Table 3
(see the Supporting Information). The active molecules have showed significant interactions with
the active sites of the three proteins and shown in 3D pictures Figs. 5, 6 & 7. (see the Supporting
Information for Figs. 6 & 7).
Significant binding energies and inhibition constants of the active molecules against the
active sites of three proteins (Dehydrosqualene synthase, Gyrase A, LpxC) indicated high affinity of
the molecules to bind to the protein leading to its inhibition, which causes cell cycle arrest finally
leads to death of bacteria. All of the compounds displayed reasonable free energies and inhibition
constants. Against S.aureus (PDB Id: 3ACX) 9a-e & 10a-e series had highest binding affinity [∆Gb
= −9.14 to −10.18 Kcal/mol] and lowest inhibition [Ki = 0.20 to 0.04] than 7a-e & 8a-e series ∆Gb
13

= −3.31 to −8.48 Kcal/mol and Ki = 3770.00 to 0.60; with E.coli (PDB Id: 1AB4) they also showed
similar trend of highest binding affinity [∆Gb = −6.86 to −7.93 Kcal/mol] and lowest inhibition [Ki
= 9.34 to 1.53] than to 7a-e & 8a-e ∆Gb = −2.74 to −4.79 Kcal/mol and Ki = 9770.00 to 307.32. By
means of P.aeruginosa (PDB Id: 5U39)] 9a-e & 10a-e and 7a-e & 8a-e exhibited binding affinities
as shown ∆Gb = −6.45 to −7.95 Kcal/mol and ∆Gb = −6.18 to −8.61 Kcal/mol respectively with
their respective inhibition shown as Ki = 18.68 to 1.48 and Ki = 29.66 to 0.49.
These results indicate that 9a-e & 10a-e possess higher affinity to bind the active site of
protein and they are in good agreement with the in vitro experimental results. Because, compounds
9a, 9e, 10a and 10b exhibited potent bacterial activity against the four tested bacterial strains. The
3D docking images (Fig. 5, 6 & 7) of ligand-protein interactions of four active compounds 9a, 9e,
10a, 10b showed good binding affinities, inhibition constants with three select proteins. The active
molecules interacted with the active sites of proteins through conventional hydrogen bonds and
hydrophobic interactions.
S.aureus (PDB Id: 3ACX), protein displayed binding affinities with ligands 9a, 9e 10a, 10b
at −9.18, −9.83, −9.52, −10.09 Kcal/mol respectively. 9a [Fig. 5(A)] has formed three hydrogen
bonds with amino acid residues. N-4 nitrogen atom of 1,3,4-oxadiazole ring in 9a act as H-bond
+
acceptor and formed one H-bond with –NH3 hydrogen atom of Gly 138 with a bond length of 2.8
Å. Another H-bond acceptor N-1 nitrogen of thieno[2,3-d]pyrimidine has formed two H-bonds with
+
-OH, -NH3 hydrogen donors of Tyr 248, Asn 168 having the bond length of 3.2 Å, 3.4 Å
respectively. In the same way compound 9e [Fig. 5(B)] has formed three H-bond interactions with
the active site. N-4, N-1 nitrogen atoms of 1,3,4-oxadiazole, thieno[2,3-d]pyrimidine rings formed
three H-bonds with Gly 138, Asn 168, Tyr 248 with bond length of 2.8 Å, 2.9 Å, 3.2 Å respectively.
Further 10a [Fig. 5(C)] involved in three H-bond interactions with Gly 138 (2.8 Å), Asn 168 (3.4
+
Å), Tyr 248 (3.2 Å) the same N-4, N-1 nitrogen atoms of ligand act as H-bond acceptor and –NH3 ,
-OH groups of protein act as H-bond donors. N-4 (1,3,4-oxadiazole ring), N-1 (thieno[2,3-
d]pyrimidine) atoms of 10b [Fig. 5(D)] interacted with active sites of enzyme by forming H-bond
with Gly 138, Tyr 248, Asn 168 at distance of 2.7 Å, 3.2 Å, 3.4 Å respectively. For the other
prominent interactions between the active site amino acid residues of Gyrase A, LpxC and the
inhibitors 9a, 9e, 10a, 10b see the Supporting Information.
14

Fig. 5. AutoDock 4.2 predicted binding mode of (A) 9a (B) 9e (C) 10a and (D) 10b docked into S.aureus C(30) carotenoid dehydrosqualene
synthase pocket. H-bonds are shown with yellow dashed lines.
15

In conclusion novel thieno[2,3-d]pyrimidine alkyne Mannich base hybrids and thieno[2,3-
d]pyrimidine 1,3,4-oxadiazole hybrids were designed, synthesized and screened for concentration
dependent neuroprotective, neurotoxic and antibacterial activities. Among all the compounds 9a,
10d showed significant neuroprotective and 9b, 9d neurotoxicity effects on the PC12 cells. 9a, 9e,
10a, 10b showed potent antibacterial activity against the four bacterial strains. Some of them
showed specific activity against one or more bacterial strains. Structure activity relationship studies
(SAR) revealed the change in the substitutions on the C4 position of thienopyrimidine ring and
terminal phenyl ring leads to substantial changes in the biological activities. In order to support the
in vitro experiments, the molecular docking studies were performed using open – source software
PyRx 0.8 implementation of AutoDock 4.2, which demonstrated that compounds 9a, 9e, 10a, 10b
showed good binding affinities with target proteins C(30) carotenoid dehydrosqualene synthase,
Gyrase A and LpxC. Hence these preliminary investigations serve as starting point to develop novel
neuroprotetive agents from thieno[2,3]pyrimidine and 1,3,4-oxadiazole based derivatives with
improved efficacy and fewer side effects. Now, further research work is going on to develop
thieno[2,3-d]pyrimidine derivatives as inhibitors for acetylcholinesterage in the laboratory.
Acknowledgement
One of the authors Mr. S. Triloknadh is thankful to University Grants Commission (UGC) New
Delhi [(Award Lt. No. F.17-142/98(SA-I)] for providing the financial assistance in the form of (JRF
& SRF).
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18

HIGHLIGHTS :
 Design and synthesis of thieno[2,3-d]pyrimidine–alkyne Mannich base and 1,3,4-oxadiazole
hybrids.
 Evaluation of neuroprotective and neurotoxic effects on PC12 cells.
 Strong bacterial activity of 9a, 9e, 10a and 10b against four bacterial strains.
 Insilico studies to demonstrate the correlation with the in vitro experimental results.
 Characterization of the synthesized compounds by spectral analysis.
19

Design, synthesis, neuroprotective, antibacterial activities and docking
studies of novel thieno[2,3-d]pyrimidine-alkyne Mannich base and
oxadiazole hybrids
Settypalli Triloknadha, Chunduri Venkata Rao*a, Kerru Nagarajua, Nallapaneni Hari Krishnaa, Chintha
Venkata Ramaiahb, Wudayagiri Rajendrab, Daggupati Trinathb, Yeguvapalli Suneethab
b
aDepartment of Chemistry, Department of Zoology, Sri Venkateswara University, Tirupati-517502,
Andhra Pradesh, India.
*a Corresponding author E-Mail: cvrsvu@gmail.com, Tel: +91 9849605140, Fax: +918772289555.
GRAPHICAL ABSTRACT:
20