Bimoqilugete Chem. lSQ1, 2, 187-194
Backbone-Substituted DTPA Ligands for
107
Radioimmunotherapy
Martin W. Brechbiel’ and Otto A. Gansow* Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. Received November 12, 1990 Four new bifunctional diethylenetriaminepentaaceticacid (DTPA) ligands were synthesized to provide an improved chelating agent for radioimmunotherapy with The new DTPA ligands contained a 4-isothiocyanatobenzyl group (pSCNBz) substituted onto the carbon backbone of DTPA for use in linkage to immunoprotein. Methyl groups were strategically incorporated onto the backbone of the ligands via a peptide route to provide 2-pSCNBz-5-Me-DTPA (2) and 3-Me-6-pSCNBz-DTPA (3). Addition of these functionalities was expected to sterically hinder the release of radiometal from the chelate. A new monosubstituted ligand, 3-pSCNBz-DTPA (41, was also prepared in order to determine whether a shift in position of the linking group had an effect on the in vivo stability of the yttrium complex. Additionally, by modification of known methods, a disubstituted DTPA ligand, 2-pSCNBz6-Me-DTPA (l),was prepared.
INTRODUCTION The radiolabeling of monoclonal antibodies for radioimmunoimaging (RII)’ and RIT is receiving increasing attention (1-4). Isotopes of iodine are currently being exploited primarily due to the convenience of protein halogenation (5,6). However, their less than optimal physical characteristics and half-lives together with problems arising from in vivo dehalogenation (7)limit the usefulness of 1811or laa1in RIT applications (8, 9). lZsI possesses many favorable attributes for RII, but lack of availability continues as a limiting factor. Metallic radionuclide complexes of ligands 1-111 have been used to expand the available choices of radionuclides
p
NCS
C02H
I1
CO2H
I
p
NCS
CO2H
CO2H CO2H
I11
with both suitable emission characteristics and half-lives. We have previously reported (IO,11) that use of ligand I11 conjugated to mAb B72.3 and labeled with W n or was superior both in tumor imaging and in biodistribution to the frequently used I, CA-DTPA, as well as EDTA analogue 11. These results were attributed to the ability of an octadentate ligand I11 to maximize the in vivo stability of the indium complex. This view is strongly supported by the recent observation that the coordination number of indium in the [In(DTPA)”] complex is indeed eight (12).
* Address for correspondence: National Inetitutes of Health, Building 10, Rm. B3B69, Bethesda, MD 20892. 1 Abbreviations: BOC, tert-butyloxycarbonyl; CA-DTPA, DTPA dianhydride; DCC, 1,3-dicy~lohexylcarbodiimide; DCU, 1,3-dicyclohexylurea; HOBT, N-hydroxybenzotriazole; mAb, monoclonal antibody; RIA, radioimmunoassay;RII, radioimmunoimaging;RIT, radioimmunotherapy;TFA,trifluoroaceticacid.
It has been along-accepted tenet of coordination chemistry that the most stable complexes are formed when the coordination number of the metal is fully saturated by ligands (13-15). A primary concern in the use of chelates linked to mAb for therapy is that undesirable localizations in vivo of free radiometal could occur from metal complex dissociation or transchelation. This would be an especially acute problem for my, which is often proposed for use in RIT (16, 19, since the principal biological site of yttrium deposition is the cortex of the bone (18,19). Should be released from the chelate and transported to the bone, radiosensitive marrow could be damaged. Clinical reports have described the failure of CA-DTPA as a ligand for RIT in that the isotope was released from the mAb conjugate and deposited on the bone (20). We did not initially attempt to employ I1 since very early studies had indicated that [Y(EDTA)]‘-dissociated in vivo (21) and was in fact an excellent reagent for selective delivery of yttrium to bone surfaces (22). Herein we continue our studies of acyclic ligands to examine the importance of ligand structure features for retarding the release of yttrium in vivo. Eight is a common coordination number for Y(II1) (15),so we initially employed the octadentate bifunctional DTPA ligand 111. Recent biodistribution studies (I1,23)with 88Y-III-B72.3 and MY-111-a-TAC confirmed that ita use markedly lowered bone dose compared to similar radioimmunoconjugates formed with I or 11. However, even for protein conjugates of 111, the %ID/g in bone was measured to be higher than that seen when lllIn or lS1Iwere used in comparable experiments (11). Thus, our reported l11In 96 ID/g bone result was set as a target to be reached for obtaining a DTPA ligand suitable for RIT with my. We therefore have attempted to further improve the stability of the complex by addition of a methyl substituent on the ligand I11 carbon backbone along with the 4-nitrobenzyl group as pictured in 1-4. Such methyl substituents have been observed to increase EDTA complex stabilities (24). Since alkylation of diethylenetriamine (dien) to obtain a DTPA is facile, preparations of the desired ligands reduced to the synthesis of the appropriate dien intermediate. The only reasonable modification of our original synthesis of I11 to produce the target compounds is the substitution of 1,2-diaminopropanefor 1,2ethylenediamine in the aminolysis of methyl I-nitrophe-
Not subJectto U.S. Copyright. Published 1991 by American Chemiccri Society
Brechblel and Gansow
188 Bbconjupte Chem., Vol. 2, No. 3, 1991
Scheme I*
nylalaninate, followed by borane reduction of the resulting amino amide to give principally the dien precursor to 1 (Scheme I). We had previously reported the DTPA produced from this triamine to be 'MX-DTPA", a mixture which is now recognized to of geometric isomers (23,25), be >95% 1 (vide infra) (26). To obtain pure samples of methyl-substituted 4-isothiocyanatobenzyl-DTPAligands 2 and 3, a peptide synthesis route to the triamines was developed (Scheme 11). The requisite blocked dipeptide amides 9 and 13were prepared from commercially available precursors by diimide coupling reactions. Deprotection and amide reduction yielded the diens 10 and 14 from which 2 and 3 were obtained by well-described methods. This methodology also allowed us to investigate the importance of having the benzyl substituent in the C-2 vs the C-3 position by providing a synthesis of 4 from amino amide 17. All ligands were synthesized both with natural carbon and with 14C-labeled bromoacetate to allow precise quantitation of the number of ligands linked to proteins. This report provides details of the syntheses of the target ligands. An earlier study reported on the in vivo stability of WY-ligand-mAb conjugates of these ligands in normal mice in a set of dual label (1311) experiments (23).
O2N
3HCI 6 /C02H CHI
a
)IN>OzH
SCN
'COeH CO2H COzH
a (a) EtaN, 1,2-diaminopropane; (b) BHs-THF, EtOH/HC1; (c) BrCH&02H, KOH, (d) H2, Pd/C, C12CS.
Scheme 11.
OtBu
10: R = pNOpBz, R1 = Me 14: R = Me. R, = PNO&
EXPERIMENTAL PROCEDURES
Materials and Methods. 4-Nitrophenylalanine (Aldrich/U.S. Biochemical) and diborane (1 M BHs-THF) (Aldrich) were used as received. All other reagents were purchased from Aldrich, Fluka, or Sigma ChemicalComp. while amino acid derivatives were obtained from US. BiochemicalCorp. or Research OrganicsInc. All solvents were dried over either LiAlH4 or CaH2 at reflux and distilled as needed. Bromo-[ 14Cz]-aceticacid (40 mCi/ mmol) was obtained from Amersham Corp. and Chemsyn Science Laboratories. Thin-layer chromatography (TLC) was performed on silica gel 60 F-254 plates from EM Reagents. TLC solvent systems used were (1)CHCls/MeOH (41); (2) 1-butanol/ H20/HOAc (4l:l); (3) EtOH/NH40H (41); (4) CH&N/ H20 (30:8);( 5 ) MeOH/10% NHrOAc(aq) (1:l). Column chromatography was performed by using Florisil (Sigma) or silica gel 60,70-230 mesh ASTM (Fluka). Ion-exchange resin (AG50WX8,200-400 mesh, H+ form) was obtained from Bio-Rad. lH NMR spectra were obtained by using either the Varian 220-MHz or Nicolet NTCBOO spectrometers. Chemical shifts are reported in ppm on the 6 scale relative to TSP (DzO) or TMS (DMSO-ds/CDCls). All coupling constants (Jvalues) are reported in Hz. 13CNMR spectra were recorded on a Varian XL300 instrument. Chemical shifts are reported in ppm on the 6 scale relative to either methanol or dioxane. Infrared spectra were recorded on either a Beckman 4220 or 4240 infrared spectrophotometer. Chemical ionization mass spectra (CI-MS) were measured with a Finnegan 3000 spectrometer. Electron impact mass spectra (ELMS) were recorded on a LKB 9OOO instrument. Plasma desorption m a s spectra (PDMS)
11 : R = pNOzBz, R1 = Me 15: R = Me, R1 = pNOpBz 19: R = H, R1= pNOzBz
2: R = pSCNBz, Rl = Me 3: R = Me, R1 = pSCNBz 4: R = H, R1= pSCNBZ
a (a) TFA; (b) THF/EtOH,EtOH/HC1; (c) BrCH&OtH,KOH, (d) H2, Pd/C; (e) C12CS.
(27)were obtained on the home-made instrument residing in the Laboratory of Chemistry in the National Heart, Lung, and Blood Institute at the National Institutes of Health. A 1000-channel scintillation counter (LS5801, Beckman, Fullerton, CA) was used for quantitative determination of 14C activity. Generally, a 50-pL aliquot of a sample containing 14C was mixed with 10 mL of scintillation cocktail (Biofluor, NEN, Du Pont). Absolute disintegration rates for W were determined by appropriate corrections for background and quench factors. Elemental analyses were measured by Galbraith Laboratories. Analytical HPLC was done on a BeckmanModel 746 system controlled by a Beckman 450data system using a 4.56 mm X 15 cm Altex 5 pm ODS Cla column with a 25-min gradient of 100% aqueous 0.05 M triethylammonium acetate to 100% methanol at a flow rate of 1mL/ min. Preparative and semipreparative HPLC were performed to isolate the 4-nitrobenzyl-DTPA ligands. This purification step was initially begun with the Beckman system operating in a semipreparative mode using a 10 mm X 25 cm Altex 10 pm ODS CIScolumn at a flow rate of 3 mL/min employing the same gradient. Preparative HPLC performed with a Waters Delta Prep 3000 system using a PrepPak 500 cartridge at a 25 mL/min flow rate
Technlcal Notes
replaced the semipreparative system midway through the syntheses. The gradients employed were Waters gradient curve 3 with 100% 0.05 M triethylammonium acetate to 50% methanol over 5 min followed by Waters gradient curve 4 from the 5050 point to 100% methanol. Syntheses of Ligands. Preparation of Geometric Isomer 1. N-(2-Aminopropyl)-pnitrophenylalaninamide (5). Methyl 4-nitrophenylalanine hydrochloride (9.80 g, 37.63 mmol) in MeOH (25 mL) was treated with EtsN (6.78 mL, 45.2 mmol) to liberate the amino ester. Ether (300 mL) was added and the precipitated hydrochloride salt was removed by filtration. After removal of the solvent, the residual oil in MeOH (5 mL) was added dropwise at room temperature to freshly distilled, vigorously stirred 1,Zdiaminopropane (50 mL). After 18 h, the solvent was removed by rotary evaporation at 50 "C a t 0.01 mm of vacuum until a constant weight was attained (10.01 g, 96%). TLC of the product in solvent system 1 on silica revealed an Rf = 0.10-0.12; lH NMR (220 MHz, DzO, pH 10.0) 6 8.06 (d, 2 H, J = 7.5), 7.41 (d, 2 H, J = 7.51, 3.72 (t, 1 H, J = 8.0), 3.18-2.73 (m, 5 H), 0.918 (m, 3 H); 13CNMR (300 MHz, CDC13) 6 173.79,173.27,146.96, 146.10,130.25,123.71,56.29,46.97,46.81,46.66,41.07,21.59 (The two amide carbonyl signals, 3:l intensity, are indicative of a mixture of geometric isomers, see also ref 26); CI-MS 267 ((M + l)/z). Anal. Calcd for ClzH18N403: C, 54.53; H, 6.77; N, 21.05. Found: C, 54.33; H, 6.76; N, 20.92. 2-Methyl-6-(pnitrobenzyl)diethylenetriamine Trihydrochloride (6). Amide 5 (9.90 g, 37.2 mmol) was dissolved in THF (200 mL) and transferred to a dried three-neck round-bottom flask fitted with condenser, stopper, and septum and under an argon atmosphere. The flask was cooled under argon in an ice bath and 1 M BHyTHF (200 mL) was injected. The reaction was then refluxed for 6 h. After cooling to room temperature, MeOH (100 mL) was added slowly, and after hydrogen evolution ceased, the resulting solution was evaporated to dryness. The residue was taken up in 100% EtOH (175 mL) and the solution was saturated with HCl(g) while being cooled in a ice bath. The saturated solution was then refluxed for 6 h during which concentrated HCl(5 mL) was added if a finely divided powder was not observed. The product precipitated cleanly and after cooling to 0 "C for 6 h was collected and dried under vacuum (11.6 g, 86.3%). TLC of the product on silica gave a single spot, Rf = 0.48 using solvent system 3; lH NMR (500 MHz, DzO, pH 11.5) 6 8.172 (d, 2 H, J = 8-01, 7.475 (d, 2 H, J = 8.0),3.226 (m, 1 H), 3.0385 (9, 1H, J = 6.01, 2.9435 (dd, 1 H, J = 13.5, 5.01, 2.699 (dd, 1 H, J = 13.5, 8.51, 2.6445 (dd, 1 H, J = 11.5, 5.0), 2.533 (m, 3 H), 1.0715 (d, 3 H, J = 6.5); (500 MHz, DzO, pH 1.0) 6 8.2715 (d, 2 H, J = 8.5), 7.6105 (d, 2 H, J = 8.5), 4.138 (m, 1 H), 3.870 (m, 1H), 3.607 (dd, 1H, J = 13.5, 7.5), 3.56-3.34 (m, 4 H), 3.239 (m, 1 H), a pair of doublets for the two diastereomeric methyl groups which integrated to a total of 3 H at 1.494 (d, J = 4.0) and 1.485 (d, J = 3.5); 13C NMR (300 MHz, CDC13) 6 147.50, 146.68, 130.17, 129.99, 123.69, 123.52,58.33, 58.16,56.14, 55.99, 52.57, 52.40, 46.82, 46.63, 42.49, 21.92, 21.85 (This spec$rum is indicative of a single geometric isomer with the doubling of peaks due to the diastereomeric nature, other weak resonances of 1 5 % intensity were seen near the reported peaks; see also ref 26); CI-MS 362 ((M + l)/z). Anal. Calcd for C12H23N402C13: C, 39.85; H, 6.65; N, 15.49. Found: C, 39.99; H, 6.64; N, 15.14. 2-Methyl-6-(p-nitrobenzyl)diethyleneN,N,N,Nf,Nf-pentaacetic Acid (7). Triamine 6 (1.00 g, 2.77 mmol) was added to bromoacetic acid (1.923 g, 13.8
Bloconlugete Chem., Vol. 2, No. 3, 1991
180
mmol) in toluene a t 0 "C with 7 N KOH (4.35 mL). The reaction was stirred a t room temperature for 24 h, after which the solution was cooled in an ice bath prior to addition of more bromoacetic acid (3.846 g, 27.6 mmol) and 7 N KOH (8.69 mL). The reaction was then stirred for 7 days a t room temperature. The solution was acidified to pH 2.0 with concentrated HBr and loaded onto a 2.6 X 25 cm AG50WX8,2Oo-400mesh, H+-form,ion-exchange column. The column was washed with water until the eluant was a t neutral pH and then the crude product was eluted with 2 M NH4OH. The product was then isolated by semipreparative HPLC with a t~ = 8.28 min. A second cation-exchange column (AG50WX8,200-400 mesh, H+ form) was used to remove the HPLC buffer, leaving the pure product (648mg, 43.25 % ): 'H NMR (500 MHz, DzO, pH 1.0) 6 8.242 (t,2 H, J = 6.51, 7.602 (d, 2 H, J = 8.01, 4.30-3.70 (complexseries of signals integrating for 12 H), 3.50-3.00 (complex broad series of multiplets integrating for 6 H), a pair of doublets for the diastereomeric methyl group integrating for a total of 3 H at 1.4175 (d, J = 6.5) and 1.342 (d, J = 6.0); (500MHz, D20, pH 13.0) 6 8.170 (d, 2 H, J = 8.0), 7.450 (d, 2 H, J = 8.0),3.40-2.00 (complex series of overlapping multiplets integrating for 18 H), a pair of doublets for the diastereomeric methyl groups integrating for a total of 3 H at 0.9545 (d, J = 6.5) and 0.721 (d, J = 6.0); 'W!f-PDMS m/z (M + K) 581, (M+ Na) 565, (M + 1) 543. Anal. Calcd for C2zHd4012: C, 48.70; H, 5.54; N, 10.33. Found C, 48.61; H, 5.62; N, 10.57. 2-(p-Aminobenzyl)-6-methyldiethylenetriamineN,N,N,",N"-pentaaceticAcid (8). Compound 7 (100 mg, 0.1845 mmol) was hydrogenated over 10% Pd/C (50 mg) at pH 8.5 (5M KOH) in an atmospheric hydrogenation apparatus. The uptake of HZwas monitored, and after the requisite amount had been consumed, the reaction was halted. The solution was filtered through a fine frit with Celite 535, then the solvent was removed and the residue dried under vacuum for 18h (89.1 mg, 94.3%): 'H NMR (500 MHz, D20, pH 1.0) 6 7.5035 (t, 2 H, J 6.5), 7.437 (m, 2 H), 4.2-3.65 (complex series of multiplets integrating for 12 protons), 3.40-3.16 (m, 4 H), 3.10 (m, 1 H), 2.9385 (9, 1, J = 10.0), 1.36-1.25 [pair of doubleta for the diastereomeric methyl groups integrating for 3 H total, 1.3415 (d, J = 6.5), 1.2675 (d, J = 6.5)]; (500 MHz, D20, pH 13.0) 6 7.11-7.04 (m, 2 H), 6.8055 (dd, 2 H, J = 8.5,8.0),3.40-2.05 (complex series of signals integrating for 18 H), 1 . 0 0 . 6 9 [series of doublets for the diastereomeric methyl groups integrating for 3 H total-0.9665 (d, J = 6.5), 0.9435 (d, J = 6.5), 0.846 (d, J = 6.0), 0.750 (d, 7.0), 0.7075 (d, J = 6.5)]; 252Cf-PDMSm/z 511 (M - 1). Anal. Calcd for C ~ Z H ~ ~ N ~ C, O 37.61; ~ O KH, ~ 3.85; : N, 7.98. Found C, 37.36; H, 3.65; N, 7.72. 2-(pIsothiocyanatobenzyl)-6-methyldiethylenetriamine-N,N,",N",N"-pentaacetic Acid (1). Aniline 8 (100 mg, 0.196 mmol) was taken up in Hz0 (5 mL) and transferred into a small round-bottom flask. The pH was adjusted to between 8.5 and 9.0 with solid NazCO3. Thiophosgene (15.0 pL, 0.196 mmol) in CHCls (10 mL) was loaded into an addition funnel fitted to the flask. The thiophosgene solution was added in one rapid addition with maximum stirring and the reaction was left to stir for 2 h. After removal of the stir bar, the organic layer was removed by room-temperature rotary evaporation. The aqueous residue was then lyophilized, leaving an off-white powder. The crude product was then purified by use of a 1 X 30 cm silica column eluted with solvent system 4 with the product coming off of the column first. The solvent was removed with minimal warming on a rotary evaporator and the remaining aqueous solution was ly-
Brechblel and Gansow
190 Bloconlugete Chem., Vol. 2, No. 3, 1991
ophilized. TLC of the product on silica using solvent system 4 gave Rf = 0.20; IR (Nujol) 2100 cm-l; lH NMR (220MHz,D20/D3CC02Na,pH 5.2) 6 7.28 (s,4 H),3.902.73 (broad complex multiplet integrating for 18H), 1.391.28 (2 br d, 3 H). The reactivity of the compound precluded obtaining chemical analyses. Preparationof Geometric Isomer 2. BOC-dl-(pnitrophenylalaniny1)-lalaninamide (9). BOC-dl-4-nitrophenylalanine (IO)(13.0 g, 41.9 mmol) was dissolved in ethyl acetate (400 mL), and triethylamine (5.84 mL, 42.0 mmol), HOBT (4.55 g, 33.6 mmol), and I-alaninamide hydrochloride (7.098 g, 42.0 mmol) were added and dissolved. DCC (8.738 g, 42.4 mmol) in ethyl acetate (50 mL) was then added and the reaction solution was stirred for 24 h. Acetic acid (1M, 1mL) was added and after 0.5 hour the suspension was filtered. The filtrate was extracted with saturated salt solution (100 mL), 1N HCl(3 X 100 mL), salt solution (100 mL), 5% NaHC03 solution (3 X 100 mL), and salt solution (100 mL). After drying over MgS04 and filtration, the solution was concentrated to 100 mL, petroleum ether was added, and the solution was held at 4 “C for 24 h. The product was collected and dried under vacuum (12.94 g, 81.3%1: lH NMR (500 MHz, DMSO-de) 6 8.1075 (d, 2 H, J = 8.5), 7.777 (d, 1H, J = 7.0), 7.4475 (d, 2 H, J = 8.5), 7.039 (s, 1H), 6.407 (s, 1H), 6.3675 (d, 1H, J = 5.5), 4.414 (br s, 2 H), 3.252 (dd, 1H, J = 13.0, 4.0), 3.024 (dd, 1 H, J = 12.5, 3.5), 1.420 (br s, 12 H); 13C NMR (300MHz, DMSO&) 6 18.43,27.95,37.80, 47.94, 55.12,78.23, 122.97,130.51, 146.12, 146.66, 155.16, 170.50,173.89; CI-MS 381 ((M + l)/z). Anal. Calcd for C1&4N406: C, 53.68; H, 6.31; N, 14.73. Found: C, 53.55; H, 6.45; N, 14.23. 3-Methyl-B-(pnitrobenzyl)diethylenetriamineTrihydrochloride (10). Dipeptide amide 9 (5.10 g, 13.42 mmol) was deprotected by treatment with trifluoroacetic acid (50 mL) for 1 h, after which the solution was rotary evaporated to near dryness. MeOH (50 mL) was added and the solution was again taken to dryness. The resulting solid was held at 0.01 mm and 50 OC for 8 h to ensure removal of residual acid. The resulting ammonium salt (5.10 g, 13.42 mmol) was taken up in THF (50 mL) and added to a flame-dried 250-mL three-neck flask fittedwith a condenser and septum and held under an argon atmosphere. The flask was cooled to 0 “C and 1 M BHrTHF (30.8 mL) was added by use of a syringe. The reaction solution was heated to vigorous reflux for 2 h and then stirred at room temperature for 2 h. The reaction flask was cooled to 0 “C and MeOH (25 mL) was slowly injected to decompose excess hydride. The solution was reduced to dryness, taken up in 100% EtOH (50 mL), and, after addition of concentrated HCl(50 mL),vigorously refluxed for 2 hand then stripped to dryness. The residue was dissolved in H20, loaded ontoa 1.5 X 20 cm AG50WX8, 200-400 mesh, H+-form, cation-exchange column, and washed with H2O until the eluant was neutral. The product was eluted from the column with concentrated HC1 (125 mL). The eluant was reduced to 10 mL and then lyophilized overnight (1.823 g, 66.2%). TLC of the solid on silica using solvent system 3 revealed one spot, Rf = 0.50; ‘H NMR (500 MHz, D20, pH 1.0) 6 8.268 (d, 2 H, J = 8.0), 7.614 (d, 2 H, J = 8.0),4.106 (m, 1H), 3.773 (m, 1H), 3.635 (dd, 1H, J = 13.0,7.5), 3.574 (dd, 1H, J = 13.5, 5.0), 3.54-3.15 (m, 4 H), 1.493 (br t, 3 H); (500 MHz, D20, pH 11.0) 6 8.091 (d, 2 H, J = 8.0), 7.438 (d, 2 H, J = 8.0), 3.167 (m, 1 H), 2.910 (m, 1 H), 2.75-2.45 (complicated m, 6 H), 1.031 (br s, 3 H); I3C NMR (300 MHz, D20, pH 1.0) 6 24.78,43.99,44.06,48.64,49.03,54.38, 54.60, 57.01, 57.62, 66.16, 66.50, 133.12, 139.31, 151.6,
155.97; (300MHz,CDCh) 6 20.23,20.40,38.04,38.17,43.05, 43.24,46.06,46.29,53.68,53.85,59.69,59.80,122.57,129.10, 145.60, 146.48; CI-MS 253 ((M + l)/z). Anal. Calcd for CI~H~SN~O C,~39.85; C ~ ~ H, : 6.36; N, 15.49. Found: C, 39.88; H, 6.36; N, 15.28.
3-Methyl-6-(pnitrobenzyl)diethylenetriamineN,N,”,”’,”-pentaacetic Acid (11). Triamine 10 (1.0 g, 3.97 mmol) was alkylated with bromoacetic acid (5.77 g, 41.51 mmol) and 7 N KOH (13.09 mL) as described above. Purification of the crude product by ion-exchange chromatography, preparative HPLC, and additional ionexchange chromatography proceeded smoothly as described for 7 with a t~ for ll by HPLC of 7.80 min (852 mg, 39.6 % ). TLC on silica using solvent system 3 revealed two spots of equal intensity, Rf = 0.092 and 0.182, while solvent system 5 revealed only one spot, Rf = 0.779; lH NMR (500 MHz, D20, pH 1.0) 6 8.246 (t, 2 H, J = 8.0), 7.583 (dd, 2 H, J = 13.5, 8.01, 4.223 (dd, 2 H, J = 39.0, 18.0),4.121 (dd, 2 H, J = 39.0, 17.01, 3.95-2.98 [complex series of complex signals integrating for 15H; decipherable signals within a complex area-3.828 (dd, 2 H, J = 37.5, 16.5) and 2.9335 (dd, 1H, J = 13.0,9.5)], two doublets for the diastereomeric methyl groups integrating for 3 H total a t 1.308 (d, J = 6.0) and 1.0475 (d, J = 4.5); (500 MHz, D20, pH 13.0) 6 8.199 (br m, 2 H), 7.55-7.40 (m, 2 H), 3.40-1.90 (complex series of signals integrating for 18H), 1.00-0.40 (complex series of apparent doubletswith a broad singlet at 0.666 for the diastereomeric methyl groups integrating for 3 H total) (The spectra of 11 vs 7 were found to be incongruent, i.e., 7 contains 95% of the product of the borane reduction of 5 to be compound 6 (and not a mixture of 6 and 10 as we had earlier thought) (23,25). NMR spectra of 1 showed t h e material referred t o earlier as t h e "MX-DTPA" equimolar mixture of 1 and 2 to be >95% 1, in agreement with t h e work of Cummins e t al. (26). T h e general synthesis of Scheme I1 for either of t h e monosubstituted DTPA's, 2- vs 3-position, will allow investigation of t h e structural influence over complex stability of t h e benzyl substituent when transposed from t h e C-2 t o t h e C-3 position without an added methyl group. Synthesis of t h e disubstituted DTPA's shouldthen provide an entry point into a vast array of potential ligands. T h e ability to control the substituents and their location on the backbone of t h e ligand should facilitate evaluation of which positions introduction of an alkyl group might be effective in maximizing the in vivo stability of t h e MYmAb conjugate, and, ultimately as well, indicate which groups might provide optimal results in vivo. We note that 1 is currently being employed for diagnosis and treatment of adult T-cell leukemia and B-cell lymphoma with lllIn and ACKNOWLEDGMENT
We would like to thank Mr. William Comstock of t h e NHLBI, Laboratory of Chemistry, for his time and efforts in obtaining the EI-MS spectra of the ligands and Drs. Greg Pippin and Tom McMurry for suggestions for improving this manuscript. We t h a n k Drs. Herman Yeh and Gui Ying Li for their assistance with NMR spectrosCOPYLITERATURE CITED (1) Strand, M., Scheinberg, D. A,, Gansow, 0. A,, et al. (1983)
Monoclonal Antibody Conjugates for Diagnostic Imaging and Therapy. In Monoclonal Antibodies and Cancer (B. D. Boss, R. Langman, I. Trowbridge, R. Dulbecco, Eds.) pp 125-131, Academic Press, Orlando. (2) Burchiel, S. W., Rhodes, B. A., Eds. (1983) Radioimmunoimaging and Radioimmunotherapy, Elsevier Science Publishing, New York. (3) Gansow, 0. A., Brechbiel, M. W., Mirzadeh, S., Colcher, D., and Roselli, M. (1990) Chelates and antibodies: Current
Bioconlugte Chem., Vol. 2, No. 3, 1991
193
Methods and New Directions. In Cancer Imaging with Radiolabeled Antibodies (D. M. Goldenberg,Ed.) Chapter 7, Kluwer Academic Press, Boston. (4) Goldenberg, D. M., Ed. (1990) Second Conference on Radioimmunodetection and Radioimmunotherapy of Cancer. Cancer Res. 50, 7748-1059s. ( 5 ) Mach, J. P., Carrel, S., Formi, M., Ribchard, J., Doneth, A., and Alberto, P. (1981) Tumor localization of radiolabeled antibodies against carcinoembryonic antigen in patients with carcinoma. New Engl. J. Med. 303, 5-10. (6) Ballou, B., Levine, G., Hakala, T. K., and Solter, D. (1979, Tumor locationdetected with radioactivelylabeled monoclonal antibody and external scintigraphy. Science 206, 844-847. (7) Scheinberg, D. A., and Strand, M. (1982) Leukemic cell targeting and therapy by monoclonal antibody in a mouse model system. Cancer Res. 42,44-49. (8) Spencer, R. P., Ed. (1978) Therapy in Nuclear Medicine, Chapters 1-3, Greene and Stratton, New York. (9) Fritzberg,A. R., Berninger, R. W., Hadley, S. W., and Wester, D. W. (1988) Approaches to radiolabeling of antibodies for diagnosis and therapy of cancer. Pharm. Res. 5, 325-334. (10) Brechbie1,M. W., Gansow, 0. A,, Atcher, R. W., Schlom, J., Esteban, J., Simpson, D. E., and Colcher, D. (1986) Synthesis of 1-@-isothiocyanatobenzyl)derivatives of DTPA and EDTA. Antibody labeling and tumor-imaging studies. Inorg. Chem. 25,2772-2781. (11) Roselli, M., Schlom, J., Gansow, 0. A., Raubitschek, A., Mirzadeh, S., Brechbiel, M. W., and Colcher, D. (1983)
Comparative biodistributions of yttrium- and indium-labe!eX monoclonal antibody B72.3 in athymic mice bearing humaii colon carcinoma xenografts. J. Nucl. Med. 30, 672-682. (12) Maecke, H. R., Riesen, A,, and Ritter, W. (1989) The r ' c " lecular structure of Indium-DTPA. J.Nucl. Med. 30,12351239. (13) Kauffman, G. B. (1966) Alfred Werner, Founder of Co-
ordination Theory; Springer, Berlin. (14) Basolo,F., and Johnson, R. (1964) Coordination Chemistry,
Benjamin/Cummings, Menlo Park, CA. (15) Greenwood, N. N., and Earnshaw, A. (1984) Chemistry of the Elements, Chapter 19, Pergamon, New York. (16) Wessels, B. W., and Rogus, R. D. (1984) Radionuclide
selection and model absorbed dose calculations for radiolabeled tumor associated antibodies Med. Phys. 11, 638-645. (17) Order, S. E., Klein, J. L., Leichner, P. K., Frincke, J., Lollo, C., and Carlo, J. (1986) W Y antiferritin. A new therapeutic radiolabeled antibody. Int. J.Rad. Oncol.Biol. Phys. 12,277281. (18) Durbin, P. W. (1960) Metabolic characteristics within chemical family. Health Phys. 2,225-238. (19) Kyker, G. C. (1962) Rare Earths. In Mineral Metabolism (C. L. Comar, and F. Bronner, Eds.) Vol. IIB, Chapter 36,
Academic Press, New York. (20) Hnatowich, D. J., Chino, M., Siebecker, D. A., Gionet, M. Griffin, T., Doherty, P. W., Hunter, R., and Kase, K. R. (1988)
Patient biodistribution of intraperitoneally administered yttrium-90-labeled antibody. J. Nucl. Med. 29, 1428-1434. (21) Kroll, H., Korman, S., Siegel, E., Hart, H. E., Rosoff, B., Spencer, H., and Laszlo, D. (1957) Excretion of yttrium and lanthanum chelates of cyclohexane l,2-trans diamine tetraacetic acid and diethylenetriamine pentaacetic acid in man. Nature 180,919-920. (22) Dudley, H. C. (1955)Influence of chelateson the metabolism of radioyttrium (Y-90). J. Lab. Clin. Med. 45, 792-799. (23) Kozak, R. W., Raubitschek,A., Mirzadeh, S., Brechbiel, M. W., Junghauns, R., Gansow,0.A., and Waldmann, T. A. (1989) Nature of the bifunctional chelating agent used for radioimmunotherapy with yttrium-90 monoclonal antibodies: Critical factors in determining in vivo survival and organ toxicity. Cancer Res. 49, 2639-2644. (24) Margerum, D. W., Cayley, G. R., Weatherburn, D. C., and Pagenkopf, G. K. (1978) Kinetics and mechanisms of complex formation and ligand exchange. In Coordination Chemistry, Monograph 174 (A. E. Martell, Ed.) Vol. 2, Chapter 1, American Chemical Society, Washington, DC.
194 Bloconlugete Chem., Voi. 2, No. 3, 1991
(25) Brechbiel, M. W. (1988)New bifunctional chelates for radioimmunoimagingand therapy. PbD. Thesis, The American University, Washington, DC. (26) Cummine, C. H., Rutter, E. W.,and Fordyce, W. A. (1991) A convenient synthesis of bifunctional chelating agents based on diethylenetriaminepentaaceticacid and their coordihation chemistry with yttrium(II1). Bioconjugate Chemietry, preceding paper in this issue. (We thank the authors for use of their work as a reference in advance of publication.) (27) Sundqvist, B., and Macfarlane, R. D. (1986)aszCf-Plasma desorption mass spectrometry. Mass Spectrom. Rev. 4,421460. (28) The specific activity increase as ligands were synthesized correlates well with a change in vendor and a change in packaging. The reagent was purchased initidly as a solid (1 mCi/ampoule) and gradually replaced with a toluene solution (3 mCi/ampoule). (29) Westerberg, D. A,, Carney,P. L., Rogers, P. E., Kline, S. J., and Johnson, D. K., (1989)Synthesis of novel bifunctional chelators and their use in preparing monoclonal antibody conjugates for tumor imaging. J. Med. Chem. 32, 236243. (30)Brown, H.C., and Kiem, P. (1973)Selective reductions. XVIII. The fast reaction of primary, secondary, and tertiary amides with diborane. A simple, convenient procedure for the conversion of amides to the corresponding amines. J.Org. Chem. 38,912-916.
Brechbkl and Q e n m
(31)Meikle, R. W. (1963)An application of paper electrophoresis to the examination of completeness of reaction and purification in the synthesisof radioactivecompounds. Nature (London) 190,61-62. (32)Floch, L., and Kovac, S. (1975)Synthesis and properties of alkyl isothiacyanatocarboxylates. Collect. Czech. Chem.Commun. 40,2845-2854. (33) Bara, Y.A., Friedrich, A., Hehlein, W., Kessler, H., Kandor, P., Molter, M., and Veith, H. J. (1978)Synthesis of the cyclic pentapeptides containing 1-phenylalanine and glycine. Chem. Ber. 111,1029-1044. (34) Lundt, B. F., Johansen, N. L., Vylund, A., and Markuesen, J. (1978)Removal of t-butyl and t-butoxycarbonyl protecting groups with trifluoroacetic acid. Znt. J.Pept. ProteinRe8. 12, 258-268. (35) Mirzadeh, S.,Brechbiel, M. W., Atcher, R. W., and Gansow, 0. A. (1990)Radiometal labeling of immunoproteins: Covalent linkage of 2-(4-isothiocyanatobenzyl)diethylenetriaminepentaacetic acid ligands to immunoglobulin. Bioconjugate Chem. 1,5945. (36) Bly, R. S.,Perkins, G. A., and Lewis, W. L. (1922)The preparation of phenylimido-phoagene,and the chlorination of formanilide. J. Am. Chem. SOC.44, 28962903.
