4-(4-Chlorophenyl)thiazol-2-amines as pioneers of potential neurodegenerative therapeutics with anti-inflammatory properties based on dual DNase I and 5-LO inhibition

Article abstract of DOI:10.1016/j.bioorg.2019.103528

Eleven new 4-(4-chlorophenyl)thiazol-2-amines were synthesized and, together with nine known derivatives, evaluated in vitro for inhibitory properties towards bovine pancreatic DNase I. Three compounds (18–20) inhibited DNase I with IC₅₀ values

Full text of DOI:10.1016/j.bioorg.2019.103528

Journal Pre-proofs
4-(4-Chlorophenyl)thiazol-2-amines as pioneers of potential neurodegenera-
tive therapeutics with anti-inflammatory properties based on dual DNase I and
5-LO inhibition
Andrija Smelcerovic, Aleksandra Zivkovic, Budimir S. Ilic, Ana Kolarevic,
Bettina Hofmann, Dieter Steinhilber, Holger Stark
PII:
S0045-2068(19)31094-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103528
YBIOO 103528
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
9 July 2019
16 December 2019
19 December 2019
Please cite this article as: A. Smelcerovic, A. Zivkovic, B.S. Ilic, A. Kolarevic, B. Hofmann, D. Steinhilber, H.
Stark, 4-(4-Chlorophenyl)thiazol-2-amines as pioneers of potential neurodegenerative therapeutics with anti-
inflammatory properties based on dual DNase I and 5-LO inhibition, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.103528
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4-(4-Chlorophenyl)thiazol-2-amines as pioneers of potential neurodegenerative
therapeutics with anti-inflammatory properties based on dual DNase I and 5-LO
inhibition
Andrija Smelcerovica,*, Aleksandra Zivkovicb, Budimir S. Ilica, Ana Kolarevicc, Bettina Hofmannd, Dieter
Steinhilberd, Holger Starkb,*
a Department of Chemistry, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000
Nis, Serbia
b Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf,
Germany
c Department of Pharmacy, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000
Nis, Serbia
d Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt/Main, Germany
*Corresponding authors
E-mail addresses: a.smelcerovic@yahoo.com (or andrija.smelcerovic@medfak.ni.ac.rs) (A. Smelcerovic),
stark@hhu.de (H. Stark)
ABSTRACT
Eleven new 4-(4-chlorophenyl)thiazol-2-amines were synthesized and, together with nine known
derivatives, evaluated in vitro for inhibitory properties towards bovine pancreatic DNase I. Three compounds
(18-20) inhibited DNase I with IC50 values below 100 µM, with compound 19 being the most potent (IC50 =
79.79 µM). Crystal violet, used as a positive control in the absence of a "golden standard", exhibited almost
5-fold weaker DNase I inhibition. Pharma/E-State RQSAR models clarified critical structural fragments
relevant for DNase I inhibition. Molecular docking and molecular dynamics simulation defined the 4-(4-
chlorophenyl)thiazol-2-amines interactions with the most important catalytic residues of DNase I. Ligand-
based pharmacophore modeling and virtual screening confirmed the chemical features of 4-(4-
chlorophenyl)thiazol-2-amines required for DNase I inhibition and proved the absence of structurally similar
molecules in available databases. Compounds 18-20 have been shown as very potent 5-LO inhibitors with
nanomolar IC50 values obtained in cell-free assay, with compound 20 being the most potent (IC50 = 50 nM).
1

Molecular docking and molecular dynamics simulations into the binding site of 5-LO enzyme allowed us to
clarify the binding mode of these dual DNase I/5-LO inhibitors. It was shown that compounds 18-20 uniquely
show interactions with histidine residues in the catalytic site of DNase I and 5-LO enzyme. In the absence of
potent organic DNase I inhibitors, compounds 18-20 represent a good starting point for the development of
novel Alzheimer's therapeutics based on dual 5-LO and DNase I inhibition, which also have anti-inflammatory
properties.
KEYWORDS
DNase I, 5-Lipoxygenase, Inhibition, Molecular dynamics, Pharmacophore modeling, Alzheimer
1. INTRODUCTION
It is widely known that the deoxyribonucleic acid (DNA) is one of the key macromolecules necessary
for the continuity of life. Bovine pancreatic deoxyribonuclease I (DNase I) [EC 3.1.21.1] is one of the best
characterized mammal endonucleases that breaks both single-stranded and double-stranded DNA producing
3'-OH/5'-P ends [1,2]. To exert its full activity, DNase I requires the presence of both Mg2+ and Ca2+ and
optimal pH between 7.0 and 8.0 [3]. DNase I is one of the major nucleases involved in DNA degradation
during apoptosis [2,4–8], acting as both cell-autonomous and waste-management nuclease [2,7], and thus
might have a crucial role in the development of many disease conditions caused by excessive cell death (Table
1). Inhibition of DNase I is an important mechanism for protecting DNA from premature degradation during
cell damage [9]. DNase I inhibitors represent an attractive potential target for the design of alternative
strategies for the prevention and/or therapy of numerous pathological conditions caused by elevated DNase I
levels and/or excessive apoptosis (Table 1).
2

Table 1
Potential therapeutic applications of DNase I inhibitors.
Conditions caused by excessive apoptosis
Reference
Autoimmune disorders
AIDS
[7,10]
Neurodegenerative disorders
Alzheimer's disease
Parkinson's disease
Huntington's disease
Amyotrophic lateral sclerosis
Retinitis pigmentosa
Spinal muscular atrophy
Cerebellar degeneration
[7,10–13]
Graft-versus-host disease
[7]
Ischemic disorders
Myocardial ischaemia
Myocardial infarction
Stroke
Acute fatty degeneration of the liver
Conditions followed by elevated DNase I levels
Myocardial dysfunction among elderly
Idiopathic dilated cardiomyopathy
Type 2 diabetes
[7,10,13–15]
[16]
[17]
[18]
[19]
Cisplatin-induced nephrotoxicity
Gamma-induced damage
[20–22]
[23]
Acetaminophen-induced hepatocellular necrosis [24]
2-Aminothiazole and its derivatives have been used as precursors for the synthesis of many compounds
with various biological activities [25], such as anticancer, anticonvulsant, antidiabetic, antihypertensive, anti-
inflammatory, antimicrobial, antioxidant, antitumor, and antiviral. Some of the drugs containing 2-
aminothiazole core are already in the market. 2-Aminothiazole scaffold is an excellent pharmacophore for the
development of many more biologically active derivatives [26].
As the number of known organic DNase I inhibitors, especially those with potential therapeutic
applications, is relatively small [27], continuing our research on finding novel ones [28–33], in the present
study a series of twenty 4-(4-chlorophenyl)thiazol-2-amines (Table 2) was evaluated for inhibitory activity
against bovine pancreatic DNase I in vitro. Some of them were previously synthesized and evaluated as ligands
for varying targets: as inhibitors of eicosanoid metabolism (1) [34], sphingosine kinase inhibitors (1, 2, 7, 15-
17) [35], and 5-lipoxygenase (5-LO) inhibitors (1, 14, 17, 18, 20) [36].
3

Having in mind that our studied molecules were built on a similar scaffold, by attaching different
groups at one or more points on the 4-(4-chlorophenyl)thiazol-2-amine, we wanted to examine DNase I
inhibitory properties of the 4-(4-chlorophenyl)thiazol-2-amines as a function of the groups at the various
attachment points. In addition, we wanted to clarify DNase I inhibitory properties of 4-(4-
chlorophenyl)thiazol-2-amines at the molecular level using molecular docking studies. Furthermore, the most
active 4-(4-chlorophenyl)thiazol-2-amines were subjected to the molecular dynamics simulations, in order to
further evaluate the stability of their binding to the active site of DNase I. Ligand-based pharmacophore
modeling approach was performed in order to reveal and confirm the chemical features of 4-(4-
chlorophenyl)thiazol-2-amines required for DNase I inhibition. Pharmacophore based virtual screening study
was carried out using PubChem compound database to find novel/potent DNase I inhibitors with 4-(4-
chlorophenyl)thiazol-2-amine moiety.
In order to perceive the multiple biological effects of the most potent DNase I inhibitors, an attempt
was made to connect their DNase I inhibitory properties with previously reported [36] and new data on their
5-LO inhibitory properties, considering the importance of these enzymes in the development of
neurodegenerative disorders in the elderly, such as is Alzheimer's disease. Molecular docking and molecular
dynamics simulations into the binding site of 5-LO enzyme allowed us to clarify the binding mode of these
novel dual DNase I/5-LO inhibitors. An in silico study of the physico-chemical, pharmacokinetic and
toxicological properties of most potent DNase I inhibitors was also performed.
2. MATERIALS AND METHODS
2.1. Synthesis of 4-(4-chlorophenyl)thiazol-2-amines
All commercial chemicals were reagent grade and used as purchased by Sigma–Aldrich
(Steinheim/Germany), ABCR (Karlsruhe/Germany), Acros Organics (Geel/Belgium), Alfa-Aesar
(Karlsruhe/Germany) and Fluorochem (Derbyshire/UK) unless otherwise indicated. Only dried solvents were
used. Reactions were monitored by thin-layer chromatography using silica gel 60 F254 aluminumbacked plate
from Merck (Darmstadt/Germany) with detection using a UV lamp and ninhydrin. Column chromatography
was performed on silica gel (SiO2, 40–63 lM). Microwave-assisted synthesis was performed using a Biotage
Initiator 2.0, 400 Watt microwave synthesizer (Biotage, Uppsala/Sweden). All products were characterized
by 1H NMR and 13C NMR. NMR spectra were recorded on a Bruker AV 250 (1H: 250 MHz; 13C: 63 MHz)
13
spectrometer (Bruker, Karlsruhe/Germany) and Bruker AV 400 (1H: 400 MHz; C: 75 MHz) spectrometer
(Bruker, Karlsruhe/Germany). Chemical shifts (d) are reported in ppm relative to tetramethylsilane (TMS) as
internal standard. Multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet; coupling constants
(J) are shown in hertz (Hz); number and assignment of protons. Mass spectra were obtained on VG Platform
II (Fisons Instruments, Ipswich/UK) using electrospray ionization (ESI). Data are listed as mass number
4

[M+H]+ or [M-H+]-. High resolution MS (HR-MS) were achieved on a LTQ Orbitrap XL (Thermo Fisher
Scientific, Waltham/USA). For final compounds the analyses were within ±5 ppm of the theoretical values.
The purity of the compounds was determined by elemental analysis (C, H, N, S) on a MicroCube instrument
(Elementar, Hanau/Germany) and was within ±0.4% of the theoretical values for all final compounds, which
corresponds to P95% purity.
Studied 4-(4-chlorophenyl)thiazol-2-amines were synthesized according to the general procedure for
the synthesis of 2-aminothiazole derivatives [35]. The cyclic condensation of an α-bromoketone (1 eq.) and
N-substituted thiourea (1 eq.) in anhydrous ethanol, stirring under microwave irradiation at 80°C for 30 min.
The precipitating hydrobromide salts were collected and washed with water and cold ethanol. If needed,
further purification was done after neutralization with saturated ammoniumhydroxide solution, stirring at
room temperature, filtration of the precipitate and washing with cold ethanol. Colum chromatography on silica
gel (eluent: hexane/ethylacetate 2:1 or dichloromethane/methanol 9:1) afforded the titled compounds in
variable yields.
N-(4-(4-Chlorophenyl)thiazol-2-yl)cinnamic acid amide (Compound 3; Table 2)
1H-NMR (250 MHz, DMSO-d6, δ): 12.58 (s, 1H, NH-C=O), 7.95 (d, J = 8,5, 2H, Cl-Ph-3-H, -5-H), 7.78 (d,
2H, Ph-2-H, -6-H), 7.65 (d, 2H, Cl-Ph-2-H, -6-H), 7.50 (m, 5H, Thiazol-5-H, Ph-3-H, -5-H, -4-H, C=O-
CH=CH), 6.98 (d, J = 15.9, 1H, C=O-CH=CH); 13C-NMR (63 MHz, DMSO-d6, δ): δ 163.48, 158.18, 147.85,
142.49, 134.26, 133.18, 132.29, 130.32, 129.10, 128.76, 128.01, 127.40, 119.52, 109.26; ESI-MS (m/z) 339.1
[M-H+]-; CHN Calculated: C 63.43%, H 3.84%, N 8.22%, S 9.41%; Found: C 63.36%, H 3.80%, N 8.16%, S
9.64%; Yield: 25 %; Colourless crystals.
N-(4-(4-Chlorophenyl)thiazol-2-yl)benzamide (Compound 4; Table 2)
1H-NMR (250 MHz, DMSO-d6, δ): 12.84 (s, 1H, NH-C=O), 8.15 (d, J = 8,0, 2H, Ph-2-H, 6-H), 7.99 (d, J =
8.6, 1H, Cl-Ph-3-H, -5-H), 7.77 (s, 1H, Thiazol-5-H), 7.67 (t, 1H, Ph-4-H), 7.57 (t, 2H, Ph-3-H, -5-H), 7.52
(d, J = 8.7, 2H, Cl-Ph -2-H, -6-H); 13C-NMR (63 MHz, DMSO-d6, δ): 165.30, 158.76, 147.97, 133.30, 132.66,
132.34, 131.96, 128.85, 128.60, 128.22, 127.52, 109.35; ESI-MS (m/z): 313.0 [M-H+]; CHN Calculated: C
61.05%, H 3.52%, N 8.90%, S 10.19%; Found: C 61.05%, H 3.60%, N 8.96%, S 10.26%; Yield: 66 %;
Colourless crystals.
4-(4-Chlorophenyl)-5-methyl-N-(pyridin-2-yl)thiazol-2-amine (Compound 5; Table 2)
5

1H-NMR (250 MHz, DMSO-d6, δ): 11.23 (s, 1H, -NH-), 8.28 (d, 1H, J = 5.0 Hz, py-6H), 7.72-7.65 (m, 3H,
Ph-Cl-2H,6H, py-4H), 7.48 (d, 2H, J = 8.5 Hz, Ph-Cl-3H,5H), 7.03 (d, 1H, J = 8.3 Hz, py-3H), 6.90 (t, 1H, J
13
= 5.2 Hz, py-5H), 2.45 (s, 3H, -CH3); C-NMR (63 MHz, DMSO-d6, δ): 155.53, 151.64, 146.29, 142.29,
137.70, 134.14, 131.30, 129.38, 128.16, 119.22, 115.66, 110.46, 11.64; ESI-MS (m/z): 302.1 [M+H+]; CHN
Calculated: C 59.70%, H 4.01%, N 13.92%, S 10.62%; Found: C 59.81%, H 3.95%, N 13.90%, S 10.54%;
Yield: 14 %; White amorphous solid.
4-(4-Chlorophenyl)-N-(2-methoxypyridin-4-yl)-5-methylthiazol-2-amine (Compound 6; Table 2)
1H-NMR (250 MHz, DMSO-d6, δ) : 10.53 (s, 1H, -NH-), 7.96 (d, 1H, J = 5.7 Hz, pyridine-6H), 7.69 (d, 2H,
J = 8.6 Hz, Ph-Cl-2H,6H), 7.53 (d, 2H, J = 8.6 Hz, Ph-Cl-3H,5H), 7.23 (sd, 1H, J = 1.7 Hz, pyridine-3H), 6.99
(dd, 1H, J = 5.8 Hz, 1.8 Hz, pyridine-5H), 3.80 (s, 3H, pyridine-CH3), 2.45 (s, 3H, thiazole-CH3); 13C-NMR
(63 MHz, DMSO-d6, δ): 164.73, 158.11, 149.14, 146.95, 144.18, 133.55, 131.77, 129.52, 128.38, 118.97,
106.49, 95.40, 52.86, 11.81; ESI-MS (m/z) 330.0 [M+H+]; CHN Calculated: C 57.91%, H 4.25%, N 12.66%,
S 9.66%; Found: C 57.85%, H 4.25%, N 12.55%, S 9.96%; Yield: 5 %; White solid.
4-(4-Chlorophenyl)-N-(2-methoxyphenyl)-5-methylthiazol-2-amin (Compound 8; Table 2)
1H-NMR (250 MHz, DMSO-d6, δ): 10.22 (s, 1H, -NH-), 7.98 (d, 1H, J = 7.8 Hz, 3H-Ph-OCH3), 7.62 (d, 2H,
J = 8.6 Hz, 2H,6H-Ph-Cl), 7.53 (d, 2H, J = 8.6 Hz, 3H,5H-Ph-Cl), 7.14 – 7.08 (m, 2H, 4H,6H-Ph-OCH3), 7.01
– 6.95 (m, 1H, 5H-Ph-OCH3), 3.86 (s, 3H, -OCH3), 2.34 (s, 3H, 5-CH3-Thiazol); 13C-NMR (63 MHz, DMSO-
d6, δ): 179.2, 163.1, 153.7, 150.5, 142.6, 132.8, 130.1, 128.5, 120.8, 120.5, 117.0, 112.4, 111.8, 55.7, 11.6;
ESI-MS (m/z): 333.8 [M+H+]; CHN Calculated: C 49.59%, H 3.92%, N 6.80%, S 7.79%; Found: C 49.32%,
H 3,80%, N 6.71%, S 7.84%; Yield: 66 %; Light yellow solid.
3-(4-(4-Chlorophenyl)-5-methylthiazol-2-ylamino)benzoic acid (Compound 9; Table 2)
1H-NMR (250 MHz, DMSO-d6, δ): 12.89 (s, 1H, -OH), 10.28 (s, 1H, -NH-), 8.25 (s, 1H, 2H-Ph-COOH), 7.94
(d, 1H, J = 7.8 Hz, 6H-Ph-COOH), 7.72 (d, 2H, J = 8.6 Hz, 2H,6H-Ph-Cl), 7.52 – 7.39 (m, 4H, 3H,5H-Ph-Cl,
4H,5H-Ph-COOH), 2.44 (s, 3H, -CH3); 13C-NMR (63 MHz, DMSO-d6, δ): 167.3, 158.9, 143.8, 141.2, 133.8,
131.5, 129.5, 129.0, 128.2, 121.6, 120.5, 117.5, 117.3, 11.8; ESI-MS (m/z): 322.2 [M+H+]; CHN Calculated:
C 52.25%, H 3.76%, N 13.06%, S 19.93%; Found: C 52.30%, H 3.87%, N 13.06%, S 20.24%; Yield: 44 %;
Light red solid.
4-(4-(4-Chlorophenyl)-5-methylthiazol-2-ylamino)benzoic acid (Compound 10; Table 2)
6

1H-NMR (250 MHz, DMSO-d6, δ): 10.59 (s, 1H, -NH-), 7.88 (d, 2H, J = 8.8 Hz, 2H,6H-Ph-Cl), 7.75 – 7.68
(m, 4H, 3H,5H-Ph-COOH, 2H,6H-Ph-COOH), 7.50 (d, 2H, J = 8.6 Hz, 3H,5H-Ph-Cl), 2.43 (s, 3H, -CH3);
13C-NMR (63 MHz, DMSO-d6, δ): 166.9, 158.5, 144.8, 143.8, 133.6, 131.7, 130.6, 129.6, 128.3, 122.6, 118.4,
115.8, 11.9; ESI-MS (m/z): 347,1 [M-H+]; CHN Calculated: C 47.96%, H 3.31%, N 6.58%, S 7.53%; Found:
C 48.21%, H 3.35%, N 6.65%, S 7.60%; Yield: 29 %; Yellow powder.
4-(4-(4-Chlorophenyl)-5-methylthiazol-2-yloxy)phenole (Compound 11; Table 2)
1H-NMR (400 MHz, DMSO-d6, δ): 9.64 (s, 1H, -OH), 7.60 (d, 2H, J = 8.6 Hz, Ph-Cl-2H,6H), 7.48 (d, 2H, J
= 8.5 Hz, Ph-Cl-3H,5H), 7.18 (d, 2H, J = 8.9 Hz, Ph-OH-3H,5H), 6.82 (d, 2H, J = 8.8 Hz, Ph-OH-2H,6H),
2.41 (s, 3H, –CH3); 13C-NMR (75 MHz, DMSO-d6, δ): 169.86, 155.55, 147.33, 142.81, 133.11, 131.95, 129.62,
128.33, 122.14, 121.58, 116.17, 12.31; ESI-MS (m/z): 318.8 [M+H+]; CHN Calculated: C 60.47%, H 3.81%,
N 4.41%, S 10.09%; Found: C 60.47%, H 3.47%, N 4.35%, S 10.06%; Yield: 29 %; Light brown solid.
2-(N-(4-(4-Chlorophenyl)-5-methylthiazol-2-yl)acetamido)phenyl acetate (Compound 12; Table 2)
1H-NMR (250 MHz, DMSO-d6, δ): 7.67 (d, 1H, J = 7.7 Hz, Ph-OH-6H), 7.58 (t, 1H, J = 7.7 Hz, Ph-OH-5H),
7.46 (d, 1H, J = 7.6 Hz, Ph-OH-3H), 7.43-7-37 (m, 5H, Ph-Cl, Ph-OH-4H), 2.47 (s, 3H, Thiazol-CH3), 2.03(s,
13
3H, O-C(O)-CH3), 1.98 (s, 3H, N-C(O)-CH3); C-NMR (63 MHz, DMSO-d6, δ): 169.89, 155.21, 152.99,
142.00, 133.64, 131.58, 130.22, 129.29, 128.19, 126.72, 122.86, 119.60, 116.71, 22.56, 11.55; ESI-MS (m/z)
401.1 [M+H+]-; CHN Calculated: C 59.92%, H 4.27%, N 6.99%, S 8.00%; Found: C 59.82%, H 4.20%, N
6.98%, S 7.92%; Yield: 46 %; Yellow needles.
2-(N-(5-Acetyl-4-(4-chlorophenyl)thiazol-2-yl)acetamido)phenyl acetate (Compound 13; Table 2)
1H-NMR (250 MHz, DMSO-d6, δ): 7.71 (d, 1H, J = 7.71 Hz, Ph-OH-3H), 7.57 (t, 1H, J = 7.77 Hz, Ph-OH-
5H), 7.47-7.39 (m, 6H, Ph-OH-4H,6H, Ph-Cl), 2.20 (s, 3H, Thiazol-C(O)-CH3), 2.06 (s, 3H, O-C(O)-CH3),
2.04 (s, 3H, N-C(O)-CH3); 13C-NMR (63 MHz, DMSO-d6, δ): 191.19, 170.60, 168.17, 159.52, 151.78, 146.42,
133.88, 133.28, 131.21, 131.00, 130.67, 130.65, 128.41, 127.99, 127.00, 124.04, 29.64, 22.92, 20.19; ESI-MS
(m/z) 429.8 [M+H+]-; CHN Calculated: C 58.81%, H 4.00%, N 6.53%, S 7.48%; Found: C 59.10%, H 4.08%,
N 6.49%, S 7.53%; Yield: 57 %; White solid.
4-(4-Chlorophenyl)-2-(4-hydroxyphenylamino)thiazole-5-carboxamide (Compound 19; Table 2)
1H-NMR (250 MHz, DMSO-d6, δ): 10.14 (s, 1H, -NH-), 9.22 (s, 1H, -OH), 7.70 (d, 2H, J = 8.5 Hz, Ph-Cl-
2H,6H), 7.47 (d, 2H, J = 8.5 Hz, Ph-Cl-3H,5H), 7.37 (d, 2H, J = 8.8 Hz, Ph-OH-3H,5H), 7.22 (br, 2H, -NH2),
7

6.74 (d, 2H, J = 8.8 Hz, Ph-OH-2H,6H); 13C-NMR (63 MHz, DMSO-d6, δ): 163.43, 162.78, 153.01, 149.92,
133.45, 132.78, 132.26, 130.65, 127.87, 120.17, 115.45, 115.01; ESI-MS (m/z) 346.8 [M+H+]; CHN
Calculated: C 55.57%, H 3.50%, N 12.15%, S 9.27%; Found: C 55.31%, H 3.50%, N 11.76%, S 9.63%; Yield:
69 %; White solid.
2.2. Evaluation of DNase I and 5-LO inhibition
4-(4-Chlorophenyl)thiazol-2-amines 1-20 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 [28–33], using
crystal violet as a positive control.
Inhibition of 5-LO activity was determined both in an intact cell system using freshly isolated
polymorphonuclear leukocytes (PMNL) and in a cell-free assay using partially purified recombinant 5-LO
and 5-LO product formation was determined by HPLC according to the method previously described [36].
2.3. In silico studies
2.3.1. Pharma/E-State RQSAR models
Pharma/E-State RQSAR models analyzed the 4-(4-chlorophenyl)thiazol-2-amines activity as a
function of the type of R-group at each attachment position, and displayed the results qualitatively in terms of
increasing, decreasing, or little effect on the activity [37]. We ran a partial-least-squares (PLS) procedure that
fitted the observed inhibitory data to the counts of the features present in each R-group at each position. For
Pharma/E-State RQSAR models, the features counted are the pharmacophore types and various E-state atom
types [37]. This was done using a 75% : 25% random training : test-set split. The procedure was repeated 100
times, picking each time 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 importance cutoff, which is a
statistic computed from all coefficients over all models.
2.3.2. Molecular docking
2.3.2.1. Ligand preparation
Selected 4-(4-chlorophenyl)thiazol-2-amines were generated using the builder panel in Maestro [37]
and subsequently optimized using the LigPrep [37]. Partial atomic charges were ascribed and possible
8

ionization states were generated at a pH of 7.0. The OPLS-2005 force field was used for optimization and the
resulting structures were used for modeling studies.
2.3.2.2. Receptor preparation
The X-ray crystallographic structures of DNase I (PDB code: 1DNK) and 5-LO (PDB code: 3O8Y),
retrieved from the Protein Data Bank, were prepared using the Protein Preparation Wizard [37]. Following the
assignment of charge and protonation state, energy minimization till RMSD value of 0.30 Å was done using
the OPLS-2005 force field. The SiteMap [37] was used to identify possible ligand-binding pockets within the
optimized structure of DNase I and 5-LO. Using Glide [37], a grid box with a 10 Å radius was generated at
the centroid of the top-ranked binding pocket obtained by SiteMap [37].
2.3.2.3. Docking protocol
Selected 4-(4-chlorophenyl)thiazol-2-amines were docked into the top-ranked binding pocket of
DNase I and 5-LO using Glide [37]. The standard precision mode without applying any constraints was used
for the flexible docking. Each enzyme/inhibitor complex with lowest Glide docking score was selected for
further study.
2.3.3. Molecular dynamics simulation
The molecular dynamics simulation of the most active 4-(4-chlorophenyl)thiazol-2-amines was carried
out using Desmond [37]. Forcefield parameters for the enzyme/inhibitor system were assigned using the
OPLS-2005 force field. The structure of the added water was based on the simple point charge (SPC) solvent
model. The system was neutralized with Na+ ions to balance the net charge of the whole simulation box to
neutral. The final system contained approximately 26100 atoms for DNase I and 136900 atoms for 5-LO. The
system was passed through a 6-step relaxation protocol before molecular dynamics simulations. The relaxed
system was simulated for 10 ns, using a normal pressure temperature (NPT) ensemble with a Nosé–Hoover
thermostat at 300 K and Martyna–Tobias–Klein barostat at 1.01325 bar pressure. Atomic coordinate data and
system energies were recorded every 1 ps. The root mean square deviation (RMSD) and root mean square
fluctuation (RMSF) of the enzyme/inhibitor complexes were analyzed with respect to the simulation time.
2.3.4. Pharmacophore modeling and virtual screening
Pharmacophore mapping was carried out using PHASE version 4.2 available in Maestro 10.1
molecular modeling package from Schrödinger [38]. Pharmacophore modeling is the qualitative picture for
9

finding the chemical feature for the active site geometry and spatial arrangements in 3D space of the ligands.
There are 6 in-built pharmacophore features available in PHASE: hydrogen bond acceptor (A), hydrogen bond
donor (D), hydrophobic group (H), negative charged group (N), positive charged group (P) and aromatic ring
(R). Common pharmacophore features were used to construct pharmacophore sites. For defining the most
active pharm - set in PHASE, an activity threshold range was selected in such a way that compounds were
active if IC50 value was below 100 µM and inactive if IC50 value was above 100 µM. The common
pharmacophore hypotheses were generated with default settings. A pharmacophore cluster was proceeded
based on the highest average similarity. The preferred hypothesis was then selected based on the survival score.
The PubChem database consisting of more than 96 million compounds was subjected to pharmacophore based
virtual screening using the top ranked hypothesis. The advanced pharmacophore screening option from
PHASE module was employed to retrieve hit compounds that fulfill the chemical moiety necessities and
spatially mapping with subsequent features in the pharmacophoric query [38].
2.3.5. In silico study of the physico-chemical, pharmacokinetic and toxicological properties
In silico study of the physico-chemical, pharmacokinetic and toxicological properties of the most
potent DNase I inhibitors was performed using Molinspiration [39], admetSAR [40], and DataWarrior [41]
prediction tools.
3. RESULTS AND DISCUSSION
3.1. Synthesis and DNase I inhibitory properties of 4-(4-chlorophenyl)thiazol-2-amines
3.1.1. Synthesis
Eleven new 4-(4-chlorophenyl)thiazol-2-amines (3-6, 8-13, 19) were synthesized by Hantsch-thiazole
synthesis illustrated in Scheme 1 [35]. Together with nine previously synthesized derivatives (1 [34], 2, 7 [35],
14 [36] 15-17 [35], 18 [36], 20 [36]), they were evaluated in vitro for the inhibitory activity against bovine
pancreatic DNase I. The obtained results were plotted and IC50 values were calculated (Table 2).
R3
O
S
S
R1
Br
N
R1
+
H2N
N
N
R2
R3
R2
Cl
Cl
Scheme 1. Synthesis of 4-(4-chlorophenyl)thiazol-2-amines.
10

3.1.2. SAR study
Starting from the scaffold of compound 1, SAR analysis of the substituted 4-(4-chlorophenyl)thiazol-
2-amines was performed focusing on the optimization. It was observed that variation of R2 substituents
(including heteroaromatic substituents) showed no differences in DNase I inhibition (compounds 1-8).
Introduction of the bulkier methyl group in R3 position accompanied with phenylcarboxylic acids (9 and 10)
or phenylcarboxilic esters (11 and 12) as R2 subtituents showed decrased IC50 values (below 150 µM). An
introduction of an electron-pulling substituent (acetyl) in R3 position of compound 12 resulted in some
improvement of the IC50 value (106.63 µM for compound 13). In the next step, the influence of different R2
aromatic subtituents on DNase I inhibition was examined (compounds 14-17), where compound 17 showed
the most promissing inhibitory properties. Therefore, this para-hydroxy-phenyl substituent was further
incorporated in compounds 18-20 which also contained electron-pulling groups in R3 position (previously
shown benefical in compound 13). As a result, IC50 values below 100 µM were reached (Table 2). Crystal
violet, used as a positive control in the absence of a "golden standard", showed almost 5-fold weaker inhibitory
effect on commercial DNase I (IC50 = 359.05 ± 7.18 µM) compared to the three most active compounds 18-
20.
Table 2
In vitro DNase I inhibitory activity of the studied 4-(4-chlorophenyl)thiazol-2-amines.
R3
S
R1
N
N
R2
Cl
DNase I inhibition
IC50 (µM) ± SD
Compd.
R1
-H
R2
R3
-H
OH
O
1 [34–36]
>200
O
O
2 [35]
-H
-H
>200
O
O
3
4
-H
-H
-H
-H
>200
>200
O
11

5
6
-H
-H
-CH3
-CH3
>200
>200
N
O
N
S
7 [35]
-H
-H
-CH3
-CH3
>200
>200
N
O
8
O
OH
9
-H
-H
-H
O
-CH3
-CH3
-CH3
136.84 ± 11.11
142.01 ± 14.87
105.21 ± 14.32
OH
10
11
O
O
O
O
O
12
13
-CH3
126.02 ± 10.53
106.63 ± 15.70
>200
O
O
O
O
OH
14 [36]
-H
-CH3
S
15 [35]
16 [35]
-H
-H
-H
-H
-CH3
-CH3
>200
O
172.12 ± 10.61
130.75 ± 11.83
81.30 ± 8.12
OH
17 [35,36]
18 [36]
-CH3
O
OH
OH
O
O
19
-H
79.79 ± 7.04
H2N
12

OH
20 [36]
-H
-CN
88.81 ± 10.26
3.1.3. R-Group analysis
As Pharma RQSAR model showed, the substantial increase in DNase I inhibition was a consequence
of hydrogen-bond acceptor substituents at 4-(4-chlorophenyl)thiazol-2-amine scaffold (Table S1, Fig. 1).
Furthermore, negatively-charged substituents at R1 position, along with hydrogen-bond donor and
hydrophobic substituents at R3 position, exerted significant enhancement to the 4-(4-chlorophenyl)thiazol-2-
amines activity (Table S1, Fig. 1). Additionally, the presence of hydrophobic substituents at R1 position
resulted in a decreased DNase I inhibition (Table S1, Fig. 1). Next, we examined the effect of the E-State
parameters of 4-(4-chlorophenyl)thiazol-2-amines on DNase I inhibition (Table S2, Fig. 2). As Table S2
shows, the introduction of aasC, dssC, aaCH, ssO, sOH and dO fragments at R1 position, dssC and dO
fragments at R2 position, as well as the introduction of aasC, dssC, tsC, sNH2, tN and dO fragments at R3
position, exerted a significant enhancement to the 4-(4-chlorophenyl)thiazol-2-amines activity. On the
contrary, the use of ssS, sCH3 and aaN fragments at R1 position, as well as the introduction of aaCH fragment
at R3 position, resulted in a decreased DNase I inhibition (Table S2, Fig. 2).
13

Fig. 1. The Pharma RQSAR model of the investigated series of 4-(4-chlorophenyl)thiazol-2-amines. The
attachment positions on the 4-(4-chlorophenyl)thiazol-2-amine 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 pharmacophore feature
type. Examined pharmacophore features: hydrogen-bond acceptor (A), hydrogen-bond donor (D),
hydrophobic (H), negatively-charged (N), positively-charged (P), and aromatic (R).
14

Fig. 2. The E-State RQSAR model of the investigated series of 4-(4-chlorophenyl)thiazol-2-amines. The
attachment positions on the 4-(4-chlorophenyl)thiazol-2-amine 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.
15

3.1.4. Insights into the molecular mechanisms of DNase I-ligand interactions
3.1.4.1. Molecular docking
Our recent in silico results showed that amino acid residues like Asn 7, Arg 9, Glu 39, Arg 111, His
134, 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 [28]. It is worth mentioning that inhibitor-binding pocket, represented
by a boxed surface map, is within the region that interacts with DNA octamer d(GGTATACC)2 (Fig. 3A). Of
note, the importance of His 134 and His 252 residues in the catalytic mechanism of DNase I has already been
highlighted [42]. 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 cleavage of scissile phosphate [43]. Furthermore, several site-directed
mutagenesis experiments on the residues surrounding His 134 and His 252 demonstrated that single mutations
on Glu 39, Asp 168 or Asp 251 residues lead to the inactive DNase I variants [44]. Additionally, the critical
role of Arg 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175 and Tyr 211 residues in DNase I activity was
confirmed, presumably because of their close proximity to the active site [44].
Fig. 3. The top ranked DNase I (A) and 5-LO enzyme (B) binding site, represented by the boxed surface maps.
The intermolecular contacts between most active 4-(4-chlorophenyl)thiazol-2-amines and DNase I
were analyzed using the ligand interaction diagram in Maestro [37]. The interaction profiles of 4-(4-
chlorophenyl)thiazol-2-amines with DNase I domain are displayed in Fig. 4. Showing interactions with
catalytic histidines (His 134 or His 252), compounds 18-20 were the most promising inhibitors of DNase I
(Table 2, Fig. 4). Additionally, these compounds exhibited some interactions with Arg 9, Glu 39, Arg 41, Tyr
76, Arg 111, Asp 168, Asn 170, Tyr 175, Tyr 211 and Asp 251 (Fig. 4), which are also implicated in a DNase
I activity [42–44]. Furthermore, compound 19 as the most active DNase I inhibitor (Table 2), uniquely showed
interactions with both catalytic histidines (Fig. 4).
16

Fig. 4. 3D/2D view of compounds 18 (A,B), 19 (C,D) and 20 (E,F) bound in the active site of DNase I.
Interactions that occur more than 10% of the molecular dynamics simulation time are shown.
3.1.4.2. Molecular dynamics simulation
The study was further extended to assess the stability of docking complexes between DNase I and the
most active 4-(4-chlorophenyl)thiazol-2-amines through molecular dynamics simulations (Animation
contents S1-S3). The RMSD and RMSF plots for DNase I and compounds 18-20 (Figs. S1 and S2) showed
that docking complexes were stable during entire simulation period. The RMSD for Cα, side chains and heavy
atoms remained within the limit of 3 Å (Fig. S1). The similar situation was noted for RMSF values (Fig. S2).
17

The obtained results indicated small structural rearrangements, less conformational changes and confirmed
stability of DNase I complexes [37]. Once the conformational stability of the systems was established, the
interaction stability of the systems was monitored. The interactions observed during 10 ns molecular
simulation confirmed the importance of Arg 9, Glu 39, Arg 41, Tyr 76, Arg 111, His 134, Asp 168, Asn 170,
Tyr 175, Tyr 211, Asp 251 and His 252 in the binding with the most active 4-(4-chlorophenyl)thiazol-2-amines
(Figs. 4-7, Animation contents S1-S3). The continuity of interactions between DNase I and the inhibitors is
clearly expressed by percentage of overall simulation time (Fig. 4). As a consequence of the number, type and
continuity of interactions with catalytic histidines (His 134 and His 252), it is distinctly clear why compound
19 is stronger DNase I inhibitor compared to compounds 18 and 20 (Figs. 4-7).
Fig. 5. Normalized stacked bar chart representation and timeline representation of interactions and contacts
between bovine pancreatic DNase I and compound 18 during the course of 10 ns molecular dynamics
simulation.
18

Fig. 6. Normalized stacked bar chart representation and timeline representation of interactions and contacts
between bovine pancreatic DNase I and compound 19 during the course of 10 ns molecular dynamics
simulation.
Fig. 7. Normalized stacked bar chart representation and timeline representation of interactions and contacts
between bovine pancreatic DNase I and compound 20 during the course of 10 ns molecular dynamics
simulation.
19

3.1.5. Pharmacophore modeling and virtual screening
The best hypothesis model for the studied 4-(4-chlorophenyl)thiazol-2-amines was selected by
analyzing the survival scores in Table S3. The selected AADHRRR.13 hypothesis had 7 features namely, 2
acceptor groups (A), 1 donor group (D), 1 hydrophobic group (H) and 3 aromatic groups (R). The structural
superposition of most active 4-(4-chlorophenyl)thiazol-2-amines with AADHRRR.13 hypothesis is presented
in Fig. 8. The angle and distances between different sites of the model are given in Tables S4 and S5,
respectively. The AADHRRR.13 hypothesis highlighted the 3D space arrangement among hydrogen-bond
substituents and aromatic rings at 4-(4-chlorophenyl)thiazol-2-amines (Fig. 8) and confirmed the importance
of selected groups in DNase I inhibition (Fig. 4). The PubChem database consisting of more than 96 million
compounds was subjected to virtual screening using the 4-(4-chlorophenyl)thiazol-2-amine scaffold. Ligand-
based virtual screening, retrieved 119 hit compounds that fulfilled the chemical moiety necessities of 4-(4-
chlorophenyl)thiazol-2-amines (Table S6). In order to find a virtual candidate for DNase I inhibition,
pharmacophore AADHRRR.13 hypothesis was applied on selected molecules. The results showed that none
of the selected molecules fulfilled the 3D space requirements of AADHRRR.13 hypothesis. These
observations could be potentially utilized to guide the rational design of novel/potent DNase I inhibitors with
4-(4-chlorophenyl)thiazol-2-amine moiety.
Fig. 8. Superimposed structures of the three most active 4-(4-chlorophenyl)thiazol-2-amines with
AADHRRR.13 hypothesis.
20

3.2. 5-LO inhibition
Compounds 18 and 20 were previously reported as potent 5-LO inhibitors [36]. In order to obtain
complete picture of 5-LO inhibitory properties of the three most potent DNase I inhibitors within the
investigated series (18-20), compound 19 was also evaluated on 5-LO inhibition. Results are shown in Table
3.
Table 3
5-LO inhibitory activity of compounds 18-20.
WT
5-LO inhibition [IC50]
(95% CI, n  3)
0.08
PMNL
5-LO inhibition [IC50]
(95% CI, n  3)
0.62
Reference on
5-LO inhibition
Compd.
18
19
20
[36]
/
(0.063 – 0.110)
0.63
(0.39 – 1.02)
0.05
(0.46 – 0.83)
36.47 ± 5.36
Residual activity at 10 µM ± SEM[%]#
1.11
[36]
(0.03 – 0.07)
(0.93 – 1.32)
Measured in cell-free assay (WT) and intact (PMNL) given as IC50 (µM) with 95% confidence intervals (CI)
#Residual activity at 10 µM ± standard error of the mean (%)
PMNL: Polymorphonuclear leukocytes; SEM: Standard error of the mean; WT: Wild-type
This (5-LO) enzyme, able to oxidize fatty acids and thereby synthesize leukotrienes, potent mediators
of oxidative and inflammatory reactions, is expressed in the central nervous system (CNS) neurons and may
participate in neurodegeneration [45–48]. It was shown that the levels of 5-LO and its metabolites
(leukotrienes) are increased in elderly subjects contributing to the development of neurodegenerative
disorders, including Alzheimer's disease, as one of the most common and most studied aging-associated
neurodegenerative disorders [45,49–52]. Thus, the inhibition of 5-LO presents a promising target for
neuroprotective therapies [45,50,52]. As aging is associated with enhanced DNA fragmentation and activation
of apoptotic machinery [53], DNase I also represents an attractive potential target do design alternative
strategies for the treatment of neurodegenerative disorders (Alzheimer's disease).
21

3.2.1. Insights into the molecular mechanisms of 5-LO-ligand interactions
3.2.1.1. Molecular docking
By combining a novel and highly effective algorithm for rapid binding-site evaluation with easy-to-
use property visualization tools, SiteMap provides researchers with an efficient means to identify and
characterize binding sites [54]. The results from the SiteMap analysis highlighted that amino acid residues
like Phe 177, Tyr 181, His 367, Leu 368, His 372, Leu 414, Phe 421, Trp 599, His 600, Ala 603 and Leu 607
constituted the top-ranked binding pocket of 5-LO enzyme (Table S7, Fig. 3B). Of note, the importance of
His 367 and His 372 residues in the catalytic site of 5-LO enzyme has already been highlighted [55,56]. It is
worth mentioning that Leu 368, Leu 414 and Leu 607 form a constellation of branched hydrophobic side
chains that envelops the region where the fatty acid must be positioned for catalysis [56]. Furthermore, it was
shown that Phe 177, Tyr 181, Phe 421, Trp 599, His 600 and Ala 603 cork the catalytic site of 5-LO enzyme
[56].
The interaction profiles of 4-(4-chlorophenyl)thiazol-2-amines with 5-LO domain are displayed in Fig.
9. Showing interactions with His 372 residue in the catalytic site of 5-LO enzyme, compounds 18-20 exhibited
potent activities with inhibitory concentration values in the nanomolar concentration range (Table 3, Fig. 9).
Additionally, these compounds exhibited some interactions with Phe 177, Tyr 181, Phe 421, Trp 599 and His
600 (Fig. 9), which are also implicated in a 5-LO activity [55,56].
22

Fig. 9. 3D/2D view of compounds 18 (A,B), 19 (C,D) and 20 (E,F) bound in the active site of 5-LO enzyme.
Interactions that occur more than 10% of the molecular dynamics simulation time are shown.
3.2.1.2. Molecular dynamics simulation
The study was further extended to assess the stability of docking complexes between 5-LO and
compounds 18-20 through the molecular dynamics simulations (Animation contents S4-S6). The RMSD and
RMSF plots for 5-LO and compounds 18-20 (Figs. S3 and S4) also confirmed that docking complexes were
stable during entire simulation period. The interactions observed during 10 ns molecular simulation confirmed
the importance of Phe 177, Tyr 181, His 367, Leu 368, His 372, Leu 414, Phe 421, Trp 599, His 600, Ala 603
23

and Leu 607 in the binding with compounds 18-20 (Fig. 9, Figs. 10-12, Animation contents S4-S6). The
continuity of interactions between 5-LO and the inhibitors are clearly expressed by percentage of overall
simulation time (Fig. 9). It is worth mentioning that compounds 18 and 20 as the most active 5-LO inhibitors
(Table 3), uniquely showed interactions with both catalytic histidines compared to less active compound 19
(Figs. 10-12). It could be concluded that compounds 18-20, as dual 5-LO and DNase I inhibitors, could be set
as a promising tool for the development of novel therapeutics used in the prevention and/or therapy of
Alzheimer's disease.
Fig. 10. Normalized stacked bar chart representation and timeline representation of interactions and contacts
between 5-LO enzyme and compound 18 during the course of 10 ns molecular dynamics simulation.
24

Fig. 11. Normalized stacked bar chart representation and timeline representation of interactions and contacts
between 5-LO enzyme and compound 19 during the course of 10 ns molecular dynamics simulation.
Fig. 12. Normalized stacked bar chart representation and timeline representation of interactions and contacts
between 5-LO enzyme and compound 20 during the course of 10 ns molecular dynamics simulation.
25

3.3. In silico study of the physico-chemical, pharmacokinetic and toxicological properties
Previously, we found that compound 18 showed no cytotoxic effects at concentration of 10 µM [36].
In order to obtain a more complete picture of the most potent DNase I inhibitors (18-20) within the studied
compounds, their physico-chemical properties were calculated using Molinspiration tool [39] (Table S8).
According to the results, the three most active compounds (18-20) fulfilled Lipinski’s rule of five and therefore
were predicted to have good oral bioavailability [57–59]. Additionally, all three compounds (18-20), as
promising leads in the treatment of Alzheimer's disease, fulfilled all the requirements necessary for a
compound to be predicted as a successful CNS drug (miLogP < 5, TPSA < 60-70 Å2, nON < 7, nOHNH < 3, nrotb
< 8, and MW < 450) [60] (Table S8). Prediction performed by admetSAR [40] confirms that compounds 18-
20 might be able to pass through blood-brain barrier (BBB) and penetrate into the CNS. Considering
toxicological properties calculated by DataWarrior [41], compounds 18-20 were predicted with no risk for
mutagenic, tumorigenic, reproductive or irritant effects.
4. CONCLUSION
A series of twenty 4-(4-chlorophenyl)thiazol-2-amines was tested on DNase I inhibition in vitro. Three
compounds (18-20) inhibited DNase I with IC50 value below 100 µM, being more potent than crystal violet,
used as a positive control. A computational approach has been exploited to reveal the relationship between
the DNase I inhibitory activity and the pharmacophore features found in the 4-(4-chlorophenyl)thiazol-2-
amines. Also, compounds 18-20 have been shown as very potent 5-LO inhibitors with nanomolar IC50 values
obtained in cell-free assay, with compound 20 as the most potent (IC50 = 50 nM). Molecular docking and
molecular dynamics simulations defined the 4-(4-chlorophenyl)thiazol-2-amines interactions with the most
important residues of DNase I and 5-LO.
Based on our in silico study, compounds 18-20 were predicted to have good oral bioavailability, might
be able to pass through BBB and penetrate into the CNS, and were also predicted as compounds with no toxic
effects. Thiazol-2-amines can be regarded as a promising scaffold for design of new DNase I inhibitors and
could have potential therapeutic applications due to the significant involvement of DNase I in pathophysiology
of many disease conditions. Compounds 18-20 could be set as a good starting point for the development of
novel dual inhibitors of 5-LO and DNase I, enzymes involved in the development of neurodegenerative
disorders among elderly, including Alzheimer's disease. Worth mentioning at this point is that 5-LO activity
is not essential for DNase I inhibition as also compounds 1, 7-10 and 14-17 are very potent 5-LO inhibitors
[35,36] but show very different activities towards DNase I.
CONFLICT OF INTEREST
26

The authors declare that there are no conflicts of interest.
ACKNOWLEDGMENTS
We thank Dr. D. Vogt, Dr. S. Woltersdorf and Dr. S. Barzen for synthesis and Dr. S. Kretschmer for
measurement of 5-LO inhibition. The financial support of this work by Ministry of Education, Science and
Technological Development of the Republic of Serbia (Grants No. OI 172044 and OI 171025) and Faculty of
Medicine of the University of Niš (Internal project No. 4) as well as the EU COST Action 13135 is gratefully
acknowledged. The authors would like to thank Schrödinger LLC for providing us the academic licenses free
of cost for this study, and the Alexander von Humboldt Foundation, Bonn, Germany, for financial support of
the visit of H. S. to A. S. in Serbia. Additional travel funding is acknowledged by HHU Funds for International
Relationships (A. Z.).
27

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