Metabolites of Antibody–Maytansinoid Conjugates - ACS Publications

Mar 31, 2015 - and in Vitro Potencies. Wayne Widdison,* Sharon Wilhelm, Karen Veale, Juliet Costoplus, Gregory Jones, Charlene Audette,. Barbara Leece...
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Metabolites of Antibody−Maytansinoid Conjugates: Characteristics and in Vitro Potencies Wayne Widdison,* Sharon Wilhelm, Karen Veale, Juliet Costoplus, Gregory Jones, Charlene Audette, Barbara Leece,† Laura Bartle, Yelena Kovtun, and Ravi Chari ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States ABSTRACT: Several antibody−maytansinoid conjugates (AMCs) are in clinical trials for the treatment of various cancers. Each of these conjugates can be metabolized by tumor cells to give cytotoxic maytansinoid metabolites that can kill targeted cells. In preclinical studies in mice, the cytotoxic metabolites initially formed in vivo are further processed in the mouse liver to give several oxidized metabolic species. In this work, the primary AMC metabolites were synthesized and incubated with human liver microsomes (HLMs) to determine if human liver would likely give the same metabolites as those formed in mouse liver. The results of these HLM metabolism studies as well as the subsequent syntheses of the resulting HLM oxidation products are presented. Syntheses of the minor impurities formed during the conjugation of AMCs were also conducted to determine their cytotoxicities and to establish how these impurities would be metabolized by HLM. KEYWORDS: ADC, antibody, conjugate, DM1, DM4, drug, liver, maytansinoid, metabolism, microsome

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INTRODUCTION An antibody−drug conjugate (ADCs) is composed of a monoclonal antibody that is conjugated to a cytotoxic compound via a linker.1−4 The antibody of the ADC binds to an antigen found on the surface of a cancer cell. Internalization of the ADC−antigen immune complex then enables the intracellular release of cytotoxic metabolites that kill the cell. ADCs typically utilize highly cytotoxic compounds because very little of the administered conjugate reaches its targeted tumor cells in vivo.5 Antibodies are known to be degraded by cells of the reticuloendothelial system (RES).6,7 It is therefore likely that RES cells degrade the majority of an administered ADC over time to release cytotoxic metabolites throughout the body. The cytotoxic metabolites of ADCs which utilize maytansinoid or auristatin payloads are ultimately excreted primarily by hepatic clearance.8,9 The types of metabolites released and the reactions that can take place during liver metabolism can depend on the linkage system utilized by the ADC.10,11 As part of understanding the properties of an ADC, it is important to determine both the cytotoxicities of metabolites generated from an ADC and the metabolic fate of any highly potent impurities that might be present in the ADC.12 Maytansinoids, of which maytansine (1) is the parent compound, are a class of antimitotic agents that are 100- to 1000-fold more cytotoxic than conventional cancer chemotherapeutic agents such as methotrexate, daunorubicin, or vincristine.13 The semisynthetic maytansinoid compounds N2′deacetyl-N2′-(3-mercapto-1-oxopropyl)maytansine (2a, DM1) and N2 ′ -deacetyl-N2 ′ -(4-mercapto-4-methyl-1-oxopentyl)maytansine (2b, DM4) are semisynthetic analogues of © XXXX American Chemical Society

maytansine possessing a C3 ester side chain bearing a nonhindered thiol or a dimethyl hindered thiol substituent respectively, Figure 1. DM1 (2a) and DM4 (2b) have been conjugated to antibodies (mAbs) via thioether bonds or disulfide bonds to give antibody−maytansinoid conjugates (AMCs).11,14,15

Figure 1. Structures of maytansine, DM1 and DM4.

Several AMCs with the mAb-SPP-DM1 (3a) design were evaluated in the clinic, and several AMCs with the generalized structures mAb-SPDB-DM4 (3b), mAb-sulfo-SPDB-DM4 (3c), and mAb-SMCC-DM1 (4) are currently in clinical trials for the treatment of various cancer indications, Figure 2.16,17 These AMCs are prepared by attaching DM1 or DM4 to lysine residues of an antibody via the cleavable disulfide linkers SPP Special Issue: Antibody-Drug Conjugates Received: November 19, 2014 Revised: March 20, 2015 Accepted: March 31, 2015

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DOI: 10.1021/mp5007757 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 2. Antibody−maytansinoid conjugates (A) and their utilized linkers (B).

Figure 3. Antibody conjugation to form AMCs and the minor side products produced using the (A) SPP, SPDB, sulfo-SPDB, or (B) SMCC linkers.

the DM1 disulfide dimer side product, DM1 dimer (7), can be formed. In contrast, DM4 (2b), presumably because of its hindered thiol, does not generate an appreciable amount of disulfide dimer during conjugation. These side products and unreacted components are then removed to varying degrees during the conjugate purification process, which often involves size exclusion chromatography (SEC) or tangential flow filtration (TFF). AMCs are administered intravenously and function by binding to the targeted surface antigen on a cancer cell after which the AMCs are internalized and processed to release one

(5a), SPDB (5b), or sulfo-SPDB (5c) or via the noncleavable linker SMCC (6) to give a thioether-linked conjugate, Figure 3. The AMC, ado-trastuzumab emtansine (Kadcyla), in which DM1 is linked to the trastuzumab antibody via the SMCC linker has been approved for marketing for the treatment of metastatic HER2-positive breast cancer in the US and other countries. The conjugation process can produce small amounts of side products, such as maytansinoid linked to a hydrolyzed linker, DM1-TPA (8a), DM1-TBA (8b), DM1-sulfo-TBA (8c), or DM1-MCC (9). When DM1 (2a) is used to prepare AMCs, B

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Figure 4. AMC metabolites generated in tumor or RES cells (primary metabolites).

Figure 5. Mouse liver metabolism of AMC primary metabolites.

the RES metabolize AMCs to give the same metabolites as produced in targeted cancer cells. AMC metabolites formed in targeted cancer cells or from RES metabolism will be referred to as primary metabolites. Experiments in mice also indicated that each of the primary AMC metabolites described is ultimately routed to the liver where S-methylation of DM1 (2a) or DM4 (2b), or oxidative phase I metabolism to convert S-Me DM1 (12a) or S-Me DM4 (12b) to the corresponding sulfones (14a) and (14b) or sulfoxides (13a) and (13b), respectively, can occur, Figure 5.9 Disulfide reduction of primary metabolites also appears to occur, but no liver phase II metabolism other than S-methylation was detected in any of the AMC-treated mice. The syntheses and in vitro cytotoxicities of primary AMC metabolites and results of their incubation with human liver microsomes (HLMs) are reported herein. Syntheses and cytotoxicities for both HLM-derived metabolites and the known conjugation side products produced during the preparation of the described AMCs are also presented.

or more metabolites which can kill the cell. The initial AMC processing step has been shown in vitro to involve proteolytic degradation of the antibody component leaving a lysine residue of the antibody attached to the linker−maytansinoid moiety as shown in Figure 4.18 Thus, lysosomal degradation of the AMCs 3a, 3b, and 3c by target cells initially gives the metabolites 10a, 10b, and 10c respectively, while degradation of AMC 4 bearing the noncleavable linker gave the Lys-Nε-SMCC-DM1 (11) metabolite. The metabolite Lys-Nε-SPP-DM1 (10a) undergoes further processing via reduction of its disulfide bond, presumably by intracellular glutathione, to release DM1 (2a). The disulfide bonds of the Lys-Nε-SPDB-DM4 (10b) and LysNε-sulfo-SPDB-DM4 (10c) metabolites are reduced in cells to give DM4 (2b). Intracellular enzymes then S-methylate a portion of the DM1 or DM4 to give metabolites S-Me-DM1 (12a) or S-Me-DM4 (12b) with formation of 12a being less favorable. The Lys-Nε-SMCC-DM1 (11) metabolite was not metabolized further. All of the AMC metabolites that have been described appear to be highly potent against the cells that produced them. The polar lysine-bearing metabolites 10a, 10b, 10c, and 11 are less cytotoxic when added exogenously to cells. In contrast, the noncharged hydrophobic metabolites DM1 (2a), DM4 (2b), S-Me-DM1 (12a), and S-Me-DM4 (12b), generated in the parent cell, are highly cytotoxic and have the potential to diffuse into, and kill, neighboring cells.19 Plasma samples taken from mice injected with AMCs have been found to contain the same metabolites as those formed in vitro in tumor cells exposed to AMCs,9 indicating that cells of

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EXPERIMENTAL SECTION N-4-(Maleimidomethyl)cyclohexane carboxylic acid (9) was purchased from Speed Chemicals. All other reagents were obtained from Sigma-Aldrich Chemical Co. or Chem Impex, unless otherwise stated. All synthetic reactions were conducted under an argon atmosphere with magnetic stirring. The compounds SPDB (5b), SPP (5a), DM1 (2a), DM4 (2b), DM1-TPA (8a), DM4-TBA (8b), and DM1 dimer (7) were C

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Syntheses of Test Compounds. N2′-Deacetyl-N2′-[4methyl-4-(3-carboxy-3-sulfopropyldithio)-1-oxopentyl]maytansine (DM4-sulfo-TBA, 8c). To a solution of DM4 (2b, 37 mg, 0.047 mmol) in a 2:1 dimethyl sulfoxide:100 mM potassium phosphate pH 8 buffer (1.2 mL) was added sulfoSPDB (5c, 19.2 mg, 0.047 mmol), and the reaction mixture was stirred for 36 h. Product was purified by preparative HPLC, and fractions containing desired product (retention time 23 min) were combined, frozen, and lyophilized to give 5 mg (11% yield) of product as a white solid. 1H NMR (400 MHz, 2:1 methanol-d4:D2O): δ 8.21 (s, 1H), 7.17 (dd, J = 5.0, 1.8 Hz, 1H), 6.74 (d, J = 11.2 Hz, 1H), 6.69−6.55 (m, 3H), 5.74 (dd, J = 15.1, 9.1 Hz, 1H), 5.51 (q, J = 6.7 Hz, 1H), 4.69 (dd, J = 12.0, 3.0 Hz, 1H), 4.21 (td, J = 10.6, 8.8, 5.8 Hz, 2H), 4.00 (d, J = 2.3 Hz, 3H), 3.65 (d, J = 12.6 Hz, 1H), 3.59 (d, J = 9.0 Hz, 1H), 3.39 (s, 3H), 3.28−3.23 (m, 3H), 2.97 (d, J = 9.7 Hz, 1H), 2.90−2.83 (m, 3H), 2.78−2.67 (m, 3H), 2.56 (dt, J = 11.8, 6.7 Hz, 2H), 2.51−2.39 (m, 2H), 2.33−2.21 (m, 2H), 2.15 (dd, J = 14.5, 3.0 Hz, 1H), 2.03−1.87 (m, 3H), 1.70 (s, 3H), 1.61−1.47 (m, 3H), 1.34−1.27 (m, 6H), 1.27−1.19 (m, 5H), 0.87 (s, 3H). HRMS: calcd for C 42H 60ClN 3 O 15 S 3 [M − H + 2Na] + 1022.2592; found 1022.2574. N2′-Deacetyl-N2′-[3-[[1-[(4-carboxycyclohexyl)methyl]-2,5dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]maytansine (DM1MCC Both Isomers, 9). DM1 (2a, 100 mg, 0.135 mmol) was dissolved in a solution of DMA (6.7 mL) and 100 mM potassium phosphate buffer pH 7.5 (2 mL). Diisopropyl ethyl amine (35 μL, 0.203 mmol) and N-4-(maleimidomethyl)cyclohexanecarboxylic acid (17, 48 mg, 0.203 mmol) were then added, and the reaction mixture was stirred for 1 h. The mixture was adjusted to pH 6 by the addition of dilute HCl, and approximately 1/2 of the volume was evaporated under vacuum. The desired isomer mixture was purified by preparative C18 HPLC, and fractions containing desired product (retention time 31 min) were combined, frozen, and lyophilized to give 80 mg (60% yield) of both isomeric product forms of 9 as a white solid. 1H NMR (400 MHz, CDCl3): δ 6.98 (s, 1H), 6.88 (s, 1H), 6.86−6.78 (m, 2H), 6.73−6.59 (m, 4H), 6.47−6.33 (m, 2H), 5.72−5.59 (m, 2H), 5.46−5.34 (m, 2H), 4.84−4.72 (m, 2H), 4.26 (t, J = 11.1 Hz, 2H), 3.98 (s, 6H), 3.82 (dt, J = 27.5, 13.8 Hz, 3H), 3.71−3.59 (m, 3H), 3.45 (t, J = 9.3 Hz, 2H), 3.34 (s, 4H), 3.33 (s, 3H), 3.31−3.22 (m, 2H), 3.19 (s, 6H), 3.16−2.92 (m, 9H), 2.86−2.80 (m, 7H), 2.67−2.53 (m, 4H), 2.39 (dd, J = 18.7, 3.6 Hz, 2H), 2.29−2.12 (m, 4H), 2.06−1.95 (m, 4H), 1.75−1.65 (m, 5H), 1.64−1.61 (m, 7H), 1.59−1.49 (m, 2H), 1.49−1.40 (m, 3H), 1.37 (d, J = 10.6 Hz, 3H), 1.33−1.24 (m, 12H), 1.24−1.16 (m, 2H), 1.07− 0.90 (m, 4H), 0.79 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 180.21, 180.16, 177.09, 176.94, 174.82, 174.73, 170.84, 170.81, 170.72, 170.59, 168.88, 156.08, 153.48, 153.28, 142.25, 141.17, 139.07, 133.09, 128.17, 125.61, 122.22, 118.89, 113.25, 88.68, 88.60, 80.86, 80.82, 78.23, 78.15, 74.34, 67.32, 67.22, 60.08, 60.04, 56.87, 56.82, 56.72, 52.67, 52.48, 46.78, 44.78, 44.60, 42.70, 42.61, 40.02, 39.82, 38.87, 36.27, 35.91, 35.79, 35.65, 35.61, 34.27, 34.03, 32.52, 30.92, 30.81, 29.75, 29.67, 28.28, 28.22, 27.69, 27.16, 15.61, 14.64, 13.63, 13.45, 12.29, 12.24. HRMS: calcd for C47H63ClN4O14S [M + Na]+ 997.3566; found 997.3647. N2′-Deacetyl-N2′-[4-[[4-[[(1S)-2-amino-1-carboxypentyl]amino]-3-oxopropyl]dithio]-1-oxopropyl]maytansine (LysNε-SPP-DM1 Both Isomers, 10a). Lysine (92 mg, 0.63 mmol) and DM1 (2a, 48 mg, 0.065 mmol) were suspended in 100 mM potassium phosphate buffer pH 7.4 (1.5 mL) and

prepared as previously described.15 The sulfo-SPDB linker (8c) was prepared as previously described.20 Human liver microsomes derived from a pool of ten human liver preparations were purchased from XenoTech. In vitro cytotoxicities were determined by clonogenic assays using KB cells purchased from ATCC (ATCC CCl-17) as previously described.21 Proton magnetic resonance (1 H NMR) and carbon magnetic resonance spectra (13C NMR) were obtained on a Bruker Avance 400 spectrometer operating at 400 and 100 MHz, respectively. The NMR chemical shifts are reported in δ values relative to the utilized NMR solvent. Figures showing HPLC traces were acquired with detection at 252 nm. Preparative and Analytical HPLC. Unless otherwise stated, preparative high performance liquid chromatography was performed on a Varian Prostar instrument using a Kromasil C8 (length, 250 mm; i.d., 20 mm) 10 μm column. The column was eluted at 21.0 mL/min with deionized water containing 0.1% formic acid and acetonitrile using programmed linear changes in mobile phase composition as follows: 15% to 32% acetonitrile from 0 to 15 min, 32% to 48% acetonitrile from 15 to 25 min, 48 to 58% acetonitrile from 25 to 35 min, 55% to 90% acetonitrile from 35 to 55 min. Synthesized metabolites and compounds generated from incubation of compounds with human liver microsomes were analyzed using a system composed of an Agilent 1100 HPLC equipped with an analytical Kromasil C8 (length, 150 mm; i.d., 2.1 mm) 5 μm column in series with a Bruker Esquire 3000 ion trap MS operating in alternating positive/negative ion mode or in a positive MS/MS2 mode. The column was eluted at a flow rate of 0.22 mL/min with deionized water containing 0.1% formic acid and acetonitrile using programmed linear changes in mobile phase composition as follows: 15% to 32% acetonitrile from 0 to 15 min, 32% to 48% acetonitrile from 15 to 25 min, 48% to 58% acetonitrile from 25 to 35 min, 58% to 90% acetonitrile from 35 to 55 min, decreasing to 15% acetonitrile from 55 to 56 min, and holding at 15% acetonitrile from 56 to 64 min. HPLC/MS data were processed using Agilent Chemstation and Bruker Daltonic Data Analysis version 3.3 software. Incubation of Primary Metabolites with Human Liver Microsomes. A nicotinamide adenine dinucleotide phosphate reduced form (NADPH) regeneration solution was prepared by dissolving NADPH (17 mg, 0.023 mmol), glucose-6-phosphate sodium salt (78 mg, 0.28 mmol), and glucose-6-phosphate dehydrogenase (100 units) in 2.0% aqueous sodium bicarbonate (8.6 mL). A human liver microsome stock solution (HLM stock) was prepared by suspending human liver microsomes (10 mg) in 50 mM tris-hydroxymethylaminomethane (TRIS)·HCl buffer, pH 7.4 (1.5 mL). Compounds to be incubated with microsomes were first dissolved in 9:1 (v/v) acetonitrile:deionized water to give a 2−3 mM stock solution, an aliquot (10 μL) of which was added to a mixture of TRIS· HCl buffer, pH 7.4 (640 μL), NADPH regeneration solution (250 μL), and HLM stock solution (100 μL), and then placed in a 37 °C water bath for 45 min. Samples were removed from the heating bath and diluted with acetonitrile (2.0 mL), vortexed for 10 s, and centrifuged in a SpeedVac without vacuum for 5 min. Supernatants (1.0 mL) were transferred to autosampler vials for HPLC analysis. Control incubations were conducted similarly except 2% aqueous sodium bicarbonate (250 μL) was used instead of the NAPDH regeneration solution. D

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Molecular Pharmaceutics

= 12.1, 2.8 Hz, 2H), 4.06 (t, J = 11.2 Hz, 2H), 3.93 (s, 6H), 3.74 (q, J = 6.0 Hz, 2H), 3.50−3.46 (m, 8H), 3.25 (s, 6H), 3.10 (s, 6H), 3.04−2.88 (m, 4H), 2.80 (d, J = 9.6 Hz, 2H), 2.71 (s, 6H), 2.65−2.55 (m, 4H), 2.08−1.98 (m, 6H), 1.86 (t, J = 12.4 Hz, 2H), 1.79−1.68 (m, 6H), 1.58 (s, 6H), 1.50−1.36 (m, 12H), 1.30−1.21 (m, 4H), 1.16 (m, 12H), 1.13−1.08 (m, 12H), 0.78 (s, 6H). 13C NMR (101 MHz, DMSO): δ 171.35, 170.98, 170.89, 170.74, 168.15, 167.84, 167.77, 155.29, 151.23, 141.28, 141.25, 138.21, 138.18, 132.58, 128.54, 125.28, 121.55, 117.06, 117.04, 113.95, 88.19, 79.99, 77.66, 73.17, 66.77, 64.48, 64.45, 60.04, 56.56, 56.11, 52.06, 51.98, 51.59, 50.03, 45.53, 37.68, 37.56, 37.51, 36.34, 35.80, 35.28, 31.93, 29.68, 29.38, 29.11, 28.56, 28.38, 28.00, 27.84, 27.69, 27.60, 26.92, 26.83, 21.29, 20.63, 15.01, 14.40, 13.01, 11.40. HRMS: calcd for C48H72ClN5O16S3 [M + H]+ 1106.3897; found 1106.3910. N2′-Deacetyl-N2′-[3-[[1-[[4-[[[(1S)-5-amino-1carboxypentyl]amino]carbonyl] cyclohexyl]methyl]-2,5dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]maytansine (Lys-NεSMCC-DM1 Both Isomers, 11). Lysine (104 mg, 0.71 mmol) and DM1 (2a, 48 mg, 0.065 mmol) were suspended in a solution of 100 mM potassium phosphate buffer pH 7.4 (2 mL) and DMA (4 mL). After 1 min a solution of SMCC (7, 24 mg, 0.072 mmol) in DMA (0.5 mL) was added and stirred at ambient temperature for 1 h. The mixture was purified by preparative C18 HPLC. Fractions containing the coeluting isomers of desired product (retention time 24 min) were combined, frozen, and lyophilized to give 41 mg (57% yield) of compound 11 as a white solid. 1H NMR (400 MHz, CD3CN): δ 7.04 (dd, J = 7.0, 5.6 Hz, 2H), 6.62−6.55 (m, 2H), 6.49 (dd, J = 10.2, 3.0 Hz, 4H), 5.63−5.50 (m, 2H), 5.35−5.25 (m, J = 6.8, 4.3 Hz, 2H), 4.53 (d, J = 9.4 Hz, 2H), 4.13 (t, J = 11.2 Hz, 3H), 4.03 (s, 7H), 3.90 (s, 6H), 3.84−3.76 (m, 1H), 3.76−3.68 (m, 1H), 3.59−3.45 (m, 6H), 3.26 (s, 6H), 3.22−2.77 (m, 26H), 2.73 (d, J = 1.0 Hz, 5H), 2.69−2.49 (m, 4H), 2.36−1.98 (m, 6H), 1.71 (s, 7H), 1.57 (s, 10H), 1.53−1.37 (m, 10H), 1.36− 1.08 (m, 21H), 0.97−0.79 (m, 3H), 0.74 (s, 3H), 0.73 (s, 3H). 13 C NMR (101 MHz, CD3CN): δ 179.31, 178.89, 177.70, 172.92, 172.77, 172.39, 172.35, 170.87, 156.85, 156.81, 154.81, 142.67, 142.65, 142.33, 142.30, 140.98, 140.94, 134.28, 128.83, 125.86, 125.83, 122.94, 118.86, 118.84, 115.15, 115.10, 100.99, 89.08, 81.45, 78.88, 75.13, 68.44, 61.57, 57.54, 57.19, 53.68, 53.58, 46.87, 45.54, 40.88, 40.66, 39.62, 39.07, 37.21, 36.56, 36.49, 36.45, 36.37, 34.95, 34.68, 33.28, 31.31, 31.25, 30.41, 29.46, 27.71, 27.52, 23.05, 15.66, 14.63, 13.68, 12.28. HRMS: calcd for C53H75ClN6O15S [M + Na]+ 1125.4597; found 1125.4506. N2′-Deacetyl-N2′-(3-methylthio-1-oxopropyl)maytansine (S-Me-DM1, 12a). Methyl iodide (34 μL, 0.64 mmol) and diisopropyl ethyl amine (144 μL, 0.82 mmol) were added to a solution of DM1 (2a, 405 mg, 0.549 mmol) in DMF (45 mL) at ambient temperature. After 1 h dithiothreitol (50 mg, 0.32 mmol) was added to destroy excess methyl iodide. After an additional 1 h the mixture was purified by preparative HPLC using a Kromasil cyano column (length, 250 mm; i.d., 21 mm) and 252 nm detection with 20 mL/min isocratic elution using hexanes:2-propanol:ethyl acetate 68:8:24. Fractions containing desired product (retention time 11 min) were combined. Solvent was removed under vacuum to give 139 mg (72% yield) of 12a as a white solid. 1H NMR (400 MHz, CDCl3): δ 6.80 (s, 1H), 6.69 (d, J = 10.9 Hz, 1H), 6.62 (s, 1H), 6.40 (dd, J = 15.2, 11.2 Hz, 1H), 6.32 (s, 1H), 5.63 (dd, J = 15.3, 9.0 Hz, 1H), 5.38 (dd, J = 13.3, 6.5 Hz, 1H), 4.74 (dd, J = 11.9, 2.8 Hz, 1H), 4.24 (t, J = 10.6 Hz, 1H), 3.95 (s, 3H), 3.63 (d, J = 12.7

DMA (4 mL) with stirring at ambient temperature. After 10 min, SPP (5a, 23 mg, 0.065 mmol) was added. The reaction was run at ambient temperature for 2 h and purified by preparative C18 HPLC. Fractions containing pure desired product (retention time 25 min) were combined, frozen, and lyophilized to give 25 mg (38% yield) of product as a white solid. 1H NMR (400 MHz, CD3CN): δ 8.38 (s, 1H), 7.14−7.01 (m, 1H), 6.69−6.44 (m, 3H), 5.59 (dd, J = 14.2, 9.1 Hz, 1H), 5.37−5.26 (m, 1H), 4.55 (dd, J = 12.1, 2.9 Hz, 1H), 4.14 (dd, J = 16.3, 5.8 Hz, 1H), 3.99−3.86 (m, 3H), 3.55−3.59 (m, 6H), 3.51 (dd, J = 10.7, 5.4 Hz, 3H), 3.28 (s, 3H), 3.16 (d, J = 3.0 Hz, 3H), 3.14−3.00 (m, 2H), 2.97−2.80 (m, 4H), 2.77 (s, 3H), 2.75−2.63 (m, 2H), 2.63−2.51 (m, 1H), 2.12 (m, 3H), 1.91− 1.69 (m, 2H), 1.69−1.55 (m, 4H), 1.54−1.29 (m, 6H), 1.22 (d, J = 6.8 Hz, 3H), 1.15 (d, J = 6.4 Hz, 3H), 1.10 (dd, J = 9.6, 6.8 Hz, 3H), 0.75 (s, 3H). HRMS: calcd for C46H68ClN5O13S2 [M + Na]+ 1020.3841; found 1020.3770. N2′-Deacetyl N2′-[4-[[4-[[(1S)-2-amino-1-carboxypentyl]amino]-4-oxobutyl]dithio]-4-methyl-1-oxopentyl]maytansine (Lys-Nε-SPDB-DM4, 10b). Lysine hydrochloride salt (38 mg, 0.173 mmol) and DM4 (2b, 20 mg, 0.025 mmol) were weighed into a 2 mL vial to which was added 100 mM potassium phosphate buffer pH 7.4 (200 μL) and DMA (200 μL). The vial was capped and vortexed for 1 min, and SPDB (5b, 12 mg, 0.037 mmol) was then added. The vial was capped and vortexed, a magnetic stir bar was added, and the reaction mixture was stirred at room temperature for 2 h. Product was purified by preparative HPLC, and fractions containing desired product (retention time 24 min) were combined, frozen, and lyophilized to give 7.3 mg (29% yield) of 10b as a white solid. 1 H NMR (CD3CN:D2O 9:1): δ 7.05 (d, J = 1.6 Hz, 1H), 6.58 (d, J = 1.6 Hz, 1H), 6.45−6.56 (m, 2H), 5.61 (dd, J = 9.2 Hz, J = 14.8, 1H), 5.33 (q, J = 6.8 Hz, 1H), 4.55 (dd, J = 2.8 Hz, J = 12 Hz, 1H), 4.13 (td, J = 10.0 Hz, J = 2.8 Hz, 1H), 3.92 (s, 3H), 3.46−3.54 (m, 4H), 3.38−3.42 (m, 1H), 3.27 (s, 3H), 3.22 (d, J = 10.1 Hz, 1H), 3.18 (s, 3H), 3.11 (t, J = 7.2 Hz, 2H), 2.93 (d, J = 9.6 Hz, 1H), 2.79 (s, 3H), 2.58−2.63 (m, 3H), 2.42−2.53 (m, 1H), 2.28−2.37 (m, 1H), 2.17 (t, J = 10.1 Hz, 2H), 2.07−2.13 (m, 1H), 1.99 (m, 1H), 1.89 (m, 2H), 1.62−1.83 (m, 6H), 1.59 (s, 3H), 1.22−1.54 (m, 8H), 1.16−1.22 (m, 12H), 0.80 (s, 3H). 13 C NMR (CD3CN:D2O 9:1): δ 173.03, 172.45, 171.06, 169.10, 155.66, 152.58, 141.33, 139.22, 132.88, 127.86, 124.84, 121.63, 113.70, 87.93, 80.20, 77.69, 73.73, 66.87, 60.07, 56.19, 55.81, 52.12, 49.79, 45.67, 38.96, 38.24, 37.92, 35.93, 35.67, 35.03, 33.99, 32.02, 29.96, 28.77, 28.34, 26.94, 26.34, 24.72, 21.91, 14.26, 13.38, 12.39, 11.13. HRMS: calcd for C48H72ClN5O13S2 [M + H]+ 1026.4335; found 1026.4355. N2′-Deacetyl N2′-[4-[[4-[[(1S)-2-Amino-1-carboxypentyl]amino]-4-oxo-3-sulfonate-butyl]dithio]-4-methyl-1oxopentyl]maytansine (Lys-Nε-sulfo-SPDB-DM4 Both Isomers, 10c). Lysine (37.5 mg, 0.256 mmol) dissolved in 1/2 saturated aqueous sodium bicarbonate (4 mL) was added to a stirring solution of DM4 (2b, 200 mg, 0.256 mmol) in DMA (4 mL); sulfo-SPDB (5c, 125 mg, 0.308 mmol) was then added. Some precipitate quickly formed, and DMA (2 mL) was added followed by deionized water (1 mL) and stirred for 45 min. Product was purified by preparative HPLC, and fractions containing desired product (retention time 21 min) were combined, frozen, and lyophilized to give 71 mg (29% yield) of 10c as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 7.72 (dt, J = 18.6, 5.8 Hz, 2H), 7.19 (dd, J = 6.1, 1.9 Hz, 2H), 6.87 (s, 2H), 6.60 (t, J = 11.5 Hz, 4H), 6.53 (d, J = 7.9 Hz, 4H), 5.59 (dd, J = 14.7, 9.0 Hz, 2H), 5.32 (q, J = 6.7 Hz, 2H), 4.53 (dd, J E

DOI: 10.1021/mp5007757 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

0.77(s, 3H), 0.76 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 170.59, 170.22, 170.08, 168.84, 156.24, 156.10, 152.40, 142.37, 142.31, 140.96, 140.91, 139.70, 139.38, 133.43, 133.29, 127.98, 127.73, 125.39, 125.19, 122.29, 121.98, 119.15, 119.07, 113.30, 100.16, 88.74, 88.67, 80.95, 78.32, 74.23, 67.21, 60.11, 56.82, 56.75, 56.70, 52.93, 52.76, 50.10, 49.41, 46.78, 46.72, 39.36, 39.19, 38.99, 36.41, 35.65, 35.54, 32.57, 31.06, 30.99, 26.94, 26.41, 15.71, 15.65, 14.72, 13.56, 12.31, 12.26. HRMS: calcd for C36H50ClN3O11S [M + Na]+ 790.2752; found 790.2751. N2′-Deacetyl-N2′-(4-methyl-4-methylsulfoxy-1-oxopentyl)maytansine (S-Me-DM4 Sulfoxide Both Isomers, 13b). To a solution of S-Me-DM4 (12b, 20 mg, 0.025 mmol) in ethanol (300 μL) were added vanadyl acetylacetonate (2.0 mg, 0.0075 mmol) and a solution of 5.5 M tert-butyl hydrogen peroxide in decane (5.0 μL, 0.025 mmol) at ambient temperature. The reaction mixture was stirred for 1 h and then purified by preparative HPLC. Fractions containing coeluting desired products (retention time 26 min) were combined, frozen, and lyophilized to give 13 mg (65% yield) of a mixture of desired product isomers as a white solid. 1H NMR (CDCl3): δ 6.82 (d, J = 1.2 Hz, 1H), 6.815 (d, J = 1.2 Hz, 1H,), 6.69 (d, J = 11.2 Hz, 2H), 6.64 (d, J = 1.2 Hz, 1H,), 6.62 (d, J = 1.2 Hz, 2H), 6.41 (dd, J = 11.2 Hz, J = 15.2 Hz, 2H,), 6.28 (s, 2H), 5.66 (dd, J = 8.8 Hz, J = 15.2 Hz, 1H,), 5.38 (q, J = 6.8 Hz, 2H,), 4.78 (dt, J = 2.4 Hz, J = 12.0 Hz, 2H,), 4.27 (dd, J = 10.8 Hz, J = 10.8 Hz, 2H), 3.98 (s, 3H), 3.97 (s, 3H,), 3.61 (d, J = 12.4 Hz, 2H), 3.49 (d, J = 9.2 Hz, 2H), 3.38−3.43 (m, 2H), 3.35 (s, 6H), 3.21 (d, J = 3.6 Hz, 6H), 3.11 (d, J = 12.4 Hz, 2H), 3.02 (d, J = 2.4 Hz, 1H), 3.00 (d, J = 2.4 Hz, 1H), 2.86 (s, 6H), 2.33 (s, 6H), 2.32−2.68 (m, 6H), 2.18 (dd, J = 2.8 Hz, J = 14.4 Hz, 2H), 2.05−2.25 (m, 1H), 1.95−2.02 (m, 2H), 1.77−1.87 (m, 1H), 1.64 (s, 6H), 1.57 (d, J = 13.6 Hz, 2H), 1.40−1.49 (m, 2H), 1.30 (d, J = 3.6 Hz, 6H), 1.28 (d, J = 2.8 Hz, 6H), 1.90− 1.28 (m, 2H), 1.177 (d, J = 1.6 Hz, 6H), 1.11 (s, 6H), 0.80 (s, 6H). 13C NMR (CDCl3): δ 171.64, 170.69, 170.66, 168.70, 156.05, 156.01, 152.25, 142.27, 142.22, 140.92, 139.24, 139.08, 133.25, 133.18, 127.82, 127.69, 125.41, 125.30, 121.95, 118.91, 118.87, 113.09, 88.48, 88.44, 80.87, 78.02, 77.99, 74.11, 66.99, 66.95, 59.99, 59.96, 56.64, 56.57, 54.74, 54.66, 52.53, 52.43, 46.63, 38.89, 36.20, 35.56, 35.50, 32.48, 32.38, 31.86, 31.31, 31.27, 30.88, 28.00, 27.82, 20.53, 20.24, 19.30, 19.19, 15.46, 14.56, 13.35, 12.19. HRMS: calcd for C39H56ClN3O11S [M + Na]+ 832.3222; found 832.3229. N 2 ′ -Deacetyl-N 2 ′ -(3-methylsulfonyl-1-oxopropyl)maytansine (S-Me-DM1 sulfone, 14a). S-Me-DM1 sulfoxide (13a, 35 mg, 0.046 mmol) was dissolved in methanol (1.0 mL) to which vanadium acetylacetonate (8.0 mg, 0.03 mmol) and 5.5 M tert-butyl hydrogen peroxide in decane (41 μL, 0.23 mmol) were added. After 2 h the mixture was purified by preparative HPLC. Fractions containing desired product (retention time 28 min) were combined, frozen, and lyophilized to give 20 mg (56% yield) of 14a as a white solid. 1H NMR (400 MHz, CDCl3): δ 6.82 (d, J = 1.7 Hz, 1H), 6.67−6.57 (m, 2H), 6.43 (dd, J = 15.3, 11.1 Hz, 1H), 6.23 (s, 1H), 5.61 (dd, J = 15.3, 9.0 Hz, 1H), 5.37 (q, J = 6.8 Hz, 1H), 4.77 (dd, J = 12.0, 3.0 Hz, 1H), 4.26 (t, J = 10.4 Hz, 1H), 3.98 (s, 3H), 3.57 (d, J = 12.7 Hz, 1H), 3.53−3.42 (m, 2H), 3.36 (s, 3H), 3.29 (d, J = 1.8 Hz, 1H), 3.20 (s, 3H), 3.18−3.07 (m, 1H), 3.00 (dd, J = 10.3, 7.5 Hz, 2H), 2.93 (s, 3H), 2.89 (s, 3H), 2.85−2.72 (m, 1H), 2.59 (dd, J = 14.4, 12.1 Hz, 1H), 2.19 (dd, J = 14.4, 3.0 Hz, 1H), 1.65 (s, 3H), 1.55 (d, J = 13.3 Hz, 1H), 1.51−1.40 (m, 1H), 1.32 (d, J = 6.9 Hz, 3H), 1.29 (d, J = 6.3 Hz, 2H), 1.27−1.19 (m, 1H), 0.79 (s, 3H). 13C NMR (101

Hz, 1H), 3.57 (s, 1H), 3.47 (d, J = 8.9 Hz, 1H), 3.33 (s, 3H), 3.17 (s, 3H), 3.08 (d, J = 12.5 Hz, 1H), 2.99 (d, J = 9.6 Hz, 1H), 2.82 (s, 4H), 2.74−2.62 (m, 2H), 2.62−2.41 (m, 2H), 2.14 (dd, J = 14.3, 2.7 Hz, 1H), 2.02 (s, 3H), 1.61 (s, 3H), 1.53 (d, J = 13.4 Hz, 1H), 1.44 (dt, J = 16.3, 8.2 Hz, 1H), 1.26 (dd, J = 8.9, 6.7 Hz, 6H), 1.20 (d, J = 13.1 Hz, 1H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 171.13, 170.88, 168.88, 156.09, 152.44, 142.25, 141.21, 139.33, 133.41, 127.84, 125.50, 122.26, 118.87, 113.31, 88.64, 80.95, 78.26, 74.25, 67.34, 60.10, 56.76, 56.72, 52.46, 46.70, 38.97, 36.32, 35.63, 33.95, 32.53, 30.83, 29.53, 16.05, 15.63, 14.71, 13.46, 12.24. HRMS: calcd for C36H50ClN3O10S [M + Na]+ 774.2803; found 774.2735. N 2′ -Deacetyl-N2 ′-(4-methyl-4-methythio-1-oxopentyl)maytansine (S-Me-DM4, 12b). Methyl iodide (91 mg, 0.64 mmol) and diisopropyl ethyl amine (112 μL, 0.64 mmol) were added to a solution of DM4 (2b, 200 mg, 0.256 mmol) in DMF (1.3 mL) at ambient temperature. After 7 h dithiothreitol (20 mg, 0.13 mmol) was added. After an additional hour of stirring the mixture was purified by preparative HPLC using a Kromasil cyano column (length, 250 mm; i.d., 21 mm), with 252 nm detection with 20 mL/min isocratic elution using hexanes:2propanol:ethyl acetate 68:8:24. Fractions containing the desired product (retention time 12 min) were combined and solvent was rotary evaporated under vacuum to give 137 mg of 12b (68% yield) as a white solid. 1H NMR (CDCl3): δ 6.813 (d, J = 1.6 Hz, 1H), 6.76 (d, J = 11.2 Hz, 1H), 6.65 (d, J = 1.6 Hz, 1H), 6.42 (dd, J = 4.4 and 11.2 Hz, 1H), 6.21 (s, 1H), 5.67 (dd, J = 6 and 9.2 Hz, 1H), 5.43 (m, 1H), 4.77 (dd, J = 3.2 and 8.8 Hz, 1H), 4.27 (t, 1H), 3.98 (s, 3H), 3.65 (d, J = 12.8 Hz, 1H), 3.50 (d, J = 8.8 Hz, 1H), 3.35 (s, 3H), 3.23 (s, 3H), 3.10 (d, J = 12.4 Hz, 1H), 3.04 (d, J = 9.6 Hz, 1H), 2.86 (s, 3H), 2.60 (dd, J = 2.4 and 12 Hz, 1H), 2.350−2.58 (m, 2H), 2.18 (dd, J = 3.2 and 11.2 Hz, 1H), 1.71−1.91 (m, 2H), 1.78 (s, 3H), 1.64 (s, 3H), 1.57 (d, J = 13.6 Hz, 1H), 1.43−1.49 (m, 1H), 1.30 (d, J = 1.2 Hz, 3H), 1.28 (d, J = 1.6 Hz, 3H), 1.24 (m, 2H), 1.21 (s, 3H), 1.19 (s, 3H), 0.80 (s, 3H). 13C NMR (CDCl3): δ 172.73, 171.18, 168.92, 156.17, 152.37, 142.50, 141.17, 139.28, 133.51, 127.92, 125.71, 122.39, 113.27, 88.66, 81.11, 78.41, 74.33, 67.48, 60.14, 56.81, 56.77, 52.54, 46.80, 43.58, 39.10, 36.32, 36.07, 35.74, 32.60, 30.96, 29.50, 28.74, 28.33, 15.67, 14.77, 13.45, 12.31, 10.72. HRMS: calcd for C39H56ClN3O10S [M + Na]+ 816.3273; found 816.3284. N 2 ′ -Deacetyl-N 2 ′ -(3-methylsulfoxy-1-oxopropyl)maytansine (S-Me-DM1 Sulfoxide Both Isomers, 13a). To a solution of S-Me-DM1 (12a, 100 mg, 0.133 mmol) in ethanol (1.0 mL) were added vanadyl acetylacetonate (11 mg, 0.041 mmol) and a solution of 5.5 M tert-butyl hydrogen peroxide in decane (24 μL, 0.13 mmol) at ambient temperature. The reaction mixture was stirred for 1 h and was purified by preparative HPLC. Fractions containing the coeluting desired products (retention time 26 min) were combined, frozen, and lyophilized to give 60 mg (59% yield) of a mixture of desired product isomers as a white solid. 1H NMR (400 MHz, CDCl3): δ 6.83−6.78 (m, 2H),6.66−6.58 (m, 4H), 6.41 (dd, J = 14.2, 12.2 Hz,2H), 6.30 (s, 2H), 5.69−5.55 (m, 2H), 5.35 (m, 2H), 4.75 (d, J = 11.7 Hz, 2H), 4.24 (m, 2H), 3.96 (s, 3H),3.95 (s, 3H), 3.68 (s, 2H), 3.58 (m, 2H), 3.47 (d, J = 9.0 Hz, 2H), 3.33 (s, 6H), 3.19 (d, J = 9.9 Hz, 6H), 3.15−3.03 (m, 4H), 3.02− 2.96(m, 2H), 2.96−2.90 (m, 2H), 2.88 (s, 3H), 2.87 (s, 3H), 2.86 (s, 2H), 2.82−2.61 (m, 4H), 2.57 (s, 3H), 2.55 (s, 3H), 2.16 (dd, J = 14.3, 2.8 Hz,2H), 1.62 (s, 6H), 1.53 (d, J = 13.3 Hz, 2H), 1.49−1.37 (m, 2H), 1.30 (d, J = 3.6 Hz, 3H), 1.29 (d, J = 3.6 Hz, 3H), 1.27 (s, 2H), 1.25 (s, 2H), 1.24−1.17 (m, 2H), F

DOI: 10.1021/mp5007757 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 6. Synthesis of S-Me-DM1, S-Me-DM4, and the oxidized metabolites of S-Me-DM1, S-Me-DM4, DM1, and DM4.

MHz, CDCl3): δ 170.48, 169.13, 168.85, 156.33, 152.30, 142.47, 140.92, 139.70, 133.45, 127.83, 125.30, 121.94, 119.28, 113.35, 88.65, 81.09, 78.45, 74.27, 67.23, 60.10, 56.89, 56.81, 52.86, 50.58, 46.87, 41.76, 39.08, 36.33, 32.68, 31.09, 26.56, 15.74, 14.79, 13.65, 12.40. HRMS: calcd for C36H50ClN3O12S [M + Na]+ 806.2701; found 806.2702. N2′-Deacetyl-N2′-(4-methyl-4-methysulfonyl-1-oxopentyl)maytansine (S-Me-DM4 Sulfone, 14b). S-Me-DM4 (12b, 20 mg, 0.025 mmol) was dissolved in ethanol (300 μL) to which vanadium acetylacetonate (2.0 mg, 0.0075 mmol) and 5.5 M tert-butyl hydrogen peroxide in decane (10 μL, 0.05 mmol) were added. After 1 h the mixture was purified by preparative HPLC. Fractions containing desired product (retention time 28 min) were combined, frozen, and lyophilized to give 15 mg (70% yield) of 14b as a white solid. 1H NMR (CDCl3): δ 6.82 (d, J = 1.6 Hz, 1H), 6.69 (d, J = 9.2 Hz, 1H), 6.61 (d, J = 1.6 Hz, 1H), 6.42 (dd, J = 10.8 and 15.2 Hz, 1H), 6.22 (s, 1H), 5.68 (dd, J = 9.2 and 15.2 Hz, 1H), 5.38 (q, J = 6.8, 1H), 4.79 (dd, J = 3.2 and 12.4 Hz, 1H), 4.26 (t, J = 10.4, 1H), 3.98 (s, 3H), 3.61 (d, J = 12.4 Hz, 1H), 3.50 (d, J = 8.8 Hz, 1H), 3.35 (s, 3H), 3.28 (s, 1H), 3.21 (s, 3H), 3.12 (d, J = 9.6 Hz, 1H), 3.02 (d, J = 9.6 Hz, 1H), 2.86 (s, 3H), 2.72 (s, 3H), 2.14−2.23 (m, 2H), 2.03−2.12 (m, 1H), 1.64 (s, 3H), 1.57 (d, J=13.6, 1H), 1.42−1.49 (m, 2H), 1.34 (s, 3H), 1.34 (s, 3H), 1.29 (d, J = 1.2 Hz, 3H), 1.27 (d, J = 1.6 Hz, 3H), 1.23 (m, 2H), 0.80 (s, 3H). 13C NMR (CDCl3): δ 171.31, 170.72, 168.66, 156.63, 152.12, 142.30, 140.83, 139.16, 133.26, 127.73, 125.37, 121.96, 118.96, 113.26, 88.47, 80.90, 78.18, 74.09, 67.09, 60.55, 59.92, 56.63, 56.58, 52.56, 46.65, 38.87, 36.14, 35.52, 34.82, 32.41, 31.51, 30.84, 28.46, 22.23, 21.47, 15.47, 14.56, 13.34, 12.18. HRMS: calcd for C39H56ClN3O12S [M + Na]+ 848.3175; found 848.3165. N2′-Deacetyl-N2′-(3-sulfonate-1-oxopropyl)maytansine (DM1 Sulfonic Acid, 15a). To a solution of DM1 (2a, 48 mg, 0.065 mmol) in methanol (4.0 mL) were added vanadyl acetylacetonate (20 mg, 0.075 mmol) and a solution of 5.5 M tert-butyl hydrogen peroxide in decane (59 μL, 0.33 mmol) at ambient temperature. The reaction mixture was stirred for 1 h and then concentrated to approximately 2 mL in vacuo. The product was purified by preparative C18 HPLC, and fractions containing desired product (retention time 23 min) were combined, frozen, and lyophilized to give 22 mg (43% yield) of 15a as a white solid. 1H NMR (400 MHz, CD3CN): δ 7.12− 7.03 (m, 1H), 6.67−6.59 (m, 1H), 6.53−6.44 (m, 2H), 5.65− 5.50 (m, 1H), 5.33−5.21 (m, 1H), 4.53 (dd, J = 12.1, 3.1 Hz, 1H), 4.21−4.08 (m, 1H), 3.92 (s, 3H), 3.54−3.42 (m, 2H), 3.27 (s, 3H), 3.19 (d, J = 12.3 Hz, 1H), 3.13 (s, 3H), 3.10−2.93 (m, 3H), 2.93−2.83 (m, 4H), 2.81 (s, 3H), 2.76−2.49 (m, 3H), 2.19−2.08 (m, 1H), 1.59 (s, 3H), 1.52−1.31 (m, 2H), 1.24 (m,

2H), 1.15 (d, J = 6.4 Hz, 3H), 0.74 (s, 3H). HRMS: calcd for C35H48ClN3O13S [M − H + 2Na]+ 830.2314; found 830.2247. N 2 ′ -Deacetyl-N 2 ′ -(4-sulfonate-4-methyl-1-oxopentyl)maytansine (DM4 Sulfonic Acid, 15b). To a solution of DM4 (2b, 25 mg, 0.032 mmol) in methanol (2.0 mL) were added vanadyl acetylacetonate (10 mg, 0.038 mmol) and a solution of 5.5 M tert-butyl hydrogen peroxide in decane (29 μL, 0.16 mmol) at ambient temperature. The reaction mixture was stirred for 30 min and then purified by preparative C18 HPLC. Fractions containing desired product (retention time 24 min) were combined, frozen, and lyophilized to give 11 mg (41% yield) of 15b as a white solid. 1H NMR (400 MHz, CD3CN): δ 8.37 (s, 1H), 7.06 (d, J = 1.6 Hz, 1H), 6.61 (d, J = 1.6 Hz, 1H), 6.55−6.45 (m, 1H), 5.67−5.53 (m, 1H), 5.33−5.25 (m, 1H), 4.55 (dd, J = 12.1, 3.0 Hz, 1H), 4.20−4.09 (m, 1H), 3.91 (s, 3H), 3.54−3.42 (m, 2H), 3.28 (s, 3H), 3.19 (d, J = 12.6 Hz, 1H), 3.13 (s, 3H), 2.89 (d, J = 9.7 Hz, 1H), 2.79 (s, 3H), 2.72− 2.51 (m, 2H), 2.37−2.27 (m, 2H), 2.12 (dd, J = 14.6, 2.4 Hz, 1H), 1.85−1.75 (m, 2H), 1.58 (s, 3H), 1.54−1.28 (m, 2H), 1.22 (d, J = 6.9 Hz, 3H), 1.15 (d, J = 6.4 Hz, 3H), 1.11 (m, 6H), 0.75 (s, 3H). HRMS: calcd for C38H54ClN3O13S [M − H + 2Na]+ 872.2783; found 872.2714.

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RESULTS The primary- and HLM-derived metabolites from the AMCs 3a, 3b, 3c, and 4, as well as side products from AMC preparation, were required for these studies and were prepared by the following methods. Reaction of DM1 (2a) or DM4 (2b) with methyl iodide in the presence of N,N-diisopropylethylamine (DIPEA) gave the corresponding S-methylation products S-Me-DM1 (12a) and S-Me-DM4 (12b), Figure 6. Oxidation of S-Me-DM1 (12a) with tert-butyl peroxide in the presence of a catalytic amount of vanadyl acetylacetonate gave the two corresponding isomers of S-Me-DM1 sulfoxide (13a). Similarly reaction of S-Me-DM4 with tert-butyl peroxide in the presence of a catalytic amount of vanadyl acetylacetonate gave the two isomers of S-Me-DM4 sulfoxide (13b) as a mixture of two isomers. Oxidation of S-Me-DM1 (12a) or S-Me-DM4 (12b) with excess tert-butyl peroxide and vanadyl acetylacetonate gave the corresponding S-Me-DM1 sulfone (14a) or SMe-DM4 sulfone (14b) repectively, via initial formation of the corresponding sulfoxides. Oxidation of DM1 (2a) or DM4 (2b) with tert-butyl peroxide in the presence of a catalytic amount of vanadyl acetylacetonate gave the sulfonic acid of DM1 (15a) or the sulfonic acid of DM4 (15b) respectively. Sulfinic acid derivatives of DM1 (15c) or DM4 (15d) were not detected by HPLC analysis of either reaction mixture. Lysine-bearing primary metabolites were prepared as shown in Figure 7. Reaction of DM1 (2a) with SPP (5a) and lysine in G

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Molecular Pharmaceutics

Figure 7. Synthesis of lysine-maytansinoid cellular metabolites.

Figure 8. Synthesis of hydrolyzed linker−maytansinoid adducts.

aqueous sodium bicarbonate gave Lys-Nε-SPP-DM1 (10a). The two lysine linked DM4 metabolites, Lys-Nε-SPDB-DM4 (10b) and Lys-Nε-sulfo-SPDB-DM4 (10c), were similarly prepared by reacting DM4 (2b) and lysine with SPDB (5b) or sulfo-SPDB (5c), respectively, in the presence of aqueous sodium bicarbonate. Reaction of a thiol, such as that of DM1 (2a), with a maleimide moiety occurs at either face of the maleimide, resulting in two isomeric products. Thus, reaction of DM1 (2a) with SMCC (6) and lysine gave Lys-Nε-SMCCDM1(11) as a 1:1 mixture of diastereomers. Preparation of the side products from the conjugation reactions, DM1-TPA (8a) and DM1 dimer (7) and DM4-TBA (8b), have been described previously.11 The side product DM4sulfo-TBA (8c) produced in the mAb-sulfo-SPDB-DM4 conjugation process was prepared by reacting DM4 (2b) with sulfo-SPDB (5c) at an alkaline pH and allowing the Nhydroxsuccinimidyl ester to hydrolyze in solution. A diastereomeric mixture of DM1-MCC (9) was prepared by reacting

DM1 (2a) with commercially available 17 in the presence of base, Figure 8. All of the synthesized compounds had the same HPLC retention times and mass spectral ion patterns as the corresponding metabolites produced by incubation of AMCs with target cells in vitro or from in vivo mouse experiments.9,16,22 We have previously shown that mouse liver metabolizes several primary AMC metabolites to give less potent compounds, and we wished to determine if such detoxification would be likely to occur in humans during liver metabolism. For these studies, the maytansinoids of interest were treated with human liver microsomes, both with and without active NADPH. The samples were analyzed by HPLC/MS, and the identities of eluting compounds were established by comparing their retention times and mass spectral profiles to synthetic standards. HPLC traces from the incubation of human liver microsomes with a small amount of acetonitrile (control) or a solution of H

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Figure 9. HPLC traces from the incubations of HLM and activated NADPH with (a) no added compound, used as a control to identify HPLC control debris (CD) peaks that are not of interest, (b) S-Me-DM1, (c) S-Me-DM4, (d) DM1, (e) DM4, and (f) DM4-SMe.

designated 13b and 13b′ and the S-Me-DM4 sulfone 14b. Incubation of DM1 (2a) with HLM and active NADPH gave DM1 disulfide dimer (7) as the major product, with the sulfonic acid of DM1 (15a) as a minor product. An HPLC peak at 26 min had a negative MS ion of 768 m/z, which was tentatively identified as the M − 1 ion of DM1 sulfinic acid (15c). DM1 (2a) incubated with HLM in the absence of active NADPH was also converted to DM1 disulfide dimer (7), however the sulfonic and sulfinic acids of DM1 (15a and 15c) respectively were not detected. No metabolites were formed

test compound in acetonitrile are shown in Figure 9. The control HPLC trace identifies HPLC peaks that are due to control debris (CD). Incubation of S-Me-DM1 (12a) with human liver microsomes (HLM) and active NADPH gave both isomers of the corresponding sulfoxides (13a and 13a′) in a 1:1 ratio as the major products along with S-Me-DM1 sulfone (14a) as a minor product. Incubation of S-Me-DM4 (12b) with HLM and active NADPH gave a single broad HPLC peak for the sulfoxide (13b) as the major product, which could be due to one or both possible isomeric forms of the sulfoxide, I

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Molecular Pharmaceutics Table 1. AMC Metabolite in Vitro Cytotoxicities toward KB Cellsa primary metabolite

IC50 (M) −11

S-Me-DM1 (12a)

2.2 × 10

S-Me-DM4 (12b)

2.6 × 10−11

DM1 (2a)

1 × 10−9c

DM4 (2b)

3 × 10−10c

Lys-Nε-SPP-DM1 (10a) Lys-Nε-SPDB-DM4 (10b) Lys-Nε-sulfo-SPDB-DM4 (10c) Lys-Nε-SMCC-DM1 (11) DM1-TPA (8a) DM4-TBA (8b) DM4-sulfo-TBA (8c) DM1-MCC (9) DM1 dimer (7)

1.0 3.3 3.0 9.7 1.8 2.3 4.0 2.9 1.4

× × × × × × × × ×

10−8 10−8 10−9 10−8 10−8 10−8 10−9 10−8 10−10

HLM metabolite(s) S-Me-DM1 sulfoxide (13a) S-Me-DM1 sulfone (14a) S-Me-DM4 sulfoxide (13b) S-Me-DM4 sulfone (14b) DM1 sulfinic acid (15c) DM1 sulfonic acid (15a) DM4 sulfinic acid (15d) DM4 sulfonic acid (15b) none none none none none none none none none

IC50 (M) −8

2.3 x10 3.5 × 10−9 2.0 × 10−9 8.0 × 10−10 ND 4.0 × 10−8 ND 2.7 × 10−8 − − − − − − − − −

fold decrease in potency

b

1050 160 77 31 ND 40 ND 90 − − − − − − − − −

ND (not determined), the compound was not yet synthesized. − indicates no value because parent compounds were not metabolized. Sulfoxides were assayed as the mixture of isomers. bFold decrease in potency = (IC50 HLM metabolite)/(IC50 primary metabolite). cIn vitro cytotoxicity varies due to thiol reactivity with cystine and other disulfide containing components present in cell media. a

charged maytansinoids were less potent with IC50 values mostly in the 2−20 nM range.

from the incubation of DM1 dimer (7) with HLM and active NADPH. Incubation of DM4 (2b) with HLM gave small amounts of metabolites, including DM4 sulfonic acid (15b) as well as a product with a major MS ion of 834 m/z in positive ion mode, presumably the (M + Na)+ ion of DM4 sulfinic acid 15d. The HPLC peak at 26.3 min was identified as the DM4− cysteine adduct because reaction of cystine with DM4 (2b) gave one product with the same HPLC retention time and MS ion pattern (data not shown). In order to understand why HLM metabolism did not occur with DM1 dimer (7) and to determine if other maytansinoids bearing disulfide bonds would be metabolized, the methyl disulfide of DM4, DM4-SMe (18), was incubated with HLM and active NADPH, Figure 9f. Several major products were formed including DM4 sulfonic acid (15b), a compound tentitively identified as DM4 sulfinic acid (15d), and one or more compounds having a major MS ion of 864 m/z in positive ion mode, presumably the M + Na+ ion of one or more mono-oxidized DM4-SMe species, data not shown. No metabolism occurred when DM4-SMe (18) was incubated with HLM in the absence of active NADPH. Each of the lysine bearing metabolites Lys-Nε-SPP-DM1 (10a), Lys-Nε-SPDB-DM4 (10b), Lys-Nε-sulfo-SPDB-DM4 (10c), and Lys-Nε-SMCC-DM1 (11) were incubated with HLM, however no microsomal metabolites were detected (data not shown). The maytansinoid−linker conjugation side products 8a, 8b, 8c, and 9 were also incubated with HLM, and again no microsomal metabolites were detected. The synthesized primary and HLM metabolites as well as synthesized side products from conjugation reactions were evaluated for their ability to suppress the proliferation of KB (human epidermoid carcinoma) cell lines in vitro. Cells were exposed to the compounds for 72 h, and the surviving fractions of cells were measured by using a clonogenic plating efficiency assay to determine IC50 values. The cytotoxic potency of these compounds is shown in Table 1, along with the fold decrease in potency that arises from the HLM transformation. All of the noncharged maytansinoids were highly cytotoxic against KB cells, with IC50 values in the sub-nanomolar range, while the

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DISCUSSION All primary maytansinoid metabolites derived from the four AMCs, mAb-SPP-DM1 (3a), mAb-SPDB-DM4 (3b), mAbsulfo-SPDB-DM4 (3c), and mAb-SMCC-DM1 (4), were synthesized and their cytotoxicities were determined on KB cancer cells. These metabolites were also incubated with HLM, and the identities of the resulting products, with the exception of the sulfinic acids, were confirmed by independent synthesis and their cytotoxicities were determined. Analysis of the primary or HLM metabolites showed that the maytansinoid macrocycle was untouched. Alterations were only observed on the maytansinoid C3 ester side chain. As expected noncharged, relatively hydrophobic maytansinoids were the most cytotoxic, while charged or more polar maytansinoids were several fold less potent. As we have discussed in previous publications, we believe that this is because charged metabolites poorly diffuse into cells. However, such metabolites may be highly potent if generated inside a cell, as occurs during ADC delivery.22 Incubation of DM1 (2a) with HLM in vitro formed the DM1 disulfide dimer (7). However, in vivo concentrations of DM1 (2a) derived by slow release from mAb-SPP-DM1 are expected to be much lower than those used in the in vitro assay with HLM. Thus, mAb-SPP-DM1 would not be expected to generate a high enough concentration of DM1 (2a) to promote DM1 disulfide (7) formation in vivo. The DM1 dimer (7) was not significantly metabolized by HLM in the presence of active NADPH while the lower molecular weight, disulfide-bearing compound DM4-SMe (18) was readily oxidized. This indicates that a disulfide moiety of a maytansinoid is not resistant to oxidation. Many P-450 enzymes present in HLM however are known to have limitations on the size of substrate they can accommodate in their active site.23 It is therefore possible that the DM1 dimer may be too large for these oxidative enzymes to process. The most cytotoxic AMC metabolites, S-Me-DM1(12a) and S-Me-DM4 (12b), were oxidatively detoxified by HLM, giving J

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carboxypentyl]amino]-4-oxo-3-sulfonate-butyl]dithio]-4-methyl-1-oxopentyl]maytansine; Lys-Nε-SPP-DM1, N2′-deacetylN 2 ′ -[4-[[4-[[(1S)-2-amino-1-carboxypentyl]amino]-3oxopropyl]dithio]-1-oxopropyl]maytansine; NADPH, nicotinamide adenine dinucleotide phosphate reduced form; RES, reticuloendothelial system; SEC, size exclusion chromatography; S-Me-DM1, N2′-deacetyl-N2′-(3-methylthio-1-oxopropyl)maytansine; S-Me-DM1 sulfone, N2′-deacetyl-N2′-(3-methylsulfonyl-1-oxopropyl)maytansine; DM1 sulfonic acid, N2′deacetyl-N2′-(3-sulfonate-1-oxopropyl)maytansine; S-Me-DM1 sulfoxide, N2′-deacetyl-N2′-(3-methylsulfoxy-1-oxopropyl)maytansine; S-Me-DM4, N2′-deacetyl-N2′-(4-methyl-4-methythio-1-oxopentyl)maytansine; S-Me-DM4 sulfone, N2′-deacetyl-N2′-(4-methyl-4-methysulfonyl-1-oxopentyl)maytansine; SMe-DM4 sulfoxide, N2′-deacetyl-N2′-(4-methyl-4-methylsulfoxy-1-oxopentyl)maytansine; SMCC, N-succinimidyl 4-(Nmaleimidomethyl) trans-cyclohexane 1-carboxylate; SPDB, Nsuccinimidyl 4-(2-pyridyldithio)butyrate; SPP, N-succinimidyl 4-(2-pyridyldithio)pentanoate; sulfo-SPDB, N-succinimidyl 4(2-pyridyldithio)-2-sulfopentanoate; TFF, tangential flow filtration

the respective sulfoxides and sulfones. The P-450 enzymes responsible for S-Me-DM4 (12b) oxidation have been identified by Davis et al.24 Maytansine is a highly potent compound, but interestingly Liu et al. found that its major HLM metabolite is 2′-N-des-methylmaytansine, which is only slightly less cytotoxic than maytansine.25 Unlike maytansine, SMe-DM1 (12a) and S-Me-DM4 (12b) contain a readily oxidized sulfide moiety which may be essential for liver detoxification of these metabolites. Microsome preparations can be used to model liver oxidation of compounds, but unlike liver itself, microsomes cannot induce phase II metabolism such as S-methylation. The thiol-bearing compounds DM1 (2a) and DM4 (2b) were not efficiently oxidized by HLM to give their sulfinic or sulfonic acids, which indicates that S-methylation would likely be an important first step for liver detoxification of DM1 (2a) or DM4 (2b). All of the metabolites or conjugation side products bearing a carboxylic acid such as Lys-Nε-SMCCDM1 (11) or DM1-MCC (9) had relatively poor cytotoxicities and were not metabolized by HLM. This is consistent with our previous work showing that mouse liver does not metabolize Lys-Nε-SMCC-DM1 to give oxidized compounds. Perfused rat liver and rat liver in vivo have been shown to reduce disulfide bonds while microsomes do not.26 The human liver would therefore be expected to convert charged disulfide-bearing maytansinoids such as Lys-Nε-SPP-DM1(10a) or Lys-NεSPDB-DM4 (10b) to DM1 (2a) or DM4 (2b) respectively, which in turn would require S-methylation before potencies can be reduced by oxidation. Overall, this study suggests that the human liver would be expected to metabolize AMCs and AMC metabolites similarly to the mouse liver, generating metabolites with markedly reduced cytotoxic potency relative to unmodified maytansine.

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REFERENCES

(1) Chari, R. V.; Miller, M. L.; Widdison, W. C. Antibody-drug conjugates: An emerging concept in cancer therapy. Angew. Chem. 2014, 53 (15), 3796−3827. (2) Ducry, L.; Stump, B. Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjugate Chem. 2010, 21 (1), 5−13. (3) Flygare, J. A.; Pillow, T. H.; Aristoff, P. Antibody-drug conjugates for the treatment of cancer. Chem. Biol. Drug Des. 2013, 81 (1), 113− 21. (4) Adair, J. R.; Howard, P. W.; Hartley, J. A.; Williams, D. G.; Chester, K. A. Antibody-drug conjugates - a perfect synergy. Expert Opin. Biol. Ther. 2012, 12 (9), 1191−206. (5) Sedlacek, H. H. Antibodies as carriers of cytotoxicity; Karger: Basel, New York, 1992; p viii, 208 pp. (6) Tabrizi, M. A.; Tseng, C. M.; Roskos, L. K. Elimination mechanisms of therapeutic monoclonal antibodies. Drug Discovery Today 2006, 11 (1−2), 81−8. (7) Tabrizi, M.; Bornstein, G. G.; Suria, H. Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease. AAPS J. 2010, 12 (1), 33−43. (8) Han, T. H.; Gopal, A. K.; Ramchandren, R.; Goy, A.; Chen, R.; Matous, J. V.; Cooper, M.; Grove, L. E.; Alley, S. C.; Lynch, C. M.; O’Connor, O. A. CYP3A-mediated drug-drug interaction potential and excretion of brentuximab vedotin, an antibody-drug conjugate, in patients with CD30-positive hematologic malignancies. J. Clin Pharmacol. 2013, 53 (8), 866−77. (9) Sun, X.; Widdison, W.; Mayo, M.; Wilhelm, S.; Leece, B.; Chari, R.; Singh, R.; Erickson, H. Design of antibody-maytansinoid conjugates allows for efficient detoxification via liver metabolism. Bioconjugate Chem. 2011, 22 (4), 728−35. (10) Abecassis, P. Y.; Amara, C. In vivo testing of drug-linker stability. Methods Mol. Biol. 2013, 1045, 101−16. (11) Kellogg, B. A.; Garrett, L.; Kovtun, Y.; Lai, K. C.; Leece, B.; Miller, M.; Payne, G.; Steeves, R.; Whiteman, K. R.; Widdison, W.; Xie, H.; Singh, R.; Chari, R. V.; Lambert, J. M.; Lutz, R. J. Disulfidelinked antibody-maytansinoid conjugates: optimization of in vivo activity by varying the steric hindrance at carbon atoms adjacent to the disulfide linkage. Bioconjugate Chem. 2011, 22 (4), 717−27. (12) Litvak-Greenfeld, D.; Benhar, I. Risks and untoward toxicities of antibody-based immunoconjugates. Adv. Drug Delivery Rev. 2012, 64 (15), 1782−99. (13) Kupchan, S. M.; Komoda, Y.; Court, W. A.; Thomas, G. J.; Smith, R. M.; Karim, A.; Gilmore, C. J.; Haltiwanger, R. C.; Bryan, R.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. † B.L. is retired.

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ABBREVIATIONS USED ADC, antibody−drug conjugate; AMC, antibody−maytansinoid conjugate; CD, control debris; DMA, N,N-dimethylacetamide; DMF, N,N-dimethylformamide; DM1, N2′-deacetylN-2′(3-mercapto-1-oxopropyl)maytansine; DM1-MCC, N2′deacetyl-N2′-[3-[[1-[(4-carboxy-trans-cyclohexyl)methyl]-2,5dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]maytansine; DM1TPA, N2′-deacetyl-N2′-[3-(3-carboxy-1-methylpropyldithio)-1oxopropyl]maytansine; DM4, N2′-deacetyl-N-2′(4-mercapto-4methyl-1-oxopentyl)maytansine; DM4 sulfonic acid, N2′deacetyl-N2′-(4-sulfonate-4-methyl-1-oxopentyl)maytansine; DM4-TBA, N2′-deacetyl-N2′-[4-methyl-4-(3-carboxypropyldithio)-1-oxopentyl]maytansine; DM4-sulfo-TBA, N2′-deacetylN2′-[4-methyl-4-(3-carboxy-3-sulfopropyldithio)-1-oxopentyl]maytansine; HLM, human liver microsomes; mAb, monoclonal antibody; Lys-Nε-SMCC-DM1, N2′-deacetyl-N2′-[3-[[1-[[4[[[(1S)-5-amino-1-carboxypentyl]amino]carbonyl] transcyclohexyl]methyl]-2,5-dioxo-3-pyrrolidinyl]thio]-1oxopropyl]maytansine; Lys-Nε-SPDB-DM4, N2′-deacetyl-N2′[4-[[4-[[(1S)-2-amino-1-carboxypentyl]amino]-4-oxobutyl]dithio]-4-methyl-1-oxopentyl]maytansine; Lys-Nε-sulfo-SPDBDM4, N2′-deacetyl-N2′-[4-[[4-[[(1S)-2-amino-1K

DOI: 10.1021/mp5007757 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/mp5007757 Mol. Pharmaceutics XXXX, XXX, XXX−XXX