Bioconjugate Chem. 1990, 1, 345-349
345
Synthesis and Evaluation of Two New Bifunctional Carboxymethylated Tetraazamacrocyclic Chelating Agents for Protein Labeling with Indium-11 1 Gerd Ruser, Walter Ritter, and Helmut R. Maecke' Department of Nuclear Medicine, University Hospital Basel, 4031 Basel, Switzerland. Received July 16, 1990
The synthesis of two new N- and C-functionalized tetraazamacrocyclic ligands intended to be covalently linked to biomolecules like monoclonal antibodies and to bind the y-emitting isotope indium111 in a thermodynamically and/or kinetically inert way is described. 12-(p-Nitrobenzyl)-1,4,7,10tetraazacyclotridecane-1,4,7,lO-tetraaceticacid (Ll) was synthesized by means of bimolecular cyclization with the appropriate malonic acid diethyl ester and triethylenetetraamine, followed by reduction with diborane and alkylation of the cyclic tetraamine with bromoacetic acid. The corresponding triscarboxymethylated ligand L2 was made by statistical alkylation of the tetraamine. Both ligands fulfill the criteria for antibody labeling using the bifunctional chelate approach, namely fast chelate formation, high radiochemical yield, and high stability under physiological conditions. Surprisingly the heptadentate ligand L2 confers higher stability to In3+ and exhibits faster complex formation than octadentate L1. 13CNMR spectra in solution indicate that the difference in stability is not due to incomplete coordination of all four carboxylate groups in In-L1.
The application of radiolabeled monoclonal antibodies (MoAb) is a potentially powerful tool in nuclear medicine for tumor diagnosis and therapy (1-6). The use of metallic radionuclides has the special advantage that the lyophilized antibody-ligand conjugates can be prepared and stored in advance. A specific radiopharmaceutical can then be prepared by simply adding a buffered solution of the radiometal prior to application. This circumventstechnical and logistical problems associated with the iodine isotopes. Many MoAb show optimal tumor localization only after few days. For this reason the y-emitting isotope lllIn was proposed (7-1 1). Its appropriate nuclear characteristics, a 68 h half-life, and y-energies of 173 keV and 247 keV make it ideally suited to tumor diagnosis. For a successful use of the isotope in antibody labeling it has to be coupled to the MoAb by using the bifunctional chelate approach. Thereby the following prerequisites have to be fulfilled: (i) fast complex formation and high radiochemical yield, (ii) high kinetic inertness, to avoid label transfer in vivo t o t h e competing serum protein transferrin and concomitant transferrin-mediated liver uptake, and (iii) preserved immunoreactivity of the antibody complex conjugate. We report here on the achievement of the first two aspects with two new bifunctional carboxymethylated tetraazamacrocycles (Ll, L2, Chart I). Our earlier work on In3+ chemistry has shown the NazIn(DTPA).SHzO complex to have coordination number eight with antiprismatic geometry (12). This geometry also holds for NaIn(DOTA).3H20 (13). The complex with the tetracarboxymethylated macrocycle now is more compact and very symmetric and the In3+-nitrogen and In3+-oxygen bonds are somewhat shorter. This is reflected in increased kinetic stability of In(D0TA)- compared to the open chain polyaminopolycarboxylato-In3+complex (14). We have therefore continued our studies toward the synthesis of C-functionalized tetraazamacrocycles and describe the approach with C-functionalized carboxymethylated derivatives of 1,4,7,10-tetraazatridecane. 1043-1802/90/2901-0345$02.50/0
Chart I
rN"7 IN
HooccH2/
N)
I
LI \ oq.mon
HomHz/
\ c%cooH
L1: R1= CHzCOOH L2: R l = H L3: Rz = CHzCOOH L4: R z = H
So far bifunctional derivatives of open chain polyaminopolycarboxylates as EDTA (15)and DTPA (7,11)were tested in animal models and patients. These ligands certainly confer high thermodynamic stability to the corresponding lllIn complexes. Because of the possible importance of kinetic inertness, bifunctional macrocycles may be of additional benefit in antibody labeling with Tn. Bifunctional DOTA has recently been shown to stabilize in human serum extremely well ( 1 6 ) and a bifunctional triazamacrocycle was proposed for antibody labeling with l111n3+(17). EXPERIMENTAL PROCEDURES
Material and Methods. All reagents and solvents were obtained from commercial sources and were used without further purification. Infrared spectra were recorded on a Perkin-Elmer 983 instrument with KBr pellets. Proton and carbon-13 nuclear magnetic resonance spectra were recorded at 25 "C on a Bruker spectrometer a t 360 and 91 MHz, respectively, and a Varian VXR-400 spectrometer a t 400 and 101 MHz. Chemical shifts reported are relative either to TMS (CDCl3) or sodium 3-(trimethylsily1)tetradeuterioproprionate (TSP, DzO). Mass measurements were obtained on a VG 70-SE mass spectrometer, using 8 kV 0 1990 American Chemical Society
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Ruser et al.
Bioconjugate Chem., Vol. 1, No. 5, 1990
Scheme I1
Scheme I
+
lll~nJ+
itooc-
COOEt
4
Trlcn
*
k
"'In
- L
(I)
i
E t O O C A COOif
+
L
transferrin (*)
- transferrin
'"in
(FAB): m / e 322 (M + H). IR (KBr): 3400 (NHz+),1600 (ar-C-C), 1510 and 1350 cm-' (N02). 'H NMR: 6 2.81 (t, 1 H), 3.04 (d, 2 H), 3.50 (m, 16 H), 7.57 (d, 2 H), 8.16 (d, 2 H). 13CN M R 6 37.92,38.52,46.43,47.44,47.74,50.71, 126.84,133.06,148.26,149.36. Elemental Anal. Calcd for C16H~7N502.1.23HzO.4HC1(489.43):C, 39.26; H, 6.89; N, 14.30; 0,10.55; C1, 28.97, HzO, 4.53. Found: C, 39.55; H, 6.71; N, 14.48; 0, 10.24; C1, 29.11; HzO, 4.54. L1 : R, = CH,COOH Tris- and tetracarboxymethylation of the tetraamine was L2 : R, = H afforded in aqueous solution at a pH of between 9.5 and 10.5. in a thioglycerol matrix. Elemental analysis were 12-CpNitrobenzyl)-1,4,7,1O-tetraazacyclotridecaneperformed a t CIBA-GEIGY Analytical Services 1,4,7,10-tetraacetic acid (Ll) was obtained at 70 "C and Laboratories. l111nC13was obtained from Mallinckrodt DipH 10.5 by the slow addition of 5 equiv of bromoacetic agnostics (10 mCi/mL in pH 1 HC1). y-Counting was acid. The pH was controlled by use of a pH-stat. After performed on a Packard A 5000 D well counter and a stirring for 3 h at 70 "C, the yellow solution was loaded homemade continuous flow-through y-detector based on onto an anion-exchange column (Dowex 1x4, formiate a Picker Model 628018 NaJ detector and a Canberra AMP form, 2.6 X 30 cm). The column was washed with water ITSCA amplifier. and the ligand was eluted with a 0.02-5 M gradient of Synthesis. The ligands L1 and L2 were synthesized formic acid. A side product (triscarboxymethylated tetaccording to Scheme I. raamine) eluted at 0.05 M HCOOH, the product at 0.5 M (pNitrobenzy1)malonic Acid Diethyl Ester 2. A HCOOH. The solution was evaporated and crystallized mixture of 58 g (0.36 mol) of diethyl malonate and 21.4 g from HCl/acetone (yield 65%). Mass spectrum (FAB): (0.18 mol) of lithium diisopropylamide in 210 mL of THF m / e 554 (M H). IR (KBr): 1725 (C=O), 1510 and 1345 was cooled to -62 "C and 39.1 g (0.18 mol) of p-nitrobencm-1 (NO2). 1H NMR: 6 2.70 (4, 1H), 2.83 (d, 2 H), 3.05zyl bromide in 30 mL of T H F was added slowly. Stirring 4.15 (m, 24 H), 7.54 (d, 2 H), 8.19 (d, 2 H). 13C NMR: 6 was continued for 1 h. A solid was removed by filtration 33.018,37.446,51.005,52.004,53.937,54.370,54.659,60.777, and the remaining solution was evaporated to dryness. 124.939, 131.156, 147.125, 147.472, 171.946. Elemental Warm ethanol was added to the solid and filtered from C, Anal. Calcd for C~4H35N501~1.85Hz0.3.5HC1(714.2): the side products (mp 160 "C, disubstituted malonic acid 40.67; H, 5.97; N, 9.86; C1,17.05. Found C, 40.93; H, 6.04; diethyl ester). The pure product was obtained by N, 9.94; C1, 17.95. crystallization from the filtrate (yield 7096, mp 58-59 "C). 12-(pNitrobenzy1)-1,4,7,1O-tetraazacyclotridecaneIR (KBr): 3110-3080 (ar-C-H), 1735 (C=O), 1600 (ar1,4,7-triacetic acid (L2) was obtained by statistical alkyc-c), 1510 and 1345 cm-' (NOz). 'H NMR: 6 1.20 (t, 8 lation of 4 with 3.5 equiv bromoacetic acid at pH 9.5, 70 H), 3.50 (m, 3 H), 4.20 (q,4 H), 7.4 (d, 2 H), 8.10 (d, 2 H). "C (3 h), and purification as above (yield 45%). Mass Elemental Anal. Calcd for C14H17N106 (295.29): C, 56.9; spectrum (FAB): m / e 496.6 (M H). IR (KBr): 1725 H, 5.8; N, 4.7; 0, 32.5. Found: C, 57.0; H, 5.9; N, 4.7; 0, (C=O), 1510 and 1345 cm-l (NOz). lH NMR: 6 2.52 (s, 32.5. 1H), 2.79 (d, 2 H), 2.83-3.93 (m, 22 H), 7.49 (d, 2 H), 8.22 12-(pNitrobenzyl)-l,4,7,lO-tetraazacyclotrid~ane(d, 2 H). '3C NMR: 6 35.251, 37.579, 46.375, 50.062ll,l%-dione(3) was synthesized by aminolysis of 38.69 g 56.468 (m), 82.194, 125.088, 131.304, 147.808, 147.712, (0.131 mol) of 2 with 19.7 (0.135 mol) of 1,4,7,10-tetraaza169.441, 176.574, 176.449. Elemental Anal. Calcd for decane. The mixture was heated in 500 mL of ethanol for C2~H33N50gl.25H20-2.8HCl(624.64): C, 42.30; H, 6.26; 5 days. The product could be crystallized by slow N, 11.26; 0,24.33; C1, 15.90; HzO, 4.33. Found: C, 42.26; evaporation of the solvent (yield 2896, mp 265 "C dec). H, 6.38; N, 11.15; 0, 24.57; C1, 16.21; H20,4.26. 1,4,8,11Mass spectrum (FAB): m / e 350 (M + H). IR (KBr): 3310 Tetraazacyclotetradecane-1,4,8,1l-tetraaceticacid (TETA, (N-H), 3310-3060 (ar-C-H), 1670 (C=O), 1510 and 1345 L3) and 1,4,8,11-tetraazacyclotetradecane-1,4,8-triacetic cm-l (NOz). lH NMR: 6 2.6-3.6 (m, 15 H), 7.41 (d, 2 H), 8.13 (d, 2 H). I3C N M R 6 34.92,39.59,46.87,47.79,57.30, acid (TESA, L4) were synthesized according to published procedures (18, 14). 128.80, 129.80, 146.60, 146.90, 169.30. Elemental Anal. Rate of Formation of l11In Chelates. The rate of Calcd for C16HzsN504 (349.39): C, 55.0; H, 6.6; N, 20.0; 1111n3+complex formation was measured by scavenging of 0, 18.3. Found: C, 54.8; H, 6.7; N, 20.0; 0, 18.1. free ll1In3+with transferrin according to Scheme 11. The 12-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclotridemeasurements were performed under relevant antibody cane (4) was obtained by reducing 7.06 g (20.2 mmol) of labeling conditions. A carrier-free solution of 150 pL of 3 in 100 mL of THF with a large excess of diborane (220 "'InC13 in 0.1 N HC1 (0.5-2 mCi indium-111, depending mmol) and heating under reflux for 24 h. Repeated on specific activity) was mixed with 2 mL of 0.05 M sodium treatment with methanol and evaporation followed by 3 citrate (pH 6.5). This solution (400 pL) was mixed with h of heating under reflux with concentrated hydrochloric 10 pL of ligand solution in HzO (final ligand concentration acid gave, after evaporation to l / 3 the volume, 71 % of the 9-50 pM). The reaction (1)was quenched after appropriate cyclic tetraamine as a tetrahydrochloride salt. The free time intervals by mixing 10-pL aliquots of this reaction tetraamine was obtained by addition of KOH (pH 12) and mixture with 200 pL of human serum or 200 p L of 2 X ' ). Mass spectrum extraction with CHzClz (yield 95 % N H ,
H N ,
(NH
H N ,
+
+
Tetraazamecrocyclic Agents for Protein Labeling
Bioconjugate Chem., Vol. 1, No. 5, 1990 347
~
i QJ
o0 ~
20 "
"
40 "
"
60 " ' 80 ~ 100 120 Time ( m i d Figure 1. Rate of formation of 111indium chelates with L1 and L2. Ligand concentrations were 10 WM1mCi 1111nC13,with hob = 8 X lo-' s-l for L1 and 3.1 X 10" s-l for L2.
M transferrin in 0.1 M carbonate buffer, pH 7.4. This causes scavenging of free In3+. The mixture was subject to gel filtration (Sephadex G-50,l X 15 cm column). The column was eluted with 0.01 M PBS buffer (pH 7.4) at a flow rate of 37 mL per h. Samples were taken for 30 s after a dead volume of 4 min. After a total of 36 samples, >98% of the radioactivity was eluted from the column. In later experiments radioactive fractions were detected with a homemade continuous flow-through y-detector based on a Picker Model 628018 sodium iodide detector and a Canberra AMP/TSCA amplifier. Transferrin and other serum proteins were detected with a flow-through cell (1 mm) and a Beckman UV/VIS spectrophotometer set at 280 nm. 1111nC13given to human serum or transferrin both eluted off the column between 5 and 9 min whereas "'In chelates eluted between 10 and 25 min. The reaction between ll1In3+and transferrin is complete within 2-3 min. The percentage of ll1In3+scavenged by transferrin was used as a measure of free In3+. I t was calculated from the radioactivity of the respective fractions (Figure 1). Rate of lllIn3+ Exchange with Transferrin in Human Serum. This rate was measured by using similar strategies as described above (19,20). After an incubation time of 30 min-2 h for L1 and L2 with indium-111 (at least 30 h for L3 a n d L4 a n d 10 times higher ligand concentrations are necessary) the complex formation was complete. A 200-pL aliquot of this mixture was given to 2-8 mL of sterile human serum and incubated up to 7 days at 37 "C in a chamber maintained at 5 % CO2 and 95% air. At appropriate time intervals, 25 pL of this mixture was taken and subjected to gel filtration as described above. The percentage of 1111n3+transferred to transferrin as a function of time is shown in Figure 2. l3C N M R of In-L1 and In-L2. Solutions of the complexes for NMR measurements were made up in D2O and the pD was adjusted with NaOD. The final pD was determined by using the equation pD = pH + 0.4 (21).L1 (20 mg, 2.84 X mol) and 7.35 mg (1.42 X mol) of In~(S04)3 were dissolved in 750 pL of D20. The pD was kept at 4.2 with NaOD. The incorporation was complete within 1 h. TSP was added and the desired pD was adjusted with NaOD or DC1, respectively. In-L2 was synthesized likewise. RESULTS
Two new bifunctional macrocyclicligands (Ll, L2) based have been on 12-(p-nitrobenzyl)-l,4,7,lO-tetraazatridecane synthesized by extensive or statistical alkylation with bromoacetic acid. The overall yield of the four-step synthesis was 9.8% for L1 and 6.9% for L2. The ligands were
0"""""""""' 0
80 100 120 140 160 180 Time (h) Figure 2. Dissociation of 111indium chelates in human serum a t 37 "C, in air/COz (95/5%). The data points are from at least two ligand concentrations between 8 X lo-' and 3 X lo+ M and/ or two complex concentrations. The deviations were between 10 and 20%. 20
40
60
characterized by the usual analytical procedures: NMR, IR, mass spectra, and elemental analysis. The analytical data are in accordance with the structures depicted in Chart I. L2 appears to be a single compound as can be judged from TLC, ion-exchange chromatography, and reversed-phase HPLC. This is somewhat surprising as two isomers are possible [ lZ(p-nitrobenzyl)-1,4,7,10-tetraazacyclotridecane-1,4,7-triaceticacid or 12-(p-nitrobenzyl)1,4,7,10-tetraazacyclotridecane-1,4,lO-triacetic acid]. Extensive NMR studies (2D, COLOC, HCCORR) did not give any conclusion as to the isomeric form. The rate of In3+ chelate formation was determined as shown in Scheme 11. The data are plotted in Figure 1.The experimental data points follow pseudo-first-order kinetics with k,bs = 8 X s-l for L1 and 3.1 X s-l for L2, respectively, a t 10 pM ligand concentration. Both complexes form at ligand concentrations >30 pM within 30 min, giving >98% radiochemical yield. The stability of these chelates in human serum is shown in Figure 2. Four complexes (In-L1 through In-L4 are compared in this plot. L2, the heptadentate bifunctional tetraazamacrocycle, loses In3+ with a pseudo-first-order rate constant of k = 1 X 10-8 s-1 (i.e.
