NW/. Med. Eiol. Vol. 19, No. 4, pp. 481489, ht. J. Radial. Appl. Ins~um. Purl B

0883s2897/92 $5.00+ 0.00 Copyright 0 1992Pergamon Press Ltd


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Synthesis, Radiochemistry and Biological Evaluation of Technetium-99m Complexes with 1&Diamine-3,6-dithiaoctane (DDO) Ligands TH. MAINA and E. CHIOTELLIS National Centre for Scientific Research “Demokritos”, Athens. Greece

153 10 Aghia Paraskevi, Attikis,

(Received 2 October 1991) The synthesis, radiochemical analysis and biological characteristics of some l,&diamine-3,6-dithiaoctane derivatives labelled with Tc-99m are reported. Analysis by HPLC shows that most of the WmTc-chelates are multicomponent. Furthermore, almost all W”Tc-complexes isolated by HPLC are lipophilic and stable in vitro. The biodistributions of the most lipophilic of these complexes were evaluated in mice. The N-morpholinylethyl and NJ’-bisalicylyl derivatives of 1,8-diamine-3,6-dithiaoctane yielded WmTccomplexes which exhibit considerable uptake and retention in organs of interest, such as the heart and the brain

Introduction Recently much effort has been focused on the design of new 99mTc-agents suitable for single photon emission computerized (SPECT) scanning of the brain (Loberg et al., 1979; Oldendorf, 1978; Gustafson et al., 1978). Radiopharmaceuticals for SPECT brain imaging have already been developed for other radionuclides; e.g. 75Se-labelled selenides (Loberg, 1980; Kung and Blau, 1980) or ‘231-labelled amines (Winchell et al., 1980; Hill et al., 1982; Kuhl et al., 1982; Holman et al., 1984; Kung et al., 1983; Uszler, 1975). However, neither of the above radionuclides has the potential for wide clinical application, due to either poor physical characteristics, or to limited availability and high cost. Therefore, 99mTc-chelates are needed. These new 99mTc-agents should be able to cross the intact blood-brain barrier (BBB) (Loberg et al., 1980; Kung and Blau, 1980; Oldendorf, 1974a; Levin, 1980); in addition they should be retained in the brain cells for a period of time sufficient for SPECT imaging (Loberg, 1980; Kung et al., 1980; Winchell et al., 1980). Recent research has revealed two groups of compounds that seem to satisfy the above requirements. The first includes a series of propylene amine oxime derivatives, which form neutral, lipophilic complexes with 99mTc(Volkert et al., 1984; Troutner et al., 1984). From this group of compounds hexamethyl propy-

lene amine oxime (HM-PAO) presented the best overall biological properties and is already in clinical use (Neirinckx et al., 1987; Hung et al., 1988; Knapp et al., 1986). Another approach involves a series of diaminedithiol (DADT) compounds, which also form neutral, lipophilic 99mTc-complexes (Burns et al., 1981; Kung et al., 1984; Lever et al., 1985; Bok et al., 1986; Johannsen and Spies, 1981; Enfange et al., 1987). The most well known of these 99mTc-chelates is 99”Tc-NEP-DADT, wherein a piperidinylethyl group is bound to one of the nitrogen atoms resulting in high uptake and retention in the brain (Chiotellis etal., 1988; Eppsetal., 1986,1987; Scheffeletal., 1988). In order to investigate the parameters involved in brain uptake and/or retention, we studied the structure-activity relationships of different 99mT~complexes containing nitrogen and sulphur donor atom sets. Therefore, we synthesized a series of 1,8diamine-3,6-dithiaoctane (DDO) derivatives of the general type N,S, (x d 2). In the DDO molecule two amine and two thioether functional groups are linked together by three ethylene bridges forming a tetradentate ligand. The major difference between the DDO derivatives and DADT is the presence of sulphur as two thioether instead of two thiol groups in the former compound. The DDO series studied in this work are developed by introducing various alkyl, phenyl (for increasing the overall lipophilicity of the final 99mTc-complexes), or alkylamine groups (expected to increase brain





retention times) on one or both nitrogen atoms of the parental molecule. Labelling with 99mTc of this new class of N,S2 compounds is investigated. The resulting 99mTc-complexes are further purified by HPLC and their biodistributions studied in mice. In this way, the detailed effects of the substituent(s) of the DDO moiety on the physico-chemical and biological characteristics of the 99mTc-DD0 complexes can be evaluated.

analyses of radiolabelled compounds were performed on a LDC/Milton Roy Chromatography System using Techsil 5 Cl8 Reverse Phase columns (HPLC Technology). Electrophoreses were conducted with a Gelman Instrument Company apparatus. Synthesis

The 1,8-diamine-3,6-dithiaoctane derivatives synthesized in this work are listed in Table 1. 1,8-Diamine-3,6-dithiaoctane

Experimental Melting points were determined in open capillaries on a Biichi apparatus and are not corrected. The hydrochlorides were prepared by conventional procedures and then purified by crystallization. All newly synthesized compounds were characterized by ‘H-NMR and ‘C-NMR, as well as by elemental analyses. NMR spectra were recorded on a FT-80A Varian spectrometer versus Me,Si as an internal standard. Proton and “C-NMR chemical shifts refer to salts in D,O, or, for compounds 4-6, to the free bases dissolved in deuterated chloroform. Elemental analyses were performed in the Centre National de la Recherche Scientifique, Service Central d’Analyse, Vernaison, France; all results are within f0.4% of the calculated values. Measurements of the radioactivity content of biological samples were made in a well-type y-counter [NaI(Tl) crystal, ICN-gamma Set 5001. HPLC

- 3.Wthiioctane


The synthesis of this compound is based on literature procedures (Dwyer and Lions, 1947, 1950; Voronkov et al., 1979). 1,2-Ethanedithiol was brought into reaction with 2-aminoethyl-chloride in ethanol; the mixture was refluxed for 4 h and the solvent was removed by evaporation. The residue was then treated with a small volume of 10% KOH and anhydrous Na,CO,. The resulting paste was extracted with CHCl, and the solvent evaporated to give 30 g of a colourless oil (52%). The corresponding hydrochloride was prepared and purified by repeated recrystallizations.


TaMe 1. Phywochemlcal data f& 1,6d!amne

of Ligands



To a well-stirred solution of n-butylamine (73 g, 1 mol) in C6H, (100 mL), a solution of ethylene sulphide (3Og, 0.05 mol) in C,H, (20 mL) was added dropwise at 50-55°C. The mixture was refluxed gently for 4 h and evaporated to remove the solvent, as well as the excess amine. (N-Butyl)-Z-aminoethylthiol.

(DDO) denvatwes: RIR~NCH~CH~~CH~CH~~C~~C~~WR,R( Andyela

Rooryatn. Compd.





n*d, K





Bp, mmll@C

Mp,oC Formula






28.45 26.36

7.16 7.51

11.06’ 10.75








CtoH?rNzSz (HCI.l/,H,O)

42.61 42.32

9.29 9.02

9.94 9.13








CwH~NzSz (HCl.ll,H@)

42.61 42.M)

9.29 9.39

9.94 9.66










1.5>100 0.2/140 21155



C1zH2eNzSz C1zHnN30Sz




39.52 39.29

6.67 6.71

6.56 6.61













42.74 42.73

7.69 7.76

7.12 6.97




C?oHzsNhSn (2HCI)

53.54 53.17

6.94 6.62

5.67 5.86





51.78 51.56

9.17 9.07

6.71 6.69






+ % Calculated, ‘* % Found values

00 6:”


Evaluation of 99mTc-complexes with DDO ligands

The residue was distilled under reduced pressure (0.2 mmHg/SO”C) to give 30 g (50%) of (N-butyl)2-aminoethylthiol as an oil. The hydrochloride was obtained in crystalline form. The same method was followed for the other aminothiols, precursors of compounds 2-6. Analytical data and physical characteristics of the thiols synthesized are reported elsewhere (Maina and Chiotellis, 1989). (N-Butyl)-1,8-diamine-3,Gdithiaoctane. A wellstirred mixture of 2-mercaptoethanol (39 g, 0.5 mol), 85% KOH (66.4g) and 95% EtOH (357 mL) was heated to boiling. Then a solution of 2-chloroethylamine hydrochloride (58.3 g, 0.5 mol) in 95% EtOH (125 mL) was added dropwise during 1 h. The mixture was refluxed for 5 h and left at room temperature for 24 h. The resulting precipitate (KCl) was removed by filtration and the filtrate was evaporated to dryness in vacua. The resulting residue was treated with water and anhydrous Na,CO,, and the paste was extracted with CHCI,. The combined organic extracts were dried over MgSO, and the CHCl, was removed in uacuo. The oily residue was then distilled under reduced pressure (1.5 mmHg/l 15C) to give 26 g of 2-aminoethylthioethanol (I) as a viscous colourless oil (45%) (Klopping, 1957, 1958). To a well-stirred, ice-cooled solution of (I) (20 g, 0.16 mol) in CHCl, (250mL), a solution of SOCIZ (53.1 g, 0.45 mol) in CHCl, (40 mL) was added dropwise. The mixture was refluxed for 2 h and left at room temperature for 24 h. The resulting crystalline solid was removed by suction filtration and rinsed with Et,0 to give 12.5 g (45%) of 2-aminoethylthioethylchloride (II) (Klopping, 1957, 1958). The free base was prepared from 12.5 g (0.07 mol) of (II) and sodium (1.63 g, 0.07 mol) in absolute ethanol (50mL). After removing the solids by filtration, the solution was added dropwise to a solution of (n-butyl)-2-aminoethylthiol sodium salt [prepared from 9.44 g of the thiol and 1.63 g of sodium in absolute EtOH (50 mL)]. The resulting mixture was refluxed for 3 h and the EtOH evaporated in vacua. The resulting residue was treated with


6.67, 7.08, 7.18, 7.25 m (5H, aromatic protons). 13C-NMR: 28.07, 38.02 (SCH2), 42.61, 55.27 (NCH,), 113.17, 117.96, 120.39 (aromatic carbons); 147.20 (C-N). For 5: ‘H-NMR: 1.44 s (NH,); 2.11, 2.16, 2.32, 2.40, 2.54 dif m (20 H, SCH2, NCH,); 3.36, 3.42 dif tr [4H,

WW,I 13C-NMR: 31.49, 32.13, 32.38, 36.23, (CH,S); 41.23, 45.60, 48.77, 53.60, 58.10 (CH,N); 66.75 P(CH,)J. For 6: ’ H-NMR: 1.48 dif m (6H, piperidinyl protons); 2.40, 2.44, 2.57, 2.65, 2.72, 2.81 dif m (20 H, SCH,, NCH,). ‘-‘C-NMR: 20.98, 27.10 (3C, piperidinyl); 31.38, 36.27 (C-S); 41.31, 43.93, 49.70, 53.50 (N-C). N,N’-Diethyl-1,8-diamine-jl&dithiaoctane


A well-stirred mixture of 1 (10 g, 0.05 mol), ethylbromide (10.5 g, 7.2 mL, 0.1 mol) and xylene (40 mL) was refluxed for 6 h. After cooling to room temperature, 10% NaOH (100 mL) was added and the organic phase removed. The aqueous phase was extracted with C6H, and the combined organic extracts were dried over MgSO,. The C,H, was evaporated in vacua and the residue distilled under reduced pressure (2 mm Hg/l50-160°C) to give 7 g of an oil (60%). The bis(oxalate) salt was also prepared and crystallized (see Table 1). 1,8-Dimorpholinyl-3,&dithiaoctane


This compound was synthesized by reacting morpholinylethyl chloride with the disodium salt of 1,2ethanedithiol in ethanol. The procedure followed was similar as for compound 1. N,N’-Disalicylyl-1,8-diamine-3,6-dithiaoctane


water and anhydrous Na,CO,, and then extracted with CHCI,. After evaporation of the organic solvent

N,N’-Bis(o-hydroxybenzylidene)-1,8-diamine-3,6dithiaoctane. A well-stirred mixture of 1 (9 g,

the oily residue was distilled under reduced pressure (0.1 mm Hg/l40”C) to yield 13 g (75%) of 2. Compounds 3-6 (Table 1) were prepared in a similar manner. Elemental analyses were not performed for compounds 4-6, due to decomposition of the hydrochloric salts of these derivatives during recrystallization. Therefore, formation of derivatives 4-6 was confirmed by ‘H-NMR and “C-NMR spectra of the free bases dissolved in CDCI,. The data are listed below and ’ H or 13Cchemical shifts are given relative to Si(CH3)4 as internal standard:

0.05 mol), salicylaldehyde (18.3 g, 0.15 mol) and MeOH (50 mL) was refluxed gently for 2 h and then left at low temperature for 24 h. The resulting precipitate was removed by suction filtration and rinsed with MeOH. After recrystallization from MeOH, 16 g of yellow crystals were obtained (yield = 82%, m.p. = 105-l 1O’C) (Dwyer and Lions, 1947, 1950).

For 4: ‘H-NMR:

1.58 (NH,); 2.49, 2.65, 2.69, 273, 2.89, 2.96 dif m (12H, NCH,, SCH,); 6.56, 6.62,



a well stirred solution of the above imine (5 g, 13 mmol) in EtOH (200mL), NaBH, (3.05 g, 78 mmol) was added in portions over 2 h. After the addition, an equal volume of HZ0 was added and the resulting precipitate (9) was removed by suction filtration to give a deliquescent white product



(75%). The dihydrochloride was then crystallized. The same procedure was compound 10 (Table 1).

prepared also

Radiochemical analysis


used for

(a) High performance liquid chromatography (HPLC). Aliquots (20 pL) of the chloroform sol-

utions of 99”Tc-labelled products were subjected to reverse phase HPLC analysis (flow rate: 1 mL/min). The solvent systems used as the mobile phase for analyses are given in Table 2. Radiometric detection was accomplished by a y-counting system (NaI crystal S 1 2 1400 V (Wildbad D7547, Berthold) calibrated for 99mT~ and the radioactive components were recorded as peaks by a properly modified recorder (Linear-Scanner II System, LB 2723, Berthold). 1 mL aliquots of the HPLC eluant were collected in separate test tubes (2112 REDIRAC Fraction Collector, LKB Bromma) and the radioactivity content of each was measured in an Isotope Calibrator (Picker). The percent of radioactivity of each peak was calculated relative to the total radioactivity recovered from the column. (b) Electrophoresis. The charge and in vitro stability, relative to formation of free pertechnetate, of HPLC-isolated 99mTc-complexes were assessed by electrophoresis (Kung et al., 1984). Cellulose strips (Sepharose III, 2.5 x 15.2 cm, Gelman Sciences Inc.) and a 0.05 M phosphate buffer, pH 7.4, were used. Electrophoresis was conducted at 200 V for 15 min.

Radiochemistry Labelling with 99mTc (a) 9h Tc-chelates 1-3 and 7-10. A mixture of each ligand (5-lOmg), NaBH, (15 mg) and l-2 mL sodium pertechnetate (20-30 mCi in 0.5 mL of normal saline) was agitated in a Vortex mixer and left to react for 30min. The aqueous phase was extracted with three successive 2 mL portions of CHCl, and the combined organic extracts were dried over MgSO, and filtered. The filtrate was evaporated to dryness under a nitrogen stream and the residue, containing the labelled product, redissolved in CHCl, (2-3 mCi/20 /*L) and kept for further study (Kung et al., 1984). The hydrochloride salt of the ligand was used in 99mTc-labelling, except for product 7, which was labelled as the bis(oxalate) salt. (b) %Tc-chelates 4-6. The free base form of the ligand was dissolved in a small portion of EtOH. To this solution, NaBH, (15 mg) and l-2 mL sodium pertechnetate (2&30 mCi in 0.5 mL of normal saline) were added. The rest of the procedure was as that utilized for chelates l-3 and 7-10.

Table2 Complexesof "Tc.


1 2





ElOetrophcfWl# Tc-bound (X) Tea!






5 75



















4.0 5.0

19 39


25 3



60 21

96 97

3 3

ai 96

19 0




4 11




7 38

11.5 14.5

A 6

39 37

3.2 4.5

10 26









3.7 5.6

















9 25












29 7 11

5 11



2.0 2.6 3.6 4.4

99 90 93 a0

13 16

5.4 6.5



6 C D

22 24

2.2 4.1


MeOHM 65ll5




6505 5

FfUHctlonCACtlVlQ x




DDO denvatws Isolated by HPLC

MeOHiHzO &I15


M&NM20 90/10


MeOHM?O 6w5




19 22 25

a0 75

0 9 10

20 18 25

4.6 5.2



54 7 6 6

64 11








10 77

a7 a7

13 13


46 li 21

93 94

Evaluation of 99m Tc-complexes with DDO ligands The strips were cut into 1 cm sections and counted in a well-type y-counter (ICN-gamma Set 500). The percent radioactivity of each section of the strip was calculated relative to the total radioactivity applied to the strip. Partition coeficient measurements The lipophilicities of HPLC isolated 99mTccomplexes were assessed by determining partition coefficient values in an n-octanol/phosphate buffer (0.1 M, pH 7.4) system. To a centrifuge tube containing 2 mL of each phase, 100 p L of the 99mTc-complex solution was added, the mixture was agitated on a Vortex mixer and finally centrifuged at 3000 rpm for 15 min. Three samples (0.2mL each) from each layer were weighed and counted in a y-counter. The partition coefficient was calculated as the mean value of each cpm/g of octanol divided by that of buffer. Each determination was repeated at least three times (Kung et al., 1984). Biodistribution Studies Biodistributions of the HPLC purified complexes were determined in male Swiss Albino mice (20 + 2 g). Most of the lipophilic 99mTc-complexes were injected as a 30% ethanolic solution (0.1 mL) through the tail vein. Groups of at least 5 animals were sacrified under ether anaesthesia by excision of the heart at each of 2, 15 and 30 min time intervals. Samples of tissues and organs were removed, weighed and their radioactivity content measured in a ycounter (ICN-gamma Set 500). Counts were decaycorrected by internal standardization to a 1% aliquot of the injected dose. The percent injected dose per organ and per gram were calculated by the following formulae: %dose/organ


%dose/g =

sample cpm x organ weight 1% standard cpm x sample weight sample cpm 1% standard cpm x sample weight.

Results and Discussion Chemistry The synthesis of the various ligands is detailed in the Experimental section and the physico-chemical characteristics of these ligands are presented in Table 1. Elemental analyses and NMR spectra confirm the compositions of the compounds newly synthesized. The ligands were isolated in the form of hydrochloride salts [compound 7 was isolated as the bis(oxalate)] and purified by repeated recrystalhzations. Compounds 4-6 were purified by fractional distillation and characterized by ‘H-NMR and ‘CNMR spectroscopy, since their decomposition at low pH did not allow isolation of the hydrochloride salts. The compounds were labelled with 99mTc at alkaline pH (Kung et al., 1984). Sodium borohydride was


used to reduce WmTc-pertechnetate in the presence of the ligand. Compounds 4-6 were labelled as free bases in 30% EtOH in order to achieve complete dissolution. Finally, the radiolabelled product was extracted into chloroform, while free pertechnetate and other radiochemical impurities were left in the aqueous phase. The radioactivity of the aqueous phase and the combined organic extracts was measured separately and the percent bound technetium calculated (yields: 30-85%). The 99mT~chelates were analysed by HPLC, as described in the Experimental section; radioanalytical data are given in Table 2. The aminothiol precusors of monosubstituted derivatives Z-6 have been labelled with 99mTc using the procedure just described and then analysed by HPLC, as described previously (Maina and Chiotellis, 1989). Chromatographic patterns as well as biodistribution of WmTc-DDO derivatives 26 were compared to those of the 99mTc-labelled aminothiols. Thus, the integrity of chelates 2-6 under the labelling conditions can be confirmed. HPLC resolved most of the 99mTc-compounds into two or more radioactive components of different retention times. However, compounds 1 and 7 yielded single peaks eluting almost at the solvent front. Chromatograms of each labelled compound (l-10) were recorded at 1 h intervals after labelling. In this way, it was possible to follow any changes in the relative amounts of 99mTc-complexes present in the mixture as a function of time. It was found, that labelled compounds 2, 5, 6 and 10 reach equilibrium 3-6 h after labelling. This phenomenon is presented in more detail for derivative 5 in Fig. 1. It is seen that soon after labelling the radioactive mixture contained four components. However, 1 h after labelling only three main radioactive peaks were observed; the system finally reached equilibrium after 3 h. The amount of the most lipophihc complex (complex C) increased with time, reaching its highest percentage (38%) at equilibrium (Maina and Chiotellis, 1990). This phenomenon can be explained by the different rates of formation and/or different stability constants of the complexes comprising the radioactive mixture. Similar results were reported in the literature for other 99mTc-chelates, such as 99mTc-HM-PA0 (Neirinckx et al., 1987; Hung et al., 1988); in this case, the main lipophilic 99mTc-complex gradually transforms to a more hydrophilic 99mTc-species. As mentioned above, most of the labelled ““‘Tcchelates were multi-component. This multiplicity presumably derives from the different combinations, in which the ligand can coordinate to the metal. Thus, the numbers and relative positions of electron donor groups in the ligand molecule generate different possible arrangements of these groups around the metal in the final complex; the ratio of ligand-to-metal molecule may also vary. In addition, since the ligands studied in this work contain both soft (S in thioether form) and hard (N in amine form) donor atoms, they




pertechnetate moved towards the anode at a distance of 3.5 cm, while the lipophilic 99mTc-species remained at the origin, a strong indication that the complexes were of neutral character. However, charged but very lipophilic molecules might also remain at the origin during electrophoresis (Kung et al., 1984). In some cases (compounds 2, 3, 4, 9 and 10 of Table 2) a considerable amount of free pertechnetate accompanied the peaks eluting at the solvent front; an indication of their decomposition, since unbound technetium from the labelling reaction was removed with the aqueous phase after extraction of the radiolabelled product in CHCI, (Experimental section). In Fig. 2, electrophoresis patterns of two representative 99mTc-complexes are shown: complex C of derivative 5, wherein all the radioactivity remained at the origin, and complex A of compound 10, wherein 75% of free pertechnetate was present. In conclusion, by electrophoresis at pH 7 most of the 99mTc-complexes tested appeared to be neutral and stable in vitro. In order to correlate the lipophilicity of 99mTcchelates with their biodistribution, octanol/buffer partition coefficients were measured. The resultant values are listed in Table 2. Lipophilicity varied with the nature of the substituent added to the parental ligand. In addition, the various HPLC-isolated 99mTccomplexes, which were derived from the same ligand, also differed in lipophilicity.



M:_ ,j@L C









8 10 12 I4


Fig. 1. HPLC patterns of compound 5 (morpholinylethyl DDO derivative) labelled with %Tc at 0, 1 and 3 h time intervals after labelling.

may stabilize

technetium in more than one valence states. The parameter mentioned above may give rise to more than one technetium complexes for each ligand. The complexes thus formed could be separated by HPLC, due to their different physico-chemi-

cal characteristics; in reverse phase HPLC, this is mainly lipophilicity. The 99mTc-complexes isolated by HPLC were further tested for their charge and stability by electrophoresis. Electrophoresis data are presented in Table 2. Under the experimental conditions used, free


All 99mTc-complexes formed from each DDO derivative were purified by HPLC and then administered to mice for biological evaluation. Biodistribution data of the most lipophilic 99mTccomplex of each DDO derivative are summarized in Table 3. Data are presented as the percent dose per organ at each of 2 and 30 min time intervals postinjection (p.i.). Biodistribution patterns varied for each compound and greatly depended upon the substituent introduced into the parental ligand molecule (compound 1). In addition, different biological behaviour resulted from the 99mTc-complexes deriving from the same ligand and separated by HPLC (results not shown). This implies, that the complexes formed by coordination of the same ligand with 99mTc


Fig. 2. Electrophoresis patterns of %Tc-DDO

TcOi m

complexes SC and 10A.


Evaluation of 99mTc-complexeswith DDO ligands Table3.Petcent



dose at 2

and3Omnn variousor~sfdlowinginbaven~lsadmns~a~ of~cDDOcomplexes,rsdatedby HPLC, WIaxe*








126.639 (5.71) bi2.768 (2.97)

20.779. (9.75) 14.308 (3!78)

0.273 (0.04) 0.161 (0.05)

0.973 (023) 2.464 (0.40)

2.600 (0.40) 1.521 (0.22)

0.622 (0.15) 0.366 (0.12)


36.208 (4.59) 16.541 (1.67)

9.791 (0.60) 11.858 (1.17)

0.907 (0.05) 0.627 (0.10)

0.636 (0.16) 2sB6(0.15)

11.416 (2.21) 0344(1.16)

1.797 (0.53) 0.664 (0.16)


33224(5.21) 11.567 (1.36)

12.912 (1.06) 10.423 (0.65)

0.247 (0.02) 0.297 (0.10)

2.640 (1.17) 15.174 (0.73)

2.64B (0.15) 4.700 (0.29)

0.672 (0.11) 0.369 (0.0s)


33.530 (9.16) 10.038 (4.06)

27.067(9.92) 30970(1.07)

0.511 (0.15) 0.305 (0.06)

0.537 (0.15) 1.273 (024)

11.746 (257) 6.451 (1.27)

1.130 (0.40) 0.619 (0.08)


2u.771(1.30) MB6 (0.07)

9.353 (1.08) 24.156 (2.35)

1.206 (0.06) 1.368 (0.32)

0.361 (0.08) 1.341 (0.43)

16.529 (3.36) 7.467 (0.72)

1.803 (0.09) 0.516 (0.13)


29.265 (0.65) 12.335 (1.46)

23.446 (2.12) 35.726 (0.24)

l.Ml(0.05) 1.071 (0.12)

0.639 (0.01) 2.666 (2.43)

9.296 (0.77) 3.074 (0.75)

1.694 (0.05) 0.403 (0.07)


36.621(2.62) 17.143 (2.19)

19.535 (1.90) 11.997 (122)

0.246 (0.03) 0.195 (003)

0.596 (0.02) 0.673 (0.21)

4.921 (0.76) 1.901 (0.15)

0.976 (0.25) 0.424 (0.06)


19.347 (1.52) 14.103 (0.6s)

12.766 (2.24) 22.965 (1.64)

3.295 (0.53) 1.468 (0.16)

1.114 (0.31) 3.964 (0.30)

35037(3.8)) 26.135 (3.26)

1.774 (0.55) 0.763 (0.19)


15.117 (1.14) 2.670 (OS)

12.641 (1.39) 14.328 (1.91)

0.899 (0.17) 0.980 (0.21)

0.633 (0.05) 0.504 (0.12)

11.656 (0.70) 6.338 (1.65)

0.991 (0.10) 0.934 (0.14)


10.322 (1.31) 3.671 (0.46)

12.075 (2.77) 16.366 (0.36)

2.119 (0.40) 1.141 (0.46)

24.932 (3.47) 15.445 (1.45)

1.334 (0.14) 0.514 (0.02)

Wee we njected M thetail wn. Values represent doseaverage 01 at least five animals, plus ofmnusstandard dewabon."Tc-Bh complexes Isolated by HPLC:Me&e biodstnbutton dataofthemost lpophhc complex ofeachDDO daftvattve arepesmted:a,b,2-30 SKIp.i. respectwely.

generated different structures and thus, different physico-chemical and biological properties. Almost all lipophilic 99mTc-DD0 complexes presented in Table 3 were shown to be stable in uivo with respect to production of free pertechnetate, since the amount of radioactivity in the stomach remained low up to 30min p.i., except however, complex 3C; 15.2% of the injected dose was detected in the stomach at 30min p.i., indicating decomposition of the complex in vivo and formation of free pertechnetate. When labelled with %“Tc the parent ligand 1,8diamine-3,6-dithiaoctane (compound 1) gave, by HPLC analysis, only one radioactive peak, peak A. The initial level of blood radioactivity of 28.8% at 2 min p.i. decreased with time and finally reached 12.8% at 30 min pi. The introduction of aliphatic or phenyl substituents onto one (compounds 2, 3 and 4) or both (compound 7) amino groups resulted in 99mTc-complexes with roughly the same biological behaviour. Thus, the dose in the blood remained high (lO-15%) at 30 min p.i. for the monosubstituted compounds, or even higher (17.1%) for the diethylDDO compound 7. Thus, uptake and retention in organs of interest, such as the brain and the heart, appeared to be rather poor for the above-mentioned complexes. Lipophilicity did not seem to substantially influence brain uptake. Piperidinylethyl-DDO (compound 6) and dimorpholinyl-DDO (compound 8) both gave lipophilic 99mTc-complexes with considerable brain uptake. However, blood clearance was very slow with 12-14% of the activity still remaining in the blood at 30min p.i. resulting in poor brain-blood ratios.

The ““Tc-complex C of comound 5, wherein a morpholinylethyl substituent was introduced onto one of the amino groups, showed considerable uptake and retention in the brain and the heart accompanied by a fast blood clearance. Detailed results are shown elsewhere (Maina and Chiotellis, 1990). The introduction of salicylyl or cyclohexyl groups onto both of the nitrogen atoms in compounds 9 and 10 generated a number of complexes for each compound. The most lipophilic of them, shown in Table 3, presented rapid blood clearance, as only 2.9 and 3.7% of the injected dose remained in the blood at 30 min p.i. Considerable uptake and retention was observed in the brain, probably due to the increased lipophilicity of these complexes. In conclusion, the 1,8-diamine-3,6-dithiaoctane derivatives studied in this work, can be labelled with 99mTc and form lipophilic and in uivo stable complexes, the biological activity of which varies with the substituent introduced into the parental ligand. Certain lipophilic 99”Tc-complexes formed in sufficient yields after labelling, in particular morpholinylethyl (5C) and bisalicylyl (9D) DDO derivatives, showed considerable uptake and retention in organs of interest, such as the brain and the heart. In this work it was shown, that the studied N,Sr ligands containing aminothioether functional groups coordinated with reduced 99mTc, forming complexes with biological interest. The presence of thioether groups in the parental DDO molecule may stabilize technetium at valence states lower than (V), as was indicated by a few preliminary experiments (results not shown). The chemistry of Tc-thioether complexes has not yet been fully understood. Therefore, we



hope that the present work may stimulate further studies on other series of Tc-complexes based on the aminothioether backbone, due to the potential of these chelates to be used as new 99mTc-radiopharmaceuticals.

der Wissenschaften der DDR, Zentralinstitut fur Kernforschung, Rossendorf, Dresden, Germany. Klopping H. L. (1957) Vinylmercaptoalkyl isothiocyanates (to E. I. DuPont de Nemours & Co.). U.S. 2,785,19o ,__) Mar. 12, CA 51: P11379g. Klopping H. L. (1958) Substituted ethyl alkyl isothiocvanates (to E. I. DuPont de Nemours & Co.). IIS , 2:824,887,‘Feb. 25, CA 52: P1191 la. Knapp W. H., von Kummer R. and Kubier W. (1986) Cerebral blood flow to blood volume imaging by SPECT (Reply). J. Nucl. Med. 21, 1139. Kuhl D. E., Barrio J. R., Huang S. C. et al. (1982) Quantifying local cerebral blood flow by N-isopropyl-p[iz3I]-iodoamphetamine (IMP) tomography. J. Nucl. Med. 23, 196. Kung H. F. and Blau M. (1980) Regional intracellular pH shift: a proposed new mechanism for radiopharmaceutical uptake in brain and other tissues. J. Nucl. Med. 21, 147. Kung H. F., Tramposch K. M. and Blau M. (1983) A new brain perfusion imaging agent: [I-123lHIPDM; N,N’trimethyl-N’-[2-hydroxy-3-methyl-5-iodobenzy~]-l.3propanediamine. J. Nucl. Med. 24, 66. Kung H. F., Molnar M., Billings J., Wicks R. and Blau M. (1984) Synthesis and biod&ribution of neutral lipid soluble Tc-99m complexes that cross the blood-brain barrier. J. Nucl. Med. 25, 326. Lever S. Z., Burns H. D.. Kervitsky T. M. er al. (1985) Design, preparation and biodistribution of a technetium-99m triaminedithiol complex to assess regional cerebral blood flow. J. Nucl. Med. 26, 1287. Levin V. A. (1980) Relationship of octanohwater partition coefficient and molecular weight to rat brain capillary permeability. J. Med. Chem. 23, 682. Loberg M. D. (1980) Radiotracers for cerebral functional imaging-a new class. J. Nucl. Med. 21, 183. Loberg M. D., Corder E. H., Fields A. T. and Gallery P. S. 11979) Membrane transport of Tc-99m-labeled radio_._ pharmaceuticals. I. Brain uptake by passive transport. J. Nucl. Med. 20, 1 I8 I. Maina Th. and Chiotelhs E. (1989) Synthesis, HPLC analysis and biodistribution of complexes of Tc-99m with thiolamine ligands. In Technetium in Chemistry and Nuclear Medicine 3 (Edited bv Nicolini M., Bandoli G _. and Mazzi U.), p. 511. Cortma International, Verona. Raven Press, New York. Maina Th. and Chiotellis E. (1990) Synthesis and biodistribution of 99mTc-morphohnyl diamine dithiaoctane complexes. Eur. J. Nucl. Med. 16, 540. Neirinckx R. D., Canning L. R., Piper I. M. et al. (1987) Technetium-99m-d.l-HM-PAO: a new radiopharmaceuti- __. cal for SPECT imaging of regional cerebral blood perfusion. J. Nucl. Med. 28, 191. Oldendorf W. H. (1974a) Blood-brain barrier permeability to drugs. Annu. Rev. Pharm. 14, 239. Oldendorf W. H. (1974b) Lipid solubility and drug penetration of the blood-brain barrier. Proc. Sot. E,Y~. Biol. Med. 147, 813. Oldendorf W. H. (1978) Need for new radiopharmaceuticals. J. Nucl. Med. 19, 1182. Scheffel U., Goldfarb H. W., Lever S. Z. et al. (1985) Comparison of technetium-99m aminoalkyldiaminodithiol (DADT) analogs as potential brain blood flow ._ imaging‘agents.’ J. NW?. Med. 29, 73. Troutner D. E.. Volkert W. A. and Hoffman T. J. (I?41 ‘-.I Neutral lipophilic complex of 99mT~ with a multidentate amine oxime. Inr. J. Abpl. Radial. Isot. 35, 467. _-__ Uszler J. M. (1975) Human CNS perfusion scanning - with “..__ ‘r3I-iodo antipyhne. Radiology il5, 197. Volkert W. A., Hoffman T. J. and Seger R. M. (1984) 9A”Tc-propylene amine oxime (99mTc-PnAO); a potential brain radiopharmaceutical. Eur. J. Nucl. Med. 9, 511. .I.

Acknowledgements-Partial financial support by Grant No. GRE/2982/R4/RB from the International Atomic Energy Agency, Vienna, is gratefully acknowledged. The authors wish also to thank Dr M. Micha-Scretta of Organometallic Chemistry Laboratory of National Research Center, Athens, Greece, for obtaining all the ‘H-NMR and “CNMR spectra of the compounds synthesized. The expert technical assistance of Mrs V. Karmi with the animal experiments is greatly appreciated. The authors are especially grateful to Professor E. Deutsch of the University of Cincinnati for kindly editing this manuscript.

References Bok B. D., Scheffel U. and Goldfarb H. W. (1986) Comparative pharmacokinetics of technetium-99m-labelled radiopharmaceuticals with cerebral tropism. J. Bioph. Biomec. 10, (2, Suppl.) 5. Burns H. D., Dannals R. F. and Dannals T. E. (1981) Synthesis of tetradentate aminothiol ligands and their technetium complexes. J. Labelled Cmpd. Radiopharm. 18, 54. Chiotelhs E.. Varvarigou A. D., Maina Th. and Stassinopoulou C. I. (1988) Comparative evaluation of 99mTc-labelled aminothiols as possible brain perfusion imaging agents. Nucl. Med. Biol. 15, 215. Dwyer F. P. J. J. and Lions F. A. (1947) A sexadentate chelate compound. J. Am. Chem. Sot. 69, 2917. Dwyer F. P. J. and Lions F. (1950) Sexadentate chelate compounds. I. J. Am. Chem. Sot. 72, 1545. Efange S. N. M., Kung H. F., Billings J., Guo Y. Z. and Blau M. (1987) Technetium-99m bis(aminoethanethiol) complexes‘ with amine sidechainslpotential brain perfusion imaging agents for SPECT. J. Nucl. Med. 28, 1012. Epps L. A., Burns H. D. and Lever S. Z. (1987) Brain imaging agents: synthesis and characterization of (Npiperidinylethyl hexamethyl-diaminodithiolate) 0x0 technetium (V) complexes. Technetium aminothiolates as brain agents. Appl. Radial. Isot. 38, 661. Epps L. A., Burns H. D., Lever S. Z., Goldfarb H. W. ef al. (1986) The chemistry and biology of technetium(V) 0x0 complexes of N-piperidinylethyl diaminodithiolate for brain imaging. In- Technetium in Chemistry and Nuclear Medicine 2(Edited bv Nicolini M.. Bandoli G. and Mazzi U.), p. 171.’ Cortina’international Verona, Raven Press, New York. Gustafson D. E. ef al. (1978) Computed transaxial imaging using single gamma emitters. Radiology 129, 187. Hill T: C.,-Hoiman B. L., Lovett R. er al. (1982) Initial exuerience with SPECT (Single-Photon Emission Computerized Tomography) of the brain using N-isopropyl I-123 p-iodoamphetamine: concise communications. J. Nucl. Med. 23, 191. Holman B. L., Lee R. G. L., Hill T. C., Lovett R. D. and Lister J. J. (1984) A comparison of two cerebral perfusion tracers, N-isopropyl 1-123 p-iodoamphetamine and 1-123 HIPDM, in the human. J. Nucl. Med. 25, 25. Hung J. C., Corlija M., Volkert W. A. and Holmes R. A. (1988) Kinetic analysis of technetium-99m d,l-HMPAQ decomposition in aqueous media. J. Nucl. Med. 29, 1568. Johannsen B. and Spies H. (1981) Chemie und Radiopharmakologie von Technetium-komplexen. Akademie

Evaluation of 99mTc-complexes with DDO ligands Voronkov M. G., Knutov V. I., Usov V. A. ef al. (1979) Heteroatomic aziridine derivatives. Khim. Geterofsikl. Soedin 11, 1474.

Winchell H. S., Horst W. D., Braun L. et al. (1980)


N-Isopropyl-[‘231)p-iodoamphetamine: single pass brain uptake and washout; binding to brain synaptosomes; and localization in dog and monkey brain. J. Nucl. Med. 21, 947.

Synthesis, radiochemistry and biological evaluation of technetium-99m complexes with 1,8-diamine-3,6-dithiaoctane (DDO) ligands.

The synthesis, radiochemical analysis and biological characteristics of some 1,8-diamine-3,6-dithiaoctane derivatives labelled with Tc-99m are reporte...
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