Selective chemotherapeutic strategies using catalytic antibodies: a common pro-moiety for antibody-directed abzyme prodrug therapy

Journal of Immunological Methods 269 (2002) 269 – 281 www.elsevier.com/locate/jim

Selective chemotherapeutic strategies using catalytic antibodies: a common pro-moiety for antibody-directed abzyme prodrug therapy Hiroyuki Kakinuma a, Ikuo Fujii b,*, Yoshisuke Nishi a,* a

Laboratory of Life Science and Bimolecular Engineering, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama 227-8512, Japan b Bimolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan Received 4 March 2002; accepted 13 March 2002

Abstract Prodrug activation by catalytic antibodies (abzymes) conjugated with anti-tumor antibodies, called antibody-directed abzyme prodrug therapy (ADAPT), has been proposed as a strategy for site-specific drug delivery systems for anti-tumor drugs. The delivery of abzymes is achieved by making a bi-specific antibody with a monovalent catalytic antibody and a monovalent binding antibody. To achieve ADAPT, we focused on specific requirements for prodrugs and catalytic antibodies, the stability of the prodrugs against natural enzymes, and the applicability of abzymes for a wide range of prodrugs. Attention was paid to the design of a pro-moiety rather than a parent drug. As a common pro-moiety, we chose vitamin B6, because the bulky vitamin B6 esters are relatively stable against hydrolytic enzymes in serum. We have generated catalytic antibodies by immunization of a vitamin B6 phosphonate transition state analog. The elicited antibodies were found to hydrolyze several anti-cancer and antiinflammatory prodrugs with the vitamin B6 pro-moiety. Finally, we evaluated antibody-catalyzed prodrug activation by examining the growth inhibition of human cervical cancer (HeLa) cells with the vitamin B6 ester of butyric acid. These results suggest that the pro-moiety of vitamin B6 ester is stable enough to resist natural enzymes in serum and is removed by the tailored catalytic antibodies. The combination of catalytic antibodies and prodrugs masked with vitamin B6 would allow hydrophobic and highly toxic drugs to be used. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Antibody-directed abzyme prodrug therapy; Prodrug; Vitamin B6; Catalytic antibody

1. Introduction * Corresponding authors. I. Fujii is to be contacted at tel.: +8166-872-7253; fax: +81-66-872-8219. Y. Nishi, tel.: +81-45-9725901; fax: +81-45-972-6205. E-mail addresses: [email protected] (I. Fujii), [email protected] (Y. Nishi).

The clinical effects of drugs depend to a great extent on their appropriate delivery to the target lesions; for example, failure to deliver anti-tumor drugs to the appropriate lesions often causes no effect, but can cause damage to normal tissues. One strategy

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 2 4 1 - 7

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for site-specific drug delivery systems for anti-tumor drugs is prodrug activation by enzymes conjugated with antibodies, which can bind to tumor-specific antigenic determinants; this technique is called antibody-directed enzyme prodrug therapy (ADEPT) (Bagshawe, 1987, 1995; Huennekens, 1994). By using enzyme –antibody conjugates, the complementary prodrugs with reduced toxicity, whose pro-moieties are specific substrates for the bacterial enzymes, are converted to the parent drugs at the tumor site by the enzyme component and then effectively damage the tumor cells without causing peripheral cytotoxicity. Clinical trials of ADEPT have been performed (Bagshawe et al., 1999; Syrigos and Epenetos, 1999) using a prodrug of a nitrogen mustard followed by administration of anti-carcinoembryonic antigen conjugated with bacterial carboxypeptidase G2, resulting in partial recovery in five of eight patients (Martin et al., 1997). The enzyme components are commonly of bacterial origin. They are designed to diminish activation by any circulating endogenous enzymes and to be specific for prodrugs. However, nonhuman enzymes generally elicit neutralizing antibodies by host immune response. This limits the repetition of treatments, unless immunosuppressants such as cyclosporin are used, which can allow two or three cycles of treatment, due to suppression of the host immunoresponse (Bagshawe and Sharma, 1996). To overcome this problem, it has been proposed that the bacterial enzymes can be replaced with humanized antibodies with catalytic functions (Bagshawe, 1995); less antigenic humanized antibodies and fully human antibodies are available through genetic engineering and transgenic mice (Green et al., 1994; Rader et al., 1998). We have demonstrated the first example of antibody-catalyzed prodrug activation (Miyashita et al., 1993). To date, some abzymes have been generated for the development of ADAPT (Campbell et al., 1994; Wentworth et al., 1996; Shabat et al., 1999). To achieve ADAPT, prodrugs and catalytic antibodies must meet specific requirements. The catalytic antibodies must manifest different catalytic properties from any endogenous natural enzymes to diminish the extent of peripheral activation of prodrugs. In other words, the prodrugs must be stable against natural enzymes and must be activated by each particular catalytic antibody. It is also of benefit if the catalytic antibodies are able to activate a wide range of pro-

drugs, because drugs showing a different spectrum may be useful for drug tolerant cells. Unfortunately, in general, the substrate specificity of catalytic antibodies is still quite restricted due to the inherent binding properties of antibodies. To satisfy these requirements, we have focused on a pro-moiety rather than a parent drug. In this work, vitamin B6 was chosen as a suitable pro-moiety for ADAPT. The prodrug derived from esterification with vitamin B6 is resistant to degradation by natural enzymes in serum due to the steric hindrance between the bulky vitamin B6 moiety and enzymes. In addition, the bulkiness and functionality of vitamin B6 seemed to be adapted to the haptenic strategy for acquiring broad substrate specificity of catalytic antibodies, in which a hapten composed of two different immunogenic regions, a strongly immunogenic aromatic ring and a nonimmunogenic unsubstituted alkyl chain, is used for immunization. Thus, immunization with a simple alkylphosphonate of vitamin B6 would elicit antibodies that strongly recognize the vitamin B6 pro-moiety, leaving the parent drug moiety free. Here, we demonstrate the generation of catalytic antibodies that can activate multiple prodrugs masked by vitamin B6. The elicited catalytic antibodies were tested in terms of their applicability to a broad range of prodrugs and have successfully inhibited the growth of a tumor cell line (HeLa) with an anti-cancer prodrug in vitro. We also discuss the possibilities and the potential problems of the catalytic antibody towards the new therapy. The important issue is to develop antibody-catalyzed prodrug activations that cannot be catalyzed by endogenous natural enzymes and that can be overcome by using vitamin B6 as a pro-moiety.

2. Materials and methods 2.1. Synthesis of TSA and prodrugs Melting points were measured with a Yanagimoto melting point aperture and were uncorrected. 1H NMR spectra were recorded with a Bruker AC-300P spectrometer for solutions in CDCl3 or MeOH-d4 with Me4Si as an internal standard. Chemical shifts of 31P NMR were referred to H3PO4 as an external standard. Flash column chromatography was performed with

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silica gel 60 (Merck). Preparative HPLC was performed with a YMC packed column (ODS, S-5, 120A, 20  2 cm). FAB mass spectra were obtained on a Kratos Mass Spectrometer CONCEPT IIH using 800 V of electron acceleration. 2.1.1. 4-(Acetylaminomethyl)-5-(hydroxymethyl)-2methyl-3-pyridyl hydrogen 5-carboxypentyl-phosphonate (1) (TSA 1) Bromotrimethylsilane (1.4 ml, 11 mmol) was added to a solution of diethyl 5-ethoxycarbonylpentylphosphonate (7) (1.0 g, 3.6 mmol) in CH2Cl2 (10 ml) under an Ar atmosphere. The mixture was stirred at 25 jC for 4 h, and then cooled to 0 jC. Methanol (10 ml) was added to the mixture to quench the excessive reagent. The mixture was evaporated and the residue was repeatedly co-evaporated with pyridine (10 ml) to give 5-ethoxycarbonylpentylphosphonic acid pyridinium salt (8) (1.1 g, quant.). 1H NMR (MeOH-d4) d 1.23 (t, 3H, J = 7.1 Hz, OCH2CH3), 1.40– 1.47 (m, 2H), 1.54 – 1.73 (m, 6H), 2.31 (t, 2H, J = 7.4 Hz, CH2PO3H), 4.10 (q, 2H, J = 7.1 Hz, OCH2CH3), 7.83 – 7.87 (m, 2H, Py), 8.31 – 8.38 (m, 1H, Py), 8.74 –8.76 (m, 2H, Py); 31PNMR (MeOH-d4) d 28.4. 4-(Acetylaminomethyl)-5-(acetoxymethyl)-3hydroxy-2-methylpyridine (403 mg, 1.60 mmol) and 2,4,6-triisopropylbenzenesulfonyl chloride (724 mg, 2.40 mmol) were added to a solution of this compound (485 mg, 1.60 mmol) in pyridine (5.0 ml). The mixture was stirred for 4 h and then evaporated. The residue was purified by flash chromatography (CHCl3/MeOH/ Et3N, 20:2:1) and the product was further purified by HPLC (MeCN/0.1% TFA, 3:7). The MeCN and TFA were evaporated, and the water was removed by lyophilization to give 4-(acetylaminomethyl)-5-(acetoxymethyl)-2-methyl-3-pyridyl hydrogen 5-ethoxycarbonyl-pentylphosphonate (9) (122 mg, 17%) as a colorless powder. 1H NMR (MeOH-d4) d 1.23 (t, 3H, J = 7.1 Hz, OCH2CH3), 1.48 – 1.54 (m, 2H, CH2), 1.62 –1.73 (m, 2H, CH2), 1.75 –1.89 (m, 2H, CH2), 1.94 (s, 3H, NHAc), 1.94 –2.01 (m, 2H, CH2), 2.09 (s, 3H, OAc), 2.34 (t, 2H, J = 7.3 Hz, CH2PO3H), 2.79 (s, 3H, Me), 4.11 (q, 2H, J = 7.1 Hz, OCH2CH3), 4.71 (s, 2H, CH2NHAc), 5.42 (s, 2H, CH2OAc), 8.48 (s, 1H, Py); 31P NMR (MeOH-d4) d 27.4; HRMS (FAB) calcd. f or C 2 0 H 3 2 N 2 O 8 P 4 59 . 1 8 9 6 ( M + 1 ) , f o u n d 459.1931(M + + 1). This compound (64 mg, 0.14 mmol) was dissolved in 1 M NaOH (1.5 ml). The

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mixture was stirred for 40 h and adjusted to pH 4.0 with 6 M HCl. The resulting mixture was purified by HPLC (MeCN/0.1% TFA, 1:4) to afford 1 (52 mg, 95%) as a colorless powder. 1H NMR (MeOH-d4) d 1.46– 1.59 (m, 2H, CH2), 1.60– 1.73 (m, 2H, CH2), 1.74– 1.89 (m, 2H, CH2), 1.94 (s, 3H, NHAc), 1.90 – 2.02 (m, 2H, CH2), 2.32 (t, 2H, J = 7.3 Hz, CH2PO3H), 2.78 (s, 3H, Me), 4.62 (s, 2H, CH2NHAc), 4.90 (s, 2H, CH2OH), 8.40 (s, 1H, Py); 31P NMR (MeOH-d4) d 27.1; HRMS (FAB) calcd. for C16H26N2O7P 389.1478 (M + 1), found 389.1494 (M+ + 1). 2.1.1.1. 4-(N-Acetylaminomethyl)-5-(hydroxymethyl)3-[[2-(4-isobutylphenyl)-propionyl]oxy]-2-methylpyridine (5). EDC (191 mg, 1.00 mmol) was added to a solution of N-acetylpyridoxamine (98 mg, 0.47 mmol) and ibuprofen (154 mg, 0.75 mmol) in Me2NCHO (3.0 ml). The mixture was stirred for 20 h at 25 jC and quenched with H2O (30 ml). The mixture was extracted with EtOAc (2  50 ml), and the combined extracts were washed with brine. The organic layer was dried over MgSO4 and then evaporated. The product was purified by flash chromatography (CHCl3/MeOH, 50:1) to give 5 (48 mg, 26%). m.p. 87 – 89 jC; 1H NMR (MeOH-d4) d 0.89 (d, 6H, J = 7.2 Hz, CHMe 2 ), 1.61 (d, 3H, J = 7.1 Hz, CHPhMe), 1.75– 2.09 (m, 7H), 2.48 (d, 2H, J = 7.2 Hz, CH2Ph), 4.13 (q, 1H, J = 7.1 Hz, CHPhMe), 4.71 (s, 2H, CH2OH), 7.18 (d, 2H, 8.2 Hz, Ph), 7.36 (d, 2H, J = 8.2 Hz, Ph), 8.28 (s, 1H, Py); HRMS (FAB) calcd. for C23H31N 2O4 399.2284 (M + 1), found 399.2294 (M+ + 1). 2.1.1.2. 4-(Acetylaminomethyl)-5-(hydroxymethyl)-2methyl-3-(butanoyl)oxypyridine (11). n-Butyric anhydride (0.276 ml, 1.69 mmol) was added to a solution of N-acetylpyridoxamine (354 mg, 1.69 mmol) in pyridine (3.5 ml). After 3 h, the mixture was evaporated. The product was purified by flash chromatography (CHCl3/MeOH, 10:1) to give 11 (260 mg, 55%). m.p. 115 –116 jC; 1H NMR (MeOH-d4) d 1.05 (t, 3H, J = 7.4 Hz, COCH2CH2Me), 2.63 (sextet, 2H, J = 7.4 Hz, COCH2CH2Me), 1.88 (s, 3H, NHAc), 2.32 (s, 3H, PyMe), 2.69 (t, 2H, J = 7.4 Hz, COCH2CH2Me), 4.41 (brs, 2H, CH2NHAc), 4.75 (s, 2H, CH2OH), 8.32 (s, 1H, Py); HRMS (FAB) calcd. for C14H21N2O4 281.1501 (M + 1), found 281.1502 (M+ + 1).

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The syntheses of other prodrugs 2– 4, 6, 10, and 12– 14 are described in Appendix A. 2.2. Measurement of half-life of the prodrugs A solution of prodrugs 2– 6 (10 mM) in DMSO was added to a solution of human serum (90 Al), and the mixture was incubated at 30 jC. After an appropriate interval, acetonitrile (100 Al) was added to the reaction mixture to remove serum protein. The resulting white precipitate was removed by centrifugation, and the supernatant was filtered through a 0.45-Am filter (Whatman, Clifton, NJ) and analyzed by HPLC using a Shimadzu 10 A VP system (Shimadzu, Kyoto, Japan) and a 250  4.6-nm ODS column (YMC, Kyoto, Japan). The compounds were detected by UV 254 nm and eluted with acetonitrile/0.1% aqueous TFA (6:4) for prodrugs 3, 5, 6, and acetonitrile/0.1% aqueous TFA (7:3) for prodrug 4, respectively. 2.3. Antibody production and purification Ten MRL/lpr autoimmune mice (Charles River Laboratories, Atsugi, Japan) were subcutaneously immunized with KLH-1 (50 Ag) mixed in complete Freund’s adjuvant on days 1 and 14. On day 28, four mice with the highest titer (1:>2  105) were subsequently boosted with KLH-1 in incomplete Freund’s adjuvant. After 3 days, the mice were killed by CO2induced anoxia, and splenocytes were extracted and fused with myeloma cells (X63.653) following the conventional protocol (Takahashi et al., 2000). The hybridomas were incubated in medium containing hypoxanthine – aminopterin – thymidine (HAT) and 10% FBS in 96-well culture plates. After an appropriate period of growth, the plates were examined by ELISA for binding to BSA-1. The bound wells were picked up and cloned. The monoclonal cells were incubated until the cells reached confluence in TIL media + 10% fetal bovine serum (FBS) (Immuno-Biological Laboratory, Gunma, Japan). The supernatants were treated with ammonium sulfate at a final concentration of 60%, and the precipitates were dissolved and dialyzed against PBS at 4 jC. Antibodies (IgG) were purified using a Hitrap protein G –Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden), followed by High Q anion exchange chromatography (Bio-Rad, Hercules, CA) with a salt

gradient from 0 to 1 M NaCl in 10 mM Tris –HCl (pH 7.5). The fractions containing antibodies were concentrated using a Centriprep Concentrator (Amicon, Beverly, MA), and the solution was changed to 25 mM Tris – HCl buffer (pH 8.0) for the kinetic assay. The concentration of antibody was determined by absorbance at 280 nm with e = 1.4 and MW = 150,000 for IgG. 2.4. Kinetic assay The kinetic parameters were determined by adding 5 Al of a stock solution of prodrugs 5, 11– 14 in DMSO to 95 Al of antibody solution (5.1 AM) in 25 mM Tris – HCl, 0.01% NaN3 (pH 8.0) at 30 jC. The initial velocities were determined by measuring the increase in fluorescence (excitation 320 nm, emission 405 nm) every 15 min for 3 h, using a Fluoroskan ascent spectrophotometer (Dainippon Pharmaceutical, Osaka, Japan). The kcat and Km values were determined by fitting the data to Lineweaver – Burk plots using KaleidaGraph software (Synergy Software, PA). All assays were performed at least in duplicate. The background hydrolyses (kuncat) were determined by initial rate analysis in the presence of mouse IgG (Funakoshi, Tokyo, Japan) under otherwise identical conditions and extrapolated to zero buffer concentration. 2.5. Growth inhibition assay of HeLa cells HeLa cells were precultured in medium (Eagle’s MEM supplemented with 10% FBS and 50 U/ml penicillin – streptomycin) (Nikken Biomedical Laboratory, Kyoto, Japan), for 48 h in a 75-cm2 culture flask at 37 jC under 5% CO2. After removing the medium, the cells were treated with 0.05% trypsin/ 0.53 mM EDTA at 37 jC for 5 min and resuspended in the medium. The cells at 1  103 were plated in a well in a 96-well tissue culture plate and incubated for 24 h at 37 jC under 5% CO2. Stock solutions (8.3 Al) of 400 mM butyric acid and the prodrug 11 in DMSO were added to the medium (991.7 Al) and the solution (300 Al) was further diluted with 25 mM Tris –HCl (pH 7.0), 0.01% NaN3 (200 Al) or 106 AM of the antibody V122 in 25 mM Tris– HCl (pH 7.0), 0.01% NaN3 (200 Al). The solutions were prepared just before they were added to the wells (100 Al to each

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of five wells in each experiment). Final concentrations of the reagents were 1.0 mM for the drugs or a combination of 1.0 mM for the prodrug and 21 AM for the antibody V122 in medium containing 20% Tris –HCl buffer and 0.25% DMSO. Control experiments were carried out without the drugs and the antibody under otherwise identical conditions. After adding the drugs, the cells were incubated for 24 h under 5% CO2 at 37 jC. The medium was removed and the cells were carefully washed twice with the medium (200 Al) and further cultured in medium (200 Al) for 48 h at 37 jC under 5% CO2. Since living cells can incorporate Neutral Red, the number of the cells was quantified by measuring the absorbed reagent (Hockley and Baxter, 1986). Neutral Red dyeing was performed as follows. After removing the medium, 200 Al of Neutral Red in the medium containing 0.5% ethanol (50 Ag/ml) was added to the cells and the cells were incubated for 3 h at 37 jC under 5% CO2. After removing the reagent, the cells were fixed with aqueous 1% formaldehyde/1% CaCl2 solution. The Neutral Red that was incorporated in the cells was extracted with 50% ethanol/1% acetic acid (100 Al). The extracts were transferred to another set of 96-well plates and the solution was measured at 540 nm with a microplate reader (Wako, Osaka, Japan). The experiment was repeated three times and each result was averaged.

3. Results and discussion 3.1. Design of prodrugs for ADAPT In ADAPT, it is important for prodrugs to satisfy two requirements: stability and specificity. Thus, prodrugs must be activated by the tailored catalytic antibodies but not by natural enzymes in serum.

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Furthermore, for practical application of ADAPT, catalytic antibodies must accept a wide range of prodrugs as a substrate; it has been pointed out that the inherent binding specificity of antibodies restricts the applicability of catalytic antibodies. These requirements would be satisfied by using vitamin B6 ester derivative as a pro-moiety to mask the activity of the drugs and by generating catalytic antibodies that recognize the pro-moiety rather than the parent drug (Fig. 1). Vitamin B6 seems highly hydrophilic and nontoxic for normal tissue in vivo (Unna and Antopol, 1940). In addition, as the vitamin B6 esters are very bulky, they are expected to be resistant to cleavage by endogenous esterases due to steric hindrance (Chen and Fang, 1997). The bulky aromatic ring of vitamin B6 is also suitable for the haptenic strategy for acquiring broad substrate specificity of catalytic antibodies, in which the hapten 1 composed of two different immunogenic regions, a strongly immunogenic aromatic ring and a nonimmunogenic unsubstituted alkyl chain, is used for immunization (Fig. 2). Thus, immunization with a simple alkylphosphonate of vitamin B6 would elicit antibodies that strongly recognize the vitamin B6 pro-moiety, leaving the parent drug moiety free. 3.2. Stability of prodrugs with vitamin B6 pro-moiety As shown in Fig. 1, vitamin B6 possesses three functional groups on a pyridine ring that are able to link to parent drugs with ester bonds. As the stability of the prodrugs strongly depends on these linkages, we examined the stability of prodrugs masked with vitamin B6 in human serum, prior to generation of catalytic antibodies. We prepared vitamin B6 ester derivatives of an anti-inflammatory drug, ibuprofen, with different linkages and tested the stability of the prodrugs. The conjugation between vitamin B6 deriv-

Fig. 1. Structure of prodrugs masked by the vitamin B6 pro-moiety and prodrug activation by catalytic antibodies.

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Fig. 2. Synthesis of the hapten 1. Reagents and conditions: (a) trimethylsilylbromide, CH2Cl2, room temperature; (b) pyridine (100% yield); (c) 4-(N-acetylaminomethyl)-3-O-hydroxy-5-(acetoxymethyl)-2-methylpyridine, 2,4,6-triisopropylbenzenesulfonyl chloride, pyridine, room temperature (17% yield); (d) 1 M NaOH, room temperature (95% yield); (e) EDC, BSA, or EDC, KLH.

atives and ibuprofen was performed with carbodiimides to give 2, 3, 5, and 6. The prodrug 4 was obtained by acetylation of compound 3. The stability was evaluated by determining the half-life of the ester derivatives at 30 jC in human serum containing 10% DMSO (Table 1). Pyridoxal-5-ylmethyl ester (2) of ibuprofen was too labile to exist in human serum and disappeared immediately. This was not due to the rapid cleavage of the ester bond of 2. Probably, the carboxaldehyde moiety in 2 reacted with amino groups of proteins existing in serum to give an imine derivative. The half-life of pyridoxine-5-ylmethyl ester (3) was 33 min and that of its diacetate derivative (4) was more stable than 3 (69 min). N-Acetylpyridoxamine derivatives showed better stability than the others did in human serum. The 3-yl ester (5) was very stable with a half-life of more than 1 day. The 5-ylmethyl ester (6) was most stable with a half-life of 6.3 days. It was found that the ester derivatives of N-acetylpyridoxamine were stable enough to be used as pro-moieties of prodrugs in ADAPT. 3.3. Design and synthesis of hapten Although the 5-ylmethy ester (6) was the most stable prodrug, we finally chose the 3-yl ester (5) as the basic structure of the prodrugs because of its greater ability to generate catalytic antibodies. In

general, phosphonate transition state analogs are commonly used as haptens to generate hydrolytic catalytic antibodies. Therefore, for generation of catalytic antibodies for the 3-yl ester (5), the corresponding pyridoxamine 3-yl phosphonate ester would be used as a hapten. On the other hand, generation of catalytic antibodies for the 5-ylmethyl ester (6) requires pyridoxamine 5-ylmethyl phosphonate ester. As such 5-ylmethyl phosphate compounds, for example pyridoxal 5-phosphate, universally exist in vivo for cofactors of transaminase for amino acid metabolism (Christen and Metzler, 1985), it might be difficult to elicit an immune response with them. In addition, the reaction product from 5, i.e. N-acetylpyridoxamine, is fluorescent when excited at 325 nm. This facilitates screening of the catalytic antibodies from a huge repertoire and measurement of the kinetic parameters. Based on the idea described above, we synthesized the hapten, transition state analog (TSA) 1, by the procedure illustrated in Fig. 2. Diethyl 5-ethoxycarbonylpentylphosphonate (7) was prepared by the Arbuzov reaction. The ethyl phosphonate (7) was selectively removed with bromotrimethylsilane and the product was neutralized with pyridine to give 8. The condensation of phosphonate ester (route c) resulted in a small loss. Only 2,4,6-triisopropylsulfonyl chloride gave the desired phosphonate ester (1), although other reagents, such as carbodiimides, and the synthesis via phosphonyl chloride resulted in failure. The TSA 1 conjugate with carrier proteins, Table 1 Half-life of ibuprofen masked by vitamin B6s in human serum

Compounds

2 3 4 5 6

Substituents R1

R2

R3

T1/2

H H Ac ibuprofen H

CHO CH2OH CH2OAc CH2NHAc CH2NHAc

ibuprofen ibuprofen ibuprofen H ibuprofen

N.D.a 33 min 69 min 28 h 6.3 days

The half-life was determined in human serum containing 10% DMSO at 30 jC. a The T1/2 was not determined.

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keyhole limpet hemocyanin (KLH), KLH-1, was used for immunization, and the TSA 1 conjugate with bovine serum albumin (BSA), BSA-1 was used for selection by an ELISA assay (Bauminger and Wilchek, 1980). 3.4. Preparation of antibodies MRL/lpr autoimmune mice were subcutaneously immunized with KLH-1 (Takahashi et al., 2000) and 33 monoclonal antibodies were obtained by standard hybridoma methods (Ko¨hler and Milstein, 1975). First, we used anti-inflammatory prodrug 5 as a substrate to test the catalytic activities of the antibodies. Two (V93 and V122) of the 33 monoclonal antibodies hydrolyzed prodrug 5. As these activities were strongly inhibited by adding an equivalent of TSA 1 to an amount of the antibody, the antibodies were confirmed to be a specific catalyst for ester hydrolysis elicited against the TSA 1. 3.5. Substrate specificity for hydrolysis of vitamin B6protected prodrugs For testing substrate specificity, the antibodies were examined with respect to their ability to hydrolyze a variety of prodrugs of anti-inflammatory (Flower and Vane, 1974; Nickander et al., 1979) and anti-cancer drugs (Fig. 3). We prepared vitamin B6 ester derivatives, 10, 11, 12, 13, and 14 from fenbufen, butyric acid, benzoic acid mustard (Ross et al., 1955), phenylacetic acid mustard (Hovinen, 1996), and chlorambucil (Roehrig et al., 1980), respectively. The prodrug 12 can be less toxic than benzoic acid mustard, however, the toxicities of compounds 13 and 14 were comparable to those of the parent drugs, probably because the carboxyl groups are not directly conjugated to the benzene rings. We confirmed that compound 13 or 14 suppressed the proliferation of HeLa cells at the same level as phenylacetic acid mustard or chlorambucil (data not shown). Here, we used these drugs (13 and 14) in order to evaluate the substrate specificity of the catalytic antibodies obtained. The kinetic parameters were measured in 25 mM Tris –HCl (pH 8.0), 5% DMSO at 30 jC for 3 h by detecting the cleaved vitamin B6 moiety with a fluorescent detector, and fitting the data to the Michaelis –Menten equation; the parameters for 10

Fig. 3. Structure of prodrugs utilized for the kinetic assay.

were not determined due to its low solubility (Table 2). The chemical stability of the mustard moiety in compounds 13 and 14 was checked with HPLC analyses in the same buffer conditions. No product other than 13 was observed, however, 20% of 14 was converted into other products. Thus, we observed the apparent kcat and Km values in 14. Two antibodies, V93 and V122, were able to hydrolyze prodrugs 10, 11, 13, and 14. In the kinetic assay, the antibodies V93 and V122 were found to possess similar degrees of catalytic activity based on the values of kcat, whereas the antibodies showed different Km values for a variety of prodrugs. In the hydrolysis catalyzed by the antibody V122, all substrates showed similar values for Km, suggesting that the vitamin B6 pro-moieties of the prodrugs are bound into the antigen-combining site and the drug moieties are outside. As expected from the hapten design, antibody V122 has the potential to apply for a variety of prodrugs masked by vitamin B6 as a common promoiety. On the other hand, antibody V93 exhibited various values of Km for the prodrugs. The Km values of prodrugs 5 and 11 were three to four times higher than that of 14. Thus, in terms of their applicability for

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Table 2 Kinetic parameters of the antibody V122 and V93 for hydrolysis of the prodrugs Prodrugs

kcat (min

1

)

Km (AM)

kcat/Km (min/M)

KI (AM)

Antibody V122 5 1.5 F 0.047  10 2 11 2.6 F 0.32  10 2 13 3.3 F 0.18  10 2 14 2.1 F 0.10  10 2

84 F 5.5 99 F 21 102 F 12 58 F 7.0

180 260 323 360

0.84 1.6 0.84 0.54

Antibody V93 5 1.4 F 0.16  10 2 11 5.6 F 0.18  10 2 14 3.4 F 0.052  10 2

242 F 49 348 F 17 81 F 3.6

58 160 420

1.1 1.2 1.4

Conditions: 25 mM Tris – HCl, 0.01% NaN3, 5% DMSO, pH 8.0 at 30 jC. The kuncat of substrates 5, 11, 13, and 14 were 1.6  10 4, 2.6  10 4, 3.5  10 4, and 2.1  10 4 min 1, respectively. The KI values were determined by Dixon plot.

various substrates, antibody V122 shows a better profile than antibody V93. Unfortunately, neither antibody was able to hydrolyze prodrug 12 of benzoic nitrogen mustard. In this case, due to a steric hindrance between the bulky benzene ring and the antibodies, prodrug 12 may not bind to the antigencombining sites.

IC50 value of butyric acid for the HeLa cells was 0.9 mM; this value was consistent with that for human promyelocytic HL60 cells (Nudelman et al., 1992). Prodrug 11 itself was less effective in inhibiting the growth of HeLa cells, exhibiting an IC50 value of more than 5 mM. At the IC50 value, the difference in the toxicity between the prodrug 11 and the drug, butyric acid, was greater than fivefold. Thus, we could perform the experiments with a combination of prodrug 11 and antibody V122. As seen in Fig. 4, 1.0 mM butyric acid suppressed cell growth by 40% of that of the control without treatment with butyric acid, whereas the same concentration of prodrug 11 was slightly suppressive but much less effective than butyric acid itself (Fig. 5). The antibody V122 alone did not suppress cell proliferation at all, and vitamin B6 of the pro-moiety itself was almost nontoxic to the cells. When the assay was done in the presence of both 1.0 mM prodrug 11 and antibody V122, significant growth inhibition up to 50% of the control without treatment with butyric acid was observed. This difference was highly significant ( P < 0.0001). Measuring the product level of hydrolysis by HPLC under the same conditions as above, the conversion of 11 to butyric acid by antibody V122 was only 33%. Compared with the cleaved level, the growth suppression of HeLa cells in this experiment was relatively

3.6. Growth inhibition assay using a HeLa cell line Finally, we evaluated the biological consequences of antibody-catalyzed prodrug activation, by examining the growth inhibition of tumor cell proliferation. As a prodrug for the assay, we chose prodrug 11 of butyric acid. Butyric acid displays anti-tumor activity, as reflected in its ability to induce growth arrest (Prasad, 1980; Januszewicz et al., 1988) and apoptosis (Lavelle et al., 1999). Preliminary trials showed that butyric acid induced partial and temporary remission in a child with acute myeloid leukemia (Novogradsky et al., 1983), although no clinical effect was detected in a case of an adult (Rephaeli et al., 1986) due to the rapid metabolism and, to a lesser extent, excretion. Developing a therapy for adults requires the development of a novel class of prodrugs of butyric acid (Nudelman et al., 1992). By using a dye incorporation assay (Hockley and Baxter, 1986) of a HeLa cell line, we determined the growth inhibition rates with IC50 values (Fig. 4). The

Fig. 4. Effect of a drug and a prodrug on the proliferation of HeLa cells. Proliferation of HeLa was determined by Neutral Red incorporation, after exposure to the drug (butyric acid) for 24 h and prodrug 11 for 24 h. Values indicate the mean F S.D. for n = 3.

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Fig. 5. Growth inhibition of HeLa cells by butyric acid and its prodrug in the presence or absence of antibody V122. Proliferation of HeLa cells was measured by Neutral Red incorporation, after 24h exposure to the drug (1 mM butyric acid, second column), its prodrug 11 (1 mM, third column), pro-moiety (1 mM, fourth column), IgG V122 (21 AM alone, fifth column), and a combination of prodrug 11 (1 mM) and IgG (21 AM, sixth column). The first column is the control without treatment with 1 mM butyric acid. Pro-moiety means N-acetylpyridoxamine. Values represent the mean F S.D. for three experiments, which were examined for n = 3 – 5 independently. Single and four stars indicate significant difference from the control experiment ( P < 0.05 and P < 0.0001, respectively).

higher than that proposed from the data in Fig. 4. We cannot explain this difference clearly, but it may be the case that it is associated with changes in permeability of the cells and/or in the metabolic rate in the cells by masking with vitamin B6. In ADAPT, the strategy of tumor targeting requires a bi-specific monoclonal antibody (bs mAB) (Kurosawa et al., 1989). The bs mAB can be provided by a hybrid hybridoma, which is obtained by fusing a hybridoma secreting the catalytic antibody with a hybridoma secreting another antibody with binding to an antigen specifically expressed on tumor cells, followed by selecting both for binding a hapten and an antigenic target. In a bi-specific manner, the second targeting antibody bound to the antigen may affect the catalytic activity of the catalytic antibody. A potential problem in this work is the poor catalytic activity of antibody V122, which is some several hundred-fold less than that seen in the case of bacterial enzymes in ADEPT (Bagshawe, 1995). The acceleration rate of V122 was some 10-fold less than that of other reported catalytic antibodies with pro-

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drug activation (Miyashita et al., 1993; Campbell et al., 1994; Wentworth et al., 1996; Shabat et al., 1999). Therefore, antibody V122 is now at an early stage towards a new therapy. The half-life of prodrug 11 is 65 h in 10% FBS, thus, the prodrug is stable enough for the in vitro studies of prodrug activation by antibody V122. In human serum, however, the half-life of 11 was shortened to 17.5 h, so hydrolyzation by V122 would apparently be overestimated in experiments using human blood samples. These results, however, suggest that vitamin B6 for a pro-moiety is a good design, because of great stability, if the antibody has a high catalytic activity. Recent efforts have made it possible to accelerate the activity by molecular evolution utilizing a phage displayed catalytic antibody (Takahashi et al., 2001).

Acknowledgements We thank Ms. N. Murakami, Ms. N. Niikura, and Ms. F. Kuratomi for excellent technical assistance. We also thank Mr. K. Hamada of Immuno-Biological Laboratories for preparing the monoclonal antibodies. We would like to thank Mr. M. Kusama for mass spectroscopic analysis of the chemicals. We are grateful to Dr. N. Takahashi and Dr. M. Shibagaki for encouragement and useful discussions. This work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO) as an R&D project within the Industrial Science and Technology Frontier Program.

Appendix A . Syntheses of prodrugs A.1 . 3-Hydroxy-5-[[2-(4-isobutylphenyl)-propionyl]oxymethyl]-2-methyl-4-pyridinecarboxaldehyde (2) 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (696 mg, 3.63 mmol) was added to a solution of pyridoxal hydrochloride (500 mg, 2.46 mmol) and ibuprofen (492 mg, 2.39 mmol) in Me2NCHO (15 ml). The mixture was stirred for 6 h at 25 jC and quenched with H2O (30 ml). The mixture was extracted with EtOAc (2  50 ml), and the combined extracts were washed with brine. The organic layer was dried over MgSO4 and then evaporated. The

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product was crystallized from hexane/EtOAc to give 2 (311 mg, 37%) as a colorless solid. m.p. 168 –170 jC; 1 H NMR (CDCl3) d 0.90 (d, 6H, J = 6.6 Hz, CHMe2), 1.65 (d, 3H, J = 6.9 Hz, CHPhMe), 1.86 (septet, 1H, J = 6.6 Hz, CHMe2), 2.20 and 2.15 (each s, 3H, Me), 2.48 (d, 2H, J = 6.6 Hz, CH2Ph), 3.98 – 4.06 (m, 1H, CHPhMe), 5.00 (d, 1H, J = 13.0 Hz, CO2CH2), 5.20 (d, 1H, J = 13.0 Hz, CO2CH2), 6.18 – 6.25 (m, 1H), 7.14– 7.18 (m, 2H, Ph), 7.26– 7.35 (m, 2H, Ph), 8.25 (s, 1H); HRMS (FAB) calcd. for C 21 H 26 NO 4 356.1862 (M + 1), found 356.1880 (M+ + 1). A.2 . 3-Hydroxy-4-(hydroxymethyl)-5-[[2-(4-isobutylphenyl)-propionyl]oxymethyl]-2-methylpyridine (3) EDC (708 mg, 3.69 mmol) was added to a solution of pyridoxine hydrochloride (500 mg, 2.43 mmol) and ibuprofen (500 mg, 2.43 mmol) in Me2NCHO (15 ml). The mixture was stirred for 6 h at 25 jC and quenched with H 2 O (30 ml). The mixture was extracted with EtOAc (2  50 ml), and the combined extracts were washed with brine. The organic layer was dried over MgSO4 and then evaporated. The product was purified by flash chromatography (CHCl3/MeOH, 20:1) to give 3 (65 mg, 7.5%): 1H NMR (CDCl3) d 0.89 (d, 6H, J = 6.6 Hz, CHMe2), 1.48 (d, 3H, J = 6.9 Hz, CHPhMe), 1.83 (septet, 1H, J = 6.6 Hz, CHMe2), 2.45 (d, 2H, J = 6.6 Hz, CH2Ph), 2.47 (s, 3H, PyMe), 3.74 (q, 1H, J = 6.9 Hz, CHPhMe), 4.59 (s, 2H, CH 2OH), 5.28 (s, 2H, CO2CH2), 7.05– 7.15 (m, 4H, Ph), 7.90 (s, 1H, Py); HRMS (FAB) calcd. for C 21 H 28 NO 4 358.2018 (M + 1), found 358.2034 (M+ + 1). A.3 . 3-Acetoxy-4-(acetoxymethyl)-5-[[2-(4-isobutylphenyl)-propionyl]oxymethyl]-2-methylpyridine (4) Ac2O (1.0 ml) was added to a solution of 3 (65 mg, 0.18 mmol) in pyridine (1.0 ml). After 1 h, the mixture was poured into aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4 and then evaporated. The product was purified by flash chromatography (hexane/EtOAc, 2:1) to give 4 (67 mg, 83%). m.p. 56– 57 jC; 1H NMR (CDCl3) d 0.89 (d, 6H, J = 6.7 Hz, CHMe 2 ), 1.45 (d, 3H, J = 7.2 Hz, CHPhMe), 1.84 (septet, 1H, J = 6.7 Hz, CHMe2), 2.01 (s, 3H, OAc), 2.25 (s, 3H, OAc), 2.39 (s, 3H,

PyMe), 2.44 (d, 2H, J = 6.7 Hz, CH2Ph), 3.64 (q, 1H, J = 7.2 Hz, CHPhMe), 4.98 –5.37 (m, 4H, CH2OAc and CO2CH2), 7.07 (d, 2H, 8.1 Hz, Ph), 7.14 (d, 2H, J = 8.1 Hz, Ph), 8.39 (s, 1H, Py); HRMS (FAB) calcd. for C25H32NO6 442.2230 (M + 1), found 442.2240 (M+ + 1). A.4 . 4-(Acetylaminomethyl)-3-hydroxy-5-[[2-(4-isobutylphenyl)-propionyl]oxymethyl]-2-methylpyridine (6) 1,3-Dicyclohexylcarbodiimide (25 mg, 0.12 mmol) was added to a solution of 4-(N-acetylaminomethyl)-3-acetoxy-5-(hydroxymethyl)-2-methylpyridine (30 mg, 0.12 mmol) and ibuprofen (25 mg, 0.12 mmol) in Me2NCHO (0.5 ml). The mixture was stirred for 20 h at 25 jC, and filtered to remove insoluble material. The filtrate was diluted with EtOAc and washed with brine. The organic layer was dried over MgSO4 and then evaporated. The product was purified by flash chromatography (CHCl3/MeOH, 50:1) to give 6 (2.3 mg, 5.0%) and 3-acetate of 6 (12 mg, 22%). 1 H NMR (MeOH-d4) of 6 d 0.91 and 0.92 (each d, each 3H, J = 6.6 Hz, CHMe2), 1.67 (d, 3H, J = 7.1 Hz, CHPhMe), 1.69 (s, 3H, NHAc), 1.87 (septet, 1H, J = 6.6 Hz, CHMe2), 2.20 (s, 3H, PyMe), 2.50 (d, 2H, J = 6.6 Hz, CH2Ph), 4.09 (q, 1H, J = 7.1 Hz, CHPhMe), 4.05 –4.25 (m, 2H), 4.55– 4.72 (m, 2H), 4.92 (brs, 1H), 7.23 (d, 2H, J = 8.1 Hz, Ph), 7.40 (d, 2H, J = 8.1 Hz, Ph), 8.24 (s, 1H, Py); HRMS (FAB) calcd. for C23H31N2O4 399.2284 (M + 1), found 399.2302 (M+ + 1). A.5 . 4-(Acetylaminomethyl)-5-(hydroxymethyl)-2methyl-3-[c-oxo-[1,1’-biphenyl]-4-butanoyl]-pyridine (10) EDC (294 mg, 1.53 mmol) was added to a solution of N-acetylpyridoxamine (200 mg, 0.952 mmol) and fenbufen (259 mg, 1.02 mmol) in Me2NCHO (1.0 ml). The mixture was stirred for 48 h at 25 jC and quenched with H 2O (20 ml). The mixture was extracted with EtOAc (2  50 ml), and the combined extracts were washed with brine. The organic layer was dried over MgSO4 and then evaporated. The product was purified by flash chromatography (CHCl3/MeOH, 20:1) to give 10 (42 mg, 10%). m.p.

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170 –171 jC; 1H NMR (MeOH-d4) d 1.91 (s, 3H, NHAc), 2.41 (s, 3H, PyMe), 3.13 (dd, 2H, J = 5.5, 6.3 Hz, CH2CO), 3.53 (dd, 2H, J = 5.5, 6.3 Hz, CH2CO), 4.47 (brs, 2H, CH2NHAc), 4.78 (s, 2H, CH2OH), 7.38 –7.49 (m, 3H, Ph), 7.66 –7.69 (m, 2H, Ph), 7.77 (d, 2H, J = 8.5 Hz, Ph), 8.12 (d, 2H, J = 8.5 Hz, Ph), 8.32 (s, 1H, Py); HRMS (FAB) calcd. for C 26 H 27 N 2 O 5 447.1920 (M + 1), found 447.1943 (M+ + 1). A.6 . 4-(Acetylaminomethyl)-3-[[ p-N,N-bis(2-chloroethyl)aminobenzoyl]oxy]-5-(hydroxymethyl)-2-methylpyridine (12) Thionyl chloride (85 ml, 1.15 mmol) was added to a solution of p-N,N-bis(2-chloroethyl)aminobenzoic acid (100 mg, 0.382 mmol) (Ross et al., 1955) in CH2Cl2 (1.0 ml) under an Ar atmosphere at 25 jC. The mixture was stirred for 1 h and then carefully evaporated to give an oil, which was coevaporated with CH2Cl2 under an Ar atmosphere to afford an acid chloride. A solution of N-acetylpyridoxamine (80 mg, 0.382 mmol) in pyridine (1.0 ml) at 0 jC was added dropwise to a solution of the residue in CH2Cl2 (1.0 ml). After stirring overnight at 25 jC, the mixture was quenched with H2O and extracted with CHCl3 (3  10ml). The combined organic layer was washed with brine and dried over MgSO4 and then evaporated. The product was purified by flash chromatography (CHCl3/MeOH, 10:1) to give 12 (158 mg, 91%). The product was recrystallized from CHCl3/Et2O to give colorless crystals. m.p. 172 – 174 jC; 1H NMR (MeOH-d4) d 1.80 (s, 3H, NHAc), 2.35 (s, 3H, PyMe), 3.75 (t, 4H, J = 6.6 Hz, NCH 2 CH 2 Cl), 3.90 (t, 4H, J = 6.6 Hz, NCH2CH2Cl), 4.43 (brs, 2H, CH2NHAc), 4.75 (s, 2H, CH2OH), 6.89 (d, 2H, J = 9.1 Hz, Ph), 8.08 (d, 2H, J = 9.1 Hz, Ph), 8.35 (s, 1H, Py); HRMS (FAB) calcd. for C21H26Cl2N3O4 454.1300 (M + 1), found 454.1326 (M+ + 1). A.7 . 4-(Acetylaminomethyl)-3-[[ p-N,N-[bis(2-chloroethyl)aminophenylacetyl]oxy]-5-(hydroxymethyl)-2methylpyridine (13) Thionyl chloride (142 ml, 1.95 mmol) was added to a solution of p-N,N-bis(2-chloroethyl)aminophenylacetic acid (Hovinen, 1996) (215 mg,

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0.779 mmol) in CH2Cl2 (3.1 ml) under an Ar atmosphere at 25 jC. The mixture was stirred for 1 h, and then carefully evaporated to give an oil, which was co-evaporated with CH2Cl2 under an Ar atmosphere to afford an acid chloride. A solution of N-acetylpyridoxamine (163 mg, 0.779 mmol) in pyridine (2.0 ml) at 0 jC was added dropwise to a solution of the residue in CH2Cl2 (3.0 ml). After stirring for overnight at 25 jC, the mixture was quenched with H2O and extracted with CHCl3 (3  10ml). The combined organic layer was washed with brine and dried over MgSO4 and then evaporated. The product was purified by flash chromatography (CHCl3/MeOH, 20:1) to give 13 (91 mg, 25%). m.p. 146 – 148 jC; 1 H NMR (MeOH-d4) d 1.87 (s, 3H, NHAc), 2.16 (s, 3H, PyMe), 3.64 – 3.69 (m, 4H, NCH2CH2Cl), 3.73 – 3.88 (m, 4H, NCH 2 CH 2 Cl), 3.88 (s, 2H, PhCH2CO), 4.38 (brs, 2H, CH2NHAc), 4.73 (s, 2H, CH2OH), 6.78 (d, 2H, J = 8.9 Hz, Ph), 7.29 (d, 2H, J = 8.9 Hz, Ph), 8.30 (s, 1H, Py); HRMS (FAB) calcd. for C22H28Cl2N3O4 468.1457 (M + 1), found 468.1469 (M+ + 1). A.8 . 4-(Acetylaminomethyl)-3-[[4-[ p-N,N-[bis(2chloroethyl)amino]-phenyl]-butanoyl]oxy]-5-(hydroxymethyl)-2-methylpyridine (14) Thionyl chloride (625 ml, 8.56 mmol) was added to a solution of chlorambucil (650 mg, 2.14 mmol) in CH2Cl2 (6.5 ml) under an Ar atmosphere at 25 jC. The mixture was stirred for 1 h, and then carefully evaporated to give an oil, which was co-evaporated with CH2Cl2 under an Ar atmosphere to afford an acid chloride. A solution of N-acetylpyridoxamine (450 mg, 2.14 mmol) in pyridine (4.5 ml) at 0 jC was added dropwise to a solution of the residue in CH2Cl2 (5.0 ml). After stirring overnight at 25 jC, the mixture was quenched with H2O and extracted with CHCl3 (3  10ml). The combined organic layer was washed with brine and dried over MgSO4 and then evaporated. The product was purified by flash chromatography (CHCl3/MeOH, 20:1) to give 14 (239 mg, 24%). m.p. 120 – 122 jC; 1H NMR (MeOH-d4) d 1.86 (s, 3H, NHAc), 1.95– 2.05 (m, 2H, CH2CH2CH2CO), 2.32 (s, 3H, PyMe), 2.64 (t, 2H, J = 7.5 Hz, CH2CH2CH2CO), 2.70 (t, 2H,

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