99mTc-DADT complexes substituted with heterocyclic amines: Effect of substitution on in vivo reactivity

Nucl. Med. Biol. Vol. 20, No. 1, pp. 105-115, 1993 Printed in Great Britain. All rights reserved

Copyright

0

0883-2897/93 55.00 + 0.00 1993 Pergamon Press Ltd

99m Tc-DADT Complexes Substituted With Heterocyclic Amines: Effect of Substitution on In Vivo Reactivity M. PAPADOPOULOS,

S. STATHAKI, S. MASTROSTAMATIS, A. VARVARIGOU and E. CHIOTELLIS*

Radiopharmaceuticals Laboratory, Institute of Radioisotopes and Radiodiagnostics, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece (Received 21 April 1992) Alkylpiperidinyl and alkylpyrrolidinyl WmTc-DADT complexes were synthesized and tested for their ability to cross the BBB. Each complex was a mixture of two epimers separated by HPLC. More lipophilic epimers were biologically evaluated in mice, at various time intervals. Similar biodistribution patterns were obtained for both piperidinyl and pyrrolidinyl DADT-complexes. Brain uptake or retention was influenced by the heterocyclic amine introduced into the DADT backbone. Subcellular concentration of selected WmTc-DADT complexes was more profound in crude nuclear and post-microsomal fractions. Moreover, interaction of 99”Tc-2,2,6,6,9,9-hexamethy1-4,7-diaza-4-(3-methy1pyrro1idiny1)-ethyl-1,lOdecanedithiol with either lipids or microsomes of whole brain was almost unaffected by time. This may suggest that a possible selective site of interaction and metabolism for DADT complexes occurs in brain.

Introduction of 99mT~agents suitable for brain perfusion imaging by SPECT, is an area for research with continuous interest. Among the technetium ligands investigated so far, aminothiol derivatives form stable, lipid soluble complexes, capable of penetrating the blood-brain barrier (Burns et al., 1981; Kung et al., 1984). Technetium ( 99Tc/ 99”Tc) diaminodithiol complexes, consist of a pentacoordinated TcO’+ core, bound to the N,S, set in a square pyramidal configuration (Epps et al., 1987; Kung et al., 1989). These complexes were neutral in blood pH and initially gave high brain uptake. Their retention in brain was found to be influenced by several substituents in the NzS2 backbone. However, substitution generally produced syn and anti isomers in relation to the central Tc = 0 core (Epps et al., 1987; Kung et al., 1989). In general diaminodithiol ligands, reported as BAT or DADT, can be classified in two major categories. The development

(1) 2,2,9,9-Tetramethyl-4,7-diaza-1, (BAT) derivatives

IO-decanedithiol

This class of ligands comprises N,S, compounds substituted by amine groups at various positions of the BAT skeleton (Efange et al., 1987; Chiotellis et al., 1988; Yamauchi et al., 1990). *Author for correspondence.

Most technetium complexes studied in animals revealed poor brain retention, despite their initially high brain uptake. (2) 2,2,6,6,9,9-Hexamethyl-4,7-diaza-1,

lo-decanedi-

thiol (DADT) derivatives In this class of aminothiols, substituents were introduced in one nitrogen of the DADT backbone (Lever et al., 1985; Scheffel et al., 1988). Animal

evaluation of the parent technetium complexes demonstrated significant brain uptake. Considerable retention in brain tissue was observed after the administration of piperidinyl derivatives, such as 99mTc-NEP-DADT and 4-methyl-NEP-DADT (Scheffel et al., 1988). A substituted BAT derivative, ethyl-cysteinate dimer (ECD), reported recently, was significantly retained in brain cells due to enzymatic hydrolysis (Cheesman et al., 1988). On the other hand, certain 99mTc-DADT complexes exhibited a marked lung uptake and thus could be proposed as new lung agents (Mahmood et al., 1990; Papadopoulos et al., 1991a). Despite the considerable number of 99mTc-DADT complexes developed so far, limited studies have been performed concerning their mechanism of uptake or retention by the brain cells (Walowitch et al., 1987; Bormans et al., 1989). Contributing to the effort of developing ““Tc-DADT complexes with improved biological 105

M. PAPAIXXOULOS et al.

106

properties, we studied the structure-activity relationship for brain and lung accumulation of new DADT derivatives. Moreover, their biodistribution characteristics within brain tissue was also studied in order to obtain information regarding their metabolic fate. The structures of the ligands synthesized is shown in Fig. 1, and mainly consist of a series of pyrrolidinyl and piperidinyl derivatives. Materials and Methods Chemical

The hydrochloric salts of DADT ligands (Fig. 1) were prepared by conventional procedures and then purified by column chromatography. All newly synthesized compounds were characterized by ’ H-NMR and “C-NMR, as well as by elemental analysis. Infrared spectra of samples, as KBr pellets, were recorded on a FT-1600 Perkin-Elmer spectrophotometer. *Tc as NaTcOd was eluted from a commercial 99Mo/99mTc generator with saline. NH,99Tc0, was obtained in 0.088 M NH,OH from Amersham. High pressure liquid chromatography (HPLC) analysis was performed on a LDC/Milton Roy Chromatography Gradient System equipped with a u.v./vis. detector (LDC/Milton Roy, MP 3000), a y-counting system (NaI, crystal S 12 1400 V Wildbard D7547, Berthold) and a Beckman 171 radioisotope detector for b- or low y-detection. Ultraviolet spectra of aqueous solutions of tech-

Rl

RI

1

H

N 3

a

H

N Ll-

I

H

N ZF

7

H

16

H

netium carrier complexes were recorded on a u.v./vis. Beckman DU-65 spectrophotometer. Measurements of the radioactivity content of biological samples were made in a well-type y-counter. Substituted heterocyclic amines were commercially available, except 3_methylpyrrolidine, 3,3-dimethylpyrrolidine, 3,4-dimethylpyrrolidine and 3-phenylpyrrolidine which were synthesized according to methods previously reported (Blicke and Chi-Jung, 1952; McCasland and Prosknow, 1954; Chigarev and Ioffe, 1967; Curphey et al., 1979). 5-#I-Methyl-chloroacety1-3,3,7,7,10,10-hexamethy1-1,2-dithia-5,8-diazacyclodecane was also synthesized and analysed by conventional analytical techniques. Synthesis of ligands listed in Fig. 1 also relied upon modified methods previously described (Lever et al., 1985; Chiotellis et al., 1988). Thus, 3,3,6,6,10,10-hexamethyl-1,2-dithia-5,8diazacyclodecane was reacted with chloroacetyl or /I-methyl-chloroacetyl-chloride to form the corresponding chloro-compounds. The latter, reacting with the various heterocyclic amines, formed the 5-(2’-aminoacetyl)-3,3,7,7,10,10-hexamethyl-1,Z dithia-5,8-diazacyclodecane derivatives. Reduction of cyclic precursors with LiAlH., in THF resulted in the corresponding DADT-derivatives 1-16 (Fig. 1). The aminothiols after purification by column chromatography (alumine oxide, ether as eluent), were converted to their hydrochloric salts by anhydrous HCl. The hydrochlorides isolated were very hydroscopic, and were kept desiccated in vacua over P,05 until use.

Rl

0

Fig. 1. Structure of DADT ligands.

RI

%Tc-DADT

complexes substituted with heterocyclic amines

terized by i.r., u.v.-vis. and Tc = 0 stretching and maximum u.v.-vis. absorptions were estimated. The red-brown Tc complexes were subjected to HPLC analysis by both radiometric and u.v.-vis. detection. In this case a Beckman 171 radioisotope detector for @- or low y-detection was used. Complexes prepared at carrier level were used as standards for the identification of Tc(V)-DADT species in 99mT~preparations.

Compound 7 (Fig. l), named 4-methyl-NEPDADT, was previously reported as a potential new %Tc brain imaging agent (Scheffel et al., 1988). This derivative is included in the present study for comparison. Radiochemical

Labelling of the ligands 1-16 (Fig. 1) with 99mTc was performed by a method outlined in the literature (Chiotellis et al., 1988). The hydrochloric salts of ligands (2-5 mg) were treated with 99”TcO; (2-10mCi) in the presence of sodium borohydrate. The DADT complexes thus formed were purified by CHCI, extraction. The solvent was evaporated to dryness, and the residue was redissolved in 1 mL methanol. The labelling yield for all ligands was over 90%. Ahquots (20 pL, 200-300 PCi) of the methanolic solution of 99mTc-DADT complexes were subjected to either normal or reverse HPLC analysis. For normal phase, p-Porasil column (CHrCl,, 1 mL/min) was used, while for reverse, C-18 Bondapack and methanol : water (85 : 15 v/v, 1 mL/min) as mobile phase. Radiometric detection was accomplished by a y-counting system calibrated for WmTc. Radioactive peaks were recorded on LDC/Milton Roy integration system MP 3000. Ahquots of 1 mL were collected and the radioactivity was measured in an isotope calibrator. The percent of radioactivity of each peak was calculated in comparison to the total radioactivity recovered from the column. Lipophilicity of %Tc-DADT isolated by HPLC was assessed by determining partition coefficient values in an octanol/phosphate buffer 0.1 M, pH 7.4 (Table 1) as previously described (Kung et al., 1984; Chiotellis et al., 1988). Ligands 1 and 7 were also labelled with technetium at carrier level. In this case Bu,N~~TcOCI, traced by %Tc was used as precursor (Papadopoulos et al., 1989). The carrier technetium complexes were characTable Code

2

3 4

5 6 6 9 10 11 12 13 14 15 16

l

1. Radiochemical

107

Biological

The solution containing the 99”Tc-DADT complexes isolated by HPLC was evaporated to dryness by a stream of N,. The residue was redissolved in methanol : water (30 : 70 v/v) for animal studies. Prior to administration, radiochemical purity of the solution was checked by HPLC. In groups of six Swiss Albino mice, 0.1 mL (1 PCi) of the solution was injected through the tail vein. The animals were sacrificed after slight ether anaesthesia by cardiectomy at 5, 15 and 30min p.i. Samples of tissues and organs were removed, weighed and their radioactivity content was measured in a y-counter. A standard of 1% of the injected dose was used and the percent dose per gram for each organ or tissue was calculated. For subcellular distribution studies female Wistar rats (150-200 g) were used. %Tc complexes (1 PCi) were administered into the femoral vein and the animals were sacrificed by asphyxiation in carbon dioxide. Brain tissue was rapidly removed and immersed in a cold homogenization medium, weighed and homogenized in a Potter-Elvehjem glass homogenizer at 0-4C and in 9 ~010.25 M sucrose containing 0.5 mM K+EDTA and 10 mM Tri-HCl, pH 7.4. The crude nuclear fraction was prepared by centrifugation in the cold (CU”C) at 800g for 10 min in a Sorvall RCSC refrigerated centrifuge. The supernatant fluid was then centrifuged at the same temperature at 15,OOOg for 10 min in the same centrifuge. The pellet thus obtained, represented the mitochondrial fraction, was

data of lipopbilic

Activity %

Column

‘75 76 63 a4 90 90 95 aa 78 60 a4 91 75 77 6a M

Reverse

%Tc-DADT EMon

9.5 12.5 14.5 13.9 4.0 4.3 a.5 9.5 3.7 12.2 15.1 12.6 3.3 14.8 4.2 3.9

I

Normal

I Reverse Normal Reverse

Normal Reverse Normal

* % Radioactivity in the eked lipophilic * P.C.: Partition coefficient values.

(mm)

fractions.

complexes PC.” 29 191 49 235 62 250 155 ii8 124 38 50 26 a4 210 262 69

M. PAPADOPOULOS et al.

108

resuspended twice in the same medium and centrifuged under the same conditions. The combined supernatants from the 15,OOOg centrifugation were centrifuged at 60,OOOg for 60min in a Beckman L2-65B Ultra Centrifuge at 04°C. The microsomal and post-microsomal fractions along with the nuclear and mitochondrial fractions were preserved at 04°C for protein content estimation and radioactivity counting. Lipid extraction of brain homogenate was carried out according to the method of Folch et al. (1957) and the total lipid extract was used for the experimental. Protein content of brain homogenates and brain subcellular fractions was determined by the BCA method (Smith et al., 1985) with bovine serum albumin as a standard. Protein binding measurements were carried out using 0.1 mL of serum. The serum protein was precipitated with 1 mL of a 10% trichloroacetic acid solution; two additional washings were performed. The radioactivity detected separately in supernatant and protein precipitate was calculated. The metabolism of 99”Tc-2,2,6,6,9,9-hexamethyl-4,7-diaza-4-(3-methylpyrrolidinyl)-ethyl-l, lodecanedithiol (3) was studied in three rats after i.v. bolus administration of 1 PCi 99mT~complex. Timed blood samples were immediately collected by heart puncture after animal sacrification and the brain removed was homogenized in physiological saline. Serum separated from RBC by centrifugation and brain homogenate, was deproteinized in 3 vol methanol then centrifuged and the supernatant was injected into the HPLC system. The whole procedure was carried out at 04°C.

Results and Discussion Synthesis of various ligands is outlined in the experimental section. Structures presented in Fig. 1 were confirmed by elemental analysis and NMR spectra (‘H-NMR, 13C-NMR). All ligands were labelled with 99mT~in high yield (over 90%) using NaBH, as reductant. Analysis by i.r. of Tc-DADT complexes (1,7) prepared at carrier level showed a stretching at 897 cm-’ while u.v.-vis. maximum absorption was at 415 nm. These data confirmed the presence of the Tc = 0 core in the complexes and are in agreement with those previously reported (Efange et al., 1987; Epps et af., 1987). 99Tc complexes were used as standards for the identification of Tc(V) species, isolated by HPLC, in 99mT~preparations. All technetium DADT complexes, prepared at either carrier or tracer level, were analysed by normal and reverse phase HPLC. Two major peaks were detected by both systems. These peaks, referred to here as epimers A and B, were found to correspond to Tc(V)--DADT species by comparative HPLC studies. Similar observations have been reported by other investigators for

technetium complexes of substituted N,S, ligands (Efange et al., 1987; Epps et al., 1987). Partition coefficients of each epimer isolated by HPLC in 99mTc preparations, were determined in octanol/O.Ol M phosphate buffer pH 7.4. In Table 1, HPLC retention times as well as partition coefficient values of the more lipophilic 99mTc-DADT complexes (epimers A) are presented. Biodistribution studies

It has been shown that incorporation of an N-alkyl-heterocyclic amine in the N, S, backbone may increase brain affinity of parent technetium complexes (Lever et al., 1985; Chiotellis et al., 1988). In this work, biodistribution data in mice for various ““Tc-labelled DADT derivatives (lipophilic epimers A) are listed in Tables 2 and 3, respectively. As is shown, all the complexes tested, crossed the blood-brain barrier in various amounts. Among the pyrrolidinyl-DADT complexes (Table 2) the ethyl-3-methyl-pyrrolidinyl derivative (3) exhibited the highest brain uptake (6.57% of injected dose per gram) at 5 min pi., followed by the isopropyl-pyrrolidinyl derivative (2) with 5.74% dose/g. Additionally, modification of the pyrrolidinyl ring by either dimethyl or phenyl substitution (4, 5 and 6, respectively) lowered brain uptake of these *ToDADT complexes. A wide range for brain uptake values fluctuating from 1.8 to 8.2% of dose/g at 5min p.i. was obtained for the piperidinyl derivatives tested (Table 3, 7-15). Furthermore, methyl substitution on either position in the piperidinyl ring (7, 8 and 9, respectively), affected brain activity (8.14, 4.40 and 4.71%). These data indicate that the position of the methyl group in the piperidine backbone may be important for the interaction of %“Tc-DADT complexes with BBB cells. By increasing the number of carbon atoms introduced to the piperidinyl ring (ll-15), a similar distribution pattern to the pyrrolidinyl derivatives was obtained. The activity detected in the brain was considerably decreased. Compound 16, which represents a piperazinylDADT derivative, showed poor brain extraction characteristics and thus the series of such compounds was not further investigated. Among the 99*Tc-piperidinyl-alkyl-DADT derivatives, compound 7 (99mTc-4-methyl-NEP-DADT) showed the highest brain uptake and retention in the brain cells. Any other substitution on the piperidinyl ring resulted in a decrease of the biological specificity of the parent Tc complex. Comparable to compound 7, high brain uptake and retention was exhibited by 99”Tc-pyrrolidinyl-ethyl-DADT (1) and the WmT~3-methylpyrrolidinyl-ethyl-DADT (3). These complexes will be referred as 99mTc-NEPY-DADT and 9h”Tc-3-methyl-NEPY-DADT, respectively.

%Tc-DADT

complexes substituted with heterocyclic amines

In all DADT complexes a considerably high amount of activity was accumulated in lungs, ranging from 6 to 46% of injected dose per gram of tissue. Subcellular distribution studies A group of DADT complexes (1, 3, 5 and 7) varying in brain transport and retention was selected for further determination of their biological behaviour in brain cells. The DADT backbone (2,2,5,5,9,9-hexamethyl-4,7-diaza-l,lO-decanedithiol) synthesized and labelled with 99mTc under the same conditions was used for comparison. Table 4 lists the subcellular distribution of 99mTc-DADT complexes in the rat brain at 10 min following a single i.v. dose. The concentration of *Tc radioactivity was found to be similarly disposed in the brain subcellular fractions. It was observed that most of the activity of either complex (1, 3, 5 and 7) was detected in the crude nuclear (64-74%) and post-microsomal (1 l-21 %) fractions. On the contrary, the DADT backbone showed a higher accumulation in the postmicrosomal fraction (54%), indicating a higher interaction with the soluble compounds of cytosol. Further, a more detailed study for biodistribution of 3-methyl-NEPY-DADT (3) was performed. The radioactive components of compound 3 present in either serum or whole brain homogenate at 2, 10 and 20 min p.i., were characterized using analytical radioHPLC (normal phase, PPorasil column) by comparing their retention times with that of the injected complex. The data obtained are presented in Fig. 2. The analysis demonstrated that in all time intervals studied only 0.3% of the activity present in the serum

109

was due to the intact complex, whereas the rest referred to a more polar substance, a possible metabolite of compound 3. In the brain homogenate however, 2.3% corresponded to the intact complex while the rest was that of the polar substance also detected in the serum. In Fig. 3 the subcellular distribution of 3-methylNEPY-DADT over the range studied is presented. In this case, data were expressed as radioactivity counted per mg protein measured in each fraction. It was revealed from the activity detected, that the crude nuclear fraction, consisting of cell nuclei and plasma membranes and microsomes might be the initial particulate sites of interaction for compound 3 at the early time. On the other hand, the lesser decline of radioactivity measured in microsomes and postmicrosomal supernatant over time, may indicate that microsomes represent a possible site of their metabolism which is subsequently in accordance with the major route of amine metabolism which occurred in lung microsomes (Moretti et al., 1987). Additionally, soluble cytosolic components of post-microsomal supernatant may facilitate the intracellular transport. A percentage over 76% of complex 3 was bound to serum proteins during the time interval studied. As has been reported elsewhere (Scheffel et al., 1988) a relationship between brain uptake of %Tc complexes and their protein binding could not be demonstrated. Further, it was found, that 84.3-74.8% of the brain radioactivity recorded at 2-20 min, accumulated in the total lipid extract of whole brain homogenate, suggesting a high degree of interaction of 3-methylNEPY-DADT with the lipid moiety of the biomolecules.

Table 2. Biodistribution of *mTc-pyrrolidinyl-alkyl-DADTderivatives. Percent dose per gram in mice* BRIBL"

LiBL"

7.96kl.7 4.53fl.l 15.41%?.1 3.03m.4

4.64 3.10 2.15

29.35 20.34 16.57

1.65m.5 1.32m.l

5.74kl.2 42.19zt10.99.34f2.1 2.72m.4 17.3M.2 4.51rn.6

0.67rn.l

i.13m.2

666fl .I

2.66m.5

3.46 2.39 1.50

25.56 13.13 9.96

1.35m.2

6.57m.6 2.67rn.4

45.63m.3

9.36mo.6

0.69m.2 0.56m.i

l.10m.2

15.9lM.4 6.75fl.5

3.54m.3 2.29rn.6

4.66 3.22 1.69

33.60 17.66 14.97

1.66rn.6 1.56m.2 0.77m.l

4.56m.7 2.6lrn.l 1.37m.i

44.66m.4 11.44m.6 37.63f2.5 7.26fl.3 13.07*1.2 4.26m.7

2.72 1.61 1.79

26.59 24.16 16.99

3.46m.6 3.16m.4

32.llm.3 2.91m.4 1.6lrn.4 15.02m.l 1.06m.3 70.W .2

2.02m.4

0.64 0.57 0.49

9.26 4.75 3.21

4.77rn.6 3.1im.2 I .91x).1

0.45 0.32 0.29

7.76 4.41 2.25

Code

Blood

1

a1.31m.1 b1.15m.l cO.93m.1

2

3

4

5

2.16rn.2 6

4.22m.5 3.69m.4 2.61rn.2

Brain

Lungs

5.24m.4

38.46k5.7

3.57m.7

23.40zt3.7

2.KktO.3

1.69m.5 1.24m.3 0.6Om.2

32.76k7.2 17.13m.5 6.3Oztl.l

Heart

7.65k1.3 3.66m.6

* The values represent the average of six animals. **Values represent brain/blood and lungs/blood ratios, respectively. a, b, c: 5, 15, 30 min time intervals studied respectively.

110

M. PAPADOPOULOS et al. Table 3. Biodistribution of 99”Tc-piperidinyl-alkyl-DADT gram in mice* Code

Brain

Blood

7

a1.47m.3 8.19fl.2 bl.02m.2 5.33m.9 2.77m.7 Q89m.3

8

1Mm.2 0.73m.l 0.42m.o

9

3.51m.2 2.18m.l 1.82m.1

10

11

Lungs

derivatives. Percent dose per

Haart

BWBL”

39.05m.l 13.94m.B 15.4ls?.3 10.7W.8 8.W2.5 8.88S.l

L/EL”

5.57 5.22 3.10

28.56 14.99 9.00

4.4Om.B 27.5b4.7 7.05m.9 1.89m.5 10.75m.3 3.19m.5 0.73m.i 5.55H.2 1som.3

3.13 2.80 1.73

19.65 14.75 13.09

4.7im.3 i.2lm.o 0.78m.l

i2.07m.9 7.04m.l 4.28m.3

7.8lm.B 3.30m.5 i .9lm.2

1.31 0.58 0.47

::: 2.83

l.Olrn.2 O.Blm.2 0.5om.l

2.3Om.3 1Mm.2 0.82m.2

8.81m.8 5.34fl.I 3.4im.5

8.72m.9 4.44rn.8 2.96m.B

2.27 2.84 1.83

8.74 8.78 8.80

1.41m.3 0.78m.l 0.88m.l

i 57m.4

i.2im.2 0.88m.o

9.78ti.7 5.58&l .4 4.03m.7

15.03m.4 8.58ti.3

1.11 1.58 1.02

8.94

12

1&to.2 0.87m.2 0.8om.l

2.5010.2 9.45m.8 1.14m.3 3.27m.5 2.71m.4 0.44m.i

8.7Om.9 1.88m.3

1.71 1.35 0.74

8.47 3.77 4.51

13

1.73m.8 1.Bom.B 8.28kl.B 1.3ozto.l 0.84m.o 4.i3m.5 1.22m.3 0.28rn.O 3.22rl.O

3.32m.4

1.02 0.50 0.23

3.83 3.18 2.83

14

1.73m.7 0.94m.2 0.85m.2

4.2om.5 38.4S12.5 8.83m.2 1.8om.3 15.34H.2 4.46zt2.0 0.75m.l 7.5M1.2 2.88m.8

2.43 1.70 1.20

22.22 18.29 11.53

15

4.81m.3 3.62m.3 2.30m.l

1Born... 0.97m.2 0.58m.i

38.70zt7.8 5.87H.9 14.32M.l 3.29m.8 8.71fl.9 2.30m.5

0.39 0.27 0.25

8.39 4.52 3.78

18

I .84m.2 I 63m.2 0.97m.2

3.4im.4 0.79m.o 0.38m.l

7.7lm.5 4.72m.8 4.29Etl.4 2.47m.4 2.78m.8 i .olm.3

1.85 0.48 0.33

4.19 2.82 2.34

3.3lrn.8

i .27m.2

I fi7m.l o.8lm.o

;::

* The values represent the average of six animals. **Values represent brain/blood and lungs/blood ratios, respectively. a, b, c: 5, IS, 30min time intervals studied respectively.

Conclusions Summarizing our results, it could be concluded that introduction of either alkylpyrrolidinyl or alkylpiperidinyl substituents into the N,Sr backbone, resulted in @“‘Tc complexes with marked brain uptake and lung accumulation.

Brain uptake was decreased when either an increased number of methyl groups or heavy substituents were introduced into the heterocyclic amine ring. Similarly the position of the methyl group on pyrrolidine or piperidine seems to play an important role in brain uptake or retention of DADT complexes. Optimum brain values were obtained with

Table 4. Subcellular distribution of @“‘Tc-DADT complexes in rat brain 10 min after i.v. administration code 1 3 5 7

DADT

Cruda nutir fmctlon

Pomnlkrasomnl -III

Mltochondfk

Mkrosomsr

71.8H.l 64.cd.5 73.8i2.8 84.7u.2

6.3d.O lO.GiO.9 9.6kl.2 17.7kl1.2

4.4ko.9 4.Ml.6 4.1flo.3 6.8M.3

17.5m.9 21.0+2.0 12.4fo.9 10.8m.7

39.41.7

3.8H.6

3.1K1.9

53.4iQ.l

All values are means SD % recovery of three rats, 1Omin p.i. Subcellular fractions and postmicrosomal supematant were obtained by differential centrifugation.

*Tc-DADT

111

complexes substituted with heterocyclic amines

BLOOD 2 min

000

10 mln

20 min

b

b b

%^ ‘; 4w

E t g lot 2 3 8 2 1

I

1

,_--d_L_ -_L 1,

0

15 0

I

a

-d?Jh_ 15 0

15

BRAIN 2 min

10min

20 mln b

b

!

b

I

a I__LJt_ 15

0

TIME (in min)

”

-_dL_ 0

a 15

Fig. 2. In uiuo metabolism of compound 3. Radioactivity elution profile by counting HPLC fractions as described in Materials and Methods. Column, PPorasil; mobile phase (i) 100% CH,CI,, 0-6min, 1 mL/min and (ii) 100% CH,OH, 615 min, 3 mL/min.

3-methyl-pyrrolidine and the 4-methyl-piperidineDADT derivatives. Regarding the biological mechanism of uptake of these complexes, it was indicated that substitution of the DADT backbone led to a higher accumulation in the subcellular components. The DADT core was more concentrated in the soluble cytosol. Moreover, 3-methyl-NEPY-DADT (3) and 4methyl-NEP-DADT (7) showed the same interaction

‘103

with the crude nuclear fraction of brain cells, while their disposition among the other subcellular components was similar. Further, the occurrence of a polar substance, a possible metabolite revealed by HPLC studies along with the evidence that microsomes represent a site of metabolism, may suggest that a metabolic pathway for the 99mT~complexes studied exists in the brain. On the other hand, their interaction with lipids may

corn/ma -. protein

5 4 3 2 1 o-

2 min @j

NUCLEAR m

20 min

10 min MITOCHONDRIAL fjfjj

MICROSOMAL

POSTMICROSOMAL

Fig. 3. Subcellular distribution of 3-methyl-NEPY-DADT (3) as a function of time. Data are expressed as radioactivity counted per mg protein measured in each fraction.

M. PAPADOPOULOS et al.

112

also indicate that the lipid integrity of the biomolccule plays an important role in the accumulation of 99mTc-DADT complexes in the brain tissue. Acknowledgements-The

authors wish to thank Dr C. I. Stassinopoulou, NMR Lab., Inst. of Biology, NCSR “Demokritos” and Dr M. Micha-Screttas. Oreanometallic Chemistry Lab., National Hellenic Research %mdation, for their support in NMR studies and for their helpful discussions.

References Blicke F. F. and Chi-Jung Lu (1952) Formulation of amines with chloral and reduction of N-formyl derivatives with lithium aluminium hydride. J. Am. Chem. Sot. 74, 3933-3934.

Bormans G., Van Nerom C., De Beukelaer C., Hoogmartens M., DeRoo M. and Verbruggen A. (1989) Comparison of 99m-Tc-ECD metabolism in organs homogenates of rat and baboon. Eur. J. Nucl. Med. 15, 423 (Abstract). Burns H. D., Dannals R. F., Dannals T. E., Kramer A. V. and Marzilli L. G. (198 1) Synthesis of tetradentate ligands and their technetium complexes. J. .&belied Crnpd. Radiopharm. 18, 5455.

Cheesman E. H., Blauchette M. A., Ganey M. V., Maheu L. J.. Miller S. J. and Watson A. D. (1988) Technetium99m’ECD. Ester derivatized diaminodithibl technetium complexes for brain perfusion. J. Nucl. Med. 29, 788 (Abstract). Chigarev A. G. and Ioffe D. V. (1967) Synthesis and reactions of b-phenyl$-cyano propionic acid. Cancer 66, 94779y. Chiotellis E., Varvarigou A. D., Maina Th. and Stassinopoulou C. I. (1988) Comparative evaluation of *Tc-labelled aminothiols as possible brain perfusion imaging agents. Nucl. Med. Biol. 15,215-223. Curphey T. J., Hung J. C. and Chu C. C. (1975) A study of the alkylation of enamines derived from sterically hindered amines. J. Org. Chem. 40, 607614. Efange S. M. N., Kung H. F., Billings J., Guo V. Z. and Blau M. (1987) Technetium-99m bis (aminoethanethiol) complexes with amine sidechains-potential brain perfusion imaging agents for SPECT. J. Nucl. Med. 28, 1012-1019.

Epps L. A., Burns H. D., Lever S. Z., Colfarb H. W. and Wagner H. N. (1987) Brain imaging agents: synthesis and characterization of (N-piperidinyl hexamethyl diaminodithiolate) 0x0 techneticum (V) complexes. Technetium aminothiolates as brain agents. Appl. Radiat. Zsot. 38, 661-664.

Folch P. J., Lees M. and Sloane Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509. Kuna H. F.. Molnar M.. Billings J.. Wicks R. and Blau M. (1684) Synthesis and’ biodi&ibution of neutral lipidsoluble Tc-99m complexes that cross the blood-brain barrier. J. Nucl. Med. 25, 326332. Kung H. G., Guo Y. Z., Yu C. C., Billings J., Subramanyam Appendix

V. and Calabrese J. C. (1989) New brain perfusion imaging agents based on 99mTc-bis(aminoethanethiol) complexes: stereoisomers and biodistribution. J. Med. Chem. 32, 433437.

Lever S. Z., Burns H. D., Kervitsky T. M., Goldfarb H. W., Woo D. V., Wong D. F., Epps L. A., Kramer A. V. and Wagner H. N. (1985) Design, preparation and biodistribution of a technetium-99m triaminedithiol complex to access regional celebral blood flow. J. Nucl. Med. 28, 1287-1294.

Mahmood A., Halpin W. A., Baidoo K. E., Sweigart D. A. and Lever S. Z. (1990) Synthesis and characterization of N-ethyl-diamine-dithiol oxotechnetate(V): a potential lung imaging agent. In Technetium and Rhenium in Chemistry and Nuclear Medicine (Edited by Nicolini M., Bandoli G. and Mazzi U.), Vol. 3, pp. 113-118. Cortina-Raven Press, New York. McCasland G. E. and Proskow S. (1954) Preparation of the epimeric 3,4 dimethylpyrrolidines. J. Am. Chem. Sci. 76, 60876088.

Moretti J. L., Holman B. L., Delmon L., Carmel D., Johnson D., Moingeon P. and Blau M. (1987) Effect of antidepressant and narcoleptic drug on N-isopropyl-piodo amphetamine biodistribution in animals. J. Nucl. Med. 28, 354-359.

Papadopoulos M. S., Mastrostamatis S. G. and Chiotellis K. (1990) Complexes of pyrrolidinyl-ethyl-DADT derivates labeled with %Tc and 99Tc. In Technetium and Rhenium in Chemistry and Nuclear Medicine (Edited by Nicolini M., Bandoli G. and Mazzi U.), Vol. 3, vv. 567-570. Cortina-Raven Press. New York. Payadopoulos M., Chiotellis E., Varvarigou A., Stathaki S. and Micha-Screttas M. (1991a) Lung uptake of neutral, lipophilic Tc(V)-aminothiol complexes. Eur. J. Nucl. Med. 18, 636, (Abstract). Papadopoulos M., Chiotellis E., Varvarigou A., Mastrostamatis S., Cotsyfakis C., Vavouraki H. and Stathaki S. (1991b) Correlation of lipophilicity to biodistribution of Tc-99m labelled aminothiols. In The Seventh Znt. Symp. on Radiopharmacology, Boston, Mass. p. 6. Scheffel II., Goldfarb H. W., Lever S. Z., Gungon R. L., Burns H. D. and Wanner H. N. (1988) Comvarison of technetium-99m amiioalkyl diaminodithiol’ (DADT) analogs as potential brain blood flow imaging agents. J. Nucl. Med. 29, 73-82.

Smith P. K., Krohn R. I., Hermanson G. I., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurements of protein using bicichoninic acid. Analyt. Biochem. 150,7685. Walovitch R. C., Tam S. W., Cheesman E. H., Watson A. D., Garrity S. T. and Williams S. J. (1987) Site selective localization of Tc-99m diamine dithiol complexes in rat brain hippocampus. J. Nucl. Med. 28, 731 (Abstract). Yamauchi H., Takahashi H., Seri S., Kawashima H., Koike H. and Kato-Azuma M. (1990) In vitro and in viuo characterization of a new series of Tc-99m complexes with N,S, ligands. In Technetium and Rhenium in Chemistry and Nuclear Medicine (Edited by Nicolini M., Bandoli G. and Mazzi U.), Vol. 3, pp. 475-502. Cortina-Raven Press, New York. opposite

BmTc-DADT complexes substituted with heterocyclic amines

113

APPENDIX Supporting

Data

Analytical data of 5-substituted 1,2-dithia-5,8-diazacyclo derivates*

Recrystallization solvent

Melting point (“C)

-

Formula

Yield (%)

C

H

N

C

H

N

I

W-LN,O%

82

II III IV V VI VII VIII IX X XI XII XIII XIV xv XVI

CwH,,N,OS, GA,WOS, C,H,,N,OS, C,HJ,N,OS, C,H,,N,OS, C,H,,N,OS, C,H,,N,OS, C,H,,N,OS, C,,H,,NrOS, C,, H,, N@S, C,, H,, N,OS, C,, H,, N,OS, GH,, N,OS, CzH,,N, OS, CWHBN,OS,

70 80 84 84 76 82 80 75 65 66 78 68 63 74 81

Ethanol Ether Ether Pet. ether Ether Pet. ether Ether Ether Pet. ether Ether Ether Ether Ether Pet. ether Pet. ether Pet. ether

146148 1333135 11&112 1299130 9698 116118 136-138 121-122 lOlLlO 168-170 174176 108-l 10 126-128 122-123 146148 128-130

57.87 58.87 58.87 59.80 59.80 64.10 59.80 59.80 59.80 59.80 60.68 60.68 60.68 60.68 64.75 56.68

9.44 9.62 9.62 9.79 9.79 8.74 9.79 9.79 9.79 9.79 9.94 9.94 9.94 9.94 8.91 9.51

11.25 10.84 10.84 10.46 10.46 9.34 10.46 10.46 10.46 10.46 10.11 10.11 10.11 10.11 9.06 13.91

57.53 58.47 58.63 60.10 59.85 63.88 59.92 59.94 60.14 60.17 60.46 60.39 61.00 60.83 65.11 57.01

9.02 9.30 9.36 9.35 9.50 8.33 9.34 9.58 9.47 9.75 9.49 9.68 9.70 9.58 8.84 9.16

11.23 10.50 10.84 10.16 10.09 9.49 10.36 10.36 10.25 10.41 10.00 9.73 10.04 9.93 8.79 14.12

Code

*Synthesis precursors of compounds

Calculated (%)

Found (%)

l-16.

R, , R, substituents referred to in Table 1. Analytical data of diaminodithiol ligands HSC(CH,),CH,NHC(CH,),CH,N[CH,C(CH,),SH]CH,CHR,R, Calculated (%) Code

R,

Formula

R,

3

Found (%)

Yield (%)

C

H

N

C

H

N

H

N

C,,H,,N,S,.3HCl

48

45.90

8.99

8.92

45.92

9.37

8.75

CH,

N 3

C,,H,,N,S1.3HCl

40

47.05

9.14

8.66

46.83

9.44

8.45

C,,H,,N,S,,3HCl,3H,O

46

42.33

9.35

7.79

42.30

9.28

8.09

C,H,,N,S,,3HCl.2.5H,O

45

44.15

9.45

7.72

44.18

9.91

7.87

C,,H,,N,S,,3HC1.1.5H,O

46

45.66

9.39

7.99

45.93

9.68

8.26

C,H,,N,S,,3HCl.2.5H,O

44

48.68

8.68

7.10

48.85

8.83

7.50

C,,H,,N,S,~3HCl.L5H,O

48

45.66

9.39

7.99

45.74

9.78

7.96

CL

N

Ph

N 3

continued ooerleaf

114

M.

PAPADOPOULOS et

al.

Appendix table--continued Calculated (%) Code

R,

Formula

R2

Found (%)

Yield (%)

C

H

N

C

H

N

8

H

C,,H,,N,S,~3HCl~OSH,O

48

47.37

9.14

8.29

47.29

9.56

8.22

9

H

C,H,N,S2.3HCl.0.5Hz0

43

47.37

9.14

8.29

47.35

9.53

8.11

10

CH,

C,H,,N,S2.3HCl.lH20

40

46.46

9.36

8.13

46.08

9.48

8.38

11

CH,

C2,H45N3S2.3HCl.lH20

46

47.49

9.49

7.91

47.40

9.81

8.26

12

C,,H,,N,S2.3HCl.IH20

46

47.49

9.49

7.91

47.45

9.61

8.32

13

C2,H,,N,S,.3HCl.lH20

40

47.49

9.49

7.91

47.83

9.72

7.96

14

C,,H,,N,S2.3HCl.0.5H20

41

48.31

9.46

7.91

48.00

9.91

8.26

C2,H,,N$,.3HCl.3H20

42

48.81

8.85

6.83

48.74

8.74

6.53

C,,H,,N,S2.4HCl.2.5H20

47

39.27

9.37

9.64

38.89

9.27

10.01

15

16

Ph

H

N

N-

The data of ‘H and 13C NMR for ligands 1-16. [(a) ‘H-NMR (b) “C-NMR]. (a) 3.11-3.63 (14H, -kH,-N), 2.09 (4H, heterocycle), 1.50, 1.52, 1.57 (18H C(CH,),. (b) 64.73, 61.28, 59.57, 54.15, 53.35, 50.57,44.48, 42.07 (CH,-N), 32.41, 30.49, 30.23, 23.35, 21.95, 21.75 (-CH,). 2.04 (4H, heterocycle), 1.30, 1.53, 1.60 (21, C-(CH,),). (2) (a) 3.09-3.92 (13H, -CH2-N), (b) 59.62, 57.28, 56.41, 55.22, 50.67, 43.83, 42.00 (CH,-N), 30.21, 24.35, 23.53, 21.99, 21.78, 15.30 (CH,). 1.70 (3H, heterocycle), 1.54, 1.58 (18H, C(CH,),), 1.08d (CH, heterocycle). (3) (a) 3.08-3.90 (14H, -CH,-N), (b) 63.95, 59.58, 56.40, 55.08, 54.24, 44.11, 42.09 (CH,-N), 32.40, 30.85, 30.22, 22.22, 21.17, 17.02 (CH,). 1.07 (2H, heterocycle), 1.48, 1.49, 1.56 (18H, C-&H,),), 1.06 (6H, CH, (4) (a) 3.11-3.96 (14H, -CH,N), heterocycle). (b) 65.80, 61.37, 59.63, 56.59, 54.41, 44.76, 42.14, 40.33 (CHIN), 30.69, 30.27, 22.36, 21.80, 15.65, 14.44 (CH,). 1.90 (2H, heterocycle), 1.48, 1.50, 1.52 (18H, C(CH,),), 1.05, 1.15 (6H, CH, (5) (a) 3.09-3.81 (14H, -CH,-N), heterocycle). (b) 63.80, 61.54, 58.90, 56.87, 53.95, 45.82, 43.23, 41.53 (CH,-N), 31.14, 29.60, 23.13, 21.34, 16.56, 15.41 (-CH,). (6) (a) 3.08-3.86 (14H, CH2-N), 1.90 (2H, heterocycle), 1.46, 1.58, 1.58 (18H, C(CH,),), 7.13 (5H phenyl). (b) 64.32, 62.18, 59.46, 57.42, 55.28, 50.82,44.26,42.28 (CH,-N) 32.84, 31.21, 29.04, 24.18, 21.86, 19.34 (C-CH,), 113.17, 117.86, 120.39, 124.18 (phenyl). 0.98d, (3H, CH, heterocycle). (7) (a) 3.09-3.93 (14H, CH2-N), 1.75 (5H, heterocycle), 1.55, 1.57, (18H, C-(CH,),), (b) 62.15, 59.63, 56.52, 54.43, 53.23, 43.58, 42.13 (
-w

Q-f,).

WY (lU

(a) 2.9e3.94 (13H, -CH,N), 1.74 (6H, heterocycle), 1.42, 1.58 (21H, C-(CH,),). (b) 59.57, 59.30, 56.45, 54.48, 52.89, 48.63, 48.50, 42.13 (CH,-N), 32.83, 30.71, 30.19, 22.82, 22.01, 21.70, 13.52 (CH,). (a) 2.80-3.90 (13H, --CH,-N), 1.80 (5H, heterocycle), 1.46, 1.56, (2lH, C-(CH,),), 0.98d (3H, CH, heterocycle). (b) 59.42, 58.80, 56.38, 55.42, 54.38, 52.08, 48.01, 47.61, (CH,-N), 32.64, 30.69, 30.12, 21.92, 21.63, 20.90, 13.71, 13.14 (CH,).

%Tc-DADT (12)

complexes substituted with heterocyclic amines

115

(a) 2.60-4.20 (14H.