Bioconjugate Chem. 1995, 6,616-623
616
Gadolinium(II1)Di- and Tetrachelates Designed for in Vivo Noncovalent Complexation with Plasma Proteins: A Novel Molecular Design for Blood Pool MRI Contrast Enhancing Agents Vladimir V. Martin, William H. Ralston,' Michael R. Hynes,' a n d John F. W. Keana" Department of Chemistry, University of Oregon, Eugene, Oregon 97403, and Mallinckrodt Medical, Inc., 675 McDonnell Boulevard., P.O. Box 5840, St. Louis, Missouri 63134. Received February 17, 1995@
A new series of gadolinium chelates designed a s blood pool contrast enhancing agents for magnetic resonance imaging applications is described. Complexes having four Gd(II1) chelate units display a significant increase in molecular relaxivity per gadolinium ion in water (9- 13 L-mmol-l-s-') compared to Gd(II1)-DTPA (5 Lsmmol-%-I). A further jump in relaxivity (25 L-mmol-lesec-l) in 4%BSA solution was observed in the case of a fatty acid-containing tetrachelate and is attributed to noncovalent binding of the tetrachelate to serum albumin. This agent was successfully used for imaging the rat circulatory system.
INTRODUCTION
Paramagnetic species enjoy wide use as contrast enhancing agents in biological and medical magnetic resonance imaging (MRI) applications owing to their ability to shorten the relaxation time of nearby water protons (1). Complexes of Gd(II1) are particularly attractive owing to the presence of seven unpaired electrons in each Gd(II1) ion and a long electron spin relaxation time (2). Compared to other paramagnetic transition ions or organic stable radicals, Gd(II1) chelates provide maximum molar relaxivity. Contrast agents designed to image the blood pool must remain in the vasculature for a t least 30 min to allow for image acquisition. Filtration by the glomeruli in the kidney defines the molecular weight of blood pool agents to be '20 000 (1, 3). Several macromolecular contrast agents have been developed and tested for blood pool MRI applications. These reagents contain multiple Gd-chelated moieties in the form of complexes with diethylenetriaminepentaacetic acid (DTPA) residues covalently linked to a macromolecular carrier. Various natural and synthetic polymers have been employed as carriers including serum albumin (31, polylysine (PL) (31, polylysine-poly(ethy1ene glycol) conjugate (MPEG-PL) (41, and functionalized dextrans (3, 5, 6). Along with relatively long retention times in the vasculature (1-4 h, compared to 15-20 min for Gd-DTPA) ( 3 )the macromolecular conjugates display a jump in molecular relaxivity up to 15 Lsmmol-l-s-l per Gd(II1) ion compared to about 6 L.mmo1-l-s-l for the Gd-DTPA complex itself (2, 3 ) . This so-called proton relaxivity enhancement effect (PRE) (7) is attributed to a lower, more optimal tumbling rate of the conjugated versus free paramagnetic unit (3, 8).
Recently, a new family of blood pool contrast agents based on Starburst dendrimer cascade polymers (9)was introduced by Wiener et al. (10). These compounds display the highest molecular relaxivity reported to date ~~
~~~
~
* To whom correspondence should be addressed:
~
~
Department of Chemistry, University of Oregon, Eugene, OR 97403. Phone: (503) 346-4609. Fax: (503) 346-4623. Internet: jkeana@ oregon.uoregon.edu. + Mallinckrodt Medical, Inc. Abstract published in Advance ACS Abstracts, July 15, 1995.
(up to 34 L.mmol-%l per Gd ion) and excellent in vivo lifetimes and may not exhibit unwanted immune reactions observed with protein covalent conjugates (1I). Syntheses of macromolecular contrast reagents are typically accomplished by labeling of multiple accessible primary amino groups on the macromolecular carrier (protein, activated dextran, or synthetic dendrimer) with a n excess of a functionalized precursor of the Gd-DTPA paramagnetic unit. DTPA anhydride (3,121 and a phenyl isothiocyanate-substitutedDTPA (10,131 have been used for this purpose. The latter has the advantage of utilizing all five DTPA carboxyls for chelation of the metal, the result being a more stable complex compared to amide derivatives derived from DTPA anhydride (14). The resulting conjugate is subjected to complexation with Gd(111) ion followed by purification using size-exclusion chromatography, ultrafiltration, or dialysis. An alternative approach utilizes a preformed Gd-DTPA chemically reactive chelate unit as the labeling reagent (6, 14). Any paramagnetic conjugate prepared by polymer labeling methodology possesses structural inhomogeneity derived from a variable degree of amino group involvement. Structural variability causes problems in synthetic reproducibility, purification, and in clinical use. Consequently, our research program has aimed at the preparation of structurally well-defined compounds. Recent results with Gd complexes (15,16)and nitroxide radicals (17,181 suggest that noncovalent binding of the contrast enhancer to a macromolecular carrier such as plasma proteins results in increases both in molecular relaxivity and intravascular retention. At the same time it is evident that MRI blood pool agents must contain more than one paramagnetic center to achieve sufficient contrast enhancement due to the limited number of binding sites on each protein molecule (17). Earlier, we introduced the notion of molecular amplifiers (MAS) designed to deliver multiple copies of a pharmacologically active group to a targeted site (6).As part of the amplifier concept, the first members of a chemically well-defined series of MAS bearing several stable nitroxide free radical groups were synthesized for MRI applications (19). The amplifiers follow the structure motif shown diagrammatically as structure 1 and consist of a central core (CC) having one or more connection points substituted with branchers (B). Each of the branchers may provide one or more sites for the
1043-1802/95/2906-0616$09.00/00 1995 American Chemical Society
Bioconjugate Chem., Vol. 6, No. 5, 1995 617
Novel Blood Pool MRI Contrast Agents
covalent attachment of active groups. Another important structural feature of the MA is the presence of a targeting group (TG) that can be designed for a particular application. Depending on the nature of the targeting group, the MA might be used to modify accessible amino groups in proteins, to concentrate the MA in a particular environment such as a membrane bilayer, or to allow selective complexation with a nucleic acid or protein. Linker groups (L) are employed for connecting the targeting group to the central core. Targeting Group
Q
Linker Central Core
Brancher Group Active
a@ lel
1
Herein, we report the synthesis of several MAS designed a s contrast enhancing agents for the blood pool through complexation with plasma proteins. We also demonstrate the utility of these compounds for blood pool MRI applications in the rat. EXPERIMENTAL PROCEDURES
General. Melting points were obtained in a ThomasHoover apparatus and are uncorrected. Infrared spectra were recorded in KBr pellets (concentration 0.2-0.5%) or in CC14or CHC13solutions (concentration 2-5%) on a Nicolet 5DX or a Nicolet Magna-IR 550 IR FT spectrometer. 'H (300 MHz) and 13C(75 MHz) NMR spectra were taken on a General Electric QE-300 FT spectrometer. Chemical shifts are reported in 6 units referenced to solvent signals. The identity of the same compounds prepared by different methods was established by comparison of their IR and lH NMR spectra and/or TLC Rf or HPLC t~ values using coelution criteria. Analytical TLC was performed on Merck plastic-backed silica gel 60 F254 plates. Preparative TLC was done on Analtech Uniplate precoated silica gel glass-backed plates (20 x 20 cm x 1mm) or on plates homemade from Merck silica gel 60 PF254 (40 x 30 cm x 3 mm). Analytical HPLC was performed on a Rainin Microsorb-MV CIS0.46 x 25 cm column. Elution utilized gradients A (HzO 0.2% TFA) - B (MeCN 0.2% TFA) with UV detection a t 230 or 254 nm. Retention times and relative percent peak areas are reported. Preparative column chromatography utilized Baker silica gel (60-200 mesh). Flash chromatography utilized 200-425 mesh Davisil silica gel (Aldrich), grade 643. Size exclusion chromatography was performed on a Pharmacia-LKB Gradifrac system (UV detection a t 280 nm) over Sephadex G-10 or Sephadex G-25 fine gels (bed 2.5 x 80 cm). Reagents were purchased from Aldrich Chemical Co. and used without purification unless noted. Solvents were purchased from Baker. Dry EtOAc and MeCN were prepared according to Perrin et al. (20). 1,5-Bis(phthalimido)-protecteddiethylenetriamine 10 was prepared by the procedure of
+
+
Sosnovsky et al. (21). The starting protected DTPA unit 14 was prepared according to Keana et al. (14, 22). All reactions were performed under a nitrogen atmosphere. Relaxivity Studies. Relaxivities were determined using the longitudinal relaxation rate ( T l ) values measured on a 20 MHz Bruker Minispec. Compounds 2-7 and Gd-DTPA were prepared in deionized water and in a 4% bovine serum albumin (BSA) solution. The relaxivity (Rl) value of each compound was calculated as a measure of the effective change in 1/T per unit of concentration. R 1 relaxivity values, in units of L-mmol-l-s-l, were determined using a t least four concentrations of each compound. Animal Imaging and Toxicity Studies. Vascular imaging was performed on a 250 g Sprague-Dawley rat. The animal was anesthetized with a n intramuscular injection of ketamine (80 mg/kg) and xylazine (12 mgl kg) and a cannula placed into a lateral tail vein. Prior to imaging a 0.1 mL maintainance dose of pentobarbital (64 mg/mL) was administered through the cannula. The rat was placed in a Siemens Magnetom SP head coil and a three-dimensional time-of-flight preinjection scout MR angiogram was acquired using a gradient echo (FISP) sequence, TR 20 ms and TE 9 ms, in a 1.5 T Siemens Magnetom. Following acquisition of the scout image, the rat was injected via the cannula with a 20 mM solution of compound 4 in sterile water a t 0.01 mmol of G a g . Images were acquired a t 15, 30, and 45 min post injection. The preliminary acute iv toxicity was evaluated in one mouse per compound a t an anticipated diagnostic dose. Conscious restrained mice, at one per compounds 2-4, were injected a t 0.01 mmol of Gd3+/kgvia a lateral tail vein and observed for 7 days. No histology was performed. 1,3-Bis(bromomethyl)d-nitrobenzene(9). A mixture of 5-nitro-m-xylene ( 8 )(3.02 g, 20 mmol), NBS (7.12 g, 40 mmol), and benzoyl peroxide (10 mg) in CC14 (50 mL) was refluxed for 16 h. The precipitated succinimide was filtered off and washed with CC14(3 x 20 mL). The combined filtrate was evaporated, and the solid residue was crystallized from 3:l hexane-EtOAc to yield 3.28 g (53%)of dibromide 9 as colorless crystals: mp 100-101 "C (from 1:lO hexane-EtOAc); IR (KBr)1540,1362, and 1215 cm-l; IH NMR (CDC13)6 4.52 ( 6 , 4H), 7.75 (8, lH), 8.19 (s, 2H); 13C NMR (CDCl3) 6 30.55, 123.66, 135.16, 140.40, 148.61; HRMS calcd for CsH7N0279Br2306.8844, found 306.8840. 1,3-Bis[[NJV-bis(2-N-phthalimidoethyl)amino]methyl]-5-nitrobenzene (11). A mixture of dibromide 9 (1.98 g, 6.4 mmol), bis(phtha1imide-protected) diethylenetriamine 10 (5.59 g, 15.4 mmol), and KzC03 (3.45 g, 25 mmol) in MeCN (100 mL) was refluxed with vigorous stirring for 4 d. The inorganic material was filtered off and washed with MeCN (4 x 20 mL). The combined filtrate was evaporated, and the residue was chromatographed over silica gel (2.5 x 30 cm) to yield 2.34 g (42%) of protected hexaamine 11 as a white powder: mp 156158 "C (from 1:5 hexane-EtOAc); IR (KBr) 1730, 1539, 1360, and 1211 cm-'; lH NMR (CDC13) 6 2.78 (t, 8H, J = 5.5 Hz), 3.51 (s, 4H), 3.73 (t, 8H, J = 5.5 Hz), 7.28 (s, lH), 7.64 (5, 2H), 7.68 (br s, 16H); 13C NMR(DMSOd6) 6 35.80 (4C), 51.73 (4C), 56.82 (2C), 122.56 ( l c ) , 123.39 ( 8 3 , 132.28 ( 8 0 , 134.83 (8C), 141.70 (lc),147.98 (lC), 168.31 ( 8 0 ; HRFAB MS calcd for (C48H40N7010+ H) 847.2836, found 874.2823. 1,3-Bis[[N,N-bis(2-aminoethyl)aminolmethyll-5nitrobenzene Hexahydrochloride (12). To a boiling suspension of hexaamine 11 (0.90 g, 1.03 mmol) in absolute EtOH (250 mL) was added hydrazine (2.5 mL,
618 Bioconjugate Chem., Vol. 6,No. 5, 1995
77 mmol) with vigorous stirring. After about 10 min the mixture became a clear solution. After a n additional 10 min reflux period it was cooled to room temperature and evaporated to dryness. The residue was evaporated twice with EtOH (30 mL) and evacuated to 0.1 Torr to remove the excess hydrazine. The solid was suspended in 2 N HC1 (50 mL), stirred for 1h, and filtered from insoluble phthalimidohydrazine. The filtrate was extracted with CHC13(3 x 30 mL, discarded) and evaporated to dryness to leave a hygroscopic crude product. Crystallization from MeOH gave 0.39 g (66%)of hexaamine hexahydrochloride 12 as a yellow powder: mp 256-258 "C (MeOH); IR (KBr) 1539, 1360, and 1211 cm-l; IH NMR (DzO) 6 2.90(t, 8H, J = 6.1 Hz), 3.18 (t, 8H, J = 6.1 Hz), 3.94(s, 4H), 7.76 (s, lH), 8.25 (s, 2H); 13C NMR (DzO) 6 36.41 (4C), 50.74 (4C), 57.65 (2C), 125.80 (2C), 137.50 (2C), 138.65 (lC), 149.50 (1C). Anal. Calcd for C16H31N70p6HCl: C, 33.58; H, 6.52; N, 17.13. Found: C, 33.58; H, 6.84; N, 17.14. 1,3-Bis[[NJV-bis(2-isothiocyanatoethyl)aminolmethyl]-5-nitrobenzene(13). To a vigorously stirred mixture of NaHC03 (4.000 g, 40 mmol) and thiophosgene (1 N in CHC13, 2 mL, 2 mmol) in CHC13 (50 mL) was introduced a solution of hexaamine hexahydrochloride 12 (0.089 g, 0.16 mmol) in HzO (1 mL) The mixture was stirred for 6 h and then MgS04 (10 g) was added to remove water. The inorganic material was filtered off and washed with CHC13 (5 x 10 mL). The combined filtrate was evaporated, and the oily residue was chromatographed on a silica gel TLC plate. Elution with CHC13gave 0.045 g (56%)of tetraisothiocyanate 13as a yellow solid: mp 62-64 "C (hexane); IR (KBr)2212,2129, 2063,1528,1440, and 1343 cm-'; IH NMR (CDCl3) 6 2.97 (t, 8H, J = 6.0 Hz), 3.62 (t, 8H, J = 6.0 Hz), 3.89 (s, 4H), 7.93 (s,lH), 8.11 (s,2H); 13C NMR (CDC13) 6 44.31 (4C), 54.55 ( 4 0 , 58.69 ( 2 0 , 123.10 ( 1 0 , 133.16 (br s, lC, C=S), 135.44 (lC), 141.75 (2C), 149.16 (1C). Anal. Calcd for CZ0H23N70zS4: C, 46.05; H, 4.44; N, 18.79; S, 24.58. Found: C, 45.85; H, 4.30; N, 18.47; S, 24.56. Reaction of Tetraisocyanate 13 with Amine 14 To Give 15. A mixture of tetraisothiocyanate 13 (0.057 g, 0.11 mmol) and amine 14 (22) (0.270 g, 0.48 mmol) in CHC13 (3 mL) was stirred for 10 d a t 50 "C and then evaporated and purified on a TLC plate (30 x 40 cm x 3 mm, eluent, 5% MeOH in CHC13) to give 0.201 g (65%) of nitro perester 15 as a yellow glassy solid: liquifies above 70 "C; IR (KBr) 1735,1538,1515,1202, and 1028 cm-'; lH NMR (DMSO-& 5%D20) 6 7.03 (d, 8H, J = 7 Hz), 7.29 (d, 8H, J = 7 Hz), 7.79 (s, lH), 7.95 (9, 2H); 'H NMR (CDC13 2% DzO) 6 2.40-3.20 (gr, 40H), 3.303.80 (gr,116H), 7.11-7.24 (br m, 8H), 7.27-7.40 (br m, 9H), 7.93 (5, 2H). Reduction of Nitro Perester 15 To Give Amine 16. A solution of 15 (0.480 g, 0.17 mmol) and SnClz (0.600 g, 3.16 mol) in MeOH (80 mL) was refluxed for 20 h, cooled to room temperature, and poured into EtOAc (250 mL). The mixture was washed with saturated NaHC03 (5 x 100 mL) and HzO (2 x 50 mL), dried (MgS04) and evaporated. The residue was chromatographed on a preparative TLC plate (30 x 40 cm x 3 mm, eluent, 8% MeOH in CHC13) to give 0.358 g (76%)of amine 16 as a glassy solid: liquifies above 50 "C; IR (KBr) 3344,2959, 1738, 1605, 1514, 1439, 1202 and 1021 cm-I. IH NMR (DMSO-&) 6 2.40-2.95 (gr, 40H), 3.20-3.80 (gr, 116H), 6.35 (s, lH), 6.45 (s, 2H), 7.09 (d, 8H, J = 8 Hz), 7.26 (d, 8H, J = 8 Hz), 7.55 (br, 4H, N-H), 9.65 (br, 4H, N-H). Acylation of Amine 16 To Give 18. The acylating reagent N-hydroxysuccinimidyl methyl sebacate 17 was prepared as follows. A mixture of sebacic acid monomethyl ester (0.216 g, 1 mmol), N-hydroxysuccinimide
+
+
Martin et al.
(0.120 g, 1.04 mmol), and DCC (0.210 g, 1.02 mmol) in THF (30 mL) was stirred for 16 h and then filtered from DCU and poured into EtOAc (150 mL). The ethyl acetate solution was washed with saturated NaHC03 (5 x 20 mL) and HzO (2 x 30 mL), dried, and evaporated to give 0.275 g (88%)of NHS ester 17 as colorless plates: mp 60-61 "C (hexane-EtOAc 1:l); IR (KBr)1820,1788,1735,1212, and 1072 cm-'; IH NMR (CDC13) 6 1.31 (br, 8H), 1.61 (m, 2H), 1.73 (m, 2H), 2.29 (t, 2H, J = 7.6 Hz), 2.59 (t, 2H, J = 7.4 Hz), 2.82 (s, 4H), 3.66 (s, 3H); I3C NMR (CDC13) 6 24.50, 24.85, 25.57 (2C), 28.64, 28.83, 28.91, 28.69, 30.90, 34.04, 51.41, 168.61, 169.13 (2C), 174.22. Anal. Calcd for C1&306N: C, 57.50; H, 7.40; N, 4.47. Found: C, 57.58; H, 7.17; N, 4.46. A mixture of amine 16 (0.245 g, 0.09 mmol) and NHS ester 17 (0.157 g, 0.5 mmol) was stirred for 10 d a t 50 "C and then cooled to room temperature and evaporated. The residual semisolid was chromatographed on a preparative TLC plate (40 x 30 cm x 3 mm, eluent, 7% MeOH in CHC13) to give 0.137 g (53%)of amide 18 as a yellowish glassy solid: liquifies above 60 "C. Amide 18 was unstable and was immediately used in the next step. Hydrolysis of Peresters 15, 16,and 18. In a 100 mL round-bottomed flask was stirred a mixture of the perester (0.060 mmol) and 1N NaOH (4 mL, 4 mmol) in MeOH (15 mL) for 23 h a t 45 "C, cooled to 25 "C, and evaporated to leave a solid residue. This crude product was suspended in MeOH (20 mL) and reprecipitated with acetone (75 mL). The product was filtered on a glass filter (F)and washed with acetone (5 x 20 mL) and ether (2 x 20 mL) to yield the corresponding sodium salt 19, 20, or 21 as a yellowish solid which was contaminated with some inorganic material, although no organic impurities were detected by NMR or HPLC. Nitro derivative 19: dec >250 "C; IR (KBr) 1598,1541, 1411, 1332, and 1116 cm-l; 'H NMR (DzO) 6 2.18 (m, 8H), 2.61 (m, 8H), 2.68 (m, 32H), 2.70-3.82 (gr,48H), 7.08 (d, 8H, J = 6 Hz), 7.25 (d, 8H, J = 6 Hz), 7.72 (s, lH), 8.14 (s,2H); HPLC (gradient, 80% A, 20% B to 95% B over 20 min) 14.4 min (95%). Amino derivative 20: dec >250 "C; IR (KBr) 1593, 1411, 1332, and 1114 cm-l; 'H NMR (DzO) 6 2.18 (br, 8H), 2.64-2.70 (br, 40H), 2.80-3.80 (gr,48H), 7.09 (d, 8H, J = 8 Hz), 7.25 (d, 8H, J = 8 Hz); HPLC (gradient, 80% A, 20% B to 95%B over 20 min) 14.3 (98%). Fatty acid derivative 21: dec '250 "C; IR (KBr) 1592, 1411, 1331, and 1114 cm-l; 'H NMR (DzO) 6 1.23 (m, 8H), 1.51 (m, 20H), 2.58 (br s, 36H), 2.65 (br s, 8H), 2.803.70 (gr,40H), 6.66 (s, 2H), 6.70 (8,lH), 7.09 (d, 8H, J = 7 Hz), 7.25 (d, 8H, J = 7 Hz); HPLC (gradient, 80% A, 20% B to 95% B over 20 min) 18.8 min (97%). Complexation of 19-21 with Gd(II1) ion. In a 20 mL three-neck flask equipped with a nitrogen inlet and pH-microelectrode was acidified a magnetically stirred solution of the sodium salt (0.08 mmol) in water (20 mL) with 0.2 N HCl to pH 6.5,and then GdC13.6H20 (0.128 g, 0.45 mmol) in HzO (1mL) was added in one portion (pH dropped to 3). The mixture was stirred for 15 min and then the pH was gradually increased to 6 over 1h and then to 9.5 over 4 h by dropwise addition of 0.1 N NaOH. The mixture was filtered through a double Metrigard prefilter (Gelman) and concentrated to 2 mL under vacuum. The concentrated solution was injected onto a size-exclusion chromatography column (Pharmacia 2.5 x 100 cm C-column packed with Sephadex G-25 Fine, bed volume 2.5 x 90 cm) and chromatographed with degassed water. The fractions containing product were ascertained by HPLC to be in the first group of peaks. Evaporation of the water gave the complexes a s a yellow glassy powder.
Novel Blood Pool MRI Contrast Agents
Bioconjugafe Chem., Vol. 6,No. 5, 1995 619
Scheme 1
p p
-OOc
COOLi
COONa
s=c,.NH
o=c:
NH
NH NH
L-GdLi2 5
$9
s=c; C -(OO-OOc
>" - ' cO/ 2 R=NOz 3 R=NH2 4 R = NHCO(CH2)&OONa
Nitro derivative 2: yield, 44%;dec 2250 "C; IR (KBr) 1598, 1404, 1321, and 1097 cm-l; HPLC (gradient, 80% A, 20% B to 95%Bover 20 min) 14.5 min (94%). Anal. C, 34.73; H, Calcd for (C109H123N23042S4Gd4Nag16H20): 4.34; N, 8.96. Found: C, 34.75; H, 4.17; N, 8.86. Amino derivative 3: yield 62%;dec >250 "C; IR (KBr) 3429,1602,1403,1323, and 1095 cm-l; HPLC (gradient, 80% A, 20% B to 95%B over 15 min) 14.3 min (100%). Anal. Calcd for (C104H12sN23040S4Gd4Na8'24H20):C, 33.66; H, 4.70; N, 8.68. Found: C, 33.69;H, 4.75;N, 8.74. Fatty acid derivative 4: yield 40%; dec >250 "C; IR (KBr) 1611, 1405, 1320, 1263, and 1095 cm-l; HPLC (Microsorb C18, 20 to 95%B in 15 min) 19.0 min (96%). Anal. Calcd for (C114H14~N23043S4Gd4Na~.28H20): C, 34.46; H, 4.97; N, 8.11. Found: C, 34.52; H, 4.88; N, 8.05. Labeling of Human Serum Albumin. To a solution of amino tetragadolinium complex 3 (6.5 mg, 2 pmol) in HEPES buffer (0.5 mL; pH 8.2) was added 1 N CSClz in CHC13 (20 pL, 20 pmol). The mixture was vigorously shaken for 7 min, and then it was extracted with CHCl3 (3 x 1 mL) to remove the excess CSC12. To the aqueous phase containing isothiocyanate 22 was added a solution of HSA (13.8 mg, 0.2 mmol) in H2O (0.5 mL), and the mixture was kept under nitrogen for 24 h and then loaded onto a Sephadex G-75 column (1.5 x 40 cm). The column was eluted with water (monitored by HPLC), and the major fraction was lyophilized to give 12.1 mg of paramagnetic (according to NMR) conjugate 23 as a white glassy solid. RESULTS
Chemistry. Three new structurally related MAS 2-4 (Scheme 1) containing four Gd-DTPA units in the molecule were used in the present study along with mono Gd-DTPA complexes 5 and 6 and bis Gd-DTPA complex 7. These latter three complexes were prepared in a n earlier model study (22). The structural abbreviations L-GdNa2 and R-4(L-GdNa2 are defined in Scheme 1 for use below owing to the complexity of the structures.
COO-
/
J-
6
HN;C=S
NH
L-GdNa2 L-GdNaz
L-GdNaz
7
Scheme 2 "02
Br
PhtN
Br
9
8
@+NPht
5
N
PhtNfJN 11
H2N-NH2
\NPht
H@/CHC13
N
