Electrophilic analogs of daunorubicin and doxorubicin - American

Mar 19, 1990 - Daunorubicin (DNR) or doxorubicin (DOX) was modified with one of four ... Bioconjugates were produced with the electrophilic DNR or DOX...
0 downloads 0 Views 808KB Size
Bioconjugate Chem. 1990, 1, 251-256

25 1

Electrophilic Analogues of Daunorubicin and Doxorubicin1 Leonard 0. Rosik and Frederick Sweet’ Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110, Received March 19, 1990

Daunorubicin (DNR) or doxorubicin (DOX) was modified with one of four “linker reagents” to produce electrophilic drug analogues for synthesis of bioconjugates. Synthesis and characterization of two new reagents [p-isothiocyanabbenzoylchloride and 3-(p-isothiocyanatophenyl)propionylchloride] are described here for the first time. Adding one of the new reagents, bromoacetyl bromide, or p(fluorosulfony1)benzoyl chloride in chloroform to an alkaline aqueous solution of DNR (or DOX) provided excellent yields of the corresponding, electrophilic 3’-N-amide analogue. The DNR and DOX analogues were characterized by thin-layer chromatography, nuclear magnetic resonance spectroscopy, and infrared spectroscopy. Bioconjugates were produced with the electrophilic DNR or DOX analogues by mixing them with bovine serum albumin (BSA),mouse IgG, or a monoclonal antibody (OC125, which specifically binds to the CA125 antigen from human ovarian carcinoma). The relative reactivity of the 3’-Nsubstituents toward protein is p(fluorosulfony1)benzoyl > phenylisothiocyanato > bromoacetyl. Overall, the new phenyl isothiocyanate acid chlorides are superior to p(fluorosulfony1)benzoyl chloride or bromoacetyl bromide as reagents with which to produce electrophilic DNR or DOX analogues for conjugation with monoclonal antibodies. The bioconjugates DNR-OC125 and DOX-OC125 are selectively toxic to two human ovarian cancer cell lines in vitro (1) and bind with high specificity to human ovarian tumor sections (2) that express the CA125 antigen.

Rapidly growing numbers of bioconjugatesderived from drugs and monoclonal antibodies have been synthesized and tested on human cancers in vitro and in vivo with varying degrees of success (3-8). Recent developments in bioconjugate chemistry focus on new methods for attaching drugs t o monoclonal antibodies while preserving characteristic drug toxicity and antibody binding. Preservation of these characteristics requires that the structural integrity of both drug and antibody be maintained (9,lO). Efforts have also been made to produce drug-antibody conjugates in which the linkage can be cleaved by t h e target cells ( 2 1 ) . The history and developmentsin bioconjugate chemistry have recently been described in excellent review articles (12, 13). Doxorubicin (DOX) is used for treating ovarian cancer, making it a logical choice for conjugation with monoclonal antibodies that bind to the cancer. DOX and daunorubicin (DNR) have tetracycline anthraquinone rings containing an amino sugar and ketonic carbonyl at the C-7and C-9-positions, respectively (Chart I). The structural difference between DNR and DOX is a hydroxy group in the side chain at the C-9-position. Hurwitz et al. earlier attempted to link DNR and DOX to an antibody by first oxidizing the carbohydrate moiety in the drugs with N d 0 4 to produce corresponding dialdehydes. The oxidized, tetracyclic anthraquinones were assumed to conjugate with amino groups in the antibody protein through formation of Schiff base, subsequently “stabilized” with NaBH4. However, opening of the amino sugar ring by NaIo4 (probably accompanied by removal of the a-hydroxyketonic side chain at C-9) caused major structural changes in the drug with consequent loss of its desired toxicity (14). 1 Presented in part a t the Fourth International Conference on Monoclonal Antibody Immunoconjugates for Cancer, San Diego, CA, March 30-April 1,1989and at the Society for Gynecologic Investigation 37th Annual Meeting, St. Louis, MO, March 2124, 1990.

1043-1802/90/2901-025 1$02.50/0

Recently, progress has been made by modifying the 3’amino group on the intact sugar moiety or the methyl ketone side chain at the C-9-position in DNR and DOX, intended for conjugation with antibodies (15-1 7). It seemed worthwhile to explore methods for modifying the 3’-amino group in DNR and DOX with new “linker reagents” so designed that at some later time a desired bioconjugate can be synthesized simply by mixing the electrophilic drug analogues with a suitable protein. These objectives have been accomplished in principle with electrophilic, affinity-labeling analogues of biologically active steroids, nucleosides, and other compounds. Electrophilic groups [e.g. bromoacetyl or p(fluorosulfony1)benzoyl] have been used in the synthesis of affinitylabeling analogues that become covalently bound to specific enzyme, transport, or receptor proteins (18). Our reason for selecting the presently reported reagents for bioconjugate synthesis is that each of them contains two different electrophilic groups. The two groups in each reagent greatly vary from one another in their rates of reaction with nucleophiles. Accordingly, during the first reaction the more reactive of the two electrophilicgroups forms a stable, covalent bond (Le., amide linkage) between the reagent and the anticancer drug. The second electrophilic group in the drug analogue remains reactive. After isolating the drug analogue, at some later time the electrophilic group can be used to form stable, covalent bonds with nucleophilic amino acid residues in proteins. In the present study, we chose the acid halide for the more rapidly reacting of the two electrophilic groups in the reagents. The relatively slower reacting, second electrophilic groups were bromacetyl, (fluorosulfony1)phenyl, and phenylisothiocyanato (Chart I). It seemed worthwhile to include the phenyl isothiocyanate group in the new reagents because other aromatic isothiocyanates have been successfully used in bioconjugate chemistry. For example,fluorescein isothiocyanate (FITC) 0 1990 American Chemical Society

Bioconjugate Chem., Vol. 1, No. 4, 1990

252

Rosik and Sweet

Chart I. Synthesis and Structures of Electrophilic Analogues of Daunorubicin and Doxorubicin

n

gg

n -

X -

0 2

1 2

0 2 0 2

OH OH c1 C1

0

RCOX

X = Cl; Br

+

DNR or DOX

& OH-

0

Compound [ I$ Yield

S02F

3 4

5 6

OH

*:y CH30

-CH2Br

Cpd -

10 [ g a l

OH 0

I

I

1 I [a81

12 I 9 5 1

(19-21) or chelator reagents for making tumor imaging substances (22-24) each produce stable conjugates when the aromatic isothiocyanate reacts with a suitable protein. In the present case, Schotten-Baumann reaction conditions were expected to cause the assorted acid halides to selectively react with the lone 3'-N-amino group in the polyfunctional DNR (or DOX) to produce the desired electrophilic analogues of the anticancer drugs. This report describes syntheses of DNR and DOX analogues that contain an electrophilic group, made by reacting two new phenyl isothiocyanate acid chlorides and also known dual electrophilic acid halides with the anthracycline antibiotics. The electrophilic analogues of DNR and DOX are sufficiently stable to be stored and then later used for producing desired monoclonal antibodyanticancer drug conjugates simply by mixing the analogues with a monoclonal antibody. Preliminary results with this approach have provided bioconjugates that exhibit specific binding to human ovarian tumor sections (2) and selective toxicity for human ovarian cancer cells (I). EXPERIMENTAL PROCEDURES

General Procedures. p-(Fluorosulfony1)benzoyl chloride and p-aminobenzoic acid were from Aldrich (Milwaukee, WI). 3-(p-Aminophenyl)propionic acid was from Pfaltz and Bauer (Waterbury, CT). Thiophosgene was from Fluka (Ronkonkoma, NY). Bromoacetyl bromide was from Eastman Kodak (Rochester, NY). Daunorubicin was from Wyeth (Princeton, NJ) and doxorubicin was from Adria (Columbus, OH). Mouse IgG was from Sigma (St.Louis, MO) and anti-CA-125monoclonal antibody (OC125) was from Centocor (Malvern, PA). Nuclear magnetic

resonance spectra were obtained with a Varian XL-300 spectrometer. Infrared spectra were performed with thinfilm NaCl plates (unless otherwise noted) and a Beckman Acculab 4 spectrophotometer. Elemental analyses were performed by Galbraith Laboratories (Knoxville,TN). Thin-layer chromatograms (TLC) were obtained with 2.5 X 7.5 cm plates of silica gel G (Eastman Kodak), developed with chloroform/methanol ( 9 5 5 v/v). Rf values are reported for characteristic mobilities of the DNR and DOX analogues. p-Isothiocyanatobenzoic Acid (3). A solution of p-aminobenzoic acid (13.7 g, 0.10 molj in 110 mL of 1 M aqueous potassium hydroxide was added dropwise during 30 min to a vigorously stirred mixture of thiophosgene (12.6 g, 0.11 mol) in 200 mL of water a t room temperature. During addition, formation of a thick mixture required that sufficient water be added to maintain smooth stirring. The total volume of the reaction mixture was ca. 1L a t the end of the addition. Then the reaction mixture was stirred for one additional hour and filtered under vacuum, and the collected white solid was washed 10 times with 100-mL portions of water. The product (16.6 g, 93 % ) was dried in a vacuum desiccator for 48 h. A sample of the dry product was recrystallized twice from ethyl acetate to provide crystalline material that melted with decomposition above 235 "C: 'H NMR (CDCl3) 6 8.10 (d, J = 8.4 Hz, 2 H), 7.31 (d, J = 8.7 Hz, 2 H); IR (KBr) 2080 (SCN), 1665 (CO), 1590, 1412, 1280, 850 cm-l. Anal. Calcd for C B H ~ N O ~C, S :53.62; H, 2.81; N, 7.82; S, 17.89. Found: C, 53.58; H, 2.73; N, 7.72; S, 17.93. 3-(p-Isothiocyanatophenyl)propionicAcid (4). Compound 4 was prepared in 77% yield from 2 by the procedure described above for the synthesis of 3. An analytical sample of 4 was recrystallized twice from benzene: mp 140-141 "C; 'H NMR (CDCl3) 6 7.20 (d, J = 8.7 Hz, 2 H), 7.16 (d, J = 8.7 Hz, 2 H), 2.95 (t, J = 7.5 Hz, 2 H), 2.67 (t, J = 7.5 Hz, 2 H); IR (KBr) 2095 (SCN), 1690 (CO), 1502, 1440, 1221, 829, cm-l. Anal. Calcd for C10H9N02S: C, 57.96; H, 4.38; N, 6.76; S, 15.47. Found: C, 58.12; H, 4.29; N, 6.75; S, 15.33. General Synthesis of Electrophilic Analogues of Anthracycline Antibiotics. A solution of an acid halide (1.1-2.1 equiv) in chloroform or benzene was added a t room temperature to DNR or DOX (1 equiv) in a vigorously stirred mixture of water, chloroform and/or benzene, and potassium bicarbonate (10-20 equiv). After stirring the resulting mixture for 10-30 min, the liquid layers were allowed to separate, the organic layer was removed, and then the aqueous layer was extracted with several portions of chloroform. The combined chloroform extracts were washed with water, dried over anhydrous sodium sulfate, and filtered, and then the solvent was removed under reduced pressure. The residue (10-15 mg) was flash chromatographed in chromatographic columns made from cotton-plugged Pasteur pipets and ca. 1 g of flash silica gel. After applying the crude material in chloroform to a column, three or four 1-mL portions of chloroform were percolated through the column, then the product was eluted with methanol/chloroform (2-3 % of methanol). The fractions containing produce (according to TLC) were pooled and the solvent was removed under reduced pressure. 3'-N-(p-Isothiocyanatobenzoyl)daunorubicin(7). Acid chloride 5 was prepared by heating a mixture of 3 (180 mg, 1 mmol), thionyl chloride (100 pL, 1.4 mmol), 4 drops of DMF, and 5 mL of chloroform, under reflux (drying tube) for 2 h. Solvents and excess thionyl chloride were removed under vacuum (water aspirator). The

Reagents for Synthesis of Bioconjugates

residue was twice dissolved in a few milliliters of chloroform and the solvent was removed under vacuum. The resulting residue was dissolved in 5 mL of chloroform. An aliquot (150 pL, 30 pmol) of the solution containing 5 was added to DNR-HCl (11 mg, 19.5 pmol) in a mixture of 500 pL of water, 400 pL of chloroform,and potassium bicarbonate (15 mg, 150 pmol), and then the mixture was stirred for 10 min a t room temperature. Flash chromatography provided 13.39 mg (99%) of pure 7: R f = 0.64; ‘H NMR (CDCl3) 6 7.71 (d, J = 8.4 Hz, 2 H), 7.22 (d, J = 8.4 Hz, 2 H), 6.48 (d, J = 7.8 Hz, 1 H, 3’-NH), 4.34 (br, 1 H 3’CH); IR 3500,3410,2940,2090 (SCN), 1719 (9 C=O), 1642 (amide), 1622 (5,12 C=) 1583,1499,1417,1290,1211,1012, 988, 716 cm-l. 3’-N-[3-(pIsothiocyanatophenyl)propionyl]daunorubicin (8). Acid chloride 6 was prepared by stirring 4 (10 mg, 48 pmol) in a solution of thionyl chloride (200 pL, 1.37 M) in benzene for 5 h at room temperature. After removal of the solvent and excess thionyl chloride under vacuum, the residue was dissolved in benzene and the solvent was removed in vacuo. Crude 6 was dissolved in chloroform (200 pL) and the resulting solution was added to a stirred mixture of DNR-HC1 (22 mg, 39 pmol) in 1.0 mL of water, chloroform (200 pL), and potassium bicarbonate (100 mg, 1mmol). After rinsing of the residual acid chloride into the reaction mixture with 200 pL of chloroform, the resulting mixture was stirred for 30 min. Flash chromatography provided pure 8 (26.2 mg, 94%); Rf = 0.61; ‘H NMR (CDC13) 6 7.15 (d, J = 9 Hz, 2 H), 7.11 (d, J 9 Hz, 2 H), 5.69 (d, J = 8.1 Hz, 1 H, 3’-NH), 4.15 (br, 1 H, 3’-CH), 2.89 (t, J = 7.6 Hz, 2 H), 2.41 (dt, J = 2.8,7.8 Hz, 2 H); IR 3490,3400, 2945, 2120 (SCN), 1720 (9 C=O), 1660 (amide), 1629 (5,12 C=O), 1590,1421,1295, 1218,1020,993,826 cm-’. 3’- N- [ 3 - ( p -I s ot h i o c y an atop hen y 1 ) prop i on y 1 3 doxorubicin (9). By the method described above, 6 was prepared from 4 (12.1 mg, 58 pmol). The a solution of 6 in 500 pL of chloroform was added to a stirred mixture of DOX.HC1 (15.7 mg, 27 pmol) in 1.0 mL of water, chloroform (200 pL), and potassium bicarbonate (24 mg, 240 pmol). Residual acid chloride was rinsed into the reaction mixture with 500 pL of chloroform and the mixture was stirred for 30 min. Flash chromatography provided pure 9 (17.2 mg, 87%): Rf = 0.37; ‘H NMR (CDCl3) 6 7.15 (d, J = 8.9 Hz, 2 H), 7.12 (d, J = 8.9 Hz, 2 H), 5.70 (d, J = 8.5 Hz, 1 H, 3’-NH), 4.12 (br, 1H, 3’-CH), 2.90 (t, J = 7.4 Hz, 2 H), 2.41 (dt, J = 3.0,7.4 Hz, 2 H); IR 3400,2950, 2120 (SCN), 1730 (9 C=O), 1660 (amide), 1630 (5, 1 2 C=O), 1590, 1419, 1296, 1219,1028,997 cm-l. 3’-N-(Bromoacetyl)daunorubicin(10). A solution of bromoacetyl bromide (110 pL, 0.11 M in benzene) was added to a stirred mixture of DNR.HCl(6.0 mg, 10.6 pmol) in 500 pL of water, 500 pL of chloroform, and potassium bicarbonate (10 mg, 100 pmol). The reaction mixture was stirred for 30 min at room temperature. Chromatography gave 6.8 mg (98%) of pure 10: Rf = 0.44; lH NMR (CDCl3) 6 6.76 (d, J = 8.2 Hz, 1 H, 3’-NH), 4.14 (br, 1 H, 3’-CH), 3.82 (s, 2 H, BrAc); IR 3490, 3400, 2940, 1711 (9 C=O), 1656 (amide), 1619 (5,12 C=O), 1580,1412,1285,1207, 1020,985,762 cm-l. 3’-N-(Bromoacetyl)doxorubicin(11). Bromoacetyl bromide solution (200 pL, 22 pmol) was added to a mixture of DOXqHCl (6.2 mg, 10.7 pmol) in 1.0 mL of water, chloroform (800 pL), and potassium bicarbonate (24 mg, 240 pmol) t h a t was s t i r r e d for 30 m i n . F l a s h chromatography provided pure 11 (6.25 mg, 8855): Rf = 0.25; ‘H NMR (CDC13) 6 6.76 (d, J = 8.1 Hz, 1 H, 3’NH), 4.14 (br, 1 H, 3’-CH), 3.82 (s, 2 H, BrAc); IR 3500,

Bioconjugate Chem., Vol. 1, No. 4, 1990 253

3400,2945,1725(9 C=O), 1661 (amide), 1625 (5,12 C=O), 1585,1416, 1290, 1214,1023, 994 cm-l. 3’-N-[p-(Fluorosulfonyl)benzoyl]daunorubicin ( 12). p(Fluorosulfony1)benzoyl chloride (5.5 mg, 25 pmol) in 0.5 mL of benzene was added to DNR-HCl(12.6 mg, 22.5 pmol) in 1 mL of water, 0.5 mL of chloroform, and potassium bicarbonate (24 mg, 240 pmol), and the mixture was stirred a t room temperature for 30 min. Flash chromatography gave 15.4 mg (95% ) of pure 12: Rf = 0.58; ‘H NMR (CDCl3) 6 8.05 (d, J = 8 Hz, 2 H), 7.95 (d, J = 8.3 Hz, 2 H), 6.61 (d, J = 8 Hz, 1 H, 3’-NH), 4.42 (br, 1 H, 3’-CH); IR 3500, 3420, 2945, 1720 (9 CEO), 1650 (amide), 1625 (5,12 C=O), 1587, 1420,1295,1220,1020, 991, 793, 768, 614 cm-l. Conjugating DNR or DOX Analogues w i t h Antibody or Other Proteins. A solution of a DNR (or DOX) analogue in acetonitrile (330-660 pM, 100 pL) was added to a mixture of 500 pL of stock antibody solution in PBS (1-2 mg/mL) and 500 pL of 0.1 M phosphate buffer (pH 8), stirred with a vortex apparatus. After standing at room temperature for 24 h, the resulting mixture was placed in a dialysis bag and dialyzed for 3 days at 0 “C against daily changes of 250 mL of a buffer, according to the following scheme: first day, 0.05 M phosphate buffer, pH 8 (containing 10% acetonitrile); second day, 0.05 M phosphate buffer, pH 7 (containing 10% acetonitrile); and third day, 0.05 M phosphate buffer, pH 7. This scheme is essential for preventing precipitation of the crude product during its purification by dialysk2 The ratios of DNR (or DOX) to antibody in the retentate were calculated from the measured light absorption at 490 nm (t = 11000) due t o DNR (or DOX) and t h e antibody protein concentration, by the method of Lowry et al. (17) from light absorption at 750 nm (referred to a standard curve from known concentrations of mouse IgG). RESULTS AND DISCUSSION

Synthesis of Electrophilic Analogues of DNR and DOX. Phenyl isothiocyanates 3 and 4 (Chart I) were obtained in good yields (93% and 77 % , respectively) by treating p-aminobenzoic acid (1) or 3-(p-aminophenyl)propionic acid (2) with thiophosgenein an aqueous alkaline solutions. Although 4 was readily converted to 6 with thionyl chloride in chloroform at room temperature, 3 remained unchanged under these conditions. Most likely, the carboxylic acid group in 3 is deactivated due to its conjugation with the electron-withdrawingphenyl isothiocyanate group. Accordingly, the more vigorously reactive DMF/thionyl chloride (Vilsmeier reagent ( 2 6 ) ) was required for converting 3 to 5. The acid chlorides that remained after removing excess reagent and the volatile byproducts served as the ”linker reagents” for synthesizing the electrophilic anthracycline antibiotic analogues. p-(Fluorosulfony1)benzoyl chloride in DMF failed to react with the 3‘-amino group in DNR. Under similar conditions bromoacetyl bromide reacts with 5’-amino-5‘deoxyadenosine in DMF to give the corresponding 5’-N(bromoacety1)amide (27), and p(fluorosulfony1)benzoyl chloride gives the corresponding 3’-ester with adenosine (28). However, adding 5,6, bromoacetyl bromide, or p-(fluorosulfony1)benzoylchloride in chloroform (or benzene) to a vigorously stirred, aqueous alkaline (potassium 2 I t was suggested during review of this paper that instead of adding a cosolvent to the dialysis mixture, addition of glutathione (or cysteine) for the bromoacetamide, and glycine (or glutamic acid) for the isothiocyanate (or sulfonyl fluoride) may facilitate efficient removal of the excess, lipophilic reagents during purification of the conjugates.

254

Bioconjugafe Chem., Vol. 1, No. 4, 1990

Rosik and Sweet

bicarbonate) solution of DNR or DOX gave excellent yields of a 3'-N-substituted electrophilic analogue of the anthracycline antibiotic (Chart I). T h e electrophilic analogues of DNR or DOX are stable for months under dry, cool, and dark conditions. A t first, crystalline hydrochlorides of DNR or DOX were used to synthesize the electrophilic anthracycline antibiotic analogues. After discovering that the lactose (added to stabilize the drugs) in pharmaceutical preparations does not interfere with the N-acylation reactions, equivalent amounts of cerubicin (DNR-HC1, buffer, and lactose) provided the same yields as aqueous solutions of crystalline DNR-HCl. While working up the acylation reaction mixtures, lactose remains in the aqueous phase and the electrophilic DNR analogues are isolated in the organic extracts. Anthracycline antibiotics are intensely red (Y490 nm; 6 = 11 000), causing less than 1 pg of the compounds to be visible on a TLC plate. Chloroform/methanol (95:5) is ideal for separating DOX (Rf = 0.03) and DNR (Rf = 0.06) from the corresponding 3'-N-amide analogues. The analogues have mobilities with Rf values of 0.25-0.37 and 0.44-0.64 for the various N-substituted derivtives of DOX and DNR, respectively. Accordingly, flash chromatography is the method of choice for final purification of the anthracycline antibiotic analogues. Characteristic 'H signals (29, 30) and IR bands (3133) from the DNR and DQX moieties of the N-substituted analogues were in complete agreement with the established spectra. Thus we reported here only those 'H NMR signals for DNR and DOX that reflect changes a t the 3'-NH (amide) and the 3'-CH (sugar) positions, in addition to the signals produced by the various 3'-N-substituents. The IR spectrum of DNR-HCl contains a characteristic pattern of bands at 1715,1623,1588,1416,1293,1217,1125, and 995 cm-'. This pattern was observed in the IR spectra of the DNR and DOX analogues. The phenyl isothiocyanate group in 3 and 4 produces characteristically strong absorption bands at 2080-2120 cm-', also observed in the IR spectra of the corresponding N-substituted analogues (7-9) of DNR and DOX. A synthesis of 3'-N-(iodoacetyl)but this doxorubicin in 75% yield has been reported material was only characterized by TLC and therefore the spectra could not be compared with those of the present DOX analogues. Reactions of Electrophilic DNR and DOX Analogues with Proteins. The DNR analogues were tested with bovine serum albumin (BSA) to assess the reactivity of the N-substituents toward proteins and the stability of t h e resulting DNR-BSA conjugates. Concentrations of BSA were 1-4 mg/mL (ca. 12-50 pM), and that of a DNR analogue were approximately 300 pM. Various conjugation reactions were carried out between pH 7 and 8 (in 0.1 M phosphate buffer, containing 10% acetonitrile as cosolvent). The best results were obtained at pH 8. 1,2-Dimethoxyethane and dioxane were tested as cosolvents, but they were not as effective as acetonitrile for dissolving the N-substituted DNR analogues at the high concentrations required for efficient conjugation reactions with proteins in aqueous solutions. Dialysis, gel-filtration chromatography, and polyacrylamide gel electrophoresis (PAGE) were used to determine the conjugation efficiency of the various DNR analogues. The conjugates prepared with DNR analogues containing a phenyl isothiocyanate group were expected to be reactive like FITC toward proteins. Thus these conjugates were only subjected to dialysis. Dialysis of the crude conjugation reaction mixture against water alone caused a major portion

(In,

of the protein and DNR analogue to precipitate. When phosphate buffer alone was used for dialysis, the DNR analogue precipitated while the DNR-protein conjugate remained in solution. However, dialysis of the reaction mixture for 2 days against phosphate buffer containing 10% acetonitrile and for an additional day against phosphate buffer alone removes all of the unreacted DNR analogue (mostly on the first day of dialysis). During the third day of dialysis, the organic cosolvent is removed. The pH of the buffer is lowered to 7 on the second and third days of dialysis because the conjugate is most stable a t the lower pH during storage. Storing a conjugate a t 0 "C for 2-3 months a t pH 8 produces a bathochromic shift in the visible spectrum (from 490 to 500-505 nm) of the anthracycline moiety. This may result from the higher rate of oxidation of the hydroquinone function in the anthracycline group promoted by the alkaline conditions. The above dialysis scheme provides a bright red solution of a DNR-protein conjugate in the retentate. The dialysates of the conjugates made from 10 and 12 appeared as light red solutions. The red material was extracted with chloroform from the dialysate of the dialyzed 10-BSA. TLC analysis of the extracted, red material showed it to be unreacted 10. The red dialysate from 12-BSA (dialyzed a t pH 8) was repeatedly extracted with chloroform, but no colored material appeared in the organic layer. After acidification of the dialysate, the red material was extracted from the aqueous solution by chloroform. Presumably, the red extract contained the sulfonic acid form of 12, that had undergone hydrolysis. When 7 or 8 were conjugated with BSA, the bright red products remained in the retentate throughout the 3 days of dialysis. Only a trace of red material could be extracted from the faintly red dialysates. Two red bands emerged from a Sephadex G25-50 column during chromatography of crude 10- or 12-BSA conjugates (eluted with 0.1 M phosphate buffered a t pH 8, containing 10% acetonitrile). The more rapidly eluted red conjugate was followed by a second red band of unconjugated starting material. Polyacrylamide gel electrophoresis was performed on the following samples: (1)BSA (reference), (2) 12-BSA (dialyzed), (3) 12-BSA (gel filtered), and (4) 12-BSA (crude reaction mixture). Lanes 2-4 exhibited the major, bright red bands that migrated with the BSA in the reference lane. Lane 4 contained a red spot a t the origin. After staining the gels for protein (CoomasieBlue), the major, migrating red bands in lanes 1-4 were vividly stained, but the red spot a t the origin did not stain. The results suggest that either dialysis or gel filtration are effective in separating the DNR-protein conjugates from the unreacted (or hydrolyzed) DNR analogue. The relative reactivity with BSA of the electrophilic substituents in the analogues of DNR and DOX is the order p(fluorosulfony1) benzoyl > phenylisothiocyanato > bromoacetyl. However, the p(fluorosulfony1)benzoylamide 12 undergoes hydrolysis in competition with protein conjugation. Unreacted 3'-N-bromoacetyl-DNR is always observed in the dialysates from the conjugation reaction mixtures. Accordingly, the phenyl isothiocyanates are ideally reactive substituents for producing bioconjugates. The resulting conjugates are entirely stable a t pH 7 for at least days at room temperature and for months a t 0 "C. DNR- and DOX-Antibody Conjugates, The electrophilic analogues of DNR were conjugated with OC125 (a monoclonal antibody that binds to the CA125 antigen in human ovarian cancer cells). During the conjugation reactions the protein concentration of OC125 was 0.5-

Reagents for Synthesis of Bioconjugates

Bioconjugate Chem., Vol. 1, No. 4, 1990

255

1.0 mg/mL (3.3-6.6 pM), the concentration of the elecsections from human ovarian cancers (2) that express the trophilic DNR analogues was 33-66 pM, and the reactions CA125 antigen. were conducted in 0.05 M phosphate buffer a t pH 8 ACKNOWLEDGMENT (containing 10% acetonitrile). The conjugates were purified by dialysis in the manner described above for the We gratefully acknowledge the technical assistance of DNR-BSA conjugates, and they exhibited similar stability. Qingxuan Chen (PAGE), in vitro toxicity testing by John The measured ratios of DNRlOC125 in the conjugates were L. Collins and Deanne Perry, the generosity of Adria between 0.5:l and 19:l. Conjugates with ratios higher than Laboratories of Columbus, OH, for providing Doxorubi1 9 1 precipitated during dialysis. DNR-OC125 conjugates cin, Wyeth Laboratories of Princeton, NJ, for daunoruused in earlier in vitro experiments had druglantibody bicin, and Centocor Laboratories of Malvern, PA, for the ratios of 3:l to 6:l (I,2). Human ovarian cancer cells were OC125 monoclonal antibody used in this study. NMR used to test the cytotoxicity of DNR-OC125 conjugates spectra were recorded at the Washington University High( I ) in which 8-OC125 had low (2-3:l) and high (10-14:l) Resolution NMR Facility that is partly funded by NIH ratios. The cytotoxicities of these bioconjugates appear Shared Instrument Grant No. 1-S10-RR02004. This to be independent of the drug/antibody ratio in that the research was supported by a grant from the Fraternal Order low and high ratio of the DNR-OC125 conjugates exhibit of Eagles (MO). equal toxicities for equivalent, nominal concentrations of DNR(34). LITERATURE CITED The relative stability of the 3’-N linkage formed between 1 or 2 and DNR is interesting. In experiments that (1) Sweet, F., Rosik, L. O., Sommers, G. M., and Collins, J. L. (1989) Daunorubicin conjugated to a monoclonal anti-CApreceded the present work, N-hydroxysuccinimide esters 125 antibody selectively kills human ovarian cancer cells. Gyof the N-trityl derivatives of 1 or 2 were condensed with necol. Oncol. 34,305-311. DNR to provide moderate yields of the corresponding 3’(2) Dezso, B., Torok, I., Rosik, L. O., and Sweet, F. (1990) Human N-substituted phenylpropionyl- or benzoylamides (34). ovarian tumors specificallybind daunorubicin-OC125 antibody Both amides were completely stable in neutral solutions. conjugates: an immunofluorescence study. Gynecol. oncol., However, when detritylation was conducted in acetic acid in press. (pH 4) the benzoyl amide derivative formed free DNR and (3) Ghose, T. I., Blair, A. H., Vaughan, K., and Kulkami, P. (1983) 1, but the detritylated 3’-N-[3-(p-aminophenyl)propionyl]Targeted Drugs (E. P. Goldberg, Ed.) pp 1-22, J. Wiley and amide-DNR remained intact. Clearly, the aromatic ring Sons, New York. that is conjugated with the carboxamide group promotes (4) Reisfeld, R. A., and Cherish, D. A. (1985) Human tumorassociated antigens: targets for monoclonal antibodyhydrolysis of the 3I-N-amide linkage. By contrast, in 3’mediated cancer therapy. Cancer Surv. 4, 271-290. N-[3-(p-aminophenyl)propionyl]amide-DNRthe two ( 5 ) Frankel, A. E., Houston, L. L., Issell, B. F., and Fathman, additional carbon atoms prevent this effect of the aromatic G. (1986) Prospects for immunotoxin thearpy in cancer. Ann. ring by electronically insulating the carboxamide group. Rev. Med. 37, 125-142. Furthermore, the aliphatic amide linkages in the 3’-NGhose, T., and Blair, A. H. (1987) The design of cytotoxic(bromoacetyl) and 3’-N-[3-(p-isothiocyanatophenyl)- (6)antigen-antibody conjugates. Crit. Rev. Ther. Drug Carrier propionyl] derivatives (8-1 1) are analogousto natural pepSyst. 3, 263-359. tide linkage systems containing alanine and phenylala(7) Pietrersz, G. A., Kanellos, J., Smyth, M. J., Zalberg, J., and nine, respectively. By this analogy, the 3’-N-amide linkage McKenzie, I. F. C. (1987) The use of monoclonal antibody in these conjugates are expected to possess greater (i.e., conjugates for the diagnosis and treatment of cancer. Im”natural”) stability in acidic environments (Le., pH 4) than munol. Cell Biol. 65, 111-125. (8) Rodwell, J. D. (1988) Antibody-Mediated Delivery Systems the corresponding 3’-N linkage in protein conjugates from Marcel Dekker, New York. 7 or 12. (9) Rodwell, J. D., Alvarez, V. L., Lee, C., Lopes, A. D., Goers, The relative toxicities in vitro of the DNR-antibody J. W. F., King, H. D. Powsner, H. J., and McKearn, T. J. (1986) conjugates made from 7 or 8 and OC125 may be related Site-specific covalent modification of monoclonal antibodies: to certain bond-stability factors (discussed above). Under in vitro and in vivo evaluations. Proc. Natl. Acad. Sei. U.S.A. equivalent conditions of concentration and incubation 83,2632-2636. periods, 8-OC125 is consistently about twice as toxic as (10) Laguzza, B. C., Nichols, C. L., Briggs, S. L., Cullinan, G. J., 7-OC125 to two different cell lines derived from human Johnson, D. A., Starling, J. J., Baker, A. L., Bumol, T. F., and ovarian tumors ( I ) . Additional experimental work must Corvalan, J. R. F. (1989) New antitumor monoclonal antibodyvinca conjugates LY203725 and related compounds: Design, be undertaken on this theme to precisely relate the preparation, and representative in vivo activity. J. Med. Chem. chemical nature of the 3’-N-amide linkage in the DNR32,548-555. antibody conjugates of 7 and 8 with the observed (11) Diener, E., Diner, U. E., Sinha, A., Xie, S., and Vergidis, differences in their toxicities. Nevertheless, the reactivity R. (1986) Specific immunosuppression by immunotoxins of the reagent, stability of the linkage, and toxicity of the containing daunomycin. Science 231, 148-150. resulting OC125 conjugate make 6 the preferred reagent (12) Means, G. E . , a n d Feeney, R. E. (1990) Chemical for producing electrophilic analogues of drugs intended Modifications of Proteins: History of Applications. Bioconfor synthesis of bioconjugates. jugate Chem.. I , 2-12. (13) Koppel, G. A. (1990) Recent Advances with Monoclonal The present report describes a variety of reagents that Antibody Drug Targeting for the Treatment of Human Cancer. efficiently provide stable electrophilic analogues of DNR Bioconjugate Chem. 1, 13-23. and DOX that can be isolated, characterized, and stored. (14) Hurwitz, E., Levy, R., Maron, R., Wilchek, M., Arnow, R., A stable anticancer drug-antibody conjugate can be and Selig, M. (1975) The covalent binding of daunomycin and produced by mixing the analogues with a monoclonal adriamycin to antibodies with retention of both drug and antibody. The potential use of the present DNRantibody activities. Cancer Res. 35, 1175-1181. OC125 bioconjugates for treating a variety of antibody(15) Gallego, J., Price, M. R., and Baldwin, R. W. (1984) targeted cancers is suggested by results from in vitro tests. Preparation of four daunomycin-monoclonal antibody 79IT/ The bioconjugatesare both selectively toxic to human ova36 conjugates with anti-tumor activity. Znt. J. Cancer 33,737rian cancer cell lines (I)and also specifically bind to tissue 744.

256

Bioconjugate Chem., Vol. 1, No. 4, 1990

(16) Yang, H. M., and Reisfeld, R. A. (1988)Doxorubicin conjugated with monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor xenographs in nude mice. Proc. Natl. Acad. Sci. U.S.A. 85,1189-1193. (17) Pietersz, C. A., Smyth, M. J., and McKenzie, I. F. C. (1988) The use of anthracycline-antibody complexes for the specific antitumor activity. In Antibody-Mediated Delivery Systems (J. D. Rodwell, Ed.) Marcel Dekker, New York. (18) Sweet, F., and Murdock, G. L. (1987)Affinity labeling of hormone-specific proteins. Endocr. Reu. 8, 154-184. (19) Riggs, J. L., Seiwald, R. J., Burckhalter, J. H., Downs, C. M., and Metcalf, T. G. (1958)Isothiocyanate compounds as fluorescent labeling agents for immune serum. Am. J . Pathol. 34,1081-1097. (20)McKinney, R. M., Spillane, J. T., and Pearce, G. W. (1964) Factors affecting the rate of reaction of fluorescein isothiocyanate with serum proteins. J . Zmmunol. 93,232-242. (21) Nairn, R. C. (1976)Fluorescent Protein Tracing Churchill Livingstone, New York. (22) Meares, C. F., McCall, M. J., Reardan, D. T., Goodwin, D. A., Diamanti, C. I., and McTigue, M. (1984)Conjugation of Antibodies with Bifunctional Chelating Agents: Isothiocyanate and Bromoacetamide Reagents, Methods of Analysis, and Subsequent Addition of Metal Ions. Anal. Biochem. 142, 68-78. (23) Westerberg, D. A., Carney, P. L., Rogers, P. E., Kline, S. J., and Johnson, D. K.(1989)Synthesis of novel bifunctional chelators and their use in preparing monoclonal antibody conjugates for tumor targeting. J . Med. Chem. 32,236-243. (24) Mirzadeh, S.,Brechbiel, M. W., Atcher, R. W., and Gansow, 0. A. (1990)Radiometal Labeling of Immunoproteins: Covalent Linkage of 2-(4-Isothiocyanatobenzyl)diethylenetriaminepentacetic Acid Ligands to Immunoglobulin. Bioconjugate Chem. 1,59-65.

Rosik and Sweet

(25) Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)Protein measurement with folin phenol reagent. J . Biol. Chem. 193,265-275. (26) Fieser, L. F., and Fieser, M. (1967)Reagents f o r Organic Synthesis pp 286-287,J. Wiley and Sons, New York. (27) Samant, B. R., and Sweet, F. (1983)5’-Bromoacetamido5’-deoxyadenosine. A novel reagent for labeling adenine nucleotide sites in proteins. J . Biol. Chem. 258,12779-12782. (28) Pal, P. K., Wechter, W. J., and Colman, R. F. (1975)Affinity labeling of a regulatory site of bovine liver glutamate dehydrogenase. Biochemistry 14,707-715. (29) Iwamoto, R. H., Lim, P., and Bhacca, N. S. (1968) The structure of daunomycin. Tetrahedron Lett. 3891-3894. (30) Arcamone, F.,Cassinelli, G., Franceschi, G., Orezzi, P., and Mondelli, R. (1968)Total absolute configuration of daunomycin. Tetrahedron Lett. 3353-3356. (31) Arcamone, F., Franceschi, G., Orezzi, P., Cassinelli, G., Barbieri, W., and Mondelli, R. (1964)Daunomycin I. The structure of duanomycinone. J . Am. Chem. SOC.86,5334-5335. (32) Wong, C. M., Schwenk, R., Popien, D., and Ho, T.-L. (1973) The total synthesis of daunomycinone. Can. J . Chem. 51,466467. (33) Arcamone, F., Franceschi, G., and Penco, S. (1969)Adriamycin, a novel antitumor antibiotic. Tetrahedron Lett. 10071010. (34) Unpublished results. Registry No. 1,150-13-0; 2,2393-17-1; 3,2131-62-6; 4,12779304-8;5, 53611-24-8;6,127793-05-9;7, 110409-19-3;8, 12779306-0;9,127793-07-1; 10,47831-81-2; 11,127793-08-2; 12,12779309-3;thiophosgene, 463-71-8;bromoacetyl bromide, 598-21-0; p(fluorosulfony1)benzoyl chloride, 402-55-1; daunorubicin hydrochloride, 23541-50-6;doxorubicin hydrochloride, 2531640-9.