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Bioconjugate Chem. 1990, 1, 132-137
Synthesis of a Diaminedithiol Bifunctional Chelating Agent for Incorporation of Technetium-99m into Biomoleculesl Kwamena E. Baidoo a n d Susan Z. Lever' Division of Radiation Health Sciences, Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205-2179. Received January 16, 1990
The synthesis of a bifunctional chelating agent (BCA), 1, based on the diaminedithiol (DADT) ligand system, is described. The six-step synthetic sequence has been accomplished in 16% overall yield, affording 1, which contains a thiolactone as a reactive moiety, which permits direct coupling to nucleophiles without the formation of byproducts. The reactivity of 1 toward benzylamine and subsequent labeling of the ligand with technetium-99m has been evaluated as a model for preparation of various bioconjugates. Both coupling and exchange labeling occur in high yield under mild conditions, and competition reactions with diethylenetriaminepentaacetic acid (DTPA) indicate the superior stability of the technetium-99m-DADT complex. Preparation of BCA 1 thus provides a new avenue into technetium-labeled radiopharmaceuticals.
Technetium-99 m (99mTc)possesses the best characteristics for scintigraphic imaging among currently available radionuclides (1,2). Its high photon yield per disintegration ensures good counting statistics and its monoenergetic y photons (140 KeV) are ideally suited for planar and single photon emission computed tomography (SPECT) instrumentation. The short half-life of 6.02 h and lack of particulate emissions generally result in a low absorbed radiation dose to patients. In addition, -Tc is inexpensive and is widely available in generator form. Due to these properties, there is considerable interest in developing protein-based radiopharmaceuticals containing this radionuclide. Initial methods relied on the native protein to offer direct stabilization for reduced technetium; however, weak nonspecific labeling, colloidal contamination, protein denaturation, and loss of label in vivo can be problematic (2-6). The bifunctional chelate approach, which provides certain advantages (7-91, has been under investigation for the labeling of proteins with 99mTc. However, the bifunctional chelating agents (BCAs) available for use with other radionuclides have had only limited success with 99mT~. For example, attempts to utilize diethylenetriaminepentaacetic acid (DTPA) coupled proteins (10-13) result in labeled products which lack sufficient in vitro and in vivo stability (2, 14). Newer chelates that form stable complexes with technetium have been synthetically modified into BCAs (Chart I). The diamidedisulfide (DADS) ligand system (15, 16) requires basic reaction conditions and higher temperatures for complex formation (17). In order to avoid these forcing conditions, preformed complexes have been coupled to the protein with a measure of success (18). The dithiosemicarbazone (CE-DTS) derivatives (19-21) require
* Address correspondence and requests for reprints to Susan 2. Lever, Ph.D., Department of Environmental Health Sciences, The Johns Hopkins University, School of Hy iene and Public Health, 2001 Hume Building, 615 N. Wolfe Et., Baltimore, Maryland 21205-2179. Taken in part from the Ph.D. thesis of K.E.B., The Johns Hopkins University, 1988. A preliminary communication of a portion of this work has been published (ref 33).
Chart I. Bifunctional Chelating Agents for Technetium ,(%I"COfi
Diamidedithiolate
Di(semithiocarbaz0ne)
(DADS)BCA
(DTS) BCA
1
Diaminedithiol
(DADT)BCA
acidic conditions for incorporation of 99mTc,which can lead to protein denaturation. In vivo, proteins labeled via this method show high liver and stomach radioactivity, suggesting label instability (21-23). Diaminedithiol (DADT) ligands represent another chelate class that forms strong complexes with technetium (24-31). As in the DADS system, DADT ligands contain two thiol groups that exhibit high affinity for technetium. However, the two coordinating nitrogens are amines rather than amides; thus, their full coordinating potential is preserved. These properties of DADT ligands result in facile complex formation and exceptional complex stability. Clearly, a BCA based on the DADT ligand system would permit efficient labeling of biomolecules, such as antibodies and other proteins, with 99mTcunder mild conditions. In this report, we describe the complete synthesis of BCA 1 (32, 33) and its coupling reaction with ben-
1043-1802/90/2901-0132$02.50/0 0 1990 American Chemical Society
Diaminedithiol Bifunctional Chelating Agent
zylamine. Incorporation of technetium into the coupled product 8 was performed under exchange-labeling conditions, and followed as a function of time. Stability of the major DADT complex was assessed in competition studies with diethylenetriaminepentaacetic acid (DTPA). EXPERIMENTAL PROCEDURES Solvents and chemicals were reagent grade and used as received. Dry tetrahydrofuran (THF) was prepared by predrying over anhydrous CaC1, followed by distillation over Na/benzophenone. [99"Tc]glucoheptonate kits ("Glucoscan") were gifts from E. I. Du Pont de Nemours Co. Inc., N. Billerica, MA. [99mTc]NaTc0,was obtained as a saline solution from a 99Mo/99"Tc generator purchased from Cintichem/Union Carbide. Melting points were determined with a ThomasHoover capillary melting point apparatus and are reported uncorrected. Spectrophotometric determinations were made on a Perkin-Elmer 399B infrared spectrophotometer. NMR spectra were recorded in deuteriochloroform solution with an NR80 FT-NMR spectrometer (IBM Instruments Inc.) operated a t 80.06 MHz for protons ('H) and 20.25 MHz for carbon-13 (I3C). Radioactivity was measured on a Capintec CRC-7 radioisotope dose calibrator. Elemental analyses were performed by Atlantic Microlabs, Norcross, GA. HPLC was performed on a Perkin-Elmer Series 2 instrument equipped with an LC-75 UV/visible detector; a 2-in. calcium fluoride flowthrough scintillation detection system; EE & G/Ortec single-channel analyzer, amplifiers, and ratemeters; and a Hewlett-Packard H P 3392A integrating recorder. Unless otherwise stated, all extractive workups utilized diethyl ether, and the organic extracts were dried over anhydrous Na,SO, and filtered prior to concentration under reduced pressure.
Hexahydro-6,6,9,9-tetramethyl-lH-imidazo[2,1-dJ[1,2,5]dithiazepine (3). Sodium borohydride (17.5 g, 0.460 mol) was added in small portions to a solution of diiminedisulfide 2 (34) (44.5 g, 0.193 mol) in EtOH (2.80 L) and the mixture was stirred for 18 h a t room temperature. Acetone (100 mL) was added to destroy the excess reducing agent, and after 15 min, volatile solvents were evaporated under reduced pressure. Aqueous sodium hydroxide (200 mL, 2.5 M) was added to the white residue. After extractive workup, the crude product was purified by shortpath silica gel chromatography (ethyl acetate), followed by recrystallization from n-pentane to afford bicyclic amine 3 as a white, crystalline solid (36.3 g, 80%: mp 63-64 "C (lit. (35)mp 65 "C); IR (KBr) 3280 cm-' (N-H); 'H NMR 6 1.23-1.32 (m, 12 H ) , 1.9 (s, 1 H) 2.50-3.60 (m, 7 H); 13C NMR 6 18.66,24.79,26.42,28.28,46.23,58.56,66.62,91.23. Anal. Calcd (C,,H,,N,S,): C, 51.72; H, 8.68; N, 12.06; S, 27.54. Found: C, 51.42; H, 9.17; N, 11.95; S, 27.50.
Hexahydro-1,6,6,9,9-pentamethyl-lH-imidaz0[2,1dJ[1,2,5]dithiazepine (4). To a solution of 3 (4.0 g, 17 mmol) in acetonitrile (40 mL) was added 50% K F / Celite (6.0 g, 51 mmol) followed by methyl iodide (2.4 mL, 5.5 g, 40 mmol). The mixture was stirred a t room temperature for 3 h and then filtered. Volatile solvents were removed by distillation under reduced pressure, aqueous sodium hydroxide (30 mL, 2.5 M) was added to the residue, and the mixture was extracted with ether (3 X 25 mL). Additional product was obtained from the Celite filter cake by treatment with aqueous NaOH (25 mL, 2.5 M) followed by reextraction. The ether solution was treated a t reflux with activated charcoal for 20 min, filtered, and evaporated under reduced pressure. Isolation by short-path silica gel chromatography (85:15 hexane/ ethyl acetate) afforded 4 as a clear oil (3.95 g, 93%): mp
Bioconjugafe Chem., Vol. 1, No. 2, 1990 133
121-122 "C (2.4 X Torr); IR (neat) no N H stretch at 3300 cm-'; 'H NMR 6 1.22-1.32 (m, 12 H), 2.48-3.10 (m, 10 H); 13C NMR 6 19.51, 24.41, 26.40, 27.92, 46.06, 54.16, 54.84, 66.88, 100.05. Anal. Calcd (C,,H,,N,S,): C, 53.65; H, 9.00; N, 11.37; S, 25.96. Found: C, 53.60; H, 8.94; N, 11.35; S, 26.00. 2,2,4,9,9-Pentamethy1-4,7-diaza1,lO-decanedithiol (5). T o a stirred solution of 4 (15.0 g, 60.6 mmol) in dry T H F (100 mL) under a slow stream of nitrogen was added LiAlH, (5.00 g, 132 mmol) in small portions. The mixture was refluxed for 18 h and cooled to room temperature. The reaction was externally cooled a t 0 "C (ice bath) and quenched by slow, dropwise addition of saturated, aqueous NH,Cl. The mixture was then quickly triturated with ethanol (4 X 50 mL) and filtered. The filtrate was adjusted to pH 3-4 with aqueous hydrochloric acid (3 N), and volatile solvents were removed by evaporation under reduced pressure. Water (50 mL) was added to the residue and the mixture was brought up to pH 8 with aqueous sodium hydroxide (2.5 M). After extractive workup, the organic layers were concentrated to about 50 mL under reduced pressure and then applied to a 50g silica gel column. The product was then eluted with ether. Concentration under reduced pressure and drying under high vacuum provided 5 as an oil: IR (neat) 3300 cm-' (N-H), 2540 cm-' (S-H); 'H NMR 6 1.35 (s, 6 H), 1.37 (s, 6 H), 1.90 (br s, 3 H), 2.38 (s, 3 H), 2.48 (s, 2 H), 2.62 (s, 2 H), 2.71 (s, 4 H); 13C NMR 6 30.48, 44.63, 45.26,46.24,48.40,59.94,63.74,72.21. For long-term storage, the free base was converted to the hydrochloride salt by redissolution of the oil in ethanol (30 mL) followed by saturation with dry HCl gas. The resulting warm solution was cooled to room temperature and the product was precipitated with ether, filtered, washed, and dried under high vacuum to afford 5.2HC1 as a white solid (14.54 g, 74%). Anal. Calcd (C,,H,,N,S,-BHCl): C, 40.75; H, 8.70; C1, 21.57; N, 8.64; S, 19.72. Found: C, 40.92; H , 8.73; C1, 21.94; N, 8.61; S, 19.75. 2,2,4,9,9-Pentamethy1-4,7-diaza1,lO-bis(p-methoxybenzy1)-1,lO-dithiadecane(6). Aqueous sodium hydroxide (50 mL, 2.5 M) was added to a stirred solution of 5 (4.00 g, 12.4 mmol) in ethanol (60 mL). Neat p-methoxybenzyl chloride (8.80 g, 56.2 mmol) was added, and the stirring was continued for 1 h a t room temperature. The ethanol was then distilled under reduced pressure. After extractive workup, the oily residue was redissolved in ethanol (10 mL). The mixture was adjusted to pH 2-3 with saturated ethanolic HC1. The warm mixture was cooled to room temperature and the product was precipitated with ether. The precipitate was filtered and washed with ether to yield 6.2HC1 as a white powder (6.9 g, 76%). For subsequent use, the free base was regenerated by treatment of the salt with aqueous sodium hydroxide (2.5 M) followed by extraction with ether: IR (free base, neat) 3300 cm-' (N-H); 'H NMR (free base) 6 1.32 (s, 12 H), 1.6 (br s, 1 H), 2.36 (s, 3 H), 2.50 (s, 2 H), 2.57 (5, 2 H), 2.61 (s, 4 H), 3.66, (s, 2 H), 3.73 (s, 2 H), 3.77 (9, 6 H), 6.80, 7.24 (AB q, J = 8.5 Hz, 8 H); I3C NMR (free base) 6 26.93,27.24,32.05,44.79,46.87,47.63,48.47,55.11,59.96, 60.11, 69.38, 113.78, 129.83, 130.38, 158.40. Anal. Calcd (C,,H,,N,O,S,): C, 66.11; H , 8.63; N, 5.71; S, 13.03. Found: C, 65.99; H , 8.64; N, 5.68; S, 13.02. 2,2,4,9,9-Pentamethy1-4,7-diaza-7(carbethoxymethyl)-l,l0-bis(p-methoxybenzyl)-l,l0-dithiadecane (7). A solution of 6 as the free base (20.0 g, 40.8 mmol) and ethyl bromoacetate (34.0 g, 203.7 mmol) in acetonitrile (75 mL) was heated a t 44 "C for 3 h. Volatile solvents were removed by evaporation under reduced pres-
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Bioconjugate Chem., Vol. 1, No. 2, 1990
sure. The mixture was brought to pH 8-9 with aqueous NaOH (2.5 M) and then extracted with ether (3 X 50 mL). Excess ethyl bromoacetate was removed via a Kugelrohr apparatus (25 OC, 0.5 X lov3Torr). The residue was chromatographed on a short-path silica gel column (89.5:10:0.5 hexane/ethyl acetate/triethylamine) to yield 7 as a clear oil (13.0 g, 55%): IR (neat) 1730 cm-' (C=O); 'H NMR 6 1.16-1.33 (m, 15 H), 2.34 (9, 3 H), 2.47-2.79 (m, 8 H), 3.56 (s, 2 H), 3.71 (s, 4 H), 3.77 (s, 6 H), 4.15 (4, 2 H), 6.80, 7.23 (AB q, J = 8.5 Hz, 8 H); 13C NMR 6 14.22,26.71,26.88,32.14,45.20,47.62,47.91,54.54, 55.14,56.74,59.32,59.97,66.74,69.47,113.34,129.37,130.35, 158.45, 171.94. Anal. Calcd (C,,H,,N,O,S,): C, 64.57; H, 8.39; N, 4.86; S 11.10. Found: C, 64.48; H , 8.42; N, 4.84; S, 11.05. 4-[2-[(2-Mercapto-2-methylpropyl)methylamino]ethyl]-6,6-dimethyl-2-thiomorpholinone(1). Anhydrous hydrogen fluoride (5.85 g, 308 mmol) was condensed into a 100-mL Teflon round-bottom flask containing 7 (2.33 g, 4.04 mmol) and anisole (0.93 g, 8.5 mmol), a t -78 OC (dry ice/acetone bath). After the addition was complete, the mixture was stirred for 1.5 h a t 0 OC (ice bath) under a positive pressure of nitrogen. The H F was then flushed with a stream of nitrogen through two KOH traps in series. Water (10 mL) was added to the residue and the mixture was brought to pH 2-3 with aqueous NaOH (2.5 M). The mixture was then washed with ether (4 X 15 mL) and the aqueous layer was brought to pH 8. After extractive workup, the crude oily residue was chromatographed on a short-path silica gel column (9O:lO hexane/ethyl acetate) to yield 1 as an oil (0.82 g, 70%): IR (neat) 1655 cm-' (C=O); 'H NMR 6 1.32 (s, 6 H), 1.48 (s, 6 H), 2.08 (s, 1 H), 2.41 (s, 3 H), 2.47, 2.68 (2 s, 8 H), 3.32 (s, 2 H); 13C NMR 6 29.89, 30.46, 45.20, 46.23, 47.65, 56.00, 57.74, 65.14, 72.22, 199.00. Anal. Calcd (Cl,H,GN,0S2): C, 53.80; H, 9.03; N, 9.65; S, 22.03. Found: C, 53.82; H, 8.79; N, 9.60; S, 22.04.
2,2,4,9,9-Pentamethy1-4,7-diaza-7-[ (benzylcarbamoyl)methyl]-l,l0-bis(p-methoxybenzyl)-l,lO-dithiadecane ( D A D T - B z A )(8). A solution of 1 (200 mg, 0.691 mmol) and benzylamine (110 mg, 1.02 mmol) in acetonitrile (5 mL) was stirred a t room temperature under nitrogen for 3 h. Concentration under reduced pressure gave a white residue, which was purified by preparative TLC on silica gel plates (2 X 2000 km; 60:40 hexane/ethyl acetate) to give 8 as a colorless oil (222 mg, 81%): IR (free base, neat) 3260, 3160 cm-' (N-H); 'H NMR (free base) 6 1.20 (s, 6 H), 1.28 (s, 6 H), 1.55 (br s, 2 H), 2.30 (s, 3 H), 2.34, (s, 8 H), 2.67 (m, 8 H), 3.38 (s, 2 H), 4.50 (d, J = 5.0 Hz, 2 H), 7.31 (s, 5 H), 7.8 (br s, 1 H). The product was stored as the dihydrochloride salt, prepared in the same manner as 5.2HC1. Anal. Calcd (C,,H,,N,OS,. 2HC1*0.5H2O): C, 50.21; H, 8.00; C1, 14.62; N, 8.78; S, 13.37. Found: C, 50.27; H , 8.05; C1, 14.71; N, 8.50; S, 13.26. Radiolabeling of 8. A solution of [99mTc0,]- (20-50 mCi, 3 mL) was added to a "Glucosan" kit, containing sodium glucoheptonate and SnCl,, mixed well, and allowed to stand for 15 min. Aliquots of this mixture containing [99mTc]glucoheptonatecomplex ( [99mTc]GH)were added to solutions of 8, to give a final DADT ligand concentration of lo-, M in a total volume of 1 mL. The mixture was analyzed by reverse-phase HPLC using an Alltech C-18 Econosil column (6.5 X 250 mm) with MeOH/O.l M ammonium formate solution (9O:lO) a t a flow rate of 8 mL/min. Activity balances were measured by comparison of the total amount of radioactivity collected with that injected. Greater than 90% of activity was recov-
Baidoo and Lever
ered from the column, and no colloidal 9 9 m T ~ was 02 detected. Within 10 min, 95% of the [99mTc]glucoheptonate complex was converted to two products (9a and 9b) with retention times of 2.2 and 4.4 min, respectively. The composition of this product mixture varied as a function of time, and only the minor complex 9b was observed from 25 min through the duration of the study (96 min). Competition Experiments with DTPA. A stock solution containing DTPA (240 mM) and 8 (2 mM) was prepared. During exchange labeling [99mTc]glucoheptonate (0.5 mL, 6 mCi) as prepared above was added to an aliquot of the stock solution (0.5mL). In a separate experiment, the major [99"Tc]DADT-BzA complex was isolated and then challenged with DTPA by incubation with 0.1 M DTPA. The mixtures were incubated for up to 3 h a t room temperature and analyzed periodically by reverse-phase HPLC as described above. The presence of [99mTc]DTPAwas assessed by comparison of retention times of an authentic sample of [99mTc]DTPA. RESULTS AND DISCUSSION Our development of a BCA based upon the DADT ligand system focused upon the synthetic features needed for coupling of the ligand to the protein. Rather than utilize an external activating group, BCA 1 was selected as the synthetic target where the thioester is derived from a portion of the DADT ligand, and incorporation of an acetate side chain provides the balance of atoms needed to form the six-membered thiolactone. Thioesters are known to react with amine nucleophiles. Therefore, in order to prevent self-condensation from intra- or intermolecular amide formation, the secondary amine was protected with a methyl substituent. The transformation of the parent DADT ligand to BCA 1 (Scheme I) therefore had to accomodate differentiation of each nitrogen to permit alkylation with nonequivalent groups. In addition, the thiol groups had to be protected throughout the alkylation reactions to prevent concomitant S-alkylation. Therefore, two features of the synthetic approach were key to the preparation of the target bifunctional chelating agent. First, use of the mild reducing agent NaBH, (2 3) afforded the bicyclic intermediate 3, where the two nitrogens were nonequivalent. In addition, the disulfide bond remained intact, serving as internal protection for the thiols. Of the two required alkylation steps, it was necessary to perform the methylation first, because the product is stable to the subsequent reduction step with lithium aluminum hydride (LiAlH,). The alkylation of 3 with methyl iodide in the presence of 50% K F on Celite afforded 4 in excellent yield (93%). K F on Celite has been reported to aid S-, 0-,and N-alkylations through extensive hydrogen bonding with the hydrogen atom bonded to the heteroatom (36). Cleavage of both rings of this bicyclic compound to the open-chained molecule was then accomplished with LiAlH, to permit elaboration of the other nitrogen. Acidification of the reaction mixture immediately after quenching is a requirement for isolation of high yields of 5. The second key feature of the synthesis utilized the acid-labile p-methoxybenzyl groups to selectively protect the thiols (5 6). Even though a large excess of pmethoxybenzyl chloride was used for the alkylation of the free thiols, there was no evidence of interfering Nalkylation when the reaction was conducted a t room temperature. The resulting p-methoxybenzyl thioethers are stable under basic conditions and permit substitution on the secondary nitrogen with ethyl bromoacetate to afford precursor 7.
-
-
Diaminedithiol Bifunctional Chelating Agent
Bioconjugate Chem., Vol. 1, No. 2, 1990
135
Scheme I
2
8
7
6
Scheme I1
100
* --b> -
80
0 a
40
;p
20
U
60
0
8
9 a, b
Our intial route for the synthesis of BCA 1 from 7 entailed a three-step sequence of deprotection, ester hydrolysis, and condensation to give the thiolactone via an intermediate dithiol carboxylic acid. In practice, however, we found that the target thiolactone BCA 1 was obtained directly from the deprotection step. Since anhydrous H F does not promote simple ester hydrolysis, the reaction is likely to involve acid-catalyzed ester interchange of the free thiol and the ethyl ester. An analogy for this reaction can be drawn from the synthesis of a six-membered thiolactone from a benzyl-protected thiol carboxylic acid by Lumma and co-workers (37)using trifluoroacetic anhydride a t reflux. The mechanism of this transformation was reported to involve acid-catalyzed debenzylation with concomitant cyclization to generate the thiolactone. With the target BCA in hand, its reactivity toward amine nucleophiles was tested by using benzylamine as a model (Scheme I). The reaction proceeded smoothly to yield the DADT-BzA adduct 8, and was practically complete in 3 h. The scope and limitations of the reaction of 1 with other nucleophiles is currently being assessed. For example, we have carried out coupling reactions with HSA in phosphate buffer and with the monoclonal antibody B72.3 in borate buffer in the presence of dimethylformamide (38). In both cases, coupling occurred readily. Therefore, under conditions required for coupling sensitive bimolecules, thiolactone 1 is sufficiently reactive. Complexation of 8 with 9 9 m Twas ~ then investigated utilizing transchelation with [99mTc]glucoheptonate (Scheme 11). Although direct labeling can also be used to achieve complex formation, many biological substrates are susceptible to damage by the relatively harsh reaction conditions used. Therefore, the mild conditions employed for ligand exchange represent a useful alternative approach to labeling with 99mT~. In addi-
0 '0
0
2
10
25
33
96
Time (mln) Figure 1. Composition of exchange labeling reaction mixture as a function of time.
tion, ligand exchange is preferred with respect to the labeling of proteins because it offers a mechanism to avoid labeling the weak binding sites on the native protein. As a consequence, an important criterion in the utility of ligands derived from BCA 1 is their ability to accept reduced 9 9 m Tfrom ~ labile complexes. Treatment of DADT-BzA 8 with preformed [99mTc]glucoheptonate ( [99m]Tc-GH)gave two [99mTc]DADT-BzAcomplexes (9a and 9b) as determined by HPLC. From activity balance measurements, the efficiency of labeling was >90% within 10 min and no colloidal 99mT~02 was detected. The two products, 9a and 9b, were obtained initially; however, by 25 min only 9b was observed (Figure l). The formation of multiple products is not unexpected, due to the presence of the side chains, which upon complexation can be syn or anti to the Tc=O core. Similar observations have been observed in other N-alkylated or Calkylated DADT complexes. In the case of C-alkylated complexes, the ratio of products is usually 5050 (31); whereas the ratio of products observed in N-alkylated complexes can vary from 80:20 to 96:4 (24,26,27,30).In the case of the N-alkylated complexes the major complex, when characterized on the "Tc scale, has been shown to be the syn isomer (27, 30). This data indicates that the syn-anti pair of isomeric C-alkylation complexes are comparable in stability, and that the syn isomer in N-
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Bioconjugate Chem., Vol. 1, No. 2, 1990
alkylated complexes is relatively more stable than the corresponding anti isomer. Since DTPA has been the most extensively used chelating agent for binding metals to proteins through the bifunctional chelate approach, we investigated the ability of the DADT-BzA ligand 8 to compete with DTPA for reduced technetium. A DTPA:DADT-BzA ratio of 120:l was used under exchange-labeling conditions. Challenge experiments were also performed in which the isolated major [99"Tc]DADT-BzA complex (9b) was incubated with 0.1 M DTPA. No evidence of the formation of a [99"Tc]DTPA complex was found in these experiments during the 3-h incubation. These results indicate, as expected, that [99mTc]DADT-BzAis substantially more stable than [99mTc]DTPA. More detailed stability measurements using DADT ligands against other BCAs are underway. The pro osed structures for the complexes formed contain a [Tc'=0I3+ core, which is similar to those derived from mono- or unsubstituted DADT ligands. The two products are presumably isomeric in nature and are expected to be cationic. Support for this hypothesis is presently based on the crystal structure of the sole N,N'disubstituted DADT-"Tc complex reported to date (39). The synthesis and characterization of these complexes on the macroscopic "Tc scale will authenticate the chelation of T c by BCA 1 and allow structural determinations. In a separate study, we have carried out preliminary studies using 1 for the preparation of 99mTc-labeledHSA and anticolorectal carcinoma antibody (B72.3) (38). BCA 1 can be coupled to both proteins under mild conditions, and exchange labeling proceeded efficiently. The 99mTclabeled products were highly stable both in vitro and in vivo, with 9 9 m Tlabeling ~ of only DADT sites, and not weak, nonspecific sites. Thus, BCA 1 provides a new route to a variety of stable products containing technetium and serves as a versatile starting material for novel 9 9 m Tradiopharmaceu~ ticals. ACKNOWLEDGMENT This research was supported in part by USPHS Grants CA32845 and SO7 RR05445. We also thank Kurt L. Loening, Director for Nomenclature of Chemical Abstracts Service, for assistance in naming 1. LITERATURE CITED (1) Clarke, M. J., and Podbielski, L. (1987) Medical diagnostic imaging with complexes of 99mTc. Coord. Chem. Reu. 78,253-
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