0883°2889/92 $5.00+ 0.00 Copyright © 1992PergamonPress Ltd
Appl. Radiat. Isot. Vol. 43, No. 11, pp. 1387-1391, 1992 Int. J. Radiat. Appl. Instrum. Part A
Printed in Great Britain.All rights reserved
Development of 3-Iodophenylisothiocyanate for Radioiodination of Monoclonal Antibodies SIYA RAM* and DONALD
J. B U C H S B A U M
Department of Radiation Oncology, University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35233-6832, U.S.A. (Received 30 January 1992)
A new radioiodination reagent, 3-iodophenylisothiocyanate (3-IPI) has been developed for coupling to monoclonal antibodies. The starting material, 3-tri-n-butylstannylphenylisothiocyanate was prepared via a reaction of hexabutylditin with 3-bromoaniline, followed by treatment with thiophosgene with an overall yield of 72%. The radioiodination of this tin precursor with Na[125I]I/iodogen in chloroform gave 3-[125I]IPI in 23-55% radiochemical yield and 81-99.6% radiochemical purity. Purification of the impure product by high pressure liquid chromatography increased the radiochemical purity of the product up to 99%. These results suggest that 3-IPI may be a useful ligand for radioiodination and coupling to a variety of monoclonal antibodies.
Introduction Direct radioiodination of monoclonal antibodies has been studied extensively in radioimmunodiagnosis and radioimmunotherapy of cancer. However, in vivo deiodination of directly labeled antibodies results in uptake of free radioiodide in the thyroid and stomach. There is also some loss of immunoreactivity of directly radioiodinated antibodies. This has led to a search to develop new radioiodination reagents which are more stable in vivo and retain greater binding to target antigens. Recently, a variety of radioiodinated reagents such as 3/4-iodobenzoic acids (Wilbur et al., 1991; Zalutsky and Narula, 1987; Vaidyanathan and Zalutsky, 1990), 5-iodo-3pyridinecarboxylates (Garg et al., 1991), and N(iodophenyl/iodophenethyl) maleimide derivatives (Srivastava et al., 1990; Wilbur et al., 1991) have been developed and antibodies coupled to these reagents have been shown to be less prone to deiodination in vivo than directly radioiodinated antibodies. A majority of these reagents possess an active ester group except the maleimide derivatives. The coupling of maleimide derivatives with antibody is only possible when the antibody possesses free-SH groups, which are generally obtained via reduction of the intrachain disulfide bonds. These observations further suggest that there is a need to develop a suitable radioiodination reagent which is free from the above shortcomings. *Author for correspondence.
In the case of aromatic acids, an active ester group of the ligand reacts with the e-amino group of lysine and forms a stable immunoconjugate via an amide linkage. Recently, a variety of compounds containing isothiocyanate groups have been synthesized and successfully coupled to monoclonal antibodies (Linder et al., 1991a,b; Kozak et al., 1989; Deshpande et al., 1990; Gansow et al., 1990; Subramanian and Meares, 1990; Levy and Dawson, 1976). These radioimmunoconjugates have been found to be stable in vivo.
Based on this information, we have designed and synthesized 3-iodophenylisothiocyanate (3-IPI) which we believe has considerable potential for radioiodination and coupling to monoclonal antibodies. This radioligand on coupling to monoclonal antibodies should generate a stable bioconjugate via a thiourea linkage.
Experimental All chemicals were of research grade and were used as obtained from the commercial suppliers. Nocarrier-added Na[~25I]I in 0.1 N NaOH was obtained from Dupont/NEN, Boston, MA. Silica gel Sep-Paks were obtained from Whatman Laboratory Division, Clifton, NJ. High pressure liquid chromatography (HPLC) (Biorad Laboratories, Richmond, CA) was performed with a 10 # m silica gel column (Adsorbosphere, Altech Associate~, Inc., Deerfield, IL), size 4.6 x 250 mm, with hexane as eluant at a flow rate of
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SIYA RAM and DONALD J. BUCHSnAUM
1 mL/min. Separated components were monitored by u.v. absorption at 280 nm and with a radioisotope detector Model 170 (Beckman Instruments, Inc., Irvine, CA). I H - N M R spectra were generated on the Brucker-WH 400MHz spectrometer in the University of Alabama at Birmingham N M R Center. Thin layer chromatography (TLC) was performed on E. Merck silica gel plates (Altech Associates, Inc., Deerfield, IL) using hexane as the mobile phase. Infrared spectra were recorded on a spectrometer Model 1605 (Perkin-Elmer, Norwalk, CT). The crude product was purified by silica gel chromatography [5 mL syringe containing 1 g of silica gel attached to a Sep-Pak cartridge (500 mg), mobile phase : hexane]. Radiochemical yields of labeled products were determined with a dose-calibrator CRC-15R (Capintec, Ramsey, N J). For the determination of the radiochemical purity of 3-[~25I]IPI, the TLC plate was cut into 10 strips (0.5cm width each). Each strip was placed in a thin walled plastic vial and counted in a Minaxi-gamma 5000 Series gamma counter (Packard, Chicago, IL). The cold 3-IPI was obtained from Fairfield Chemical Company (Blythewood, SC). The GC analysis was performed with a DB5, fused-silica coated capillary column, 4 m x 320 p m i.d., temperature programmed for 125 to 250°C with a rate of 8°C/min, flow of helium gas 15mL/min (Varian 3400 Gas Chromatograph, Sugarland, TX). Mass spectra were taken with a VG-70S high resolution mass spectrometer (VG Analytical, U.K.).
Synthesis of 3-tri-n-butylstannylaniline (2) Compound 2 was prepared by a modification of a previously described procedure (Khawli et al., 1991). To a solution of 3-bromoaniline, 1 (1.72 g, 0.01 mol) in triethylamine (12mL), tetrakis(triphenylphosphine)palladium(0) (0.2 g, 1.73 x 10 -4mOl) was added, followed by the addition of hexabutylditin (7.4 g, 0.013 mol) under an argon atmosphere. The resulting reaction mixture was stirred at 100°C for 16h. The resulting black color solution was diluted with CH2CI 2 (80 mL) and filtered through a celite pad. The filtrate was evaporated under reduced pressure to afford crude stannane 2. The crude product was purified by silica gel charomatography (column size 2 cm x 45 cm, silica gel, 30 g, 100-200 mesh) using hexane:ethyl acetate (100-90:0-10) mixture as the eluant. The appropriate fractions were combined and evaporated under vacuum to produce 1.6g (70.2%) of 2. TLC/silica gel [hexane: CHC13 (3 : 2)], Rr for bromoaniline = 0.682, Rf for product = 0.473, 1H-NMR (CDC13) 60.9 (t, 9H, 3 x CH3, J = 7.3 Hz) 1.0-1.04 (m, 5H, C---CH2---C), 1.32 (q, 8H, ---CH2, J = 7 . 5 H z ) , 1.45-1.58 (m, 5H, --CH2), 6.63 (dd, IH, A r - - H , J = 1.0Hz), 6.78 (d, 1H, A r - - H , J = 2 . 4 H z ) , 6.85 (d, 1H, Ar--H, J = 0.8 Hz), 7.13 (t, IH, A r - - H , J = 7.53 Hz).
Synthesis of 3-tri-n-butylstannylphenylisothiocyanate (3) To an ice cold suspension of calcium carbonate (0.4 g, 0.004 mol) and thiophosgene (0.182 g, 0.0016 mol) in water/CHCl 3 (1:1) (16 mL), a solution of amino compound 2 (0.6 g, 0.0016 mol) in CHCI 3 (2mL) was added dropwise. The progress of the reaction was monitored by TLC/silica gel/hexane. After stirring for 2 h, CHC13 (30 mL) was added, followed by the addition of 1 N HC! solution (10 mL). The organic layer was removed quickly from the aqueous layer. The organic layer was washed with water (10 mL), dried over Na2SO4, and filtered. The organic layer on evaporation under reduced pressure, afforded 0.49 g (74%) of 3 as a brown oil. TLC/silica gel/hexane, Rf for product = 0.778, R r for starting material=0.067; i.r. (neat) 2956 ( - - C H 2 ~ H 2 ) , 2104 (--NCS), 781 and 688 ( A r - - H ) cm-J. I H - N M R (CDCI3) 60.9 (t, 9H, 3 × CH3, J = 7.3 Hz), 1.07 (m, 6H, 3 x CH2), 1.34 (q, 6H, 3 x C---CHE, J = 7.4 Hz), 1.52 (m, 6H, C - - C - - C H 2 ) , 7.15 (dt, 1H, A r - - H , J = 1.5 Hz, J = 1.9 Hz), 7.27-7.38 (m, 3H, A r - - H ) ; H R M S (re~e) calcd for CIsH22NSSn 368.045, found 368.0462.
Synthesis of 3-[12sI]IPI (4) A mixture of 3 (300/~g, 6.97 x 10 -7 mol), Iodogen (300/~g, 6.97x 10-7mol), acetic acid (5/~L) and Na[125I]I (1.93 mCi, 20/~L solution of 0.1 N NaOH) in chloroform (30~0/~L) was stirred for 1 h at room temperature. Then a 5°/'0 aqueous solution of sodium metabisulfite (6/~ L) was added. The resulting reaction mixture was loaded over a silica gel column [5 ml syringe containing 1 g of silica gel attached to a Sep-Pak (500mg)] and eluted with hexane (5 mL). The hexane layer was evaporated by passing an argon stream through the septum using two 18 G 1!,, "2 needles for the inlet and outlet. The outlet needle was connected with a 3 mL syringe which was filled with charcoal in order to adsorb any volatile iodine. After evaporation, 1.07 mCi (55%) of the product with a radiochemical purity of 99.6% was obtained as determined by TLC. The radiochemical purity of 3-[nsI]IPI prepared in this way was also checked by HPLC under the conditions reported above.
Purification of 4 by' HPLC The crude 3-[125I]IPI was purified by HPLC under the experimental conditions described in the Experimental section. The retention time for 3-[125I]IPI was 4.5 min. During elution of the radioactive peak, 2min fractions were collected. The radioactivity in each fraction was measured in a dose calibrator. The first fraction showed the majority of activity and the second fraction possessed a trace amount of activity. For example, the purification of crude 3-[125I]IPI (229/~Ci) by HPLC provided 116#Ci of pure 4 with a radiochemical purity of 990.
Synthesis of 3-iodophenylisothiocyanate
Results and Discussion
Synthesis of 3-IPI and precursor for radiolabeling The synthetic strategy for the preparation of 3-IPI is illustrated in Scheme 1. The starting material, 3-tri-n-butylstannylphenylisothiocyanate 3 was prepared in two steps from 3-bromoaniline. The literature procedure (Khawli et al., 1991) for the preparation of 3-tri-n-butylstannylaniline 2 was unsuccessful in our hands. However, reaction of 3-bromoaniline with hexabutylditin/(PPh3)4Pd(o ) in triethylamine at 100°C, followed by purification over a silica gel column gave compound 2 in 70% yield. The 3-tri-n-butylstannylaniline on treatment with CHClflCaCO3 afforded 3-tri-n-butylstannylphenylisothiocyanate 3 in 74% yield. The formation of compound 3 was confirmed by i.r. which gave clear evidence for the formation of an isothiocyanate group (2104cm - I ) and ~H-NMR. The high resolution mass spectrum of the purified compound 3 showed molecular ion peaks (M-57) at 364, 366 and 368 in the appropriate abundance for H6Sn, HaSn and 12°Sn isotopic contributions. The conversion of compound 3 into 3-IPI was studied under a variety of conditions. The reaction of iodine, and NaI N-chlorosuccinimide (NCS) with 3 in CHCI 3 was monitored by TLC and GC under the conditions as described in the Experimental section. The retention times for laboratory synthesized 3-IPI and the starting material were 4.72 and 11.89 min, respectively. The retention time for authentic 3-IPI was 4.75 min. In the case of iodine, the reaction was over within 7 min, while NaI/NCS required 10 min for the completion of the reaction, when 1.4 mol of NaI was used for 1 mol each of NCS and the tin compound. The use of iodogen instead of NCS provided a good yield of 3-IPI under the conditions reported above. In order to optimize the reaction conditions, the effect of solvent was studied. When
NH2
NH2 [PPh3]4Pd I Et3N
SnBu3
Br 1
2 /CSCI2/CaC03
NCS
NCS
*1
SnBu3
4
3 *1 • 131D2Sl
Scheme 1. Synthetic strategy for the preparation of 3-[12~/~311]IPI.
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chloroform or chloroform containing 7-10% acetic acid was used as solvent, a good yield of 3-IPI was obtained (Table 1). However, use of either excess of acetic acid in CHCI3 or acetic acid as a solvent in place of CHC13 formed a complex mixture which was untractable. Similar results were obtained when the reaction mixture was heated at 80°C. In the demetallation of 3-tri-n-butylstannylphenylisothiocyanate 3 with an electrophilic iodine, one might expect that iodine can form an addition complex with the isothiocyanate group. However, in the experimental conditions reported here, no such complex formation was observed. These results are further supported by Dewanjee et aL (1990) who recently reported the preparation of radiolabeled 3-iodo-4-methoxyphenylisothiocyanate via electrophilic iodination of 4-methoxyphenylisothiocyanate in good yield. The identity of the isolated product was established by spectroscopic data (1HN M R and i.r.) and chromatographic data (TLC, GC and HPLC). These observations suggest that a chloroform: acetic acid system and iodogen are ideal for this reaction.
[12sI]-Labeling The radiochemical yield and purity data for 3-[~25I]IPI prepared on separate occasions (N = 11) are shown in Table 1. The radiochemical yields obtained in CHC13/AcOH were 23-55% with a radiochemical purity in the range of 81-99.6%. The time required for manual synthesis and purification was 1.5 h after adding Na[~25I]I. For example, 129/~Ci of Na[ ~25I]I on reaction with the tin compound provided 51.5#Ci of final product within 1.25h. Initially, compound 4 was purified over a silica gel column using CHCI 3 as eluant. The radiochemical purity of 3-[~25I]IPI was found to be between 81-93% as determined by TLC. When we replaced the chloroform with hexane the purity of 3-[~25I]IPI was increased (Table 1). These results indicate that unreacted trace radioiodide is eluted along with the desired compound which decreased the radiochemical purity of the final product. Alternatively, radioiodide is more soluble in polar solvents such as chloroform while the solubility of iodide is negligible in nonpolar solvents such as hexane (Table 1). Based on these observations, hexane was selected as the preferred solvent for the purification of this compound. The final product, 3-[125I]IPI, was characterized on the basis of its chromatographic behavior (HPLC, TLC) by comparing it with an authentic sample of cold 3-IPI. The effect of substrate/iodogen molar ratio was also studied. The results showed that a substrate/iodogen ratio of 1 is ideal for this reaction (Table 1). On HPLC cold 3-IPI had a retention time of 4.2 min under the conditions described in the Experimental section. However, HPLC of 3-[tzsI]IPI showed one peak at 4.5 min. The Rr values of cold 3-IPI and its precursor on TLC using silica gel, and
S1YARAM and DONALDJ. BUCHSBAUM
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Table I. Effectof solvent and oxidizingagent on the radiochemicalyield of 3-[12sIIIPI prepared by reaction of Na[TM I]I and compound 3 Substrate lodogen Reaction Radiochemical Radiochemical Entry Solvent (#mol) (#mol) time (min) yield (%)b purity (%)c 1 2 3 4 5 6 7 8 9 10 11
CHCI3 CHCI3 CHC13 CHCI3 CHCI3 CHCI3/AcOH~ CHCI3/AeOHa CHC13/AcOH a CHC13/AcOH a CHCI3/AcOH a CHCI3/AcOH a
2.32 3.39 3.49 1.70 1.0 0.93 0.93 0.70 0.70 0.70 0.70
1.07 1.85 1.37 4.63 6.94 2.78 2.78 2.78 0.70 0.70 0.70
25 15 25 20 50 225 60 20 20 40 72
39 49 35 37 27 23 40 26 31 46 55
81d 81d 87d 89d 84d 93a 92d 88d 93d 98.5~ 99.6e
' 5 ~ #L acetic acid was added. bIsolated yields. CRadiochemical purity was determined by TLC. dColumn was eluted with CHCI3. ~Column was eluted with hexane.
hexane as a mobile phase were 0.70 and 0.78, respectively. The Rf value o f the synthesized 3-IPI, as well as 3-[ 125I]IPI were identical to an authentic sample o f 3-IPI. The above physicochemical data unequivocally d e m o n s t r a t e the identity, chemical purity and radiochemical purity o f our product. The radiochemical purity o f 3-[125I]IPI was also determined by T L C and H P L C . It should also be n o t e d that this new radioiodination reagent for coupling to m o n o c i o n a l antibodies does not require H P L C separation. The coupling o f this new ligand with 17-1A and D612 m o n o c l o n a l antibodies reactive with h u m a n colon cancer has been studied ( R a m et al., 1992a,b) in our laboratory. The r a d i o i m m u n o c o n j u g a t e s 3-['25I]IPI17-1A and 3-[125I]IPI-D612 showed similar uptake in LS174T colon cancer xenografts and m o s t normal tissues in athymic nude mice c o m p a r e d to the directly radioiodinated 17-1A and D612 antibodies, respectively. However, thryoid uptake for b o t h new radioi m m u n o c o n j u g a t e s was significantly lower than 1311-17-1A and 131I-D612. The detailed results o f this study will be reported elsewhere ( R a m et al,, 1992b).
Acknowledgements--This work was supported by the NIH Grant CA 44173. We acknowledge the support of Dr Merle M. Salter, valuable assistance from Dr M. B. Khazaeli, and Ms Elisa Fleming for technical help. We thank Mrs Renee Kite for her efforts in preparing this manuscript and the UAB NMR Center for ~H-NMR data.
References Deshpande S. V., Subramanian R., McCall M. J., DeNardo S. J., DeNardo G. L. and Meares C. F. (1990) Metabolism of indium chelates attached to monoclonal antibody: minimal transchelation of indium from benzyl-EDTA chelate in vivo. J. Nucl. Med. 31, 218. Dewanjee M. K., Ghafouripour A. K., Ganz W., McCarthy K., Serafini A. and Sfakianakis G. (1990) Radioiodination of proteins by a new conjugation technique with activated methoxyphenylisothiocyanate.J. Nucl. Med. 31, 907 (abstract). Gansow O. A., Brechbiel M. W., Mirzadeh S., Colcher D. and Roselli M. (1990) Chelates and antibodies: current
methods and new directions. In Cancer Imaging with Radiolabeled Antibodies (Ed. Goldenberg D. M.), p. 153. Kluwer Academic Publishers, Boston. Garg S., Garg P. and Zalutsky M. R. (1991) N-succinimidyl 5-(trialkylstannyl)-3-pyridine carboxylates. A new class of reagents for protein radioiodination. Bioconjugate Chem. 2, 50. Hylarides M. D., Wilbur D. S, Reed M. W., Hadley S. W., Schroeder J. R. and Grant L. M. (1991) Preparation and in vivo evaluation of an N-(p-[125I]iodophenethyl) maleimide-antibody conjugate. Bioconjugate Chem. 2, 435. Khawli L. A., Chen F.-M., Alauddin M. M. and Epstein A. L. (1991) Radioiodinated monoclonal antibody conjugates: synthesis and comparative evaluation. Antibody, Immunoconjugates Radiopharmac. 4, 163. Kozak R. W., Raubitschek A., Mirzadeh S., Brechbiel M. W., Junghaus R., Gansow O. A. and Waldman 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. Levy N. L. and Dawson J.R. (1976) Radiolabeling of immunoglobulin without loss of antibody activity-use of ~4C-phenylisothiocyanate with human cytotoxic antibody. J. Immunol. 116, 1526. Linder K. E., Wen M. D., Nowotnik D. P., Malley M. F., Gougoutas J. Z., Nunn A. D. and Eckelman W. C. (1991a) Technetium labeling of monoclonal antibodies with functionalized BATOs. 1. TcCI(DMG)3 PITC. Bioconjugate Chem. 2, 160. Linder K. E., Wen M. D., Nowotnik D. P., Ramalingam K., Sharkey R. M., Yost F., Narra R. K., Nunn A. D. and Eckelman W. C. (1991b) Technetium labeling of monoclonal antibodies with functionalized BATOs: 2. TcCI(DMG)3 CPITC labeling of B72.3 and NP-4 whole antibodies and NP-4 F(ab')2. Bioconjugate Chem. 2, 407. Ram S., Fleming E. and Buchsbaum D. J. (1992a) Development of radioiodinated 3-iodophenylisothiocyanate for coupling to monoclonal antibodies. J. Nucl. Med. 33, 1209 (abstract) Ram S., Khazaeli M. B., Harrison P., Fleming E., Bright S., Jones P. and Buchsbaum D. J. (1992b) Biodistribution and tumor localization of 3-[125I]iodophenylisothiocyanate labeled monoclonal antibodies 17-1A and D612 in nude mice bearing colon cancer xenografts. Antibody, Immunoconjugates Radiopharmac (in press). Srivastava P. C., Buchsbaum D. J., Allred J. F., Brubaker P. G., Hanna D. E. and Spicker J. K. (1990) A new conjugating agent for radioiodination of proteins: low
Synthesis of 3-iodophenylisothiocyanate in vivo deiodination of a radiolabeled antibody in a tumor model. BioTechniques g, 536. Subramanian R. and Meares C. F. (1990) Bifunctional chelating agents for radiometal-labeled monoclonal antibodies. In Cancer Imaging with Radiolabeled Antibodies (Ed. Goldenberg D. M.), p. 183. Kluwer Academic Publishers, Boston. Vaidyanathan G. and Zalutsky M. R. (1990) Radioiodination of antibodies via N-succinimidyl 2, 4-dimethoxy-
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3-(trialkylstannyl)benzoates. Bioconjugate Chem. 1, 387. Wilbur D. S., Hadley S. W., Grant L. M. and Hylarides M. D. (1991) Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T labeled Fab. Bioconjugate Chem. 2, I l l . Zalutsky M. R. and Narula A. S. (1987) A method for the radiohalogenation of proteins resulting in decreased thryoid uptake of radioiodine. Appl. Radiat. lsot. 38, 1051.
Appl. Radiat. Isot. Vol. 43, No. 11, pp. 1387-1391, 1992 Int. J. Radiat. Appl. Instrum. Part A
Printed in Great Britain.All rights reserved
Development of 3-Iodophenylisothiocyanate for Radioiodination of Monoclonal Antibodies SIYA RAM* and DONALD
J. B U C H S B A U M
Department of Radiation Oncology, University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35233-6832, U.S.A. (Received 30 January 1992)
A new radioiodination reagent, 3-iodophenylisothiocyanate (3-IPI) has been developed for coupling to monoclonal antibodies. The starting material, 3-tri-n-butylstannylphenylisothiocyanate was prepared via a reaction of hexabutylditin with 3-bromoaniline, followed by treatment with thiophosgene with an overall yield of 72%. The radioiodination of this tin precursor with Na[125I]I/iodogen in chloroform gave 3-[125I]IPI in 23-55% radiochemical yield and 81-99.6% radiochemical purity. Purification of the impure product by high pressure liquid chromatography increased the radiochemical purity of the product up to 99%. These results suggest that 3-IPI may be a useful ligand for radioiodination and coupling to a variety of monoclonal antibodies.
Introduction Direct radioiodination of monoclonal antibodies has been studied extensively in radioimmunodiagnosis and radioimmunotherapy of cancer. However, in vivo deiodination of directly labeled antibodies results in uptake of free radioiodide in the thyroid and stomach. There is also some loss of immunoreactivity of directly radioiodinated antibodies. This has led to a search to develop new radioiodination reagents which are more stable in vivo and retain greater binding to target antigens. Recently, a variety of radioiodinated reagents such as 3/4-iodobenzoic acids (Wilbur et al., 1991; Zalutsky and Narula, 1987; Vaidyanathan and Zalutsky, 1990), 5-iodo-3pyridinecarboxylates (Garg et al., 1991), and N(iodophenyl/iodophenethyl) maleimide derivatives (Srivastava et al., 1990; Wilbur et al., 1991) have been developed and antibodies coupled to these reagents have been shown to be less prone to deiodination in vivo than directly radioiodinated antibodies. A majority of these reagents possess an active ester group except the maleimide derivatives. The coupling of maleimide derivatives with antibody is only possible when the antibody possesses free-SH groups, which are generally obtained via reduction of the intrachain disulfide bonds. These observations further suggest that there is a need to develop a suitable radioiodination reagent which is free from the above shortcomings. *Author for correspondence.
In the case of aromatic acids, an active ester group of the ligand reacts with the e-amino group of lysine and forms a stable immunoconjugate via an amide linkage. Recently, a variety of compounds containing isothiocyanate groups have been synthesized and successfully coupled to monoclonal antibodies (Linder et al., 1991a,b; Kozak et al., 1989; Deshpande et al., 1990; Gansow et al., 1990; Subramanian and Meares, 1990; Levy and Dawson, 1976). These radioimmunoconjugates have been found to be stable in vivo.
Based on this information, we have designed and synthesized 3-iodophenylisothiocyanate (3-IPI) which we believe has considerable potential for radioiodination and coupling to monoclonal antibodies. This radioligand on coupling to monoclonal antibodies should generate a stable bioconjugate via a thiourea linkage.
Experimental All chemicals were of research grade and were used as obtained from the commercial suppliers. Nocarrier-added Na[~25I]I in 0.1 N NaOH was obtained from Dupont/NEN, Boston, MA. Silica gel Sep-Paks were obtained from Whatman Laboratory Division, Clifton, NJ. High pressure liquid chromatography (HPLC) (Biorad Laboratories, Richmond, CA) was performed with a 10 # m silica gel column (Adsorbosphere, Altech Associate~, Inc., Deerfield, IL), size 4.6 x 250 mm, with hexane as eluant at a flow rate of
1387
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SIYA RAM and DONALD J. BUCHSnAUM
1 mL/min. Separated components were monitored by u.v. absorption at 280 nm and with a radioisotope detector Model 170 (Beckman Instruments, Inc., Irvine, CA). I H - N M R spectra were generated on the Brucker-WH 400MHz spectrometer in the University of Alabama at Birmingham N M R Center. Thin layer chromatography (TLC) was performed on E. Merck silica gel plates (Altech Associates, Inc., Deerfield, IL) using hexane as the mobile phase. Infrared spectra were recorded on a spectrometer Model 1605 (Perkin-Elmer, Norwalk, CT). The crude product was purified by silica gel chromatography [5 mL syringe containing 1 g of silica gel attached to a Sep-Pak cartridge (500 mg), mobile phase : hexane]. Radiochemical yields of labeled products were determined with a dose-calibrator CRC-15R (Capintec, Ramsey, N J). For the determination of the radiochemical purity of 3-[~25I]IPI, the TLC plate was cut into 10 strips (0.5cm width each). Each strip was placed in a thin walled plastic vial and counted in a Minaxi-gamma 5000 Series gamma counter (Packard, Chicago, IL). The cold 3-IPI was obtained from Fairfield Chemical Company (Blythewood, SC). The GC analysis was performed with a DB5, fused-silica coated capillary column, 4 m x 320 p m i.d., temperature programmed for 125 to 250°C with a rate of 8°C/min, flow of helium gas 15mL/min (Varian 3400 Gas Chromatograph, Sugarland, TX). Mass spectra were taken with a VG-70S high resolution mass spectrometer (VG Analytical, U.K.).
Synthesis of 3-tri-n-butylstannylaniline (2) Compound 2 was prepared by a modification of a previously described procedure (Khawli et al., 1991). To a solution of 3-bromoaniline, 1 (1.72 g, 0.01 mol) in triethylamine (12mL), tetrakis(triphenylphosphine)palladium(0) (0.2 g, 1.73 x 10 -4mOl) was added, followed by the addition of hexabutylditin (7.4 g, 0.013 mol) under an argon atmosphere. The resulting reaction mixture was stirred at 100°C for 16h. The resulting black color solution was diluted with CH2CI 2 (80 mL) and filtered through a celite pad. The filtrate was evaporated under reduced pressure to afford crude stannane 2. The crude product was purified by silica gel charomatography (column size 2 cm x 45 cm, silica gel, 30 g, 100-200 mesh) using hexane:ethyl acetate (100-90:0-10) mixture as the eluant. The appropriate fractions were combined and evaporated under vacuum to produce 1.6g (70.2%) of 2. TLC/silica gel [hexane: CHC13 (3 : 2)], Rr for bromoaniline = 0.682, Rf for product = 0.473, 1H-NMR (CDC13) 60.9 (t, 9H, 3 x CH3, J = 7.3 Hz) 1.0-1.04 (m, 5H, C---CH2---C), 1.32 (q, 8H, ---CH2, J = 7 . 5 H z ) , 1.45-1.58 (m, 5H, --CH2), 6.63 (dd, IH, A r - - H , J = 1.0Hz), 6.78 (d, 1H, A r - - H , J = 2 . 4 H z ) , 6.85 (d, 1H, Ar--H, J = 0.8 Hz), 7.13 (t, IH, A r - - H , J = 7.53 Hz).
Synthesis of 3-tri-n-butylstannylphenylisothiocyanate (3) To an ice cold suspension of calcium carbonate (0.4 g, 0.004 mol) and thiophosgene (0.182 g, 0.0016 mol) in water/CHCl 3 (1:1) (16 mL), a solution of amino compound 2 (0.6 g, 0.0016 mol) in CHCI 3 (2mL) was added dropwise. The progress of the reaction was monitored by TLC/silica gel/hexane. After stirring for 2 h, CHC13 (30 mL) was added, followed by the addition of 1 N HC! solution (10 mL). The organic layer was removed quickly from the aqueous layer. The organic layer was washed with water (10 mL), dried over Na2SO4, and filtered. The organic layer on evaporation under reduced pressure, afforded 0.49 g (74%) of 3 as a brown oil. TLC/silica gel/hexane, Rf for product = 0.778, R r for starting material=0.067; i.r. (neat) 2956 ( - - C H 2 ~ H 2 ) , 2104 (--NCS), 781 and 688 ( A r - - H ) cm-J. I H - N M R (CDCI3) 60.9 (t, 9H, 3 × CH3, J = 7.3 Hz), 1.07 (m, 6H, 3 x CH2), 1.34 (q, 6H, 3 x C---CHE, J = 7.4 Hz), 1.52 (m, 6H, C - - C - - C H 2 ) , 7.15 (dt, 1H, A r - - H , J = 1.5 Hz, J = 1.9 Hz), 7.27-7.38 (m, 3H, A r - - H ) ; H R M S (re~e) calcd for CIsH22NSSn 368.045, found 368.0462.
Synthesis of 3-[12sI]IPI (4) A mixture of 3 (300/~g, 6.97 x 10 -7 mol), Iodogen (300/~g, 6.97x 10-7mol), acetic acid (5/~L) and Na[125I]I (1.93 mCi, 20/~L solution of 0.1 N NaOH) in chloroform (30~0/~L) was stirred for 1 h at room temperature. Then a 5°/'0 aqueous solution of sodium metabisulfite (6/~ L) was added. The resulting reaction mixture was loaded over a silica gel column [5 ml syringe containing 1 g of silica gel attached to a Sep-Pak (500mg)] and eluted with hexane (5 mL). The hexane layer was evaporated by passing an argon stream through the septum using two 18 G 1!,, "2 needles for the inlet and outlet. The outlet needle was connected with a 3 mL syringe which was filled with charcoal in order to adsorb any volatile iodine. After evaporation, 1.07 mCi (55%) of the product with a radiochemical purity of 99.6% was obtained as determined by TLC. The radiochemical purity of 3-[nsI]IPI prepared in this way was also checked by HPLC under the conditions reported above.
Purification of 4 by' HPLC The crude 3-[125I]IPI was purified by HPLC under the experimental conditions described in the Experimental section. The retention time for 3-[125I]IPI was 4.5 min. During elution of the radioactive peak, 2min fractions were collected. The radioactivity in each fraction was measured in a dose calibrator. The first fraction showed the majority of activity and the second fraction possessed a trace amount of activity. For example, the purification of crude 3-[125I]IPI (229/~Ci) by HPLC provided 116#Ci of pure 4 with a radiochemical purity of 990.
Synthesis of 3-iodophenylisothiocyanate
Results and Discussion
Synthesis of 3-IPI and precursor for radiolabeling The synthetic strategy for the preparation of 3-IPI is illustrated in Scheme 1. The starting material, 3-tri-n-butylstannylphenylisothiocyanate 3 was prepared in two steps from 3-bromoaniline. The literature procedure (Khawli et al., 1991) for the preparation of 3-tri-n-butylstannylaniline 2 was unsuccessful in our hands. However, reaction of 3-bromoaniline with hexabutylditin/(PPh3)4Pd(o ) in triethylamine at 100°C, followed by purification over a silica gel column gave compound 2 in 70% yield. The 3-tri-n-butylstannylaniline on treatment with CHClflCaCO3 afforded 3-tri-n-butylstannylphenylisothiocyanate 3 in 74% yield. The formation of compound 3 was confirmed by i.r. which gave clear evidence for the formation of an isothiocyanate group (2104cm - I ) and ~H-NMR. The high resolution mass spectrum of the purified compound 3 showed molecular ion peaks (M-57) at 364, 366 and 368 in the appropriate abundance for H6Sn, HaSn and 12°Sn isotopic contributions. The conversion of compound 3 into 3-IPI was studied under a variety of conditions. The reaction of iodine, and NaI N-chlorosuccinimide (NCS) with 3 in CHCI 3 was monitored by TLC and GC under the conditions as described in the Experimental section. The retention times for laboratory synthesized 3-IPI and the starting material were 4.72 and 11.89 min, respectively. The retention time for authentic 3-IPI was 4.75 min. In the case of iodine, the reaction was over within 7 min, while NaI/NCS required 10 min for the completion of the reaction, when 1.4 mol of NaI was used for 1 mol each of NCS and the tin compound. The use of iodogen instead of NCS provided a good yield of 3-IPI under the conditions reported above. In order to optimize the reaction conditions, the effect of solvent was studied. When
NH2
NH2 [PPh3]4Pd I Et3N
SnBu3
Br 1
2 /CSCI2/CaC03
NCS
NCS
*1
SnBu3
4
3 *1 • 131D2Sl
Scheme 1. Synthetic strategy for the preparation of 3-[12~/~311]IPI.
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chloroform or chloroform containing 7-10% acetic acid was used as solvent, a good yield of 3-IPI was obtained (Table 1). However, use of either excess of acetic acid in CHCI3 or acetic acid as a solvent in place of CHC13 formed a complex mixture which was untractable. Similar results were obtained when the reaction mixture was heated at 80°C. In the demetallation of 3-tri-n-butylstannylphenylisothiocyanate 3 with an electrophilic iodine, one might expect that iodine can form an addition complex with the isothiocyanate group. However, in the experimental conditions reported here, no such complex formation was observed. These results are further supported by Dewanjee et aL (1990) who recently reported the preparation of radiolabeled 3-iodo-4-methoxyphenylisothiocyanate via electrophilic iodination of 4-methoxyphenylisothiocyanate in good yield. The identity of the isolated product was established by spectroscopic data (1HN M R and i.r.) and chromatographic data (TLC, GC and HPLC). These observations suggest that a chloroform: acetic acid system and iodogen are ideal for this reaction.
[12sI]-Labeling The radiochemical yield and purity data for 3-[~25I]IPI prepared on separate occasions (N = 11) are shown in Table 1. The radiochemical yields obtained in CHC13/AcOH were 23-55% with a radiochemical purity in the range of 81-99.6%. The time required for manual synthesis and purification was 1.5 h after adding Na[~25I]I. For example, 129/~Ci of Na[ ~25I]I on reaction with the tin compound provided 51.5#Ci of final product within 1.25h. Initially, compound 4 was purified over a silica gel column using CHCI 3 as eluant. The radiochemical purity of 3-[~25I]IPI was found to be between 81-93% as determined by TLC. When we replaced the chloroform with hexane the purity of 3-[~25I]IPI was increased (Table 1). These results indicate that unreacted trace radioiodide is eluted along with the desired compound which decreased the radiochemical purity of the final product. Alternatively, radioiodide is more soluble in polar solvents such as chloroform while the solubility of iodide is negligible in nonpolar solvents such as hexane (Table 1). Based on these observations, hexane was selected as the preferred solvent for the purification of this compound. The final product, 3-[125I]IPI, was characterized on the basis of its chromatographic behavior (HPLC, TLC) by comparing it with an authentic sample of cold 3-IPI. The effect of substrate/iodogen molar ratio was also studied. The results showed that a substrate/iodogen ratio of 1 is ideal for this reaction (Table 1). On HPLC cold 3-IPI had a retention time of 4.2 min under the conditions described in the Experimental section. However, HPLC of 3-[tzsI]IPI showed one peak at 4.5 min. The Rr values of cold 3-IPI and its precursor on TLC using silica gel, and
S1YARAM and DONALDJ. BUCHSBAUM
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Table I. Effectof solvent and oxidizingagent on the radiochemicalyield of 3-[12sIIIPI prepared by reaction of Na[TM I]I and compound 3 Substrate lodogen Reaction Radiochemical Radiochemical Entry Solvent (#mol) (#mol) time (min) yield (%)b purity (%)c 1 2 3 4 5 6 7 8 9 10 11
CHCI3 CHCI3 CHC13 CHCI3 CHCI3 CHCI3/AcOH~ CHCI3/AeOHa CHC13/AcOH a CHC13/AcOH a CHCI3/AcOH a CHCI3/AcOH a
2.32 3.39 3.49 1.70 1.0 0.93 0.93 0.70 0.70 0.70 0.70
1.07 1.85 1.37 4.63 6.94 2.78 2.78 2.78 0.70 0.70 0.70
25 15 25 20 50 225 60 20 20 40 72
39 49 35 37 27 23 40 26 31 46 55
81d 81d 87d 89d 84d 93a 92d 88d 93d 98.5~ 99.6e
' 5 ~ #L acetic acid was added. bIsolated yields. CRadiochemical purity was determined by TLC. dColumn was eluted with CHCI3. ~Column was eluted with hexane.
hexane as a mobile phase were 0.70 and 0.78, respectively. The Rf value o f the synthesized 3-IPI, as well as 3-[ 125I]IPI were identical to an authentic sample o f 3-IPI. The above physicochemical data unequivocally d e m o n s t r a t e the identity, chemical purity and radiochemical purity o f our product. The radiochemical purity o f 3-[125I]IPI was also determined by T L C and H P L C . It should also be n o t e d that this new radioiodination reagent for coupling to m o n o c i o n a l antibodies does not require H P L C separation. The coupling o f this new ligand with 17-1A and D612 m o n o c l o n a l antibodies reactive with h u m a n colon cancer has been studied ( R a m et al., 1992a,b) in our laboratory. The r a d i o i m m u n o c o n j u g a t e s 3-['25I]IPI17-1A and 3-[125I]IPI-D612 showed similar uptake in LS174T colon cancer xenografts and m o s t normal tissues in athymic nude mice c o m p a r e d to the directly radioiodinated 17-1A and D612 antibodies, respectively. However, thryoid uptake for b o t h new radioi m m u n o c o n j u g a t e s was significantly lower than 1311-17-1A and 131I-D612. The detailed results o f this study will be reported elsewhere ( R a m et al,, 1992b).
Acknowledgements--This work was supported by the NIH Grant CA 44173. We acknowledge the support of Dr Merle M. Salter, valuable assistance from Dr M. B. Khazaeli, and Ms Elisa Fleming for technical help. We thank Mrs Renee Kite for her efforts in preparing this manuscript and the UAB NMR Center for ~H-NMR data.
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