Wioconjugate Chem. 1992, 3, 563-509
563
Large-Scale Synthesis of the Bifunctional Chelating Agent 2- (p-Nitrobenzyl)- 1,4,7,10-tetraazacyclododecane-~,~,~’,~’’-tetraaceti~ Acid, and the Determination of Its Enantiomeric Purity by Chiral Chromatography Oliver Renn and Claude F. Meares’ Department of Chemistry, University of California, Davis, California 95616. Received May 15, 1992
The attachment of radiometals to monoclonal antibodies for medical applications requires extreme stability under physiological conditions, with no significant release of metal. Chelators that can hold radiometals like ll1In,67Ga,and ”Y with high stability under these conditions are essential for radiotherapy or immunoscintigraphy. 2- @-Nitrobenzyl)-l,4,7,10-tetraazacyclododecane-N~’,”’,N’’’-tetraaceticacid (nitrobenzyl-DOTA) is one of the most promising bifunctional chelating agents. A large-scale synthesis of nitrobenzyl-DOTA is described. The overall yield for the nine-step synthesis sequence starting from nitrophenylalanine is 5.6%. Synthesis of nitrobenzyl-DOTA according to the new procedure yields up to 10 g without special apparatus. Both enantiomers of the chiral chelate nitrobenzyl-DOTA have been prepared, and their enantiomeric purity has been checked by chiral chromatography.
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INTRODUCTION Radiolabeled monoclonal antibodies (mAbs)l have shown considerable promise in the detection and therapy of cancer (1-5). Radionuclides of interest for radioimmunotherapy and radioimmunoimaging are metals such as “‘In, ”Y,67Cu,55C0,and e8Ga (6-9). Chelators that can hold these radiometals with high stability under physiological conditions are essential to avoid excessive radiation damage to nontarget cells (10). Bifunctional chelating agents (11, 12) are compounds with a strong metal-chelatinggroup a t one end and a reactive functional group at the other. When conjugatedto mAbs,these agents act as carriers of radiometals for tumor targeting and radiotherapy. Methods to attach these chelates to mAbs have gained growing attention in the last several years (13, 14) and are now well-developed (15). Among the metallic radionuclides for therapy, yttrium90 is of particular interest due to its superior properties, including pure 8-emission and the high dose yield per nanomole (16). We have recently developed a new macrocyclic bifunctraazational chelating agent, 2- @-nitrobenzyl)- 1,4,7,10-te cyclododecane-NJV’,”N’’’-tetraacetic acid (10, nitrobenzyl-DOTA,Figure l),that holds yttrium with extraordinary stability under physiological conditions in human serum (17).
Radiopharmaceuticals prepared from this DOTA analog have gained growing attention. Nitrobenzyl-DOTA binds an interesting variety of metals better than any other chelator we have studied so far. It binds both yttrium and indium with superior stability, so it should be possible to use DOTA chelates of the y-emitting l1l1n as a tracer for DOTA chelates of the 8-emitting “Y to gain accurate measurements of radiation dosimetry.
* Address correspondence to Claude F. Meares, Department of Chemistry, University of California, Davis, CA 95616. Telephone: 916-752-0936. FAX: 916-752-8938. ‘Abbreviations used: BOC, tert-butoxycarbonyl; BOC-ON, 2- [ [(tert-butoxycarbonyl)oxy]imino]-2-phenylacetonitrile;DMF, NJV-dimethylformamide; DOTA, 1,4,7,10-tetraazacyclododecane-NJV’,”’JV-tetraacetic acid; mAb, monoclonal antibody; THF, tetrahydrofuran; TFA, trifluoroacetic acid; TsC1, p-toluenesulfonyl chloride.
HOOC
COOH
(S)-nitrobenzyi-DOTA 10s
COOH
(R)-nitrobenryl-DOTA lob
Figure 1. Schematic structures of (R)-and (S)-nitrobenzyl-
DOTA.
In addition, the stability of nitrobenzyl-DOTA chelates with other metals such as copper (Cu-nitrobenzyl-DOTA has shown a better serum stability than any other copper chelate studied so far) suggest that it will be useful in a variety of applications to biological systems. NitrobenzylDOTA has also been used as a precursor for the synthesis of a new chelate conjugate for pretargeted diagnosis and therapy (18), which is now in clinical trials. The growing demand for large amounts of 10encouraged us to develop a large-scale synthesis. This compound is prepared in a nine-step synthesis from nitrophenylalanine. The synthesis, described by Moi and Meares was originally devised for lab-scale, giving final yields of less than 100 mg. In this report we describe a large-scale synthesis procedure that produces amounts up to 10 g. Like many of the drugs in current use, 10 incorporates a chiral center. The majority of such compounds are marketed as racemates. Generally differences in activity may occur between enantiomers. Thus it is important to control enantiomeric purity. Of particular importance in the case of 10, which is prepared either from L- or D-nitrophenylalanine, different immunogenicity could result when the protein-conjugated chelate is administered as a radiopharmaceutical (19). We have prepared (S)nitrobenzyl-DOTA (loa)as well as (E)-nitrobenzyl-DOTA (lob) and have explored several chromatographic methods for chiral discrimination. Chromatography with a chiral stationary phase (Cyclobondcolumn) gave good resolution of 10a and lob, showing no detectable contamination by one enantiomer in preparations of the other.
(In,
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0 1992 American Chemical Society
584 Bioconlugete Chem., Vol. 3, No. 6, 1992
EXPERIMENTAL PROCEDURES All reagents and solvents were the purest commercially available and were used without further purification, if not stated otherwise. Borane-THF (1 M) in Sure/Seal bottles was purchased from Aldrich. Pure water (18 M a cm-l) was employed throughout. Metal-free conditions were maintained during synthetic chemistry. All glass labware was washed with a mixed acid solution and thoroughly rinsed with deionized, distilled water (20). All plastic labware was washed with 3 M HC1 and thoroughly rinsed. Reagent-grade THF was distilled from benzophenone sodium ketyl immediately before use. Thin-Layer Chromatography. TLC was run on plastic backed silica gel plates (0.2 mm thick silica gel 60 F254, E. Merck, Germany) using a 10% (w/v) aqueous ammonium acetate/CHaOH (1:lv/v) solution as the eluent. In this system, unchelated cobalt and conjugates remain at the origin while free chelates migrate to Rf 0.5-0.7. High-Performance Liquid Chromatography. HPLC was carried out on a Rainin Rabbit HPX System (Rainin Instrument, Woburn, MA) equipped with titanium piston washing pump heads. Solvents were mixed using a Dynamax dual-chamber dynamic mixer (Titanium). UV absorbance was measured using an absorbance/fluorescence monitor (ISCO Model UA-5) at 254 or 345 nm. A Gilson Model 201 Fraction collector (Gilson Medical Electronics, Middleton, WI) was used. The HPLC system was controlled by Dynamax software on a Macintosh Plus computer. Normal-phase HPLC was performed at room temperature with a Dynamax 21.4 X 250 mm silica column using a gradient of CH2Clz and CH3CN. For detailed descriptions of the gradients, see below. Reversed-phase HPLC was performed at room temperature with a Dynamax 21.4 X 250 mm CIS column, generally using gradients of CH30H or CH3CN and 0.1 M ammonium acetate (pH 6) with a flow rate of 12.5 mL/ min. All solvents for HPLC and reaction mixtures were filtered through a nylon 66 Millipore filter (0.45 pm) prior to use. Chiral Chromatography. This was done using the system described above. The following procedures were tried: (i) Chromatography was performed on a Pirkle covalent D-phenylglycinecolumn (250 X 4.6 mm), obtained from Regis Chemical Co. (Morton Grove, IL). Mobile phases of mixtures of dimethyl sulfoxide, CH2Cl2, CH3CN, and 2-propanol at 1 mL/min were used. (ii) Chromatography was performed on ASTEC Cyclobond I columns (250 X 4.6 mm) with Cyclobond guard columns, purchased from Rainin Instruments (Woburn, MA). Mobile phases of aqueous ammonium acetate, ammonium phosphate, triethylammonium acetate, and ammonium citrate buffers (0.1 to 0.01 M or 1% to 0.1 % ) with varying amounts of CH30H and CH3CN at different pH values were used. Flow rates were between 0.7 and 1.4 mL/min. (iii) For chiral derivatization, Marfey's reagent [ (l-fluoro2,4-dinitrophenyl-5)-~-alanine amide] from Peptides International, Louisville, KY, was used. The derivatization of (R)-and (5')-aminobenzyl-DOTA with Marfey's reagent was carried out as recommended by Peptides International; however the reaction temperature and time had to be increased to 65 "C and to 2 h. Chromatography was performed on a CISreversed-phase column, with mobile phases of 0.1 M ammonium acetate and varying amounts of CH3CN or CH30H at a flow rate of 12.5 mL/min. (iv) Chromatography was performed on a CISreversed-phase
Renn and Meares
column, with a mobile phase of aqueous L-lysine (1X 10-2 M) as a chiral additive, at a flow rate of 12.5 to 5 X mL/min. Ultraviolet Spectrophotometry. Optical density measurements at 280 nm were made on a Gilford Model 250 spectrophotometer using a 1-cmpathlength microcell. Optical densities were measured at dilutions which gave absorbance readings of 0.1-1.0. Radiation Counting. y-counting was done in a Beckman Model 310 counter with the appropriate energy windows set for 57C0. TLC plates containing radiolabeled materials were visualized with an AMBIS radioanalytical imaging system. NMR Spectroscopy. Proton NMR spectra were obtained on a GE QE 300 Spectrometer a t 300 MHz or a Bruker AC 200 at 200 MHz. Chemical shifts were relative to either HDO (4.70 ppm) or residual CHCl3 (7.24 ppm). For D20 solutions, 0.4 unit was added to the pH meter reading. Mass Spectroscopy. Mass spectra and exact mass measurements were obtained on a ZAB-HS-2F mass spectrometer (VGAnalytical, Wythenshawe,UK). During mass spectroscopic measurements, either 3-nitrobenzyl alcohol or dithiothreitol/dithioerythritol(3:1w/w) was used as a matrix along with small amounts ofp-toluenesulfonic acid. High-resolutionFAB spectra contained polyethylene glycol or polyethylene glycol methyl ether as reference compound. Preparation of 2-(pNitrobenzyl)-1,4,7,1O-tetraazacyclododecane-Nfl,N",h""-tetraacetic Acid (10). Total yields for each step are given in Table I. (S)-pNitrophenylalanine. (24)-Phenylalanine(80 g, 0.48 mol) was dissolved in 215 mL of concentrated sulfuric acid over the course of 5 h at 0 "C. Concentrated nitric acid (27 mL) was added dropwise over the course of 2 h at 4-8 "C. The reaction mixture was poured over 300 mL of ice and carefully neutralized with small additions of (NH&C03. On neutralization, (SI-p-nitrophenylalanine precipitated. The precipitate was collected and recrystallized from water. Yield: 61.63 g (61%1. 'H NMR (300 MHz, D20, pH 6): 6 3.18 (4, 1H), 3.23 (4, 1 H), 3.90 (t, 1 H), 7.40 (d, 2 H), 8.10 (d, 2 H). N-(tert-B utoxycarbony1)-(S)-p-nitrophenylalanine (1). (S)-p-Nitrophenylalanine (61.63 g, 293.2 mmol in 176 mL of H20) was reacted with BOC-ON (79.5 g, 323 mmol) in 171 mL of dioxane and 62 mL of triethylamine (4 h, room temperature, N2 atmosphere). The reaction mixture was poured into 500 mL of water and extracted with ether (5 X 400 mL) and the pH of the aqueous layer adjusted to 1with 6 M HCl. This solution was extracted with ethyl acetate (5 X 400 mL), and the extracts were taken to dryness. The yellow material was dissolved in 300 mL ether, the solvent was removed under reduced pressure, and the residue was lyophilized to give the product, N-(tert-butoxycarbony1)-(S)-p-nitrophenylalanine (1) as a white powder. Yield: 77.97 g (86%). lH NMR (300 MHz, CDC13: 6 1.30 (d, 9 H), 3.10 (m, 2 H), 4.50 (9, 1 H), 7.40 (d, 2 H), 9.80 (8, 1 H). N-( tert-B utoxycarbony1)-( S )-p-nitrophenylalanine N-Hydroxysuccinimide Ester (2). N-Hydroxysuccinimide (29 g, 252 mmol) was added to a solution of 1 (77.97 g, 252 mmol) and dicyclohexylcarbodiimide (57 g, 275 mmol) in 1250 mL of dioxane (room temperature, 17 h, Nz atmosphere). The resulting precipitate was filtered off and the filtrate taken to dryness. The residue was dissolved in lo00 mL of 2-propanoland the undissolved material was filtered off. The filtrate was taken to dryness, giving N-(tert-butoxycarbony1)-(SI-p-nitrophenylalanine
BioconJugate Chem., Vol. 3, No. 8, 1992 565
Technlcal Notes
N-hydroxysuccinimide ester (2). Yield: 87.03 g (82%1. ‘H NMR (300 MHz, CDC13): 6 1.40 ( ~ , H), 9 2.90 ( ~ , H), 4 3.40 (m, 2 H), 5.00 (8, 1 H), 7.50 (d, 2 H), 8.20 (d, 2 H).
N-( tert-Butoxycarbonyl)-(S)-p-nitrophenylalanylglycylglycylglycine (3). Compound 2 (93.00 g, 219 mmol) in 415 mL of DMF was added to a solution of 58.25 g (307 mmol) of triglycine and 34.42 g of sodium bicarbonate in 820 mL of water and the solution stirred for 5 h at room temperature (nitrogen atmosphere). The reaction mixture was filtered and 1770 mL of water was added. The product, N-(tert-butoxycarbony1)-(&nitrophenylalanylglycylglycylglycine(31, was precipitated from solution by the addition of concentrated HC1 (final pH 1). The mixture was held at 4 “C for 12 h, and the precipitate was filtered off, washed with ether, and dried in a desiccator under reduced pressure. Yield: 104.09 g (999% ). ‘H NMR (300 MHz, D20, pH 11): 6 1.10 ( ~ , H), 3 1.25 ( ~ , H), 6 3.15 (m, 2 H), 3.65 (8, 3 H), 3.85 (8, 3 H), 4.40 (m, 1 H), 7.25 (d, 2 H), 8.05 (d, 2 H). FAB-MS: mle calcd (M + Na+) 504, found 504. (S)-pNitrophenylalanylglycylglycylglycine (4). Compound 3 was dissolved in trifluoroacetic acid at 0 “C and stirred for 1 h. The solvent was removed under reduced pressure to give (SI-p-nitrophenylalanylglycylglycylglycine (4)as the trifluoroacetate salt in quantitative yield. This material is very hygroscopic; the preparation of 4 was always done immediately before the reduction, or the sample was stored under high vacuum. FAB-MS: mle calcd (M H+) 382, found 382. (S)-1l-(pNitrobenzyl)-3,6,9,12-tetraazadodecanol (5,6). Caution: BHrTHF can cause explosions andlor fire i f not used properly. Protect from sparks, water, etc. Handle only in a hood behind sufficient shielding. Use a fiberglass tray under the reaction apparatus. In our hands, reductions with u p to 1.6 L BHrTHF (1 M) can be done safely using the following equipment and precautions. Glassware was carefully checked and oven-dried at 200 “C prior to use. A 5-L three-neck round-bottom flask was equipped with a water-cooled condenser (5 X 60 cm) and an addition funnel (500 mL). The addition funnel was sealed with a rubber septum. A low nitrogen flow was applied through this septum into the reaction flask. A large gas bubbler (1.0-cm diameter) filled with mineral oil was connected to the condenser with Tygon tubing. Due to the possible high pressures during the addition of borane and the reduction, the system has to be carefully checked for a safe release of high pressure. All glassware was fixed with metal clamps. Tubing must be secured too, because the tubing material may weaken and become kinked during the reaction, resulting in a closed system. Also, the tubing softens during the HC1 workup. Borane from SureISeal bottles was added using the double-needle technique (see Aldrich information: Handling Air Sensitive Reagents) through the septum into the addition funnel. A 500-mL addition funnel was chosen to keep the volume of BHrTHF near the reaction flask low; addition of BHyTHF solution in 500-mL aliquota was repeated as necessary. For efficient stirring of the large volume (up to 2.3 L), it was necessary to use an egg-shaped stir bar and a magnetic stirrer with electronic speed control (ModelRET, IKA,Germany). A heating mantle was used for heating the reaction mixture. The 5-L round-bottom flask was doubly clamped on a rigid support high enough to change the ice bath and heating mantle easily, without moving the heavy reaction vessel. Compound 4 (44.52 g, 90 mmol) was dissolved in 420 mL of freshly distilled THF, and after cooling to 0 “C,
+
1600mL of 1M BHcTHF complex (1600 mmol) was added slowly. The mixture was refluxed for 17 h, cooled to 0 OC, quenched with 200 mL of CHaOH, saturated with HCl gas, and refluxed again for 17 h. The solvent was removed by evaporation under reduced pressure. The remaining viscous, colorless solution was decanted. The white, gummy precipitate was washed with CHCb, dissolved in 1000 mL of water, and washed again with 3 X 200 mL CHCl3. The solvent was evaporated under reduced pressure and lyophilized to give 5 as a white powder. Yield: 42.5 g (93%1. The resulting hydrochloride salt 5 was dissolved in a minimum amount of water by adjusting the pH to 10 with 10 M NaOH. The basic solution was continuously extracted with CHC13 for 8 h. The organic layer was taken to dryness under reduced pressure to give (S)-ll-(p-nitrobenzyl)-3,6,9,12-tetraazadodecanol (6). Yield: 95%. ‘H NMR (300MHz, D20, pH 11):6 2.25 (br s, 6 H), 2.50-3.00 (m, 14 HI, 3.10 (m, 1 H), 3.60 (t, 2 H), 7.35 (d, 2 H), 8.15 (d, 2 HI. FAB-MS: mle calcd (M H+) 326, found 326. (S)-1 l-(pNitrobenzyl)-N~~,”’,O-penta.kis(tolylsulfonyl)-3,6,9,12-tetraazadodecanol(7):Freshly prepared 6 (30.9 g, 94 mmol) was dissolved in 480 mL of CH3CN, 240 mL of triethylamine, and 240 mL of CH2C12. p-Toluenesulfonyl chloride (90.0 g, 471 mmol) was added and the mixture was stirred for 5 h under N2. The solvent was removed under reduced pressure. The residue was taken up in CHC13, washed with aqueous HC1, and prepurified by open silica gel chromatography (4 X 8 cm, 60-200 mesh); the eluent was CHC13,followed by CH3CN. Final purification was done by normal-phase HPLC to give the product ( S ) - l l - ( p - n i t r o b e n z y l ) - N ~ J V ” ~ - O pentakis(tolylsulfonyl)-3,6,9,12-tetraazadodecanol(7). Yield: 26.80 g, (26% after HPLC purification). Normalphase HPLC: solvent A, CH2C12; solvent B, CH3CN; 245% B, 0-21 min; 5-100% B, 21-23 min; 100-2% B, 25-29 min; UV detection at 345 nm; product peak, 26 min; 245 runs (172 h) to purify 41.5 mmol of 7. lH NMR (300 MHz, CDC13): 6 2.30 (8, 3 HI, 2.50 (m, 12 HI, 2.60-3.50 (m, 14 H), 3.75 (m, 1H), 4.20 (t, 2 H), 5.30 (d, 1H), 7.00-7.90 (m, 24 H). High-resolution FAB-MS: mle calcd (M H+) 1096.263; found 1096.265. (S)-2-(pNitrobenzyl)-N,N,N’,”’-tetrakis( tolylsulfonyl)-1,4,7,lO-tetraazacyclododecane(8). Compound 7 (45.43 g, 41.5 mmol) was dissolved in 4200 mL of DMF, CsCO3 (13.4g, 42 mmol) was added, and the mixture was stirred at 60 “C for 5 h under Nz. The solvent was removed under reduced pressure, and the residue was taken up in CHCl3 and washed with 0.1 M HCl(3 X 250 mL). The solvent was again removed under reduced pressure to give the crude compound 8. The residue was dissolved in 200 mL of CHCL and purified by open silica gel chromatography (4 X 8 cm, 60-200 mesh, eluent CHC13). The volume was reduced to =lo0 mL, and CH30H and ethyl acetate ( ~ 2 0 mL 0 each) were slowly added in equal portions until the first precipitate appeared. (S)-2-@Nitrobenzyl)-N~JV”~-tetrakis(tolylsulfonyl)1,4,7,10tetraazacyclododecane (8) was collected after cooling to 4 “C. Yield: 22.05 g (58%). Analytical normal-phase HPLC: solvent A, CH2C12; solvent B, CHsCN; 2-3.7 % B, 0-12min;3.7-4.1% B, 12-14min;4.1-100% B, 14-16min; 100% B, 16-17 min; 100-2s B, 17-18 min; 2% B, 18-21 min; product peak, 18 min. 1H NMR (300 MHz, CDCl3): 6 2.40-2.60 (m, 12 HI, 3.00-4.20 (m, 16 H), 4.50 (m, 1 H), 7.00-7.50 (m, 12 H), 7.60-7.90 (m, 6 HI, 8.15 (d, 2 H). FAB-MS: mle calcd (M + H+) 924.24; found 924.25. (S)-2-(pNitrobenzyl)- 1,4,7,10-tetraazacyclododecane (9). Compound 8 (13.03 g, 14.1 mmol) was dissolved
+
+
Renn and Meares
500 Bloconjugate Chem., Vol. 3, No. 6, 1992
in 600 mL of concentrated H2S04, and 22.5 g of phenol was added. The mixture was heated to 100 "C and stirred for 56 h under N2. The mixture was poured on to 2 L of ice and neutralized with ~ 3 . 3kg of barium hydroxide. During neutralization, more water must be added to make the mixture easy to stir, yielding a final volume of 16 L. The neutral suspension was held at 4 "C for 12 h. The large amount of barium sulfate could be removed easily by filtration through funnels with fritted discs (9-cm diameter) of decreasing porosity (coarse, medium, fine). The remaining solution (9.5 L) was evaporated to a small volume (about 300 mL) under reduced pressure and purified by reversed-phase HPLC to give the product (SI2-@-nitrobenzyl)-1,4,7,lG~traamcyclododecane (9). Yield after HPLC purification: 6.28 g of 9.2.5CF3COOH (84%). Reversed-phase HPLC: solvent A, 0.1 % trifluoroacetic acid in water; solvent B, CH3CN; 15-2096 B, 0-20 min; 2 0 4 0 % B, 20-21 min; 60-1576 B, 21-23 min; 15% B, 23-31 min; product peak, 18 min; 121 runs (64 h) to purify 16.6 mmol of 9. lH NMR (300 MHz, D20, pH 2): 6 2.803.50 (m, 17 H), 7.50 (d, 2 H), 8.20 (d, 2 H).FAB-MS: mie calcd (M H+) 308.210, found 308.209. (5)-2- (pNit roben zyl) 1,4,7,10- tetraazacyclododecane-N,N',N",N"'-tetraaceticAcid (10). Bromoacetic acid (10.14 g, 73.0 mmol) was added to a solution of 9 (8.83 g of 9-2.5CF3COOH, 16.6 "019) in 45 mL of water at pH 11. The mixture was stirred at 70 "C, while the pH was maintained at 10 using a pH-stat with 3 M NaOH. After 5 h the solution was neutralized with 6 M HC1and purified by reversed-phase HPLC to give the product (SI-2-pnitrobenzyl-1,4,7,10-tetraazacyclododecane-N,","',"''tetraacetic acid (10). Yield: 6.50 g (73 % 1, based on 57C02+ metal-binding assay (14). Reversed-phase HPLC: solvent A, 0.1 M ammonium acetate, pH 6; solvent B, CH30H; 15-30% B, 0-20 min; 30-100% B, 20-25 min; 100-15% B, 25-30 min; product peak, 10 min; 123 runs (70 h) to purify 12.05 mmol of 10. 'H NMR (200 MHz, D20, pH 5.8): 6 2.80-3.96 (complex multiplet, 25 H), 7.54 (d, 2 H, J = 8.39 Hz), 8.25 (d, 2 H, J = 8.57 Hz). FAB-MS: mle calcd (M + H+) 540, found, 540.
+
TFA 0 %
93%
o z N w ~ ~
'NH
'NH
'OH
NHZ
NHz TFA
1 ) BH, THF
rdbr
93%
-
RESULTS AND DISCUSSION
Large-Scale Synthesis of Nitrobenzyl-DOTA (Figure 2). Originally, 2-@-nitrobenzyl)-DOTA (Figure 1) was synthesized by a nine-step procedure (17). The last four steps required preparative HPLC to purify the products. We tried to avoid these time-consuming, costly HPLC purification procedures and develop a synthesis procedure that gives a final yield of several grams of 10 without special apparatus. Since nitrobenzyl-DOTA has one chiral carbon atom, starting from L-phenylalanine or D-phenylalanine results in either the S- or R-enantiomer of 2-@-nitrobenzyl)DOTA. We have prepared both enantiomers and developed an analytical method to check their enantiomeric purity by chiral chromatography. (8)-p-nitrophenylalanineis commercially available or can be prepared using common procedures from phenylalanine. After protection of the a-amino group with BOCON, the carboxyl terminus of 1 was activated using dicyclohexylcarbodiimide and N-hydroxysuccinimidecoupling to give 2. The tetrapeptide 3 was prepared in 99 % yield by reaction with triglycine. The t-BOC group was removed using neat CF3COOH to give 4 in quantitative yield. The first critical step of the synthesis is the borane reduction of 4 to the polyamino alcohol 5. On the original scale, the reduction step required 68 mL of 1M BHrTHF.
(9)
(10)
Figure 2. Reaction scheme for the large-scale synthesis of nitrobenzyl-DOTA (10) from nitrophenylalanine. Starting the synthesiswith 0.29 mol of phenylalanine gives about 140 g (0.22 mol) of deprotected tetrapeptide. Since four C=O groups must be reduced, a total of 4.8 L of 1 M BHrTHF is necessary for 0.22 mol of 4. To reduce 0.22 mol, it proved most convenient to carry out three 0.7-mol reductions of 4. From readily available components, we designed a reduction apparatus where reductions with up to 1.6 L BHsTHF can be done safely. Particular caution was taken regarding possible high H2 pressures in the apparatus, which might occur when handling these large amounts of material. High pressure has to be released safely; it is therefore very important that the reaction mixture, which may be viscous at the beginning, is always stirred efficiently. General precautions and procedures are described in the experimental part. The workup was improved to give 5 in yields up to 93 5% as the hydrochloride, a white powder. Several attempts were made to convert the hydrochloride 5 directly to the tosylated polyamino alcohol 7. However, none of the attempts to generate the free base 6 by ion exchange or by the use of Ag20 gave satisfactory results, partly due to the instability of the polyamine. Finally, 6 was prepared using continuous liquid-liquid extraction into an organic phase. The tosylation of polyamino alcohol 6 is a multistep reaction, which involves the alkylation of four amines and a primary alcohol. This reaction is a difficult step in the
Technlcal Notes
Bloconlugete Chem., Vol. 3, No. 6, 1992 567
Table I. Stepwise Yields for Nitrobenzyl-DOTA Synthesis
product N-(tert-butoxycarbony1)-p-nitrophenylalanine(1) N-(tert-butoxycarbony1)-p-nitrophenylalanine N-hydroxysuccinimideester (2) N-(tert-butoxycarbonyl)-p-nitrophenyl~anylglycylglycy~lycine (3) p-nitrophenylalanylglycylglycylglycine(4) 11-@-nitrobenzyl)-3,6,9,12-tetraazadodecanolhydrochloride (5) ll-@-nitrobenzyl)-3,6,9,12-tetraazadodecanol (6) 11-@-nitrobenzyl)-N~~~”,O-pentakia(nyl)-3,6,9,12-tetraazadodecanol(7) 2-@-nitrobenzyl-N~~~-tetrakis(toly~~onyl)-1,4,7,10-tetraazacyclododecane (8) 2-@-nitrobenzyl)-l,4,7,10-tetraazacyclododecane (9) 2-@-nitrobenzyl)-1,4,7,lO-~traazacyclododec~e-N~~~”’-~traacetic acid (10) overall yield
synthesis,since the yield is low due to side reactions (interand intramolecular condensation reactions involving partially tosylated intermediates are possible). We explored different solvents, bases, reaction temperatures, and procedures [e.g., different concentrations of 6 or addition of a catalyst (21)l. On this scale, tosylation of 6 using p-toluenesulfonyl chloride in a mixture of triethylamine/ CH&N/CHZClz (1:2:1)a t room temperature gave the best yields (26%). The large number of side products (up to eight according to HPLC) requires purification by HPLC. Reasonable HPLC runtime was made possible by using a prepurification step (open silica column) to remove the oily byproducts. The cyclization step was carried out in DMF (0.01 M 7 at 60 OC). In the original synthesisprocedure preparative HPLC was necessary for purification of the cyclic tetratosylamide; on this scale, attempts to precipitate 8 were successful and 8 could be obtained as a clean, white powder. Removal of the tosylamide to generate the free cyclic tetraamine in neat HzS04 with phenol gives the detosylated product. Neutralization of the sulfuric acid solution with barium hydroxide results in large amounts of precipitated barium sulfate. However, we found that barium sulfate could be removed easily after aging the precipitate, by using a series of three glass frits with decreasing porosity. Most of the barium sulfate was retained by the coarse filter, allowing a short filtration time. Preparative HPLC was used to separate the product 9 from the phenol in the mixture. The concentrated solutioncontained the product 9 in good purity, so that relatively large amounts of the crude product could be injected. Finally,the cyclic amine was alkylated with bromoacetic acid under basic conditions to give the product, nitrobenzyl-DOTA. From the reaction mixture, the tetraalkylated compound was obtained in high yield. For medical applications, 10 was purified by HPLC. Table I gives the yields of all the steps involved in the reaction sequence. The overall yield is 5.6% ;starting with 300 mmol of p-nitrophenylalanine gives a yield of 9.15 g (17 mmol) nitrobenzyl-DOTA. The reaction sequence involves three preparative HPLC purification steps: the first step was improved in terms of separation, with a 30% reduction of run time so that the relative amounts of solvents were considerably less than previously reported (17). The second HPLC purification was done under aqueous conditions; CHJCN was decreased by 60%, resulting from a change in the solvent gradient and a 30% reduction of run time. While this paper was in preparation, a synthesis of bifunctional tetraazamacrocycleswas published (22).That report describes the synthesis of 10 by two different routes; both start from nitrophenylalanine, but in each case the macrocycle is formed by bimolecular cyclization. Yields of the cyclization steps are lower (40% and 44% ) than for the intramolecular cyclizationdescribed here (58%1. The bimolecular cyclization step is also a limiting factor for
yield ( % ) 86 82 99 99 93 95 26 58 84
73 5.6
scaling up those synthesesbecause these reactionsare done under high dilution and by using a specialapparatus. Thus final yields of 1 or 2 g, depending on the route, result (22). The cyclized products in ref 22 are reduced to yield the substituted macrocyclic tetraamine. We experienced generally higher yields when the linear molecule 5 was reduced; reduction of a macrocyclic amide with borane may be hindered sterically,resulting in lower yields. Thus a reduction followed by a cyclization step seems to be a better reaction sequence, giving yields of 93 % (reduction of 5) and 58% (cyclization of 7) compared to 40% (44%) and 55% (37%) when the reduction is done after the cyclization. However, the low yield of the tosylation step in our synthesis scheme lowers the overall yield. For the final alkylation to prepare nitrobenzyl-DOTA, the yields differ considerably. In ref 22 the reported conditions (pH 8.5, excess of BrCH2COZH) give a 48% yield, compared with 73% for the alkylation of 9 at pH 11. Determination of the Enantiomeric Purity of 10 by Chiral Chromatography. The introduction of a side chain into DOTA, giving a C-functionalized macrocycle, results in a chiral molecule since the C(2) atom is chiral (Figure 1). Nitrobenzyl-DOTA, when reduced and conjugated to a tumor-targeting antibody, could thus form diastereomeric radiopharmaceuticals. These might have different biological properties, including immunogenicity (19). The determination of enantiomericpurity is of particular interest, since it is known that the enantiomeric forms of a drug may have different biological activities (24). Methods available to analyze enantiomers include chromatography on chiral stationary phases, the use of chiral additives in the mobile phase, derivatization to yield diastereomers, chiral detectors, NMR, and enantiomer specific immunoassays. Until chiral stationary phases were developed, chiral derivatization was the most common method. As diastereomers differ in their physical properties, they may be separated on achiral stationary phases. However, for derivatizationthe reagent must be obtainable in chemically and optically pure form, and the derivative must be formed in high yield. For simplicity, the direct use of chiral liquid chromatography (25-27)is the most common first approach to enantiomeric analysis. We tried four different chiral HPLC systems to determine the enantiomeric purity of 10. Chiral stationary phases tested included a Pirkle column and a Cyclobond column. For the resolution of enantiomers on an achiral stationary phase, chiral derivatization using Marfey’s reagent (28)was investigated. In addition, we briefly examined chiral ion-pair chromatography. Chiral Stationary Phases for Enantiomeric Resolution of 10. When choosing a chiral stationary phase for enantiomeric resolution, the different characteristics of the phase have to be considered in order to match the structural features of the solute and the mobile phase. We
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first tried a ?r-electron acceptor column. The first commercially available chiral stationary phase for HPLC was a 3,5-dinitrobenzoyl-~-phenyl glycine “Pirkle”column (291, bound covalently to a silica support. However, our attempts to develop a method for the analysis of the enantiomeric purity of 10 using a Pirkle column were unsuccessful. Strongly anionic or cationic complexeswith 10 are not easily resolved on this stationary phase, which may make derivatization necessary. Since 10 is only soluble in polar solvents such as water, dimethyl sulfoxide or DMF, it proved impossible to add enough nonpolar organic modifier into the mobile phase. We next tried a cyclodextrin-based column, used in the reversed-phase mode. Cyclodextrins form chiral cavities that are bound to a silica support through a spacer. The cyclodextrin structure can be viewed as a cone open at both ends; the interior of the cone is relatively hydrophobic. If a chiral molecule fits into the cavity and has functional groups that interact with the secondary hydroxyl groups at the wider opening of the cone, then chiral separation may occur. Cyclodextrin columns are available in three different sizes, which allow separation of enantiomers of different sizes. The nitrobenzyl group of 10 fits well into the cavity of a 8-cyclodextrin, so a Cyclobond I column was chosen. Besides the nitrobenzyl group, nitrobenzyl-DOTA has two other types of functional groups: four carboxylic moieties and four tertiary amine moieties. In reported stereochemical resolutions of chiral molecules containing a carboxylic moiety, this function has to be attached to the C*, which appears to be the major structural requirement. In 10,the amine group is closer to the chiral C*. However,the flexibility of the carboxymethyl group might make its involvement in the process of chiral recognition possible. Steric hindrance at the C* may increase the stereochemical resolution. I t is recommended (30) that the aromatic moiety is at the C*, as with nitrobenzylDOTA. Mobile phase additives such as buffer salts can help improve column efficiency and resolution. We therefore investigated a number of different buffers: ammonium acetate, ammonium nitrate, ammonium phosphate, ammonium citrate, and triethylammonium acetate. When determining the optimum pH, the nearest hydrogen bonding group to the stereogenic center has to be considered. As an amine group is closest to C*, buffers of low pH were investigated first. Using gradients down to pH 4.0, the time difference between the elution of the two isomers was increased, but the a-value increased only slightly (from 1.08 to 1.09). However, this also resulted in long retention times (71 and 78 min for 95:5 (v/v) 0.1 M ammonium acetate pH 4.75/CH3CN), and the peaks became extremely broad and flat. CH3CN as the organic modifier gave generally better results than CH30H. We also studied the influence of buffers with higher pH, as recommended for the resolution of carboxylic acids. A pH 7.5 ammonium acetate buffer containing 10% CH3CN gave a 1.23 and sufficiently sharp peaks (Figure 3). The S-isomer 10a elutes first at 11.6 min; the R-isomer 10b elutes at 13.6 min. As can be seen from Figure 3,no cross-contaminationcould be detected, demonstratingthat this synthesis does not involve significant racemization. Among all buffers investigated, ammonium acetate seemd to work best. Triethylammonium acetate buffers caused strong retention of 10 on the column. An ammonium phosphate buffer might also work well, but closer investigation was impossible due to the relatively short lifetime of the Cyclobond columns. To achieve these
Renn and Meeres
Figure 3. Determination of the enantiomeric purity of (S)nitrobenzyl-DOTA (loa) (dashed line), and (R)-nitrobenzylDOTA (lob)(solidline) usinga Cyclobondcolumn: mobile phase, isocratic 90% 0.1 M ammonium acetate, pH 7.5, 10% CHsCN; flow rate, 1.0 mL/min. The small peaks in front of loa and 10b are unidentified impurities that are also evident on achiral CIS HPLC. They may be metal chelates formed from trace metal impurities in the system.
results, two Cyclobond columns were consumed. This clearly demonstrates the major disadvantageof Cyclobond columns: the short lifetime, especiallywhen gradients with high aqueous content are used.
Chiral Derivatization for Enantiomeric Resolution. Because of the short lifetime of Cyclobond columns, we also investigated other methods. Marfey’s reagent (B), which provides a quantitative method of separating Land D-aminO acids, has been used to analyze the optical purity of peptides. In one study, 22 derivatives of 11 amino acids were studied on a CIScolumn in a single reversedphase chromatographic run (31). Even though the complete separation of all 22 derivatives studied was not achieved, the derivative of each enantiomer was well resolved from that of its enantiomeric counterpart. This prompted us to test Marfey’s reagent for chiral derivatization. As an amine function is necessary to form the covalent bond to the reagent, we used 2-@aminobenzyl)DOTA, which was prepared from 10a and 10b according to ref 23. However, no resolution could be obtained even with extremely shallow solvent gradients. Chiral Ion-Pair Chromatography. We also investigated chiral ion-pair chromatography (32,33). The separation of enantiomers on chiral stationary phases involves the reversible formation of diastereomeric complexes. In favorable cases, it is possible to achieve separation by adding a chiral reagent to the mobile phase and using nonchiral stationary phases. Organic acids and bases are able to interact with ion-pairing reagents in the mobile phase by forming less polar ion pairs. In some cases it is assumed that in situ coating of the column to form a temporary stationary phase is responsible for separation. Chiral mobile phases work with less expensive column packing, since the normal achiral materials can be used. However, due to the large amounts of the chiral additive needed, chiral ion-pair chromatography can be costly. A study of the mechanism of anionic chelate retention in ion-pair reversed-phase chromatography was recently published (34). We tried the conditions recommended there and used L-lysine as a chiral counterion, but we were unable to resolve 10 using this system. Ion-pair chromatography in reversed-phase systems may be not sufficient
Technlcal Notes
for some enantiomeric separations, since the undesired dissociation and solvation effects of water present in the eluent affectthe intimate contact betweenthe hydrophobic surfaces of the solute and the reagent molecule (35). ACKNOWLEDGMENT
We thank Min Li and Douglas P. Greiner for helpful discussions. Thiswork was supported by NIH-NCI Grants CA 16861 and CA 47829. LITERATURE CITED (1) Yuanfang, L., and Chuanchu, W. (1991) Radiolabeling of
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