Convenient synthesis of bifunctional tetraaza macrocycles

Buck E. Rogers, Carolyn J. Anderson, Judith M. Connett, Li Wu Guo, W. Barry Edwards, Elizabeth L. C. Sherman, Kurt R. Zinn, and Michael J. Welch...
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Bioconjugate Chem. 1002, 3, 108-117

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Convenient Synthesis of Bifunctional Tetraaza Macrocycles Thomas J. McMurry,’ Martin Brechbiel, Krishan Kumar, and Otto A. Gansow Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Building 10, Room B3-B69, Bethesda, Maryland 20892. Received August 5,1991

A convenient synthesis of 4-nitrobenzyl-substitutedmacrocyclic tetraamines and their conversion to bifunctional poly(amino carboxylate) chelating agents is described. Cyclization of (4-nitrobenzy1)ethylenediamine with appropriate BOC-protected amino disuccinimido esters in dioxane at 90 “C resulted in the formation of 12- and 14-membered ring diamides in 40% and 44% yield, respectively. A 12membered macrocyclic triamide was also prepared in 44 % yield by cyclization of N-(2-aminoethyl)4-nitrophenylalaninamide with disuccinimidyl N-(tert-butoxycarbony1)iminodiacetate. Deprotection (HCl/dioxane) and reduction with borane gave the substituted macrocyclic amines which were then alkylated with either bromoacetic acid or tert-butyl bromoacetate. Preparation of the isothiocyanate derivatives and 14Clabeled chelating agents are described. Attempts to prepare a 9-membered macrocyclic diamide using this cyclization technique resulted instead in a 20% yield of a 1O:l mixture of isomeric fused 5,6 ring acylamidines. Deprotection (HCl/dioxane) and reduction with borane gave a substituted piperazine derivative in 55 % yield.

INTRODUCTION

The notion that a tumor-seeking monoclonal antibody (mAb) can act as a biological “magic bullet” has prompted extensive investigations to determine the utility of radiolabeled antibody in cancer diagnosis and therapy (I). Radioiodination was first employed to label antibodies (2)and has been used to prepare [l3lI1mAbconjugates for clinical studies (3). However, neither 1311 nor lz5I has proved to be ideal for scintigraphy or therapy (4). Fortunately, exploitation of the broad array of decay properties available within the metallic radionuclides is possible provided the antibody can be altered to incorporate a strong metal-binding site. Methodology required to introduce a exogenous metal-binding site into protein was demonstrated in 1974 by Meares and co-workers,who reported the synthesis of a bifunctional EDTA (5)molecule bearing a p-diazophenyl moiety capable of functionalizing serum albumin and fibrinogin (6-8).Linkage of a metal complex to mAb and the subsequent tumor localization of lllIn in mice was first achieved (9) using the mixed anhydride DTPA-isobutylcarboxylic carbonic anhydride (IO),which reacts with proteins to form a modified tetraacetic acid ligand. Tetraacetic acid chelates of the type noted above did not have sufficient thermodynamic and kinetic stability to prevent in vivo loss of radionuclides such as wY, an energetic P emitter proposed for use in cancer therapy (11-13). Because dissociated yttrium accumulates in the bone (14) and would provide an undesirable dose to the radiosensitive marrow, the need to efficiently sequester the ion is apparent. Efforts to improve the thermodynamic and kinetic stability of the radionuclide complexes has led to the development of DTPA chelating agents which satisfy the requirements of the typically 8-9 coordinate (I5,16)yttrium ion (17,18). Biological experiments have clearly demonstrated the advantages of these DTPA chelatesfor in vivo sequestration of wy(19)and lllIn (17,18). We have recently reported the synthesis of a series of modified DTPA ligands in which backbone alkyl substituents have been added to increase the kinetic inertness of the chelates (20). Concurrent with that work, we have been exploring the chemistry of macrocyclic poly(amino

* Author to whom correspondence should be addressed. Not subject to U S . Copyright.

carboxylates) known to form unusually stable metal complexes (21-23). Complexes of macrocyclic ligands typically show enhanced thermodynamic and kinetic stability relative to an open-chain analogue. The thermodynamic “macrocyclic effect” is attributed to the enthalpic and entropic contributions resulting from the “preorganization” (24) enjoyed by the macrocycle (25). The 9-, 12-, and 14membered ring poly(amino carboxylates) 1,4,7-triazacyclononane-N,”,N”-triacetic acid (NOTA), 1,4,7,10-tetr a a z a c y c l o d o d e c a n e - N , ” ~ ~ - t e ~ ~ c eacid t i c (DOTA), and 1,4,8,11-tetraazacyclotetradecane-N,”~~-tetraacetic acid (TETA) form an attractive series of ligands in which the variation in cavity size and the number of donor atoms confer unique properties on the metal complexes (21, 26-28). For example, DOTA has been shown to form remarkably stable complexes of both divalent and trivalent metals, including yttrium and the lanthanides (29, 301, prompting the synthesis of several C- and N-functionalized derivatives of DOTA (33-36) and TETA (37, 38) suitable for linkage to mAb. Biological experiments with 88Y-labeled DOTA/Lym-1 conjugates have confirmed that the Y [DOTAI- complex exhibits high stability in vivo (39). Our interest in synthesizing a bifunctional DOTA ligand was stimulated by the observation that the complexes [Pb(DOTA)l2-and [Bi(DOTA)]were kinetically inert within the pH ranges of 4-10 (40), suggesting the utility of DOTA as a chelating agent for the CY emitter 212Biand its parent zlzPb(41). We report here the synthesis of the macrocyclic bifunctional poly(amino carboxylates) 2 and 3 by a synthetic route employing a diamide cyclization reaction. Our attempts to prepare 1 were thwarted by the propensity of the 9-membered macrocyclic diamide precursor to undergo a transannular reaction, resulting in the formation of a fused 5,6 ring acyl amidine. EXPERIMENTAL PROCEDURES

Materials and Methods. Anhydrous solvents (THF, dioxane, and DMF) were obtained from Aldrich. Triethylamine was distilled from CaH2. Diborane was used as received (Aldrich). Ethylenediamine-N,h”-dipropionic acid dihydrochloride was obtained from American Tokyo Kasei. @-Nitrobenzy1)ethylenediamine dihydrochloride

Published 1992 by American Chemical Society

Bloconjugate Chem., Vol. 3, No. 2, 1992 10s

Synthesis of Bifunctional Tetraaza Macrocycles

1

2

PNCS-Bz-NOTA

p-NCS-Bz-DOTA

3 p-NCS-Bz-TETA

(4) and N-(2-aminoethyl)-4-nitrophenylalaninamide(5) were prepared as described previously (I7).Ion-exchange resins were purchased from Bio-Rad; the AG50W-X8resin was further purified by literature methods (42). Silica used for flash chromatography was purchased from Fluka (silica gel 60, 230-400 mesh). Proton and 13C NMR spectra were obtained using a Varian 300XL instrument unless otherwise noted. Chemical shifts are reported in ppm on the 6 scale relative to TMS, TSP, or the following internal references (noted in text): t-BuOD (IH, 1.25; l3C, 31.1), CD3CN (lH, 1.93; l3C, 1.30), CD3CN (13C, 118.2), dmso-de solvent (lH, 2.49; 13C, 39.5), CDCl3 solvent (l3C, 77.0). Proton chemical shifts are annotated as follows: ppm (multiplicity, integral, couplingconstant (Hz)). Chemical ionization mass spectra (CI-MS) were obtained on a Finnegan 3000 instrument. Electron impact (EI-MS) mass spectra were recorded on an LKB9000 instrument. Fast atom bombardment (FABMS) mass spectra were taken by the Mass Spectroscopy Laboratory, College of Chemistry, University of California, Berkeley, CA. Elemental analyses were performed by Galbraith Laboratories (Knoxville, TN) or Atlantic Microlabs (Atlanta, GA). Carbon-14-labeled compounds prepared in this work were not submitted for elemental analysis, exact mass measurement, or NMR due to the possibility of radioactive contamination. However, chromatographic behavior of the carbon-14-labeledcompounds was identical to that observed for the unlabeled analogues. Analytical HPLC was performed using a Beckman gradient system equipped with Model 114M pumps controlled by System Gold software and a Model 165dualwavelength detector operating at 254 and 280 nm. A Waters C-18 reverse-phase column (15-pm particles, 100A pore size, 3.9 X 300 mm) and a binary gradient of 0100%B/25 min (solvent A = 0.05 M AcOH/EtsN, pH 5.5, solvent B = MeOH) at 1.0 mL/min were used for all analyses. N,N'B i s ( tert- b ut o x y ca r b o n y 1 ) et h y 1 e ne d i amine-Nfl-diacetic Acid (6). Triethylamine (22.3 mL, 0.16 mol) was added to a suspension of ethylenediamineN,"-diacetic acid (Aldrich, 7.0 g, 39.7 mmol) in 50% aqueousdioxane (200mL). To theclear solution, 2-[[(tertbutoxycarbonyl)oxylimino]-2-phenylacetonitrile (Aldrich BOC-ON, 20.1 g, 81.5 mmol) was added. The reaction was stirred for 5 days at room temperature and then extracted with EtOAc (200 mL). The layers were separated, and the aqueous solution was extracted with EtOAc (2 X 200 mL). The aqueous solution was cooled in an ice bath and acidified with 3 N HC1 to pH 2.0. The cloudy solution was extracted with EtOAc (3 X 200 mL), and the combined organic fractions were dried with MgS04, filtered, and concentrated to approximately 50 mL. The product was isolated by adding petroleum ether to effect a cloudy solution, refrigerating (4 "C) for 18 h, and

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collecting the white powdery solid by filtration (12.82 g, 86%1: IR (Nujol) 3548,3420,1746,1730,1686; 1H NMR (dmso-d6) 6 3.841 (d, 2 H, J = 6.5), 3.806 (s,2 H), 3.27 (m, 4 H), 1.384,1.368,1.322 (3 s, total 18 H, ratio 1:1:1,67);13C NMR (dmsod6, ref = solvent) 6 171.25, 171.11, 154.71, 154.50,79.00,78.87,49.10,48.29,45.93,45.71,45.64,45.32, 27.86,27.78; MS (CI/NHd m/e 221 (M-BOC-CH~C(CH~)Z + l), 177 (M - 2BOC + 1). Anal. Calcd for ClsH2eN208: C, 51.06; H, 7.45; N, 7.45. Found: C, 51.31; H, 7.37; N, 7.38. Disuccinimidyl Nfl-Bis( tert-butoxycarbony1)ethylenediamine-Nfl-diacetate(7). N-Hydroxysuccinimide (7.34 g, 63.8 mmol), compound 6 (11.71 g, 31.1 mmol), and EDC [1-[3-(dimethylamino)propyll-3-ethylcarbodiimide hydrochloride] (12.24 g, 63.8 mmol) were dissolved in a 4:l mixture of EtOAc and DMF (300 mL). The reaction was stirred at room temperature for 18 h. The resulting white precipitate was filtered, washed with hexanes, and vacuum dried to give 14.80 g (83% ) of the disuccinimido ester: mp 212-214 "C dec; IR (Nujol) 1840, 1803,1759 (sh), 1708 cm-l; lH NMR (dmso-de) 6 4.372 (m, 4 H), 3.360 (m, 4 H), 2.798 (s, 8 H), 1.407, 1.395, 1.363 (3 s, total 18 H, ratio 1:0.71:2); l3C NMR (dmso-de, ref = solvent) 6 169.76,166.45,166.20,154.41,154.03,80.05,47.03, 46.38,45.98,45.75,45.50,45.26,27.50,25.37;MS (CI/NH3) m/e 588 (M NH4), 571 (M 1). Anal. Calcd for C24H34N4012: C, 50.52; H, 6.01; N, 9.82. Found: C, 50.45; H, 6.01; N, 9.67. N,N'- B i s ( ter t -but o x y ca r b on y 1 ) et h y 1 e ne d i amine-N,N-dipropionic Acid (8). EthylenediamineN,"-dipropionic acid dihydrochloride (15.5 g, 56.0 mmol) was reacted with Et3N (32 mL, 0.23 mol) and BOC-ON (28.8 g, 0.117 mol) in 50% aqueous dioxane (250 mL) as described for 6. After extractive workup and acidification, a precipitate was collected and dried (20.2 g, 89%): 'H NMR (D20, pH = 9.5) 6 3.424 (m, 8 H), 2.404 (m, 4 H), 1.454 (s,18 H); 13CNMR (D20, pH 9.5, ref = CD3CN) 6 179.26, 155.87, 80.17, 43.53, 43.04, 34.97, 26.79; MS (CI/ NH3) m/e 405 (M + 1). Anal. Calcd for CleH32NzOe: C, 53.45; H, 7.97; N, 6.93. Found: C, 53.33; H, 7.97; N, 6.97. Disuccinimidyl Nfl-Bis( tert-butoxycarbony1)ethylenediamine-N,W-dipropionate(9). Dicarbamate 8 (9.0 g, 22.3 mmol) and N-hydroxysuccinimide (5.25g, 45.7 mmd) were suspended in 50% DMF/EtOAc (300 mL) and stirred until a clear solution formed. EDC (8.75 g, 45.6 mmol) was added and the reaction was stirred for 18 h under argon. The white precipitate was collected and vacuum dried (11.35 g, 85%): 'H NMR (dmS0-d6)6 3.438 (m, 4 H), 3.310 (m, 4 H, partially obscured by D2O in solvent), 2.917 (t, 4 H, J = 7.5),2.813 (s,8 H), 1.393 (s, 18 H); 13C NMR (dmSO-ds) 6 169.95, 167.31, 154.31, 79.13, 54.78,27.87, 25.37; MS (CI/NH3) m/e 599 (M + 1). Anal. Calcd for C26H38N4012: C, 52.17; H, 6.36; N, 9.37. Found: C, 51.92; H, 6.46; N, 9.27. N-( tert-Butoxycarbony1)iminodiaceticAcid (10). Iminodiacetic acid (15.0 g, 0.113 mol) was treated with Et3N (39.2 mL, 0.282 mol) and BOC-ON (Aldrich, 27.8 g, 0.113 mol) as described for 6. After aqueous workup, the combined EtOAc fractions were dried over MgSO,, filtered, and rotary evaporated to dryness (23.5 g, 89%): lH NMR (220 MHz, dmSO-ds) 6 3.90 (s,2 H), 3.86 ( 8 , 2 H), 1.35 (s, 9 H); MS (CI/NH3) m/e 234 (M + 1). Anal. Calcd for CgHlbN06: C, 46.35; H, 6.44; N, 6.00. Found: C, 46.62; H, 6.52; N, 5.87. Dis uccinimidyl N-( tert-Butoxycarbonyl)iminodiacetate (11). Compound 10 (19.14 g, 82 mmol), N-hydroxysuccinimide (18.98 g, 165 mmol), and EDC (32.51 g, 169.6 mmol) were combined in EtOAc (300mL) and stirred for 18 h. The solution was transferred to a separatory funnel and extracted sequentially with saturated NaCl

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solution (2 X 200 mL), 5 % NaHC03 (3 X 200 mL), and salt solution again (200 mL). The organic solution was dried over MgS04, filtered, and concentrated to about 50 mL with some precipitation of product. Hexanes (50 mL) were added to the suspension, and the flask was cooled at -20 "C for 3 h. The precipitate was collected, washed with hexanes, and dried in vacuo (30.9 g, 88%1. 'H NMR (dmSO-da) 6 4.546 ( 8 , 2 H), 4.528 (9, 2 H), 2.812 (s, 8 H), 1.396 (s,9 H); MS (CI/NH3) m / e 428 (M + 1). Anal. Calcd for C17H21N3O10: C, 47.78; H, 4.95; N, 9.83. Found: C, 47.49; H, 5.03; N, 9.68. 1,4-Bis(tert-butoxycarbonyl)-8(4-nit robenzy1)6,11-dioxo-1,4,7,10-tetraazacyclododecane(12). A 5L three-neck flask was equipped with a stirbar, two 500mL Normag water-jacketed addition funnels, and a Claison adapter fitted with a thermometer and reflux condenser. After purging with argon, anhydrous dioxane was introduced and the temperature was increased to 90 "C. @Nitrobenzy1)ethylenediamine dihydrochloride (4,2.68 g, 10 mmol) and Et3N (4.2 mL, 30 mmol) were added to a 250-mL round-bottom flask containing anhydrous DMF (200 mL). The solution was stirred 5 min, filtered, and then transferred to addition funnel "A". DMF was added to increase the total volume to 250 mL. Diester 7 (5.88 g, 10 mmol) was dissolved in DMF (250 mL) by heating to 60 "C and then transferred to funnel "B" (preheated to 60 "C). The contents of the addition funnels were added simultaneously to the stirring dioxane solution over an 8-h period. The temperature of the solution was maintained at 90 "C for 12 h, after which time the reaction was cooled to room temperature and the solvent was removed by rotary evaporation. The residual brown oil was redissolved in EtOAc (200 mL) and extracted with H2O (2 X 200 mL), 5% NaHC03 (2 X 200 mL), and saturated NaCl(2 X 100 mL). The EtOAc was dried (NazS04) and evaporated to a brown solid. Recrystallization from hot MeOH gave a tan precipitate which was further purified by flash chromatography (Fluka, 230-400 mesh silica, 41 X 250 mm, 2% MeOH/CH2C12) to give 2.19 g (4.0 mmol, 40%): IR (Nujol) 3290,3080 (w), 1710 (s), 1693 (sh), 1655 ( 8 ) cm-l; lH NMR (dmSO-ds) 6 8.29 (d, 1 H, J = 6.71, 8.20 (d, 1 H, J = 10.3), 8.16 (d, 2 H, J = 8.641, 7.49 (d, 2 H, J = 8.641, 4.18 (m, 3 H), 3.803 (m, 3 H), 3.2 (m, 2 H), 2.944 (m, 2 H), 2.744 (m, 3 H), 1.405 ( s , 9 H), 1.379 (s,9 H); MS (CI/NH3) m / e 536 (M + l),480, 436, 424,406, 380, 336, 306. Anal. Calcd for C25H37N50~0.5H20:C, 55.14; H, 7.03; N, 12.86. Found: C, 55.08; H, 7.01; N, 12.82. I-( tert-Butoxycarbonyl)-5-(4-nitrobenzyl)3,6,1l-trioxo-1,4,7,1O-tetraazacyclododecane (13). Diester ll (12.81 g, 30 mmol) and N-(2-aminoethyl)-4-nitrophenylalaninamide (5, 7.56 g, 30 mmol) were each dissolved in DMF (100 mL) and one-half of each solution was loaded into a 50-mL gas-tight syringe. The two syringes were locked onto a syringe pump and the two reactants were added to ca. 4 L of efficiently stirred anhydrous dioxane under argon at 95 "C such that the addition was complete after 24 h. The syringe-pump addition was then repeated using the second half of the two solutions. After the second addition had finished, the reaction was heated for an additional 18 h and then cooled to room temperature. The solution was concentrated to about 300 mL during which a large amount of precipitate formed. The flask was cooled at 4 "C for 18 h, after which the gelatinous crude product was collected on a Buchner funnel with constant trituration with petroleum ether (300 mL). The resulting light tan solid was dried under high vacuum (8.50 9). The crude product was suspended in CHCL (300 mL) and heated to boiling. MeOH was slowly added until the solution was clear. The flask was lightly stoppered and the solution was cooled to

McMurry et el.

room temperature and then to -20 "C for 48 h. The offwhite precipitate was collected and dried in vacuo. The filtrate was concentrated and a second crop was collected (5.97g, 44%): lH NMR (dmso-de)6 8.583 (br t, 1 H), 8.145 (d, 2 H, J = 8.51, 7.699 (m, 1 H), 7.519 (d, 2 H, J = 8.5), 7.137 (m,1 H), 4.371 (9, 1 H, J = 15.0, 9.0), 3.986 (dt, 2 H, J = 15.5, 3.6), 3.885 (br t, 2 H, J = 16.01,3.580 (m, 1 H), 3.365 (m, 1 H), 3.160 (dd, 1 H, J = 14.0,6.0),2.950 (m, 3 H), 1.384 ( ~ , H); 9 13CNMR (dmS0-d6)6 170.71, 169.34, 154.72, 146.37, 146.16, 130.32, 123.14, 79.63, 54.62, 54.38, 50.65,50.32,49.98,49.79,37.99,37.91,36.55,36.43,34.83, 27.80; MS (CI/NHs) mle 450 (M + 1). Anal. Calcd for C20H27N5O7:C, 53.45; H, 6.06;N, 15.58. Found: C, 53.35; H, 6.13; N, 15.52. 2 44-Nitrobenzy1)-1,4,7,10-tetraazacyclododecane Trihydrochloride (14). Route A (via Diamide 12). In a 200-mL Schlenk flask, compound 12 (2.06 g, 3.79 mmol) was suspended in anhydrous dioxane (20 mL) and cooled to ca. 10 "C. HC1-saturated dioxane (10 mL) was added and the reaction was stirred at room temperature for 14 h protected from atmospheric water by a Drierite-filled drying tube. The HCl/dioxane was then evaporated in vacuo by transferring to a liquid nitrogen trap, After vacuum drying at 0.1 mmHg for 48 h, the diamide dihydrochloride was obtained as a tan solid (1.6 g). This solid (1.6 g) was suspended in anhydrous THF (150 mL) and cooled to 0 "C before adding BHrTHF (1.0 M, 19.4 mL, 19.4 mmol). The reaction was stirred at 0 "C for 1 h and 45 "C for 48 h. The solution was cooled in an ice bath, quenched with MeOH (150 mL), and then evaporated to dryness. The residue was redissolved in MeOH (60 mL), heated to boiling, and evaporated. This procedure was repeated once with MeOH and once with absolute EtOH. The residue was then taken up in absolute EtOH (90 mL), cooled in an ice bath, and saturated with HCl(g). The flask was equipped with a reflux condenser and drying tube, and the reaction refluxed for 24 h. The EtOH solution was concentrated to ca. 30 mL and cooled to 4 "C. The resulting white precipitate was filtered on a medium frit under argon, washed with Et20, and vacuum dried. Recrystallization was achieved by dissolving in boiling MeOH and allowing the solution to stand at room temperature for 24 h. The white solid (0.91 g, 55%) was isolated as a trihydrochloride salt: 'H NMR (DzO, pH = 1.0) 6 8.230 (d, 2 H, J = 8.5), 7.530 (d, 2 H, J = 8.5), 3.443 (m, 1H), 3.25-2.90 (m, 16 H); 13C NMR (D20, pH = 1.0, ref = tert butyl alcohol) 6 148.14, 146.17, 131.74, 125.47, 55.59,49.05,45.98,45.58,45.29,44.42,43.89,42.19,38.16; MS (CI/NH3) m / e 308 (M + 1). Anal. Calcd for C~~H~~N~O~-~H C,C41.44; ~ * HH, ZO 6.91; : N, 16.12; C1, 24.48. Found: C, 41.42; H, 6.94; N, 15.92; C1, 24.09. Route B (viaTriamide 13). Triamide 13 (5.62 g, 12.5 mmol) was added to a solution of anhydrous dioxane (300 mL) previously saturated with HCl(g) and then stirred for 18 h under argon. Diethyl ether (100 mL) was added and the suspension was cooled at 4 "C for 6 h. The solid was collected and dried under vacuum for 18 h. The deprotected material was washed into a threenecked round-bottom flask with THF (50 mL) and the solution was cooled in an ice bath. Diborane/THF (1 M, 75 mL) was added via syringe and the reaction was heated at 50 "C for 48 h. The solution was cooled to 0 "C and MeOH (50 mL) was added. After stirring at room temperature for 1 h, the solution was evaporated to dryness. The residue was taken up in 100% EtOH and saturated with HCl. The saturated solution was then refluxed for 6 h under argon. After stirring at room temperature for 12 h, the reaction was left at 4 "C for 24 h. The white precipitate was collected and vacuum dried. The trihydrochloride salt was recrystallized by dissolving in hot

Synthesls of Bifunctional Tetraara Macrocycles

100% MeOH (65 mL) and being allowed to cool slowly to 4 OC (2.03 g, 37 %). Analytical data was indistinguishable from that described above for the product obtained via route A. 2-(4-Nitrobenzyl)-1,4,7,10-tetraazacyclododecaneNJT,”,”’-tetraacetic Acid Hydrochloride (15). Tetraazacyclododecane 14 (1.25 g, 2.87 mmol) was dissolved in H2O (15 mL) at 47 “C. The solution was neutralized with 5 M NaOH (0.625 mL) prior to adding BrCH2C02H (3.25 g, 23.7 mmol). The solution was then neutralized with 5 M NaOH, the pH stat endpoint set to pH = 8.5, and the reaction stirred for 14 h. The pH was lowered to 1.0 by the addition of concentrated HC1 and the solution applied to a AG50W-XB cation-exchange resin (H+ form, 200-400 mesh, 16 X 200 mm). The column was washed with water (ca. 500 mL) prior to elution of the amino carboxylate with 2 M NH40H. The fractions containing product were evaporated to near dryness and then applied to an AG1-X2 resin (200-400 mesh, acetate form, 20 X 200 mm). The column was washed with water (500 mL), and the product eluted with an acetic acid gradient (0-1.5 M over 2 L). Twenty-milliliter fractions were collected, with the product 2 eluted in tubes 25-38. The fractions were combined,concentrated to ca. 20 mL by rotary evaporation, and then lyophilized to give a pale yellow, fluffy solid. Residual acetic acid was removed by relyophilizing twice from 30 mL of water to give 0.8 g (1.39 mmol, 48%): MS (EI, exhaustively silylated with N,O-Bis(trimethylsily1)trifluoroacetamide/CH3CN)m/e 827 (M+),812 (M - 15). Anal. Calcd for C23H33N5010’2H20: C, 48.00; H, 6.48; N, 12.17. Found: C, 47.70; H, 6.18; N, 12.57. A portion of 15 was purified by recrystallization from aqueous HC1. Pale yellow 15 (0.7 g) was suspended in ca. 1mL of water. Approximately 6 mL of concentrated HC1 was added. The solution was heated to reflux briefly and then allowed to stand for 14 h. The resulting pale yellow crystals were collected on a fine frit and washed with concentrated HC1 and then 2-propanol before drying at 75 “C (0.1 mmHg) for 48 h (0.7 g, 95%, 46% conversion from 14): lH NMR (D20, pH = 11.2, NDdOD, ref = tBuOD) 6 8.20 (d, 2 H, J = 8.57), 7.49 (d, 2 H, J = 8-57), 3.652 (s,2 H), 3.561 (s,2 H), 3.48-3.06 (m, 11H), 3.04-2.80 (m, 6 H), 2.74-2.58 (m, 2 H); 13CNMR (D20, pH = 11.2, NDdOD, ref = CD3CN) 6 179.36, 178.71, 175.88, 175.62, 148.66, 147.15, 131.19, 124.81, 59.93, 58.65, 57.66, 57.12, 55.42, 53.61, 53.00, 52.45, 49.99, 47.45; MS (FAB, m-nitrobenzyl alcohol) m/e 540 (M + 1). Anal. Calcd for C23H33N501~~1.6HC1*0.5H20: C, 45.52; H, 5.91; N, 11.54; C1, 9.35. Found: C, 45.39; H, 5.94; N, 11.52; C1, 9.41. 2-(4-Aminobenzyl)-l,4,7,lO-tetraazacyclododecaneN,N”N”,N”-tetraaceticAcid (16). In a 25-mL threeneckflask,recrystallizedcompound15 (130mg,0.21 mmol) was dissolved in HzO (6 mL). Following the addition of 130 mg of 10% Pd/C catalyst, the flask was attached to an atmospheric hydrogenation apparatus. After hydrogen uptake ceased (ca. 5 h), the suspension was filtered (Whatman glass-fiber filter) and lyophilized to give a pure white solid (110 mg, 85%): lH NMR (D20, pH = 11.2, ND40D) 6 7.084 (d, 2 H, J = 7.82), 6.806 (d, 2 H, J = 7.81), 3.60-2.5 (m, 23 H), 2.30 (br m, 2 H); lH NMR (D20, pH = 2.0) 6 7.429 (m, 4 H), 4.13-3.87 (m, 4 H), 3.79-3.33 (m, 12 H), 3.27-2.83(m,BH),2.710(dd,lH, J=12.6,9.9);l3CNMR (D20,pH = 2, ref = TSP) 6 176.43,173.06,140.80,133.75, 131.78,126.46,58.70,58.42,58.15,57.24,56.42,54.68,54.28, 53.55, 52.92, 52.37, 50.05, 49.36; MS (FAB, thioglycerol/ glycerol) m/e 510 (M + H), 532 (M + Na), 554 (M + 2Na - H); analytical HPLC t~ = 8.42 min. Anal. Calcd for C23HaaNsOs-2HCl.2H20: C, 44.66; H, 6.68; N, 11.32. Found C, 44.75; H, 6.74; N, 11.37. 2-(4-Isothiocyanatobenzyl)-1,4,7,lO-tetraaza-

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cyclododecane-N,N,lV,N“’-tetraaceticAcid (2). In a 2.5-mL polypropylene vial, compound 16 (35 mg, 0.57 pmol) was dissolved in H20 (250 pL). A 1M solution of thiophosgene in CHCl3 (77 pL) was added and the twophase reaction shaken vigorously for 30 s and then stirred for 2 h. The aqueous phase was removed via pipet and the CHCl3 washed with H20 (3 X 200 pL). The combined aqueous washings were lyophilized to give a pale yellow solid (34 mg, 86%1: IR (Nujol) 2080 cm-l; lH NMR (D20, pH = 1.3) 6 7.416 (s,4 H), 4.46-3.34 (m, 17 H), 3.32-2.70 (m, 8 H); MS (FAB, thioglycerol/glycerol) m/e 552 (M + H), 574 (M + Na); analytical HPLC t~ = 17.79min. Anal. Calcd for C24H33N50~S.2HC1.4H20: C, 41.38; H, 6.22 N, 10.05. Found: C, 41.59; H, 6.04; N, 10.28. 2-(4-Nitrobenzyl)-1,4,7,10-tetraazacyclododecaneN,N,N”,1\P”-tetra(l-14C)acetic Acid Hydrochloride (15-W). The synthesis of 14C-labeled2 was performed as described above for 15 by alkylating 14 (0.63 g, 1.44 mmol) with (lJ4C) bromoacetic acid (2.0 mCi) prior to adding nonradioactive bromoacetic acid (1.36 g, 9.92 mmol). Compound 15-14Cwas purified by ion-exchange chromatography and recrystallized from HCl as described above. The specific activity of the chelate was determined by dissolving a known weight of chelate in a volumetric flask, calculating the exact concentration by spectrophotometry (E = 8260 L mol-’ cm-l, pH = 6.2 MesC1, determined for analytically pure 151,and averaging the counts of three 50-pL aliquots: 3.51 X lo6 dpm/pmol. 2-(4-Aminobenzyl)- 1,4,7,1O-tetraazacyclododecaneNJT,N’,N”-tetra( l-14C)acetic Acid (16-W). Compound 15-’4c (115 mg) was dissolved in H20 (5 mL) and hydrogenated as described above for the preparation of 16: analytical HPLC t~ = 8.23 min (purity > 95%). 2- (4-Isot hiocyanatobenzy1)-1,4,7,10- t e t r a a z a cyclododecane-NJTJV”JV”-tetra(lJ4C)aceticAcid (2W). In a 2-mL polypropylene vial, compound 16-l4C(37 mg) was dissolved in H2O (0.5 mL). Thiophosgene (109 pL of a 1M CHC13 solution) was added and the reaction shaken vigorously for 30 s and then stirred at room temperature for 2 h. The product was isolated as described above for the preparation of 2: analytical HPLC t~ = 17.76 min (purity > 96%). 1,4-Bis( tert-butoxycarbonyl)-9-(4-nitrobenzyl)7,12-dioxo-1,4,8,1 l-tetraazacyclotetradecane(17). A 5-L three-necked round bottom flask was fitted with a condenser, two 0.5-L water-jacketed Normag addition funnels, one of which was heated to 60 “C, and an argon inlet. The flask was charged with anhydrous 1,4-dioxane (ca. 3.5 L) and heated to 90 “C. Diester 9 (8.97 g, 15mmol) was dissolved in hot anhydrous DMSO (120 mL) and transferred into the heated addition funnel. In anhydrous DMSO (100 mL), 4 (4.02 g, 15 mmol) was dissolved and treated with triethylamine (4.17 mL, 30 mmol). The volume of the solution was adjusted to 120 mL with dry DMSO and transferred into the second addition funnel. The two reactants were added dropwise over ca. 8 h and the reaction was stirred for 18 h. An identical set of additions was made to the heated solution on each of the following two days, 45 mmol total in each reactant. After cooling, the reaction solution was concentrated to about 0.5 Land transferred to a separatory funnel. Water (200 mL) and CHC13 (400 mL) were added and the layers were separated. The organic layer was then extracted with 1N HCl(3 X 300 mL), saturated NaCl solution (3 X 300 mL), 5% NaHC03 (3 X 300 mL), and saturated NaCl solution again (3 X 300 mL). After drying over MgS04 and filtering, the solution was concentrated to about 150 mL. One-third of the solution was loaded onto a 3.7 X 50 cm silica column in CHC13. The product was eluted with

112

Bloconjugate Chem., Vol. 3, No. 2, 1992

1% MeOH in CHCl3. The remaining two-thirds of the crude product solution was similarly chromatographed. The purified product solutions were combined and concentrated to a light yellow solid (11.14g, 44%): TLC on silica (5% MeOH/CHCl3) Rf = 0.20;‘H NMR (CDC13) 6 8.072 (d, 2 H, J = 8.5),7.324 (d, 2 H, J = 8.5),7.05 (br m, 2 H), 4.101 (m, 1 H), 3.70-3.20 (m, 8 H), 2.98 (m, 4 H), 2.751 (dd, 1H, J = 14.0,7.0),2.35 (m, 4 H), 1.387 (e, 9 H), 1.367( ~ ,H); 9 ‘3C NMR (CDC13) 6 172.89,171.27,154.99, 154.65,145.96,144.39,129.09,122.77,79.31,79.15,52.06, 48.06,47.36,44.85,44.15,42.19,37.90,36.16,35.88,27.45; MS (CI/NH3) m/e 564 (M 1). Anal. Calcd for C27H41N508: C, 57.53;H, 7.33;N, 12.42.Found: C, 57.60; H, 7.32;N, 12.33. 2-(4-Nitrobenzyl)-194,8,1 l-tetraazacyclotetradecane Tetrahydrochloride (18). Diamide 17 (5.0g, 8.88mmol) was added to anhydrous 1,4-dioxane (200mL) previously saturated with HCl(g) and stirred under argon for 18 h. Diethyl ether (100mL) was added and the flask was held at 4 “C for 4 h. The solid was filtered off and vacuum dried for 18 h. The dried solid was washed into a three-necked round bottom flask with THF (150mL). The flask was cooled in an ice bath and BHeTHF (1 M, 62 mL) was injected. The reaction was heated at 50 “C for 48 h after which additional borane (10mL) was added. After 48 h, the solution was cooled, MeOH (100mL) was added, and the solution was rotary evaporated to dryness. The solid was taken up in 100% EtOH (110 mL) and saturated with HCl(g) while cooling in an ice bath. The solution was then refluxed for 4 h under argon and left stirring at room temperature for 18 h. The flask, now containing a fine white precipitate, was stoppered and held at 4 “C for 18 h. The solid was collected, washed with ether, and vacuum dried (2.88g, 66%): ‘H NMR (DzO, pH = 1.0)6 8.251 (d, 2 H,J = 8.5),7.569 (d, 2 H, J = 8.51, 3.77 (m, 1 H), 3.55-2.96(m, 17 H), 2.20-1.92(m, 4 H); 13C NMR (D20, pH = 1.0,ref = CD3CN) 6 146.43,139.84, 129.70,123.31,52.22,42.61,42.36,41.04,40.79,40.15,38.34, 37.95,34.34,18.82,18.48;MS (CI/NH3) mle 336 (M + 1). Anal. Calcd for C ~ ~ H ~ ~ N ~ O T ~ H C ~C,. 41.65; O . ~ HH, ~O: 6.99;N, 14.28;C1, 28.95. Found: C, 41.50;H, 6.96;N, 14.02;C1, 28.79. 2-(4-Nitrobenzyl)-l,4,8,1 l-tetraazacyclotetradecane-N,h”JV”JV”’-tetraaceticAcid (19).Tetraamine 18 (0.5 g, 1.02 mmol) and NaZC03 (0.492g, 4.64 mmol) were added to a 50-mLflask with DMF (20mL) and heated to about 80 “C after which tert-butyl bromoacetate (1.04 g, 5.3mmol) was added. The reaction was heated for 18, h and after cooling, the solution was poured into a separatory funnel with CH2Clz (60mL). The solution was extracted with water (3 X 100 mL), separated, dried over MgSO4, filtered, and concentrated under high vacuum to a thick dark oil. The oil was stirred with trifluoroacetic acid (20mL) for 18h under argon. The solution was rotary evaporated and the residue dissolved in water (25 mL). The solution was loaded onto a column of AG50W-X8 cation-exchange resin (H+ form, 200-400 mesh, 2.6 X 15 cm) and washed with water until the eluant was neutral. The crude product was then eluted from the column with 2M NH4OH (1L). The solution was taken to dryness and the solid was redissolved in water (5mL) and then loaded onto an AG1-X8 anion-exchange column (acetate form, 200-400 mesh, 1.2 X 30 cm). The column was washed with water to collect 11 fractions (18 X 150 mm tubes). The column was then eluted with a 2-L gradient of acetic acid, 0.0-2.0M. Fractions 60-82 were combined and upon concentration a white precipitate formed. After the total volume was reduced to about 10 mL, the flask was left at 4 “C for 1 week. The resulting fine white power was collected on a small Hirsh funnel and then dried at 90 “C

+

McMurry et el.

(0.01mmHg) for 18 h (178mg, 29% 1: ‘H NMR (DzO, pH = 11.5) 6 8.195 (d, 2 H, J = 8.5),7.499 (d, 2 H, J = 8.5), 3.38-3.04 (m, 8 H), 2.94-2.28 (m, 17 H), 1.84-1.56 (m, 4 H); l3C NMR (DzO,pH = 11.5,ref = TSP) 6 183.03,181.82, 152.54,148.72,133.05,126.51,62.39,62.15,61.97,58.55, 56.54,54.44,54.32,54.05,52.28,52.13,51.49,35.79,27.13, 26.42;MS (FAB, glycerol/thioglycerol) m/e 568 (M + 1). Anal. Calcd for C ~ ~ H ~ ~ N ~ O ~C,V49.75; ~ H H, Z O6.85; : N, 11.60. Found: C, 49.46;H, 6.59;N, 11.09. 2-(4-Aminobenzyl) - 1,4,8,1l-tetraazacyclotetradecane-N,N,N”JV”-tetraaceticAcid (20). Tetraacid 19 (73mg, 0.121mmol) was suspended in HzO (2mL) and 1 drop of concentrated NH40H was added to affect a clear pale yellow solution. The solution was added to a suspension of 10% Pd/C in HzO (3mL) presaturated with Hz attached to an atmospheric hydrogenation apparatus. After Hz uptake had ceased, the catalyst was filtered off with a fine frit and Celite 577. The filtrate was lyophilized to a fine white powder (68mg, 98% 1: ‘H NMR (DzO, pH = 1.0)6 7.460 (d, 2 H, J = 8.5),7.395(d, 2 H, J = 8.5), 4.20-2.90 (m, 24 H), 2.735 (dd, 1 H, J = 13.5,8.5),2.15 (m, 2 H), 1.95 (m, 2 H); (D20, pH = 13.0)6 7.103 (d, 2 H, J = 9.0),6.817(d, 2 H, J = 9.0),3.40-2.40 (m, 22 H), 2.24 (m, 3 H), 1.70(m, 4 H); 13CNMR (D20, pH = 13.0,ref = CD3CN) 6 181.01,180.14,179.79,132.16,144.89,130.88, 117.39,60.58,60.39,60.17,56.32,55.32,52.77,52.55,50.85, 49.35,31.68,25.70,24.85;MS (FAB, thioglycerol/glycerol) mle 538 (M + 1); analytical HPLC t R = 9.35 min (purity > 98%). 2-(4-Isothiocyanatobenzyl)-1,4,8,1l-tetraazacyclotetradecane-N,N’,iV’,N”’-tetraaceticAcid (3). Aniline 20 from above was dissolved in H2O (2mL), and thiophosgene (20 pL, 0.26 mM) in CHCl3 (10 mL) was added in one portion with maximum stirring. After 2 h, the organic phase was removed by rotary evaporation without heating. The aqueous residue was lyophilized to leave an off-white powder (57.5 mg, 78%): IR (Nujol) 2110 cm-l; ‘H NMR (D20, pH = 3.5) 6 7.297 (s, 4 H), 3.8-2.5 (m, H), 1.70 (m, 4 H);MS (FAB, thioglyceroll glycerol) m/e 580 (M + 11,602(M + Na); analytical HPLC t~ = 17.99 min (purity > 98%). 2-(4-Nitrobenzyl)-1,4,7,10-tetraazacyclotetradecane-N,W,N”,N”’-tetra(1-W)aceticAcid (19-W). A modified procedure for the preparation of 19 was followed. Tetraamine 18 (0.86g, 1.79mmol) and Na2C03 (1.7g, 16 mmol) were combined in DMF (20 mL) and heated to ca. 80 “C. Tertiary butyl bromo(l-14C)acetate (Amersham, 2 mCi, 12 mCi/mmol) was added with two l-mL rinses of DMF for each ampoule. After 2 h, tertbutyl bromoacetate (1.56g, 8 mmol) was added and the reaction was continued for 18 h. After cooling to room temperature, the solution was extracted into CHzClz (30 mL) and washed with water (2 X 100 mL). The organic phase was dried over MgS04 and filtered into a Schlenk flask. The contents were then frozen in liquid nitrogen. The flask was then attached to two liquid nitrogen traps in line and the system was evacuated. After disconnecting the vacuum system, the frozen material was allowed to reach room temperature and eventually was heated gently to transfer any volatile radioactive residue to the traps. With the transfer completed, trifluoroacetic acid (25mL) was added to the contents of the Schlenk flask and the solution was stirred for 18 h. The solution was concentrated to a thick oil, dissolved in HzO, and loaded onto an AG50W-X8 cation-exchange column (H+ form, 200-400 mesh, 26 X 300 mm). The column was washed with water until the eluant was neutral. The crude product was then eluted from the column with 1 L of 4 M NH40H and the solution was rotary evaporated to leave a solid. The solid was taken up in water and loaded onto an AGl-X8anion-

Synthesis of Bifunctional Tetraaza Macrocycles

exchange column (acetate form, 200-400 mesh, 1.6 X 30 cm). The column was washed with water and twenty fractions (ca. 20 mL) were taken, after which the column was eluted with a 0.0-2.0 M HOAc gradient (2 L). Fractions 60-82 were combined and concentrated to 50 mL, during which a fine white precipitate formed. The flask was stoppered and left at 4 "C for 48 h. The product was collected on a small Hirsh funnel, transferred to a vial, and dried for 18 h (0.02 mmHg, 90 "C). The specific activity was determined by counting three aliquota (100,200, and 300 pL) of a solution of the ligand (pH = 7.4,O.l M Po43-buffer, 0.05% NaNd with the 14C standards and correcting for quenching. The concentration of the solution was determined via UV (e = 7730 L mol-1 cm-1). The averaged result was 2.72 X lo6 dpm/ mol. Analytical HPLC t~ = 11.95 min (purity = 95%). 2-(4-1sothiocyanatobenzy1)-1,4,8,1l - t e t r a a z a cyclotetradecane-N","N , "-et'rta( 1-W)acetic Acid (3-W). Compound 19-'4c (52 mg) was dissolved in H2O (3mL) and hydrogenated as described for the preparation of 20. Compound 20-l4C (45 mg) was taken up in H20 (3 mL) and thiophosgene (15 mL in 10 mL of CHC13) was added with vigorous stirring. The stirring was continued for 2 h at room temperature and the product was isolated as described for 3. 1,4y7-Triaza-3(2)-(4-nitrobenzyl)-7-( tert-butoxycarbonyl)-9-oxobicyclo[4.3.0]-4-nonene(21a and 21b). In a50-mL volumetric flask, 4 (1.5g, 5.6 mmol) was dissolved in anhydrous DMF (30 mL). Et3N (3.9 mL, 28 mmol) was added, and the volume adjusted to 50 mL with DMF. The resulting suspension was sonicated for 5 min, cooled to 4 "C, and then filtered to remove insoluble triethylamine hydrochloride. The filtrate was taken up in a 50-mL gastight syringe. Diester 11 (2.39 g, 5.6 mmol) was dissolved in anhydrous DMF (50 mL) and taken up with a 50 mL gas-tight syringe. A 5-L three-neck flask was placed under an argon atmosphere, charged with dioxane (4 L), and heated to 90 "C. The contents of the two syringes were added via an Orion Model 355 syringe pump to the efficiently stirred dioxane over an 8-h period. This addition procedure was repeated three more times over the next 48 h (22.4 mmol total). The dioxane was evaporated to an oil, which was concentrated to a thick slurry on a vacuum rotary evaporator and vacuum dried for 14 h. The residue was taken up in EtOAc (300 mL) and extracted with 5% NaHC03 (4 X 150 mL). A final extraction was performed with H2O (150 mL), using saturated NaCl to break up the resulting emulsion. The EtOAc was dried (Na2S04) and evaporated to give a brownish-orange solid. Purification was achieved using flash chromatography (230-400 mesh silica, 40 X 300 mm) and a gradient of 0-1% MeOH/CH2Cl2 (1L of CH2Cl2,2 L of 0.25% MeOH/CH2C12,2 L of 0.5% MeOH/CH2C12,2 L of 0.75% MeOH/CH2C12,1L of 1% MeOH/CH2C12). The fractions containing product were evaporated to give a 1 0 1 mixture of 21a and 21b (pale yellow foam, 17.6 g, 4.7 mmol, 21 7%): MS (CI/NH3) m/e 375 (M + HI, 319 (M - CHzC(CH3)2+ H), 275 (M BOC + H). Anal. Calcd for ClaH22N405: C, 57.75; H, 5.92; N, 14.96. Found: C, 57.30; H, 5.92; N, 14.79. Separation of 21a from 21b was achieved using HPLC (Waters 10 pm, 125 A, 19 X 300 mm silica, flow = 17 mL/ min) and a 25-min linear gradient of 25 % CHzCl$hexane to 100% IPA. Fraction 21a eluted at 15.7 min and was evaporated to give 1.4 g of pure 1,4,7-triaza-3-(4-nitrobenzyl)-7-(tert-butoxycarbonyl)-9-oxobicyclo[4.3.Ol-4-nonene (21a): lH NMR (dmso-d6) 6 8.235 (d, 2 H, J = 8-57), 7.633 (d, 2 H, J = 8.571, 4.507 (m, 1 H), 4.401 (d, 2 H, J = 9.94h4.145 (d, 2 H, J = 18.6),3.816 (dd, 1H, J = 10.4, 11),3.450 (dd, 1H, J = 6.7, 11.2), 3.050 (d, 2 H, J = 6.91,

Bloconlugate Chem., Voi. 3, No. 2, 1992

113

1.472 ( ~ , H); 9 13CNMR (CDC13) 6 164.34,154.16,153.36, 147.09, 145.03, 130.20, 123.80, 82.09, 65.91, 47.27, 47.03, 41.71,41.61,28.25; MS (CI/NH3) m/e 375 (M H), FAB (CHCl$m-N02BzOH) m/e 375 (M + H), 319 (M - CH2C(CH3)2 + H), (E11 m/e 375 (M + H), 374 (M+),301 (M O(CH3)3), 238 (M - CH2PhN02), 182 (M - CHzPhNOz CH2C(CH3)2). Anal. Calcd for CleH22N405: C, 57.75; H, 5.92; N, 14.96. Found: C, 57.13; H, 6.04; N, 14.61. Fraction 21b eluted at 17.5 min and was evaporated to give 0.16 g of 1,4,7-taiaza-2-(4-nitrobenzyl)-7-(tert-butoxycarbonyl)-9-oxobicyclo[4.3.0]-4-nonene(21b): 1H NMR (dmso-d6) 6 8.105 (d, 2 H, J = 8.671, 7.289 (d, 2 H, J = 8.67), 4.545 (m, 1H), 4.38-4.00 (m, 4 H), 3.858 (dd, 1H, J = 9.59,15.66), 3.646 (dd, 1H, J = 4.85,15.66), 3.330 (dd, 1H, J = 3.12, 13.41, 2.901 (dd, 1H, J = 8.83, 13.4), 1.472 ( ~ , H); 9 13CNMR (CDCl3)6 164.75,153.95,153.34,147.25, 143.74, 130.29, 123.78, 82.09, 58.89, 56.63, 47.26, 41.80, 41.69,38.23, 28.25; MS (CI/NH3) m/e 375 (M H), 319 (M- CH2C(CH3)2 + H), 275 (M - BOC + H). Anal. Calcd for ClaH22N40~0.5H20:C, 56.39; H, 6.05; N, 14.29. Found C, 56.39, H, 6.05, N, 14.61. 1,4,7-Triaza-3- (4-nitrobenzyl)-9-oxobicyclo[4.3.014-nonene Hydrochloride (22). In a 250-mL Schlenk flask, the mixture of 21a + 21b (1.1g, 2.94 mmol) was dissolved in 30 mL of anhydrous dioxane under argon. Dioxane saturated with HC1 gas (30 mL) was added and the reaction vessel sealed under positive argon pressure. The reaction was stirred for 14 h at room temperature, at which time a white precipitate was evident. HC1 gas and dioxane were removed by trap-to-trap distillation under vacuum. The solid residue was dried for 24 h a t 0.1 mmHg (0.95 g): IR (Nujol) 3400 (w, br), 1760 (w), 1707 (8) cm-l; lH NMR (dmso-de) 6 9.67 (br s, 3 H), 8.199 (d, 2 H, J = 8-77),7.592 (d, 2 H, J = 8.77), 4.574 (mult, 1H), 4.216 (s, 2 H), 3.935 (8, 2 H), 3.870 (dd, 1H, J = 11.1,10.5), 3.570 (s, dioxane impurity, 0.14 equiv), 3.497 (dd, 1H, J = 11.4, 6.4); 13CNMR (dmS0-d6) 6 161.20,160.91,152.63, 146.22, 146.03,144.56,130.75,130.49,123.46,123.28,66.28,64.39, 55.60,47.29,44.57,40.39,37.20; MS (CI/NH3): 275 (M + HI. N-[2-A m i n o - 3- (4 - n i t r o p he n y 1) p r o p y 1]pi per azine (23). In a 200-mL Schlenk flask, 22 (0.87g, 2.8 mmol) was suspended in anhydrous THF (50 mL) under argon. After cooling to 0 "C, 1M BH3-THF (14 mL) was added. The reaction was warmed to room temperature for 1hand then 45 "C for 2 h. The reaction was quenched by the addition of MeOH (20 mL) and evaporated to a brown oil. The oil was redissolved in MeOH (20 mL), heated to boiling, and evaporated to an oil. This procedure was repeated two more times with MeOH and once with anhydrous EtOH. The oil was dissolved in anhydrous EtOH (50 mL) and saturated with HC1 gas at 0 "C. The suspension was warmed to 40 "C for 6 h and then cooled to 4 "C. The resulting precipitate was collected on a medium frit under a blanket of argon, washed with Et20, and vacuum-dried. This procedure yielded 0.6 g of product which was converted to the free base using 40% NaOH, extracted into CHC13, and then flash chromatographed ( C H ~ C ~ Z / C H ~ O H / N H24/8/1,230-400 ~OH mesh silica, 20 X 200 mm) to give a brown oil. The oil was dissolved in absolute EtOH and precipitated with anhydrous HCl(g) to give 0.5 g of the hydrochloride salt. An additional 0.1 g of this pure material was obtained by evaporation of the original HCl/EtOH filtrate, giving a total of 0.6 g (1.53 mmol, 55%): lH NMR (D20, pH = 1.6, ref = t-BuOD) 6 8.272 (d, 2 H, J = 8-67],7.580 (d, 2 H, J = 8.67), 3.897 (m, lH),3.363(t,4H,J=5.3),3.225-3.160(m,2H),3.12-3.01 (m, 2 H), 2.94-2.84 (m, 4 H); 13C NMR (D20, pH = 1.6, CD3CN) 148.04,144.02,131.37,125.06,58.98,50.19,50.04, 43.81,37.11; MS (CI/NHa) m/e 265 (M + 1). Anal. Calcd

+

+

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Blocon]ugate Chem., Vol. 3, No. 2, 1992

for C13H20N40~-3HC1.H20: C, 39.86; H, 6.43; N, 14.30;C1, 27.15. Found: C, 40.14; H, 6.61; N, 14.05; C1, 26.95.

McMurry et al.

Scheme I 0

RESULTS AND DISCUSSION

Because poly(amino carboxylates) are readily prepared from the corresponding polyamine by alkylation with haloacetic acids or their esters, the preparation of pure macrocyclic polyamines constitutes the most crucial aspect of synthesizing these bifunctional macrocyclic poly(amino carboxylates). Representative examples of important cyclization procedures which have been used to form macrocyclic polyamines include the aminolysis of malonates with polyamines (43),high dilution acylation of diamines by acid chlorides and their derivatives (44-481, intramolecular reaction of amines with active esters (49,50),metalmediated cyclization reactions (51), the "crablike" cyclization of a bis(cr-chloroamide) with amines (521, and the cyclization of deprotonated tosylamides with tosylates or mesylates in aprotic solvents (53). The last reaction, known as the Richman-Atkins cyclization, provides an efficient and convenient means of preparing unsubstituted macrocyclicpolyamines, Yields of 40-50 % are commonly obtained for bimolecular cyclizations without resorting to high dilution. While several groups have synthesized functionalized macrocyclic polyamines employing a bimolecular Richman-Atkins cyclization step, none have incorporated the versatile aryl nitro group (5458). However, Moi et al. have described a unique intramolecular variant of this technique to prepare p-NO2Bz-DOTA, 15 (33). Our initial efforts to prepare the substituted DOTA precursor 2-(4-nitrobenzyl)tolylsulfonyl)-l,4,7,l0-tetraazacyNJv'~~-tetrakis@ clodececane by a bimolecular Richman-Atkins reaction of 3,6-bis@-tolylsulfonyl)-1,8-bis[(tolylsulfonyl)oxyl-3,6diazaoctane with N,"-disodio-N,"-bis@-tolylsulfonyl)1-(4-nitrobenzyl)ethylenediaminein dry DMF at 80 'C was not synthetically useful, and produced only trace amounts of product. In contrast, cyclization of N,"-disodidV,W-(p-tolylsulfonyl)-1-benzylethylenediaminewith

R= NO,, decomposition R= H, 45%

the same ditosylate was facile, forming 2-benzylN,"JV",iV"'-tetrakisw-tolylsulfonyl)- 1,4,7,10-tetraazacyclododecane)in 45 % yield, thus implicating the nitro group and not adverse steric interactions as the culprit in the failed cyclization attempt (59). These preliminary results prompted the search for another cyclization procedure which would allow convenient entry into nitroaryl-functionalized macrocyclic polyamines of varying ring size. The reaction of (4-nitrobenzy1)ethylenediaminewith carbamate-protected amino disuccinimido esters 7 and 9 under high-dilution conditions (90 OC, dioxane, Et3N) afforded the macrocyclic diamides 12 and 17 in 41% and 44% yield, respectively, after purification by flash chromatography or recrystallization (Scheme I). No higher molecular weight cyclic oligomerswere isolated. The ready availability of carbamate-protected amino diesters from the corresponding aminoacetic acids as well as the substituted diamine (17) makes this an efficient route for constructing C-functionalized polyaza macrocyclic rings. In contrast to some other cyclizations employing disuccinimido esters and diamines which work efficiently at 0 OC (601, reaction temperatures lower than 90 OC were found to adversely affect the yield of the macrocycles. Sheh and

7 n.1

0"

9 n.2

12 n.1, 4we 17 n-2. 44%

4

C0.H

13

'

HCI

14 "-1. 55% 18 n-2. 68%

n '

~O,H

2 n.l,34% 3 n=2.21%

Mokotoff have reported the preference for monomeric cyclotetrapeptide formation in an intramolecular pentafluorophenyl ester cyclization was enhanced at 80-90 "C (61). Deprotection of the BOC protecting groups was effected by stirring the macrocycles in anhydrous HCl(g)/dioxane for 14 h. Following removal of the solvent and drying, the resulting diamide amine hydrochloride was reduced to the corresponding amine with borane/THF. Purification of the amine was accomplished by crystallization of the hydrochloride salt. The 12-membered ring polyamine 14 was also prepared by an independent route employing the same cyclization chemistry but with a different diamine starting material. 0

w

0

5

11

13

Thus, ester 11 was reacted with 5 in dioxane at 90 "C to form the macrocyclictriamide 13 in 44 % yield. While the cyclization yield is similar to the above reaction employing diamine 4, this approach had two practical advantages for preparing the 12-membered ring tetraamine. First, ester 11 was much more soluble in a variety of solvents than ester 7, thus allowing use of a syringe pump instead of a thermostated addition funnel used for the preparation of diamide 12. Secondly, the product triamide conveniently precipitated upon concentration of the reaction solution, which facilitated purification. Conversion to the tetramine was effected uneventfully by procedures described above for the macrocyclic diamides. With the pure cyclic carbon substituted amines in hand, alkylation of the macrocyclic amines to form the poly(amino carboxylates) (62) was accomplished in one step by using BrCHzC02H (pH = 8.6) or in two steps by using BrCHzCOO-t-Bu (Na2C03/DMF) followed by deprotection with TFA. Preparation of an internally labeled 14C ligand was most conveniently performed at this step using either bromo( 1-14C)aceticacid or tert-butylbromo(l-14C)acetate, thus permitting an accurate and sensitive determination of the number of ligands ultimately attached to the mAb. Following the alkylation, the crude ligand was

Synthesis of Bifunctional Tetraara Macrocycles

Bloconjugate Chem., Voi. 3, No. 2, 1992

Scheme I1

L

J "01 i.DI.1.d

11

4

.3HCI 23

then subjected to cation-exchange chromatography to remove acid and salt (AG50W-X8 column). The organic components were then further purified by anion-exchange chromatography on an AGl-X8(2) resin. We, and others ( B )have , noted that these macrocyclic poly(amino carboxylates), particularly p-N02-Bz-DOTA (151, need to be handled carefully to avoid adventitious sequestering of divalent metals, especially calcium. For that reason, the alkylation step and subsequent steps were performed in acid-washed glassware or metal-free plasticware. In addition, 15 was recrystallized from aqueous HC1 to insure metal-free purity. Catalytic reduction of the 4-nitrobenzyl-substituted macrocycles in the presence of 10% Pd/C (HzO)proceeded to give the aniline derivatives in nearly quantitative yield. Conversion to the isothiocyanate was accomplished by vigorously stirring an aqueous solution of the aniline with thiophosgene in CHCl3. Conversion typically was complete in 1-2 h and was readily verified by HPLC (see Experimental Procedures). The product isothiocyanate was characterized by a strong stretch at 2100 cm-I in the infrared spectrum and a singlet for the aromatic protons in the 300-MHz proton NMR spectrum. MAb/bismuth(111)complex conjugates using 2 have been shown to be stable in vivo, as evidenced by indistinguishable biodistribution data for both 205/2mBi-DOTA/103A and biosynthetically labeled 35S-103A(63). Attempts to prepare a 9-membered ring triaza diamide by the reaction of 11with 4 followed an unexpected course deserving of additional comment (Scheme 11). Following aqueous workup, a product with chromatographic behavior similar to the 12- and 14-membered ring BOC-protected macrocycles was isolated and purified by flash chromatography. Proton NMR revealed the presence of a minor component 21b (ca. 103'% ), which was ultimately separated from the major fraction 21a by HPLC. Both fractions exhibited the following mass spectral features, indicating that fractions 21a and 21b were isomeric. Electron impact mass spectroscopy revealed a parent ion at m / e 374, 18 mass units less than the molecular weight calculated for the expected macrocyclic diamide product, as well as a significant ion at mle 375 attributed to self chemical ionization. Fragments consistent with loss of carbamate (M- OC(CH3)3,M - BOC) and nitrobenzyl group (M 136) were observed. In addition, both CIlNH3 and FAB mass spectra gave molecular ions at 375 (M + H)+, consistent with a molecular weight of 374 and supported by elemental analysis. Although an amide stretch was apparent in the IR (1680 cm-l),examinationofthe 'HNMRof21aand21brevealed no DzO exchangeable amide protons, suggesting a lack of secondary amides. In the carbonyl region of the carbon13 NMR, each isomer was characterized by one amide resonance (164.338 (21a)/164.749 (21b)), one carbamate resonance (154.160/153.945), and a resonance (153.355/ 153.343) not characteristic of the larger BOC-protected

115

macrocycles. Upon reaction with 1.55equiv of NaCNBH3 at pH = 6, the unique resonance disappeared, suggesting the presence of an imine bond. Consistent with this interpretation, the CI/NH3 mass spectrum of the reduced material exhibited a parent ion at m/e 377. Taken together, these results provide convincing evidence for the structure of the cyclization products as being the isomeric acyl amidines 21a and 21b. Presumably the products are derived from the initial macrocyclic diamide cyclization product by transannular acylation of an amide nitrogen, followed by dehydration of an intermediate azacyclol. Although unanticipated, literature precedence exists for the transannular reaction of lactam nitrogens with ketones (64) and amides (65, 66) in medium-ring compounds. Interestingly, the distribution of products was identical when the cyclization was performed at 45 "C, although the yield dropped to less than 1% . Our observations may offer an explanation why the "crablike" cyclizationof N,"bis(a-chloroacetamide)ethylenediaminewith benzylamine failed to give an identifiable 9-membered ring diamide, while the corresponding N-methyl tertiary amide derivative gave the anticipated triazacyclononane in 44 % yield (52).

Following deprotection with HCVdioxane,two attempts were made to reductively expand the fused ring system to the macrocyclic triamine. First, reduction of the mixture of the deprotected 21a and 21b with borane/THF converted the fused 6,Bmembered ring acylamidine into N[2-amino-3-(4-nitrophenyl)propyll piperazine, which was purified by chromatography (silica,CH&l2/CH30H/NH4OH 24/8/1) and isolated in 55% yield as a trihydrochloride salt. The structure of the triamine was confirmed by acylation with 3.3 equiv of acetyl chloride in CH& and characterization of the product diamide. The efficient production of piperazine 23 at the expense of a macrocyclic triazacyclononane suggests that reductive cleavage of the neighboring C-N bond is preferred over that of the central C-N bond for this system. Such a result would be predicted on the basis of the results obtained by Takeuchi et al. for the preparation of benzoannelated cyclic 1,5 diamines from fused ring quinazolinones (67). In that work, the moderate yield of a medium-ring cyclic diamine was explained by the high strain energy of the resulting 11membered ring. A second attempt to reductively cleave the central C-N bond using DIBAH (68) in refluxing toluene resulted in the formation of many products and was therefore deemed not useful for synthetic purposes. Thus, a noteworthy limitation of the synthetic route described herein is the inability to prepare medium-ring diamides due to the formation of fused-ring acylamidines under the reaction conditions employed. However, larger ring macrocyclic diamides are prepared in good yield from readily accessiblestarting materials and are easily purified, thus providing an efficient means of preparing the bifunctional macrocyclic poly(aminocarboxylates)p-NCSBz-DOTA (2) and p-NCS-Bz-TETA (3). The macrocyclic chelating agents described here have been synthesized in sufficient quantity and high purity to permit the study of the thermodynamic and kinetic properties of the metal complexes of interest, as well as biological evaluation of the mAb conjugates. ACKNOWLEDGMENT

We thank Dr. Henry Fales for insightful discussions concerning the structure and mass spectrum of compound 21. LITERATURE CITED (1) Parker, D. (1990) Tumor targeting with radiolabeled macrocycle-antibody conjugates. Chem. SOC.Rev. 19, 271-291.

118 Bloconlugete Chem., Vol. 3, No. 2, 1992

McMurry et

81.

(2) Pressman, D., and Kieghtley, G. J. (1948)The zone of activity

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tetraazacycloalkane-N,”,N”,N”’-tetraacetic acids as a function of ring size. Angew. Chem. Znt. Ed. Engl. 15, 686. (23) Stetter, H., Frank, W., and Mertens, R. (1981) Tetrahedron 37,767-772. (24) Cram, D. J., Kaneda, T., Helgeson, R. C., Brown, S. B., Knobler, C . B., Maverick, E., and Trueblood, K. N. (1985) Host guest complexation. 35. Spherands, the first completely preorganized ligand systems. J. Am. Chem. SOC.107, 3645. (25) Hancock, R. D., and Martell, A. E. (1988) The chelate, cryptate, and macrocycliceffects. Comments Znorg. Chem. 6 , 237. (26) Weighardt, K., Bossek, U., Chaudhuri, P., Herrmann, W., Menke, B. C., and Weiss, J. (1982) 1,4,7-triazacyclononaneN,N”N”-triacetate (TCTA), a hexadentate ligand for divalent and trivalent metal ions. Crystal structures of [ C P (TCTA)], [FeTTCTA)I, and N~[CU~~(TCTA)I-~N~B~.~H~O. Znorg. Chem. 21, 4308-4314. (27) van der Merwe, M. J., Boeyens, J. C. A., and Hancock, R. D. (1983) Optimum ligand hole sizes for stabilizing nickel(111). Structure of the nickel(II1) complex of 1,4,7-triazacyclononane-N,”,N”-triacetate. Znorg. Chem. 22, 3489-3990. (28) Broan, C. J., Cox, J. P. L., Craig, A. S., Kataky, R., Parker, D., Harrison, A., Randall, A. M., and Ferguson, G. (1991) Structure and solution stability of indium and gallium complexesof 1,4,7-triazacyclononanetriacetate and of yttrium complexes of 1,4,7,10-tetraazacyclododecanetetraacetateand related ligands: kinetically stable complexesfor use in imaging and radioimmunotherapy. X-ray molecular structure of the indium and gallium complexesof 1,4,7-triazacyclononane-1,4,7triacetic acid. J. Chem. SOC.Perkin Trans. 2, 87-99. (29) Loncin, M. F., Desreux, J. F., and Merciny, E. (1986) Coordination of lanthanides by two polyamino polycarboxylic macrocycles: Formation of highly stable lanthanide complexes. Znorg. Chem. 25,2646-2648. (30) Cacheris, W. P., Nickle, S. K., and Sherry, A. D. (1987) Thermodynamic study of lanthanide complexes of 1,4,7-triazacyclononane-N,”,N”-triacetic acid and 1,4,7,10-tetraazacyclododecane-N,”,N”J?”’-tetraacetic acid. Znorg. Chem.26, 958-960. (31) Riesen,A.,Zehnder, M., andKaden,T. A. (1986)219. Metal complexes of macrocyclic ligands. Part XXIV. Binuclear complexes with tetraazamacrocycle-N,”,N”J?”’-tetraacetic acids. Helu. Chim. Acta 69, 2074-2080. (32) Moi, M. K., Yanuck, M., Deshpande, S. V., Hpoe, H., DeNardo, s.J., and Meares, C. F. (1987) X-ray crystal structure of a macrocyclic copper chelate stable enough for use in living systems. Copper(I1) dihydrogen 6-@-nitrobenzy1)-1,4,8,11tetraazacyclotetradecane-1,4,8,1l-tetraacetate.Znorg. Chem. 26,3458-3463. (33) Moi, M. K., Meares, C. F., and DeNardo, S. J. (1988) The peptide way to macrocyclic bifunctional chelating agents: Synthesis of 2-@-nitrobenzyl)-1,4,7,lO-tetraazacyclododemeN,”,N”,N”’-tetraacetic acid and study of its yttrium(II1) complex. J. Am. Chem. SOC.110, 6266-6267. (34) Cox, J. P. L., Craig, A. S., Helps, I. M., Jankowski, K. J., Parker, D., Eaton, M. A. W., Millican, A. T., Millar, K., Beeley, N. R. A,, and Boyce, B. A. (1990) Synthesis of C- and N-functionalized derivatives of 1,4,7-triazacyclononane-1,4,7triyltriacetic acid (NOTA), 1,4,7,10-tetra-azacyclododecane1,4,7,10-tetrayltetra-acetic acid (DOTA), and diethylenetriaminepenta-acetic acid (DTPA): Bifunctional complexing agents for the derivatisation of antibodies. J.Chem. SOC.Perkin Trans. 1 2567-2576. (35) Kline, S. J., Betebenner, D. A., and Johnson, D. K. (1991) Carboxymethyl-substituted bifunctional chelators: Preparation of aryl isothiocyanate derivatives of 3-(carboxymethyl)3-azapentanedioic acid, 3,12-bis(carboxymethyl)-6,9-dioxa3,12-diazatetradecanedioic acid, and 1,4,7,10-tetraazacyclododecane-N,”,N”,N”’-tetraacetic acid for use as protein labels. Bioconjugate Chem. 2, 26-31. (36) McCall, M. J., Diril, H., and Meares, C. F. (1991)Simplified method for conjugating macrocyclic bifunctional chelating agents to antibodies via 24misothiolane. Bioconjugate Chem. 1, 222-226. (37) Moi, M. K., Meares, C. F., McCall, M. J., Cole, W. C., and DeNardo, S. J. (1985) Copper chelates as probes of biological

Synthesis of Blfunctlonal Tetraara Macrocycles systems: stable copper complexes with a macrocyclic bifunctional chelating agent. Anal. Biochem. 148, 249-253. (38) Morphy, J. R., Parker, D., Alexander, R., Bains, A., Carne, A. F., Eaton, M. A. W., Harrison, A., Millican, A,, Rhind, S. K., Titmas, R., and Weatherby, D. (1988) Antibody labelling with functionalized cyclam macrocycles. J.Chem. SOC.Chem. Commun. 156-158. (39) Deshpande, S. V., DeNardo, S. J., Kukis, D. L., Moi, M. K., McCall, M. J., DeNardo, G. L., and Meares, C. F. (1990) Yttrium-90-labeled monoclonal antibody for therapy: Labeling by a new macrocyclic bifunctional chelating agent. J. Nucl. Med. 31, 473-479. (40) Kumar, K., Magerstadt, M., and Gansow, 0. (1989) Lead(11) and bismuth(II1) complexes of the polyazacycloalkaneN-acetic acids nota, dota, and teta. J. Chem. SOC.,Chem. Commun. 3, 145. (41) Gansow, 0. A.,Atcher, R. W., Link,D. C.,Friedman, A. M., Seevers, R. H., Anderson, W., Scheinberg, D. A., and Strand, M. Radionuclide Generators: New Systems for Nuclear Medicine Applications. In ACS Symposium Series #241 (F. F. Knapp, Jr., and T. A. Butler, Eds.) pp 215-227, American Chemical Society, Washington, DC. (42) Buhr, J. D. (1978) Ph.D. Dissertation, Stanford University. (43) Tabushi, I. (1977) Preparation of C-alkylated macrocyclic amines. Tetrahedron Lett. 12, 1049-1052. (44) Stetter, H., and Marx, J. (1957). Liebigs Ann. Chem. 59, 607. (45) Stetter, H., and Mayer, K.-H. (1961). Chem. Ber. 94,14101416. (46) Vellaccio, F., Jr., Penzar, R. V., and Kemp, D. S. (1977)The reaction of dialkylmalonyl dichlorides with 1,3-diaminopropanes: a new route to macrocyclic polyamides and polyamines. Tetrahedron Lett. 6, 547-550. (47) Nagao, Y., Seno, K., Miyasaka, T., and Fujita, E. (1980) Monitored aminolysis of 3-acylthiazolidine-2-thione:a new synthesis of macrocyclic amides. Chem. Lett. 159-162. (48) Uoto, K., Tomohiro, T., and Okuno, H. (1990) Preparation of large macrocyclic tetraamines consisting of a methylene backbone and a cyclophane type skeleton. J. Heterocycl. Chem. 27,893. (49) Schmidt, U., Griesser, H., Lieberknecht, A., and Talbiersky, J. (1981)A novel method for preparation of ansapeptides, synthesis of model peptide alkaloids. Angew. Chem. Znt. Ed. Engl. 3, 280-281. (50) Schmidt,U., Lieberknecht, A., Griesser,H., Utz,R.,Beuttler, T., and Bartkowiak, B. (1986) Amino acids and peptides, 55. Synthesis of biologically active cyclopeptides, 7. Total synthesis of chlamydocin. Synthesis 361-366. (51) For example, see: Barefield, K. E., Wagner, F., and Hodges, K. D. (1976) Synthesis of macrocyclic tetramines by metal ion assisted cyclization reactions. Znorg. Chem. 15, 1370-1377. (52) Krakowiak, K. E., Bradshaw, J. S., and Izatt, R. M. (1990) Preparation of a variety of macrocyclic di- and tetraamides and their peraza-crown analogs using the crab-like cyclization reaction. J. Heterocycl. Chem. 27, 1585. (53) Richman, J. E., and Atkins, T. J. (1974) Nitrogen analogs of crown ethers. J. Am. Chem. SOC.96, 2268-2270. (54) Craig, A. S., Helps, I. M., Jankowski, K. J., Parker, D., Beeley, N. R. A., Boyce, B. A., Eaton, M. A. W., Millican, A. T., Millar, K., Phipps, A., Rhind, S. K., Harrison, A,, and Walker, C. (1989) Towards tumor imaging with indium-111 labeled macrocycle-antibody conjugates. J.Chem. SOC.Chem. Commun. 794-796.

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(55) Cox, J. P. L., Jankowski, K. J., Kataky, R., Parker, D., Beeley, N. R. A., Boyce, B. A., Eaton, M. A. W.,Millar, K., Millican, A. T., Harrison, A., and Walker, C. (1989) Synthesis of a kinetically stable yttrium-90 labelled macrocycle-antibody conjugate. J. Chem. SOC.Chem. Commun. 797-798. (56) Deutsch, J., Gries, H., Conrad, J., and Weinmann, H.-J. (1988) International Patent Application, WO 88/08422,1988. (57) Marecek, J. F., and Burrows, C. J. (1986) Synthesis of an optically active spermine macrocycle, (S)-6-(hydroxymethyl)1,5,10,14,-tetraazacyclooctadecane,and its complexation at ATP. Tetrahedron Lett. 27, 5943-5946. (58) Wagler, T. R., and Burrows, C. J. (1987) Synthesis of an optically active C-functionalized cyclam: (S)-b(hydroxymethyl)-l,4,8,11-tetra-azacyclotetradecane and ita nickel(I1) complex. J. Chem. SOC.Chem. Commun. 277-278. (59) Nitro groups are known to activate aryl groups to nucleophilic attack; see: Strauss, M. J. (1970) Anionic sigma complexes. Chem. Reu. 70, 667-712. (60) Diederich, F., Dick, K., and Griebel, D. (1986) Complexation of arenes by macrocyclic hosts in aqueous and organic solution. J. Am. Chem. SOC.108,2273-2286. (61) Sheh, L., and Mokotoff, M. (1985) Cyclization studies of tetrapeptide homologs. Tetrahedron Lett. 26, 5755-5758. (62) Desreux, J. F. (1980) NMR spectroscopy of lanthanide complexesof a tetraacetate tetraaza macrocycle. Unusual conformational properties. Znorg. Chem. 19, 1319-1324. (63) Reugg,C. L., Anderson-Berg,W. T., Brechbiel,M. W., Minadeh, S. M., Gansow, 0. A., and Strand, M. (1990) Improved in vivo stability and tumor targeting of bismuth-labeled antibody. Cancer Res. 50,4221-4226. (64) Cohen, L. A., and Witkop, B. (1955)Transannular reactions of peptides. The peptide nitrogen in a 10-membered ring. J. Am. Chem. SOC.6595-6600. (65) Shemyakin, M. M., Antonov, V. K., Shkrob, A. M., Shchelokov, V. I., and Agadzhanyan, Z. E. (1965) Activation of the amide group by acylation. Tetrahedron 21,3537-3372. (66) Antonov, V. K., and Shemyakin, M. M. (1965) Further data on aminoacyl incorporation in peptides. Acta. Chim. Hung. Tomus 44,93-98. (67) Takeuchi, H., Matsushita, Y., and Eguchi, S. (1991) Novel ring enlargement of lactams via quinazolinone annelation. A facile route to benzoannelated large-membered cyclic 1,5-diamines. J. Org. Chem. 56, 1535-1537. (68) Yamamoto, H., and Maruoka, K. (1981) Regioselectivecarbonyl amination using diisobutylaluminum hydride. J.Am. Chem. SOC.103,4186-4186. Ragistry No. 2, 127985-74-4;2J4C isomer, 138878-33-8;3, 138878-17-8;3J4C isomer, 138878-35-0;4,105359-57-7; 6,5304903-9; 7, 53049-04-0; 8, 138878-18-9;9, 138878-19-0; 10, 5607420-5; 11,123317-48-6;12,138878-20-3;13,138878-21-4;14,13887822-5; 14 base, 135825-00-2; 15, 138878-23-6; 15-14C isomer, 138878-32-7; 16, 123317-52-2; 16-14C isomer, 138898-85-8; 17, 138878-24-7; 18, 138878-25-8; 19, 138878-26-9; 19-14C isomer, 138878-34-9;20,138878-27-0;21a, 138878-28-1;21b, 138878-316; 22,138878-29-2;23,138878-30-5;Nfl-ethylenediaminediacetic acid, 5657-17-0; bromoacetic acid, 79-08-3; a-[[(tert-butoxycarbonyl)oxy]imino]-2-phenylacetonitrile,80994-44-1; 1-[3(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride, 25952-53-8;ethylenediamine-Nfl-dipropionicacid dihydrochloride, 32705-91-2; iminodiacetic acid, 142-73-4; N-hydroxysuccinimide, 6066-82-6.