Technetium complexes of pentadentate amine-phenol ligands

NW/. Med. Biol. Vol. 20, No. 2, pp. 21 I-216, 1993 Printed in Great Britain. All rights reserved

0883-2897/93 $6.00 + 0.00 Copyright 0 1993 Pergamon Press Ltd

Technetium Complexes of Pentadentate Amine-Phenol Ligands M. R. A. PILLAIl*, C. S. JOHN’, J. M. L03, D. E. TROUTNER4, W. A. VOLKERT’ and R. A. HOLMES’

M. CORLIJA5,

‘Isotope Division, Bhabha Atomic Research Center, Bombay 400085, India, *Department of Radiology, Radiopharmaceuticals Chemistry, George Washington University Medical Center, Washington, DC 20037, U.S.A., %stitute of Nuclear Sciences, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China, “Zynaxis Cell Sciences Inc., 371 Phoenixville Pike, Malvern, PA 19355 and ‘Department of Radiology, University of Missouri and Research Services, H.S. Truman VA Hospital, Columbia, MO 65211, U.S.A. (Received 3 hdy 1992) We have synthesized five new pentadentate amine-phenol ligands as part of a study to develop bifunctional chelating agents for labeling antibodies with %Tc. These ligands were prepared by condensing various triamine ligands with salicylaldehyde and reducing the Schifl’ bases obtained to get the amine-phenol ligands. Radiochemical studies of 99mTcand 99Tccomplexes were performed with the precursor imine-phenols and the amine-phenol ligands. Technetium complexes were prepared at total technetium concentrations of 0.1-100 PM at ligand concentrations of 100 PM. Complexation yields and radiochemical purities were estimated by chromatographic techniques, paper electrophoresis and by solvent extraction into CHCl,. Complexation was achieved with both imine- and amine-phenol ligands. The amine-phenol complexes were found to be stable over a period of 24 h. The lipophilicities and stability of the amine-phenol complexes were higher than those of the imine-phenol complexes. Biodistribution studies with two of the amine-phenol complexes were carried out in Sprague-Dawley rats. A large portion of the injected activity was taken up by the liver and the clearance rate of the chelates from blood was slow.

Introduction

zyldiethylenetriamine, and studied its complexation with Co(H). In the present studies, a total of eight ligands were synthesized and studied for their complexation with 99mTc Three ligands contained two imine bonds (Schiff bases), i.e. ligands la, IIIa and Va (Fig. I),

Earlier studies demonstrated that tetradentate amine-phenol ligands form neutral lipophilic technetium complexes in high yields (Pillai et al., 1990a,b). The excellent complexation of the amine-phenol ligands with 99mT~and the high stability of the resultant complexes prompted us to study the feasibility of developing bifunctional ligands of the amine-phenol type for labeling antibodies with 99mTc. The amine-phenol ligands in the previous

while the imine groups were reduced to form two secondary amine groups in five other ligands (Fig. 2). Ligands II-IV were derivatives of ligand I, with ligand IV being a precursor of a BFCA in which the benzylamine group could be converted to an arylisothiocyanate group for conjugation. Ligand V was synthesized to study the effect of having a backbone other than the diethylenetriamine.

work were synthesized by condensing diamines with two equivalents of salicylaldehyde and reducing the resultant Schiff bases with NaBH,. For the current studies, we selected a triamine, diethylenetriamine, for the synthesis of ligands since the central nitrogen can be readily functionalized for development of bifunctional chelating agents (BFCAs) and further coordinating groups can be added to the primary amines. Refosco et al. (1988) reported the synthesis and characterization of 99Tc complexes of the Schiff base forms of similar pentadentate ligands. Motekaitis et al. (1984) synthesized a pentadentate amine-phenol ligand, 1,7-bis(2-hydroxybenzyl)4-ben-

Materials and Methods 3,3’-diamino-N-methyldiDiethylenetriamine, propylamine and salicylaldehyde were purchased from Aldrich Chemical Co. Sodium borohydride and stannous tartrate were from Sigma Chemical Co. 99mTcwas eluted from a Mallinckrodt generator after a 24 h growth and the activity ranged from 50 to 75 MBq/mL. 9vc as NH,TcO, was obtained from New England Nuclear and diluted in 0.9% saline

*Author for correspondence. 211

M. R. A.

212

PILLAl

Ligand Ia

Ligand UIa OH

H

et al.

Sodium borohydride, 1 g (0.027 mol), was added to this and mixed at room temperature for 24 h. The solvent was removed by rotary evaporation and the contents dissolved in about 150 mL of deionized water, and extracted with 2 x 150 mL portions of CHCl,. The chloroform layer was combined and back extracted with 150 mL of water to remove water soluble impurities. The CHCl, layer was dried by standing with -40 g of anhydrous sodium sulfate powder for about 30 min, filtered and the CHCl, removed by rotary evaporation. The product was isolated as white powder and stored in a desiccator. ‘H-NMR (CDCl,) 6 2.7(s) (8H), 3.93(s) benzylic CH, (4H), 6.6-7.2(m) aromatic (8H). r3C-NMR (CDCl,) 6 48.13, 48.34, 52.73, 116.82, 117.89, 123.97, 128.74, 129.28, 159.18 Syntheses and characterization of ligands II, III and IV are described elsewhere (Pillai et al., 199Oc). Ligands Va and V were synthesized in the same manner as that of ligands Ia and I by using 3,3’-diamino-N-methyldipropylamine instead of diethylenetriamine. The products formed were oils. Ligand Va:

Ligand I

Ligand Va

Fig. 1. Imine-phenol

ligands.

solution to the required concentration. Flexible silica gel plates (7.5 x 2.5 cm, coating thickness 0.25 mm) were from J. T. Baker Chemical Co. Whatman 3MM chromatography paper (30 x 2.5 cm) was used for paper electrophoresis. Proton and “C-NMR spectra were obtained in a JEOL FX 90Q spectrometer.

Ligsnd II

X=H

Ligand IIl

X=NOs

Ligand IV

x=NH*

Synthesis Ligand Ia. Diethylenetriamine, 4.12 g (0.04 mol), was dissolved in 200 mL of absolute ethanol. Salicylaldehyde, 9.8 g (0.08 mol), was added to this and the solution turned bright yellow. It was refluxed for 24 h and the solvent was removed by rotary evaporation. The Schiff base was isolated as an oil. ‘H-NMR (CDCl,) 6 2.7-3 (t) CH2 (4H), 3.5-3.8 (t) CH2 (4H), 6.7-7.4(m) aromatic (8H), 8.3 imine CH (2H). 13CNMR(CDCl,)S47.47, 59.28, 116.76, 118.39, 131.17, 132.04, 160.91, 165.90. Ligund I. The Schiff base (la), 4 g (0.013 mol), prepared above was dissolved in 100 mL ethanol.

Ligand V

Fig. 2. Amine-phenol

ligands.

wmTc complexes of pentadentate Table

I. Preparation of technetium complexes Volume

Reagent

or solvent

0.5 M NaHCO, Normal saline 5 mM ligand 0.00054.5 mM NH,TcO, V9mTc,50-75 MBq/mL

(mL)

Final concentration

0.5 3.0 0. I 1.0 0.2

0.05 M 0.15M 0.1 mM 0.0001-O. I mM 2-3 MBq/mL

Above solutions were reduced with 0.2mL (2mL for 0.1 mM technetium) of a saturated solution of stannous tartrate.

‘H-NMR (CDCl,) 6 1.6-2.0 (p) CH, (4H), 2.2 (s) methyl (3H), 2.3-2.5 (t) CH, (4H), 3.4-3.7 (t) CHr (4H), 6.7-7.3(m) aromatic (8H), 8.2 imine CH (2H). “C-NMR (CDCQ 6 28.29, 41.84, 54.95, 57.11, 116.82, 118.22, 130.95, 131.88, 161.13, 164.76. Ligand V: ‘H-NMR (CDCI,) 6 1.5-1.9 (p) CH2 (4H), 2.2 (s) methyl (3H), 2.3-2.5 (t) CH, (4H), 2.6-2.8 (t) CH, (4H), 3.9 (s) benzylic CH2 (4H) 6.7-7.3(m) aromatic (8H). ‘-‘C-NMR (CDCl,) 6 26.88, 42.0, 47.47, 52.73, 56.14, 116.22, 118.82, 122.56, 128.19, 128.51, 158.31. Complexation

The complexation reactions were carried out as per the protocol given in Table 1. Typically a 5 mL saline solution containing 0.1 mL(5 x lo-” mmol) of an ethanolic solution of the ligand, 0.5 mL of 0.5 M bicarbonate buffer, pH 9, 1 mL of 99T~Op of varying concentration and 0.2 mL of 99mTCO; (- l&l5 MBq) was reduced at room temperature with 0.2 mL of a saturated solution of stannous tartrate. Quality control techniques Thin layer chromatography. TLC was done using flexible silica gel plates and 0.9% saline solution as solvent. A 5 PL portion of the complex was applied 1 cm from the lower end of the strip. The strips were developed up to 7 cm from the bottom and cut into 10 equal segments after drying. The fraction of the activity at the first four segments and the last six segments were calculated separately as R, < 0.5 and R, > 0.5, respectively. Paper electrophoresis. Paper electrophoresis was done in a Gelman Deluxe chamber and power supplied for 1 h at 300 V in a pH 9, 0.01 M NaHCO, solution. Samples were spotted 1Ocm from the cathode. Following the run the strips were dried and cut into l-cm segments and their radioactivity measured. Solvent extraction. Solvent extraction was done by mixing 1 mL of the reaction mixture with 1 mL of CHClr for about 2 min over a vortex mixer. Equal aliquots of the aqueous and organic layers were withdrawn and counted for radioactivity. The geometry of the counter was suitably modified such that the count rates were low enough to minimize dead time loss. A second extraction was carried out by extracting 0.~5mL of the aqueous layer again with CHCl,. 0.5 mL of the organic layer was back extracted with

ligands

213

0.5 mL of 0.05 M bicarbonate buffer to estimate the distribution ratio. The above three types of measurements were typically done at 1 and 24 h after preparation of the complexes. HPLC. Liquid chromatography separations were carried out in a Beckman Series 332 dual pump gradient elution system equipped with a radiation detector connected to a strip chart recorder and an integrator. A Hamilton PRP-1 reversed phase column, 25 cm, 10 PM was used for separation and the mobile phases were water and acetonitrile. Initial elution was for 2min with 90% Hz0 and 10% CHrCN. During the next 2 min a gradient was applied so that at the end of that time the eluting solution was 30% Hz0 and 70% CH,CN. This was maintained for 14min after which another gradient was applied to return the column back to the original condition. A flow rate of 2 mL/min was maintained throughout the separation. Biodistribution studies. Biodistribution studies were carried out in Sprague-Dawley rats weighing 175-250 g anesthetized with sodium pentobarbital, 50mg/kg weight of the body. The complexes (0.05-O. 1 mL) were injected into the right jugular vein and the rats were sacrificed at 15 s, 5, 10 and 30 min after the injection. The organs were excised, blotted to remove blood and counted for radioactivity. (All the animal experiments were carried out in compliance with the relevant national laws relating to the conduct of animal experimentation.)

Results and Discussion The pentadentate imine-phenol and the aminephenols studied here showed complexation with 99mTc. All the complexes prepared showed extractability into CHC& and hence the solvent extraction technique could bc used for the estimation of complexation yield. The second extraction and the back extraction results could be additionally used for calculating the total amount of extractable complex formed. These complexes remained at the point of spotting in TLC studies with 0.9% saline as solvent, whereas the 99mTc0; moved with the solvent front, and hence this method was used for the estimation of 99mTc0,. Table 2 gives the results of the complexation studies of the imine-phenol ligands at pH 9. The complexation yields with these groups of ligands were low with initial yields varying from 13 to 79%. The solvent extraction yield for ligand Ia varied from 73 to 79% at different concentrations of TcO,. The back extraction of these complexes showed about 28% of the activity in the aqueous layer suggesting a distribution ratio of about 2.5. The complexes were not exceptionally stable over a period of 24 h. The major impurity formed during storage was TcO,. The complexes of ligand IIIa were formed in lower yields than those of the complexes of ligand Ia. The

M. R. A. PILLAI er al.

214

Table 2. Complex yields* and % free TcOi impurity at different concentrations imine-nhenol ligands. Complexation at pH 9 % Extraction yield

WXI

IO-’

--

In ula Va

% Free. TcO,10-s

10-l

10-d

10-r

1

24

1

24

1

24

1

24

1

24

1

24

13 72 22

60 61 16

19 61 21

25 41 15

19 42 13

64 30 15

1

I5 33 8

I 16 17

60 48 8

19 32 22

33 48 15

h

Ligand

10-e

of TcO< for

15 12

All reaction mixtures contained lo-’ M @and and pH 9 buffer. *Complexation yield measured by extraction into CHCl,.

back extraction studies gave a distribution ratio of about 6 suggesting that it is more lipophilic than the complexes of ligand Ia. The increased lipophilicity may be due to the substitution of the middle nitrogen with the nitro benzyl group. The stability of this complex was comparable to ligand Ia. The complex with ligand Va was formed in low yields and was less lipophilic with a distribution ratio of < 1. The amine-phenol ligands were studied for complexation at different pHs and found to produce higher complexation yields at pHs 9 and 10. Results of studies at pHs 9 and 10 are given in Tables 3 and respectively. The distribution ratio in 4, CHC&/O.O5 M NaHCOS solutions were >20 for the complexes of ligands I-IV and about 10 for the complex with ligand V. Hence, all the complex yields reported are the first extraction yield from the reaction mixture into CHCl,. Complexation yields were generally high at lo-‘M TcO; for all the ligands. Complexation yields were >85% for ligand II at all concentrations of TcO; studied. Ligands III and V showed similar results except that the yields were lower at 1O-4 M TcO;. Ligands I and IV showed only about 45 and 3 1% complexation, respectively, at low4 M TcO; . The stability of complexes was moderate to good throughout the concentration range studied. The stability of all the complexes was excellent at 10m7M of TcO; .The results of the complexation studies at pH 10 are shown in Table 4. The 1 h complexation yields at lo-’ and low6 M TcO; were comparable to that of the results obtained at pH 9 (Table 3). However, the stability of the complexes was poor at pH 10 with the complexes being converted to TcO; on standing. The extractability of the complexes at different pHs was also studied. Complexes prepared at pH 9 were

adjusted to different pHs by diluting a small portion of the complex with buffer at an appropriate pH (acetate buffer was used for pHs 3-7 and bicarbonate/carbonate buffer for pHs 9 and 10). These solutions were extracted with CHCl,. The results of the extraction studies are provided in Fig. 3. The extractions of Tc-complexes with ligands II, III and IV were high at all pHs. However, for ligands I and V the extractions were low between pHs 3 and 5. In order to investigate whether the low extraction efficiency of complexes with ligands I and V at low pHs was due to the lower distribution ratio or the formation of free TcO, impurity, complexes were prepared at pH 9, extracted into CHCl,, and then back extracted into different pH buffers. The results of the back extraction studies are given in Fig. 4. The results are in agreement with the extraction results at different pH and suggest a lower distribution ratio at low pH for complexes of ligands I and V. More detailed complexation studies at different pHs were carried out for ligand IV and its two analogs, ligands II and III. Complexes of these ligands were prepared at different pHs. The complexation yields as estimated by solvent extraction studies are given in Fig. 5. The complexation yields varied at different pHs and the best yields were seen at pH 9. At lower pHs the main impurity was TcO;. All the complexes studied were neutral as indicated by paper electrophoresis. TcO; levels estimated from the paper electrophoresis method were consistent with the solvent extraction data. Complexes of all ligands were analyzed by HPLC and the results of three such studies are given in Fig. 6. The HPLC studies showed that most of these ligands form more than one complex species (Fig. 6(a) and (b)]. For ligand Ia, part of the complex was

Table 3. Complex yields* for amine-phenol ligands complexed and incubated at pH 9

Table 4. Complex yields* for amine-phenol ligands complexed and incubated at pH 10 Complex yield (%)

Complex yield (%) [TcO;] Ligand 1 II III IV V

h

10-e _________

lo-’

10-J

10-r

(Tco;]

1

24

1

24

1

24

1

24

Ligand

94 91 91 88 90

84 98 98 89 87

12 98 96 88 89

70 98 98 79 87

13 98 97 80 87

47 94 89 69 88

45 85 67 31 58

25 92 74 35 61

I II III IV V

All reaction mixtures contained 10e4 M ligand and pH 9 buffer. *The yields reported are obtained from the first extraction of the complex from buffer into CHCl,.

h

10-b

lo-’

10-r -___

10’

1

24

1

24

I

24

1

24

87 91 84 77 90

79 42 1 23 87

85 92 90 11 89

28 38

52 94 89 27 87

14 40 1 8 88

21 80 62 21 58

4 66 82 13 61

I 18 87

All reaction mixtures contained lo-” M @and. *The yields reported are obtained from the first extraction of the complex from buffer into CHCl,.

%Tc complexes of pentadentate ligands

215

(al so,000 1 60,000

-

40,000

-

Cl x

0 II

5

. III

t 4

0

2

4

6

6

10

12

10

12

10

12

Retention time, min Fig. 3. Complex yields as estimated by solvent extraction of the complexes of amine-phenol ligands at different pHs. Complexes were prepared at pH 9, adjusted to a different pH by incubating with different buffers and extracted with CHCl,.

(b) ao.000 , 60,000

-

20,ooo

-

I

P. c-

0

2

4

Retention

6

time,

6

mln

Cc) 20

2

I

I

I

I

4

6

8

10

I 12

PH

100,000 1

ao,ooo -

Fig. 4. Back extraction of the complexes of amine-phenol ligands at different pHs. Complexes were prepared at pH 9, extracted with CHCl, and back extracted with different buffers.

-x

-

x-‘7L /

a0

2

a

E 60

x

-.

2

2

II

cl Ill

,\”

p: x

40

. 20

1 2

.

IV

/ I 4

I 6

I 8

I 10

4

Retention

I 12

PH

Fig. 5. Complexation of ligands II, III and IV at different pHs.

eluted at a very low retention time, R, = 1.3 min, immediately after the elution of TcO,- impurity. As this complex was eluted in 10% CH&N it was a

hydrophilic species. The complex eluted at the longer retention time was the one which was extractable into CHCI,. The complex of ligand I (figure not given)

6

6

time, rnln

Fig. 6. (a) HPLC profile of the complex of ligand IP, (b) HPLC profile for the complex of ligand Va and (c) HPLC profile for the complex of ligand V. HPLC separations in a PRP-1 column. Gradient elution using acetonitrile and water. &2 min, 10% CH,CN and 90% H,O. 2-4 min, linear gradient of lO-70% CH,CN in H,O. 4-18 min 70% CH,CN and 30% H,O. 18-20 min, linear gradient of 3&90% H,O in CH,CN. Flow rate 2 mL/min.

also showed two peaks for the complex in addition to the TcO; peak, the complexes had retention times of 6.8 and 8.2 min, and hence both of them were extracted into CHCI,. Although HPLC studies of the complexes of ligands II, III and IV showed multiple peaks (figure not given), most of the activity (> 80%) was seen in the major peak. The HPLC profiles for the complexes of ligand V showed single species [Fig. 6(c)]. The complex of ligand Va was eluted at a

M. R. A. PILLAI etal.

216

Table 5. Biodistribution studies of the *Tc complex of ligand II in Suraau+Dawley rats

Time Brain Blood Heart Lung Liver Spleen Stomach Large intestine Small intestine Kidney Muscle

% Dose/organ

Organ

% Dose/organ

Organ/

Table 6. Biodistribution studies of the %Tc complex of ligand V in SpragueDawley rats

15s

5 min

10 min

30 min

0.14 37.1 1.3 2.9 25.0 1.0 0.94 1.0 4.6 7.1 0.13

0.05 13.2 0.8 2.4 38.3 1.2 1.5 I.1 11.2 7.8 0.10

0.05 14.7 0.89 2.7 33.1 1.6 1.7 1.0 13.7 8.4 0.05

0.09 13.0 0.62 2.0 31.9 1.3 0.96 0.76 23.2 7.9 0.06

Time Brain Blood Heart Lune Live; Spleen Siomach Large intestine Small intestine Kidney Muscle

15s

5 min

10 min

30 min

0.15 49.8 0.89 1.67 27.0 0.74 1.12 1.5 5.6 5.2 0.11

0.05 10.7 0.47 0.87 29.6 0.48 2.2 I .06 21.9 5.4 0.11

0.05 12.5 0.43 0.79 28.7 0.59 0.99 1.12 23.9 5.0 0.14

0.05 11.4 0.41 0.87 25.8 0.58 1.8 I .04 32.1 5.1 0.09

The values given are average distribution in 2 rats.

The values given are average distribution in 2 rats.

shorter retention time (5 min) than the complex of ligand V (6.5 min) confirming the higher lipophilicity of the 99mTc-amine-phenol complexes compared to the 99mTc-imine-phenol complexes. The formation of multiple species with these ligands can be explained as there is a possibility that the central nitrogen atom in some cases may not be coordinating and in the event that it is coordinating there is more than one isomer possible due to the orientation of the substituent group in the central nitrogen atom. Biodistribution studies were carried out with the complexes of ligands II and V. Only two rats were used for each time point, as these studies were mostly carried out as preliminary tests to determine the possibility of significant uptake in brain, as might be expected for neutral, lipophilic complexes (Tables 5 and 6). The complexes did not exhibit measurable uptake in brain and most of the injected activity was accumulated by the liver with apparent hepatobiliary clearance’ into the intestine with time. The blood clearance rate was relatively slow.

4-aminobenzyl (ligand IV) and hydrogen (ligand I) substitutions. The stability of complex with ligand IV at tracer levels of WmTc is adequate for protein labeling studies and hence we used this ligand for labeling y-globulins with 99mTc. Preliminary results showed that human y-globulins can be labeled with preformed complexes of ligand IV in good yields with 99mTc remaining attached to the proteins for 24 h (Troutner et al., 1990). Acknowledgements-This

work was supported by the US Department of Energy, Grant No. DE FGO2-86ER60400 and the University of Missouri Research Council.

References Motekaitis R. J., Martel A. E. and Nelson D. A. (1984) Formation and stabilities of cobalt(H) chelates of N-benzyl triamine Schiff bases and their dioxygen complexes. Inorg. Chem. 23, 275-283. Pillai M. R. A., John C., Lo J. M. and Troutner D. E. (1990a) Radiochemical studies of technetium complexes of tetradentate amine-phenols. Appl. Radiat. Isot. 41, 557-561.

Pillai M. R. A., John C. S., Lo J. M., Schlemper E. 0 and Troutner D. E. (1990b) p(- Oxo-Bis-(0x0) Dinuclear Conclusion complexes of technetium (V) with amine-phenol ligands: syntheses, characterization and X-ray structure. Inorg. The pentadentate amine-phenol ligands reported Chem. 29, 18561860. here show excellent complexation with technetium. Pillai M. R. A., John C. and Troutner D. E. (1990~) The stability of the resultant complexes over a period Labeling of human gamma globulin with [losRh] rhodium of 24 h at pH 9 are excellent where TcO, is used at using a new bifunctional ligand. Bioconj. Chem. 2, 191-197. lo-‘M concentrations. The stability of the 99mTc Refosco F. J., Tisato F., Mazzi U., Bandoli G. and Nicolini complex with ligand V was excellent over the entire M. (1988) Technetium (V) and rhenium (V) complexes concentration range of TcO< studied. The high stabwith Schiff-base ligands containing the ONNNO donor ility seen for the complexes with this ligand may be atom set. Crystal structure of [N,N’-3-Azapentane-l-5due to the formation of four favoured six-membered diyl bis (salicylidineiminato) (3-)-O,O’,N,N’,N”]-oxotechnetium (V). Chem. Sot. Dalton Trans. 611415. rings upon complexation with technetium. In comTroutner D. E., Pillai M. R. A., John C., Lo J. M. and plexes formed from ligands with a diethylene triamine Misellati K. (1990) Pentadentate amine-phenol complexes backbone, the maximum stability was observed for of 99mTc.In Technetium and Rhenium in Chemistry and complexes with the ligand having benzyl substitution Nuclear Medicine 3 (Edited by Nicolini, Bandoli and Ma@, pp. 585-593. Cortina International, Italy. (ligand II), followed by nitrobenzyl (ligand III),