Preparation, radiolabeling and biodistribution of a new class of bisaminothiol (BAT) ligands as possible imaging agents

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NW/. Med. Viol. Vol. 18, No. 5, pp. 551-556, 1991 hr. J. Radial. Appl. In&urn. Part B Printed in Gnat Britain

Pergamon Press plc

Preparation, Radiolabeling and Biodistribution of a New Class of Bisaminothiol (BAT) Ligands as Possible Imaging Agents CHRISTY

S. JOHN, ME&PING HANK

KUNG, JEFFREY

BILLINGS

and

F. KUNG*

Department of Radiology, Division of Nuclear Medicine, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. (Received

23 May

1990; received for publication

12 October

I990)

In developing new ligands as potential brain and heart perfusion imaging agents two ligands based upon N,S, donor atoms with the biphenyl backbone were synthesized. Biphenyl-2,2’-bis(N-l-amino-Zmethylpropane-2-thiol) (BP-BAT-TM) and biphenyl-2,2’-bis(N-1-amino-2-ethyl-butane-2-thiol) (BP-BAT-TE) form stable, neutral and lipid soluble complexes with [%Tc]pertechnetate in the presence of tin(I1) tartarate as a reducing agent. The [*Tc]BP-BAT-TM complex penetrates the blood-brain barrier following iv. injection into rats. Washout from the brain is fast, indicating no retention. The biodistribution of [%Tc]BP-BAT-TE in rats showed an intitial heart uptake (0.8% /organ, at 2 min) and a slow washout (0.74% at 15 min). No brain uptake was found (0.05%). Significant uptake and retention in liver was observed. An imaging study of [*Tc]BP-BAT-TE in a monkey showed no brain uptake and a clear indication of liver uptake and gall bladder clearance. These results indicate that this ligand system may be suitable as the basic core structure for the development of new imaging agents. Further studies with structural variations in the biphenyl backbone are warranted to develop new *Tc imaging agents for clinical applications.

Introduction There is a current interest in developing new %Tc labeled radiopharmaceuticals for use in nuclear medicine clinics. This stems from the fact that 99mT~is an ideal nuclide, having a half life of 6 h and a low y energy (140 kev). It is readily available in nuclear medicine clinics at instant demand and is economical to use. One class of brain perfusion agents (*Tc-amineoxime) was first invented by Troutner et al. (Troutner et al., 1984; Volkert et al., 1984), and later developed by researchers at Amersham International, Inc. (Jurisson et al., 1986; Neirinckx et al., 1987). The final product, [%“Tc]d, I- HMPAO, is currently being used in nuclear medicine clinics for the study of regional cerebral perfusion (rCBF). A second category of brain and heart perfusion agents is BAT0 complexes (Boronic Acid Adducts of Technetium Oximes), (Linder et al., 1987, 1988; Narra et al., 1989) being *All correspondence should be addressed to: H. F. Kung, Ph.D., Division of Nuclear Medicine, Department of Radiology, Hospital of University of Pennsylvania, Philadelphia, PA 19104, U.S.A. pel. (215)-662.30961.

developed by researchers at Squibb Medical Research Institute. This class of compound is currently undergoing clinical trials. Another class of *Tc brain perfusion agents is based on the N,S, donor atoms, bisaminothiol (BAT) or diaminodithiol (DADT). A considerable amount of work has been reported on bisaminothiol (BAT) ligands (Dannals, 1981; Epps et al. 1983; Epps, 1984; Kung et al., 1984, 1985). It has been shown that BAT ligands form neutral, stable and lipid soluble complexes with 99mT~which are able to cross the blood-brain barrier reflecting regional perfusion. However, their retention in the brain is rather poor and therefore excludes their use for single photon emission computed tomography (SPECT). Later on several other derivatives containing amino group as side chains on basic BAT skeleton were synthesized and labeled with %Tc (Efange et al., 1987; Lever et a[., 1985; Kung et al., 1989). The biodistribution studies showed that the compounds did not have optimum brain retention suitable for SPECT imaging. The most promising BAT derivative is L&-ethylene cysteinate dimer (ECD) developed by researchers at DuPont Co. (Cheeseman et al., 1989; Walovitch et al., 1989; Holman et al., 1989). The

551

CHRISTYS. JOHNet

al.

R = Me, Et

2, R = Me, 52% 3, A = Et, 49%

4, R = Me, 63% 5, R = Et, 67%

Scheme 1. Synthesis of biphenyl BAT ligands. [99”Tc]ECD appears to be trapped in the brain by an in vitro enzyme hydrolytic process. The monoacidic compound is retained in brain tissue for prolonged periods. As part of our effort in investigating the structureactivity relationship of N,S2 ligands of the use as possible brain agents, a new series of the N2S2 ligands with a biphenyl backbone was prepared. Herein we report the synthesis, radiolabeling and biodistribution of this class of BAT ligands.

Experimental Melting points were determined with a Meltemp (Laboratory Devices) and are reported uncorrected. Infra-red spectra were obtained with a Mattson Polaris FT-IR spectrometer. NMR spectra were recorded on either a Varian EM 360A spectrometer or a Bruker 360 MHz instrument. Chemical ionization MS were obtained on a VG ZAB-E mass spectrometer using isobutane as the reactant gas. Elemental analyses were performed by Atlantic Microlabs, Inc., of Atlanta, Georgia. 2,2’-Diaminobiphenyl, 1 To the hydrogenation cell was added 7.0 g, 0.021 mol of 2,2’-dinitrobiphenyl in ethyl acetate (100 mL). A catalytic amount of palladium absorbed on carbon (0.2 g) was added to the cell. The hydrogenation was carried out on a Parr hydrogenator at 50 psi for 6-8 h. At the end of this period the solution became clear; the catalyst was filtered and the filtrate was evaporated to give a colorless oil which solidified on cooling. The compound was recrystallized from ethanol to give a solid (4.8g, 98%). m.p. 80°C (77-78°C Melby). ‘H-NMR (CDCI,): 6 3.5-3.7 (bs, 4.0 H, NH,); 6.4-7.1 (m, 8.0 H, arom.).

3,3,12,12-Tetramethyl-1,2-dithia-5,lO-diazabiphenylcyclododeca -4, lo-diene, 2 A round bottomed flask was charged with 2,2’diaminobiphenyl (5.7 g, 0.031 mol), 2,2’-dithiobis(Zmethylbutyna1) (7.9 g, 0.031 mol) and toluene (125 mL). The mixture was refluxed overnight (14 h) with a Dean-Stark trap. After cooling, the volatile materials were removed in vacua to give compound 2 as a yellowish oil (8.4 g, 73%). The attempts to purify the product on silica gel column resulted in the hydrolysis of bisimine. ‘H-NMR(CDC1,): 6 1.2-l .5 (m, 12.8 H, CH,); 6.6-6.9 (m, 2.0H, CH); 7.0-7.9 (m, 8.0H, arom.). i.r. (neat) o(C=N) 164Ocm-‘. The crude oil was used for the subsequent reduction step. 3,3,12,12- Tetraethyl- 1,2-dithia- 5, lo-diazabiphenylcyclododeca -4, lOdiene, 3 To a round bottomed flask was added 2,2’diaminobiphenyl (5.7 g, 0.031 mol), 2,2’-dithiobis(2-ethylbutynal) (8.1 g, 0.031 mol) and toluene (150 mL). The mixture was refluxed overnight (14 h) with a Dean-Stark trap. After this period the volatiles were removed under vacuum to give a yellowish gummy solid. The residue was washed with hexane and recrystallized with hexane/chloroform 60/40 to give a white solid, 3 (6.Og, 48%). m.p, 170°C. i.r. (KBr): u(C=N) 1642cm-‘. m/e =411 (M+ l)+. ‘H-NMR (CDCI,): S 0.5-1.0 (m, 12 H, CH,); 1.3-1.9 (m, 8H, CH,); 6.6-6.9 (m, 2H, CH): 7.0-7.9 (m, 8 H, arom.). Anal. Calcd for Cz4H3,,N2Sl: C, 70.24; H, 7.32; N, 6.83; S, 15.61. Found: C, 70.20; H, 7.40; N, 6.81; S, 15.55. Biphenyl-2,2’-bis(Nthiol), 4

I-amino-2-methyl-propane-2-

To the crude bisimine, 3, (6.0 g, 0.017 mol) in THF (150 mL) lithium aluminum hydride (3.7 g, 0.10 mol) was added. The mixture was refluxed for 6 h, cooled,

553

Bisaminothiol ligands as imaging agents and then a saturated solution of potassium hydrogen tartarate (20 mL) was added carefully dropwise. The solid suspension was extracted with ethylacetate (200 mL) by heating the mixture. The suspension was filtered and the filtrate was evaporated to give a yellowish viscous oil. The crude product was purified by passing through a silica gel column and eluted with hexanes/EtOAc 80/20 to give compound 4 as a colorless oil (3.2 g, 63%). ‘H-NMR(CDC1,): al.3 (s, 12 H, CH,); 1.6 (s, 2 H, NH); 3.0-3.2 (d, 4H, CH,); 3.9-4.2 (m, 2 H, SH); 6.6-7.3 (m, 8 H, arom.). The dihydrochloride salt of the ligand was prepared by heating the free amine with an ethanolic solution of HCl for 10 min. The solvents evaporated and the residue obtained was triturated with ether several times and recrystallized with an ether-ethanol mixture to give a white solid, m.p. 130-132°C. Biphenyl-2,2’-bis(N-l-amino-3-ethylbutane-3-thiol),

5

The bisimine, 3, (2.Og) was reduced by the same procedure as described above. 1.3 g (67%) of the desired compound, 5, was obtained as a colorless viscous oil. i.r. (neat): u(SH) 2560cm-‘, u(NH) 3450cm-‘. ‘H-NMR(CDC1,): S 0.7-1.0 (t, 12 H, CH,); 1.2-1.7 (m, 10 H,CH,); 3.0-3.2 (bs, 4 H, CH,); 3.7-4.0 (m, 2 H, SH); 6.5-7.2 (m, 8 H, arom.). The dihydrochloride salt of the free amine was prepared by a method similar to above. The crude solid obtained was recrystallized with an ether-ethanol mixture to give a beige-white solid, m.p. 106-109°C. Preparation of [*Tc]BP-BAT

complexes

The dihydrochloride salt of ligand compound 4 or 5 (5 mg) was dissolved in deionized water (1 mL). To 100 p L of this solution was added 500 p L ethanol and 200 /IL of [99mTc]sodium pertechnetate. A saturated solution of tin(H) tartarate in water (1OOpL) was added to the solution and the resulting mixture was heated at 90°C for 15 min. The product was extracted into chloroform (2 x 2 mL); the compound extracts were dried under a stream of nitrogen. The complex was dissolved in saline solution and the radiochemical purity and in vitro stability were determined by reverse phase HPLC (PRP-1 column; 10% 5 mM DMGA (pH 7.0)/90% CH,CN). After incubation at room temperature for as long as 4 h, the same HPLC procedure was repeated to determine the in vitro stability. Animal distribution studies

Male Sprague-Dawley rats (200-300 g) were injected intravenously (under ether as anesthesia) with 0.2 mL of a saline solution containing *Tc complex of 4 or 5 (l-20 p Ci). At various time points following injection, blood samples were collected by cardiac puncture and the rats were sacrificed immediately thereafter by cardiectomy while under ether anesthesia. The organs of interest were subsequently excised, weighed, and the radioactivity counted using Beckman automatic 7 counter (Model 5500). The percent

Tc-99

BP-BAT-TE

I-uv

I

A gamma tIL

Tc-99

BP-BAT-TE

Fig. 1. HPLC profiles of [BmTc]BP-BAT-TEand [99mTc]BPBAT-TE with reverse phase column (PRP-I), solvent system of acetonitrile:dimethyl glutaric acid (0.5 mM) 90: 10; flow rate = I .Omlimin. dose/organ suggests that an identical species of similar chemical structure was obtained under both chelating conditions. Reverse phase HPLC data of carrier-added and no-carrier-added [99mTc]BP-BATTE are shown in Fig. 1. The no-carrier-added 99mT~ complexes were found to be stable at room temperature for up to 4 h (no change in HPLC profile) when dissolved in either chloroform or saline solution. Single crystal x-ray diffraction studies of [99mTc]BPBAT-TE have shown that a square pyramidal Tc=ON, S2 complex is formed. This pattern of complexation is identical to that for reported BAT ligands (Epps, 1984; Kung et al., 1989). An ORTEP diagram of [99mT~]BP-BAT-TE is shown in Fig. 2 (John, unpublished data). The crystallographic details will be published elsewhere. The two complexes [99mTc]BP-BAT-TM and [99mTc]BP-BAT-TE showed quite different HPLC retention times on a reverse phase PRP-1 column; the complex [99mT~]BPBAT-TM had a retention time of 8.0min in MeCN/DMGA 90/10 solvent at a flow rate of 1.OmL/min. Under similar conditions, the complex [99mTc]BP-BAT-TE had a retention time of 11.Omin. This significant difference in retention time of the two compounds is also reflected in octanol/saline partition coefficients (pH 7.0). The partition coefficient for BP-BAT-TM is 303 + 61 whereas the partition coefficient for BP-BAT-TE is 3206 +_809 (Table 1). The biodistribution studies in rats showed that [99mTc]BP-BAT-TM is able to cross the blood-brain barrier. At 2 min postinjection the brain/blood ratio

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al.

Fig. 2. ORTEP diagram of [*mTc]BP-BAT-TE. is 0.275. At 15 min, these ratio values were determined by comparison of the tissue radioactivity with suitably diluted aliquots of the injected dose. The percent dose/g values were computed from the percent dose/organ values and the corresponding mean organ weights (mean organ weights: heart, 0.85 g; brain, 165 g; blood, 18 g; liver, 9 g; kidneys, 1.9 g; lungs, 1.6 g). Finally, the brain/blood ratio was calculated from the corresponding percent dose/g values.

were then radioactivity counted in a well counter. The partition coefficient was calculated as the ratio of the counts per minute in the octanol layer divided by that in the aqueous layer. Samples from the I-octanol layer were then subsequently repartitioned until a consistent partition coefficient was achieved. This was usually achieved in three runs.

Results and Discussion Imaging study in monkey A male cynomologus monkey (5 lb) was sedated with ketamine (40-50 mg), cannulated with a 25 g buttefly and anesthetized with sodium pentobarbitol 10 mg/kg i.v., with additional doses given as needed. A dose of 5 mCi [99mTc]BP-BAT-TE in 1 cm3 saline was injected i.v. and immediately planar whole body images (30 s/frame for 2 h) were obtained using a Picker Digital Dyna Camera on line to a GE Star II Computer system with a 20% window set at an energy peak of 140 keV. The dynamic planar images of the trunk area were summed (10 min x 12) and the liver activity was qualitatively analyzed for relative distribution of radioactivity. Determination of partition coeficients The partition coefficients were determined by mixing the [*Tc]BP-BAT complex with 3 g each of I-octanol and buffer (pH 7.O:O.l M phosphate) in several test tubes. The tubes were vortexed for 3 min at room temperature and subsequently centrifuged for 5 min to separate the layers. Two weighed samples (0.5 g each) from the octanol and the aqueous layers Table

I. Partition coefficients and HPLC retention [*mT~]BP-BAT-TM and [*Tc]BP-BAT-TE*

[PR”Tc]BP-BAT-TM r*TclBP-BAT-TE

times of

Partition coefficient

Retention timet

303 f 61 3206 f 809

6.0 min 11.6min

*Octanol/pH 7.0 phosphate buffer. tHPLC was performed with acetonitrile:dimcthylglutaric (0.5 mM) 90: 10, flow rate = 1.0 ml/mitt.

acid

The synthesis of BP-BAT ligands was accomplished by the reduction of 2,2’-dinitrobiphenyl with hydrogen under pressure (50 psi) in the presence of Pd/C as catalyst. Almost quantitative yields of 2,2’diaminobiphenyl were obtained. Condensation of diamine with 2,2’-dithiodialdehyde in toluene gave the bisimine, 2, and 3 in about 50% yields. The bisimines were reduced in anhydrous tetrahydrofuran (THF) using excess lithium aluminum hydride to afford modest yields (about 65%) of 4 and 5. An aqueous ethanolic solution of ligand (l-3 mg) as a dihydrochloride salt was allowed to react with 99mTc0; in the presence of tin(H) tartarate as a reducing agent. The resulting complex was extracted into chloroform. The HPLC of carrier-added (u.v. detector) and no-carrier added (y detector) complexes exhibited an identical profile increase to 1.6 and then the activity washes out and the brain/blood ratio declines to 1.1 (Table 2). This indicates that there is Table 2. Biodistribution of [@‘“Tc]BP-BAT-TM in rats after an i.v. injection (% dose/organ, average of 3 rats f SD) Organ Blood Heart Muscle Lung Kidney Spleen Liver Skin Brain Brain blood*

2 min

ISmin

30 min

23.34 f 5.07 1.330 2 0.08 14.22 k 5.00 2.03 +_0.28 2.73 f 0.47 1.40 f 0.54 31.22 f 7.01 3.95 * 0.55 0.42 f 0.03 0.275 & 0.05

2.202 + 0. I72 0.36 -+ 0.02 13.95 + 3.31 0.89 +_0.30 0.95+0.13 2.43 10.120 54.67 * 6.91 4.39 f 0.02 0.225 * 0.03 I .59 f 0.28

I.293 It 0.25 0.20 * 0.07 8.97 -+ 0.99 0.55 f 0.03 0.67 _+0. I I 2.14 f 0.46 52.37 -+ 4.50 5.85 f 2.00 0.10+0.03 1.09f0.13

*Percent dose/gram ratio.

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Bisaminothiol ligands as imaging agents Table 3.

Biodistribution of [*Tc]BP-BAT-TE in rats after an i.v.

injection(% dose/organ,averageof 3 rats f SD) 30 min 15min 2 min orr?an 24.70 + 6.36 0.80 + 0.04 10.60 + 1.72 2.66 + 0.62 1.47 + 0.23 1.42 I 0.08 59.86 + 10.38 6.10 + 0.58 0.09 + 0.008 0.05 IO.02

Blood Heart MlJSCle

Lung Kidnev

Spleei Liver Skin Brain Brain blood*

2.44 * 0.74 + 14.09 + I .45 * I .48 + 0.78 ; 64.59 i 4.50 * 0.08 + 0.48 ;

0.51 0.06 2.21 0.21 0.29 0.36 4.89 1.19 0.01 0.04

1.79 * 0.33 0.39 * 0.05 12.24 k 0.85 0.85 + 0.26 0.84 + 0.10 0.34;0.12 44.97 _+2.68 4.65 5 1.30 0.05 + 0.002 0.59 + 0.11

‘Percent dose/gram ratio.

free diffusion of the chelate across the blood-brain barrier. Additionally, it shows that it is a reversible process which depends upon the relative concentration gradient between the brain tissue and the blood. The biodistribution of [99mTc]BP-BAT-TE showed very little or no uptake by the brain (Table 3). However, at 2 min postinjection the uptake by heart is about 0.8%. The heart activity washes out at 30 min postinjection to 0.4%. The monkey imaging study clearly showed uptake by heart, with a significant amount of activity in the liver. The activity washes out from the heart rapidly and the majority of activity was cleared from the hepatobiliary system. The wash out curve for liver is shown in Fig. 3. In conclusion, two new ligands based upon N2Sz donor atom with a rigid biphenyl backbone have been synthesized. These ligands form neutral, lipid soluble and very stable complexes with 99mTc.Unfortunately, neither complex displayed retention in brain or heart suitable for single photon emission computed tomographic (SPECT) imaging studies. However, the fact that they form neutral complexes of high in vitro stability suggests that the structural variations of this biphenyl backbone N,S, ligand system may be a basic ligand system suitable for the development of new perfusion imaging agents.

z-

5

E

1

L’

0

.

10

I

20

30

Time

(mitt)

40

50

60

Fig. 3. Liver uptake and washout of [99mT~]BP-BAT-TE in

a monkey.

Acknowledgement-This work was supported in part by a grant NS-18509, awarded by NIH.

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