Design and synthesis of novel protein kinase CK2 inhibitors on the base of 4-aminothieno[2,3-d]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2016.03.004

An extension of our previous research work has resulted in a number of new ATP-competitive CK2 inhibitors that have been identified among 4-aminothieno[2,3-d]pyrimidine derivatives. The most active compounds obtained in the course of the research are 3-(5-p-tolyl-thieno[2,3-d]pyrimidin-4-ylamino)-benzoic acid, 5e (NHTP23, IC₅₀ = 0.01 μM), 3-(5-phenyl-thieno[2,3-d]pyrimidin-4-ylamino)-benzoic acid, 5g (NHTP25, IC₅₀ = 0.065 μM) and 3-(6-methyl-5-phenyl-thieno[2,3-d]pyrimidin-4-ylamino)-benzoic acid, 5n (NHTP33, IC₅₀ = 0.008 μM). Structure-activity relationships of the tested 4-aminothieno[2,3-d]pyrimidine derivatives have been studied and their binding mode with ATP-acceptor site of CK2 has been proposed. A negative effect of intramolecular hydrogen bonding in the compounds' structure is discussed.

Full text of DOI:10.1016/j.ejmech.2016.03.004

Accepted Manuscript
Design and synthesis of novel protein kinase CK2 inhibitors on the base of 4-
aminothieno[2,3-d]pyrimidines
Olga V. Ostrynska, Anatoliy O. Balanda, Volodymyr G. Bdzhola, Andriy G. Golub, Igor
M. Kotey, Olexander P. Kukharenko, Andrii A. Gryshchenko, Nadiia V. Briukhovetska,
Sergiy M. Yarmoluk
PII:
S0223-5234(16)30178-7
10.1016/j.ejmech.2016.03.004
EJMECH 8429
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2015
Revised Date: 20 January 2016
Accepted Date: 2 March 2016
Please cite this article as: O.V. Ostrynska, A.O. Balanda, V.G. Bdzhola, A.G. Golub, I.M. Kotey, O.P.
Kukharenko, A.A. Gryshchenko, N.V. Briukhovetska, S.M. Yarmoluk, Design and synthesis of novel
protein kinase CK2 inhibitors on the base of 4-aminothieno[2,3-d]pyrimidines, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.004.
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ACCEPTED MANUSCRIPT
Design and synthesis of novel protein kinase CK2 inhibitors on the
base of 4-aminothieno[2,3-d]pyrimidines
Olga V. Ostrynskaa, Anatoliy O. Balandaa, Volodymyr G. Bdzholaa, Andriy G.
Golubb, Igor M. Koteya, Olexander P. Kukharenkoa, Andrii A. Gryshchenkoa, Nadiia V.
Briukhovetskaa, Sergiy M. Yarmoluka, *
a
Department of Medicinal Chemistry, Institute of Molecular Biology and Genetics
of National Academy of Sciences of Ukraine, 150 Zabolotnogo str., 03680 Kyiv, Ukraine
b
Otava Ltd, 400 Applewood Crescent, Unit 100, Vaughan, Ontario L4K 0C3,
Canada
* Corresponding author. E-mail address: sergiy@yarmoluk.org.ua (S. M.
Yarmoluk)
Abstract. An extension of our previous research work has resulted in a number of
new ATP-competitive CK2 inhibitors that have been identified among 4-aminothieno[2,3-
d]pyrimidine derivatives. The most active compounds obtained in the course of the
research are 3-(5-p-tolyl-thieno[2,3-d]pyrimidin-4-ylamino)-benzoic acid, 5e (NHTP23,
IC50 = 0.01 µM), 3-(5-phenyl-thieno[2,3-d]pyrimidin-4-ylamino)-benzoic acid, 5g
(NHTP25, IC50 = 0.065 µM) and 3-(6-methyl-5-phenyl-thieno[2,3-d]pyrimidin-4-
ylamino)-benzoic acid, 5n (NHTP33, IC50 = 0.008 µM). Structure–activity relationships
of the tested 4-aminothieno[2,3-d]pyrimidine derivatives have been studied and their
binding mode with ATP-acceptor site of CK2 has been proposed. A negative effect of
intramolecular hydrogen bonding in the compounds’ structure is discussed.

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Keywords: Protein kinase CK2; Inhibitor; Aminothieno[2,3-d]pyrimidine; Drug
design; Docking; Intramolecular hydrogen bond.
1. Introduction. Protein kinase CK2 is a well-known highly conserved and
constitutively active serine/threonine protein kinase that plays an important role in many
cellular processes, such as cell cycle progression, apoptosis, cell differentiation and
transcription. Overexpression and hyperactivation of this enzyme has been observed in a
wide variety of human diseases [1]. It has been proven that CK2 inhibition is critical for
treatment and prevention of inflammatory processes, infection and cancer. The history of
CK2 as a drug target began with the identification of its inhibitor DRB in 1986 [2]. To
date, a significant number of active CK2 inhibitors from different chemical classes have
been developed [3-4] (the most famous and active ones are presented in Table 1).
However, only one compound (CX-4945 [5], IC50=1 nM) has entered phase ІІ clinical
trials so far (ClinicalTrials.gov Identifier: NCT02128282). Therefore, discovery of
specific, low toxic and highly active inhibitors of protein kinase CK2 is important for
development of novel therapeutic agents.
Taking into consideration the significance of this matter we have been actively
searching for CK2 inhibitors during the last decade [6] and identified potent compounds
among different chemical classes (Table 1) such as 3-carboxy-4(1H)-quinolone derivatives
[7], 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones [8], flavone derivatives [9, 10] and
(thieno[2,3-d]pyrimidine-4-ylthio)carboxylic acid derivatives [11]. High CK2 inhibitory
activity of the compounds from the last class induced us to study thieno[2,3-d]pyrimidines

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thoroughly. In this work, we focus on the synthesis and evaluation of 4-aminothieno[2,3-
d]pyrimidine derivatives.
Table 1
Chemical structure and inhibitory activity of well-known inhibitors of protein kinase
CK2
I
OH
Cl
Cl
N
N
OH
O
OH
I
I
N
HO
O
N
N
O
HO
N
H
O
OH
O
OH
HO
I
DRB [2]
ТІВІ [12]
Quinalizarin [13]
IQA [14]
Ki = 23 ꢀM
Кі = 0.023 ꢀM
Ki = 0.06ꢀM
Кі = 0.17 ꢀM
Cl
OH
O
O
O
O
OH
OH
CN
HN
O
CH3
HN
N
N
HO
O
OH
N
N
OH
N
N
N
O
HO
O
H
H
OH
OH
N
Fisetin [15]
Кі = 0.17 ꢀM
Ellagic acid [16]
Ki = 0.06 ꢀM
Pyrazolotriazine 15а [17]
СХ-4945 [5]
Кі = 0,00038 ꢀM
Кі = 0,00026 ꢀM
O
O
I
Cl
O
O
Br
O
OH
H3C
I
I
Cl
CH3
N
OH
O
S
Br
OH
N
OH
N
O
O
Cl
I
O
S
H3C
N
Quinolone 7 [7]
TID 46 [8]
FNH79 [10]
Compound 6a [11]
Ki = 0.06 ꢀM
Ki = 0.1 ꢀM
Ki = 0.0018 ꢀM
Ki = 0.04 ꢀM
2. Experimental section
2.1. Molecular docking
Ligands were prepared for docking with energy minimization and converted into
pdbqt format with Vega ZZ [18]. Docking was done in crystal structure of CK2 (PDB ID:

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4GRB [19]) using AutoDock 4.2 with default parameters. The crystal structure chosen for
the docking is one of the recent human CK2-inhibitor complexes resolved. It has sufficient
resolution, (2.15 Å), doesn’t contain mistakes or sequence alterations, and the conservative
water molecule W1 located near Lys68 in ATP-binding site is preserved. Resulted docking
complexes were analysed with AutoDockTools-1.5.6 [20].
2.2. Biology
Сompounds were tested using in vitro kinase assay[21]. Each test was done in
triplicate in a reaction volume of 30 ꢀL, containing 6 ꢀg of peptide substrate
RRRDDDSDDD (New England Biolabs); 10 units of recombinant human CK2
32
holoenzyme (New England Biolabs); 50 ꢀM ATP and γ-labeled P ATP, diluted to
specific activity 100 ꢀCi/ꢀM; CK2 buffer (20 mM Tris-HCl, pH 7.5; 50 mM KCl; 10 mM
MgCl2) and inhibitor in varying concentrations. Incubation time was 20 min at 30o C. The
reaction was stopped by adding an equal volume of 10 % o-phosphoric acid and the
reaction mixture was loaded onto 20-mm discs of phosphocellulose paper (Whatman).
Disks were washed three times with 1 % o-phosphoric acid solution, air-dried at room
temperature, and counted by the Cherenkov method in a beta-counter (LKB). As negative
control an equal volume of DMSO was added to the reaction mixture. Percent inhibition
was calculated as ratio of substrate-incorporated radioactivity in the presence of inhibitor
to the radioactivity incorporated in control reactions, i.e. in the absence of inhibitor. Serial
dilutions of inhibitor stock solution were used to determine its IC50 concentration. The IC50
values represent means of triplicate experiments with SEM never exceeding 15%.

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Selectivity tests were performed on protein kinases ASK1, JNK3, c-Met, Aurora А,
ROCK1, FGFR1 and Tie2 according to the supplier’s recommendations (Millipore).
Kinase residual activity, determined in the presence of 10 µM of inhibitor is expressed as a
percentage of the control without inhibitor. Final concentration of ATP in the experiment
was 100 µM.
2.3. Chemistry
All studied 4-aminothieno[2,3-d]pyrimidine derivatives were synthesized starting
from the corresponding carbonyl compounds, according to Scheme 1. The first three steps
of chemical synthesis were described by our research group previously [22].
Nuclear magnetic resonance spectra were recorded with Varian Mercury VRX-400
spectrometer using DMSO-d6 as solvent and tetramethylsilane as internal standard.
Chemical shift values (δ) are quoted in ppm, and coupling constants (J) in Hz.
2.3.1. Substituted 2-amino-3-carbethoxythiophene (a). A mixture of ethyl
cyanoacetate (107 mL, 1 mol), elemental sulfur (32 g, 1 mol) and the corresponding
ketone (1 mol) in ethanol (200 mL) was stirred at room temperature. To this mixture,
diethylamine (80 mL) was added dropwise during 12 h with stirring. Then the reaction
mixture was left on 14 hours; the precipitate was filtered, washed by aqueous alcohol (1:1)
and dried in air. Yield 50-88%.
2.3.2. Substituted thienopyrimidinones (b). A mixture of formamide (2 mol) and 2-
aminothiophene (1 mol) was heated at 160-170o C for 24 h. The hot mixture was diluted

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with isopropyl alcohol (80 mL) and cooled. The solid product was collected by filtration
and washed with isopropyl alcohol (60 mL) and water (60 mL) and dried at 60o C. Yield
85-90%.
2.3.3. Substituted thienopyrimidinones (c). Corresponding aminothiophene (0.01
mol) was supplemented with dioxane (30 mL), acetonitrile (0.85 mL) and dioxane (30
mL) saturated with hydrogen chloride. The mixture has been stirred for 12 h at room
temperature and neutralized by ammonia. The solid product was filtered, washed with
isopropanol alcohol (4 mL) and water (10 mL). Yield 85- 90%.
2.3.4. Substituted 4-chloro-thieno[2,3-d]pyrimidines (d). A mixture of
thienopyrimidinone (0.01 mol), POCl3 (11 mL) and PCl5 (1.5 g) was quickly heated to
105o C and boiled for 5-6 hours. The solvent was evaporated. Dry product was dissolved
in methylene chloride (12 mL) and neutralized by cold solution of NaOH (0.5 N). The
organic phase was separated and dried over Na2SO4, filtered, and then solvent was
evaporated in vacuum. The residue was crystallized from isopropyl alcohol. Yield 80%.
2.3.5. Substituted 4-aminothieno[2,3-d]pyrimidine (e). A mixture of corresponding
amino acid (0.011 mol) dissolved in the minimum amount of water, ethanol (15 mL),
triethylamine (0.5 mL) and starting compound (0.01 mol) was heated to 100o C and boiled
for 24 h. The solvent was evaporated; the residue was recrystallized from ethanol and
water. Yield 30-80%.
2.3.6. Substituted 4-aminothieno[2,3-d]pyrimidine (f). A mixture of 4-
chlorothieno[2,3-d]pyrimidine (0.01 mol), aminobenzoic acid (0.011 mol) and DMF
(dimethylformamide) (2 mL) was heated to 150o C and boiled for 6 hours. The precipitate
was filtered and washed with acetone (4 mL). To obtain corresponding bases,

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hydrochloride was dissolved in ethanol and supplemented with triethylamine (0.01 mol).
Yield 60-80%.
O
R1
ii for R3 = H
iii for R3 = CH3
O
N
R1
R1
O
O
i
O
+
NH
O
N
R2
R2
R2
NH2
S
R3
S
2
1
R4
R1
HN
N
R2
v
S
R3
N
R1
Cl
N
4a-w
iv
N
R2
R5
R4
S
R3
R1
S
N
3
vi
R2
N
N
R3
5a-p
Scheme 1. Scheme of synthesis for compounds 4a-w, 5a-p. Reagents and
conditions: (i) S, (C2H5)2NH, C2H5OH, r.t., 12 h, 50-88 %; (ii) HCONH2, 160-170o C, 24
h, 85-90%; (iii) CH3CN, dioxane, HCl, r.t., 12 h, 85-90 %; (iv) PCl5, POCl3, 105o C, 5-6 h,
80 %; (v) R4NH2, H2O, C2H5OH, Et3N, 100o C, 24 h, 30-80 %; (vi) R4NH2, DMF, 150o C,
6 h, 60-80 %.
Table 2
Structure and inhibitory activity of 4-aminothieno[2,3-d]pyrimidine derivatives
prior to chemical optimization

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R4
R1
HN
5
4
R2
N
6
3
S
2
1 N
R3
IC50,
ꢀM
Compound №
R1
R2
R3
R4
OH
4a
Н
Н
O
O
>33
9.5
Hetaryl
Hetaryl
heteroatom
OH
4b
heteroatom
heteroatom
heteroatom
OH
OH
OH
4c
4d
СН3
СН3
Н
Н
O
O
O
>33
>33
Hetaryl
4e
4f
Н
Н
Н
16
Hetaryl
heteroatom
heteroatom
heteroatom
OH
OH
OH
OH
O
O
O
O
СН3
СН3
>33
>33
4g
H
H
4h
4i
Н
Н
>33
>33
heteroatom
heteroatom
O
СН3
O

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OH
OH
O
O
4j
H
Н
>33
>33
F
heteroatom
heteroatom
4k
H
Н
OH
O
4l
H
Н
>33
heteroatom
OH
4m
4n
H
H
СН3
Н
Н
O
>33
>33
heteroatom
OH
O
heteroatom
OH
OH
O
4o
H
H
Н
>33
heteroatom
O
H2N
OH
4p
4q
CH2-CH3
H
Н
Н
O
O
>33
>33
heteroatom
heteroatom
O
O
OH
OH
O
4r
H
СН3
>33
heteroatom
O
H2N
Table 3
Structure and inhibitory activity of 4-aminothieno[2,3-d]pyrimidine derivatives after
chemical optimization

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R5
R4
R1
N
5
4
R2
N
6
3
S
2
1 N
R3
IC50,
ꢀM
Compound №
R1
R2
R3
R4
R5
heteroatom
4s
H
Н
>33
>33
>33
O
O
HO
HO
heteroatom
4t
H
H
СН3
СН3
O
OH
4u
heteroatom
O
OH
4v
H
СH3
>33
>33
heteroatom
4w
СН3
COOH
СН3
heteroatom
O
OH
5a
Н
H
>33
Hetaryl
heteroatom

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O
O
OH
OH
5b
5c
СН3
СН3
Н
Н
Н
3.8
heteroatom
H
Н
0.26
heteroatom
O
OH
5d [22]
H
H
H
H
H
Н
Н
Н
Н
Н
Н
Н
Н
Н
Н
0.65
0.01
1.1
heteroatom
O
OH
5e [22]
(NHTP23)
heteroatom
O
OH
5f
5g (NHTP25)
5h
heteroatom
O
OH
0.065
0.83
heteroatom
O
OH
Cl
heteroatom

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O
OH
5i
H
СН3
Н
>33
heteroatom
O
OH
OH
5j
H
СН3
Н
Н
1.5
6.5
heteroatom
O
O
O
СН3
СН3
5k [22]
СН3
Н
Н
heteroatom
O
OH
5l
Н
Н
1.5
heteroatom
OH
O
O
OH
5m
H
Н
>33
heteroatom
O
OH
5n (NHTP33)
СН3
СН3
Н
Н
Н
Н
0.008
0.31
heteroatom
OH
O
OH
5o
heteroatom

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O
OH
5p
СН3
COOH
Н
H
>33
heteroatom
2.3.1.
N-(5,6,7,8-Tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)glycine
1
hydrochloride (4a) H NMR (400 MHz, DMSO-d6): δ: 1.82 (4H, s, CH2), 2.76 (2H, s,
CH2), 2.92 (2H, s, CH2), 4.11 (2H, d, CH2), 6.89 (1H, s, NH), 8.25 (1H, s, 2-H), 12.60 (bs,
13
COOH). C NMR (100 MHz, DMSO-d6): δ: 22.27, 25.24, 26.02, 42.82, 115.92, 126.73,
132.11, 152.84, 156.84, 164.97, 171.98. ). Anal. Calc. for C12H14ClN3O2S: C, 48.08; H,
4.71; S, 10.70. Found: C, 48.04; H, 4.68; S, 10.67.
2.3.2.
N-(5,6,7,8-Tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)-β
-alanine
1
hydrochloride (4b) H NMR (400 MHz, DMSO-d6): δ: 1.81 (4H, s, CH2), 2.42-2.46 (2H,
m, CH2), 2.74 (2H, s, CH2), 2.91 (2H, s, CH2), 3.60-3.63 (2H, m, CH2), 6.88 (1H, s, NH),
8.25 (1H, s, 2-H), 12.64 (bs, COOH). ). Anal. Calc. for C13H16ClN3O2S: C, 49.76; H, 5.14;
S, 10.22. Found: C, 49.73; H, 5.13; S, 10.19.
1
2.3.3. N-(5,6-Dimethylthieno[2,3-d]pyrimidin-4-yl)glycine hydrochloride (4c) H
NMR (400 MHz, DMSO-d6): δ: 2.39 (3H, s, CH3), 2.44 (3H, s, CH3), 4.12 (2H, s, CH2),
7.05 (1H, s, NH), 8.24 (1H, s, 2-H), 12.60 (bs, COOH). 13C NMR (100 MHz, DMSO-d6):
δ: 13.39, 14.40, 42.88, 116.95, 124.61, 128.98, 152.68, 156.88, 164.25, 172.00. Anal.
Calc. for C10H12ClN3O2S: C, 43.88; H, 4.42; S, 11.71. Found: C, 43.86; H, 4.41; S, 11.69.
2.3.4. N-(7-Methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)glycine
1
hydrochloride (4d) H NMR (400 MHz, DMSO-d6): δ: 1.05 (3H, d, CH3), 1.43-1.48(1H,
m, CH), 1.89-1.94 (2H, m, CH2), 2.33-2.40 (1H, m, CH), 2.82-3.04 (3H, m, CH), 4.10
(2H, d, CH2), 6.89 (1H, s, NH), 8.24 (1H, s, 2-H). 13C NMR (100 MHz, DMSO-d6): δ:
21.38, 25.75, 28.68, 30.32, 33.15, 42.88, 115.78, 126.38, 131.68, 152.87, 156.81, 165.08,
171.98. Anal. Calc. for C13H16ClN3O2S: C, 49.76; H, 5.14; S, 10.22. Found: C, 49.74; H,
5.13; S, 10.20.
2.3.5.
N-(7-Methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)-β-
1
alanine hydrochloride (4e) H NMR (400 MHz, DMSO-d6): δ: 1.03 (3H, d, CH3), 1.39-

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1.45 (1H, m, CH), 1.85-1.90 (2H, m, CH2), 2.30-2.37 (1H, m, CH), 2.56-2.60 (2H, m,
CH2), 2.78-3.00 (3H, m, CH), 3.67-3.71 (2H, m, CH2), 6.63 (1H, s, NH), 8.25 (1H, s, 2-H),
13
12.22 (bs, COOH). C NMR (100 MHz, DMSO-d6): δ: 21.35, 25.56, 28.64, 30.32, 33.13,
33.78, 36.78, 115.73, 126.38, 131.35, 153.07, 156.92, 164.94, 173.83. Anal. Calc. for
C14H18ClN3O2S: C, 51.29; H, 5.53; S, 9.78. Found: C, 51.28; H, 5.52; S, 9.76.
2.3.6. N-(5,6-Dimethylthieno[2,3-d]pyrimidin-4-yl)-β-alanine hydrochloride (4f) 1H
NMR (400 MHz, DMSO-d6): δ: 2.36 (3H, s, CH3), 2.40 (3H, s, CH3), 2.59 (2H, t, CH2),
13
3.67-3.71 (2H, m, CH2), 6.79(1H, s, NH), 8.26 (1H, s, 2-H), 12.33 (bs, COOH). C NMR
(100 MHz, DMSO-d6): δ: 13.35, 14.27, 33.74, 36.82, 116.87, 124.63, 128.61, 152.94,
156.94, 164.09, 173.85. Anal. Calc. for C11H14ClN3O2S: C, 45.91; H, 4.90; S, 11.14.
Found: C, 45.90; H, 4.88; S, 11.12.
2.3.7. N-[5-(4-Methylphenyl)thieno[2,3-d]pyrimidin-4-yl]-β-alanine hydrochloride
(4g) 1H NMR (400 MHz, DMSO-d6): δ: 2.38 (3H, s, CH3), 2.43-2.45 (2H, m, CH2), 3.56-
3.58 (2H, m, CH2), 5.65 (1H, s, NH), 7.31 (4H, s, C6H4), 7.41 (1H, s, 6-H), 8.41 (1H, s, 2-
13
H), 12.27 (bs, COOH). C NMR (100 MHz, DMSO-d6): δ: 21.27, 33.55, 36.52, 113.65,
121.06, 129.00, 129.93, 132.74, 134.96, 138.14, 153.89, 157.07, 166.77, 173.47. Anal.
Calc. for C16H16ClN3O2S: C, 54.93; H, 4.61; S, 9.17. Found: C, 54.92; H, 4.59; S, 9.15.
1
2.3.8. N-(5-Phenylthieno[2,3-d]pyrimidin-4-yl)-β-alanine hydrochloride (4h) H
NMR (400 MHz, DMSO-d6): δ: 2.43-2.46 (2H, m, CH2), 3.56-3.60 (2H, m, CH2), 5.61
13
(1H, s, NH), 7.45-7.51 (6H, m, aromatic-H), 8.42 (1H, s, 2-H), 12.29 (bs, COOH). C
NMR (100 MHz, DMSO-d6): δ: 33.51, 36.51, 113.60, 121.43, 128.80, 129.14, 129.37,
134.98, 135.65, 153.93, 157.05, 166.82, 173.48. Anal. Calc. for C15H14ClN3O2S: C, 53.65;
H, 4.20; S, 9.55. Found: C, 53.64; H, 4.18; S, 9.51.
2.3.9.
N-[6-(Ethoxycarbonyl)-5-methylthieno[2,3-d]pyrimidin-4-yl]glycine
hydrochloride (4i) 1H NMR (400 MHz, DMSO-d6): δ: 1.30 (3H, t, J = 7.1 Hz, CH3, C2H5),
2.89 (3H, s, CH3), 4.15 (2H, d, CH2),4.29 (2H, q, J = 7.1 Hz, CH2, C2H5), 7.48 (1H, s,
13
NH), 8.39 (1H, s, 2-H), 12.61 (bs, COOH). C NMR (100 MHz, DMSO-d6): δ: 14.47,
15.89, 43.04, 61.59, 117.00, 139.83, 155.82, 158.54, 162.45, 166.61, 171.52. Anal. Calc.
for C12H14ClN3O4S: C, 43.44; H, 4.25; S, 9.66. Found: C, 43.42; H, 4.24; S, 9.63.

ACCEPTED MANUSCRIPT
1
2.3.10. N-[5-(4-Fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]-β-alanine (4j) H NMR
(400 MHz, DMSO-d6): δ: 2.43-2.46 (2H, m, CH2), 3.58-3.61 (2H, m, CH2), 5.70 (1H, s,
NH), 7.31 (2H, t, C6H4), 7.48 (3H, s, C6H4, 6-H), 8.41 (1H, s, 2-H), 12.32 (bs, COOH). 13C
NMR (100 MHz, DMSO-d6): δ: 33.53, 36.47, 113.57, 116.11, 116.33, 121.64, 131.36,
131.93, 133.90, 153.95, 157.01, 161.28, 163.72, 166.81, 173.68. Anal. Calc. for
C15H12FN3O2S: C, 56.77; H, 3.81; S, 10.10. Found: C, 56.75; H, 3.80; S, 10.9.
2.3.11.
N-[5-(3,4-Dimethylphenyl)thieno[2,3-d]pyrimidin-4-yl]glycine
1
hydrochloride (4k) H NMR (400 MHz, DMSO-d6): δ: 2.32 (6H, s, 2CH3), 4.03 (2H, d,
CH2), 5.85 (1H, s, NH), 7.16-7.25 (4H, m, C6H3(CH3)2, 6-H), 8.30 (1H, s, 2-H). 13C NMR
(100 MHz, DMSO-d6): δ: 19.59, 42.81, 113.69, 121.17, 126.40, 130.18, 130.50, 133.11,
135.12, 136.82, 137.53, 153.72, 156.93, 166.91, 171.49. Anal. Calc. for C16H16ClN3O2S:
C, 54.93; H, 4.61; S, 9.17. Found: C, 54.92; H, 4.60; S, 9.15.
2.3.12.
4-{[5-(3,4-Dimethylphenyl)thieno[2,3-d]pyrimidin-4-yl]amino}butanoic
acid hydrochloride (4l) 1H NMR (400 MHz, DMSO-d6): δ: 1.62 (2H, t, CH2), 2.10 (2H, t,
CH2), 2.33 (6H, s, 2CH3), 3.42 (2H, t, CH2), 5.24 (1H, s, NH), 7.19-7.22 (4H, m,
C6H3(CH3)2, 6-H), 8.30 (1H, s, 2-H), 11.98 (bs, COOH). Anal. Calc. for C18H20ClN3O2S:
C, 57.21; H, 5.33; S, 8.48. Found: C, 57.20; H, 5.31; S, 8.46.
1
2.3.13. N-(6-Methylthieno[2,3-d]pyrimidin-4-yl)glycine (4m) H NMR (400 MHz,
DMSO-d6): δ:2.53 (3H, s, CH3), 4.11 ( 2H, d, CH2), 7.27 (1H, s, NH), 8.20 (1H, s, 5-H),
13
8.27 (1H, s, 2-H),12.63 (bs, COOH). C NMR (100 MHz, DMSO-d6): δ: 16.33, 42.22,
116.93, 136.63, 153.21, 156.26, 165.39, 172.03. Anal. Calc. for C9H9N3O2S: C, 48.42; H,
4.06; S, 14.36. Found: C, 48.40; H, 4.02; S, 14.32.
1
2.3.14. N-(6-Phenylthieno[2,3-d]pyrimidin-4-yl)serine (4n) H NMR (400 MHz,
DMSO-d6): δ: 3.89-3.91 ( 2H, m, CH2), 4.80-4.81 (1H, m, CH), 5.16 (bs, OH), 7.37-7.41
(1H, m, C6H5), 7.48-7.52 (2H, m, C6H5), 7.70-7.72 (2H, m, C6H5), 8.04 (1H, d, NH), 8.29
13
(1H, s, 5-H), 8.34 (1H, s, 2-H), 12.74 (bs, COOH). C NMR (100 MHz, DMSO-d6): δ:
56.65, 61.57, 116.08, 117.97, 125.98, 128.96, 129.75, 133.53, 138.66, 154.00, 156.77,
165.50, 172.45. Anal. Calc. for C15H13N3O3S: C, 57.13; H, 4.16; S, 10.17. Found: C,
57.10; H, 4.14; S, 10.15.

ACCEPTED MANUSCRIPT
1
2.3.15. N2-(6-Phenylthieno[2,3-d]pyrimidin-4-yl)asparagine (4o) H NMR (400
MHz, DMSO-d6): δ: 2.67-2.81 (2H, m, CH2), 5.04-5.08 (1H, m, CH), 6.98 (1H, s, C6H5),
7.37-7.51 (4H, m, C6H5), 7.69 (2H, d, NH2), 8.10 (1H, s, 5-H), 8.16 (1H, d, NH), 8.35 (1H,
13
s, 2-H), 12.84 (bs, COOH). C NMR (100 MHz, DMSO-d6): δ: 37.09, 50.61, 115.66,
117.88, 126.04, 128.99, 129.75, 133.46, 138.82, 154.02, 156.52, 165.51, 171.45, 173.65.
Anal. Calc. for C16H14N4O3S: C, 56.13; H, 4.12; S, 9.37. Found: C, 56.10; H, 4.09; S, 9.35.
1
2.3.16. N-(6-Ethylthieno[2,3-d]pyrimidin-4-yl)glycine (4p) H NMR (400 MHz,
DMSO-d6): δ: 1,35 (3H, t, J = 7.3 Hz, CH3, C2H5), 2.90 (2H, q, J = 7.3 Hz, CH2, C2H5),
4.10 (2H, d, CH2), 7.28(1H, s, 5-H), 8.01 (1H, t, NH), 8.19 (1H, s, 2-H), 12.37 (bs,
COOH). Anal. Calc. for C10H11N3O2S: C, 50.62; H, 4.67; S, 13.51. Found: C, 50.59; H,
4.64; S, 13.49.
1
2.3.17. N-[5-(3,4-Dimethoxyphenyl)thieno[2,3-d]pyrimidin-4-yl]-β-alanine (4g) H
NMR (400 MHz, DMSO-d6): δ: 2.42-2.46 (2H, m, CH2), 3.60-3.63 (2H, m, CH2), 3.82
(3H, s, OCH3), 3.85 (3H, s, OCH3), 5.73-5.77 (1H, s, 6-H), 6.90-7.03 (3H, m,
C6H3(OCH3)2), 7.24 (1H, s, NH), 8.31 (1H, s, 2-H),12.12 (bs, COOH). Anal. Calc. for
C17H17N3O4S: C, 56.81; H, 4.77; S, 8.92. Found: C, 56.79; H, 4.74; S, 8.90.
2.3.18. N2-(2-Methyl-6-phenylthieno[2,3-d]pyrimidin-4-yl)asparagine (4r) 1H NMR
(400 MHz, DMSO-d6): δ: 2.45 (3H, s, CH3), 2.71-2.73 (2H, m, CH2), 5.02-5.07 (1H, m,
CH), 6.82 (1H, s, C6H5), 7.32-7.44 (4H, m, C6H5), 7.66 (2H, d, NH2), 7.82 (1H, d, NH),
7.99 (1H, s, 5-H), 12.64 (bs, COOH). ). Anal. Calc. for C17H16N4O3S: C, 57.29; H, 4.53; S,
9.00. Found: C, 57.25; H, 4.51; S, 9.00.
1
2.3.19. 1-(6-Penylthieno[2,3-d]pyrimidin-4-yl)proline (4s) H NMR (400 MHz,
DMSO-d6): δ: 2.14-2.33 (4H, m, H-proline), 4.04-4.16 (2H, m, H-proline), 4.70-4.78 (1H,
m, H-proline), 7.34 (1H, t, C6H5), 7.43 (2H, t, C6H5), 7.72 (2H, d, C6H5), 7.85 (1H, s, 5-H),
8.25 (1H, s, 2-H),12.44 (bs, COOH). Anal. Calc. for C17H15N3O2S: C, 62.75; H, 4.65; S,
9.85. Found: C, 62.73; H, 4.62; S, 9.83.
1
2.3.20. 1-(2-Methyl-6-phenylthieno[2,3-d]pyrimidin-4-yl)proline (4t) H NMR (400
MHz, DMSO-d6): δ: 2.11-2.31(4H, m, H-proline), 2.43 (3H, s, CH3), 4.01-4.13 (2H, m, H-
proline), 4.71-4.77 (1H, m, H-proline), 7.32 (1H, t, C6H5), 7.42 (2H, t, C6H5), 7.70 (2H, d,

ACCEPTED MANUSCRIPT
C6H5), 7.79 (1H, s, 5-H), 12.33 (bs, COOH). 13C NMR (100 MHz, DMSO-d6): δ: ). Anal.
Calc. for C18H17N3O2S: C, 63.70; H, 5.05; S, 9.45. Found: C, 63.69; H, 5.01; S, 9.43.
2.3.21.
1-(2-Methyl-6-phenylthieno[2,3-d]pyrimidin-4-yl)piperidine-3-carboxylic
1
acid (4u) H NMR (400 MHz, DMSO-d6): δ: 1.63-1.69 (1H, m, H-piperidine), 1.83-1.90
(2H, m, H-piperidine), 2.03-2.08 (1H, m, H-piperidine), 2.49 (3H, s, CH3), 2.60-2.65 (1H,
m, H-piperidine), 3.39-3.52 (2H, m, H-piperidine),4.22-4.26 (1H, m, H-piperidine), 4.45-
4.48 (1H, m, H-piperidine), 7.34-7.45 (3H, m, C6H5), 7.71 (2H, d C6H5), 7.81 (1H, s, 5-H),
12.37 (bs, COOH). Anal. Calc. for C19H19N3O2S: C, 64.57; H, 5.42; S, 9.07. Found: C,
64.55; H, 5.41; S, 9.03.
2.3.22.
1-(2-Methyl-6-phenylthieno[2,3-d]pyrimidin-4-yl)piperidine-4-carboxylic
1
acid (4v) H NMR (400 MHz, DMSO-d6): δ: 1.69-1.78 (2H, m, H-piperidine),1.98-
2.02(2H, m, H-piperidine), 2.47 (3H, s, CH3), 2.55-2.61(1H, m, H-piperidine), 3.28-3.35
(2H, m, H-piperidine), 4.49-4.52 (2H, m, H-piperidine), 7.32 (1H, t, C6H5), 7.41 (2H, t,
C6H5), 7.68 (1H, s, 5-H), 7.72 (2H, d, C6H5), 12.16 (bs, COOH). Anal. Calc. for
C19H19N3O2S: C, 64.57; H, 5.42; S, 9.07. Found: C, 64.54; H, 5.41; S, 9.05.
2.3.23. 2,5-Dimethyl-4-piperidin-1-ylthieno[2,3-d]pyrimidine-6-carboxylic acid
1
(4w) H NMR (400 MHz, DMSO-d6): δ: 1.64-1.70 (6H, m, H-piperidine), 2.49 (3H, s,
CH3), 2.72 (3H, s, CH3), 3.36-3.40 (4H, m, H-piperidine), 13.08 (bs, COOH). Anal. Calc.
for C14H17N3O2S: C, 57.71; H, 5.88; S, 11.00. Found: C, 57.69; H, 5.86; S, 11.00.
2.3.24.
4-[(7-Methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-
yl)amino]benzoic acid hydrochloride (5a) 1H NMR (400 MHz, DMSO-d6): δ: 1.06 (3H, d,
CH3), 1.42-1.48 (1H, m, CH), 1.91-1.94 (2H, m, CH2), 2.37-2.44 (1H, m, CH), 2.87-2.90
(1H, m, CH), 3.14-3.19 (2H, m, CH2), 7.79 (2H, d, J = 8.3 Hz, C6H4), 7.90 (2H, d, J = 8.3
13
Hz, C6H4), 8.44 (1H, s, NH), 8.46 (1H, s, 2-H), 12.66 (bs, COOH). C NMR (100 MHz,
DMSO-d6): δ: 21.37, 25.24, 28.70, 30.33, 33.33, 117.82, 120.40, 124.79, 126.55, 130.38,
133.70, 144.04, 152.23, 154.45, 166.77, 167.37. ). Anal. Calc. for C18H18ClN3O2S: C,
57.52; H, 4.83; S, 8.53. Found: C, 57.50; H, 4.81; S, 8.52.
2.3.25.
3-[(5,6-Dimethylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic
acid
1
hydrochloride (5b) H NMR (400 MHz, DMSO-d6): δ: 2.36 (3H, s, CH3), 2.40 (3H, s,
CH3), 7.44(1H, t, J = 7.6 Hz, C6H4), 7.71 (1H, d, J = 7.6 Hz, C6H4), 7.92 (1H, d, J = 7.6

ACCEPTED MANUSCRIPT
Hz, C6H4), 8.20 (1H, s, C6H4), 8.44 (1H, s, NH), 8.72 (1H, s, 2-H), 12.68 (bs, COOH).
Anal. Calc. for C15H14ClN3O2S: C, 53.33; H, 4.77; S, 9.49. Found: C, 53.32; H, 4.74; S,
9.46.
2.3.26. 3-[(6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic acid hydrochloride
(5c) 1H NMR (400 MHz, DMSO-d6): δ: 7.39 (1H, t, J = 7.6 Hz, C6H4), 7.51 (3H, t, J = 7.3
Hz, C6H5), 7.69 (1H, d, J = 7.6 Hz, C6H4), 7.72 (2H, d, J = 7.3 Hz, C6H5), 8.27 (1H, d, J =
7.6 Hz, C6H4), 8.30 (1H, s, C6H4), 8.43 (1H, s, NH), 8.53 (1H, s, 5-H), 9.80 (1H, s, 2-H),
13.03 (bs, COOH). 13C NMR (100 MHz, DMSO-d6): δ: 115.64, 118.82, 121.88, 124.19,
125.27, 126.17, 129.24, 129.78, 131.61, 133.33, 139.76, 139.98, 153.61, 154.56, 166.34,
167.33. Anal. Calc. for C19H14ClN3O2S: C, 59.45; H, 3.68; S, 8.35. Found: C, 59.44; H,
3.66; S, 8.32.
2.3.27. 4-{[5-(4-Methylphenyl)thieno[2,3-d]pyrimidin-4-yl]amino}benzoic acid
hydrochloride (5d) [22].
2.3.28. 3-{[5-(4-Methylphenyl)thieno[2,3-d]pyrimidin-4-yl]amino}benzoic acid
hydrochloride (5e) [22].
2.3.29. 4-[(5-Phenylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic acid hydrochloride
1
(5f) H NMR (400 MHz, DMSO-d6): δ: 7.44(2H, d, J = 8.1 Hz, C6H4), 7.50 (1H, s, NH),
7.59 (5H, s, C6H5), 7.72 (1H, s, 6-H), 7.84 (2H, d, J = 8.1 Hz, C6H4), 8.65 (1H, s, 2-H),
13
12.72 (bs, COOH). C NMR (100 MHz, DMSO-d6): δ: 115.16, 119.10, 123.31, 125.16,
129.28, 129.43, 129.73, 130.76, 134.43, 135.55, 142.81, 153.27, 154.54, 167.17, 167.83.
Anal. Calc. for C19H14ClN3O2S: C, 59.45; H, 3.68; S, 8.35. Found: C, 59.43; H, 3.66; S,
8.32.
2.3.30. 3-[(5-Phenylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic acid hydrochloride
(5g) 1H NMR (400 MHz, DMSO-d6): δ: 7.39-7.42 (2H, m, aromatic-H), 7.58-7.61 (7H, m,
aromatic-H), 7.69 (1H, s, NH), 8.02 (1H, s, 6-H), 8.61 (1H, s, 2-H), 13.04 (bs, COOH).
13C NMR (100 MHz, DMSO-d6): δ: 114.73, 121.12, 122.97, 124.39, 124.61, 129.13,
129.36, 129.70, 131.86, 134.60, 135.66, 139.02, 153.39, 154.94, 167.40, 167.67. Anal.
Calc. for C19H14ClN3O2S: C, 59.45; H, 3.68; S, 8.35. Found: C, 59.44; H, 3.67; S, 8.34.
2.3.31. 4-{[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-yl]amino}benzoic acid
1
hydrochloride (5h) H NMR (400 MHz, DMSO-d6): δ: 7.56-7.61 (6H, m, aromatic-H),

ACCEPTED MANUSCRIPT
7.64 (1H, s, NH), 7.74-7.79 (3H, m, aromatic-H), 8.61 (1H, s, 2-H), 12.43 (bs, COOH).
Anal. Calc. for C19H13Cl2N3O2S: C, 54.56; H, 3.13; S, 7.67. Found: C, 54.55; H, 3.10; S,
7.65.
2.3.32.
4-[(2-Methyl-6-phenylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic
acid
1
hydrochloride (5i) H NMR (400 MHz, DMSO-d6): δ: 2.44 (3H, s, CH3), 7.33-7.45 (3H,
m, C6H5), 7.62 (2H, d, J = 7.6 Hz, C6H5), 7.90 (2H, d, J = 8.3 Hz, C6H4), 8.01 (2H, d, J =
8.3 Hz, C6H4), 8.18 (1H, s, NH), 9.71 (1H, s, 5-H), 12.64 (bs, COOH). 13C NMR (100
MHz, DMSO-d6): δ: 26.13, 115.44, 116.67, 119.92, 124.78, 126.04, 129.03, 129.74,
130.56, 133.44, 138.64, 144.21, 154.13, 162.57, 167.40. Anal. Calc. for C20H16ClN3O2S:
C, 60.37; H, 4.05; S, 8.06. Found: C, 60.35; H, 4.03; S, 8.04.
2.3.33.
3-[(2-Methyl-6-phenylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic
acid
1
hydrochloride (5j) H NMR (400 MHz, DMSO-d6): δ: 2.46 (3H, s, CH3), 7.35-7.48 (4H,
m, C6H5), 7.60-7.66 (3H, m, C6H5, C6H4), 8.21 (1H, s, C6H4), 8.33 (1H, d, C6H4), 8.40
(1H, s, NH), 9.66 (1H, s, 5-H), 12.97 (bs, COOH). 13C NMR (100 MHz, DMSO-d6): δ:
26.15, 115.51, 116.37, 121.63, 123.87, 124.87, 126.00, 128.96, 129.19, 129.73, 131.70,
133.53, 138.37, 140.26, 154.39, 162.60, 167.09, 167.71. Anal. Calc. for C20H16ClN3O2S:
C, 60.37; H, 4.05; S, 8.06. Found: C, 60.36; H, 4.02; S, 8.05.
2.3.34. 3-{[6-(Ethoxycarbonyl)-5-methylthieno[2,3-d]pyrimidin-4-yl]amino}benzoic
acid (5k) [22].
2.3.35.
4-[(6-Methyl-5-phenylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic
acid
hydrochloride (5l) 1H NMR (400 MHz, DMSO-d6): δ: 2.34 (3H, s, CH3), 6.93 (1H, s, NH),
7.27 (2H, d, J = 8.3 Hz, C6H4), 7.54-7.56 (2H, m, C6H5), 7.65-7.68 (3H, m, C6H5), 7.80
13
(2H, d, J = 8.3 Hz, C6H4), 8.59 (1H, s, 2-H), 12.67 (bs, COOH). C NMR (100 MHz,
DMSO-d6): δ: 14.13, 116.47, 118.54, 124.95, 129.66, 129.82, 130.68, 130.83, 134.34,
142.64, 152.49, 153.33, 165.06, 167.12. Anal. Calc. for C20H16ClN3O2S: C, 60.37; H, 4.05;
S, 8.06. Found: C, 60.35; H, 4.02; S, 8.04.
1
2.3.36. 5-[(6-Methylthieno[2,3-d]pyrimidin-4-yl)amino]isophthalic acid (5m) H
NMR (400 MHz, DMSO-d6): δ: 2.62 (3H, s, CH3), 7.57 (1H, s, NH), 8.19 (1H, s, C6H3),
8.44 (1H, s, C6H3), 8.73 (1H, s, C6H3), 8.73 (1H, s, 5-H), 9.63 (1H, s, 2-H). Anal. Calc. for
C15H11N3O4S: C, 54.71; H, 3.37; S, 9.74. Found: C, 54.69; H, 3.35; S, 9.72.

ACCEPTED MANUSCRIPT
2.3.37.
3-[(6-Methyl-5-phenylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic
acid
1
hydrochloride (5n) H NMR (400 MHz, DMSO-d6): δ: 2.33 (3H, s, CH3), 6.82 (1H, s,
aromatic-H), 7.29-7.37 (2H, m, aromatic-H), 7.55-7.64 (6H, m, aromatic-H), 7.93 (1H, s,
13
NH), 8.55 (1H, s, 2-H), 13.04 (bs, COOH). C NMR (100 MHz, DMSO-d6): δ: 14.12,
116.11, 120.35, 123.80, 124.17, 129.43, 129.52, 129.75, 129.90, 130.69, 131.79, 134.05,
134.47, 138.88, 152.66, 153.70, 164.84, 167.28. Anal. Calc. for C20H16ClN3O2S: C, 60.37;
H, 4.05; S, 8.06. Found: C, 60.34; H, 4.03; S, 8.02.
2.3.38. 2-Hydroxy-5-[(6-methyl-5-phenylthieno[2,3-d]pyrimidin-4-yl)amino]benzoic
acid hydrochloride (5o) 1H NMR (400 MHz, DMSO-d6): δ: 2.34 (3H, s, CH3), 6.60 (1H, s,
aromatic-H), 6.87 (1H, d, J = 8.8 Hz, aromatic-H), 7.20 (1H, dd, J = 2.7 Hz, J = 8.8 Hz,
aromatic-H), 7.54-7.66 (5H, m, C6H5), 7.79 (1H, d, NH), 8.49 (1H, s, 2-H). Anal. Calc. for
C20H16ClN3O3S: C, 58.04; H, 3.90; S, 7.75. Found: C, 58.00; H, 3.86; S, 7.73.
2.3.39. 4-[(3-Carboxyphenyl)amino]-5-methylthieno[2,3-d]pyrimidine-6-carboxylic
1
acid (5p) H NMR (400 MHz, DMSO-d6): δ: 3.08 (3H, s, CH3), 7. 43 (1H, t, J = 7.8 Hz,
aromatic-H), 7.69 (1H, d, J = 7.6 Hz, aromatic-H), 7.93 (1H, d, J = 7.6 Hz, aromatic-H),
8.22 (1H, s, aromatic-H), 8.40 (1H, s, NH), 8.61 (1H, s, 2-H). Anal. Calc. for
C15H11N3O4S: C, 54.71; H, 3.37; S, 9.74. Found: C, 54.69; H, 3.35; S, 9.73.
3. Results and Discussion
3.1. In vitro activity of the first set of 4-aminothieno[2,3-d]pyrimidine derivatives
The 4-aminothieno[2,3-d]pyrimidines were synthesized as analogs of compounds
described previously [11]. Taking into account high potency of the substituted (thieno[2,3-
d]pyrimidine-4-ylthio)carboxylic acids, it was expected that the obtained derivatives
(Table 2) would also inhibit the protein kinase CK2 activity in submicromolar range.
Besides this, new compounds in which atom of Sulfur in fourth position of the thieno[2,3-

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d]pyrimidine heterocycle is replaced to Nitrogen should display higher metabolic stability
[23] and thus they could be considered as more perspective drug candidates.
Biochemical tests in vitro revealed that only 2 out of 18 new compounds were active
in micromolar range. These are 4b (IC50 = 9.5 ꢀM) – 3-(5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d]pyrimidin-4-ilamino)propanoic acid and 4e (IC50 =16 ꢀM) – 3-(7-
methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-ilamino)propanoic acid.
It is interesting that compound 4g (Fig. 1), which structure differs by only one atom
(S is replaced by N) if compare to previously studied compound 6a [11] (IC50 = 0.1 ꢀM)
(Fig. 1), showed no activity.
Fig. 1. Chemical structure of previously described compound 6a [11] and newly
obtained compounds.
3.2. Molecular models of the first set of 4-aminothieno[2,3-d]pyrimidine derivatives
bound to CK2 ATP-binding site

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When analyzing data obtained in the in vitro tests, we were keen to know why
compounds, which are close structural analogs of previously reported CK2 inhibitors,
displayed no activity. Trying to provide an explanation for this phenomenon, we
investigated molecular complexes of the tested compounds with the CK2 obtained with
docking. It turned out that all newly synthesized compounds have a binding mode similar
to the one we described previously [11]. The thieno[2,3-d]pyrimidine core contributed to
tight ligand fixation in the ATP-binding site making hydrophobic contacts with Val53,
Val66, Val116, Met163, Ile174 and hydrogen bond with Val116 of the hinge region. Other
hydrogen bonds are formed in the phosphate-binding region between the compounds’
carboxylic acid moiety and Lys68 and/or Asp175.
The obtained complexes didn’t provide an explanation why the studied compounds
are inactive compared to 4-mercaptothieno[2,3-d]pyrimidines. The activity loss might be
caused by decreased van der Waals interactions and shortening bond length (C-S: 1.8 Å vs
C-N: 1.4 Å) due to the replacement of Sulfur atom to Nitrogen atom. However, in our
previous work [11] it was shown that shortening of linker`s length by about 1 Å (in the
investigated compounds, the distance between Sulfur atom and Oxygen atom of carboxyl
group in acetic acid is about 3.5 Å; the distance between Sulfur atom and Oxygen atom of
carboxyl group in propionic acid is about 4.7 Å) led to significant decrease of inhibitory
activity but not to a total activity loss. Here, the distance shortening by 0.4 Å (1.8 Å minus
1.4 Å) is much lower than 1 Å, but the activity loss is total. Therefore, there must be other
factors contributing to the effect of such activity change. Taking into account a minor
difference between these derivatives, most likely the reason of activity loss was an

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introduction of a hydrogen bond donor. We suppose that the compounds with amino group
could form intramolecular hydrogen bond NHꢁꢁꢁO=C (Fig. 1, compound 4g). It is
presumed that this interaction causes 4-aminothieno[2,3-d]pyrimidine pseudo-cyclization
resulting in reduction of the ligand’s overall length and inability to form high binding
affinity complexes with CK2 due to lack of intermolecular hydrogen bonds with Lys68
and Asp175. It should be noted that the molecular docking program we used in this study
did not predict the pseudo-cyclization, and during the ligand position and conformation
search the carboxylic group switches to intermolecular H-bonds with Lys68 and Asp175,
which is more energetically and sterically favourable in terms of the docking algorithm
and scoring function. Thus, formation of internal hydrogen bonds in small molecule
ligands should be considered at various stages of inhibitor design, and the docking
software settings should be adjusted appropriately when possible.
3.3. Inhibitory potency and structure-activity relationships of the second set of 4-
aminothieno[2,3-d]pyrimidine derivatives
Based on the results obtained with the first set of molecules, the second set of 4-
aminothieno[2,3-d]pyrimidine derivatives with R4 = carboxyphenyl and 4-piperidine-1-yl-
thieno[2,3-d]pyrimidine derivatives was synthesized (Fig. 1, compound 5e). We were
guided by the fact that in this case these substituents cannot form any intramolecular
hydrogen bonds and thus they are within optimal length in order to be involved into
formation of H-bonds with amino acid residues in the depths of the CK2 ATP-binding site.

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Structure and activity data for the obtained inhibitors is represented in Table 3. As
one can see, about 60% of the newly synthesized compounds inhibit CK2 activity in the
range from 0.008 ꢀM to 10 ꢀM. The most active CK2 inhibitors are compounds 3-(5-p-
tolyl-thieno[2,3-d]pyrimidin-4-ylamino)benzoic acid, 5e (NHTP23, IC50 = 0.01 ꢀM), 3-(5-
phenylthieno[2,3-d]pyrimidin-4-ylamino)benzoic acid, 5g (NHTP25, IC50 = 0.065 ꢀM)
and 3-(6-methyl-5-phenyl-thieno[2,3-d]pyrimidin-4-ylamino)benzoic acid, 5n (NHTP33,
IC50 = 0.008 ꢀM).
Kinetic studies of 5e, 5g and 5n have shown that the activity of 4-aminothieno[2,3-
d]pyrimidine derivatives is a result of competitive binding in ATP-acceptor site of CK2
(Fig. 2). Inhibition constants (Ki) for these compounds are 4.5 nM, 12.7 nM and 4 nM,
respectively.
A
B
C
Fig. 2. Lineweaver–Burk plots of CK2 inhibition by 5e (A), 5g (B) and 5n (C). Ki
values are 4.5 nM, 12.7 nM and 4 nM, respectively. Concentration of compounds 5e, 5g
varied from 0 nM to 108 nM, concentration of compound 5n varied from 0 nM to 60.75
nM. Enzyme activities were tested as described in Materials and Methods.

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As it was expected, the replacement of aliphatic carboxylic acids to aromatic ones in
ligands’ structure led to increased inhibitory activity. It is demonstrated by compounds 5b
(R4 = C6H4COOH-m), 4c (CH2COOH) and 4f (C2H4COOH) with IC50 values of 3.8 ꢀМ, >
33 ꢀМ and > 33 ꢀМ, respectively, and also by compounds 5k (R4 = C6H4COOH-m) and 4i
(CH2COOH) with IC50 of 6.5 ꢀМ and > 33 ꢀМ, respectively. The position of carboxyl
group of phenyl substituent R4 is also important. Comparison of the ligands with aromatic
carboxylic acids and identical R1, R2, R3 has shown that the most active inhibitors have R4
= C6H4COOH-m. For example, the compound 5i (R4 = C6H4COOH-p) is inactive, while 5j
(R4 = C6H4COOH-m) has IC50 1.5 ꢀM. The activity of compound 5l (R4 = C6H4COOH-p)
is 200 times lower than the one of compound 5n (R4 = C6H4COOH-m) (IC50 is 1.5 ꢀM and
0.008 ꢀM, respectively). The activity of compounds 5e and 5g with R4 = C6H4COOH-m is
by an order of magnitude higher if compare to 5d and 5f with R4 = C6H4COOH-p (IC50
values are 0.01 ꢀM, 0.065 ꢀM and 0.65 ꢀM, 1.1 ꢀM, respectively). It should be noted that
introduction of additional substituents in phenyl moiety leads to activity loss. For example,
compound 5o which contains an additional hydroxyl group in the carboxyphenyl
substituent has IC50= 0.31 ꢀM, whereas compound 5n with R4 = C6H4COOH-m has IC50 =
0.008 ꢀM.
Substituents R1 and R2 have less effect on the compounds’ activity. The R1 effect is
increased in the range C6H5 < 4-ClC6H4 < 4-CH3C6H4 (5f, 5h, 5d, IC50 = 1.1 ꢀM, 0.83 ꢀM
and 0.65 ꢀM, respectively and 5g, 5e IC50 = 0.065 ꢀM and 0.01 ꢀM, respectively).
Substituent R2 affects the activity more than R1 that can be seen from pairs of compounds
5f-5l, 5b-5k, 5g-5n having IC50 values 1.1-1.5 ꢀM, 3.8-6.5 ꢀM and 0.065-0.008 ꢀM,
respectively.

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All the tested compounds have hydrogen atom or methyl group as R3 substituent.
Presence of methyl group shows negative effect on ligands’ activity that is demonstrated
by assessment of compounds 5c and 5j (IC50 is 0.26 ꢀM and 1.5 ꢀM, respectively).
3.4. Selectivity
To check the inhibitors’ selectivity towards CK2 the effect of compounds 5e, 5g and
5n was studied on a limited number of protein kinases available in our laboratory
immediately. Kinase profiling included four serine/threonine (ASK1, JNK3, Aurora A and
ROCK1) and three tyrosine protein kinases (FGFR1, c-Met and Tie2). The aim of this
research was to quickly assess if our compounds display wide spectrum protein kinase
inhibition effect similar to known inhibitors, for example, staurosporine [24].
The results of the test showed that the compounds at concentration 10 ꢀM did not
inhibit activity of these kinases significantly (Table 4) with the exception of Aurora A
kinase.
Table 4
Kinase profiling of 5e, 5g and 5n*
№
5e
5g
5n
CK2
ASK1
JNK3 FGFR1 c-Met Aurora A
Tie2
ROCK1
105,8 %
88,6 %
1.14 % 122 % 85,7 % 60,5 % 53,7 %
1.51 % 122 % 89,5 % 78,9 % 71,5 %
0.91 % 112 % 70,3 % 73,9 % 87,2 %
36,2 %
38,9 %
15,3 %
109 %
119,7 %
84,2 %
101,5 %

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* kinase residual activity, determined in the presence of 10 µM of inhibitor is expressed as a percentage of
the control without inhibitor. Final concentration of ATP in the experiment was 100 µM.
3.5. Molecular modeling of the second set of 4-aminothieno[2,3-d]pyrimidine
derivatives bound to CK2 ATP-binding site
Inspection of docking poses of compounds 4s-w, 5a-p (Fig. 3) has shown that 4-
aminothieno[2,3-d]pyrimidine derivatives have the same positions in the CK2 ATP-
binding site as compounds 4a-r. A number of hydrophobic contacts between the ligands
and amino acid residues Leu45, Val53, Val66, Met163, Ile174 and Phe113 (π-π stacking)
have been identified. In addition, it has been observed that the thienopyrimidine core is
located in the hinge region of the active site and forms a hydrogen bond with the main
chain NH group of Val116. Also, carboxyl group of the ligands is oriented towards
phosphate-binding site and forms hydrogen bonds with Asp175 and Lys68. Furthermore, it
is connected via water-mediated hydrogen bonds with carboxyl group of Glu81 and main
chain NH group of Trp176. Substituents R1 and R2 are oriented outward of the ATP-
binding site, and form hydrophobic contact with Leu45. Since the substituent R3 is located
close to the hinge region, one should take into account that bulky substituents in this
position may create steric hindrance and result in total activity loss of the compounds. We
also assume that presence of carboxyphenyl substituents in R4 could provide tight ligand
fixation between Phe113 and Ile174 due to π-π stacking that could explain higher activity

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of structurally optimized 4-aminothieno[2,3-d]pyrimidines if compared to 4-
mercaptothieno[2,3-d]pyrimidines [11].
Fig. 3. Binding mode of compound 5n obtained with molecular docking.
Intermolecular hydrogen bonds are indicated with dotted lines. Amino acid residues that
participate in hydrophobic interactions have dark blue labels; amino acid residues that
form H-bond with the ligand have green labels.
Also, we made superposition of two crystal structures - PDB ID: 4GRB which have
been used for docking and PDB ID: 3PE1 [25] complexed with inhibitors 5n and CX-
4945, respectively (Fig. 4). Obtained data showed that all active site amino acid side
chains in both CK2 crystal structures have very similar positions, and both inhibitors
orient their carboxyl acid residues towards Lys68 forming H-bonds with Lys68, Asp175
and Val116. It is observed that thieno[2,3-d]pyrimidine core of 5n and
benzo[c][2,6]naphthyridine of CX-4945 partially overlap. This indicates that the

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compounds have similar hydrophobic contacts with CK2 ATP-acceptor site residues. Also,
inhibitor 5n displays π-π stacking interaction with Phe113 residue, while CX-4945
doesn’t.
Fig. 4. Superposition of inhibitors 5n (red) and CX-4945 (yellow) complexed with
CK2 crystal structures 4GRB and 3PE1.
4. Conclusions
Rational design of biologically valuable compounds requires deep knowledge about
structure of both small molecule ligands and corresponding macromolecular target as well
as their mode of interaction. In the course of this work, new CK2 inhibitors among 4-
aminothieno[2,3-d]pyrimidines have been identified. The foundation of this study is based
on our previously published results for substituted (thieno[2,3-d]pyrimidin-4-
ylthio)carboxylic acids. During structure-activity relationship analysis, the important role
of intramolecular hydrogen bonding has been proposed. Intramolecular hydrogen bonds
are abundant in medicinal chemistry, and in most cases they produce positive effect by