Synthesis and DNase I inhibitory properties of some 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines

Article abstract of DOI:10.1016/j.bioorg.2018.07.009

A series of six novel and six known thieno[2,3-d]pyrimidin-4-amines 2–13 were synthesized, and further were used as a starting material for preparation of a small series of eight novel thieno[2,3-d]pyrimidin-4-phthalimides 14–21. Eight compounds, five amine and three phthalimide derivatives, inhibited bovine pancreatic DNase I with an IC₅₀ below 200 μM, being more effective than referent inhibitor crystal violet. Phthalimide derivatives 16, 18 and 19 exhibited higher DNase I inhibitory activity compared to their amine precursors 7, 10 and 11. Compound 19, as the most potent (IC₅₀ = 106 ± 16 μM), offers a good starting point for a design of new DNase I inhibitors. The Pharma RQSAR model showed a significant enhancement of thieno[2,3-d]pyrimidines activity using aryl substituents at R1 position. The E-State RQSAR model clarified the most important structural fragments relevant for DNase I inhibition. Molecular docking and Site Finder module defined the thieno[2,3-d]pyrimidines interactions with the most important catalytic residues of DNase I, including Glu 39, His 134, Asp 168 and His 252. We also found that steric effects and increase of molecular volume play a vital role in DNase I inhibition.

Full text of DOI:10.1016/j.bioorg.2018.07.009

BioorganicChemistry80(2018)693–705
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and DNase I inhibitory properties of some 5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidines
Anelia Ts. Mavrovaa,⁎, Stefan Dimova, Denitsa Yanchevab, Ana Kolarevićc, Budimir S. Ilićd,
Gordana Kociće, Andrija Šmelcerovićd,⁎
a
University of Chemical Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Science, Acad. G Bonchev Str., Build. 9, 1113 Sofia, Bulgaria
b
c
Department of Pharmacy, Faculty of Medicine, University of Niš, 18000 Niš, Serbia
d
Department of Chemistry, Faculty of Medicine, University of Niš, Bulevar Dr Zorana Đinđića 81, 18000 Niš, Serbia
e
Institute of Biochemistry, Faculty of Medicine, University of Niš, Bulevar Dr Zorana Đinđića 81, 18000 Niš, Serbia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Thieno[2,3-d]pyrimidines
Synthesis
DNase I inhibition
R-Group analysis
Molecular docking
SAR analysis
A series of six novel and six known thieno[2,3-d]pyrimidin-4-amines 2–13 were synthesized, and further were
used as a starting material for preparation of a small series of eight novel thieno[2,3-d]pyrimidin-4-phthalimides
14–21. Eight compounds, five amine and three phthalimide derivatives, inhibited bovine pancreatic DNase I
with an IC50 below 200 µM, being more effective than referent inhibitor crystal violet. Phthalimide derivatives
16, 18 and 19 exhibited higher DNase I inhibitory activity compared to their amine precursors 7, 10 and 11.
Compound 19, as the most potent (IC50 = 106
16 µM), offers a good starting point for a design of new DNase
I inhibitors. The Pharma RQSAR model showed a significant enhancement of thieno[2,3-d]pyrimidines activity
using aryl substituents at R1 position. The E-State RQSAR model clarified the most important structural frag-
ments relevant for DNase I inhibition. Molecular docking and Site Finder module defined the thieno[2,3-d]
pyrimidines interactions with the most important catalytic residues of DNase I, including Glu 39, His 134, Asp
168 and His 252. We also found that steric effects and increase of molecular volume play a vital role in DNase I
inhibition.
1. Introduction
The bioisosteric relation of thieno[2,3-d]pyrimidine-4-ones with
quinolones, cytosine and uracil led to the generation of compounds
Deoxyribonuclease I (DNase I) is one of the best characterized en-
donuclease among mammals [1]. It is predominantly found in the
exocrine pancreas [2]. DNase I catalyzes DNA hydrolysis by producing
3′-OH/5′-P ends and exerts its full activity in the presence of both Ca2+
and Mg2+ under neutral pH conditions [1]. On the other hand, DNase I
inhibitors are compounds able to control or modify this process. Both
DNase I and its inhibitors can be used as compounds for diagnosis,
monitoring and therapy of various diseases [3–5]. It was shown that
DNase I plays a fundamental role in apoptosis as possible mediator of
internucleosomal DNA fragmentation [6–8]. This makes DNase I in-
hibitors an attractive potential target to design alternative strategies for
the treatment of various disease conditions related with excessive
apoptosis, including neurodegenerative diseases, ischemia-reperfusion
injury, graft-versus-host disease and autoimmune disorders [8,9]. Re-
cently, we reported ascorbic acid as a pioneer of substrate-based DNase
I inhibitors [10].
with various biological properties, such as antibacterial [11,12], anti-
parasitic [13], analgesic [14], anti-inflammatory [15], anticancer [16],
antioxidant [17], antiemetic [18], antiviral [19] and antihistaminic
[20]. The structural biosterism with quinazoline analogue has been
recently demonstrated to play an important role in the antibacterial and
central nervous system (CNS) activity of thienopyrimidines [21,22].
Additionally, phthalimide derivatives exhibit multifaceted biological
activity, such as anti-mycobacterium tuberculosis [23], anticonvulsant
and neurotoxicity [24,25], analgesic and anti-inflammatory [26], as
well as α-glucosidase inhibition [27] activities.
Taking into consideration the bioisosteric relationship between
thieno[2,3-d]pyrimidines and the pyrimidine nucleosides, as well as
their structural similarity (Fig. 1), in this study we synthesized a small
library of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyr-
imidines and evaluated their inhibitory activity against bovine pan-
creatic DNase I in vitro. R-Group analysis and molecular docking are in
⁎
Corresponding authors.
E-mail addresses: anmav@abv.bg (A. Ts. Mavrova), andrija.smelcerovic@medfak.ni.ac.rs (A. Šmelcerović).
https://doi.org/10.1016/j.bioorg.2018.07.009
Received 23 May 2018; Received in revised form 8 July 2018; Accepted 9 July 2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

A. Ts. Mavrova et al.
BioorganicChemistry80(2018)693–705
Fig. 1. Structural similarity of the synthesized thieno[2,3-d]pyrimidines and pyrimidine nucleoside deoxycytidine.
silico drug design methods used for better understanding of drug
properties and target interaction that hypothesize the designing of
novel drug candidates [28]. Having in mind that our studied molecules
were built on a similar scaffold, by attaching different groups at one or
more points on the thieno[2,3-d]pyrimidine, we wanted to examine
DNase I inhibitory property of the thieno[2,3-d]pyrimidine-4-amines as
a function of the groups at the various attachment points. In addition,
we wanted to clarify DNase I inhibitory properties of thieno[2,3-d]
pyrimidines at the molecular level using the Site Finder module and
molecular docking studies. Finally, in silico study of the physico-che-
mical and toxicological properties of the studied compounds was per-
formed.
The 2-aminothiophene-3-carbonitrile 1 was used as a starting ma-
terial synthesized by Gewald reaction [29,30] which is a very often
used reaction method for 2-aminothiophene synthesis. The 2-sub-
stituted thieno[2,3-d]pyrimidine-4-amines 2–13 were obtained by
passing a stream of dry HCl gas through a dry dioxane solution of
equimolar ratio of 2-aminothiophene 1 and alkyl or aryl nitriles for 6 h
[31]. The bipharmacophoric derivatives 14–21 were synthesized by
reaction of thieno[2,3-d]pyrimidine-4-amines 2–13 and phthalic an-
hydride in glacial acetic acid under reflux (method A) or in toluene/
acetic anhydride/pyridine mixture (method B).
The chemical structure of the compounds was established by FT-IR,
1H NMR and elemental analysis (1H NMR and FT-IR data are provided
in the Experimental part and some of the spectra in the Supplementary
material – Figs. S1–S27).
2. Materials and methods
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bioorg.2018.07.009.
Melting points (mp) were determined on an Electrothermal AZ 9000
3MK4 apparatus and were uncorrected. The thin layer chromatography
(TLC, Rf values) was performed on F254 or silica gel plates F254
2.1. Synthesis
The synthesis of thieno[2,3-d]pyrimidine-4-amine derivatives con-
taining phthalimide moiety is illustrated in Scheme 1.
OH
O
O
O
N
H2N
O
N
N
N
N
C
N
b
HN
S
a
R
R
N
N
(or c)
S
NH2
R
S
S
2 - 13
14 - 21
1
Comp.
2
R
Comp.
R
Comp.
14
R
Comp.
20
R
Cl
8
9
Cl
OMe
OMe
OMe
CF3
O
CH3
3
15
21
OEt
N
CH3
4
5
10
11
16
17
N
NO2
O
CH3
N
OMe
Cl
CH3
OMe
OMe
6
7
12
13
18
19
CF3
NO2
N
Scheme 1. Synthesis of phthalimide derivatives of thieno[2,3-d]pyrimidine-4-amines: a) RCN, dry HCl gas, dioxane, 6 h; b) phthalic anhydride, acetic acid, reflux –
method A; c) toluene, phthalic anhydride, acetic anhydride, pyridine – method B.
694

A. Ts. Mavrova et al.
BioorganicChemistry80(2018)693–705
(Merck, 0.2 mm thick) and visualization was effected with ultraviolet
light. IR spectra were recorded on a Bruker Equinox 55 spectro-
photometer as potassium bromide discs. All 1H NMR spectra were re-
corded on a Bruker Avance DRX 250 spectrometer (Bruker, Faelanden,
Switzerland) operating at 250 MHz and a Bruker Avance DRX 600
spectrometer (Bruker, Faelanden, Switzerland) operating at 600 MHz.
Chemical shifts were expressed relative to tetramethylsilane (TMS) and
were reported as δ (ppm). The measurements were carried out at am-
bient temperature (300 K). The microanalyses for C, H, N and S were
performed on PerkinElmer elemental analyzer. The UV–VIS absorption
spectra were recorded on a spectrophotometer Hewlett Packard 8452A.
2.1.2.4. 2-(Morpholinomethyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-amine (5). The formed pyrimidine was recrystallized
from MeOH. Yield: 40%; Mp. 224–225 °C; IR (KBr): ν(cm−1) 3445
(NH), 3309 (NH), 3241 (NH), 3091 (ArH), 3010 (ArH), 2945 (CH),
2884 (CH), 2847 (CH), 1654 (NH), 1114 (CeO); 1H NMR (DMSO-d6, δ):
1.84 (t, J = 2.8 Hz, 4H, (CH2)2), 2.50 (m, 4H, CH2–O–CH2), 2.88 (d,
J = 2.1 Hz, 2H, CH2), 3.00 (d, J = 2.0 Hz, 2H, CH2), 3.52 – 3.60 (m, 4H,
CH2–N–CH2), 3.73 (s, 2H, CH2). 1H NMR (CDCl3, δ) 1.71–1.82 (m, 4H,
(CH2)2), 2.45 (s, 4H, CH2–O–CH2), 2.72 (t, J = 6.0 Hz, 2H, CH2), 2.85
(t, J = 6.1 Hz, 2H, CH2), 3.41 (s, 2H, CH2), 3.52–3.61 (m, 4H,
CH2–N–CH2), 12.03 (s, 1H, NH, exchange with D2O); Analysis: Calc.
for C15H20N4OS: C, 59.18; H, 6.62; N, 18.41; O, 5.26; S, 10.53; Found:
C, 59.20; H, 6.62; N, 18.40; O, 5.27; S, 10.53.
The fluorescence spectra were taken on
fluorimeter.
a Scinco FS-2 spectro-
2.1.2.5. 2-(3,4,5-Trimethoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (6). The formed precipitate was recrystallized
from pyridine and washed with MeOH. Yield: 43%; Mp. 214–215 °C; IR
(KBr): ν(cm−1) 3542 (NH), 3397 (NH), 2986 (CH), 2930 (CH), 2834
(CH), 1645 (NH), 1106 (CeO); 1H NMR (DMSO-d6, δ): 1.82 (s, 4H,
(CH2)2), 2.76 (s, 2H, CH2), 2.93 (s, 2H, CH2), 3.72 (s, 3H, OCH3), 3.87
(s, 6H, 2OCH3), 6.86 (s, 2H, NH2), 7.69 (s, 2H, ArH); Analysis: Calc. for
C19H21N3O3S: C, 61.44; H, 5.70; N, 11.31; O, 12.92; S, 8.63; Found: C,
61.41; H, 5.73; N, 11.28; O, 12.90; S, 8.63.
2.1.1. Synthesis of 2-aminothiophene-3-carbonitrile 1
The synthesis of 2-aminothiophene-3-carbonitrile 1 was performed
by cyclocondensation of cyclohexanone with malononitrile and sulfur
in equimolar quantity with catalyst HNEt2 as described by Sabnis et al.
[30].
2.1.2. General procedure for the synthesis of 5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amines 2–8, 10–13
The corresponding 2-aminothiophene 1 (0.0094 mol), the appropriate
cyano derivative RCN (0.0094 mol) and 20 mL of dioxane were mixed in a
round flask whereat a stream of dry hydrogen chloride was passed through
the solution for 6 h. The reaction mixture was allowed to stay for 12 h at
room temperature. It was then poured into a beaker containing crushed ice
and neutralized to pH ∼ 8 using 10% (v/v) NH4OH. The precipitate was
filtered, washed many times with water and dried.
2.1.2.6. 2-(Pyridin-2-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-amine (7). The compound was recrystallized with system of
solvents Benzene/MeOH = 1:1. Yield: 45%; Mp. 264–266 °C (lit.
249–251 °C [31]); IR (KBr): ν(cm−1) 3487 (NH), 3270 (NH), 3138
(NH), 2925 (CH), 2835 (CH), 1635 (NH), 740 (Ar-R); 1H NMR (DMSO-
d6, δ): 1.83 (s, 4H, (CH2)2), 2.79 (s, 2H, CH2), 2.95 (s, 2H, CH2), 6.99
(bs, 2H, NH2), 7.45 (dd, J = 6.7, 4.8 Hz, 1H, ArH), 7.91 (td, J = 7.8,
1.7 Hz, 1H, ArH), 8.32 (d, J = 7.9 Hz, 1H, ArH), 8.67 (d, J = 4.2 Hz,
1H, ArH); Analysis: Calc. for C15H14N4S: C, 63.80; H, 5.00; N, 19.84; S,
11.36; Found: C, 63.81; H, 5.02; N, 19.81; S, 11.39.
2.1.2.1. 2-Benzyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
amine (2). The neutralisation resulted in resinous product, which was
crystallized with pyridine. The formed precipitate was recrystallized
from pyridine. Yield: 41%; Mp. 201–203 °C (lit. 193–195 °C [31]); IR
(KBr): ν(cm−1) 3382 (NH), 3293 (NH), 3187 (NH), 3023 (ArH), 2934
(CH), 2833 (CH), 1619 (NH), 718 (ArH); 1H NMR (DMSO-d6, δ): 1.78
(d, J = 2.7 Hz, 4H, (CH2)2), 2.71 (s, 2H, CH2), 2.86 (s, 2H, CH2), 3.94
(s, 2H, CH2), 6.56–7.13 (bs, 2H, NH2), 7.15–7.21 (m, 1H, ArH),
7.23–7.28 (m, 4H, ArH); Analysis: Calc. for C17H17N3S: C, 69.12; H,
5.80; N, 14.22; S, 10.85; Found: C, 69.09; H, 5.79; N, 14.22; S, 10.90.
2.1.2.7. 2-(2-Chloroethyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-amine (8). The formed precipitate was recrystallized from
MeOH to give 20% yield as a white crystals. Mp. 170–172 °C; IR (KBr):
ν(cm−1) 3488 (NH), 3301 (NH), 3112 (NH), 2938 (CH3), 2840 (CH2),
1644 (NH); 1H NMR (DMSO-d6, δ): 1.78 (d, J = 2.8 Hz, 4H, (CH2)2),
2.71 (s, 2H, CH2), 2.86 (s, 2H, CH2), 3.07 (t, J = 6.6 Hz, 2H, CH2), 4.01
(t, J = 6.6 Hz, 2H, CH2), 6.75 (bs, 2H, NH2); Analysis: Calc. for
2.1.2.2. Ethyl
2-(4-amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
C12H14ClN3S: C, 53.82; H, 5.27; Cl, 13.24; N, 15.69; S, 11.97; Found:
pyrimidin-2-yl)acetate (3). The neutralisation led to resinous product,
which was crystallized with EtOAc. The formed precipitate was
recrystallized from EtOAc. Yield: 52%; Mp. 170–172 °C (lit.
170–171 °C [31]); IR (KBr): ν(cm−1) 3460 (NH), 3314 (NH), 3150
(NH), 2993 (CH), 2973 (CH), 2853 (CH), 1719 (C]O), 1650 (NH),
1196 (CeO); 1H NMR (DMSO-d6, δ): 1.17 (t, J = 7.1 Hz, 3H, CH3), 1.79
(s, 4H, (CH2)2), 2.73 (s, 2H, CH2), 2.88 (d, J = 1.8 Hz, 2H, CH2), 3.67
(s, 2H, CH2), 4.08 (q, J = 7.1 Hz, 2H, CH2), 7.04–7.26 (bs, 2H, NH2);
Analysis: Calc. for C14H17N3O2S: C, 57.71; H, 5.88; N, 14.42; O, 10.98;
S, 11.00; Found: C, 57.66; H, 5.85; N, 14.44; O, 10.97; S, 11.02.
C, 53.86; H, 5.31; Cl, 13.24; N, 15.67; S, 11.90.
2.1.2.8. 2-Ethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
amine (10). The formed precipitate was recrystallized from MeOH.
Yield: 37%; Mp. 174–176 °C; IR (KBr): ν(cm−1) 3491 (NH), 3446 (NH),
3303 (NH), 3133 (NH), 2943 (CH3), 2841 (CH2), 1639 (NH); 1H NMR
(DMSO-d6, δ): 1.20 (t, J = 7.6 Hz, 3H, CH3), 1.79 (t, J = 2.6 Hz, 4H,
(CH2)2), 2.63 (q, J = 7.6 Hz, 2H, CH2), 2.71 (d, J = 2.0 Hz, 2H, CH2),
2.87 (d, J = 1.9 Hz, 2H, CH2), 6.71 (bs, 2H, NH2); Analysis: Calc. for
C
12H15N3S: C, 61.77; H, 6.48; N, 18.01; S, 13.74; Found: C, 61.79; H,
6.51; N, 18.01; S, 13.74.
2.1.2.3. 2-(Pyridin-3-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-amine (4). The residue was recrystallized with dioxane and
then it was isolated by preparational chomatography in a system of
solvents EtOAc/Chloroform = 5:1. Yield: 30%; Mp. 286–288 °C (lit.
206–208 °C [31]); IR (KBr): ν(cm−1) 3298 (NH), 3170 (NH), 3075
(ArH), 3008 (ArH), 2935 (CH), 1646 (NH); 1H NMR (DMSO-d6, δ): 1.80
(ddd, J = 16.6, 9.2, 4.9 Hz, 4H, (CH2)2), 2.77 (t, J = 6.0 Hz, 2H, CH2),
2.91 (t, J = 6.0 Hz, 2H, CH2), 7.55 (dd, J = 8.0, 4.8 Hz, 1H, ArH),
8.40–8.47 (m, 1H, ArH), 8.72 (dd, J = 4.7, 1.4 Hz, 1H, ArH), 9.23 (d,
J = 1.9 Hz, 1H, ArH), 12.73 (s, 1H, NH, exchange with D2O); Analysis:
Calc. for C15H14N4S: C, 63.80; H, 5.00; N, 19.84; S, 11.36; Found: C,
63.84; H, 4.07; N, 19.79; S, 11.32.
2.1.2.9. 2-(4-Nitrobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-amine (11). The mixture was left for 12 h after
neutralization to crystallize. The product was recrystallized with
system of solvents DMSO/MeOH = 5:1. Yield: 51%; Mp. 248–250 °C
(lit. 320–322 °C [31]); IR (KBr): ν(cm−1) 3491 (NH), 3300 (NH), 3102
(NH), 2942 (CH2), 2835 (CH2), 1642 (NH), 1517 (NO2), 1343 (NO2),
723 cm−1 (ArH); 1H NMR (DMSO-d6, δ): 1.78 (s, 4H, (CH2)2), 2.71 (s,
2H, CH2), 2.86 (s, 2H, CH2), 4.11 (s, 2H, CH2), 6.83 (bs, 2H, NH2), 7.53
(d, J = 8.8 Hz, 2H, ArH), 8.11–8.17 (m, 2H, ArH); Analysis: Calc. for
C
17H16N4O2S: C, 59.98; H, 4.74; N, 16.46; O, 9.40; S, 9.42; Found: C,
59.93; H, 4.75; N, 16.46; O, 9.43; S, 9.41.
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2.1.2.10. 2-(3-Chlorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-amine (12). The mixture was titrated up to alkaline pH and
left for a night. In result the resinous product crystallized. The product
was recrystallized with pyridine and washed with MeOH. Yield: 48%;
Mp. 210–212 °C; IR (KBr): ν(cm−1) 3491 (NH), 3306 (NH), 3094 (ArH),
2928 (CH2), 2834 (CH2), 1646 (NH), 783, 740, 682 (ArH); 1H NMR
(DMSO-d6, δ): 1.78 (s, 4H, (CH2)2), 2.71 (s, 2H, CH2), 2.87 (s, 2H, CH2),
3.96 (s, 2H, CH2), 6.80 (bs, 2H, NH2), 7.22 (d, J = 7.5 Hz, 1H, ArH),
7.24–7.27 (m, 1H, ArH), 7.31 (dd, J = 10.3, 5.0 Hz, 2H, ArH); Analysis:
Calc. for C17H16ClN3S: C, 61.90; H, 4.89; Cl, 10.75; N, 12.74; S, 9.72;
Found: C, 61.90; H, 4.88; Cl, 10.75; N, 12.72; S, 9.71.
ArH), 7.31 (t, J = 7.6 Hz, 2H, ArH), 7.42 (d, J = 7.3 Hz, 2H, ArH), 7.88
(dd, J = 5.4, 3.1 Hz, 2H, ArH), 8.03 (dd, J = 5.5, 3.0 Hz, 2H, ArH);
Analysis: Calc. for C25H19N3O2S: C, 70.57; H, 4.50; N, 9.88; O, 7.52; S,
7.54; Found: C, 70.53; H, 4.49; N, 9.83; O, 7.52; S, 7.53. Method B:
Yield: 63%.
2.1.4.2. 2-(2-(3,4,5-Trimethoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-yl) isoindoline-1,3-dione (15). Method A: The
reaction mixture was refluxed for 28 h. After cooling, the compound
crystallized as white crystals. The product was purified by double
recrystallization from acetic acid. Yield: 21%; Mp. = 269–270 °C; IR
(KBr): ν(cm−1) 3003 (ArH), 2965 (CH), 2935 (CH), 2835 (CH), 1788
2.1.2.11. 2-(3-(Trifluoromethyl)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amine (13). The mixture titrated up to alkaline
pH and left for a night. In result the resinous product crystallized. The
product was recrystallized with acetic acid. Yield: 57%; Mp.
244–246 °C; IR (KBr): ν(cm−1) 3476 (NH), 3279 (NH), 3162 (NH),
2945 (CH2), 2846 (CH2), 1646 (ArH), 1117 (C − F), 691 (ArH); 1H
NMR (DMSO-d6, δ): 1.82 (s, 4H, (CH2)2), 2.78 (s, 2H, CH2), 2.94 (s, 2H,
CH2), 7.02 (bs, 2H, NH2), 7.72 (t, J = 7.8 Hz, 1H, ArH), 7.83 (d,
J = 7.7 Hz, 1H, ArH), 8.60–8.65 (m, 2H, ArH); Analysis: Calc. for
(C]O),
1726
(C]O),
718
(ArH);
1H
NMR
(CDCl3):
δ(ppm) = 1.75–1.96 (m, 4H, (CH2)2), 2.62 (dt, J = 27.0, 6.1 Hz, 2H,
CH2), 2.93 (dd, J = 12.4, 6.3 Hz, 2H, CH2), 3.96 (dd, J = 28.8, 16.6 Hz,
9H, 3OCH3), 7.18, 7.83 (ds, 2H, ArH), 7.90 (ddd, J = 15.8, 5.4, 3.1 Hz,
2H, ArH), 8.06 (ddd, J = 20.2, 5.4, 3.1 Hz, 2H, ArH); Analysis: Calc. for
C
27H23N3O5S: C, 64.66; H, 4.62; N, 8.38; O, 15.95; S, 6.39; Found: C,
64.70; H, 4.64; N, 8.38; O, 15.96; S, 6.37; Method B: Yield: 51%.
2.1.4.3. 2-(2-(Pyridin-2-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-yl)isoindoline-1,3-dione (16). Method A: The reaction
mixture was refluxed for 30 h. The product was purified by eluent
system of Benzene/MeOH = 1:3. Yield: 15%; Mp. = 282–285 °C; IR
(KBr): ν(cm−1) 3066 (ArH), 2932 (CH), 2884 (CH), 2837 (CH), 1783
(C]O), 1721 (C]O), 721 (ArH); 1H NMR (DMSO-d6, δ): 1.71 (dd,
J = 7.6, 4.0 Hz, 2H, CH2), 1.82 (dd, J = 7.5, 3.9 Hz, 2H, CH2), 2.55 (t,
J = 6.0 Hz, 2H, CH2), 2.96 (t, J = 6.1 Hz, 2H, CH2), 7.56 (ddd, J = 7.5,
4.7, 1.1 Hz, 1H, ArH), 8.00 (td, J = 7.8, 1.8 Hz, 1H, ArH), 8.05 (dd,
J = 5.4, 3.1 Hz, 2H, ArH), 8.14 (dd, J = 5.5, 3.0 Hz, 2H, ArH), 8.45 (dt,
J = 8.0, 1.0 Hz, 1H, ArH), 8.76 (ddd, J = 4.7, 1.8, 0.9 Hz, 1H, ArH);
Analysis: Calc. for C23H16N4O2S: C, 66.97; H, 3.91; N, 13.58; O, 7.76; S,
7.77; Found: C, 66.99; H, 3.92; N, 13.56; O, 7.76; S, 7.77; Method B:
Yield: 44%.
C
17H14F3N3S: C, 58.44; H, 4.04; F, 16.31; N, 12.03; S, 9.18; Found: C,
58.45; H, 4.07; F, 16.33; N, 12.01; S, 9.10.
2.1.3. Synthesis of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
amine 9
A steam of dry hydrogen chloride gas was passed through the so-
lution of compound 1 (0.01 mol) and 30 mL dry acetonitrile for 6 h. The
reaction mixture was allowed to stay for 12 h at room temperature.
Then the mixture was poured into a beaker containing crushed ice and
neutralized to pH ∼ 8 using 10% NH2OH. The product was re-
crystallized with system of solvents benzene/MeOH = 1:1. Yield: 81%;
Mp. 233–234 °C (lit. 224–225 °C [31]); IR (KBr): ν(cm−1) 3446 (NH),
3303 (NH), 3133 (NH), 2931 (CH3), 2951 (CH2), 2907 (CH2), 1639
(NH); 1H NMR (DMSO-d6, δ): 1.79 (t, J = 2.5 Hz, 4H, (CH2)2), 2.36 (s,
3H, CH3), 2.71 (d, J = 1.9 Hz, 2H, CH2), 2.86 (d, J = 1.9 Hz, 2H, CH2),
6.70 (bs, 2H, NH2); Analysis: Calc. for C11H13N3S: C, 60.24; H, 5.97; N,
19.16; S, 14.62; Found: C, 60.24; H, 5.99; N, 19.16; S, 14.62.
2.1.4.4. 2-(2-Methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-yl)isoindoline-1,3-dione (17). Method A: The reaction
mixture was refluxed for 46 h. The product was isolated by hot
filtration with MeOH, whereupon by cooling of the filtrate the pure
2.1.4. General procedure for the synthesis of 5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-phthalimides 14–21
product crystallized. Yield: 17%; Mp. = 201–202 °C; IR (KBr): ν(cm−1
)
2934 (CH), 2864 (CH), 1784 (C]O), 1722 (C]O), 718 (ArH); 1H NMR
(DMSO-d6, δ): 1.67 (dt, J = 5.7, 4.6 Hz, 2H, CH2), 1.78 (dt, J = 8.7,
4.7 Hz, 2H, CH2), 2.45 (t, J = 6.1 Hz, 2H, CH2), 2.75 (s, 3H, CH3), 2.88
(t, J = 6.0 Hz, 2H, CH2), 8.01 (dd, J = 5.5, 3.1 Hz, 2H, ArH), 8.09 (dd,
J = 5.5, 3.0 Hz, 2H, ArH); Analysis: Calc. for C19H15N3O2S: C, 65.31; H,
4.33; N, 12.03; O, 9.16; S, 9.18; Found: C, 65.31; H, 4.35; N, 12.03; O,
9.18; S, 9.18; Method B: Yield: 65%.
Method A. The corresponding thieno[2,3-d]pyrimidin-4-amine 2–13
(0.0017 mol) and 0.28 g phthalic anhydride (0.0019 mol) in 25 mL
glacial acetic acid were mixed in a round-bottom flask. The solution
was refluxed and the reaction was monitored by TLC. After the reaction
was completed the mixture was poured into water and neutralized with
10% (v/v) NH4OH to pH ∼ 7. The precipitate formed was filtered and
washed thoroughly with water.
Method B. To a solution of 0.0012 mol phthalic anhydride in to-
luene, 0.001 mol of the corresponding thieno[2,3-d]pyrimidin-4-amine
was added in and the reaction mixture was refluxed for 5 h. The reac-
tion solution was cooled to room temperature, 0.002 mol of acetic an-
hydride and 0.008 mol of pyridine were added and the refluxing was
continued for 1 to 3 h. The solution was cooled in an ice bath, water was
added to the reaction mixture, the organic layer was separated and after
drying concentrated under reduced pressure, whereby the iso-
indoldiones crystallized.
2.1.4.5. 2-(2-Ethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
4-yl)isoindoline-1,3-dione (18). Method A: The reaction mixture was
refluxed for 34 h. The product was isolated by hot filtration with MeOH,
then after crystallization the product was twice recrystallized from
MeOH. Yield: 16%; Mp. = 156–157 °C; IR (KBr): ν(cm−1) 3056 (ArH),
2940 (CH), 2871 (CH), 2839 (CH), 1786, 1764 (C]O), 1724 (C]O),
714 (ArH); 1H NMR (DMSO-d6, δ): 1.32 (t, J = 7.6 Hz, 3H, CH3),
1.63–1.71 (m, 2H, CH2), 1.75–1.82 (m, 2H, CH2), 2.46 (t, J = 6.1 Hz,
2H, CH2), 2.88 (t, J = 6.1 Hz, 2H, CH2), 3.03 (q, J = 7.6 Hz, 2H,
CH2CH3), 8.02 (dd, J = 5.5, 3.1 Hz, 2H, ArH), 8.09 (dd, J = 5.5, 3.0 Hz,
2H, ArH); Analysis: Calc. for C20H17N3O2S: C, 66.10; H, 4.71; N, 11.56;
O, 8.80; S, 8.82; Found: C, 66.09; H, 4.75; N, 11.50; O, 8.83; S, 8.83;
Method B: Yield: 45%.
2.1.4.1. 2-(2-Benzyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
4-yl)isoindoline-1,3-dione (14). Method A: The reaction mixture was
refluxed for 40 h. The product crystallized after cooling. The compound
was purified by double recrystallization from acetic acid. Yield: 12%;
Mp. = 222–225 °C; IR (KBr): ν(cm−1) 3082 (ArH), 3059 (ArH), 3028
(ArH), 2949 (CH), 1784 (C]O), 1725 (C]O), 721 (ArH); 1H NMR
(CDCl3, δ): 1.72–1.92 (m, 4H, (CH2)2), 2.56 (t, J = 6.2 Hz, 2H, CH2),
2.88 (t, J = 6.1 Hz, 2H, CH2), 4.44 (s, 2H, CH2), 7.23 (t, J = 7.4 Hz, 1H,
2.1.4.6. 2-(2-(4-Nitrobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-yl)isoindoline-1,3-dione (19). Method A: The reaction
mixture was refluxed for 8 h. After the neutralization, the product
was purified by TLC developed with EtOAc/n-Hex = 2:5. Yield: 12%;
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A. Ts. Mavrova et al.
BioorganicChemistry80(2018)693–705
Mp. = 223–225 °C; IR (KBr): ν(cm−1) 3082 (ArH), 3059 (ArH), 3028
(ArH), 2949 (CH), 1784 (C]O), 1725 (C]O), 721 (ArH); 1H NMR
(DMSO-d6, δ): 1.66 (dd, J = 7.5, 3.9 Hz, 2H, CH2), 1.75–1.81 (m, 2H,
CH2), 2.47 (t, J = 6.2 Hz, 2H, CH2), 2.89 (t, J = 6.0 Hz, 2H, CH2), 4.54
(s, 2H, CH2), 7.61 (d, J = 8.8 Hz, 2H, ArH), 8.02 (dd, J = 5.5, 3.1 Hz,
2H, ArH), 8.09 (dd, J = 5.5, 3.0 Hz, 2H, ArH), 8.16–8.20 (m, 2H, ArH);
Analysis: Calc. for C25H18N4O4S: C, 63.82; H, 3.86; N, 11.91; O, 13.60;
S, 6.82; Found: C, 63.83; H, 3.90; N, 11.91; O, 13.62; S, 6.80; Method B:
Yield: 54%.
2.1.4.7. 2-(2-(3-Chlorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-yl)isoindoline-1,3-dione (20). Method A: The reaction
mixture was refluxed for 30 h. After cooling the crystallized
thienopyrimidine 12 was filtrated and the mixture neutralized. The
residue was purified by recrystallization from acetic acid, giving the
desired compound 20. Yield: 10%; Mp. = 191–193 °C; IR (KBr):
ν(cm−1) 3058 (ArH), 2931 (CH), 2856 (CH), 1787 (C]O), 1720 (C]
O), 716 (ArH); 1H NMR (DMSO-d6, δ): 1.66 (dd, J = 7.4, 4.0 Hz, 2H,
CH2), 1.78 (dd, J = 7.5, 4.0 Hz, 2H, CH2), 2.47 (t, J = 6.1 Hz, 2H, CH2),
2.88 (t, J = 6.0 Hz, 2H, CH2), 4.38 (s, 2H, CH2), 7.27–7.31 (m, 2H,
ArH), 7.32–7.36 (m, 1H, ArH), 7.39 (s, 1H, ArH), 8.01 (dd, J = 5.5,
3.1 Hz, 2H, ArH), 8.09 (dd, J = 5.5, 3.0 Hz, 2H, ArH); Analysis: Calc.
for C25H18ClN3O2S: C, 65.28; H, 3.94; Cl, 7.71; N, 9.14; O, 6.96; S, 6.97;
Found: C, 65.25; H, 3.98; Cl, 7.71; N, 9.19; O, 6.98; S, 6.96; Method B:
Yield: 47%.
Fig. 2. The 2-aminopyridine and 2-substituted-4-amino-thieno[2,3-d]pyr-
imidines acetic acid complexes.
partial-least-squares (PLS) procedure that fitted the observed inhibitory
data to the counts of the features present in each R group at each po-
sition. For Pharma/E-State RQSAR models, the features counted are the
pharmacophore types and various E-state atom types [28]. This was
done using a 75%: 25% random training: test-set split. The procedure
was repeated one hundred times, each time picking the model that does
the best job without overfitting. For each model, each pharmacophore/
E-state feature type at each R-group position was given a coefficient
that reflects how much it contributes to the property being modeled. A
feature type was deemed significant (colored red or blue) if the absolute
value of the mean of its coefficients over the models exceeded the im-
portance cutoff, which is a statistic computed from all coefficients over
all models.
2.1.4.8. 2-(2-(3-(Trifluoromethyl)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-yl) isoindoline-1,3-dione (21). Method A: The
reaction mixture was refluxed for 29 h and after cooling the desired
imide product 21 crystallized. The residue was recrystallized from
acetic acid. Yield: 35%; Mp. = 233–234 °C; IR (KBr): ν(cm−1) 3073
(ArH), 3026 (ArH), 2940 (CH), 2837 (CH), 1788 (C]O), 1718 (C]O),
724, 660 (ArH); 1H NMR (DMSO-d6, δ): 1.70 (dt, J = 5.6, 4.5 Hz, 2H,
CH2), 1.81 (dt, J = 8.9, 4.8 Hz, 2H, CH2), 2.54 (t, J = 6.1 Hz, 2H, CH2),
2.95 (t, J = 6.0 Hz, 2H, CH2), 7.80 (t, J = 7.8 Hz, 1H, ArH), 7.94 (d,
J = 7.7 Hz, 1H, ArH), 8.04 (dd, J = 5.4, 3.1 Hz, 2H, ArH), 8.13 (dd,
J = 5.5, 3.0 Hz, 2H, ArH), 8.66 (s, 1H, ArH), 8.71 (d, J = 8.0 Hz, 1H,
ArH); Analysis: Calc. for C25H16F3N3O2S: C, 62.62; H, 3.36; F, 11.89; N,
8.76; O, 6.67; S, 6.69; Found: C, 62.62; H, 3.37; F, 11.89; N, 8.76; O,
6.66; S, 6.70; Method B: Yield: 65%.
2.3.2. Importance analysis
Importance analysis addressed the question of how sensitive the
DNase I inhibitory property of thieno[2,3-d]pyrimidines was to R group
variation at specific position. A position was more important than an-
other if varying the R group at that position led to greater property
differences than are observed when the R group at the other position is
varied. The importance value of a position was the range of the prop-
erty over the R groups at specific position, averaged over all structures
containing a given R group at that position.
2.4. Molecular docking
2.4.1. Ligand preparation
2.1.5. Theoretical calculations
Examined thieno[2,3-d]pyrimidines were built with ChemBioDraw
Ultra 13.0 (PerkinElmer, Inc.) and their geometries have been opti-
mized with ChemBio 3D Ultra 13.0 (PerkinElmer, Inc.) using MM2
The quantum chemical calculations were performed using the
Gaussian 09 package [32] of programs. Geometry optimization was
carried out by analytical gradient technique without any symmetry
constraint in gas phase. The results were obtained at DFT level of theory
using B3LYP/6-311+G** [33,34]. The stationary points found on the
molecular potential energy hypersurfaces were characterized using
standard harmonic vibrational analysis.
2.2. Evaluation of DNase I inhibition
Thieno[2,3-d]pyrimidine-4-amines 2, 3, 5–13 and their phthalimide
derivatives 14, 16, 18–21 were investigated for the inhibitory effect
towards bovine pancreatic DNase I. The in vitro evaluation of DNase I
inhibition is based on spectrophotometric measurement of acid-soluble
nucleotides formation at 260 nm according to the method previously
described [10], using crystal violet as a positive control.
2.3. R-Group analysis
2.3.1. Pharma/E-State RQSAR models
Fig. 3. Absorbance (dashed line) and emission (solid line) spectra of compound
3 in benzene with addition of acetic acid (AA).
Pharma/E-State RQSAR models, analyzed the thieno[2,3-d]pyr-
imidines activity as a function of the kind of R-group at each attach-
ment position, and displayed the results qualitatively in terms of in-
creasing, decreasing, or little effect on the activity [28]. We ran a
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BioorganicChemistry80(2018)693–705
Fig. 4. DFT optimized structure of representative thieno[2,3-d]pyrimidine-4-amine 7 and its phthalimide derivative 16.
Table 1
In vitro DNase I inhibitory activity of the studied thieno[2,3-d]pyrimidines.
Comp.
IC50 (µM)
SD
Comp.
IC50 (µM)
> 200
SD
Comp.
IC50 (µM)
SD
2
3
5
6
7
8
197
13
9
16
18
19
20
134
15
> 200
> 200
> 200
156
10
11
12
13
14
195
14
148
106
19
16
128
> 200
148
17
27
> 200
> 200
383
29
21
> 200
> 200
Crystal violet
49
force field until a minimum 0.100 Root Mean Square (RMS) gradient
was reached. Subsequently all compounds structures were followed by
energy minimization with MMFF94x force field in the Molecular
Operating Environment (MOE) Software package 2014.0901 [35].
Conformational search for preparation of the ligands was carried out by
MOE LowModelMD method which performs molecular dynamics per-
turbations along with low frequency vibrational modes with energy
window 7 kcal/mol, and conformational limits of 1000.
3. Results and discussion
3.1. Synthesis
The 2-aminothiophene precursor has been prepared via a multi-
component condensation between appropriate malononitrile, cyclo-
hexanone, sulfur and diethyl amine in conditions represented in the
literature by Gewald [29]. The formation of the pyrimidine ring was
performed by passing a stream of dry HCl gas through a dry dioxane
solution [31]. The phthalimide derivatives 14–21 were synthesized by
reaction of amines 2–13 with phthalic anhydride under reflux.
It is known that 2-aminopyrimidines form a cyclic complex with
acetic acid via hydrogen bonds (Fig. 2) [37]. This leads to tautomerism
and proton transfer in an excited state, resulting in the molecule going
into an imino-form [38,39]. It is known that complex formation is a
temperature-dependent process [40], as with increasing temperature
the quantity of the complex increases. At the same time, it has been
reported that in a polar environment the rising of temperature (energy)
leads to displacement of equilibrium to imino-tautomer [38,41,42].
Based on these facts and the results obtained, it can be concluded
that the low reactivity of 4-amino-5,6,7,8-tetrahydrobenzothieno[2,3-
d]pyrimidines is due to a complex formation and tautomerisation which
obstruct the process of acylation at high temperature. It can be assumed
that the low yield of imide compounds 14–21 is related to the fact that
4-amino-pyrimidine and 2-amino-pyridine derivatives exhibit double
H-bond 1:1 complex formation with acetic acid [37,39].
2.4.2. Receptor preparation
The X-ray crystallographic structure of a complex between DNase I
and the self-complementary octamer duplex d(GGTATACC)2 (PDB code:
1DNK) was obtained from the Protein Data Bank [36]. The errors of the
DNase I were corrected by the Structure Preparation process in MOE.
After the correction, hydrogens were added and partial charges (Gas-
teiger methodology) were calculated. Energy minimization (AM-
BER12:EHT, RMS gradient: 0.100) was performed.
2.4.3. Docking protocol
The molecular docking study was performed using MOE to under-
stand the ligand protein interactions in detail. The Site Finder module
of the MOE was used to identify ligand-binding pocket within the op-
timized structure of DNase I. The default Triangle Matcher placement
method was used for the induced fit docking. GBVI/WSA dG scoring
function which estimates the free energy of binding of the ligand from a
given pose was used to rank the final poses. Each ligand protein com-
plex with lowest relative binding free energy (ΔG) was selected. A more
negative score indicates that ligand is more likely to dock with the
receptor and achieve more favorable interactions.
Spectrophotometric analysis of the 4-aminothiopyrimidine 3 was
performed to establish the possibility of tautomerization of the reagent
in acetic acid (AA) medium. For the experiment, compound 3 was
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BioorganicChemistry80(2018)693–705
Fig. 5. The Pharma RQSAR model of the
investigated series of thieno[2,3-d]pyr-
imidine-4-amines. The attachment positions
on the thieno[2,3-d]pyrimidine scaffold
were labeled with a list of pharmacophore
features, colored by significance: blue for
significant positive contributions, red for
significant negative contributions, and gray
for insignificant contributions to DNase I
inhibition. If a pharmacophore feature was
absent from an attachment position,
a
lower-case letter was used for the pharma-
cophore feature type. Examined pharmaco-
phore features: hydrogen-bond acceptor (A),
hydrogen-bond donor (D), hydrophobic (H),
negatively-charged (N), positively-charged
(P), and aromatic (R).
dissolved in non-polar solvents benzene (ε = 2.3). Fluorescence spectra
were recorded at wavelength of excitation (λex) corresponding to the
absorption maximum of the compound in benzene.
thieno[2,3-d]pyrimidine-4-amines and their phthalimide derivatives is
illustrated in Fig. 4 by compounds 7 and 16 bearing aryl substituent (2-
pyridyl ring) at 2C position of the pyrimidine ring. Fig. S28 in the
Supplementary material provides theoretically calculated bond lengths
of the thienopyrimidine moiety for representative thieno[2,3-d]pyr-
imidine-4-amines and phthalimides within the studied series. The IR
spectra of the thieno[2,3-d]pyrimidine-4-amines in solid state showed
bands within the region 3350–3100 cm−1 confirming the presence of
primary amino groups.
It was observed (Fig. 3) that upon addition of AA to the analyte
solution, the fluorescence band A corresponding to the analyte in a non-
polar solvent was displaced batochromatically with large Stokes shift
(B) and at the same time only insignificant batochromatic shift of ab-
sorbance of 6 nm was observed, which indicates the formation of a
complex with the acetic acid. This result is in accordance with the
spectroscopic evidences for acetic acid-2-iminopyridine complex for-
mation previously reported [37–40].
Considering the above facts, we have set ourselves the aim to create
a new approach for the synthesis of isoindoldiones in order to increase
the yields and reduce the reaction time. The synthesis of isoindoldiones
proceeded in good yields in one step if the interaction between the
amine and phthalic anhydride was carried out by refluxing in toluene
and without isolation of the formed intermediate acid. Acetic anhydride
and pyridine were added and the reaction solution was refluxed 1–3 h
more. After completion of the reaction, water was added, the aqueous
solution was extracted with toluene and the organic layers were dried
and concentrated under reduced pressure. This new approach results in
the production of thienopyrimidine-isoindolones with significantly
higher yields than the reaction in an acetic acid medium, and moreover
the reaction time is repeatedly reduced.
Based on the DFT optimization, the phthalimide fragment in the
thienopyrimidine-isoindolones has almost perpendicular orientation
(68–71°) in respect to the adjacent thienopyrimidine plane (Fig. 4B).
The introduction of the phthalimide fragment leads to alteration of the
bond lengths in the pyrimidine ring. On the other hand, the size and
nature of the substituents attached at 5-position of the pyrimidine ring
has little influence on the structural parameters of the phthalimide
fragment (Fig. S28 Supplementary material). Accordingly, the IR
spectra of all the thienopyrimidine-isoindolones displayed two char-
acteristic bands for the stretching vibrations of phthalimide C]O
groups varying in very narrow intervals – app. 1784 and 1722 cm−1
.
To the best of our knowledge, the synthesis of compounds 5, 6, 8,
10, 12–21 within this paper has been reported for the first time.
3.2. DNase I inhibition
Molecular structure optimization at DFT B3LYP/6-311++G** level
of theory predicted planar structure of the thienopyrimidine moiety
with approximately equal C-N distances in the ring. The structure of the
Seventeen thieno[2,3-d]pyrimidines (2, 3, 5–14, 16 and 18–21)
were tested in vitro on inhibitory activity towards bovine pancreatic
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A. Ts. Mavrova et al.
BioorganicChemistry80(2018)693–705
Fig. 6. The E-State RQSAR model of the investigated series of thieno[2,3-d]pyrimidine-4-amines. The attachment positions on the thieno[2,3-d]pyrimidine scaffold
were labeled with a list of letters representing the E-state atom types (Table S2) colored by significance: blue for significant positive contributions, red for significant
negative contributions, and gray for insignificant contributions to DNase I inhibition. If an E-state atom type was absent from an attachment position, it was not
included in the annotation for that position.
DNase I. The obtained results were plotted and IC50 values were cal-
culated (Table 1). Eight compounds, including five amino (2, 7, 10, 11
and 13) and three phthalimide (16, 18 and 19) derivatives, inhibited
bovine pancreatic DNase I with IC50 values below 200 µM. Phthalimide
derivative 19 and its amine precursor 11 showed the most potent in-
derivatives 7, 10 and 11. However, the examined phthalimides 14 and
21 showed lower DNase I inhibitory activity in respect to the thieno
[2,3-d]pyrimidine-4-amines 2 and 13. No difference in DNase I in-
hibition was observed between the two 3-chlorobenzoyl derivatives 12
and 20. Crystal violet, a known organic DNase I inhibitor [43], was
used as a standard and exhibited weaker inhibitory effect on commer-
hibitory activity against DNase I, with IC50 values of 106
16 and
128 17 μM, respectively (Table 1). From a structural point of view,
cial DNase I (IC50 = 383
49 µM) compared to the studied com-
this affinity could be explained by the presence of p-NO2 group in the
side chain, which may contribute to better binding to the active enzyme
center analogously to N]O derivatives. It can be noted that some of the
phthalimide derivatives (16, 18 and 19) exhibited higher inhibitory
effect towards DNase I in comparison to the structurally related amino
pounds. Furthermore, the same thieno[2,3-d]pyrimidines exhibited a
much higher DNase I inhibitory activity than natural DNase I inhibitor
neomycin (completely inhibiting degradation of plasmid DNA in vitro at
a concentration of 2 mM) [44] and synthetic DNase I inhibitor 2-nitro-
5-thiocyanobenzoic acid (inhibiting DNase I with affinity constant of
Fig. 7. The importance analysis histogram of
the investigated series of thieno[2,3-d]pyr-
imidines.
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A. Ts. Mavrova et al.
BioorganicChemistry80(2018)693–705
cleavage of scissile phosphate. Furthermore, several site-directed mu-
tagenesis experiments on the residues surrounding His 134 and His 252
demonstrated that single mutations on Glu 39 or Asp 168 residues,
resulted in a very low activities on a DNA molecule [47]. The effects of
these mutations also confirmed the active role of Glu 39 and Asp 168 in
the IV catalytic site. In addition, as it was shown in the case of actin,
steric effects may play a crucial role in DNase I inhibition [48].
The interaction profiles of thieno[2,3-d]pyrimidines with DNase I
domain are shown in Figs. 9 and 10 and Table S3. Compound 11, which
corresponds to one of the most promising inhibitors in the non-phtha-
limide series, showed interactions with two catalytic histidines, His 134
and His 252. Furthermore, similar interactions with His 134 or His 252
were also observed for compounds 2, 10 and 13 (Fig. 9, Table S3).
Additionally, these compounds showed H-donor interactions with re-
sidues Glu 39 or Asp 168 (Table S3), which are also implicated in the
cleavage of scissile phosphate [47]. The results from Table S3 indicate
that thieno[2,3-d]pyrimidines 7, 16, 18 and 19, were not found to
possess any similar interactions as previously discussed compounds.
Furthermore, these compounds showed generally increased DNase I
inhibition compared to other thieno[2,3-d]pyrimidines (Table 1). Al-
though interactions with catalytic residues are clearly important de-
terminant of DNase I inhibitors [46,47], the results from Table 1 and
Table S3 suggest that steric effects also play a vital role in DNase I
inhibition [48]. For these reasons, the molecular size and flexibility of
thieno[2,3-d]pyrimidines were calculated, and SAR analysis was ac-
complished. It was found that increase of molecular volume and weight
leads to inhibitory enhancement (Fig. 11), and also confirms the im-
portance of steric effects in DNase I inhibition (Table 1, Table S3).
Fig. 8. The top ranked DNase I binding site, represented by a grey-red surface
map.
approximately 16.7 mM) [45].
3.3. In silico studies
3.3.1. R-Group analysis
As Pharma RQSAR model showed, the presence of aryl substituents
at R1 position of the studied thieno[2,3-d]pyrimidines have substantial
increased DNase I inhibition (Table S1, Fig. 5). Furthermore, hydrogen-
bond acceptor/donor substituents at all examined positions exerted an
insignificant enhancement to the thieno[2,3-d]pyrimidines activity
(Table S1). Additionally, the presence of hydrophobic substituents at R1
position resulted in a decreased DNase I inhibition (Table S1, Fig. 5).
Next, we examined the effect of the E-State parameters of thieno[2,3-d]
pyrimidines on the DNase I inhibition (Table S2, Fig. 6). As Table S2
shows, the introduction of aaCH, aaN, dO and sOm fragments at R1
position, as well as the introduction of aaCH fragment at R2 or R3
position, exerted a significant enhancement to the thieno[2,3-d]pyr-
imidines activity. On the contrary, the use of ssCH2, sCH3, ssO and sCl
fragments at R1 position, resulted in a decreased DNase I inhibition
(Table S2, Fig. 6). Additionally, we wanted to examine how sensitive
DNase I inhibitory property of thieno[2,3-d]pyrimidines was to R group
variation at specific position. As Fig. 7 shows, variation of substituents
at R1 position had substantial effects on DNase I inhibition, compared
to R2 or R3 position.
3.3.3. SAR analysis
Noting that variations of the substituents at 2C position of the
pyrimidine ring lead to slight changes in DNase I inhibition has drawn
our attention to investigate the structure–activity relationship (SAR) of
the amine and phthalimide derivatives that showed DNase I inhibition
with IC50 below 200 µM. The mechanism of DNase I inhibition by
crystal violet is based on its binding to the DNA matrix, thereby in-
hibiting its interaction with the enzyme [49]. Therefore, it is very im-
portant the studied thieno[2,3-d]pyrimidines to possess favourable
pharmacokinetic properties, such as sufficient bioavailability and per-
meability through the different membranes to the desired receptor
binding site, optimal metabolization and elimination profile. Thus, the
calculated molecular properties, including lipophilicity, molecular size,
flexibility and presence of hydrogen bond donors and acceptors could
provide useful informations about SAR. According to the SAR analysis
performed by Molinspiration tool [50], it could be seen that most of the
compounds showing DNase I inhibition with IC50 below 200 µM are in
accordance with Lipinski’s “Rule of five” [51], having logP, MW, NHA
and NHD values less than 5, 500, 10 and 5, respectively (Table 2). It
appeared that compounds 13 and 19 are not in compliance with Li-
pinski’s rule in regard of partition coefficient (logP), but possess rela-
tively high DNase I inhibition activity. However, it has been reported
that the lipophilicity of DNase I inhibitors is extended in a very broad
range [52]. Moreover, all of the tested thienopyrimidine derivatives
possess TPSA values lower than 140–150 Å2 usually required for ac-
ceptable bioavailability (Table 2) [53]. Another important factor for the
optimal bioavailability is moderate conformational flexibility which is
described by the number of rotatable bonds (Nrotb) [54]. None of the
compounds showing DNase I inhibition with IC50 below 200 µM has
more than 10 rotatable bonds (Table 2) which is regarded as another
sign of the expected good oral bioavailability.
3.3.2. Molecular docking
The binding site residues in DNase I have been identified using the
Site Finder module implemented in the MOE software [35]. The results
from the analysis highlighted that amino acid residues like Asn 7, Arg 9,
Glu 39, Tyr 76, Glu 78, Arg 111, His 134, Ala 136, Pro 137, Asp 168,
Asn 170, Thr 203, Thr 205, Thr 207, Tyr 211, Asp 251 and His 252
constituted the binding pocket of the DNase I structure [10]. Our results
are consistent with a recent study highlighting the conservation of the
amino acids involved in the identified cation-binding sites across DNase
I and DNase I-like protein [46]. It is worth mentioning that inhibitor-
binding pocket, represented by a grey-red surface map, is within the
region that interacts with DNA octamer d(GGTATACC)2 (Fig. 8).
The intermolecular contacts between thieno[2,3-d]pyrimidines and
DNase I were analyzed using the ligand interaction diagram of MOE
suite. It illustrates the existence of hydrogen bond, pi-cation and H-pi
interactions (Table S3). Additionally, the bond distances, bond energy
and relative binding free energy between the inhibitor and receptor
atoms were also examined. Of note, the importance of His 134 and His
252 residues in the catalytic mechanism of DNase I has been already
highlighted [46]. It was confirmed that catalytic residues His 134 and
His 252 are a part of the ion binding site IV, which is implicated in the
It was found that the increase of molecular volume (Vol) leads to the
enhancement of DNase I inhibition (Fig. 11A). The same effect was
observed with the increase of sum of H-bond donor (NHD) and H-bond
acceptor (NHA) groups multiplied with the molecular weight (MW)
(Fig. 11B).
Considering toxicological properties calculated by DataWarrior
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BioorganicChemistry80(2018)693–705
Fig. 9. 3D/2D view of compounds 2 (A, B), 7 (C, D), 10 (E, F), 11 (G, H) and 13 (I, J) bound in the active site of DNase I. The polar part of the active site is shown as a
pink surface, hydrophobic part as a green surface, while the solvent exposed part is shown as a red surface.
702

A. Ts. Mavrova et al.
BioorganicChemistry80(2018)693–705
Fig. 10. 3D/2D view of compounds 16 (A, B), 18 (C, D) and 19 (E, F) bound in the active site of DNase I. The polar part of the active site is shown as a pink surface,
hydrophobic part as a green surface, while the solvent exposed part is shown as a red surface.
Fig. 11. SAR relationship of thieno[2,3-d]pyrimidines showing DNase I inhibition with IC50 below 200 µM.
703

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BioorganicChemistry80(2018)693–705
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Table 2
Calculated molecular properties of thieno[2,3-d]pyrimidines showing DNase I
inhibition with IC50 below 200 µM.
logPa
MWb
TPSAc
Vold
NHA
NHD
Nrotb
e
f
g
Comp.
2
7
4.20
3.05
2.74
4.10
5.07
4.85
4.54
5.39
295.4
282.4
233.3
340.4
349.4
412.5
363.4
472.5
51.81
64.70
51.81
97.63
51.81
77.75
64.86
109.29
266.3
245.3
211.5
289.6
280.8
345.4
311.5
395.6
3
4
3
6
3
6
5
5
2
2
2
2
2
0
0
1
2
1
1
3
2
2
2
4
10
11
13
16
18
19
a
Octanol-water partition coefficient, calculated by the methodology devel-
oped by Molinspiration.
b
Molecular weight.
c
Topologic polar surface area [Å2].
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d
Molecular volume [Å3].
Number of hydrogen bond acceptors (O and N atoms).
Number of hydrogen bond donors (OH and NH groups).
Number of rotatable bonds.
e
f
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4. Conclusion
A small library of twelve 2-substituted thieno[2,3-d]pyrimidin-4-
amines were synthesized and used as precursors for preparation of eight
new bipharmacophoric derivatives via cyclocondensation with phthalic
anhydride. Evaluating DNase I inhibitory properties, eight of the amine
and imide derivatives (2, 7, 10, 11, 13, 16, 18 and 19) inhibited bovine
pancreatic DNase I activity in vitro with an IC50 below 200 µM and si-
multaneously exhibited higher inhibitory activity than crystal violet,
used as positive control. Tested phthalimide derivatives 16, 18 and 19
exhibited higher DNase I inhibition than their amine precursors 7, 10
and 11. The Pharma RQSAR model showed a significant enhancement
of thieno[2,3-d]pyrimidines activity using aryl substituents at R1 po-
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fragments relevant for DNase I inhibition. Furthermore, the thieno[2,3-
d]pyrimidines interactions with the most important catalytic residues of
DNase I, including Glu 39, His 134, Asp 168 and His 252, were shown.
It was found that steric effects and increase of molecular volume play a
vital role in DNase I inhibition. These observations could be potentially
utilized to guide the rational design and optimization of new thieno
[2,3-d]pyrimidine inhibitors. As the most potent, compound 19 offers a
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Acknowledgements
The financial support of this work by Science Fund/Chemical
Technology and Metallurgy University (Grant 11523), Ministry of
Education, Science and Technological Development of the Republic of
Serbia (Grants No. OI 172044, OI 171025 and TR 31060) and Faculty of
Medicine of the University of Niš (Internal project No. 4) is gratefully
acknowledged. The authors would like to thank Chemical Computing
Group and Schrödinger LLC., for providing us the academic licenses free
of cost for this study.
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Conflict of interest
The authors declare that there are no conflicts of interest.
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