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

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: DOI: Reference:

S0968-0896(16)30875-6 http://dx.doi.org/10.1016/j.bmc.2016.12.004 BMC 13431

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

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Ewa Gniazdowska , Przemysław Koźmiński , Marek Wasek , Marek Bajda , Joanna Sikora , Elżbieta Mikiciuk-Olasik5, Paweł Szymański6** 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: [email protected]

**

Corresponding author: Phone +48 42 6779290; E-mail: [email protected]

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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 within the range from 0.92 to 1.56 For the selected radioconjugate

99m

Tc(NS3)(CN-

NH(CH2)7Tac) (LogD = 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

99m

Tc(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

99m

Tc(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.

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; NS3, tris(2-mercaptoethyl)-amine; CN-BFCA, isocyanobutyric acid succinimidyl ester; n.c.a., no

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carrier added; HPLC, high-performance liquid chromatography; RT, retention time; PBS, Phosphate buffered saline; TFA, trifluoroacetic acid; RCP, radiochemical purity; RCY, radiochemical yield; Et3N, triethylamine; Hynic, 6-hydrazinenicotinic acid; DTNB, 5,5'dithiobisnitrobenzoic acid; SD, standard deviation

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 is no cure for this disease, which worsens as it progresses, and leads to death1,2. Tacrine belongs to the class of drugs which are cholinesterase inhibitors3,4. 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 agent able to define the specific site of action in the brain 4-6. However, the use of tacrine is limited due to significant appearance of hepatotoxicity and cardiovascular system impairment, in spite of its mild cognitive benefits which do not alter the course of the disease 7. Therefore, the search for new tacrine analogues is still of interest for scientists involved in AD research8-12. Due to presence of AChE in liver and intestines, tacrine and its analogues labeled

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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 define abnormalities in the tissue structures or in the functioning of the studied organs 13. 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 (T1/2 = 6 h, Eγ = 140 keV) and of rich coordination chemistry14-16. 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 exchange in vivo17-19. The radioconjugates

99m

Tc(NS3)(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

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probe able to determine indirectly the level of the neurotransmitter in the brain and as the result the areas of neurodegeneration. To verify that 99mTc(NS3)(CN-NH(CH2)7Tac) radioconjugate has been synthesized in n.c.a. scale, the non-radioactive ‘cold’ rhenium reference compound, Re(NS3)(CN-NH(CH2)7Tac), has been synthesized and characterized by mass spectrometry, elemental analysis and 1H and 13

C NMR.

2. Methods 2.1. 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. Na99mTcO4 was eluted from the commercially available

99

Mo/99mTc generator (Radioisotope

Centre POLATOM, Institute of Atomic Energy, Poland). Mass Spectrometry (MS): Mass spectra were measured on the Bruker 3000 Esquire mass spectrometer equipped with ESI.

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Infrared (IR): IR spectra in solid KBr pellets (investigated species amount to about 1% of the pellet) were recorded in the range 4.000 - 600 cm-1 using Bruker Equinox 55 FT-IR spectrophotometer. All spectra were registered independently at least three times with 50 scans each and with spectral resolution of 1 cm-1. 1

H NMR and 13C NMR spectra were obtained on a 400 MHz Varian Mercury spectrometer at

room temperature. In the case of 1H NMR chemical shifts were reported as δ values relative to the internal TMS. 2.2. Synthesis The compounds (NS3, CN-BFCA and Re(NS3)(PMe2Ph)) were characterized by different methods, i.e. Elemental Analysis (EA), Mass Spectrometry (MS), Infrared (IR), 1H and

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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 NS3 ligand was prepared by reaction of tris(2-chloroethyl)amine hydrochloride with potassium thioacetate, followed by reduction with LiAlH4. The final product was precipitated as the oxalate salt and applied as such in further reactions. Detailed conditions are given in ref20. EA, Found (Calculated): C 36.52 (36.51); H 7.27 (7.66); N 7.62 (7.10) 1

H NMR (CDCl3)  (ppm): 1.80 (s, 3H, SH); 2.72 (m, 12H, NCH2 and SCH2)

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C NMR (CDCl3)  (ppm): 822.8 (H2CS); 57.0 (NCH2)

IR (KBr plates) (cm-1): 2987 (CH); 2425 (SH) 2.2.2. Isocyanobutyric acid succinimidyl ester The aliphatic linker CN-BFCA (BFCA = Bifunctional Coupling Agent) - monodentate isocyanide ligand was synthesized according to the procedure described in ref21.

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EA, Found (Calculated): C 51.82 (51.43); H 4.72 (4.80); N 13.44 (13.33) 1

H NMR (CD3CN)  (ppm): 2.07 (m, 6H, CH2CH2CH2); 2.76 (t, 2H, CH2CO); 3,56 (tt, 2H,

CH2NC) 13

C NMR (CD3CN)  (ppm): 25.6 (CH2CH2CH2); 40.4 (CN); 170.2 (CO)

IR: (KBr plates), cm-1: 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(NS3)(PMe2Ph) The “cold rhenium precursor” Re(NS3)(PMe2Ph) was synthesized according to the procedure described in ref22. 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 (CDCl3)  (ppm): 7.42 and 7.92 (m, 5H, C6H5); 2.9 and 3.1 (m, 12H, 3xCH2S and

3xNCH2); 1.91 (d, 6H, 2xCH3) MS (m/z): Calculated: 518.72, Found: 519.68 [M+H+] 2.2.4. Tacrine derivatives, NH2(CH2)nTac Tacrine derivatives were synthesized according to the procedure described in ref23,24. The synthesis of all tacrine derivatives (1a-1h) concerned obtainment of 9-chloro-1,2,3,4tetrahydroacridine by direct heating the mixture of anthranilic acid and cyclohexanone in fresh POCl3. In the next step of synthesis coupling 9-chloro-1,2,3,4-tetrahydroacridine with 2 equivalents of the appropriate diamine and catalytic amounts of sodium iodide in the presence of phenol at 180 oC 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 %.

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NH2(CH2)2Tac (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 (CDCl3) ( ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 5.1 (s, 1H, NH), 3.5

(t, 2H, NHCH2), 3.0 (s, 2H, in cyclohexane ring), 2.8–2.9 (m, 2H, CH2NH2), 2.6 (s, 2H, in cyclohexane ring), 2.0 (s, 2H, NH2), 1.8 (t, 4H, in cyclohexane ring). MS (m/z): Calculated: 241.34, Found: 242.22 [M+H+] NH2(CH2)3Tac (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 (CDCl3) ( ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 5.0 (s, 1H, NH), 3.5

(t, 2H, NHCH2), 2.9 (s, 2H, in cyclohexane ring), 2.6–2.8 (m, 2H, CH2NH2), 2.6 (s, 2H, in cyclohexane ring), 2.0 (s, 2H, NH2), 1.8 (t, 4H, in cyclohexane ring), 1.6–1.7 (m, 2H, CH2CH2CH2). MS (m/z): Calculated: 255.36, Found: 256.23 [M+H+] NH2(CH2)4Tac (1c, N-(1,2,3,4-Tetrahydroacridin-9-yl)butane-1,4-diamine) IR (KBr) ν (cm-1): 1580.3, 2859.3, 2931.8, 3060.8, 3350.3; 1

H NMR (CDCl3) (δ ppm.): 8.0 - 7.4 (4H, in aromatic ring of benzene), 4.2 (br, 1H, NH), 3.5

(t, 2H, NHCH2), 3.1 (s, 2H, in cyclohexane ring), 2.8-2.7 (4H, in cyclohexane ring and CH2NH2), 1.9-1.8 (m, 6H, in cyclohexane ring and NH2), 1.9 (q, 4H, CH2CH2CH2CH2 in aliphatic hydrocarbon chain). MS (m/z): Calculated: 269.39, Found: 270.2 [M+H+] NH2(CH2)5Tac (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, NHCH2), 3.0 (s, 2H, in cyclohexane ring), 2.7–2.6 (m, 4H, in cyclohexane ring and

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CH2NH2), 1.9–1.8 (m, 6H, in cyclohexane ring and NH2), 1.6 (q, 2H, NHCH2CH2), 1.5–1.3 (m, 4H,CH2CH2CH2CH2 in aliphatic hydrocarbon chain). MS (m/z): Calculated: 283.42, Found: 284.21 [M+H+] NH2(CH2)6Tac (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 (CDCl3) ( ppm.): 8.0 - 7.4 (4H, in aromatic ring of benzene), 4.1 (s, 1H, NH), 3.6

(t, 2H, NHCH2), 3.1 (s, 2H, in cyclohexane ring), 2.7 (d, 2H, CH2NH2), 2.6 (s, 2H, in cyclohexane ring), 2.1 (s, 2H, NH2), 1.9 (t, 4H, in cyclohexane ring ), 1.5–1.7 (m, 4H, NHCH2CH2CH2 in aliphatic hydrocarbon chain), 1.2–1.4 (br, 4H, CH2CH2 in aliphatic hydrocarbon chain). MS (m/z): Calculated: 297.44, Found: 298.23 [M+H+] NH2(CH2)7Tac (1f, N-(1,2,3,4-Tetrahydroacridin-9-yl)heptane-1,7-diamine) IR (KBr) ν (cm-1): 1580.3, 2854.9, 2927.2, 3059.5, 3307.4; 1

H NMR (CDCl3) (δ ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 3.9 (s, 1H, NH), 3.4

(t, 2H, NHCH2), 3.0 (s, 2H, in cyclohexane ring), 2.7 (d, 4H, CH2CH2NH2), 2.6 (s, 2H, in cyclohexane ring), 2.0 (s, 2H, NH2), 1.8–1.9 (m, 4H, in cyclohexane ring), 1.5 - 1.6 (m, 2H, NHCH2CH2 in aliphatic hydrocarbon chain), 1.2–1.4 (br, 6H, CH2CH2 in aliphatic hydrocarbon chain). MS (m/z): Calculated: 311.47, Found: 312.24 [M+H+] NH2(CH2)8Tac (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 (CDCl3) ( ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 3.9 (s, 1H, NH), 3.4

(t, 2H, NHCH2), 3.0 (s, 2H, in cyclohexane ring), 2.6 (d, 4H, CH2CH2NH2), 2.5 (s, 2H, in cyclohexane ring), 2.1 (s, 2H, NH2), 1.8 (p, 4H, in cyclohexane ring), 1.5–1.6 (m, 2H, NHCH2CH2), 1.2–1.3 (br, 8H, CH2CH2CH2CH2 in aliphatic hydrocarbon chain).

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MS (m/z): Calculated: 325.50, Found: 326.20 [M+H+] NH2(CH2)9Tac (1h, N-(1,2,3,4-Tetrahydroacridin-9-yl)nonane-1,9-diamine) IR (KBr) ν (cm-1): 1562.4, 2853.3, 2925.9, 3059.8, 3289.1; 1

H NMR (CDCl3) (δ ppm.): 7.9 - 7.3 (4H, in aromatic ring of benzene), 3.9 (s, 1H, NH), 3.4

(t, 2H, NHCH2), 3.0 (s, 2H, in cyclohexane ring), 2.6 (d, 4H, CH2CH2NH2), 2.5 (s, 2H, in cyclohexane ring), 2.1 (s, 2H, NH2), 1.9 (m, 4H, in cyclohexane ring), 1.5–1.6 (m, 2H, NHCH2CH2), 1.4–1.1 (br, 10H, CH2CH2 in aliphatic hydrocarbon chain). MS (m/z): Calculated: 339.52, Found: 340.28 [M+H+]

2.2.5. Syntheses of CN-NH(CH2)nTac The coupling reaction of the isocyanide linker CN-BFCA with NH2(CH2)nTac is shown in Scheme 1.

Scheme 1. Coupling of CN-BFCA linker with NH2(CH2)nTac. In the case of all tacrine derivatives (1a-1h) the coupling reaction of CN-BFCA linker with NH2(CH2)nTac was performed according to the same procedure. Solution containing about 0.5 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 NH2(CH2)nTac, so that the ratio of CN-BFCA to Et3N and to NH2(CH2)nTac was approximately equal to 1.2 : 2 : 1 (slight excess of CN-BFCA and about two times excess of Et 3N in relation to the tacrine derivative

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NH2(CH2)nTac. 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]+ 2.2.6. Preparation of 99mTc(NS3)(CN-NH(CH2)nTac) For labeling CN-NH(CH2)nTac with 99mTc a two-step procedure17 was applied (Scheme 2).

Scheme 2. Labeling of CN-NH(CH2)nTac with technetium-99m (using the 99mTc complex of the type’4+1’). In the first step 1 mL of eluate from the

99

Mo/99mTc generator (200 - 1000 MBq) was

added to a kit formulation containing 1 mg of Na2EDTA, 5 mg of mannitol and 0.1 mg of SnCl2 in the freeze-dried form under nitrogen. The mixture was allowed to stand at room

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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 99m

Tc-EDTA/mannitol migrates with the solvent front in water but remains at the origin in

acetone. In the second step the intermediate

99m

Tc-EDTA/mannitol compound reacted with

300 µg of the NS3 ligand and with about 50 µg of the isocyanide-modified tacrine derivative CN-NH(CH2)nTac. The reaction progress and radiochemical purity were controlled by HPLC (System 2). The radiochemical yield of the

99m

Tc-labeled radioconjugates was approximately

95%. 2.2.7. Synthesis of Re(NS3)(CN-NH(CH2)7Tac) The non-radioactive rhenium reference compound Re(NS3)(CN-NH(CH2)7Tac) was prepared in two steps (Scheme 3). In the first step 4.02 mg (7.74 μmol) of ‘cold rhenium precursor’ Re(NS3)(PMe2Ph) and 2.46 mg (11.46 μmol) of isocyanobutyric succinimidyl ester were dissolved in 1 mL of CHCl3 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 3)(CN-BFCA) and 0.91 mg (2.89 μmol) of NH2(CH2)7Tac dissolved in 160 μL of DMF we have added 0.24 μL (1.72 μmol) of 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(NS3)(CN-NH(CH2)7Tac).

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The compound was characterized by HPLC in system 2 and 1H-NMR. Data for Re(NS3)(CN-BFCA), C15H22N3O4S3Re: 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(NS3)(CN-NH(CH2)7-Tac), C31H46N5OReS3: 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 (CD3CN)  (ppm): 7.89 - 7.29 (4H, CHCHCH in aromatic ring of benzene); 3.91 (s,

1H, NH); 3.58 (m, 2H, CNCH2CH2); 3.41 (t, 2H, NHCH2CH2); 3.09 (m, 6H, 3xNCH2CH2S); 3.03 (s, 2H, in cyclohexane ring); 2.92 (t, 2H, CH2CH2C(O)N); 2.76 (t, 6H, 3xNCH2CH2S); 2.69 (d, 4H, CH2CH2CH2NH); 2.62 (s, 2H, in cyclohexane ring); 2.01 (m, 2H, CH2CH2CH2); 1.8-1.9 (m, 4H, in cyclohexane ring); 1.5-1.6 (m, 2H, NHCH2CH2CH2); 1.2 - 1.4 (br, 6H, CH2CH2CH2 in aliphatic hydrocarbon chain) 13

C NMR (CDCl3) (δ ppm.): 170.2 (1C, C(O)), 157.2 (1C, CN), 158.5, 150.7, 147.5, 128.7,

128.2, 123.5, 122.9, 120.2, 115.8 (9C, C, CH in aromatic rings), 58.7 (3C, NCH2CH2S), 43.8 (3C, CH2S), 49.4, 41.9, 40.5, (3C, CNH2, CNH, CNCH2 in aliphatic chain), 34.0, 33.1, 31.7, 29.8, 29.2, 26.9, 26.7, 24.8, 24.1, 23.0, 22.8 (11C, CH2 in aliphatic chain and cyclohexane ring). MS (m/z): Calculated: 787.14, Found: 788.23[M+H]+ 2.3. In vitro stability studies Stability studies of 99mTc(NS3)(CN-NH(CH2)nTac). The radioconjugate isolated from the reaction mixture (using HPLC System 1), present in the solution in concentration no higher than 10-4 mM, was incubated at 37 oC 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. 2.3.1. Stability studies of 99mTc(NS3)(CN-NH(CH2)nTac) in human serum

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For that purpose 0.1 mL of solution of the isolated

99m

Tc(NS3)(CN-NH(CH2)nTac)

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 incubated at 37 oC. 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 by HPLC for the content of the 99mTc(NS3)(CN-NH(CH2)nTac) complex. 2.3.2. Stability studies of 99mTc(NS3)(CN-NH(CH2)nTac) in cerebrospinal fluid About 0.05 mL of the solution of isolated

99m

Tc(NS3)(CN-NH(CH2)nTac) radioconjugate

in the 0.1 M PBS buffer, pH 7.4, was added to 0.5 mL of cerebrospinal fluid (obtained from hospital) and incubated at 37 oC. 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 99mTc(NS3)(CN-NH(CH2)nTac) 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. The activity of each layer (which shows concentration of the

99m

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.

14

2.5. In vitro biological activity studies of 99mTc(NS3)(CN-NH(CH2)7Tac) In vitro biological activity studies of the

99m

Tc(NS3)(CN-NH(CH2)7Tac) radioconjugate

and other intermediate species in respect to inhibition of AChE activity, were performed using modified Ellman’s method25,26. 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 spectrophotometric Ellman’s method25,26. 1 mL of diluted erythrocytes was incubated (5 min, 37 oC) 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

15

37°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 coefficient of the [TNB-] ion, , was found to be 10.6 mM-1cm-1. 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 ((A0-AIC)*100%)/A0, where A0 means acetylcholinesterase activity in the control sample, 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]. 2.6. Biodistribution study of 99mTc(NS3)(CN-NH(CH2)7Tac) 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 99m

Tc(NS3)(CN-NH(CH2)nTac), 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 injected dose per gram of blood. The standard source of 99mTc was measured together with the samples in order to perform decay correction.

16

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, 2CKM, respectively). The three-dimentional structures of 3f (radioconjugate) and 2f (tacrinebased 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

17

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. Results and discussion 3.1. Tacrine derivatives labeling, stability and lipophilicity studies The first step in our experimental work was preparation of a new series of tacrine derivatives, NH2(CH2)nTac, containing different number (n) of methylene groups in aliphatic chain (Fig. 1A), and coupling these compounds with bifunctional linker CN-BFCA to obtain monodentate isocyanide ligands containing tacrine derivatives, CN-NH(CH2)nTac (Fig. 1B).

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 derivatives; C - structure of 99mTc(NS3)(CN-NH(CH2)nTac) radioconjugates.

18

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 CNNH(CH2)nTac compound was located at higher retention time (RT) than that of NH2(CH2)nTac (Table 1). Table 1 presents also the RT values of tacrine derivatives labeled with technetium99m – 99mTc(NS3)(CN-NH(CH2)nTac) radioconjugates (Fig. 1C).

Table 1. Retention time values of the peaks (recorded on HPLC chromatograms) corresponding to the compounds synthesized in the present work. Retention time, RT, [min] compound

NH2(CH2)nTac

(CH2)n

99m

Tc(NS3)(CN-NH(CH2)nTac)

(system 1)

(system 1)

(system 2)

1. 7.9 8.0 9.9 10.4 10.9 11.6 12.2 12.9

2. 13.1 13.2 13.5 14.3 15.0 16.0 16.8 17.8

3. 14.2 14.3 14.2 14.9 15.6 16.2 17.0 17.9

a: n = 2 b: n = 3 c: n = 4 d: n = 5 e: n = 6 f: n = 7 g: n = 8 h: n = 9 All

CN-NH(CH2)nTac

99m

Tc-labeled tacrine derivatives,

99m

Tc(NS3)(CN-NH(CH2)nTac), were synthesized

with radiochemical yield (RCY) higher than 95%, radiochemical purity (RCP) higher than 98% and specific activity of the radioconjugate of about 30 GBq/mol. The HPLC 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) – chromatogram of

99m

Tc(NS3)(CN-NH(CH2)7Tac) with the peak at RT = 16.2 min, and (B) –

chromatogram of ‘cold’ rhenium reference compound Re(NS3)(CN-NH(CH2)7Tac) with the peak at RT = 16.4 min. Practically, the same positions of these peaks can be a proof of the existence of the 99mTc(NS3)(CN-NH(CH2)7Tac) species synthesized in n.c.a. scale.

19

B

A 140000

99m

1000

Tc(NS3)(CN-NH(CH2)7Tac)

Re(NS3)(CN-NH(CH2)7Tac)

120000 800

100000 600

mV

cpm

80000 60000

400

40000 200

20000 0

0

0

5

10

15

20

25

30

0

5

10

15

20

25

30

t [min]

t [min]

Figure 2. HPLC chromatogram of reaction mixture (A):

99m

Tc(NS3)(CN-NH(CH2)7Tac)

radioconjugate and (B): Re(NS3)(CN-NH(CH2)7Tac) which was ‘cold’ rhenium reference compound (system 2). Results of stability and lipophilicity (LogD) studies of

99m

Tc(NS3)(CN-NH(CH2)nTac)

radioconjugates isolated from the reaction mixture are presented in Table 2.

Table 2. Results of stability and lipophilicity studies of

99m

Tc(NS3)(CN-NH(CH2)nTac)

radioconjugates isolated from the reaction mixture.

(CH2)n

n=2 n=3 n=4 n=5 n=6 n=7 n=8 n=9

% of intact 99mTc(NS3)(CN-NH(CH2)nTac) radioconjugate after 24 h of incubation at 37 oC in solution of: PBS Cysteine Histidine human buffer -3 -3 10 M 10 M serum pH 7.4 93.5  0.2 In all cases the 93.4  0.2 99m Tc(NS3)(CN-NH(CH2)nTac) 95.4  0.3 radioconjugates were entirely stable. 92.2  0.3 HPLC chromatograms recorded in different time intervals, up to 24 h, 93.4  0.3 showed only one peak of RT 95.0  0.5 corresponding to that of the studied 92.9  0.4 radioconjugate. 92.6  0.3

Stability studies of

LogD

1.28 0.08 1.41  0.06 1.43  0.07 1.35  0.07 1.45  0.08 1.56  0.06 1.43  0.05 0.92  0.05

99m

Tc(NS3)(CN-NH(CH2)nTac) radioconjugates showed that these

compounds do not undergo the ligand exchange reaction with amino acids or other strongly

20

competing natural ligands (containing SH or NH groups), present in excess concentrations in human body fluids (the so called ‘challenge experiments’). Stability studies of 99m

Tc(NS3)(CN-NH(CH2)nTac) radioconjugates in human serum showed also that about 5 to

8 % of studied radioconjugates have been bound by serum protein components. The obtained solid residue which precipitated after ethanol addition and sample centrifugation contained 5 - 8% of initial technetium-99m radioactivity. Lipophilicity

(LogD

values)

determined

for

99m

Tc(NS3)(CN-NH(CH2)nTac)

radioconjugates are in the range from 0.92 to 1.56. The relationship between LogD value and the number of CH2 groups (n) passes through a maximum for n = 7. The changes of LogD 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 increases the LogD values by about 0.65 per each CH2 group27. It is also known that CH2 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 groups into the LogD value smaller28. Moreover, the hydrocarbon chains containing more than seven CH2 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 hydrocarbon chains containing the same number of CH2 groups but having linear structure29. Therefore, the introduction of CH2 groups (n from 2 to 7) into aliphatic chain of 99m

Tc(NS3)(CN-NH(CH2)nTac) radioconjugates causes the systematic increase of LogD

value, while introduction of the eighth and ninth CH2 group results the decrease of the LogD value.

21

According to literature data the LogD values of radiopharmaceuticals, suitable for crossing the blood-tissue barrier, should be within the range from 1 to 430. 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 from 1.5 to 2.531. Therefore, for the subsequent biological studies (stability in cerebrospinal fluid, biological activity towards acetylcholinesterase and multiorgan biodistribution) the compound 99mTc(NS3)(CN-NH(CH2)7Tac), characterized by the highest LogD value, has been selected. Stability studies of

99m

Tc(NS3)(CN-NH(CH2)7Tac) 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 99m

Tc(NS3)(CN-NH(CH2)7Tac)

cpm

40

20

0 0

5

10

15

20

25

30

t [min]

Figure 3. HPLC chromatogram of

99m

Tc(NS3)(CN-NH(CH2)7Tac) radioconjugate after

incubation in cerebrospinal fluid for 24 h (system 2). 3.2. Biological activity studies In vitro biological activity studies of the

99m

Tc(NS3)(CN-NH(CH2)7Tac) radioconjugate

and other intermediate species in respect to inhibition of AChE activity, were performed using modified Ellman’s method25,26. The dependence of acetylcholinesterase inhibition obtained

22

during incubation of human erythrocytes of healthy volunteers with various concentrations of

Percent of acetylcholinesterase inhibition (%)

examined compounds, as well as IC50 values, are presented in Figure 4 and Table 3.

100

80

60

1f 2f 3f Tac

40

20

0 1E-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);

Tacrine, Tac

IC50 M 0.226 ± 0.095

NH2(CH2)7Tac ,1f

0.108 ± 0.011

CN-NH(CH2)7Tac, 2f

0.013 ± 0.005

99m

0.045 ± 0.003

Compound

Tc(NS3)(CN-NH(CH2)7Tac), 3f

Comparison of IC50 values in Table 3 showed that biological activity of 3f (99mTc(NS3)(CNNH(CH2)7Tac) radioconjugate) is, indeed, about three times lower than that of 2f (CNNH(CH2)7Tac – tacrine derivative coupled with bifunctional linker), however about two times higher than that of 1f (NH2(CH2)7Tac – tacrine derivative) and about five times higher than that of Tac (tacrine – the standard AChE enzyme inhibitor). Generally, one can say that labeling of tacrine with technetium-99m (using the

99m

Tc complex of ’4+1’ type) does not

cause the decrease of biological activity of the labeled biomolecule. It is also worth to notice

23

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 technetium-99m cation, IC50 = 17.60 ± 0.8 nM)4. According to "Clarke's Analysis of Drugs and Poisons" there are different data concerning therapeutic concentrations of tacrine for the treatment of Alzheimer's disease32. 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 newly synthesized compounds CN-NH(CH2)7Tac and

99m

Tc(NS3)(CN-NH(CH2)7Tac) 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 (99mTcradioconjugate) 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

24

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 Hbond 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.

25

3.4. Biodistribution studies Biodistribution studies performed with

99m

Tc(NS3)(CN-NH(CH2)7Tac) are presented in

Table 4 and Figure 6.

Table 4. Biodistribution studies of

99m

Tc(NS3)(CN-NH(CH2)7Tac) in normal male BALB/c

mice at 30 and 60 min postinjection (n = 3, % ID/g±S.D.). %ID/g Organ

30 min

60 min

Heart

0.73 ± 0.04

0.45 ± 0.02

Kidneys

2.89 ± 0.18

1.41 ± 0.11

Liver

5.69 ± 0.15

3.99 ± 0.19

Lungs

3.49 ± 0.3

2.54 ± 0.3

Spleen

1.02 ± 0.9

2.16 ± 0.5

Brain

0.065 ± 0.009

0.026 ± 0.008

Stomach

0.305 ± 0.03

0.52 ± 0.08

0.77 ± 0.1

0.24 ± 0.09

Blood

6

30 min 60 min

4

od B lo

ch

in

ma Sto

B ra

n lee Sp

er L iv

Lu

ne y K id

He

ngs

0,07 0,06 0,05 0,04 0,03 0,02 0,01 0,00 a rt

% ID/g

2

Figure 6. Biodistribution studies of 99mTc(NS3)(CN-NH(CH2)7Tac) in BALB/c mice after 30 and 60 min post-injection.

The significantly higher uptake of

99m

Tc(NS3)(CN-NH(CH2)7Tac) in liver (3.99 - 5.69

%ID/g) than in kidney (1.41 - 2.89 %ID/g) indicates the clearance of the radioconjugate

26

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 found in human lung)33. The ascertained presence of the radioconjugate in brain (0.065 ± 0.009 and 0.026 ± 0.008 %ID/g after 30 min and 60 min postinjection, respectively) indicates its possibility to cross the blood-brain barrier.

4. Conclusions The 99mTc-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 99m

Tc(NS3)(CN-NH(CH2)nTac) radioconjugates are satisfactorily stable also in human serum.

The labeling procedure of tacrine derivative NH2 (CH2)7Tac with technetium-99m (using the 99m

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, 99m

Tc(NS3)(CN-NH(CH2)7Tac), 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. In conclusion one can say that the

99m

Tc(NS3)(CN-NH(CH2)7Tac) 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

27

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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Graphical abstract

31

Highlights 

Labeling procedure of tacrine derivatives with technetium-99m.



Determination of physicochemical properties of the conjugates.



In vitro and in vivo studies of biological properties of the conjugates.



99m

Tc-tacrine derivative conjugates as diagnostic tool for cholinesterase level evaluation

32