Synthesis, physicochemical and biological studies of technetium-99m labeled tacrine derivative as a diagnostic tool for evaluation of cholinesterase level

Article abstract of DOI:10.1016/j.bmc.2016.12.004

In the present work the synthesis and physicochemical investigations of new tacrine analogues labeled with technetium-99m are reported. All obtained novel radioconjugates showed high stability in the presence of an excess of standard amino acids cysteine or histidine, as well as in human serum. Lipophilicity (Log?D values) of these compounds is within the range from 0.92 to 1.56. For the selected radioconjugate^99mTc(NS₃)(CN-NH(CH₂)₇Tac) (Log?D?=?1.56) the biological activity studies in the course of inhibition of acetylcholinesterase action have been performed (IC₅₀?=?45.0?nM, estimated by means of Ellman's method). Biodistribution studies of this compound showed its uptake in brain on the level of 0.07%ID/g and its clearance through the hepatic and renal route in comparable degree. The ascertained presence of the radioconjugate in brain indicates its possibility to cross the blood-brain barrier. Molecular modeling of^99mTc(NS₃)(CN-NH(CH₂)₇Tac) radioconjugate showed that the main structural fragment is tacrine moiety which is responsible for most interactions within catalytic and peripheral active sites and provides the anti-acetylcholinesterase activity. The^99mTc(NS₃)(CN-NH(CH₂)₇Tac) radioconjugate may be considered to be a diagnostic tool for patients suffering from Alzheimer's disease as well as a marker to determine the physiological condition of liver and intestines.

Full text of DOI:10.1016/j.bmc.2016.12.004

Accepted Manuscript
Synthesis, physicochemical and biological studies of technetium-99m labeled
tacrine derivative as a diagnostic tool for evaluation of cholinesterase level
Ewa Gniazdowska, Przemysław Koźmiński, Marek Wasek, Marek Bajda,
Joanna Sikora, Elżbieta Mikiciuk-Olasik, Paweł Szymański
PII:
S0968-0896(16)30875-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.004
BMC 13431
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 October 2016
17 November 2016
3 December 2016
Please cite this article as: Gniazdowska, E., Koźmiński, P., Wasek, M., Bajda, M., Sikora, J., Mikiciuk-Olasik, E.,
Szymański, P., Synthesis, physicochemical and biological studies of technetium-99m labeled tacrine derivative as
a diagnostic tool for evaluation of cholinesterase level, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.12.004
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Synthesis, physicochemical and biological studies of technetium-99m
labeled tacrine derivative as a diagnostic tool for evaluation of
cholinesterase level
1
1*
2
3
4
Ewa Gniazdowska , Przemysław Koźmiński , Marek Wasek , Marek Bajda , Joanna Sikora ,
5
6**
Elżbieta Mikiciuk-Olasik , Paweł Szymański
1
Centre of Radiochemistry and Nuclear Chemistry, Institute of Nuclear Chemistry and
Technology, Warsaw, Poland;
2
Department of Bioanalysis and Drug Analysis, Medicinal University of Warsaw, Warsaw,
Poland
3
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian
University Medical College, Krakow, Poland;
4
Laboratory of Bioanalysis, Department of Pharmaceutical Chemistry, Drug Analyses and
Radiopharmacy, Medical University, Lodz, Poland;
5
Department of Pharmaceutical Chemistry, Drug Analyses and Radiopharmacy, Medical
University, Lodz, Poland;
6
Laboratory of Radiopharmacy, Department of Pharmaceutical Chemistry, Drug Analyses
and Radiopharmacy, Medical University, Lodz, Poland.
*
Corresponding author: Phone +48 22 5041011; E-mail: p.kozminski@ichtj.waw.pl
**
Corresponding author: Phone +48 42 6779290; E-mail: pawel.szymanski@umed.lodz.pl
1

ABSTRACT
In the present work the synthesis and physicochemical investigations of new tacrine
analogues labeled with technetium-99m are reported. All obtained novel radioconjugates
showed high stability in the presence of an excess of standard amino acids cysteine or
histidine, as well as in human serum. Lipophilicity (LogD values) of these compounds is
9
9m
within the range from 0.92 to 1.56 For the selected radioconjugate
Tc(NS
3
)(CN-
NH(CH Tac) (LogD = 1.56) the biological activity studies in the course of inhibition of
2 7
)
acetylcholinesterase action have been performed (IC50 = 45.0 nM, estimated by means of
Ellman’s method). Biodistribution studies of this compound showed its uptake in brain on the
level of 0.07 %ID/g and its clearance through the hepatic and renal route in comparable
degree. The ascertained presence of the radioconjugate in brain indicates its possibility to
9
9m
cross the blood-brain barrier. Molecular modeling of
Tc(NS
3
)(CN-NH(CH
2
7
) Tac)
radioconjugate showed that the main structural fragment is tacrine moiety which is
responsible for most interactions within catalytic and peripheral active sites and provides the
9
9m
anti-acetylcholinesterase activity. The Tc(NS
3
)(CN-NH(CH
2
7
) Tac) radioconjugate may be
considered to be a diagnostic tool for patients suffering from Alzheimer’s disease as well as a
marker to determine the physiological condition of liver and intestines.
Keywords: tacrine, cholinesterase inhibitor, radiopharmaceuticals technetium-99m,
biodistribution, molecular modeling studies
Abbreviations: Tac, tacrine (1,2,3,4-tetrahydroacridin-9-amine); AD, Alzheimer's disease;
AChE, acetylocholinesterase; CNS, central nervous system; IC50, half maximal inhibitory
concentration; BM, biologically active molecule; BFCA, Bifunctional Coupling Agent; NS ,
3
tris(2-mercaptoethyl)-amine; CN-BFCA, isocyanobutyric acid succinimidyl ester; n.c.a., no
2

carrier added; HPLC, high-performance liquid chromatography; R
Phosphate buffered saline; TFA, trifluoroacetic acid; RCP, radiochemical purity; RCY,
radiochemical yield; Et N, triethylamine; Hynic, 6-hydrazinenicotinic acid; DTNB, 5,5'-
dithiobisnitrobenzoic acid; SD, standard deviation
T
, retention time; PBS,
3
1
. Introduction
Tacrine (1,2,3,4-tetrahydroacridin-9-amine, tetrahydroacridine) is an oral medicament used
to treat patients with Alzheimer's disease (AD) – the most common form of dementia. There
1
,2
is no cure for this disease, which worsens as it progresses, and leads to death . Tacrine
3
,4
belongs to the class of drugs which are cholinesterase inhibitors . Cholinesterase inhibitors
inhibit the action of acetylcholinesterase (AChE) which is the enzyme responsible for the
degeneration of acetylcholine (ACh). Acetylcholine is one of several neurotransmitters in
central nervous system (CNS) – chemicals used by nerve cells to communicate with one
another. Reduced the level of ACh in the brain is believed to be responsible for some of the
symptoms of Alzheimer's disease. By blocking the enzyme that hydrolyses ACh, the
concentration of ACh in the brain increases, which results in the improvement in thinking and
alleviation of disease clinical symptoms. Tacrine in the form of monohydrochloride was the
first drug approved by the United States Food and Drug Administration in 1993 for palliative
treatment of AD. Tacrine and its analogues labeled with diagnostic radionuclide (e.g. Tc-99m,
I-125, C-11) were also studied from the view-point of their application as potential diagnostic
4
-6
agent able to define the specific site of action in the brain . However, the use of tacrine is
limited due to significant appearance of hepatotoxicity and cardiovascular system impairment,
7
in spite of its mild cognitive benefits which do not alter the course of the disease . Therefore,
the search for new tacrine analogues is still of interest for scientists involved in AD
8
-12
research . Due to presence of AChE in liver and intestines, tacrine and its analogues labeled
3

with diagnostic radionuclide can also play a role of markers to determine the physiological
condition of these organs.
The more and more important role in modern medicine is played by radiopharmaceuticals,
the compounds labeled with various short-lived radionuclides which are introduced into the
body for either diagnostic or therapeutic purposes. The radiopharmaceuticals selectively
distributed within given tissues or organs, are usually coordination compounds with the
radiometal as the central ion, firmly attached to the biologically active molecule (BM). The
measurement of space distribution of intensity of the gamma rays emitted from the body
makes possible to determine localization of the radiopharmaceuticals within the body and to
1
3
define abnormalities in the tissue structures or in the functioning of the studied organs . The
diagnostic methods of nuclear medicine make possible detection of diseases at their early
stage, much earlier than the accompanying morphological changes could be detected by
methods of classical medicine. Among the most important diagnostic radiopharmaceuticals
are those labeled with technetium-99m – the radionuclide of nearly ideal nuclear properties
1
4-16
(
T
1/2 = 6 h, E
γ
= 140 keV) and of rich coordination chemistry
.
The goal of this work was to synthesize and investigate the radioconjugates of the newly
designed tacrine analogues with the ‘4+1’ mixed-ligands technetium(III) complex. These
complexes are very stable not only in the thermodynamic sense, but also towards ligand
17-19
99m
exchange in vivo . The radioconjugates
Tc(NS
3
)(CN-BM) consist of the central metal
ion Tc(III) coordinated by the tetradentate NS
3
tripodal chelator (tris(2-mercaptoethyl)-amine)
and a monodentate isocyanide ligand CN-BFCA (isocyanobutyric succinimidyl ester, BFCA -
Bifunctional Coupling Agent), previously coupled with the biomolecule. The technetium-99m
labeled tacrine analogue introduced into the body accumulates in areas of high concentration
of cholinesterase (mainly in the central nervous system) and thereby becomes a diagnostic
4

probe able to determine indirectly the level of the neurotransmitter in the brain and as the
result the areas of neurodegeneration.
9
9m
To verify that Tc(NS
scale, the non-radioactive ‘cold’ rhenium reference compound, Re(NS
has been synthesized and characterized by mass spectrometry, elemental analysis and H and
3
)(CN-NH(CH
2
)
7
Tac) radioconjugate has been synthesized in n.c.a.
3
)(CN-NH(CH Tac),
2 7
)
1
1
3
C NMR.
2
2
. Methods
.1. General
All solvents and commercially available substances were of reagent grade and used
without further purification. Deionized water was prepared in a Hydrolab water purification
system (Hydrolab, Poland)
The 5,5'-dithiobisnitrobenzoic acid (DTNB), acetylthiocholine iodide (enzyme substrate) and
tacrine were obtained from Sigma-Aldrich (Munich, Germany).
The gradient and HPLC conditions were as follows: solvent A, 0.1% (v/v) TFA in water;
solvent B, 0.1% (v/v) trifluoroacetic acid (TFA) in acetonitrile;
System 1: semi-preparative Phenomenex Jupiter Proteo column, 4 μm, 90 Å, 250  10 mm,
UV/Vis detection at 220 nm, gradient elution: 0 - 20 min 20 to 80% solvent B, 20 min 80%
solvent B; 2 mL/min.;
System 2: analytical Phenomenex Jupiter Proteo column, 4 μm, 90 Å, 250  4.6 mm, -
detection, gradient elution: 0 - 20 min 20 to 80% solvent B, 20 min 80% solvent B; 1 mL/min.
9
9m
99
99m
Na TcO was eluted from the commercially available Mo/ Tc generator (Radioisotope
4
Centre POLATOM, Institute of Atomic Energy, Poland).
Mass Spectrometry (MS): Mass spectra were measured on the Bruker 3000 Esquire mass
spectrometer equipped with ESI.
5

Infrared (IR): IR spectra in solid KBr pellets (investigated species amount to about 1% of the
-1
pellet) were recorded in the range 4.000 - 600 cm using Bruker Equinox 55 FT-IR
spectrophotometer. All spectra were registered independently at least three times with 50
-1
scans each and with spectral resolution of 1 cm .
1
13
H NMR and C NMR spectra were obtained on a 400 MHz Varian Mercury spectrometer at
1
room temperature. In the case of H NMR chemical shifts were reported as δ values relative to
the internal TMS.
2
.2. Synthesis
The compounds (NS
3
, CN-BFCA and Re(NS
3
)(PMe Ph)) were characterized by different
2
1
13
methods, i.e. Elemental Analysis (EA), Mass Spectrometry (MS), Infrared (IR), H and C
NMR. The purity of these compounds, determined by Elemental Analysis and analytical
HPLC in system 2, was higher than 97%.
2
.2.1. Tris(2-mercaptoethyl)-amine
The tetradentate NS ligand was prepared by reaction of tris(2-chloroethyl)amine
hydrochloride with potassium thioacetate, followed by reduction with LiAlH . The final
product was precipitated as the oxalate salt and applied as such in further reactions. Detailed
3
4
2
0
conditions are given in ref .
EA, Found (Calculated): C 36.52 (36.51); H 7.27 (7.66); N 7.62 (7.10)
1
H NMR (CDCl
3
)  (ppm): 1.80 (s, 3H, SH); 2.72 (m, 12H, NCH
)  (ppm): 822.8 (H CS); 57.0 (NCH
IR (KBr plates) (cm ): 2987 (CH); 2425 (SH)
.2.2. Isocyanobutyric acid succinimidyl ester
The aliphatic linker CN-BFCA (BFCA = Bifunctional Coupling Agent) - monodentate
2
and SCH
2
)
1
3
C NMR (CDCl
3
2
2
)
-1
2
2
1
isocyanide ligand was synthesized according to the procedure described in ref .
6

EA, Found (Calculated): C 51.82 (51.43); H 4.72 (4.80); N 13.44 (13.33)
1
H NMR (CD
NC)
C NMR (CD
3
CN)  (ppm): 2.07 (m, 6H, CH
2
CH
2
CH
2
); 2.76 (t, 2H, CH CO); 3,56 (tt, 2H,
2
CH
2
1
3
3
CN)  (ppm): 25.6 (CH
2
CH
2
CH ); 40.4 (CN); 170.2 (CO)
2
-1
IR: (KBr plates), cm : 2951, 2926 (CH); 2152 (CN); 1814, 1785, 1729 (CO) (succinimidyl
ester)
+
MS (m/z): Calculated: 210.21, Found: 211.1 [M+H ];
2
.2.3. “Cold rhenium precursor”, Re(NS
3
)(PMe
2
Ph)
Ph) was synthesized according to the
The “cold rhenium precursor” Re(NS
3
)(PMe
2
2
2
procedure described in ref .
EA, Found (Calculated): C 32.48 (32.42); H 4.42 (4.47); N 2.53 (2.70); S 18.79 (18.54)
1
H NMR (CDCl
xNCH ); 1.91 (d, 6H, 2xCH
MS (m/z): Calculated: 518.72, Found: 519.68 [M+H ]
.2.4. Tacrine derivatives, NH (CH Tac
Tacrine derivatives were synthesized according to the procedure described in ref . The
3
)  (ppm): 7.42 and 7.92 (m, 5H, C
6
H
5
); 2.9 and 3.1 (m, 12H, 3xCH
2
S and
3
2
3
)
+
2
2
)
2 n
2
3,24
synthesis of all tacrine derivatives (1a-1h) concerned obtainment of 9-chloro-1,2,3,4-
tetrahydroacridine by direct heating the mixture of anthranilic acid and cyclohexanone in
fresh POCl . In the next step of synthesis coupling 9-chloro-1,2,3,4-tetrahydroacridine with 2
3
equivalents of the appropriate diamine and catalytic amounts of sodium iodide in the presence
o
of phenol at 180 C was performed. All compounds were purified by flash chromatography to
get the final products. The purity of final products was determined by TLC. Some compounds
required additional purification by HPLC method. Finally, the purity of all tacrine derivatives
was higher than 97 %.
7

NH
2
(CH
2
)
2
Tac (1a, N-(1,2,3,4-Tetrahydroacridin-9-yl)ethane-1,2-diamine)
-
IR (KBr)  (cm 1): 1562.5, 2854.1, 2930.8, 3058.1, 3358.8;
1
H NMR (CDCl
t, 2H, NHCH ), 3.0 (s, 2H, in cyclohexane ring), 2.8–2.9 (m, 2H, CH
cyclohexane ring), 2.0 (s, 2H, NH
3
) ( ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 5.1 (s, 1H, NH), 3.5
(
2
2
NH ), 2.6 (s, 2H, in
2
2
), 1.8 (t, 4H, in cyclohexane ring).
+
MS (m/z): Calculated: 241.34, Found: 242.22 [M+H ]
NH
2
(CH
2
)
3
Tac (1b, N-(1,2,3,4-Tetrahydroacridin-9-yl)propane-1,3-diamine)
-
IR (KBr)  (cm 1): 1579.1, 2856.1, 2928.0, 3059.5, 3313.1;
1
H NMR (CDCl
t, 2H, NHCH ), 2.9 (s, 2H, in cyclohexane ring), 2.6–2.8 (m, 2H, CH
cyclohexane ring), 2.0 (s, 2H, NH
CH CH CH ).
3
) ( ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 5.0 (s, 1H, NH), 3.5
(
2
2
NH ), 2.6 (s, 2H, in
2
2
), 1.8 (t, 4H, in cyclohexane ring), 1.6–1.7 (m, 2H,
2
2
2
+
MS (m/z): Calculated: 255.36, Found: 256.23 [M+H ]
NH
2
(CH
2
4
) Tac (1c, N-(1,2,3,4-Tetrahydroacridin-9-yl)butane-1,4-diamine)
-1
IR (KBr) ν (cm ): 1580.3, 2859.3, 2931.8, 3060.8, 3350.3;
1
H NMR (CDCl
t, 2H, NHCH ), 3.1 (s, 2H, in cyclohexane ring), 2.8-2.7 (4H, in cyclohexane ring and
CH NH ), 1.9-1.8 (m, 6H, in cyclohexane ring and NH ), 1.9 (q, 4H, CH CH CH CH in
aliphatic hydrocarbon chain).
MS (m/z): Calculated: 269.39, Found: 270.2 [M+H ]
3
) (δ ppm.): 8.0 - 7.4 (4H, in aromatic ring of benzene), 4.2 (br, 1H, NH), 3.5
(
2
2
2
2
2
2
2
2
+
NH
2
(CH
2
)
5
Tac (1d, N-(1,2,3,4-tetrahydroacridin-9-yl)pentane-1,5-diamine)
-
IR (KBr)  (cm 1): 1562.8, 2854.7, 2930.6, 3065.2, 3275.1;
1
H NMR (CDCl)(  ppm): 7.8 - 7.3 (4H, in aromatic ring of benzene), 3.9 (br, 1H, NH), 3.4
(
t, 2H, NHCH
2
), 3.0 (s, 2H, in cyclohexane ring), 2.7–2.6 (m, 4H, in cyclohexane ring and
8

CH
m, 4H,CH
MS (m/z): Calculated: 283.42, Found: 284.21 [M+H ]
2
NH
2
), 1.9–1.8 (m, 6H, in cyclohexane ring and NH
2
), 1.6 (q, 2H, NHCH
2
CH ), 1.5–1.3
2
(
2
CH CH CH in aliphatic hydrocarbon chain).
2
2
2
+
NH
2
(CH
2
)
6
Tac (1e, N-(1,2,3,4-Tetrahydroacridin-9-yl)hexane-1,6-diamine)
-
IR (KBr)  (cm 1): 1563.0, 2856.0, 2929.0, 3063.0, 3354.0;
1
H NMR (CDCl
t, 2H, NHCH ), 3.1 (s, 2H, in cyclohexane ring), 2.7 (d, 2H, CH
cyclohexane ring), 2.1 (s, 2H, NH ), 1.9 (t, 4H, in cyclohexane ring ), 1.5–1.7 (m, 4H,
NHCH CH CH in aliphatic hydrocarbon chain), 1.2–1.4 (br, 4H, CH CH in aliphatic
hydrocarbon chain).
MS (m/z): Calculated: 297.44, Found: 298.23 [M+H ]
NH (CH Tac (1f, N-(1,2,3,4-Tetrahydroacridin-9-yl)heptane-1,7-diamine)
3
) ( ppm.): 8.0 - 7.4 (4H, in aromatic ring of benzene), 4.1 (s, 1H, NH), 3.6
(
2
2
NH ), 2.6 (s, 2H, in
2
2
2
2
2
2
2
+
2
2 7
)
-1
IR (KBr) ν (cm ): 1580.3, 2854.9, 2927.2, 3059.5, 3307.4;
1
H NMR (CDCl
t, 2H, NHCH ), 3.0 (s, 2H, in cyclohexane ring), 2.7 (d, 4H, CH
cyclohexane ring), 2.0 (s, 2H, NH ), 1.8–1.9 (m, 4H, in cyclohexane ring), 1.5 - 1.6 (m, 2H,
NHCH CH in aliphatic hydrocarbon chain), 1.2–1.4 (br, 6H, CH CH in aliphatic
hydrocarbon chain).
MS (m/z): Calculated: 311.47, Found: 312.24 [M+H ]
3
) (δ ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 3.9 (s, 1H, NH), 3.4
(
2
2
CH NH ), 2.6 (s, 2H, in
2
2
2
2
2
2
2
+
NH
2
(CH
2
)
8
Tac (1g, N-(1,2,3,4-Tetrahydroacridin-9-yl)octane-1,8-diamine)
-
IR (KBr)  (cm 1): 1562.0, 2854.0, 2926.0, 3061.0, 3347.0;
1
H NMR (CDCl
t, 2H, NHCH ), 3.0 (s, 2H, in cyclohexane ring), 2.6 (d, 4H, CH
cyclohexane ring), 2.1 (s, 2H, NH ), 1.8 (p, 4H, in cyclohexane ring), 1.5–1.6 (m, 2H,
NHCH CH ), 1.2–1.3 (br, 8H, CH CH CH CH in aliphatic hydrocarbon chain).
3
) ( ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 3.9 (s, 1H, NH), 3.4
(
2
2
CH NH ), 2.5 (s, 2H, in
2
2
2
2
2
2
2
2
2
9

+
MS (m/z): Calculated: 325.50, Found: 326.20 [M+H ]
NH (CH Tac (1h, N-(1,2,3,4-Tetrahydroacridin-9-yl)nonane-1,9-diamine)
2
)
2 9
-1
IR (KBr) ν (cm ): 1562.4, 2853.3, 2925.9, 3059.8, 3289.1;
1
H NMR (CDCl
t, 2H, NHCH ), 3.0 (s, 2H, in cyclohexane ring), 2.6 (d, 4H, CH
cyclohexane ring), 2.1 (s, 2H, NH ), 1.9 (m, 4H, in cyclohexane ring), 1.5–1.6 (m, 2H,
NHCH CH ), 1.4–1.1 (br, 10H, CH CH
3
) (δ ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 3.9 (s, 1H, NH), 3.4
(
2
2
CH NH ), 2.5 (s, 2H, in
2
2
2
2
2
2
2
in aliphatic hydrocarbon chain).
+
MS (m/z): Calculated: 339.52, Found: 340.28 [M+H ]
2
.2.5. Syntheses of CN-NH(CH
The coupling reaction of the isocyanide linker CN-BFCA with NH
in Scheme 1.
2
)
n
Tac
2
(CH ) Tac is shown
2
n
Scheme 1. Coupling of CN-BFCA linker with NH
In the case of all tacrine derivatives (1a-1h) the coupling reaction of CN-BFCA linker
with NH (CH Tac was performed according to the same procedure. Solution containing about
.5 mg (2.3 μmol) of CN-BFCA dissolved in 100 μL of DMF and about 0.5 μL (3.6 μmol) of
triethylamine was added to the appropriate amount of NH (CH Tac, so that the ratio of
CN-BFCA to Et N and to NH (CH Tac was approximately equal to 1.2 : 2 : 1 (slight excess
of CN-BFCA and about two times excess of Et N in relation to the tacrine derivative
2
(CH
2
)
n
Tac.
2
)
2 n
0
2
)
2 n
3
2
)
2 n
3
1
0

NH
2
(CH
2
n
) Tac. The mixture was allowed to stay overnight at room temperature and then the
solvent was removed under vacuum. The residue was dissolved in a mixture of 50 μL of
acetonitrile and 100 μL of water. The crude product was purified by semi-preparative HPLC
(
System 1), alkalized and lyophilized. Yield ≥ 95%.
+
+
MS of 2a, (m/z): Calculated: 336.16, Found: 337.21 [M+H]
MS of 2b, (m/z): Calculated: 350.18, Found: 351.21 [M+H]
+
MS of 2c, (m/z): Calculated: 364.20, Found: 365.22 [M+H]
MS of 2d, (m/z): Calculated: 378.21, Found: 379.23 [M+H]
+
+
MS of 2e, (m/z): Calculated: 392.23, Found: 393.27 [M+H]
+
MS of 2f, (m/z): Calculated: 406.58, Found: 407.24 [M+H]
MS of 2g, (m/z): Calculated: 420.26, Found: 421.27 [M+H]
+
+
MS of 2h, (m/z): Calculated: 434.27, Found: 435.29 [M+H]
9
9m
2
.2.6. Preparation of Tc(NS
3
)(CN-NH(CH
2
)
n
Tac)
9
9m
17
For labeling CN-NH(CH Tac with Tc a two-step procedure was applied (Scheme 2).
2 n
)
9
9m
Scheme 2. Labeling of CN-NH(CH
2
) Tac with technetium-99m (using the Tc complex of
n
the type’4+1’).
9
9
99m
In the first step 1 mL of eluate from the Mo/ Tc generator (200 - 1000 MBq) was
added to a kit formulation containing 1 mg of Na EDTA, 5 mg of mannitol and 0.1 mg of
SnCl in the freeze-dried form under nitrogen. The mixture was allowed to stand at room
2
2
1
1

temperature for 20 min. The radiochemical purity was checked by HPLC (System 2) and TLC
methods. When Merck 60 WF254 aluminium sheets are used the intermediate complex
9
9m
Tc-EDTA/mannitol migrates with the solvent front in water but remains at the origin in
9
9m
acetone. In the second step the intermediate
00 µg of the NS ligand and with about 50 µg of the isocyanide-modified tacrine derivative
CN-NH(CH Tac. The reaction progress and radiochemical purity were controlled by HPLC
System 2). The radiochemical yield of the
Tc-EDTA/mannitol compound reacted with
3
3
)
2 n
9
9m
(
Tc-labeled radioconjugates was approximately
9
5%.
.2.7. Synthesis of Re(NS
The non-radioactive rhenium reference compound Re(NS
prepared in two steps (Scheme 3). In the first step 4.02 mg (7.74 μmol) of ‘cold rhenium
precursor’ Re(NS )(PMe Ph) and 2.46 mg (11.46 μmol) of isocyanobutyric succinimidyl ester
were dissolved in 1 mL of CHCl and stirred at room temperature for 2 h. In the second step
to the mixture containing 0.78 mg (1.32 μmol) of Re(NS )(CN-BFCA) and 0.91 mg (2.89
μmol) of NH (CH Tac dissolved in 160 μL of DMF we have added 0.24 μL (1.72 μmol) of
2
3
)(CN-NH(CH
2
) Tac)
7
3
)(CN-NH(CH
2
7
) Tac) was
3
2
3
3
2
2 7
)
triethylamine. The mixture was allowed to stand overnight at room temperature. After
completion of the reaction, DMF was removed under vacuum and the residue was dissolved
in 200 μL of acetonitrile/water (1:1) mixture. The products obtained in both steps were
purified on a semipreparative HPLC column under conditions described above (System 1) and
lyophilized. Yield ≈ 45%.
Scheme 3. Reaction route to Re(NS
3
)(CN-NH(CH
2
7
) Tac).
1
2

1
The compound was characterized by HPLC in system 2 and H-NMR.
Data for Re(NS )(CN-BFCA), C15 Re:
3
H
22
N
3
O S
4 3
EA, Found (Calculated): C 30.48 (30.50); H 3.76 (3.75); N 7.15 (7.11); S 16.48 (16.28)
+
MS (m/z): Calculated: 590.76, Found: 591.72 [M+H]
Data for Re(NS
3
)(CN-NH(CH
2
)
7
-Tac), C31
H
46
N
5
OReS :
3
EA, Found (Calculated): C 47.18 (47.30); H 5.96 (5.89); N 8.95 (8.90); S 12.48 (12.22)
1
H NMR (CD
H, NH); 3.58 (m, 2H, CNCH
.03 (s, 2H, in cyclohexane ring); 2.92 (t, 2H, CH
.69 (d, 4H, CH CH CH NH); 2.62 (s, 2H, in cyclohexane ring); 2.01 (m, 2H, CH
.8-1.9 (m, 4H, in cyclohexane ring); 1.5-1.6 (m, 2H, NHCH CH CH ); 1.2 - 1.4 (br, 6H,
CH CH in aliphatic hydrocarbon chain)
C NMR (CDCl ) (δ ppm.): 170.2 (1C, C(O)), 157.2 (1C, CN), 158.5, 150.7, 147.5, 128.7,
28.2, 123.5, 122.9, 120.2, 115.8 (9C, C, CH in aromatic rings), 58.7 (3C, NCH CH S), 43.8
3
CN)  (ppm): 7.89 - 7.29 (4H, CHCHCH in aromatic ring of benzene); 3.91 (s,
1
3
2
1
2
CH
2
); 3.41 (t, 2H, NHCH
CH C(O)N); 2.76 (t, 6H, 3xNCH
CH
2
CH
2
); 3.09 (m, 6H, 3xNCH
2
CH
2
S);
S);
2
2
2
CH
2
2
2
2
2
2
CH );
2
2
2
2
CH
2
2
2
1
3
3
1
2
2
(
3C, CH
2
S), 49.4, 41.9, 40.5, (3C, CNH
2
, CNH, CNCH
2
in aliphatic chain), 34.0, 33.1, 31.7,
in aliphatic chain and cyclohexane
2
9.8, 29.2, 26.9, 26.7, 24.8, 24.1, 23.0, 22.8 (11C, CH
2
ring).
+
MS (m/z): Calculated: 787.14, Found: 788.23[M+H]
2
.3. In vitro stability studies
9
9m
Stability studies of Tc(NS
3
)(CN-NH(CH
2
n
) Tac). The radioconjugate isolated from the
reaction mixture (using HPLC System 1), present in the solution in concentration no higher
-4
o
than 10 mM, was incubated at 37 C with 10 mM solutions of histidine or cysteine in the
PBS buffer (pH 7.4). HPLC analyses of the incubated solutions were performed at different
time periods from 0.5 h up to 24 h, since staring the incubation.
99m
2
.3.1. Stability studies of Tc(NS
3
)(CN-NH(CH
2
n
) Tac) in human serum
1
3

9
9m
For that purpose 0.1 mL of solution of the isolated
Tc(NS
3
)(CN-NH(CH
2
n
) Tac)
radioconjugate in the 0.1 M PBS buffer, pH 7.4, was added to 0.9 mL of human serum
(
obtained from the Centre of Radiobiology and Biological Dosimetry, INCT Warsaw) and
o
incubated at 37 C. At specified time intervals small samples (0.1 - 0.2 mL) of the mixture
were withdrawn, mixed in the Eppendorf tube with ethanol (0.3 - 0.5 mL) and vigorously
shaken to precipitate proteins. Then, the samples were centrifuged (14000 rpm, 5 min) and the
supernatant was separated. The radioactivity of both supernatant and precipitate was
measured using the well-type NaI(Tl) detector. To check if the radioconjugates did not
convert into other water-soluble radioactive species, aliquots of the supernatant were analyzed
9
9m
by HPLC for the content of the Tc(NS
3
)(CN-NH(CH
)(CN-NH(CH Tac) in cerebrospinal fluid
Tc(NS )(CN-NH(CH Tac) radioconjugate
2
)
n
Tac) complex.
99m
2
.3.2. Stability studies of Tc(NS
3
)
2 n
9
9m
About 0.05 mL of the solution of isolated
3
)
2 n
in the 0.1 M PBS buffer, pH 7.4, was added to 0.5 mL of cerebrospinal fluid (obtained from
o
hospital) and incubated at 37 C. After that, the stability study procedure was the same as in
the case of stability studies in human serum.
2
.4. Lipophilicity studies
Lipophilicity (expressed as LogD) of the Tc(NS
9
9m
3
)(CN-NH(CH
2
n
) Tac) radioconjugates,
which is an important factor affecting the distribution of drug molecules in the organism, was
characterized by their distribution coefficients, D, in the system n-octanol/PBS buffer pH 7.4.
9
9m
The activity of each layer (which shows concentration of the
Tc species in the layer) was
determined by measuring -radiation, with a well-type NaI(Tl) detector. Distribution
coefficient D was calculated as the ratio of activity of organic to that of aqueous phase (as an
average value from at least three independent measurements). Immediately after the
distribution experiments the aqueous phases were analyzed by HPLC to check whether the
studied radioconjugate has not decomposed during the experiment.
1
4

9
9m
2
.5. In vitro biological activity studies of Tc(NS
3
)(CN-NH(CH
Tc(NS )(CN-NH(CH
and other intermediate species in respect to inhibition of AChE activity, were performed using
2
)
7
Tac)
9
9m
In vitro biological activity studies of the
3
2 7
)
Tac) radioconjugate
2
5,26
modified Ellman’s method
. IC50 value was estimated by plotting the percent inhibition as
a function of the inhibitor concentration and fitting the corresponding concentration–response
curve.
2
.5.1. Erythrocytes preparation
Blood was extracted from healthy donors of Blood Donation Centre in Lodz and
collected in vacuum tubes containing solution of potassium EDTA. Erythrocytes were
separated from plasma and from leucocytes by centrifugation (3000 x g, 10 min, 4 °C) in
Micro 22R centrifuge (Hettich ZENTRIFUGEN). Next, erythrocytes were washed three times
with 0.9% saline at 4 °C (1:1, v/v, centrifugation 3000 x g, 10 min) and stored at -30 °C. The
frozen erythrocytes were restored in a water bath at 37 °C for 15 minutes immediately before
each experiment and diluted (1:400) with phosphate buffer, pH 8. Once thawed erythrocytes
were not frozen again and were not used in following experiments.
2
.5.2. Activity of human erythrocytes acetylcholinesterase
The evaluation of acetylcholinesterase activity was determined by the modified
2
5,26
spectrophotometric Ellman’s method
. 1 mL of diluted erythrocytes was incubated (5 min,
o
3
7 C) with 10 µL of DTNB (1 µmol/mL) and with 10 - 70 µL of aqueous solution of the
studied compound (0.005 - 0.4 µg/mL) or with 10 µL of the aqueous solution of tacrine
0.0002-2.4 µg/mL). As a control sample 1 mL of diluted erythrocytes was incubated with 10
(
µL of DTNB and 10 - 70 µL of distilled water. After incubation was stopped 10 µL of
acetylthiocholine iodide (0.75 µmol/L) was added to each sample and a 3-minutes continuous
recording of enzyme activity was initiated. The continuous measurements of absorbance at
1
5

3
7°C for 3 minutes were performed at wavelength λ = 436 nm by means of a
spectrophotometer (Cecil CE2021, London, England). Registration and evaluation of the
results were performed using the software for kinetic studies (DATA STREAM CE3000 5.0).
For measurement under experimental conditions, 37 °C and λ = 436 nm, the absorption
-
-1
-1
coefficient of the [TNB ] ion, , was found to be 10.6 mM cm . The activity of human
erythrocytes acetylcholinesterase was expressed as the rate of the enzymatic reaction [A/min].
2
.5.3. Analysis of the results
Acetylcholinesterase inhibition, expressed in %, was calculated using the equation
(
(A
0
-AIC)*100%)/A
0
, where A means acetylcholinesterase activity in the control sample,
0
expressed as the rate of the enzymatic reaction [A/min], AIC – acetylcholinesterase activity in
the sample of the examined compound or the reference standard, expressed, as above, as the
rate of the enzymatic reaction [A/min].
99m
2
.6. Biodistribution study of Tc(NS
3
)(CN-NH(CH
2
)
7
Tac) in normal mice
Male BALB/c mice (provided by the Maria Skłodowska-Curie Institute of Oncology,
Warsaw, Poland), aged 12 weeks and weighing between 23 and 29 g, were used in all studies
according to the relevant national regulations. Each mouse was injected with 100 - 130 L of
9
9m
Tc(NS
3
)(CN-NH(CH
2
n
) Tac), 2.01 - 2.55 MBq, through the tail vein. The mice were
sacrificed at 30 min and 1 h post-injection. Then, tissues were dissected, washed free from
blood, blotted dry and weighed in an analytic balance. Associated radioactivity was counted
using gamma counter. The accumulated radioactivity in the tissue of organs was calculated in
terms of percentage of injected dose per gram (%ID/g) of tissue, as well as percentage of
9
9m
injected dose per gram of blood. The standard source of Tc was measured together with the
samples in order to perform decay correction.
1
6

2
.7. Docking studies
Docking studies were performed for the most promising radioconjugate 3f and its parent
tacrine-based complexing agent 2f (tacrine derivative coupled with the bifunctional linker).
The data of docking studies for Donepezil, tacrine and bis-(7)-tacrine were extracted from
crystals of their complexes with acetylcholinesterase (Protein Data Bank codes: 4EY7, 1ACJ,
2
CKM, respectively). The three-dimentional structures of 3f (radioconjugate) and 2f (tacrine-
based complexing agent) were drawn into the GaussView 5.0 and subsequently optimized by
application of DFT-B3LYP method with LANL2DZ basis set in Gaussian 09 software.
Further studying of 2f and 3f compounds (called as ligands in this part of studies), including
checking the atoms, bond types and protonation states, was performed with Sybyl X 1.1.
Finally, 2f and 3f ligands were saved in mol2 format.
Human acetylcholinesterase of resolution 2.35 Å (complex with donepezil, PDB code: 4EY7)
was split into single monomers and chain A was utillized for docking. The protein was
prepared by Hermes 1.5. All histidine residues were treated as HSE tautomers i.e. with
hydrogen at N atom. Missing hydrogen atoms were added and ligand molecules removed.
The binding site was defined to consist from all amino acid residues within 10 Å from
donepezil. Two residues (Tyr337 and Trp286), which are known to occur in the form of two
main rotamers, were treated as flexible.
Dockings were performed with Gold 5.1. A standard set of genetic algorithm with a
population size of 100 and the number of operations of 100 000 was applied. GoldScore and
visual inspection were used for evaluation of the docking results. For each ligand the final
results involved 10 configurations, arranged on the ranking list according to the scoring
function values. Docking of the studied compounds to human AChE was validated on the
basis of reference compounds. Gold Suite could reproduce original orientation of ligands with
low rmsd (root-mean square deviation) values below 2 Å. Since Gold software did not contain
1
7

parameters for technetium, it was parameterized on the basis of manganese. Results were
visualized with PyMOL 0.99rc6.
Software
GaussView 5.0, Semichem Inc., Shawnee Mission, KS, USA, 2009
Gaussian 09, Gaussian Inc., Wallingford, CT, USA, 2009
Sybyl X 1.1, Tripos: St. Louis, MO, USA, 2010
Hermes 1.5, CCDC Software Limited: Cambridge, UK, 2011
Gold 5.1, CCDC Software Limited: Cambridge, UK, 2011
PyMOL 0.99rc6, DeLano Scientific LLC: Palo Alto, CA, USA, 2006
3
3
. Results and discussion
.1. Tacrine derivatives labeling, stability and lipophilicity studies
The first step in our experimental work was preparation of a new series of tacrine
derivatives, NH
chain (Fig. 1A), and coupling these compounds with bifunctional linker CN-BFCA to obtain
monodentate isocyanide ligands containing tacrine derivatives, CN-NH(CH Tac (Fig. 1B).
2
(CH
2
)
n
Tac, containing different number (n) of methylene groups in aliphatic
)
2 n
Figure 1. A – structure of tacrine derivatives containing different number of CH
2
groups in
aliphatic chain; B – structure of monodentate isocyanide ligands containing tacrine
9
9m
derivatives; C - structure of Tc(NS
3
)(CN-NH(CH ) Tac) radioconjugates.
2
n
1
8

The coupling of tacrine derivatives with CN-BFCA linker leads to obtain compounds of
higher lipophilicity – on the recorded HPLC chromatogram (system 1) the peak of the CN-
NH(CH
2
)
n
Tac compound was located at higher retention time (R
Table 1). Table 1 presents also the R values of tacrine derivatives labeled with technetium-
Tac) radioconjugates (Fig. 1C).
T
) than that of NH
2
(CH ) Tac
2
n
(
T
9
9m
9
9m – Tc(NS
3
)(CN-NH(CH
)
2 n
Table 1. Retention time values of the peaks (recorded on HPLC chromatograms)
corresponding to the compounds synthesized in the present work.
Retention time, R , [min]
T
compound
9
9m
NH
2
(
(CH
2
)
n
Tac CN-NH(CH
2
)
n
Tac
Tc(NS
3
)(CN-NH(CH
2
n
) Tac)
system 1)
(system 1)
2.
(system 2)
(
CH
)
2 n
1
.
3.
a: n = 2
b: n = 3
c: n = 4
d: n = 5
e: n = 6
f: n = 7
g: n = 8
h: n = 9
7.9
8.0
9.9
10.4
10.9
11.6
12.2
12.9
13.1
13.2
13.5
14.3
15.0
16.0
16.8
17.8
99m
14.2
14.3
14.2
14.9
15.6
16.2
17.0
17.9
9
9m
All
Tc-labeled tacrine derivatives,
Tc(NS
3
)(CN-NH(CH
2
) Tac), were synthesized
n
with radiochemical yield (RCY) higher than 95%, radiochemical purity (RCP) higher than
8% and specific activity of the radioconjugate of about 30 GBq/mol. The HPLC
9
chromatogram of reaction mixture of randomly chosen tacrine derivative labeling reaction is
shown in Figure 2A. There are two chromatograms presented in this figure. (A) –
9
9m
chromatogram of
chromatogram of ‘cold’ rhenium reference compound Re(NS
peak at R = 16.4 min. Practically, the same positions of these peaks can be a proof of the
existence of the Tc(NS
Tc(NS
3
)(CN-NH(CH
2
)
7
Tac) with the peak at R
T
= 16.2 min, and (B) –
3
)(CN-NH(CH
2 7
)
Tac) with the
T
9
9m
3
)(CN-NH(CH ) Tac) species synthesized in n.c.a. scale.
2
7
1
9

A
B
140000
120000
100000
99m
1000
Tc(NS )(CN-NH(CH ) Tac)
Re(NS )(CN-NH(CH ) Tac)
3
2
7
3
2
7
800
600
400
200
0
80000
60000
40000
20000
0
0
5
10
15
t [min]
20
25
30
0
5
10
15
20
25
30
t [min]
9
9m
Figure 2. HPLC chromatogram of reaction mixture (A):
Tc(NS
3
)(CN-NH(CH
2
7
) Tac)
radioconjugate and (B): Re(NS
compound (system 2).
3
)(CN-NH(CH
2
7
) Tac) which was ‘cold’ rhenium reference
9
9m
Results of stability and lipophilicity (LogD) studies of
Tc(NS
3
)(CN-NH(CH
2
)
)
n
Tac)
radioconjugates isolated from the reaction mixture are presented in Table 2.
9
9m
Table 2. Results of stability and lipophilicity studies of
radioconjugates isolated from the reaction mixture.
Tc(NS
3
)(CN-NH(CH
2
n
Tac)
9
9m
%
of intact Tc(NS
3
)(CN-NH(CH
2
)
n
Tac)o
radioconjugate after 24 h of incubation at 37 C
in solution of:
PBS
LogD
(
2 n
CH )
Cysteine
Histidine
human
serum
buffer
-3
-3
1
0 M
10 M
pH 7.4
n = 2
n = 3
n = 4
n = 5
n = 6
n = 7
n = 8
n = 9
93.5  0.2 1.28 0.08
93.4  0.2 1.41  0.06
95.4  0.3 1.43  0.07
92.2  0.3 1.35  0.07
93.4  0.3 1.45  0.08
95.0  0.5 1.56  0.06
92.9  0.4 1.43  0.05
92.6  0.3 0.92  0.05
In all cases the
Tc(NS )(CN-NH(CH ) Tac)
radioconjugates were entirely stable.
HPLC chromatograms recorded in
different time intervals, up to 24 h,
99m
3
2
n
showed only one peak of R
T
corresponding to that of the studied
radioconjugate.
9
9m
Stability studies of
Tc(NS
3
)(CN-NH(CH
2
)
n
Tac) radioconjugates showed that these
compounds do not undergo the ligand exchange reaction with amino acids or other strongly
2
0

competing natural ligands (containing SH or NH groups), present in excess concentrations in
human body fluids (the so called ‘challenge experiments’). Stability studies of
9
9m
Tc(NS
% of studied radioconjugates have been bound by serum protein components. The obtained
solid residue which precipitated after ethanol addition and sample centrifugation contained
3
)(CN-NH(CH
2
)
n
Tac) radioconjugates in human serum showed also that about 5 to
8
5
- 8% of initial technetium-99m radioactivity.
9
9m
Lipophilicity (LogD values) determined for
Tc(NS
3
)(CN-NH(CH
2
n
) Tac)
radioconjugates are in the range from 0.92 to 1.56. The relationship between LogD value and
the number of CH groups (n) passes through a maximum for n = 7. The changes of LogD
2
value can be explained in terms of hydrophobic hydration of aliphatic hydrocarbons.
Introduction of hydrophobic species (e.g. hydrocarbon chains) into water causes disruption of
the hydrogen bonding network between water molecules. Then in bulk water a cavity is
formed able to accommodate inside a ‘guest’ hydrophobic molecule. It is known that in the
homologues series of aliphatic hydrocarbons and their derivatives the hydrophobic hydration
2
7
increases the LogD values by about 0.65 per each CH
groups located in the  and  positions in respect to the oxygen atom (as well as to nitrogen
atom) are less hydrophobic than those more distant, which makes contribution of these CH
2
group . It is also known that CH
2
2
2
8
groups into the LogD value smaller . Moreover, the hydrocarbon chains containing more
than seven CH groups (n>7) have a tendency to bend and even to curl up, what can result in
their smaller hydrophobic properties compared to those, naturally estimated theoretically, of
2
2
9
hydrocarbon chains containing the same number of CH
Therefore, the introduction of CH groups (n from 2 to 7) into aliphatic chain of
Tac) radioconjugates causes the systematic increase of LogD
2
groups but having linear structure .
2
9
9m
Tc(NS
3
)(CN-NH(CH
)
2 n
value, while introduction of the eighth and ninth CH
2
group results the decrease of the LogD
value.
2
1

According to literature data the LogD values of radiopharmaceuticals, suitable for
3
0
crossing the blood-tissue barrier, should be within the range from 1 to 4 .
Radiopharmaceuticals designed for imaging of the central nervous system, suitable for
crossing the blood-brain barrier, should be characterized with LogD value within the range
3
1
from 1.5 to 2.5 . Therefore, for the subsequent biological studies (stability in cerebrospinal
fluid, biological activity towards acetylcholinesterase and multiorgan biodistribution) the
9
9m
compound Tc(NS
3
)(CN-NH(CH
2
7
) Tac), characterized by the highest LogD value, has been
selected.
9
9m
Stability studies of
Tc(NS
3
)(CN-NH(CH
2
7
) Tac) radioconjugate after incubation in
cerebrospinal fluid up to 24 h (Fig. 3) showed that about 99 % of radioconjugate exists in the
solution in the intact form (about 1 percent of studied radioconjugate has been bound by
cerebrospinal fluid protein components).
60
40
20
0
9
9m
Tc(NS )(CN-NH(CH ) Tac)
3
2 7
0
5
10
15
t [min]
20
25
30
9
9m
Figure 3. HPLC chromatogram of
Tc(NS
3
)(CN-NH(CH
2
7
) Tac) radioconjugate after
incubation in cerebrospinal fluid for 24 h (system 2).
3
.2. Biological activity studies
9
9m
In vitro biological activity studies of the
Tc(NS
3
)(CN-NH(CH
2
7
) Tac) radioconjugate
and other intermediate species in respect to inhibition of AChE activity, were performed using
2
5,26
modified Ellman’s method
. The dependence of acetylcholinesterase inhibition obtained
2
2

during incubation of human erythrocytes of healthy volunteers with various concentrations of
examined compounds, as well as IC50 values, are presented in Figure 4 and Table 3.
100
80
60
40
20
0
1
2
3
f
f
f
Tac
1
E-4
1E-3
0,01
0,1
1
10
concentration [µM]
Figure 4. Inhibition of acetylcholinesterase activity by various concentrations of the examined
compounds (Tac, 1f, 2f, 3f).
Table 3. The IC50 values for activities towards human erythrocytes acetylcholinesterase (mean
±
SD);
IC50
Compound

M
Tacrine, Tac
NH (CH Tac ,1f
CN-NH(CH Tac, 2f
)(CN-NH(CH
0.226 ± 0.095
0.108 ± 0.011
0.013 ± 0.005
0.045 ± 0.003
2
2 7
)
)
2 7
9
9m
Tc(NS
3
2
7
) Tac), 3f
9
9m
Comparison of IC50 values in Table 3 showed that biological activity of 3f ( Tc(NS
3
)(CN-
Tac) radioconjugate) is, indeed, about three times lower than that of 2f (CN-
Tac – tacrine derivative coupled with bifunctional linker), however about two
(CH Tac – tacrine derivative) and about five times higher
than that of Tac (tacrine – the standard AChE enzyme inhibitor). Generally, one can say that
NH(CH
NH(CH
2
2
)
)
7
7
times higher than that of 1f (NH
2
)
2 7
9
9m
labeling of tacrine with technetium-99m (using the
Tc complex of ’4+1’ type) does not
cause the decrease of biological activity of the labeled biomolecule. It is also worth to notice
2
3

that the biological activity of 2f (the compound containing tacrine derivative and ready to
coordinate technetium-99m cation, IC50 = 0.013 ± 0.005 M) is very similar to biological
activity of other acetylcholinesterase inhibitors, e.g. cyclopenta[b]quinoline derivative
coupled with Hynic (the compound, as before, containing biomolecule and used to coordinate
4
technetium-99m cation, IC50 = 17.60 ± 0.8 nM) .
According to "Clarke's Analysis of Drugs and Poisons" there are different data concerning
3
2
therapeutic concentrations of tacrine for the treatment of Alzheimer's disease . Generally,
depending on the duration of treatment and on the dose, the tacrine concentration in the
plasma in the range from 2.6 μg/L to 34 μg/L (0.011 μM - 0.145 μM) is observed. However,
the concentration of tacrine in serum higher than 20 μg/L is associated with a high risk of
adverse reactions. To conclude one can say that our results of in vitro study confirmed that the
9
9m
newly synthesized compounds CN-NH(CH
2
)
7
Tac and
Tc(NS
3
)(CN-NH(CH
2
7
) Tac) are
strong inhibitors of human erythrocytes acetylcholinesterase activity and due to their high
inhibitory activity may prove to be potential candidates for treatment or diagnosis of
Alzheimer's disease.
3
.3. Molecular modeling studies
Binding mode of compounds 2f (tacrine derivative with bifunctional linker) and 3f ( Tc-
9
9m
radioconjugate) with human AChE was studied by docking to the enzyme active sites. It has
been observed that both derivatives were extended along the active gorge of
acetylcholinesterase and interacted with both catalytic and peripheral active sites. Due to this
they can influence not only hydrolysis of acetylcholine but also perform extra functions such
as interaction with amyloid-beta. The main structural fragment of both compounds was
tacrine moiety. It was responsible for most interactions within catalytic active sites and it
provided the anti-acetylcholinesterase activity. Tacrine was located in the anionic subsite near
2
4

the catalytic triad. It formed a characteristic sandwich by - stacking interactions with Trp86
and Tyr337. Additionally, due to the protonation of nitrogen atom in the cyclic system,
hydrogen bond with the carbonyl group of His447 and cation -  interaction with Trp86 and
Tyr337 could be formed. The amine group attached to the tacrine moiety was engaged in H-
bond with water molecule. The alkyl linker was located in the middle part of the gorge of
enzyme and provided hydrophobic interactions with Tyr124 and Tyr341 located at the
peripheral anionic site (PAS). The amide group was located in the PAS over the side chain of
Trp286 and formed simultaneously hydrogen bond with Tyr72. However, there was a small
difference between 2f and 3f compounds. Derivative 2f interacted through CO group with
Tyr72 (H-bond: oxygen from CO – hydrogen from OH of Tyr72) while radioconjugate 3f
formed the hydrogen bond due to NH fragment of the amide group (H-bond: hydrogen from
NH – oxygen from OH of Tyr72). The alkyl chain of cyanobutyrate was engaged in
hydrophobic interactions at the entry to the gorge, mainly with Leu76. The part of 3f
molecule, containing technetium complex, was located on the surface of the enzyme near the
entry to the active site and was interacting with Thr75 and Pro78. The binding mode of both
compounds is presented in Figure 5.
Figure 5. Binding mode of compounds 2f (left) and 3f (right) within the active site of human
acetylcholinesterase.
2
5

3
.4. Biodistribution studies
9
9m
Biodistribution studies performed with
Tc(NS
3
)(CN-NH(CH
2
7
) Tac) are presented in
Table 4 and Figure 6.
9
9m
Table 4. Biodistribution studies of
Tc(NS
3
)(CN-NH(CH
2
7
) Tac) in normal male BALB/c
mice at 30 and 60 min postinjection (n = 3, % ID/g±S.D.).
%
ID/g
Organ
3
0 min
60 min
Heart
Kidneys
Liver
0.73 ± 0.04
2.89 ± 0.18
5.69 ± 0.15
3.49 ± 0.3
0.45 ± 0.02
1.41 ± 0.11
3.99 ± 0.19
2.54 ± 0.3
2.16 ± 0.5
Lungs
Spleen
Brain
1.02 ± 0.9
0.065 ± 0.009
0.305 ± 0.03
0.77 ± 0.1
0.026 ± 0.008
0.52 ± 0.08
0.24 ± 0.09
Stomach
Blood
6
4
2
3
6
0 min
0 min
0
0
0
0
0
0
0
0
,07
,06
,05
,04
,03
,02
,01
,00
t
y
r
s
n
h
d
o
r
e
e
in
ac
a
e
g
le
e
n
iv
n
a
r
lo
H
i
d
L
u
p
B
m
o
B
L
S
t
K
S
99m
Figure 6. Biodistribution studies of Tc(NS )(CN-NH(CH ) Tac) in BALB/c mice after
3
2 7
3
0 and 60 min post-injection.
9
9m
The significantly higher uptake of Tc(NS )(CN-NH(CH ) Tac) in liver (3.99 - 5.69
3
2 7
%
ID/g) than in kidney (1.41 - 2.89 %ID/g) indicates the clearance of the radioconjugate
2
6

mainly through the hepatic route. The relatively high uptake in lungs (2.54 - 3.49 %ID/g) can
be caused by presence of a considerable amount of cholinesterase enzymes in this tissue
(
under normal physiological conditions the presence of cholinesterase enzymes has been
3
3
found in human lung) . The ascertained presence of the radioconjugate in brain (0.065 ±
.009 and 0.026 ± 0.008 %ID/g after 30 min and 60 min postinjection, respectively) indicates
its possibility to cross the blood-brain barrier.
0
4
. Conclusions
9
9m
The Tc-labeled tacrine radioconjugates show high stability in the presence of an excess
of standard amino acids cysteine or histidine. The proof of their stability is that no
transchelation reactions have been observed in the challenge experiments. The
9
9m
Tc(NS
3
)(CN-NH(CH
2
)
n
Tac) radioconjugates are satisfactorily stable also in human serum.
(CH Tac with technetium-99m (using the
Tc complex of the type’4+1’) does not cause the decrease of the biological activity of the
labeled biomolecule. The biodistribution studies performed for the selected radioconjugate,
The labeling procedure of tacrine derivative NH
2
)
2 7
9
9m
9
9m
Tc(NS
3
)(CN-NH(CH
2
7
) Tac), have shown its clearance through the hepatic and renal route
in comparable degree. The ascertained presence of the radioconjugate in brain indicates its
possibility to cross the blood-brain barrier.
9
9m
In conclusion one can say that the
Tc(NS
3
)(CN-NH(CH
2
7
) Tac) radioconjugate may be
considered to be a diagnostic tool for patients suffering from Alzheimer’s disease as well as
for determine physiological condition of liver and intestines.
Acknowledgements and notes
The work has been supported by the statutory activity of the Institute of Nuclear
Chemistry and Technology, Warsaw, Poland and Medical University of Lodz, Poland (Grant
2
7

no. 503/3-015-01/503-31-002).
The authors thank Prof. S. Siekierski (INCT, Warsaw, Poland) for valuable discussion
and review of the manuscript.
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