Pyrrolobenzodiazepine Dimer Antibody–Drug Conjugates

Article pubs.acs.org/jmc

Pyrrolobenzodiazepine Dimer Antibody−Drug Conjugates: Synthesis and Evaluation of Noncleavable Drug-Linkers Stephen J. Gregson,*,† Luke A. Masterson,† Binqing Wei,‡ Thomas H. Pillow,*,‡ Susan D. Spencer,‡ Gyoung-Dong Kang,† Shang-Fan Yu,‡ Helga Raab,‡ Jeffrey Lau,‡ Guangmin Li,‡ Gail D. Lewis Phillips,‡ Janet Gunzner-Toste,‡ Brian S. Safina,‡ Rachana Ohri,‡ Martine Darwish,‡ Katherine R. Kozak,‡ Josefa dela Cruz-Chuh,‡ Andrew Polson,‡ John A. Flygare,‡,§ and Philip W. Howard† †

Spirogen, QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States

‡

S Supporting Information *

ABSTRACT: Three rationally designed pyrrolobenzodiazepine (PBD) druglinkers have been synthesized via intermediate 19 for use in antibody−drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11−48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the antiHER2 ADCs (HER2 0 MCF7, all inactive) and 0.10−1.73 μg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5−1 mg/kg, 1 mg/kg, and 3−6 mg/ kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for antiCD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.

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potency.7 One such PBD dimer, SJG-136, was evaluated as a standalone agent in phase I/II clinical trials for cancer.5,8 With the resurgence of interest in ADC technology, it became apparent that potent PBD dimers were ideal candidates for the cytotoxic component. The number of potential chemical linking positions in PBDs offers great flexibility for antibody connection, and this is apparent in the two PBD drug-linker molecules [3 (SGD1910)9,10 and 4 (SG3249),11−13 Figure 1] which feature in numerous ADCs being evaluated in clinical trials.14−19 3 is linked via the C2 position of the PBD and 4 via the N10 position and to the antibody through a maleimide− cysteine connection. Both molecules contain a dipeptide (valine−alanine) trigger that is cleaved by cathepsin in the lysosome. A self-immolative PAB spacer is necessary in 4 to liberate the bis-imine DNA cross-linking PBD dimer upon catabolism of the ADC, while the PEG group aids water solubility. An indolobenzodiazepine (IBD) 5 (IMGN779) is also in clinical development as an ADC drug-linker and is linked to the antibody through an aryl tether in the IBD.20 Again, the linkage in 5 is cleavable (disulfide trigger) but it is linked to the antibody through a lysine connection. Given that a noncleavable linker is employed in the approved drug 1, we wanted to apply this notion to the PBD dimer platform and envisaged that linking through an aryl tether

INTRODUCTION

There is a great deal of pharmaceutical industry interest in the development of antibody−drug conjugates (ADCs) for the treatment of cancer.1 The majority of ADCs have a common design concept that generally consists of three variable parts: a tumor antigen targeting monoclonal antibody, a covalent linking group, and a cytotoxic warhead.2 Two ADCs have been on the market for several years (Figure 1): trastuzumab emtansine (1, HER2 [human epidermal growth factor receptor 2] targeting, noncleavable linker, emtansine warhead) and brentuximab vedotin (2, CD30 targeting, cleavable dipeptide linker, self-immolative p-aminobenzyl [PAB] spacer, monomethyl auristatin E warhead).2 At present there are >60 ADCs3,4 in clinical trials and several of these contain the pyrrolo[2,1-c][1,4]benzodiazepine (PBD) ring system as the cytotoxic component of the ADC (Figure 1).5 PBDs were originally discovered in the 1960s and a number of natural products containing the ring system were isolated from various Streptomyces species.6 The mode of antitumor action was later established and shown to involve sequence-selective alkylation in the minor groove of DNA, where an N2 of guanine forms a covalent bond with the electrophilic N10/C11 imine on the PBD.5 Advances in PBD synthesis enabled the preparation of many synthetic derivatives including dimeric DNA cross-linking PBDs with extended base sequence recognition capability and increased cytotoxic © 2017 American Chemical Society

Received: May 18, 2017 Published: November 7, 2017 9490

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

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Figure 1. Structures of marketed ADC drug-linkers trastuzumab emtansine (1) and brentuximab vedotin (2) and a standalone PBD dimer and PBD (3 and 4)/IBD (5) ADC drug-linkers in clinical trials and development. Notable PBD atom numbers are shown in 3.

Figure 2. PBD dimer ADCs evaluated in this study. The drugs were conjugated to either anti-HER2 (thio Hu anti-HER2 4D5 HC-A118C) or antiCD22 (thio Hu anti-CD22 10F4v3 HC-A118C) cysteine engineered antibodies.

and primate plasma models, some are unstable in rodent plasma.22 Therefore, premature drug release prior to internalization may affect efficacy and tolerability data when assessing cleavable ADCs in rodents at the preclinical stage. As proof of principle for the novel noncleavable ADCs, HER2 (found in many solid tumors) and CD22 (found in many hematological cancers) were attractive antigen targets to investigate. There are several next generation anti-HER2 ADCs

would provide a symmetrical molecule and facilitate the possibility of a noncleavable linkage to an antibody. A noncleavable linker similar to that of 1 could give active metabolites from lysosomal antibody degradation that may be less membrane permeable and decrease potential toxicities in neighboring healthy tissue through lack of bystander effect.21 Another motive for investigating noncleavable PBD-ADCs was that although dipeptide cleavable linkers are stable in human 9491

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Figure 3. (a) Structure of pentyldioxy tethered PBD dimer 4a. (b) Schematic representation of an aryl tether PBD dimer bound to 10-nucleotide long DNA duplex (5′-AACCATCGTT-3′). (c) Model of 6 (carbon atoms in cyan) bound in the DNA minor groove, where the molecular surface of the drug-linker cysteine adduct is shown in yellow. (d) Alternative view of the same complex with 6 looking down the minor groove. The covalent bonds between C11/C11′ of the PBD and the exocyclic N2 atom of two deoxyguanines are marked by solid lines and labeled with the bond length values (in Å), and potential hydrogen bonds between N10/N10′ of the PBD and an adjacent DNA base are marked by dashed lines. (e) Model of 7 (carbon atoms in brown) bound in the DNA minor groove. (f) Model of 8 (carbon atoms in green) bound in the DNA minor groove.

in development including topoisomerase 1 inhibitor-based23 and duocarmycin-based payloads24 and a biparatopic ADC containing a tubulysin warhead.25 For CD22, inotuzumab ozogamicin has demonstrated promising clinical activity and has recently received FDA approval for patients with relapsed or refractory acute lymphoblastic leukemia.26 We report molecular modeling, synthesis, and efficacy evaluation of the first examples of noncleavable anti-HER2 (trastuzumab, 4D5) and anti-CD22 PBD ADCs based on three drug-linker molecules 6, 7, and 8 (Figure 2). The compounds described here consisted of an alkyne (6), triazole (7), or piperazine (8) incorporated in the PBD aryl tether and a maleimide connecting group for site-specific antibody cysteine conjugation. A PEG spacer was included in the linkers to enhance conjugation solubility in aqueous media. These molecules were accessible from the key iodoaryl intermediate 19 using Sonogashira, click, or Buchwald chemistry. The belief was the active drugs released from ADCs of 6, 7, and 8 would be cysteine adducts, and this hypothesis was based on published evidence for other noncleavable ADCs: In the case of marketed noncleavable ADC 1, catabolism in the lysosome yields the released drug as a lysine adduct of the cytotoxin emtansine;27,28 similarly the noncleavable ADC

cAC10-maleimidocaproyl-monomethylauristatin F releases a cysteine adduct of monomethylauristatin F.29

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RESULTS Molecular Modeling. Previous studies have shown that PBD dimer 4a, containing a pentyldioxy tether, efficiently forms DNA interstrand cross-links in a sequence-dependent fashion and exhibited subnanomolar antiproliferative activities against a panel of cancer cell lines (Figure 3a).30−32 On the basis of the solution structure of a PBD monomer covalently bound in a standard B-DNA helix (PDB entry 2KTT33), we modeled how PBD dimers, including 4a, would bind in a 10nucleotide long DNA duplex (5′-AACCATCGTT-3′). In a similar fashion to the pentyl tether in 4a, modeling predicted that released linker-cysteine adducts containing a 1,3bismethylbenzene tether would fit equally as well into the DNA minor groove (Figure 3b). Furthermore, modeling suggested that substitution at the 5-position of the benzene tether could directly protrude out of the minor groove without interfering with the DNA backbone and provide a novel way of linking a symmetrical PBD dimer to the antibody. Candidate noncleavable drug-linkers were evaluated in terms of their steric and electrostatic interactions complementary to the minor groove of DNA (particularly the phosphate backbone), and 9492

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

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Scheme 1. Synthesis of 19a

Reagents and conditions: (a) (1) (COCl)2, DCM, DMF, rt, 16 h; (2) methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate·HCl, DCM, TEA, −20 °C, 16 h, 68% yield; (b) LiBH4, THF, 0 °C, 4 h, 90% yield; (c) SnCl2, MeOH, reflux, 3 h, 12 carried through crude; (d) (Boc)2O, THF, reflux, 16 h, 60% yield; (e) TEMPO, DAIB, DCM, rt, 16 h, 46% yield; (f) 10% w/w Pd−C, EtOH, rt, 4 h, 53% yield; (g) DHP, PTSA, EtOAc, rt, 1 h, 65% yield; (h) PPh3CH3Br, KOtBu, THF, 0 °C to rt, 18 h, 72% yield; (i) THF, AcOH, H2O, rt, 3 h, 87% yield; (j) 1,3-bis(bromomethyl)-5-iodobenzene, K2CO3, TBAI, DMF, 60 °C, 3 h, 91% yield. a

Scheme 2. Synthesis of 6a

a

Reagents and conditions: (a) propargylamine, EDCI, DCM, rt, 16 h, 71% yield; (b) 19, Pd(PPh3)4, CuI, diethylamine, DMF, 4 Å molecular sieves, microwave at 100 °C, 26 min, 70% yield; (c) (1) TBSOTf, 2,6-lutidine, DCM, rt, 16 h; (2) TBAF, THF, rt, 1 h, 23 carried through crude; (d) EDCI, N-maleoyl-β-alanine, DCM, rt, 3 h, 24 carried through crude; (e) 95% TFA/H2O, 0 °C, 1 h, 60% yield from 22 (in 4 steps).

contribute to binding. Alkyne substitution on the central benzene allows a tether linker to reach above the phosphate rim of the DNA minor groove. The rest of the linker provides ample separation between the antibody attachment point (maleimide−cysteine, Figure 2) and the DNA minor groove. Assuming the ethylene glycol moieties adopt an energetically favored gauche conformation,34 the separation was estimated to be >10 Å. For drug-linker compounds 7 and 8, the triazole and piperazine moieties were predicted to be largely coplanar with the benzene tether due to conjugation and were well

their conformational preferences. The search identified three diverse scaffolds that contain an alkyne, a triazole, or a piperazine moiety, respectively (6, 7, and 8 in Figure 2). For example, in the model of compound 6 bound to DNA (Figure 3c and Figure 3d), two covalent bonds (bond length 1.47−1.49 Å) between the C11/C11′ of the PBD and the exocyclic N2 atoms of two deoxyguanines, separated by four base pairs on opposing DNA strands, serve to anchor the PBD dimer snugly inside the minor groove. Potential hydrogen bonds between the N10 of the PBD and an adjacent DNA base may also 9493

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Figure 4. Structures of compounds to assist discussion of Scheme 2.

Scheme 3. Synthesis of 7a

Reagents and conditions: (a) TMS-acetylene, Pd(PPh3)4, CuI, diethylamine, DMF, microwave at 100 °C, 30 min, 95% yield; (b) K2CO3, MeOH, rt, 3 h, 78% yield; (c) 11-azido-3,6,9-trioxaundecan-1-amine, CuSO4·5H2O, sodium ascorbate, tBuOH, H2O, rt, 16 h, 27 carried through crude; (d) 6-maleimidohexanoic acid N-hydroxysuccinimide ester, DCM, rt, 3 d, 45% yield; (e) 95% TFA/H2O, 0 °C, 1 h, 53% yield. a

of the nitro group. The crude aniline 12 was Boc protected to give 13 in 60% yield, which was followed by simultaneous ring closure and secondary alcohol oxidation to provide ketone 14. Debenzylation by hydrogenolysis, followed by protection of the free phenol and C11-OH with THP, gave 16 which was isolated as a mixture of diastereoisomers arising from the THP group. Wittig olefination followed by selective cleavage of THP from the phenol furnished 18, in which the C11 THP group

accommodated in the DNA minor groove (Figure 3e and Figure 3f). A similar separation of the DNA and the antibody point for that observed with 6 was also realized for 7 and 8. Chemistry. The known nitrobenzoic acid 935 was converted to its corresponding acid chloride and coupled to methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate·HCl to give amide 10 in 68% yield (Scheme 1). The methyl ester was reduced to the diol 11 in 90% yield, followed by SnCl2 mediated reduction 9494

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

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Scheme 4. Synthesis of 8a

Reagents and conditions: (a) tert-butyl piperazine-1-carboxylate, RuPhos Pd G1 methyl tert-butyl ether adduct, RuPhos, Cs2CO3, THF, 85 °C, 4 h, 34% yield; (b) 95% TFA/H2O, 0 °C, 30 min, 30 carried through crude; (c) EDCI, 1-maleimido-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid, DCM, rt, 2 h, 32% yield. a

was still required to prevent racemization at C11a36,37 during the subsequent dimerization step. A DMF solution of known 1,3-bis(bromomethyl)-5-iodobenzene 38 (obtained in two steps39 by LiBH4 reduction and then Appel halogenation from commercially available dimethyl 5-iodoisophthalate) was reacted at 60 °C with 2 equiv of phenol 18 in the presence of K2CO3 and TBAI to provide the key iodoaryl intermediate 19 in 91% yield. The synthesis of alkyne drug-linker 6 from intermediate 19 is shown in Scheme 2. Initially, a linear approach was explored starting with a Sonogashira reaction on 19 with propargylamine, followed by amide coupling with 1-maleimido-3-oxo7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid. This was unsatisfactory due to the instability of the resulting arylpropargylamine 22a affording small quantities of 24 in low overall yield (Scheme 2 and Figure 4). Instead, a more convergent approach was adopted, which involved reacting propargylamine with 2,2dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid (20) to give the Sonogashira coupling partner 21 in 71% yield. The subsequent Sonogashira reaction on 19 was performed under microwave conditions and gave 22 in 70% yield. Attempted triple Boc deprotection of 22 to give 23b with 95% TFA/H2O was unsuccessful and instead resulted in polymerization using these conditions. Therefore, selective primary N-Boc deprotection was investigated as an alternative approach.40 This involved a two-stage process where the Boc carbamate was converted to a TBS carbamate followed by TBAF cleavage to reveal the desired amine. Although the Boc removal was not as selective as anticipated (side products 23a and 23b resulting from secondary N-Boc cleavage were observed by LC−MS), recovery was respectable and crude material was carried through to the next step. Amine 23 (and side products 23a and 23b) was coupled to N-maleoyl-βalanine to provide a mixture of predominantly 24, plus 24a and 6. Treatment with 95% TFA afforded drug-linker 6 in 60% yield over the four steps. The synthesis of triazole-linked drug-linker 7 is shown in Scheme 3. Alkyne 25 was obtained in 95% yield using Sonogashira on 19 under microwave conditions at 100 °C, and

subsequent TMS cleavage with K2CO3 gave click substrate 26 in 78% yield. Copper-catalyzed click reaction of 26 with 11azido-3,6,9-trioxaundecan-1-amine provided triazole 27. Amide coupling of 27 with 6-maleimidohexanoic acid N-hydroxysuccinimide ester was slow and gave precursor 28 in 45% yield. Cleavage of Boc and THP protecting groups with 95% TFA furnished 7 in 53% yield. Buchwald chemistry41 was chosen for installation of the piperazine in drug-linker 8; a solution of 19 in the presence of base, Boc-protected piperazine, and RuPhos/RuPhos Pd G1 methyl tert-butyl ether adduct was heated at 85 °C in a sealed tube to give 29 in 34% yield (Scheme 4). The formation of unwanted dehalogenated 19 was minimized by using cesium carbonate as base rather than sodium tert-butoxide. Simultaneous THP cleavage and triple Boc deprotection with 95% TFA gave 30, which was carried through without purification. Finally, EDCI coupling of 1-maleimido-3-oxo-7,10,13,16tetraoxa-4-azanonadecan-19-oic acid with 30 gave 8 in 32% yield. Antibody Conjugation. The maleimide PBD drug-linkers 6, 7, and 8 were conjugated to mutated anti-HER2 and antiCD22 antibodies at position HC-A118C. Site specific cysteine engineered antibodies were chosen which contain engineered cysteines to give a theoretical drug/antibody ratio (DAR) of 2 for each ADC.42 Average DAR, aggregation analysis, and percentage remaining free drug for the six ADCs are shown in Table 1. In summary, the maleimides were reacted in 6- to 8fold molar excess with deblocked, reoxidized, cysteine engineered antibodies. The crude ADCs were then purified by cation exchange chromatography and characterized by UV spectroscopy to determine protein concentration. Analytical size-exclusion chromatography (SEC) was used for aggregation analysis and LC−MS determined DAR. In Vitro Cytotoxicity. The anti-HER2 ADCs were evaluated for their in vitro cytotoxicity in three breast cancer cell lines with differing levels of HER2 expression based on immunohistochemistry: MCF7 (HER2 0), KPL-4 (HER2 3+), and SK-BR-3 (HER2 3+). As anticipated, the anti-HER2 ADCs displayed no activity in the HER2 0 MCF7 negative28,43 control 9495

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Table 1. Analysis of ADCs

Table 2. In Vitro Cytotoxicity Data in HER2 Models a

PBD

mAb

DAR

% monomer

6

anti-HER2b anti-CD22c anti-HER2b anti-CD22c anti-HER2b anti-CD22c

1.9 1.7 1.8 1.8 1.8 1.8

95.6 98.6 95.6 95.0 95.6 94.2

7 8

d

% free drug

e

IC50 (ng/mL)

10000

11.14 22.90 15.00 >10000

a

Thio Hu anti-HER2 4D5 HC-A118C. bThio Hu anti-CD22 10F4v3 HC-A118C. cHER2 0. dHER2 3+.

a

DAR = drug/antibody ratio determined by LC−MS. bThio Hu antiHER2 4D5 HC-A118C. cThio Hu anti-CD22 10F4v3 HC-A118C. d Determined by SEC. eDetermined by LC−MS.

in BJAB cells and 1.22 and 1.73 μg/mL, respectively, in WSUDLCL2 cells (Figure 6a,b). Anti-CD22 7 and the control antiHER2 ADCs did not show any activity in the CD22 3+ lines. In Vivo Efficacy. Investigation of the in vivo single-dose efficacy for the three noncleavable PBDs was performed in the Founder 5 (Fo5) mammary tumor transplant model (Figure 7a−c, Table 4) for the anti-HER2 conjugates and in the WSUDLCL2 lymphoma xenograft model (Figure 7d, Table 4) for the anti-CD22 ADCs. The anti-CD22 ADCs acted as controls in the Fo5 experiments, and similarly the anti-HER2 compounds were used as negative controls for WSU-DLCL2. The three anti-HER2 ADCs showed good efficacy in the Fo5 model which was dose-responsive. Anti-HER2 6 and 8 were tested at 0.3, 1, and 3 mg/kg, and the doses to give tumor stasis were 0.3−1 mg/kg and 1 mg/kg, respectively (Figure 7a,b). In

cell line (Figure 5c, Table 2). 8 and 6 both showed good potency in HER2 3+ KPL-4 with IC50 values of 28 ng/mL and 48 ng/mL, respectively; 7 was inactive in KPL-4 (Figure 5b). All three anti-HER2 ADCs showed potent activity in HER2 3+ SK-BR-3 line with IC50 values of 11, 15, and 23 ng/mL for 6, 8, and 7 (Figure 5a), respectively. The control anti-CD22 ADC for 8 was inactive across all lines (Figure 5a−c). Similarly, the anti-CD22 ADCs were tested in two CD22 3+ lymphatic lines (BJAB and WSU-DLCL2) and one CD22 0 hematological line (Jurkat). The anti-CD22 ADCs and antiHER2 controls were inactive at low doses in the CD22 0 line Jurkat, which was as expected (Figure 6c, Table 3). Anti-CD22 6 and 8 gave IC50 values of 0.10 and 0.18 μg/mL, respectively,

Figure 5. In vitro cytotoxicity data for the PBD ADCs in HER2 models (a) SK-BR-3, (b) KPL-4, and (c) MCF7. 9496

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Figure 6. In vitro cytotoxicity data for the PBD ADCs in CD22 models (a) BJAB, (b) WSU-DLCL2, and (c) Jurkat.

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CONCLUSION Noncleavable linkers in ADCs are known to change the active metabolite and thus can greatly influence the safety and efficacy profiles for this class of drugs. As the parameters that make up a successful ADC are currently not well-understood and are dependent on the antigen target and disease indication, the largest possible toolbox of linker types for a given drug class is of great value. Six examples of PBD dimer ADCs possessing noncleavable linkers were designed and successfully synthesized. Molecular modeling studies, with a 10-nucleotide long DNA duplex (5′AACCATCGTT-3′), suggest that linking the antibodies through aryl tethers would not have a detrimental effect on the ability of the released PBD cysteine catabolites to bind in the minor groove of DNA. In vitro cytotoxicity evaluation showed the anti-HER2 PBD-ADCs to have potent activity in the HER2 3+ cell line SKBR-3 (IC50 values of 11−23 ng/mL); however, a slight reduction in activity in HER2 3+ KPL-4 (IC50 28−48 ng/mL, 7 inactive) was observed. Complete abolition of cytotoxicity in the HER2 0 MCF7 line demonstrated selectivity in vitro with these ADCs for higher HER2-expressing cell lines. Similar results were obtained in the CD22 in vitro models for anti-CD22 6 and 8 with IC50 of 0.10−0.18 μg/mL in BJAB CD22 3+ cells and 1.22−1.73 μg/mL in WSU-DLCL2 CD22 3+ cells, while no activity was observed in the CD22 0 Jurkat line for anti-CD22 6, 7, and 8 at low doses. Anti-CD22 7 had poor activity against CD22 3+ expressing lines. For both HER2

Table 3. In Vitro Cytotoxicity Data in CD22 Models IC50 (μg/mL) PBD 6 7 8 6 7 8

mAb a

anti-CD22 anti-CD22a anti-CD222a anti-HER2b anti-HER2b anti-HER2b

Jurkatc

WSU-DLCL2d

BJABd

>20 >20 >20 >20 >20 >20

1.22 >20 1.73 >20 >20 >20

0.10 >20 0.18 >20 >20 >20

a

Thio Hu anti-CD22 10F4v3 HC-A118CThio. bHu anti-HER2 4D5 HC-A118C. cCD22 0. dCD22 3+.

the cases of 6 and 8, the control anti-CD22 ADCs at 3 mg/kg did show activity at a similar level to anti-HER2 6 at 0.3 mg/kg and anti-HER2 8 at 1 mg/kg. From the in vitro experiments, anti-HER2 7 was less active, so it was tested at 3 and 6 mg/kg in Fo5 and the dose to give tumor stasis was 3−6 mg/kg; however, there was more of a differential from the control ADC at 6 mg/kg (Figure 7c). Anti-CD22 6 was tested at 0.5 and 2 mg/kg in WSU-DLCL2 and showed good efficacy at the higher dose level versus the anti-HER2 control at 2 mg/kg (Figure 7d). Anti-CD22 7 did not show any activity at 5 mg/kg; similarly anti-CD22 8 did not show efficacy at 0.5 mg/kg in WSU-DLCL2. 9497

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Figure 7. In vivo efficacy data for the PBD ADCs, single dose at day 0: (a) 6, (b) 8, and (c) 7 in the Fo5 model and (d) 6, 7, and 8 in the WSUDLCL2 model.

Overall, the ADCs performed better in HER2 targeted models than CD22 in vitro and in vivo; for both targets ADCs derived from alkyne 6 and piperazine 8 were more potent than triazole 7. Given the modeling suggests steric factors would be similar between the drug-linkers, we wondered whether polarity may explain the difference in activity for 7. The calculated (MarvinSketch 15.1.12.0) log D values at pH 7.4 of the three drug-linkers were 3.49 (7), 1.72 (6), and 1.48 (8), which suggests 7 would penetrate cell membranes better than 6 and 8 and thus be more potent; however, the reverse was observed with 6 and 8 being the most active. The multifactorial nature of ADC constructs (e.g., linker type, polarity, warhead potency, internalization potential, ability to reach drug target, etc.) make it difficult to discriminate in terms of biological activity between three drug-linker molecules which broadly come in the same class. Structure−activity relationships of PBDs5 have determined how to fine-tune PBD warhead activity, but the effects of the linking group from the aryl tether of a PBD-ADC on biological activity are not clearly understood at the present time. From a chemistry perspective, the syntheses of 7 and 8 were facile from the key iodoaryl intermediate 19. The synthesis of 6 was the most challenging, and although the methodology was not ideal (unwanted partial secondary N-Boc cleavage), the recovery of material for antibody conjugation was respectable. Iodoaryl intermediates of type 19 are versatile and could be

Table 4. In Vivo Efficacy Summary tumor stasis dose (mg/kg) a

PBD

Fo5

6 7 8

0.3−1 3−6 1

WSU-DLCL2b 2 >5 >0.5

a

Thio Hu anti-HER2 4D5 HC-A118C. bThio Hu anti-CD22 10F4v3 HC-A118C.

and CD22 models, negative control ADCs did not display activity in the 3+ expressing cell lines. The anti-HER2 ADCs of the three compounds showed promising in vivo antitumor efficacy in the HER2-expressing Fo5 breast model. The rank order of potency from the in vitro studies was reflected in vivo with HER2 conjugates of 6 and 8 requiring similar doses to reach tumor stasis (0.3−1 mg/kg for 6 and 1 mg/kg for 8), with 7 being less active (3−6 mg/kg). In vivo activity in the Fo5 model with the control anti-CD22 ADCs was more pronounced with 6 and 8 than 7, which could simply be due to 7 being less efficacious. However, it should be noted that the targeted anti-HER2 ADC of 6 was active at about 1/10 the dose of control and in the case of 8 about 1/3 the dose of control. In the CD22 model WSU-DLCL2, only antiCD22 6 showed activity (2 mg/kg) compared to negative control HER2 (2 mg/kg) at the doses tested. 9498

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Journal of Medicinal Chemistry

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spray spectra were obtained at 20−30 V using 50% acetonitrile in water and 0.1% formic acid as a solvent. The instrument was calibrated with [Glu1]-fibrinopeptide B immediately prior to measurement, and accurate mass calculations were performed using Xcalibur 3.1 software. N-(3-(3,5-Bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11atetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)-1-(3-(2,5-dioxo-2,5-dihydro1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan15-amide (6). A solution of 95:5 v/v TFA/H2O (5 mL) was added to a crude sample of the Boc/THP-protected compound 24 (∼470 mg, 0.32 mmol) at 0 °C (ice/acetone). After stirring at 0 °C for 1 h, the reaction was deemed complete as judged by LC−MS, desired product peak at retention time 1.32 min (ES+) m/z 1070 [M + H]+•. The reaction mixture was kept cold and added dropwise to a chilled saturated aqueous solution of NaHCO3 (120 mL). The mixture was extracted with DCM (3 × 40 mL), and the combined organic layers were washed with brine (50 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to provide the crude product. Purification by flash chromatography (100% CHCl3 to 96% CHCl3/MeOH) gave 6 as an orange foam (204 mg, 0.19 mmol, 60% yield in four steps from 22): [α]21D +351° (c 0.47, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.66 (d, 2H, J = 4.4 Hz), 7.52 (s, 2H), 7.45−7.40 (m, 3H), 6.98−6.94 (m, 1H), 6.80 (s, 2H), 6.66 (s, 2H), 6.55−6.50 (m, 1H), 5.22−5.07 (m, 8H), 4.30−4.22 (m, 6H), 3.96 (s, 6H), 3.91−3.85 (m, 2H), 3.82 (t, 2H, J = 7.2 Hz), 3.76 (t, 2H, J = 5.8 Hz), 3.65−3.43 (m, 14H), 3.38 (dd, 2H, J = 5.3, 10.2 Hz), 3.16−3.08 (m, 2H), 2.94 (d, 2H, J = 15.7 Hz), 2.54−2.44 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 171.2, 170.5, 169.8, 164.6, 162.8, 150.3, 148.0, 141.6, 140.6, 137.1, 134.2, 130.2, 125.9, 123.7, 120.4, 111.7, 111.2, 109.4, 86.4, 82.2, 70.5, 70.3, 70.2, 70.1, 69.7, 67.1, 56.2, 53.8, 51.4, 39.3, 36.8, 35.5, 34.5, 34.3, 29.7; IR (CHCl3) 3311, 3082, 3004, 2868, 1706, 1653, 1623, 1601, 1507, 1453, 1433, 1375, 1245, 1094, 1003, 874, 827, 793, 695 cm−1; MS (ES+) m/z (relative intensity) 1070 ([M + H]+•, 100). HRMS (ESI): m/z calculated for C57H63N7O14 + H+ [M + H+], 1070.450 58. Found: 1070.449 83. N-(2-(2-(2-(2-(4-(3,5-bBs((((S)-7-methoxy-2-methylene-5oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (7). A solution of 95:5 v/v TFA/H2O (5 mL) was added to a sample of the Boc/THP-protected compound 28 (428 mg, 0.29 mmol) at 0 °C (ice/acetone). After stirring at 0 °C for 1 h, the reaction was deemed complete as judged by LC−MS, desired product peak at retention time 2.72 min (ES+) m/z 1055 [M + H]+•. The reaction mixture was kept cold and added dropwise to a chilled saturated aqueous solution of NaHCO3 (100 mL). The mixture was extracted with DCM (3 × 30 mL), and the combined organic layers were washed with H2O (20 mL), brine (40 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to provide the crude product. Purification by flash chromatography (100% CHCl3 to 96% CHCl3/MeOH) gave 7 as an orange foam (163 mg, 0.15 mmol, 53% yield): [α]21D +441° (c 0.15, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.00 (s, 1H), 7.88 (d, 2H, J = 1.1 Hz), 7.65 (d, 2H, J = 4.4 Hz), 7.52 (s, 2H), 7.50 (br s, 1H), 6.85 (s, 2H), 6.66 (s, 2H), 6.10−6.05 (m, 1H), 5.27−5.16 (m, 8H), 4.59 (t, 2H, J = 5.0 Hz), 4.28 (s, 4H), 3.95−3.85 (m, 10H), 3.65−3.46 (m, 12H), 3.39−3.35 (m, 2H), 3.11 (dd, 2H, J = 9.1, 15.9 Hz), 2.93 (d, 2H, J = 15.8 Hz), 2.12 (t, 2H, J = 7.5 Hz), 1.66−1.53 (m, 4H), 1.32− 1.24 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ 172.7, 170.8, 164.7, 162.7, 150.5, 148.1, 147.0, 141.7, 140.6, 137.5, 134.0, 131.7, 125.8, 124.5, 121.3, 120.4, 111.7, 111.4, 109.4, 70.7, 70.6, 70.5 (x 2), 70.1, 69.8, 69.5, 56.2, 53.8, 51.4, 50.4, 39.1, 37.7, 36.3, 35.5, 28.3, 26.4, 25.1; IR (CHCl3) 3313, 3084, 2934, 2864, 1703, 1601, 1505, 1454, 1432, 1370, 1246, 1127, 1095, 1052, 1001, 875, 827, 793, 695 cm−1; MS (ES+) m/z (relative intensity) 1055 ([M + H]+•, 60), 527 (100). HRMS (ESI): m/z calculated for C56H63N9O12 + H+ [M + H+], 1054.466 89. Found: 1054.466 19. N-(15-(4-(3,5-Bis((((S)-7-methoxy-2-methylene-5-oxo2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8yl)oxy)methyl)phenyl)piperazin-1-yl)-15-oxo-3,6,9,12tetraoxapentadecyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-

useful for the introduction of antibody linking groups using other palladium chemistries. The data presented here support the molecular modeling hypothesis that noncleavable linking of an ADC through the aryl tether of a PBD dimer does not interfere with PBD-DNA binding and biological activity. Mode of action studies on this class of noncleavable PBD-ADCs are underway, and the results will be published elsewhere. Two PBD drug-linker molecules containing dipeptide cleavable linkers are currently being evaluated in the clinical setting.5 The work described here suggests that noncleavable PBD dimer ADCs show potential and warrant further evaluation for PBD-ADC toolbox inclusion.

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EXPERIMENTAL SECTION

Molecular Modeling. Modeling was based on the solution structure of a PBD monomer covalently bound to a 10mer B-DNA duplex (PDB entry 2KTT33). The DNA sequence was mutated to 5′AACCATCGTT-3′ to introduce two dG nucleotides at an appropriate configuration for alkylation with the PBD dimers. The C11(S) geometry was adopted for all PBD molecules studied, as this stereochemistry is known to lead to energetically favored adducts with the exocyclic C2-NH2 group of an embedded guanine in the context of a B-DNA duplex. The length of the covalent bond between the C11 of the PBD and the exocylic amino N atom of deoxyguanine was constrained to a value between 1.47 and 1.49 Å, consistent with the observations from PBD structure 2KTT. Local minimizations of the complex including the above constraints were performed in MOE (version 2015, Chemical Computing Group) using the default parameters. The models were visually examined, and manual adjustments were made as needed in MOE to correct some artifacts of the local minimization. Synthesis of Compounds. Fine chemicals and reaction solvents were purchased from Sigma-Aldrich, and chromatography solvents were bought from Fisher Scientific. MAL-dPEG4-CO2H (1-maleimido3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid) and t-Boc-Namido-dPEG4-acid (20, 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5azaicosan-20-oic acid) were obtained from Quanta BioDesign, Ltd. Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. IR spectra were recorded on a Bruker Alpha Platinum-ATR. 1H and 13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ = 0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dd (doublet of doublets), or m (multiplet), with coupling constants given in hertz (Hz). TLC was performed on silica gel aluminum plates (Merck 60, F254), and flash chromatography utilized silica gel (Merck 60, 230−400 mesh). The LC−MS conditions for compounds in Schemes 1, 3, and 4 were as follows: Waters Alliance 2695 HPLC, Waters 2996 PDA, and Waters Micromass ZQ in electrospray mode using a mobile phase of water (A, 0.1% formic acid) and acetonitrile (B, 0.1% formic acid) with a Phenomenex Onyx Monolithic C18 50 mm × 4.60 mm column at 50 °C. Initial composition was 5% B for 1.0 min, then gradient from 5% B to 95% B over 3 min, held for 30 s at 95% B, then returned to 5% B in 18 s and held for 12 s; the total method run time was 5 min at a flow rate of 3.0 mL/min. Detection range was between 220 and 400 nm. The LC−MS conditions for compounds in Scheme 2 and Figure 3 were as follows: Shimadzu Nexera/Prominence LC−MS 2020 in electrospray mode using a mobile phase of water (A, 0.1% formic acid) and acetonitrile (B, 0.1% formic acid) with a Waters Acquity UPLC BEH Shield RP18 1.7 μm, 2.1 mm × 50 mm column at 50 °C. Initial composition was 5% B for 25 s, then gradient from 5% B to 100% B over 1 min 35 s, held for 50 s at 100% B, then returned to 5% B in 5 s and held for 5 s; the total method run time was 3.0 min at a flow rate of 0.8 mL/min. Detection was at 214 and 254 nm. Accurate mass measurements were performed using a Q-Exactive Orbitrap (ThermoFisher Scientific) with mass spectrometry performed on a ThermoQuest Navigator (Thermo Electron). Electro9499

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

Journal of Medicinal Chemistry

Article

propanamide (8). EDCI (1.1 equiv, 35 mg, 0.18 mmol) was added to a solution of compound 30 (1.0 equiv, 116 mg, 0.165 mmol) and MAL-dPEG4-acid (1.0 equiv, 69 mg, 0.165 mmol) in anhydrous DCM (5 mL) under an argon atmosphere. The resultant solution was stirred at room temperature for 2 h where analysis by LC−MS showed formation of desired product at retention time 2.65 min (ES+) m/z 1101 [M + H]+•. The reaction mixture was diluted with DCM (50 mL), washed with water (100 mL), saturated aqueous NaHCO3 solution (100 mL), water (100 mL), brine (100 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to give the crude product. Purification by flash chromatography (100% CHCl3 to 95% CHCl3/MeOH) gave the product 8 as a yellow glass (58 mg, 0.053 mmol, 32% yield): [α]18D +628° (c 0.25, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.66 (d, 2H, J = 4.4 Hz), 7.52 (s, 2H), 6.99 (s, 1H), 6.96−6.91 (m, 2H), 6.83 (s, 2H), 6.68 (s, 2H), 6.39−6.32 (m, 1H), 5.22−5.08 (m, 8H), 4.28 (s, 4H), 3.96 (s, 6H), 3.89−3.72 (m, 8H), 3.69−3.55 (m, 14H), 3.52 (t, 2H, J = 5.0 Hz), 3.43−3.37 (m, 2H), 3.28−3.08 (m, 6H), 2.94 (d, 2H, J = 15.8 Hz), 2.67 (t, 2H, J = 6.7 Hz), 2.50 (t, 2H, J = 7.3 Hz); 13C NMR (CDCl3, 100 MHz) δ 170.5, 169.9, 169.5, 164.7, 162.6, 151.6, 150.6, 148.0, 141.6, 140.6, 137.8, 134.2, 120.2, 117.9, 114.8, 111.6, 111.3, 109.4, 71.0, 70.4, 70.1, 69.8, 67.4, 56.2, 53.8, 51.4, 49.3, 49.0, 45.5, 41.4, 39.2, 35.5, 34.5, 34.4, 33.5; IR (CHCl3) 3312, 3083, 3003, 2868, 1705, 1624, 1600, 1506, 1433, 1377, 1244, 1094, 995, 874, 827, 793, 695 cm−1; MS (ES+) m/z (relative intensity) 1101 ([M + H]+•, 35), 551 (100). HRMS (ESI): m/z calculated for C58H68N8O14 + H+ [M + H+], 1101.492 78. Found: 1101.492 49. N-[4-Benzyloxy-5-methoxy-2-nitrobenzoyl]-(2S,4R)-[4hydroxypyrrolidine-2-carboxylate] (10). A catalytic amount of dry DMF (2 drops) was added to a solution of acid 9 (1.0 equiv, 5 g, 16.4 mmol) in dry DCM (70 mL) and oxalyl chloride (1.1 equiv, 1.6 mL, 2.3 g, 18.1 mmol), and then the reaction mixture was stirred overnight under a nitrogen atmosphere. The resulting solution was added dropwise to a solution of the methyl (2S,4R)-4-hydroxypyrrolidine-2carboxylate·HCl (1.1 equiv, 5.74 g, 18.1 mmol) and TEA (3.0 equiv, 6.84 mL, 4.96 g, 49.2 mmol) in dry DCM (100 mL) at −20 °C (ethylene glycol/dry ice). The reaction mixture was allowed to warm to room temperature and stirred overnight at which point TLC (EtOAc) revealed that the reaction was complete. The mixture was washed with 1 N HCl (50 mL), water (50 mL), saturated aqueous NaHCO3 (50 mL), brine (50 mL), dried (MgSO4), and filtered. The solvents were removed by rotary evaporation under reduced pressure to leave a yellow solid which was triturated with cold EtOAc to yield pure ester 10 (4.84 g, 11.2 mmol, 68% yield): [α]28D −57° (c 0.20, CHCl3); 1H NMR (CDCl3, 400 MHz) (rotamers) δ 7.69 (s, 1H), 7.46−7.32 (m, 5H), 6.85 (s, 1H), 6.80 (s, 1H), 5.20 (s, 2H), 4.83 (t, 1H, J = 8.0 Hz), 4.59−4.52 (m, 2H), 4.49−4.42 (m, 2H), 4.20−4.09 (m, 4H), 3.97 (s, 3H), 3.93 (s, 3H), 3.80 (s, 3H), 3.75−3.71 (m, 3H), 3.53−3.49 (m, 3H), 3.47 (s, 3H), 3.18−3.10 (m, 3H), 2.45−2.10 (m, 1H), 1.38−1.22 (m, 1H); 13C NMR (CDCl3, 100 MHz) (rotamers) δ 172.5, 172.4, 167.0, 166.7, 154.9, 154.5, 148.3, 148.2, 137.3, 135.3, 135.2, 128.8, 128.5, 127.6, 127.3, 126.5, 109.7, 109.1, 71.4, 70.0, 69.2, 59.0, 57.2, 56.8, 56.6, 56.3, 54.6, 52.5, 52.3, 39.4, 38.0; IR (neat) 3433, 2950, 1742, 1626, 1577, 1521, 1454, 1432, 1336, 1277, 1213, 1073, 750 cm−1; MS (ES+) m/z (relative intensity) 431 ([M + H]+•, 100). N-[4-Benzyloxy-5-methoxy-2-nitrobenzoyl]-(2S,4R)-[4-hydroxy-2-(hydroxymethyl)pyrrolidine] (11). A solution of the ester 10 (1.0 equiv, 3.8 g, 8.83 mmol) in THF (100 mL) was cooled to 0 °C and treated with LiBH4 (1.5 equiv, 0.29 g, 13.25 mmol) in portions. After stirring for 30 min at 0 °C, the reaction mixture was allowed to warm to room temperature and stirred under a nitrogen atmosphere for 4 h at which time TLC (EtOAc) revealed complete consumption of ester 10. The mixture was cooled to 0 °C again, and water (100 mL) was carefully added followed by 1 N HCl (250 mL) which provoked vigorous effervescence. After evaporation of the THF by rotary evaporation under reduced pressure, the residue was neutralized to pH 7 with 1 N NaOH. The aqueous solution was then extracted with EtOAc (4 × 70 mL), and the combined organic layers were washed with brine (50 mL), dried (MgSO4), filtered, and evaporated by rotary evaporation under reduced pressure to furnish the pure diol

11 (3.2 g, 7.96 mmol, 90% yield), which was used in the subsequent reaction without further purification: [α]29D −96° (c 0.15, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.74 (s, 1H), 7.50−7.29 (m, 5H), 6.86 (s, 1H), 5.20 (m, 2H), 4.61−4.46 (m, 2H), 4.38−4.30 (m, 1H), 4.04− 3.85 (m, 5H), 3.82−3.70 (m, 1H), 3.20−3.11 (m, 1H), 3.39−3.25 (m, 1H), 2.28−2.09 (m, 1H), 1.99−1.85 (m, 1H); 13C NMR (CDCl3, 100 MHz) δ 155, 148.2, 137.2, 135.2, 128.8, 128.5, 127.7, 127.6, 109.3, 109.1, 71.4, 69.4, 65.0, 59.9, 56.9, 56.8, 37.1; IR (neat) 3357, 2938, 1614, 1576, 1520, 1454, 1434, 1333, 1276, 1220, 1069, 1048, 1006, 730, 698, 647 cm−1; MS (ES+) m/z (relative intensity) 403 ([M + H]+•, 100). N-[2-Amino-4-benzyloxy-5-methoxybenzoyl]-(2S,4R)-[4-hydroxy-2-(hydroxymethyl)pyrrolidine] (12). A mixture of diol 11 (1.0 equiv, 26 g, 64.6 mmol) and SnCl2·2H2O (5.0 equiv, 72.9 g, 323 mmol) in MeOH (300 mL) was heated at reflux and the progress of the reaction monitored by TLC (EtOAc). After 3 h, the MeOH was evaporated by rotary evaporation under reduced pressure and the resulting residue was cooled and treated carefully with saturated NaHCO3 (400 mL). The mixture was diluted with EtOAc (800 mL), and after 12 h stirring at room temperature the inorganic precipitate was removed by filtration through Celite. The organic layer was separated, washed with brine (100 mL), dried (MgSO4), filtered, and evaporated by rotary evaporation under reduced pressure to give a brown solid 12 which was used in the next reaction without further purification: [α]29D −69° (c 0.13, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.36−7.29 (m, 7H), 6.78 (s, 1H), 6.26 (s, 1H), 5.10 (s, 2H), 4.60−4.47 (m, 1H), 4.72−4.26 (m, 1H), 3.85−3.52 (m, 9H), 2.19− 2.06 (m, 1H), 1.82−1.70 (m, 1H); 13C NMR (CDCl3, 100 MHz) δ 172.8, 151.0, 141.9, 140.0, 136.5, 128.6, 128.0, 127.1, 112.9, 112.4, 103.4, 70.7, 69.4, 37.0, 66.2, 59.0, 58.5, 57.1; IR (neat) 3358, 2939, 1619, 1589, 1512, 1455, 1432, 1408, 1263, 1231, 1171, 1113, 1012, 787, 735, 698, 644 cm−1; MS (ES+) m/z (relative intensity) 373 ([M + H]+•, 70), 256 (100). N-[4-Benzyloxy-5-methoxy-2-(tert-butyloxycarbonylamino)benzoyl]-(2S,4R)-[4-hydroxy-2-(hydroxymethyl)pyrrolidine] (13). A solution of aniline 12 (1.0 equiv, 24 g, 64.6 mmol) and (Boc)2O (1.0 equiv, 14.1 g, 64.6 mmol) in THF (300 mL) was heated at reflux overnight. The reaction mixture was allowed to cool to room temperature and the THF was removed by rotary evaporation under reduced pressure to give the crude product. The residue was subjected to flash column chromatography (30% EtOAc/hexane) to afford the product 13 (18.3 g, 38.8 mmol, 60% yield) as a yellow oil: [α]25D −46° (c 0.15, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.72 (s, 1H), 7.49− 7.26 (m, 6H), 6.79 (s, 1H), 5.15 (d, 2H, J = 4.54 Hz), 4.43−4.26 (m, 1H), 3.94−3.77 (m, 4H), 3.76−3.54 (m, 3H), 2.78 (bs, 1H), 2.24− 2.15 (m, 1H), 1.84−1.71 (m, 1H), 1.46 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 153.6, 150.1, 145.1, 136.2, 130.4, 128.6, 128.1, 127.6, 118.8, 111.5, 108.1, 80.8, 70.8, 69.7, 59.4, 56.7, 37.2, 28.3; IR (neat) 3358, 2971, 1717, 1597, 1519, 1454, 1432, 1404, 1367, 1241, 1157, 1118, 1016, 773, 698 cm−1; MS (ES+) m/z (relative intensity) 473 ([M + H]+•, 100). (11S,11aS)-8-Benzyloxy-10-(tert-butyloxycarbonyl)-11-hydroxy-7-methoxy-2-oxo-1,2,3,10,11,11a-hexahydro-5Hpyrrolo[2,1-c][1,4]benzodiazepine-5-one (14). BAIB (5.0 equiv, 17.0 g, 53.0 mmol) and TEMPO (0.1 equiv, 0.17 g, 1.05 mmol) were added to a solution of Boc protected aniline 13 (1.0 equiv, 5.0 g, 10.6 mmol) in DCM (50 mL), and the mixture was allowed to stir overnight. When the reaction was complete as indicated by TLC (50% EtOAc/hexane), the reaction mixture was diluted with DCM (100 mL) and washed with saturated Na2S2O3 (60 mL). The aqueous layer was extracted with DCM (2 × 50 mL), and the combined organic layers were washed with brine (50 mL) and dried (MgSO4). Filtration, followed by removal of solvent by rotary evaporation under reduced pressure, afforded a crude solid which was washed with cold EtOAc to give cyclized compound 14 (2.3 g, 4.9 mmol, 46% yield) as a white solid: [α]25D +117° (c 0.14, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.45−7.33 (m, 5H), 7.23 (s, 1H), 6.68 (s, 1H), 5.69−5.55 (m, 1H), 5.26−5.03 (m, 2H), 4.46−4.22 (m, 1H), 4.01−3.75 (m, 6H), 3.02− 2.90 (m, 1H), 2.73−2.68 (m, 1H), 1.28 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 207.9, 167.7, 150.5, 149.0, 136.2, 130.3, 129.1, 128.8, 9500

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

Journal of Medicinal Chemistry

Article

128.6, 128.2, 127.0, 123.9, 114.6, 110.7, 86.0, 71.1, 56.6, 56.2, 52.6, 40.3, 28.0; IR (neat) 3389, 2978, 1762, 1700, 1637, 1603, 1511, 1456, 1431, 1368, 1329, 1256, 1221, 1157, 1118, 1059, 767 cm−1; MS (ES+) m/z (relative intensity) 469 ([M + H]+•, 27), 413 (100). (11S,11aS)-10-(tert-Butyloxycarbonyl)-8,11-dihydroxy-7-methoxy-2-oxo-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (15). A catalytic amount of 10% w/w palladium on carbon (0.23 g) was added to a solution of cyclized compound 14 (2.3 g, 4.9 mmol) in absolute alcohol (50 mL). The reaction mixture was hydrogenated for 4 h at 30 psi. When the reaction was complete as indicated by TLC (50% EtOAc/hexane), the reaction mixture was filtered through Celite, and removal of solvent under reduced pressure afforded the phenol 15 (4.70 g, 2.64 mmol, 53% yield) as a white solid: [α]25D +115° (c 0.10, MeOH); 1H NMR (CDCl3, 400 MHz) δ 7.20 (s, 1H), 6.73 (s, 1H), 6.02 (s, 1H), 5.71− 5.53 (m, 1H), 4.35−4.22 (m, 1H), 4.06−3.84 (m, 6H), 3.02−2.87 (m, 1H), 2.80−2.68 (m, 1H), 1.39 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 208.1, 167.8, 148.2, 145.9, 129.9, 123.1, 115.9, 110.1, 85.9, 56.7, 56.2, 52.6, 40.4, 28.1; IR (neat) 3355, 2977, 1760, 1685, 1606, 1515, 1469, 1415, 1369, 1332, 1297, 1212, 1162, 1132, 1042, 767 cm−1; MS (ES+) m/z (relative intensity) 379 ([M + H]+•, 93), 364 (100). (11S,11aS)-10-(tert-Butyloxycarbonyl)-7-methoxy-2-oxo8,11-di(tetrahydroxypyran-2-yloxy)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (16). A catalytic amount of PTSA was added to a solution of DHP (10.0 equiv, 4.8 mL, 52.8 mmol) in EtOAc (10 mL) at 0 °C. After stirring for 10 min, the phenolic compound 15 (1.0 equiv, 2.0 g, 5.28 mmol) was added portionwise to the mixture and stirred until the disappearance of starting material was observed by TLC (50% EtOAc/hexane). The mixture was diluted with EtOAc (100 mL), washed with saturated NaHCO3 (30 mL), brine (30 mL), and dried (MgSO4). Filtration, followed by removal of solvent under reduced pressure, afforded a crude solid which was subjected to flash column chromatography (30% EtOAc−hexane) to give the protected compound 16 (1.8 g, 3.4 mmol, 65% yield, mixture of diastereoisomers from THP): [α]24D +110° (c 0.10, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.22−7.15 (3s, 3H), 6.90−6.86 (2s, 1H), 6.01−5.90 (m, 1H), 5.86−5.77 (m, 1H), 5.50−5.43 (m, 1H), 5.41−5.26 (m, 1H), 5.20−5.12 (m, 1H), 5.06− 4.96 (m, 1H), 4.44−4.23 (m, 2H), 4.05−3.82 (m, 14H), 3.66−3.51 (m, 4H), 3.00−2.82 (m, 3H), 2.65−2.55 (m, 1H), 2.14−1.83 (m, 6H), 1.81−1.42 (m, 18H), 1.34 (s, 18H); 13C NMR (CDCl3, 100 MHz) δ 208.5, 208.3, 168.2, 168.1, 148.7, 148.6, 129.6, 129.5, 125.9, 118.2, 110.9, 110.5, 100.4, 96.6, 91.2, 88.3, 81.5, 64.7, 63.9, 63.6, 62.1, 62.0, 61.6, 57.0, 56.9, 56.2, 52.8, 52.7, 40.8, 40.4, 31.2, 31.1, 30.9, 30.6, 30.1, 30.0, 28.2, 28.1, 28.0, 27.9, 25.2 (×2), 25.1, 20.6, 20.2, 20.0, 18.8, 18.6, 18.2; IR (neat) 2941, 1762, 1702, 1649, 1604, 1508, 1454, 1429, 1393, 1367, 1324, 1256, 1197, 1162, 1115, 1072, 1021, 960, 904, 870, 731 cm−1; MS (ES+) m/z (relative intensity) 547 ([M + H]+•, 56), 261 (100). (11S,11aS)-10-(tert-Butyloxycarbonyl)-7-methoxy-2-methylidene-8,11-di(tetrahydroxypyran-2-yloxy)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (17). Potassium tert-butoxide (10.0 equiv, 4.1 g, 36 mmol) was added portionwise to a suspension of methyltriphenylphosphonium bromide (10.0 equiv, 12.8 g, 36 mmol) in THF (50 mL) at 0 °C, under a nitrogen atmosphere. After stirring for 2 h at 0 °C, a solution of the ketone 16 (1.0 equiv, 2.0 g, 3.6 mmol) was added dropwise and the mixture allowed to warm to room temperature. After stirring overnight, the reaction mixture was diluted with EtOAc (250 mL) and water (250 mL) and the organic layer separated, washed with brine, dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to give a dark red oil, which was shown to contain a mixture of several components by TLC (50% EtOAc/hexane). Purification by flash chromatography (30% EtOAc/ hexane) isolated the pure olefin 17 as a white solid (1.4 g, 2.59 mmol, 72% yield, mixture of diastereoisomers from THP): [α]22D +105° (c 0.20, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.20−7.10 (3s, 3H), 6.90−6.77 (2s, 1H), 5.87−5.75 (m, 1H), 5.71−5.62 (m, 1H), 5.48− 5.22 (m, 2H), 5.19−4.96 (m, 6H), 4.38−4.23 (m, 2H), 1.32 (s, 18H), 4.18−4.04 (m, 2H), 4.03−3.82 (m, 10H), 3.72−3.51 (m, 6H), 2.95−

2.69 (m, 3H), 2.59−2.50 (m, 1H), 2.14−1.83 (m, 6H), 1.81−1.40 (m, 18H); 13C NMR (CDCl3, 100 MHz) δ 168.2, 168.1, 155.2, 149.4, 148.8, 148.1, 147.9, 142.3, 129.8, 129.7, 129.6, 127.3, 120.3, 118.1, 110.7, 110.3, 109.5, 109.4, 100.3, 99.6, 98.4, 96.7, 96.5, 96.3, 96.0, 90.9, 88.0, 81.2, 81.1, 64.5, 63.6, 63.4, 62.1, 62.0, 61.6, 60.0, 59.8, 56.2, 50.7, 35.5, 35.1, 31.3, 31.2, 31.0, 30.7, 30.4, 30.2, 30.1, 28.2, 28.0, 27.9, 25.3, 25.2, 25.1, 20.6, 20.0, 19.9, 18.8, 18.7, 18.3; IR (neat) 2940, 2866, 1702, 1639, 1604, 1508, 1453, 1431, 1393, 1367, 1324, 1198, 1162, 1114, 1072, 1019, 960, 906, 870, 727, 644 cm−1; MS (ES+) m/z (relative intensity) 545 ([M + H]+•, 38), 343 (100). (11S,11aS)-10-(tert-Butyloxycarbonyl)-8-hydroxy-7-methoxy-2-methylidene-11-(tetrahydroxypyran-2-yloxy)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (18). A solution of THF/AcOH/H2O (2:1:1, 5 mL) was added to the olefin 17 (1.8 g, 3.3 mmol), and the resulting mixture was stirred for 3 h at which point TLC (50% EtOAc/hexane) revealed the reaction was complete. The mixture was then neutralized with saturated NaHCO3 and extracted with EtOAc (3 × 30 mL), and the combined organic layers were washed with brine (50 mL) and dried (MgSO4). Removal of solvent by rotary evaporation under reduced pressure gave the crude product. The residue was subjected to flash chromatography (50% EtOAc/hexane) to afford the product 18 as a white solid (1.34 g, 2.90 mmol, 87% yield, mixture of diastereoisomers from THP): [α]27D +81° (c 0.16, CHCl3); 1 H NMR (CDCl3, 400 MHz) δ 7.21 (s, 1H), 7.17 (s, 1H), 6.92 (s, 1H), 6.63 (s, 1H), 6.00 (s, 1H), 5.97 (s, 1H), 5.88−5.75 (d, 1H), 5.72−5.65 (d, 1H), 5.19−4.96 (m, 6H), 4.38−4.23 (m, 2H), 4.18− 4.04 (m, 2H), 4.00−3.87 (m, 8H), 3.67−3.51 (m, 4H), 2.96−2.84 (m, 2H), 2.80−2.68 (m, 1H), 2.58−2.50 (m, 1H), 1.84−1.66 (m, 4H), 1.64−1.45 (m, 8H), 1.32 (s, 18H); 13C NMR (CDCl3, 100 MHz) δ 167.5, 167.3, 147.7, 147.5, 146.0, 145.8, 142.0, 130.5, 130.3, 117.0, 116.4, 109.8, 109.4, 100.7, 96.1, 91.0, 88.0, 81.5, 64.6, 63.6, 60.0, 59.8, 56.2, 56.1, 50.7, 35.4, 35.1, 31.3, 30.8, 29.7, 28.1, 28.0, 25.2, 25.1, 20.7, 20.0; IR (neat) 2940, 2851, 1703, 1630, 1512, 1467, 1440, 1407, 1391, 1367, 1326, 1200, 1161, 1118, 1072, 1019, 910, 730 cm−1; MS (ES+) m/z (relative intensity) 461 ([M + H]+•, 75), 259 (100). (11S,11aS,11′S,11a′S)-Di-tert-butyl 8,8′-(((5-Iodo-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) (19). 1,3-Bis(bromomethyl)-5-iodobenzene (1 equiv, 2.00 g, 5.20 mmol) was added to a stirred solution of Boc/THP-protected PBD capping unit 18 (2 equiv, 4.75 g, 10.3 mmol), TBAI (0.1 equiv, 190 mg, 0.52 mmol), and K2CO3 (2 equiv, 1.42 g, 10.3 mmol) in dry DMF (60 mL). The reaction mixture was heated to 60 °C and stirred under an argon atmosphere for 3 h at which point analysis by LC−MS revealed substantial product formation at retention time 4.15 min (ES+) m/z 1171 [M + Na]+•. The reaction mixture was allowed to cool to room temperature, and the DMF was removed by rotary evaporation under reduced pressure. The resulting residue was partitioned between water (50 mL) and EtOAc (50 mL), and the aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with water (2 × 20 mL), brine (50 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to provide the crude product. Purification by flash chromatography (50% EtOAc/hexane to 80% EtOAc/hexane) gave the bis-ether 19 as a white foam (5.42 g, 4.72 mmol, 91% yield, mixture of diastereoisomers from THP): [α]21D +44° (c 0.29, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.76 (s, 2H), 7.51 (br s, 1H), 7.25 (s, 1H), 7.21 (s, 1H), 6.87 (s, 1H), 6.53 (s, 1H), 5.81−5.78 (m, 1H), 5.68 (d, 1H, J = 9.3 Hz), 5.13−4.97 (m, 10H), 4.34−4.27 (m, 2H), 4.15−4.08 (m, 2H), 3.93−3.88 (m, 8H), 3.62− 3.57 (m, 3H), 3.51−3.47 (m, 1H), 2.94−2.86 (m, 2H), 2.74 (d, 1H, J = 16.0 Hz), 2.54 (d, 1H, J = 16.1 Hz), 1.77−1.21 (m, 30H); 13C NMR (CDCl3, 100 MHz) δ 167.3, 167.1, 154.8, 149.7, 149.5, 149.3, 148.9, 142.1, 139.6, 139.4, 135.7, 135.6, 129.8, 129.6, 127.4, 126.8, 125.0, 116.0, 115.8, 110.9, 110.3, 109.6, 109.5, 100.8, 96.1, 94.8, 91.1, 88.3, 81.5, 81.0, 70.4, 70.0, 64.9, 63.5, 60.0, 59.9, 56.2, 56.1, 50.7, 35.5, 35.2, 31.4, 31.0, 28.2, 28.1, 25.3, 20.9, 20.1; IR (CHCl3) 2942, 2864, 1702, 1638, 1604, 1510, 1455, 1431, 1398, 1367, 1326, 1274, 1246, 1202, 9501

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

Journal of Medicinal Chemistry

Article

1161, 1118, 1072, 1014, 965, 904, 858, 639 cm−1; MS (ES+) m/z (relative intensity) 1171 ([M + Na]+•, 10), 947 (100). tert-Butyl (15-Oxo-3,6,9,12-tetraoxa-16-azanonadec-18-yn1-yl)carbamate (21). EDCI (1 equiv, 263 mg, 1.37 mmol) was added to a stirred solution of t-Boc-N-amido-dPEG4-acid (20) (1 equiv, 500 mg, 1.37 mmol, Stratech Scientific Limited) and propargylamine (1 equiv, 88 μL, 76 mg, 1.37 mmol) in dry DCM (10 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 16 h at which point analysis by LC−MS showed a substantial amount of desired product at retention time 1.26 min (ES+) m/z 403 [M + H]+•, 425 [M + Na]+•. Note that both starting material and product had weak UV absorption (214 and 254 nm) and were best detected on ES+ TIC. The reaction mixture was diluted with DCM (100 mL) and washed with H2O (30 mL), brine (40 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to provide the crude product. Purification by flash chromatography (100% DCM to 98% DCM/MeOH) gave the amide 21 as an oil (392 mg, 0.97 mmol, 71% yield): 1H NMR (CDCl3, 400 MHz) δ 6.99−6.83 (m, 1H), 5.09−4.96 (m, 1H), 4.04 (dd, 2H, J = 2.6, 5.4 Hz), 3.74 (t, 2H, J = 5.7 Hz), 3.70− 3.59 (m, 12H), 3.54 (t, 2H, J = 5.1 Hz), 3.35−3.26 (m, 2H), 2.50 (t, 2H, J = 5.7 Hz), 2.21 (t, 1H, J = 2.5 Hz), 1.45 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 171.3, 156.0, 80.1, 79.2, 71.0, 70.6, 70.5, 70.3 (x 2), 70.2, 67.0, 40.4, 36.7, 28.9, 28.4; IR (CHCl3) 3307, 2870, 1696, 1657, 1526, 1454, 1391, 1365, 1272, 1249, 1169, 1093, 1042, 942, 862, 781, 650 cm−1; MS (ES+) m/z (relative intensity) 425 ([M + Na]+•, 30), 403 ([M + H]+•, 40), 303 (100). Di-tert-butyl 8,8′-(((5-(2,2-Dimethyl-4,20-dioxo-3,8,11,14,17pentaoxa-5,21-diazatetracos-23-yn-24-yl)-1,3-phenylene)bis(methylene))bis(oxy))(11S,11aS,11′S,11a′S)-bis(7-methoxy-2methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine10(5H)-carboxylate) (22). A catalytic amount of Pd(PPh3)4 (0.02 equiv, 23.0 mg, 19.5 μmol) was added to a mixture of the iodoaryl compound 19 (1 equiv, 1.02 g, 0.89 mmol), Boc-acetylene 21 (1.1 equiv, 393 mg, 0.98 mmol), CuI (0.04 equiv, 7.4 mg, 39.1 μmol), diethylamine (22 equiv, 2.02 mL, 1.43 g, 19.5 mmol), and oven-dried 4 Å molecular sieve pellets in dry DMF (9 mL) in an oven-dried sealable vessel. The mixture was degassed in vacuo and flushed with argon 3 times and then heated in a microwave at 100 °C for 26 min at which point analysis by LC−MS revealed substantial product formation at retention time 1.89 min (ES+) m/z 1446 [M + Na]+•, 1424 [M + H]+•. The reaction mixture was allowed to cool to room temperature and was then filtered through a sinter to remove the sieves (washed with DMF). The filtrate was subjected to rotary evaporation under reduced pressure and the resulting residue dissolved in DCM (100 mL) and washed with H2O (20 mL), brine (30 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to give the crude product. Purification by flash chromatography (100% DCM to 97% DCM/MeOH) provided the alkyne 22 as a yellow foam (882 mg, 0.62 mmol, 70% yield, mixture of diastereoisomers from THP): [α]21D +33° (c 0.24, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.54−7.49 (m, 1H), 7.47 (br s, 2H), 7.25 (s, 1H), 7.21 (s, 1H), 6.97−6.79 (m, 2H), 6.54 (br s, 1H), 5.85−5.74 (m, 1H), 5.69 (d, 1H, J = 9.2 Hz), 5.17−4.94 (m, 11H), 4.35−4.23 (m, 4H), 4.16−4.08 (m, 2H), 3.96−3.85 (m, 8H), 3.74 (t, 2H, J = 5.7 Hz), 3.68−3.56 (m, 15H), 3.55−3.44 (m, 3H), 3.34−3.24 (m, 2H), 2.96− 2.84 (m, 2H), 2.74 (d, 1H, J = 15.9 Hz), 2.59−2.49 (m, 1H, obscured by t at 2.52), 2.52 (t, 2H, J = 5.7 Hz), 1.82−1.17 (m, 39H); 13C NMR (CDCl3, 100 MHz) δ 171.2, 167.3, 167.1, 156.0, 154.9, 149.6, 149.2, 148.8, 142.1, 137.5, 137.4, 130.1, 129.8, 129.6, 127.1, 126.6, 125.8, 123.9, 115.7, 115.5, 110.8, 110.2, 109.6, 100.8, 96.0, 91.1, 88.3, 86.4, 82.1, 81.5, 81.0, 79.2, 70.6 (x 2), 70.5, 70.4, 70.3 (×2), 70.2, 67.0, 64.9, 63.5, 60.0, 59.9, 56.1 (×2), 50.7, 40.4, 36.8, 35.5, 35.2, 31.4, 30.9, 29.6, 28.4, 28.2, 28.1, 25.3, 20.8, 20.0; IR (CHCl3) 3326, 2939, 2868, 1703, 1639, 1604, 1511, 1432, 1395, 1367, 1327, 1274, 1246, 1203, 1162, 1116, 1073, 1016, 965, 904, 860, 637 cm−1; MS (ES+) m/z (relative intensity) 1446 ([M + Na]+•, 100), 1424 ([M + H]+•, 5). Di-tert-butyl 8,8′-(((5-(1-Amino-15-oxo-3,6,9,12-tetraoxa-16azanonadec-18-yn-19-yl)-1,3-phenylene)bis(methylene))bis(oxy))(11S,11aS,11′S,11a′S)-bis(7-methoxy-2-methylene-5-

oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) (23). TBSOTf (10 equiv, 1.42 mL, 1.64 g, 6.20 mmol) was added to a stirred solution of the tri-Boc protected compound 22 (1 equiv, 882 mg, 0.62 mmol) and 2,6-lutidine (13 equiv, 0.96 mL, 883 mg, 8.25 mmol) in dry DCM (15 mL) at room temperature. The reaction mixture was allowed to stir under an argon atmosphere for 16 h during which time analysis by LC−MS revealed formation of the desired TBS carbamate at retention time 2.09 min (ES+) m/z 1504 [M + Na]+•. The reaction mixture was diluted with DCM (60 mL) and washed with saturated NH4Cl (2 × 20 mL), H2O (20 mL), brine (30 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to give the crude TBS carbamate. The product was redissolved in THF (15 mL) and treated with a solution of TBAF (1.2 equiv, 744 μL of a 1.0 M solution in THF, 0.744 mmol) at room temperature. The reaction mixture was allowed to stir for 1 h at room temperature at which point analysis by LC−MS revealed substantial formation of 23 at retention time 1.45 min (ES+) m/z 1324 [M + H]+• along with 23a at retention time 1.29 min (ES+) m/z 1121 [M + H]+•, 1138 [M + H2O]+ and 23b at retention time 1.12 min (ES+) m/z 919 [M + H]+•, 937 [M + H2O]+•, 955 [M + 2H2O]+•. The THF was removed by rotary evaporation under reduced pressure and the resulting residue redissolved in DCM (60 mL) and washed with saturated NH4Cl (2 × 20 mL), H2O (20 mL), brine (30 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to give the key amine crude mixture 23, 23a, and 23b as a pinkish foam (∼800 mg). Di-tert-butyl 8,8′-(((5-(1-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazatricos-22-yn-23yl)-1,3-phenylene)bis(methylene))bis(oxy))(11S,11aS,11′S,11a′S)-bis(7-methoxy-2-methylene-5-oxo-11((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1Hpyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (24). EDCI (1 equiv, 61 mg, 0.32 mmol) was added to a stirred solution of N-maleoyl-β-alanine (1 equiv, 53 mg, 0.32 mmol) and crude amine 23 (1 equiv, ∼418 mg, 0.32 mmol) in dry DCM (6 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 h at which point analysis by LC−MS showed a substantial amount of 24 at retention time 1.80 min (ES+) m/z 1475 [M + H]+•, 1497 [M + Na]+•, along with 24a at retention time 1.56 min (ES+) m/z 1273 [M + H]+•, 1295 [M + Na]+ and 6 at retention time 1.31 min (ES+ M+• not observed). The reaction mixture was diluted with DCM (30 mL) and washed with H2O (15 mL), brine (20 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to provide the crude mixture 24, 24a, and 6 as a foam (∼470 mg). (11S,11aS,11′S,11a′S)-Di-tert-butyl 8,8′-(((5-((Trimethylsilyl)ethynyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine10(5H)-carboxylate) (25). A catalytic amount of Pd(PPh3)4 (0.02 equiv, 15.0 mg, 13.1 μmol) was added to a mixture of the bis-ether 19 (1 equiv, 750 mg, 0.65 mmol), TMS-acetylene (3 equiv, 278 μL, 191 mg, 1.96 mmol), CuI (0.04 equiv, 5.0 mg, 26.1 μmol), diethylamine (20 equiv, 1.35 mL, 956 mg, 13.1 mmol), and oven-dried 4 Å molecular sieve pellets in dry DMF (5.6 mL) in an oven-dried sealable vessel. The mixture was degassed in vacuo and flushed with argon 3 times and then heated in a microwave at 100 °C for 30 min at which point analysis by LC−MS revealed complete consumption of starting material with formation of 19 at retention time 4.37 min (ES+) m/z 1142 [M + Na]+•. Peak at retention time 3.97 min (ES+) m/z 1069 [M + Na]+• observed which corresponds to TMS cleavage under LC− MS conditions. The reaction mixture was allowed to cool to room temperature and was then filtered through a sinter to remove the sieves (washed with DMF). The filtrate was subjected to rotary evaporation under reduced pressure and the resulting residue purified by flash chromatography (50% EtOAc/hexane to 80% EtOAc/hexane) to provide the TMS-acetylene 25 as a yellow foam (691 mg, 0.62 mmol, 95% yield, mixture of diastereoisomers from THP): [α]21D +49° (c 0.23, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.55−7.46 (m, 3H), 7.24 (s, 1H), 7.20 (s, 1H), 6.88 (s, 1H), 6.53 (s, 1H), 5.80−5.78 9502

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

Journal of Medicinal Chemistry

Article

through to the next step without further purification. [α]22D +26° (c 0.31, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.11−7.83 (m, 3H), 7.57−7.53 (m, 1H), 7.25 (s, 1H), 7.21 (s, 1H), 6.94 (br s, 1H), 6.60 (br s, 1H), 5.80−5.75 (m, 1H), 5.68 (d, 1H, J = 8.1 Hz), 5.24−4.96 (m, 10H), 4.65−4.55 (m, 2H), 4.34−4.27 (m, 2H), 4.15−4.08 (m, 2H), 3.93−3.85 (m, 10H), 3.72−3.38 (m, 16H), 2.94−2.86 (m, 2H), 2.74 (d, 1H, J = 15.2 Hz), 2.54 (d, 1H, J = 15.6 Hz), 1.73−1.11 (m, 34H); 13C NMR (CDCl3, 100 MHz) δ 167.4, 167.2, 154.8, 150.0, 149.8, 149.2, 148.9, 142.1, 137.8, 132.2, 132.1, 131.9, 129.9, 129.6, 128.6, 128.4, 126.6, 125.4, 124.3, 115.8, 115.6, 110.8, 110.2, 109.5, 109.4, 100.7, 96.1, 91.1, 88.3, 81.4, 81.0, 71.2, 70.8, 70.6, 70.5, 69.7, 69.6, 69.2, 64.8, 63.6, 61.6, 60.0, 59.9, 56.2, 50.7, 50.4, 35.5, 35.2, 31.4, 31.0, 28.2, 28.1, 25.3, 20.8, 20.0; IR (CHCl3) 2932, 2858, 1704, 1635, 1602, 1511, 1454, 1432, 1404, 1368, 1327, 1272, 1255, 1203, 1163, 1116, 1073, 1017, 964, 904, 860, 837, 793 cm−1; MS (ES+) m/z (relative intensity) 1266 ([M + H]+•, 80), 532 (100). (11S,11aS,11′S,11a′S)-Di-tert-butyl 8,8′-(((5-(1-(18-(2,5Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-13-oxo-3,6,9-trioxa-12azaoctadecyl)-1H-1,2,3-triazol-4-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1Hpyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) (28). Solid 6-maleimidohexanoic acid N-hydroxysuccinimide ester (1.05 equiv, 327 mg, 1.06 mmol) was added to a stirred solution of the primary amine 27 (1 equiv, 1.28 g, 1.01 mmol) in dry DCM (30 mL) at room temperature. Progress was monitored by LC−MS and after 3 days stirring the reaction proceeded no further, a substantial amount of desired product was observed at retention time 3.65 min (ES+) m/z 1459 [M + H]+• accompanied by unreacted starting material at retention time 3.15 min. The reaction mixture was treated with silica gel, and the solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash chromatography (100% DCM to 97% DCM/MeOH) to give the maleimide 28 as a foam (658 mg, 0.45 mmol, 45% yield, mixture of diastereoisomers from THP): [α]22D +41° (c 0.82, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.02 (s, 1H), 7.91 (br s, 2H), 7.57−7.54 (m, 1H), 7.26 (s, 1H), 7.22 (s, 1H), 6.95 (s, 1H), 6.67 (s, 2H), 6.61 (br s, 1H), 6.21 (br s, 1H), 5.80−5.78 (m, 1H), 5.69 (d, 1H, J = 9.2 Hz), 5.25−4.97 (m, 10H), 4.60 (t, 2H, J = 4.8 Hz), 4.34−4.27 (m, 2H), 4.16−4.08 (m, 2H), 3.96−3.89 (m, 10H), 3.69−3.54 (m, 11H), 3.49−3.41 (m, 5H), 3.39−3.37 (m, 2H), 2.95−2.86 (m, 2H), 2.74 (d, 1H, J = 15.7 Hz), 2.55 (d, 1H, J = 16.2 Hz), 2.15 (t, 2H, J = 7.5 Hz), 1.73−1.21 (m, 36H); 13C NMR (CDCl3, 100 MHz) δ 173.0, 170.8, 167.2, 154.9, 150.1, 149.8, 149.2, 148.9, 142.0, 137.9, 137.8, 134.1, 131.7, 129.9, 129.6, 127.0, 126.4, 125.5, 124.2, 121.3, 115.8, 115.5, 110.8, 110.2, 109.6, 109.5, 100.7, 96.0, 91.1, 88.3, 81.4, 81.0, 72.4, 71.1, 70.8, 70.6, 70.5 (×2), 70.4, 70.2, 70.1, 69.8, 69.5, 64.8, 63.5, 61.6, 60.1, 59.9, 56.2, 56.1, 50.7, 50.5, 39.2, 37.6, 36.3, 35.5, 35.1, 31.4, 30.9, 28.3, 28.1 (x 2), 26.4, 25.3, 25.1, 20.8, 20.0; IR (CHCl3) 3349, 2941, 2866, 1703, 1636, 1603, 1511, 1455, 1432, 1403, 1368, 1327, 1274, 1255, 1204, 1161, 1117, 1072, 1017, 965, 905, 869, 828, 793, 695 cm−1; MS (ES+) m/z (relative intensity) 1459 ([M + H]+•, 30), 528 (100). (11S,11aS,11′S,11a′S)-Di-tert-butyl 8,8′-(((5-(4-(tertB u t o x y c a r b o n y l ) p i p e r a z i n - 1 - y l )- 1 , 3 - p h e ny l e n e ) b i s (methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1Hpyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) (29). RuPhos (0.2 equiv, 18 mg, 38 μmol), RuPhos Pd G1 methyl tertbutyl ether adduct (0.12 equiv, 18 mg, 22 μmol), cesium carbonate (5.0 equiv, 0.36 g, 1.1 mmol), and iodo derivative 19 (1.0 equiv, 0.307 g, 0.27 mmol) were placed in a microwave vial which was evacuated and flushed with argon 3 times. Anhydrous THF (5 mL) was added followed by tert-butyl piperazine-1-carboxylate (1.1 equiv, 70 mg, 0.37 mmol), and the resulting mixture was heated at 85 °C for 4 h and then overnight at room temperature. LC−MS analysis showed desired product at retention time 4.12 min, (ES+) m/z 1208 [M + H]+•. The reaction mixture was diluted with saturated NaHCO3 (100 mL) and extracted with EtOAc (3 × 100 mL). The combined EtOAc extracts were washed with brine (100 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by flash chromatography (100%

(m, 1H), 5.67 (d, 1H, J = 9.4 Hz), 5.17−4.96 (m, 10H), 4.34−4.27 (m, 2H), 4.15−4.08 (m, 2H), 3.93−3.89 (m, 8H), 3.61−3.57 (m, 3H), 3.49−3.47 (m, 1H), 2.94−2.86 (m, 2H), 2.78−2.71 (m, 1H), 2.54 (d, 1H, J = 16.6 Hz), 1.77−1.19 (m, 30H), 0.24−0.22 (m, 9H); 13C NMR (CDCl3, 100 MHz) δ 167.4, 167.2, 154.9, 150.0, 149.8, 149.3, 149.0, 142.2, 137.8, 137.6, 132.3, 132.2, 132.0, 130.3, 129.9, 129.7, 128.7, 128.5, 127.3, 126.7, 125.9, 124.4, 116.0, 115.9, 115.8, 110.9, 110.3, 109.6, 109.5, 104.4, 101.0, 96.2, 95.4, 91.3, 88.4, 81.5, 81.1, 71.0, 70.6, 65.0, 63.7, 60.5, 60.1, 60.0, 56.2 (x 2), 50.8, 35.6, 35.3, 31.5, 31.1, 28.2 (×2), 25.4, 21.0, 20.2, 0.0; IR (CHCl3) 2942, 2864, 2155, 1703, 1643, 1604, 1510, 1454, 1430, 1397, 1367, 1326, 1275, 1248, 1202, 1162, 1118, 1072, 1016, 966, 904, 843, 795, 698 cm−1; MS (ES+) m/z (relative intensity) 1142 ([M + Na]+•, 30), 918 (100). (11S,11aS,11′S,11a′S)-Di-tert-butyl 8,8′-(((5-Ethynyl-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) (26). Solid K2CO3 (2 equiv, 383 mg, 2.77 mmol) was added to a stirred solution of the TMS-protected compound 25 (1 equiv, 1.55 g, 1.39 mmol) in MeOH (20 mL). After 3 h stirring at room temperature the reaction was deemed to be complete as judged by LC−MS, desired product peak at retention time 4.00 min (ES+) m/z 1047 [M + H]+•. The MeOH was removed by rotary evaporation under reduced pressure, and the resulting residue was partitioned between water (60 mL) and EtOAc (60 mL). The layers were separated, and the aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with water (30 mL), brine (30 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to provide the crude product. Purification by flash chromatography (50% EtOAc/hexane to 80% EtOAc/hexane) gave the acetylene 26 as an orange foam (1.13 g, 1.08 mmol, 78% yield, mixture of diastereoisomers from THP): [α]22D +91° (c 0.24, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.57−7.53 (m, 3H), 7.25 (s, 1H), 7.21 (s, 1H), 6.88 (s, 1H), 6.53 (s, 1H), 5.80−5.78 (m, 1H), 5.68 (d, 1H, J = 9.4 Hz), 5.17−4.96 (m, 10H), 4.34−4.27 (m, 2H), 4.15− 4.08 (m, 2H), 3.93−3.88 (m, 8H), 3.62−3.57 (m, 3H), 3.51−3.47 (m, 1H), 3.09−3.08 (m, 1H), 2.94−2.86 (m, 2H), 2.74 (d, 1H, J = 16.1 Hz), 2.54 (d, 1H, J = 16.3 Hz), 1.76−1.08 (m, 30H); 13C NMR (CDCl3, 100 MHz) δ 167.3, 167.1, 154.9, 149.8, 149.6, 149.2, 148.9, 142.1, 137.8, 137.6, 132.2, 132.1, 131.9 (x 2), 130.4, 129.8, 129.6, 128.6, 128.4, 127.2, 126.7, 126.1, 123.2, 115.9, 115.7, 110.8, 110.3, 109.5, 109.4, 100.8, 96.1, 91.1, 88.3, 82.9, 81.4, 81.0, 78.0, 70.7, 70.4, 64.9, 63.6, 60.0, 59.9, 56.1 (x 2), 50.7, 35.5, 35.2, 31.4, 31.0, 28.1 (x 2), 25.3, 20.9, 20.0; IR (CHCl3) 3246, 2942, 2865, 1702, 1638, 1604, 1510, 1455, 1431, 1398, 1368, 1326, 1274, 1246, 1202, 1161, 1117, 1072, 1014, 965, 904, 861, 794 cm−1; MS (ES+) m/z (relative intensity) 1069 ([M + Na]+•, 20), 845 (100). (11S,11aS,11′S,11a′S)-Di-tert-butyl 8,8′-(((5-(1-(2-(2-(2-(2Aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) (27). Solid CuSO4·5H2O (0.05 equiv, 13.0 mg, 52.0 μmol) and (+)-sodium L-ascorbate (0.20 equiv, 41.0 mg, 0.21 mmol) were added to a stirred solution of 11-azido-3,6,9-trioxaundecan-1-amine (1 equiv, 227 mg, 207 μL, 1.04 mmol) and the alkyne 26 (1 equiv, 1.09 g, 1.04 mmol) in tert-BuOH (6 mL) and H2O (6 mL) at room temperature. A color change from yellow to green was observed as the reaction progressed. After stirring for 16 h, analysis by LC−MS revealed a substantial of amount of desired product formed corresponding to peak at retention time 3.12 min (ES+) m/z 1266 [M + H]+•. [NOTE: On some occasions reaction progress stalled; however, the reaction was driven to completion upon addition of further CuSO4·5H2O (0.05 equiv) and (+)-sodium L-ascorbate (0.20 equiv).] The reaction mixture was partitioned (without shaking of the separating funnel) between water (50 mL) and EtOAc (50 mL). The aqueous phase was extracted with EtOAc (3 × 15 mL), and the combined organic layers were washed with water (30 mL), brine (50 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to provide the crude product 27 as a green foam (1.32 g, 1.04 mmol, 100% crude yield). The crude product was carried 9503

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

Journal of Medicinal Chemistry

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CHCl3 to 98.5% CHCl3/MeOH) to provide 29 as an orange film (111 mg, 0.09 mmol, 34% yield, mixture of diastereoisomers from THP): [α]22D +49° (c 0.36, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.24 (s, 2H), 7.20 (s, 1H), 7.00 (s, 1H), 6.99−6.94 (m, 2H), 6.89 (s, 1H), 6.55 (s, 1H), 5.85−5.73 (m, 1H), 5.68 (d, 1H, J = 9.4 Hz), 5.17−4.93 (m, 10H), 4.35−4.24 (m, 2H), 4.17−4.06 (m, 2H), 3.99−3.83 (m, 8H), 3.65−3.52 (m, 7H), 3.51−3.43 (m, 1H), 3.19−3.08 (m, 4H), 2.96− 2.84 (m, 2H), 2.74 (d, 1H, J = 16.1 Hz), 2.54 (d, 1H, J = 15.3 Hz), 1.82−1.15 (m, 39H); 13C NMR (CDCl3, 100 MHz) δ 167.3, 167.2, 154.9, 154.7, 152.1, 150.1, 149.8, 149.1, 148.8, 142.1, 138.1, 129.8, 129.6, 126.8, 126.4, 117.1, 115.7, 115.5, 114.7, 110.6, 110.1, 109.5 (×2), 100.5, 96.0, 91.1, 88.2, 81.3, 80.9, 80.0 (x 3), 71.5, 71.1, 64.6, 63.5, 60.0, 59.9, 56.1 (×2), 50.7, 49.1 (x 2), 49.0, 35.5, 35.2, 31.3, 30.9, 28.4, 28.1 (×2), 25.3 (×2), 20.7, 20.0; IR (CHCl3) 2975, 2939, 2856, 1699, 1640, 1602, 1510, 1454, 1430, 1393, 1367, 1326, 1273, 1246, 1202, 1161, 1118, 1072, 1015, 997, 965, 904, 861, 816, 792, 638 cm−1; MS (ES+) m/z (relative intensity) 1208 ([M + H]+•, 25), 483 (100). (11aS,11a′S)-8,8′-(((5-(Piperazin-1-yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one) (30). A cold (ice bath) solution of 95:5 v/v TFA/H2O (4 mL) was added to compound 29 (0.2 g, 0.165 mmol) which had been cooled in an ice bath. The solution was stirred at 0 °C for 30 min when reaction was shown to be complete by LC−MS retention time 2.33 min (ES+) m/z 703 [M + H]+•. The reaction mixture was added dropwise to a mixture of ice and saturated NaHCO3 solution to neutralize the TFA. The mixture was extracted with DCM (4 × 75 mL) and the combined extracts were washed with water (100 mL), saturated brine (100 mL), dried (MgSO4), filtered, and the solvent was removed by rotary evaporation under reduced pressure to give 30 as a yellow solid which was used without further purification (0.116 g, 0.165 mmol, 100% yield). Reduction/Oxidation of THIOMABs for Conjugation. Full length cysteine engineered monoclonal antibodies42,44−46 (THIOMABs) were expressed in CHO cells as cysteine and glutathione capped intermediates. The engineered cysteines were made reactive by reduction with a 50-fold excess of DTT in 50 mM Tris, pH 7.5, with 2 mM EDTA for 3 h at 37 °C or overnight at room temperature.47 The reduced THIOMAB antibody was purified by cation exchange chromatography (SPHP resin, GE) using an AKTA Prime purification system. Samples were acidified by the addition of 10% acetic acid to a final concentration of 0.5% and then diluted 6-fold with 10 mM succinate, pH 5. The antibody was then loaded onto the column and washed with 10 column volumes of succinate buffer. The column was eluted with 50 mM Tris, pH 7.5, and 150 mM NaCl. The eluted, reduced THIOMAB antibody was treated with a 15-fold molar excess of dehydroascorbic acid (DHAA) to re-form the interchain disulfides. Reoxidation was complete in 2−3 h, as monitored by LC−MS. The reoxidized antibody was again purified by cation exchange chromatography using the conditions already described. The purified, reoxidized THIOMAB antibody was dialyzed into 20 mM sodium succinate, pH 5, 150 mM NaCl, 2 mM EDTA and stored frozen at −20 °C. Conjugation of THIOMAB Antibodies with Compounds to Prepare ADCs. The deblocked, reoxidized THIOMAB antibodies were reacted with 2.5- to 6-fold molar excess of compounds 6, 7, and 8 (from a DMSO stock at a concentration of 20 mM) in 50 mM Tris, pH 8, with 10% DMSO v/v final, until the reaction was complete (1− 16 h) as determined by LC−MS analysis of the reaction mixture. The crude ADCs were then applied to a cation exchange column after dilution with 20 mM histidine acetate, pH 5.5. The column was washed with at least 10 column volumes of 20 mM histidine acetate, pH 5.5, and the antibody was eluted with 20 mM histidine acetate, pH 5.5, and 300 mM NaCl. The ADCs were formulated into 20 mM histidine acetate, pH 5.5, with 240 mM sucrose using gel filtration columns (Sephacryl S200, GE), pooling the main monomer peak. The ADCs were characterized by UV spectroscopy to determine protein concentration, analytical SEC for aggregation analysis, and LC−MS before and after treatment with lysine C endopeptidase. SEC was performed using a Shodex KW802.5 column in 0.2 M potassium

phosphate, pH 6.2, with 0.25 mM potassium chloride and 15% IPA at a flow rate of 0.75 mL/min. The aggregation state of the conjugate was determined by integration of eluted peak area absorbance at 280 nm. LC−MS analysis was performed using an Agilent quadrupole time-offlight 6520 ESI instrument. As an example, an ADC generated using this chemistry was treated with 1:500 w/w endoproteinase Lys C (Promega) in Tris, pH 7.5, for 30 min at 37 °C. The resulting cleavage fragments were loaded onto a 1000 Å, 8 μm PLRP-S column heated to 80 °C and eluted with a mobile phase of water (A, 0.05% TFA) and acetonitrile (B, 0.04% TFA), gradient 30% B to 40% B in 5 min; the flow rate was 0.5 mL/min. Protein elution was monitored by UV absorbance detection at 280 nm prior to electrospray ionization and MS analysis. Chromatographic resolution of the unconjugated Fc fragment, residual unconjugated Fab, and drugged Fab was usually achieved. The obtained m/z spectra were deconvoluted using Mass Hunter software (Agilent Technologies) to calculate the mass of the antibody fragments and drug to antibody ratio (DAR). In Vitro Cytotoxicity.28,48 Human breast tumor lines SK-BR-3 and MCF7 were obtained from the American Type Culture Collection. KPL-4 breast cancer cells were provided by Prof. Junichi Kurebayashi, Kawasaki Medical Hospital, Kurashiki, Okayama, Japan. Cells were plated in black-walled 96-well plates (4000 for SK-BR-3; 1300 for KPL-4; 5000 for MCF7) and allowed to adhere overnight at 37 °C in a humidified atmosphere containing 5% CO2. The medium was then removed and replaced by fresh culture medium containing different concentrations of each ADC. Cell Titer-Glo (Promega Corp.) was added to the wells at 5 days after ADC administration, and the luminescent signal was measured using EnVision multilabel plate reader (PerkinElmer). BJAB, Jurkat, and WSU-DLCL2 cell lines were seeded at 4−7000 cells/well and grown on Corning 384-well flat clear bottom white polystyrene TC-treated microplates in cysteine-free RPMI or DMEM Ham’s F-12 media containing 10% FBS, 2 mM L-glutamine, and 0.015 g/L methionine supplemented with 50 μM fresh cystine to confluence. The ADCs were incubated for 4 days before cell viability was determined using Cell Titer-Glo (Promega Corp.) and the luminescent intensity measured on an EnVision multilabel plate reader (PerkinElmer). In Vivo Efficacy.28 The efficacy of anti-HER2 ADCs was investigated in an allograft model of MMTV-HER2 Fo5 mouse mammary tumors. The Fo5 model is a transgenic mouse model in which the human HER2 gene, under transcriptional regulation of the murine mammary tumor virus promoter (MMTV-HER2), is overexpressed in mammary epithelium. The overexpression causes spontaneous development of mammary tumors that overexpress the human HER2 receptor. The mammary tumor of one of the founder animals has been propagated in subsequent generations of FVB mice by serial transplantation of tumor fragments. Before being used for an in vivo efficacy study, the MMTV-HER2 Fo5 transgenic mammary tumor was surgically transplanted into the no. 2/3 mammary fat pad of nu/nu mice (Charles River Laboratories) in fragments that measured approximately 2 × 2 mm. The efficacy of anti-CD22 ADCs was evaluated in a xenograft model of WSU-DLCL2 human non-Hodgkin lymphoma (from DSMZ, German Collection of Microorganisms and Cell Cultures). Cells were maintained in RPMI 1640 supplemented with 10% FBS (Sigma) and 2 mM L-glutamine. To establish a subcutaneous xenograft model for an in vivo efficacy study, female C.B-17 SCID mice (from Charles River Laboratories) were each inoculated in the flank area with the tumor cells (20 million cells in 0.2 mL of Hank’s balanced salt solution; HyClone). When tumors reached desired volumes, the tumor-bearing mice were randomized and given a single dose by iv injection of the ADC. Results were plotted as mean tumor volume ± SEM of each group over time. 9504

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

Journal of Medicinal Chemistry

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Article

ASSOCIATED CONTENT

TCEP, tris(2-carboxyethyl)phosphine hydrochloride; TEA, triethylamine; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00736. Atomic coordinates for models (PDB) Molecular formula strings and some data (CSV)

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REFERENCES

(1) Thomas, A.; Teicher, B. A.; Hassan, R. Antibody-drug conjugates for cancer therapy. Lancet Oncol. 2016, 17 (6), e254−262. (2) Flygare, J. A.; Pillow, T. H.; Aristoff, P. Antibody-drug conjugates for the treatment of cancer. Chem. Biol. Drug Des. 2013, 81 (1), 113− 121. (3) Lambert, J. M.; Morris, C. Q. Antibody-drug conjugates (ADCs) for personalized treatment of solid tumors: a review. Adv. Ther. 2017, 34 (5), 1015−1035. (4) Beck, A.; Goetsch, L.; Dumontet, C.; Corvaia, N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug Discovery 2017, 16 (5), 315−337. (5) Mantaj, J.; Jackson, P. J.; Rahman, K. M.; Thurston, D. E. From anthramycin to pyrrolobenzodiazepine (PBD)-containing antibodydrug conjugates (ADCs). Angew. Chem., Int. Ed. 2017, 56 (2), 462− 488. (6) Gerratana, B. Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines. Med. Res. Rev. 2012, 32 (2), 254−293. (7) Antonow, D.; Thurston, D. E. Synthesis of DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepines (PBDs). Chem. Rev. 2011, 111 (4), 2815−2864. (8) Hartley, J. A. The development of pyrrolobenzodiazepines as antitumour agents. Expert Opin. Invest. Drugs 2011, 20 (6), 733−744. (9) Jeffrey, S. C.; Burke, P. J.; Lyon, R. P.; Meyer, D. W.; Sussman, D.; Anderson, M.; Hunter, J. H.; Leiske, C. I.; Miyamoto, J. B.; Nicholas, N. D.; Okeley, N. M.; Sanderson, R. J.; Stone, I. J.; Zeng, W.; Gregson, S. J.; Masterson, L.; Tiberghien, A. C.; Howard, P. W.; Thurston, D. E.; Law, C. L.; Senter, P. D. A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology. Bioconjugate Chem. 2013, 24 (7), 1256−1263. (10) Kung Sutherland, M. S.; Walter, R. B.; Jeffrey, S. C.; Burke, P. J.; Yu, C.; Kostner, H.; Stone, I.; Ryan, M. C.; Sussman, D.; Lyon, R. P.; Zeng, W.; Harrington, K. H.; Klussman, K.; Westendorf, L.; Meyer, D.; Bernstein, I. D.; Senter, P. D.; Benjamin, D. R.; Drachman, J. G.; McEarchern, J. A. SGN-CD33A: a novel CD33-targeting antibodydrug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML. Blood 2013, 122 (8), 1455−1463. (11) Tiberghien, A. C.; Levy, J. N.; Masterson, L. A.; Patel, N. V.; Adams, L. R.; Corbett, S.; Williams, D. G.; Hartley, J. A.; Howard, P. W. Design and synthesis of tesirine, a clinical antibody-drug conjugate pyrrolobenzodiazepine dimer payload. ACS Med. Chem. Lett. 2016, 7 (11), 983−987. (12) Saunders, L. R.; Bankovich, A. J.; Anderson, W. C.; Aujay, M. A.; Bheddah, S.; Black, K.; Desai, R.; Escarpe, P. A.; Hampl, J.; Laysang, A.; Liu, D.; Lopez-Molina, J.; Milton, M.; Park, A.; Pysz, M. A.; Shao, H.; Slingerland, B.; Torgov, M.; Williams, S. A.; Foord, O.; Howard, P.; Jassem, J.; Badzio, A.; Czapiewski, P.; Harpole, D. H.; Dowlati, A.; Massion, P. P.; Travis, W. D.; Pietanza, M. C.; Poirier, J. T.; Rudin, C. M.; Stull, R. A.; Dylla, S. J. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Sci. Transl. Med. 2015, 7 (302), 302ra136. (13) Flynn, M. J.; Zammarchi, F.; Tyrer, P. C.; Akarca, A. U.; Janghra, N.; Britten, C. E.; Havenith, C. E.; Levy, J. N.; Tiberghien, A.; Masterson, L. A.; Barry, C.; D’Hooge, F.; Marafioti, T.; Parren, P. W.; Williams, D. G.; Howard, P. W.; van Berkel, P. H.; Hartley, J. A. ADCT-301, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting CD25-expressing hematological malignancies. Mol. Cancer Ther. 2016, 15 (11), 2709−2721. (14) Rudin, C. M.; Pietanza, M. C.; Bauer, T. M.; Ready, N.; Morgensztern, D.; Glisson, B. S.; Byers, L. A.; Johnson, M. L.; Burris, H. A., 3rd; Robert, F.; Han, T. H.; Bheddah, S.; Theiss, N.; Watson, S.; Mathur, D.; Vennapusa, B.; Zayed, H.; Lally, S.; Strickland, D. K.; Govindan, R.; Dylla, S. J.; Peng, S. L.; Spigel, D. R. Rovalpituzumab

AUTHOR INFORMATION

Corresponding Authors

*S.J.G.: e-mail, [email protected]; phone, (+44) 2037496251. *T.H.P.: e-mail, [email protected]; phone, (+01) 6502251652. ORCID

Stephen J. Gregson: 0000-0002-6859-9772 Present Address §

J.A.F.: Merck, 630 Gateway Boulevard, South San Francisco, CA 94080, United States. Author Contributions

The manuscript was written through contributions of all authors. Notes

The authors declare the following competing financial interest(s): S.J.G., L.A.M., G.-D.K., and P.W.H. are employees of Spirogen (now owned by Medimmune Inc.). B.W., T.H.P., S.D.S., S.-F.Y., J.L., G.L., G.D.L.P., J.G.-T., B.S.S., R.O., M.D., K.R.K., J.d.C.-C., and A.P. are employees of Genentech Inc. J.A.F. and H.R. are former employees of Genentech Inc. All animal studies were carried out in compliance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Genentech, Inc. Authors will release the atomic coordinates and experimental data upon article publication.

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ACKNOWLEDGMENTS Neki Patel of Spirogen is thanked for editorial assistance on this manuscript. ABBREVIATIONS USED AcOH, acetic acid; ADC, antibody−drug conjugate; (Boc)2O, di-tert-butyl dicarbonate; t-Boc-N-amido-dPEG4-acid, 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid; br s, broad singlet; DAIB, (diacetoxyiodo)benzene; DAR, drug/ antibody ratio; DHAA, dehydroascorbic acid; DHP, 3,4dihydro-2H-pyran; EDCI, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; EtOAc, ethyl acetate; EtOH, ethanol; Fo5, Founder 5; FVB, Friend virus B; HER2, human epidermal growth factor receptor 2; IBD, indolobenzodiazepine; IPA, propan-2-ol; mAb, monoclonal antibody; MALdPEG4-acid, 1-maleimido-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid; MeOH, methanol; KOtBu, potassium tertbutoxide; PAB, p-aminobenzyl; PBD, pyrrolobenzodiazepine; RuPhos, 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl; RuPhos Pd G1 methyl tert-butyl ether adduct, chloro-(2dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2aminoethyl)phenyl]palladium(II)−methyl tert-butyl ether adduct; SEC, size-exclusion chromatography; TBAI, tetrabutylammonium iodide; TBSOTf, tert-butyldimethylsilyl trifluoromethanesulfonate; t BuOH, 2-methyl-2-propanol; 9505

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tesirine, a DLL3-targeted antibody-drug conjugate, in recurrent smallcell lung cancer: a first-in-human, first-in-class, open-label, phase 1 study. Lancet Oncol. 2017, 18 (1), 42−51. (15) Bauer, T. M.; Spigel, D.; Ready, N.; Morgensztern, D.; Glisson, B. S.; Byers, L. A.; Burris, H.; Robert, F.; Strickland, D. K.; Pietanza, M. C.; Govindan, R.; Dylla, S. J.; Peng, S.; Rudin, C. ORAL02.01: Safety and efficacy of single-agent rovalpituzumab tesirine, a DLL3-targeted ADC, in recurrent or refractory SCLC. J. Thorac. Oncol. 2016, 11 (11S), S252−S253. (16) Bixby, D. L.; Stein, A. S.; Fathi, A. T.; Kovacsovics, T. J.; Levy, M. Y.; Erba, H. P.; Lancet, J. E.; Jillella, A. P.; Ravandi, F.; Walter, R. B.; Stein, E. M.; Faderl, S.; DeAngelo, D. J.; Ho, P.; O’Meara, M. M.; Voellinger, J. L.; Advani, A. Vadastuximab talirine monotherapy in older patients with treatment naive CD33-positive acute myeloid leukemia (AML). Blood 2016, 128 (22), 590. (17) Erba, H. P.; Levy, M. Y.; Vasu, S.; Stein, A. S.; Fathi, A. T.; Maris, M. B.; Advani, A.; Faderl, S.; Smith, S. E.; Wood, B. L.; Walter, R. B.; Yang, J.; Donnellan, W. B.; Feldman, E. J.; Voellinger, J. L.; Ravandi, F. A phase 1b study of vadastuximab talirine in combination with 7+3 induction therapy for patients with newly diagnosed acute myeloid leukemia (AML). Blood 2016, 128 (22), 211. (18) Fathi, A. T.; Erba, H. P.; Lancet, J. E.; Stein, E. M.; Ravandi, F.; Faderl, S.; Walter, R. B.; Advani, A.; DeAngelo, D. J.; Kovacsovics, T. J.; Jillella, A. P.; Bixby, D. L.; Levy, M. Y.; O’Meara, M. M.; Ho, P.; Stein, A. S. Vadastuximab talirine plus hypomethylating agents: a welltolerated regimen with high remission rate in frontline older patients with acute myeloid leukemia (AML). Blood 2016, 128 (22), 591. (19) Yang, J.; Ravandi, F.; Advani, A.; Vasu, S.; Walter, R. B.; Faderl, S.; Stein, A. S.; Erba, H. P.; Fathi, A. T.; Donnellan, W. B.; Levy, M. Y.; Smith, S. E.; Wood, B. L.; Feldman, E. J.; Voellinger, J. L.; Maris, M. B. A phase 1b study of vadastuximab talirine as maintenance and in combination with standard consolidation for patients with acute myeloid leukemia (AML). Blood 2016, 128 (22), 340. (20) Miller, M. L.; Fishkin, N. E.; Li, W.; Whiteman, K. R.; Kovtun, Y.; Reid, E. E.; Archer, K. E.; Maloney, E. K.; Audette, C. A.; Mayo, M. F.; Wilhelm, A.; Modafferi, H. A.; Singh, R.; Pinkas, J.; Goldmacher, V.; Lambert, J. M.; Chari, R. V. A new class of antibody-drug conjugates with potent DNA alkylating activity. Mol. Cancer Ther. 2016, 15 (8), 1870−1878. (21) Polakis, P. Antibody drug conjugates for cancer therapy. Pharmacol. Rev. 2016, 68 (1), 3−19. (22) Dorywalska, M.; Dushin, R.; Moine, L.; Farias, S. E.; Zhou, D.; Navaratnam, T.; Lui, V.; Hasa-Moreno, A.; Casas, M. G.; Tran, T. T.; Delaria, K.; Liu, S. H.; Foletti, D.; O’Donnell, C. J.; Pons, J.; Shelton, D. L.; Rajpal, A.; Strop, P. Molecular basis of valine-citrulline-PABC linker instability in site-specific ADCs and its mitigation by linker design. Mol. Cancer Ther. 2016, 15 (5), 958−970. (23) Ogitani, Y.; Aida, T.; Hagihara, K.; Yamaguchi, J.; Ishii, C.; Harada, N.; Soma, M.; Okamoto, H.; Oitate, M.; Arakawa, S.; Hirai, T.; Atsumi, R.; Nakada, T.; Hayakawa, I.; Abe, Y.; Agatsuma, T. DS8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a promising antitumor efficacy with differentiation from T-DM1. Clin. Cancer Res. 2016, 22 (20), 5097− 5108. (24) Menderes, G.; Bonazzoli, E.; Bellone, S.; Black, J.; Altwerger, G.; Masserdotti, A.; Pettinella, F.; Zammataro, L.; Buza, N.; Hui, P.; Wong, S.; Litkouhi, B.; Ratner, E.; Silasi, D. A.; Huang, G. S.; Azodi, M.; Schwartz, P. E.; Santin, A. D. SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows promising antitumor activity in epithelial ovarian carcinoma with HER2/Neu expression. Gynecol. Oncol. 2017, 146 (1), 179−186. (25) Li, J. Y.; Perry, S. R.; Muniz-Medina, V.; Wang, X.; Wetzel, L. K.; Rebelatto, M. C.; Hinrichs, M. J.; Bezabeh, B. Z.; Fleming, R. L.; Dimasi, N.; Feng, H.; Toader, D.; Yuan, A. Q.; Xu, L.; Lin, J.; Gao, C.; Wu, H.; Dixit, R.; Osbourn, J. K.; Coats, S. R. A biparatopic HER2targeting antibody-drug conjugate induces tumor regression in primary models refractory to or ineligible for HER2-targeted therapy. Cancer Cell 2016, 29 (1), 117−129.

(26) Kantarjian, H. M.; DeAngelo, D. J.; Stelljes, M.; Martinelli, G.; Liedtke, M.; Stock, W.; Gokbuget, N.; O’Brien, S.; Wang, K.; Wang, T.; Paccagnella, M. L.; Sleight, B.; Vandendries, E.; Advani, A. S. Inotuzumab ozogamicin versus standard therapy for acute lymphoblastic leukemia. N. Engl. J. Med. 2016, 375 (8), 740−753. (27) Shen, B. Q.; Bumbaca, D.; Saad, O.; Yue, Q.; Pastuskovas, C. V.; Khojasteh, S. C.; Tibbitts, J.; Kaur, S.; Wang, B.; Chu, Y. W.; LoRusso, P. M.; Girish, S. Catabolic fate and pharmacokinetic characterization of trastuzumab emtansine (T-DM1): an emphasis on preclinical and clinical catabolism. Curr. Drug Metab. 2012, 13 (7), 901−910. (28) Lewis Phillips, G. D.; Li, G.; Dugger, D. L.; Crocker, L. M.; Parsons, K. L.; Mai, E.; Blattler, W. A.; Lambert, J. M.; Chari, R. V.; Lutz, R. J.; Wong, W. L.; Jacobson, F. S.; Koeppen, H.; Schwall, R. H.; Kenkare-Mitra, S. R.; Spencer, S. D.; Sliwkowski, M. X. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibodycytotoxic drug conjugate. Cancer Res. 2008, 68 (22), 9280−9290. (29) Doronina, S. O.; Mendelsohn, B. A.; Bovee, T. D.; Cerveny, C. G.; Alley, S. C.; Meyer, D. L.; Oflazoglu, E.; Toki, B. E.; Sanderson, R. J.; Zabinski, R. F.; Wahl, A. F.; Senter, P. D. Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjugate Chem. 2006, 17 (1), 114−124. (30) Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. Design, synthesis, and evaluation of a novel pyrrolobenzodiazepine DNAinteractive agent with highly efficient cross-linking ability and potent cytotoxicity. J. Med. Chem. 2001, 44 (5), 737−748. (31) Gregson, S. J.; Howard, P. W.; Gullick, D. R.; Hamaguchi, A.; Corcoran, K. E.; Brooks, N. A.; Hartley, J. A.; Jenkins, T. C.; Patel, S.; Guille, M. J.; Thurston, D. E. Linker length modulates DNA crosslinking reactivity and cytotoxic potency of C8/C8′ ether-linked C2exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers. J. Med. Chem. 2004, 47 (5), 1161−1174. (32) Hartley, J. A.; Hamaguchi, A.; Suggitt, M.; Gregson, S. J.; Thurston, D. E.; Howard, P. W. DNA interstrand cross-linking and in vivo antitumor activity of the extended pyrrolo[2,1-c][1,4]benzodiazepine dimer SG2057. Invest. New Drugs 2012, 30 (3), 950−958. (33) Rettig, M.; Weingarth, M.; Langel, W.; Kamal, A.; Kumar, P. P.; Weisz, K. Solution structure of a covalently bound pyrrolo[2,1c][1,4]benzodiazepine-benzimidazole hybrid to a 10mer DNA duplex. Biochemistry 2009, 48 (51), 12223−12232. (34) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. Molecular conformation in oligo(ethylene glycol)-terminated self-assembled monolayers on gold and silver surfaces determines their ability to resist protein adsorption. J. Phys. Chem. B 1998, 102 (2), 426−436. (35) Althuis, T. H.; Hess, H. J. Synthesis and identification of the major metabolites of prazosin formed in dog and rat. J. Med. Chem. 1977, 20 (1), 146−149. (36) Tercel, M.; Stribbling, S. M.; Sheppard, H.; Siim, B. G.; Wu, K.; Pullen, S. M.; Botting, K. J.; Wilson, W. R.; Denny, W. A. Unsymmetrical DNA cross-linking agents: combination of the CBI and PBD pharmacophores. J. Med. Chem. 2003, 46 (11), 2132−2151. (37) Wells, G.; Martin, C. R.; Howard, P. W.; Sands, Z. A.; Laughton, C. A.; Tiberghien, A.; Woo, C. K.; Masterson, L. A.; Stephenson, M. J.; Hartley, J. A.; Jenkins, T. C.; Shnyder, S. D.; Loadman, P. M.; Waring, M. J.; Thurston, D. E. Design, synthesis, and biophysical and biological evaluation of a series of pyrrolobenzodiazepine-poly(N-methylpyrrole) conjugates. J. Med. Chem. 2006, 49 (18), 5442−5461. (38) Alberico, D.; Rudolph, A.; Lautens, M. Synthesis of tricyclic heterocycles via a tandem aryl alkylation/Heck coupling sequence. J. Org. Chem. 2007, 72 (3), 775−781. (39) Peterle, T.; Ringler, P.; Mayor, M. Gold nanoparticles stabilized by acetylene-functionalized multidentate thioether ligands: building blocks for nanoparticle superstructures. Adv. Funct. Mater. 2009, 19 (21), 3497−3506. 9506

DOI: 10.1021/acs.jmedchem.7b00736 J. Med. Chem. 2017, 60, 9490−9507

Journal of Medicinal Chemistry

Article

(40) Sakaitani, M.; Ohfune, Y. Selective transformation of N-tbutoxycarbonyl group into N-alkoxy-carbonyl group via N-carboxylate ion equivalent. Tetrahedron Lett. 1985, 26 (45), 5543−5546. (41) Maiti, D.; Fors, B. P.; Henderson, J. L.; Nakamura, Y.; Buchwald, S. L. Palladium-catalyzed coupling of functionalized primary and secondary amines with aryl and heteroaryl halides: two ligands suffice in most cases. Chem. Sci. 2011, 2 (1), 57−68. (42) Junutula, J. R.; Raab, H.; Clark, S.; Bhakta, S.; Leipold, D. D.; Weir, S.; Chen, Y.; Simpson, M.; Tsai, S. P.; Dennis, M. S.; Lu, Y.; Meng, Y. G.; Ng, C.; Yang, J.; Lee, C. C.; Duenas, E.; Gorrell, J.; Katta, V.; Kim, A.; McDorman, K.; Flagella, K.; Venook, R.; Ross, S.; Spencer, S. D.; Lee Wong, W.; Lowman, H. B.; Vandlen, R.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Mallet, W. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 2008, 26 (8), 925−932. (43) Verma, V. A.; Pillow, T. H.; DePalatis, L.; Li, G.; Phillips, G. L.; Polson, A. G.; Raab, H. E.; Spencer, S.; Zheng, B. The cryptophycins as potent payloads for antibody drug conjugates. Bioorg. Med. Chem. Lett. 2015, 25 (4), 864−868. (44) Dornan, D.; Bennett, F.; Chen, Y.; Dennis, M.; Eaton, D.; Elkins, K.; French, D.; Go, M. A.; Jack, A.; Junutula, J. R.; Koeppen, H.; Lau, J.; McBride, J.; Rawstron, A.; Shi, X.; Yu, N.; Yu, S. F.; Yue, P.; Zheng, B.; Ebens, A.; Polson, A. G. Therapeutic potential of an antiCD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood 2009, 114 (13), 2721− 2729. (45) Shen, B.-Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.; Yu, S.-F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.; Eigenbrot, C.; Nguyen, T.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel, D.; Spencer, S. D.; Khawli, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.; Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 2012, 30 (2), 184−189. (46) Junutula, J. R.; Bhakta, S.; Raab, H.; Ervin, K. E.; Eigenbrot, C.; Vandlen, R.; Scheller, R. H.; Lowman, H. B. Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. J. Immunol. Methods 2008, 332 (1−2), 41−52. (47) Getz, E. B.; Xiao, M.; Chakrabarty, T.; Cooke, R.; Selvin, P. R. A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal. Biochem. 1999, 273 (1), 73−80. (48) Zhang, D.; Pillow, T. H.; Ma, Y.; Cruz-Chuh, J. D.; Kozak, K. R.; Sadowsky, J. D.; Lewis Phillips, G. D.; Guo, J.; Darwish, M.; Fan, P.; Chen, J.; He, C.; Wang, T.; Yao, H.; Xu, Z.; Chen, J.; Wai, J.; Pei, Z.; Hop, C. E.; Khojasteh, S. C.; Dragovich, P. S. Linker immolation determines cell killing activity of disulfide-linked pyrrolobenzodiazepine antibody-drug conjugates. ACS Med. Chem. Lett. 2016, 7 (11), 988−993.

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