Latent Warheads for Targeted Cancer Therapy: Design and Synthesis

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Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Latent Warheads for Targeted Cancer Therapy: Design and Synthesis of pro-Pyrrolobenzodiazepines and Conjugates Iontcho R. Vlahov,* Longwu Qi, Paul J. Kleindl, Hari K. Santhapuram, Albert Felten, Garth L. Parham, Kevin Wang, Fei You, Jeremy F. Vaughn, Spencer J. Hahn, Hanna F. Klein, Marilynn Vetzel, Joseph A. Reddy, Melissa Nelson, Jeff Nicoson, and Christopher P. Leamon Endocyte Inc., 3000 Kent Avenue, West Lafayette, Indiana 47906, United States S Supporting Information *

ABSTRACT: Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds, and are currently under development as novel payloads in antibody−drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed diazepine-ring-opened conjugated prodrugs lacking the imine moiety. Once the prodrug (pro-PBD) conjugate enters a targeted cell, cleavage of the linker system triggers the generation of a reactive intermediate possessing an aldehyde and aromatic amine. An intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine and, as a result, the diazepine ring. In our pro-PBDs, we mask the aldehyde as a hydrolytically sensitive oxazolidine moiety which in turn is a part of a reductively labile self-immolative linker system. To prove the range of applications for this new class of latent DNA-alkylators, we designed and synthesized several novel latent warheads: pro-PBD dimers and hybrids of pro-PBD with other sequence-selective DNA minor groove binders. Preliminary preclinical pharmacology studies showed excellent biological activity and specificity.



INTRODUCTION Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antibiotics are a class of natural products produced by various actinomycetes bacteria, and are known for their antibiotic and antitumor properties.1−5 PBDs and their synthetic C-8/C-8′-tethered dimers (Figure 1) are sequence selective DNA-cross-linking alkylating agents and

are considerably more potent than systemic chemotherapeutic agents. All PBD-based derivatives bind in a specific manner to DNA after recognition of a three-base-pair sequence. Insertion of the PBD dimer into the minor groove results in two interstrand cross-linking covalent aminal bonds,6 formed as the result of the nucleophilic attacks of both exocyclic N-2/N-2′ amines presented by two guanine (G) bases, integrated in the opposing DNA-strands, on the electrophillic C-11/C-11′ imines of the PBD dimer. Two additional hydrogen bonds are formed between the ring nitrogen N-3 of the 3′-adenines adjacent to the aminal-modified guanine and both N-10, N-10′ protons of the PBD units. The perfect fit of a PBD-dimer in the DNA minor groove results in negligible distortion of the DNA helix, thus potentially avoiding DNA-repair mechanisms and the common phenomenon of drug resistance.7,8 Since the discovery of natural PBDs, various synthetic PBDmonomers, PBD-hybrids, and PBD-dimers have been designed, prepared, and explored, improving our understanding of the mode of action of this important class of compounds.9−12 A synthetic PBD-dimer, SJG-136 (Figure 1), has undergone clinical evaluation as a monotherapy.10 In recent years, Received: August 9, 2017 Revised: November 21, 2017

Figure 1. General structure of PBD, interconvertible PBD forms in water/alcohol, and the structure of the PBD-dimer SG-136. © XXXX American Chemical Society

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DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Scheme 1. Concept of pro-PBDs as Latent Warheads for Targeting Cancer Cells

Scheme 2. Synthesis of pro-PBD Model Compound 10a

a

Reagents and conditions: (a) DMP, DCM, rt; (b) (1) ethanolamine, MgSO4, DCM, rt; (2) mixed carbonate 11, Et3N, rt, 46% for the 3 steps; (c) TFA/DCM, rt; (d) 2-amino-4,5-dimethoxybenzoic acid, PyBOP, DIPEA, DCM, rt, 25% for the 2 steps.

turned our attention to modifying the PBD dimer within the conjugate construct with the hope of reducing the observed offtarget toxicities. Several groups in the PBD-prodrug and ADC arena have addressed these concerns in their own ways. Thurston and Howard synthesized PBD prodrugs and conjugates with cleavable linkers/side-chains at the N-10 position of PBD.5,17,18 By doing so, they effectively blocked the imine moiety and modified the structure so as to prevent DNA alkylation or cross-linking. They demonstrated that “protection” in this manner significantly reduced the toxicities observed in a number of PBD prodrugs. In another paper, Miller et al. noted that the delayed toxicities that they observed with their antibody−PBD dimer conjugates could be managed by reducing one of the imines to an amine.5,19 The resulting monoimine can alkylate DNA but can no longer act as a crosslinker. It was found to be only two to four times less potent than its di-imine precusor. Interestingly, the completely reduced diamine still exhibits subnanomolar activity as the result of its ability to perfectly noncovalently bind within the DNA minor groove. Taken together, these studies suggest that masking the imine and perturbing the core PBD structure in such a way as to prevent binding might maximize therapeutic efficiency in a SMDC setting. Jeffrey et al. have shown that the heterocyclic iminium moiety in several anthracycline conjugates can be masked by replacing this moiety with its synthetic precursors: namely, an 1,3-oxazolidine carbamate and an amine.20,21 The oxazolidine carbamate provided an attachment point for the drug to the antibody through one of several enzymatically labile linkers. Applying this strategy to PBDs satisfied our design criteria. Once prodrug (pro-PBD) conjugate 1 (Scheme 1) enters a targeted cell, and endosomal reductive cleavage and fragmentation of the linker system triggers the generation of 1,3-

synthetic PBD-dimers have emerged as a new class of warheads in the field of antibody−drug conjugates (ADCs) and small molecule drug conjugates (SMDCs).5,13−15 One of the major challenges for developing this class of compounds as stand-alone and/or conjugated therapeutic agents is the highly electrophilic character of the imine at the C-11 position (Figure 1). From a synthetic prospective, this functional group is notoriously reactive and generates several closely related derivatives during the course of synthetic manipulations and/or purification procedures. Depending upon the solvents, nucleophiles present in the medium, and isolation conditions, a complex equilibrium of interconverting forms, such as imine, carbinolamine, and carbinolamine ether are observed. The equilibrium does not affect the biological activity of the PBDs since all forms possess an electrophilic C11 moiety, enabling each PBD construct to alkylate the NH2 group of guanine in the minor groove of DNA.2 In addition, the stability of a carbinolamine-containing PBD is related to the type and pattern of aromatic A-ring substituents and other structural features such as the degree of C-ring saturation.2,16 In general, carbinolamines of PBDs (or their equivalents) are easily generated during the final synthetic and/or purification step. Furthermore, any reaction conditions capable of causing racemization at the C-11a position must be avoided in order to maintain the correct chirality/configuration to provide isohelicity with the minor groove of DNA. Therefore, N-10 should be protected as a carbamate (or masked as a NO2group) throughout the synthesis until the imine-forming step. In our hands, several PBD dimer (di-imine) containing SMDCs, although highly potent, displayed high (in some cases delayed) toxicities (unpublished results). Modification of the linker or spacer systems in this series of conjugates did little to improve the very tight therapeutic window. As a result, we B

DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Scheme 3. Conjugation of the pro-PBD Model Compounda

a

Reagents and conditions: (a) MeOH/H2O (1:1), Et3N, rt, 60% yield.

oxazolidine 2. The latter, being hydrolytically labile, upon spontaneous reaction with water, forms a reactive aldehyde− amine intermediate 3. Subsequently, once the aromatic amine in 3 is in close proximity to the newly generated aldehyde, a seven-membered ring as carbinolamine 4 is formed. Spontaneous loss of a molecule of water results in the expected imine, thus ultimately generating the diazepine ring and providing in situ the PBD molecule 5.



RESULTS AND DISCUSSION Our initial proof-of-concept study involved the design and synthesis of model compound 10 (Scheme 2). Commercially available N-(Boc)-prolinol 6 was converted to the corresponding aldehyde 7 using the Dess−Martin periodinane (DMP) reagent. Treatment of 7 with ethanolamine in the presence of anhydrous MgSO4 resulted in the expected 1,3-oxazolidine.20,22 This labile intermediate 9 was reacted with the heterobifunctional mixed carbonate 1123,24 to form the stable compound 8 as a diastereomeric mixture. Removal of the Boc-protecting group and acylation of the secondary amine 9 with 2-amino4,5-dimethoxybenzoic acid provided the model pro-PBD 10. Next, 10 was attached to folic acid (FA) via a water-soluble peptide spacer unit.23 Once administered in vivo, such a conjugate targets folate-receptor (FR)-overexpressing cancer cells, and after internalization to the reducing endosomal environment, releases the pro-PBD. As indicated in Scheme 3, the conjugate 13 can be assembled by tethering FA-Spacer unit 12 to the pro-PBD unit 10 in a disulfide-exchange reaction. The peptide-based spacer 12 was designed to be bifunctional containing both acidic (Asp) and basic (Arg) amino acids to provide the best potential for water solubility of the conjugate under physiologic conditions. This unit was assembled using standard fluorenylmethyloxycarbonyl-based solid phase peptide synthesis (Fmoc SPPS) as described previously.23 Incubation of a 1 mM PBS-solution of the diastereomeric conjugate 13 with 20 equiv of dithiothietol (DTT) at 37 °C, resulted in the reduction of the disulfide bond within 120 min (LC/MS studies − Figure 2).23 After the disulfide-exchange reaction (Scheme 4, route a and b), fast formation of 14 and 15 was observed (Figure 2, t = 15 min). The desired PBD 18 was initially detected within 1 h as a result of several consecutive transformations: (1) generation of the self-immolative species 16, (2) its conversion to unstable 1,3-oxazolidine 17a and

Figure 2. LC profile for the release study of model conjugate 13.

subsequently to the ring-open imine 17b (both 17a and 17b exist in dynamic equilibrium), (3) next, 17b is trapped by the aniline amino group to lead to 17c, and (4) finally, spontaneous loss of ethanolamine resulting in the formation of PBD construct 18. Importantly, during these transformations the oxazolidene diastereomeric center disappears and a PBD possessing the sp2 hybridized C-11 center is created. PBD DTT adduct 19 was also observed, providing additional evidence of PBD formation and the high reactivity of its imine moiety. Encouraged by the results of this model study, we applied the same methodology to prepare a series of latent warheads and corresponding conjugates. One of our synthetic approaches began with the formation of the tethered 21 though a simple double alkylation of methyl vanilinate 20 with 1,5-dibromopentane (Scheme 5). Dinitro intermediate 22 was prepared by regioselective bisnitration of 21.25 Then, subsequent hydrolysis and hydrogenation resulted in 24. The bis-acid 24 was used further for preparation of bis-proPBD folate conjugate 31 (Scheme 6). Compound 31 is a first in class example, where two pro-PBDs are tethered together and serves as masked and/or latent bis-PBD. In brief, secondary amine 28 was prepared under similar conditions as described above for 9. Coupling of 28 with 24 in the presence of PyBOP provided the bis-pro-PBD molecular architecture of 29. Carbohydrate-based folate-spacer unit 30 was synthesized using standard Fmoc-SPPS protocols as described in our C

DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Scheme 4. Formation of PBD 18 as the Result of Reaction of Model Conjugate 13 with DTTa

a

Reagents and conditions: (a) DTT, PBS buffer, 37 °C.

Scheme 5. Synthesis of Key Intermediate 24a

Reagents and conditions: (a) 1,5-dibromopentane, K2CO3, DMF, 60 °C, 80%; (b) Cu(NO3)2·3H2O, Ac2O, 0 °C, 82%; (c) aq. NaOH (1 M), THF, 40 °C; (d) Pd/C, H2, MeOH, 81% for 2 steps.

a

previous publication.26 Treatment of 30 with 29 in the presence of triethylamine under an inert atmosphere yielded the desired monoconjugate 31. After targeting FR-expressing cancer cells, such a conjugate would be expected to experience FR-mediated endocytosis resulting in on-site-generation of bisPBD (as depicted under the green dotted line in Scheme 6). As we have already seen, reducing one of the imine moieties to an amine can greatly improve the toxicity profiles of PBD dimers. Likewise, replacing one of the imine moieties in a bis-

PBD with an amido functionality also reduces the observed offtarget toxicity.27 Interestingly, amino and amido derivatives of PBD have also been found in nature.28 We designed a hybrid of a pro-PBD tethered to a 1,4-diazepine lactam and its corresponding folate conjugate. Our synthesis (Scheme 7) started with Boc-deprotection of the N-atom in 25 under acidic conditions to form 32, followed by coupling with a half equivalent of 24. The resulting monoester 33 was stirred overnight at room temperature, cyclizing smoothly to 1,4D

DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Scheme 6. Synthesis of Bis-pro-PBD Folate Conjugatea

a Reagents and conditions: (a) DIBAL, THF, −78 °C−rt; (b) (1) ethanolamine, MgSO4, DCM, rt; (2) mixed carbonate 11, Et3N, rt, 46% for 3 steps; (c) TFA/DCM (25%), rt; (d) 24, PyBOP, TEA, DCM, 88%; (e) TEA, DMSO/H2O (9:1), 24%.

Scheme 7. Synthesis of pro-PBD-Diazepine-Lactam Hybrid and Its Folate Conjugatea

a

Reagents and conditions: (a) TFA/DCM (25%) rt; (b) 24, PyBOP, DIPEA, DMF, rt; (c) stirring overnight, 86% for 3 steps; (d) 28, PyBOP, DIPEA, DCM, 58%; (e) 12, TEA, DMSO, 22%.

binds to the minor groove of double-stranded B-DNA with a preference for sequences rich in adenine and thymine.30 Consequently, we designed a hybrid pro-PBD/Hoechst dye 33258 as a novel latent warhead for targeted therapies. Phenol 20 was reacted with 1,5-dibromopentane in the presence of K2CO3 in refluxing acetone to afford the monoether 37. Regioselective nitration of 37 resulted in intermediate 38, which in turn was treated with Hoechst 33258 to provide bisether 40 in moderate yield. Methyl ester hydrolysis of 40 resulted in corresponding acid 41 and hydrogenation of the

diazepine lactam 34. The second coupling between 34 and amine 28 provided the pro-PBD-diazepine-lactam hybrid 35, activated for further conjugation. Conjugate 36 was assembled by tethering FA-spacer unit 12 to hybrid unit 35 in a disulfideexchange reaction similar to that described for the synthesis of conjugate 13. Although small in size, PBD monomers are known to be extremely sequence selective toward DNA, with the 5′-PuGPu sequence being most reactive.29 On the other hand, many years ago it was found that the dye Hoechst 33258 (Scheme 8, 39) E

DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Scheme 8. Synthesis of Hybrid of pro-PBD/Hoechst Dye 33258 and Its Conjugationa

Reagents and conditions: (a) 1,5-dibromopentane, K2CO3, acetone, reflux, 85%; (b) Cu(NO3)2·3H2O, Ac2O, 0 °C, 96%; (c) K2CO3, DMF, 60 °C, 18%; (d) LiOH, THF/MeOH/H2O; (e) Pd/C, H2, MeOH. (f) 28, PyBOP, DIPEA, DCM/DMF, 85%; (g) 30, MeOH/H2O, Et3N, 30%.

a

In addition, the activity of 44 against FR(+)-KB tumors in female nude mice was also investigated (Figure 3). Compound 44 showed excellent antitumor activity, with complete cures observed in five of the five mice in the study cohort. No weight loss was observed, a demonstration of the tolerance of this PBD conjugate at a dosage that would have been impossible with the free PBD-Hoescht hybrid 45. In conclusion, we have successfully applied the 1,3oxazolidine carbonate-iminium masking strategy to the synthesis of conjugated prodrug forms of PBDs, bis-PBDs, and PBD hybrids with other DNA-binders. Initial model studies clearly demonstrated that under reductive conditions, rapid linker cleavage takes place, resulting ultimately in the formation of a PBD construct. Preliminary preclinical pharmacological studies, which showed excellent biological activity and specificity, heralded the potential of our approach. Further studies on a series of such conjugates will be reported in due course. The examples presented were all folate-SMDCs. Based on our modular design concept,23 it is possible to envision a variety of conjugates which incorporate alternative targeting vectors. In an era of targeted medicines, we believe that choosing the right combination of latent warheads and selfimmolative linker systems might pave the way toward novel, highly specific therapeutic approaches.

nitro group formed intermediate 42. Freshly prepared 28 was condensed with 42 in the presence of PyBOP to provide the desired product 43. Treatment of 30 with 43 in MeOH/H2O with trimethylamine under inert atmosphere yielded conjugate 44. After targeting FR-expressing cancer cells, such a conjugate would be expected to experience FR-mediated endocytosis resulting in on-site-generation of pro-PBD−Hoechst 33258 hybrid 45. All of the folate conjugates were evaluated in vitro on folate receptor positive KB (FR(+)-KB) cells (Table 1) with and Table 1. Activity of Folate Conjugates on FR(+)-KB cells, 2 h Pulse at 37 °C compound

IC50 (nM)

specificitya (multiples)

31 36 44

1.30 1.00 0.70

17 240 96

a Specificity compares the activity of the compound alone against the activity of the compound with added folate competitor. e.g., 31 is 17 times more active alone than with folate competition.

without added folate competitor. All of the compounds showed activity in the 1 nM range. Importantly, the activity of compounds 36 and 44 was significantly suppressed (2 orders of magnitude or more) in competition experiments using 100-fold excess of folic acid, clearly demonstrating that the conjugate is targeting the FR. The relative lack of competition exhibited by 31 may be attributed to the two disulfide moieties present within this conjugate. We have found such constructs to be relatively unstable compared to conjugates containing a single disulfide bond and, as a result, have a tendency to release drug payloads prematurely. Compound 36 was also studied in the MDA-MB-231 cell line and found to have an IC50 of 0.44 nM.



EXPERIMENTAL SECTION (R)-2-(Pyridin-2-yldisulfanyl)ethyl-2-((S)-1-(tertbutoxycarbonyl)pyrrolidin-2-yl)oxazolidine-3-carboxylate (8). Prolinol (106.1 mg, 0.53 mmol) in DCM (2 mL) was treated with Dess-Martin periodinane (268.0 mg, 0.63 mmol) at rt and the reaction was stirred for 4 h. Then the reaction was quenched with MeOH/H2O (1:1, 2 mL), and extracted with DCM (2 mL, 3×). The organic phase was washed with brine (3 F

DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 3. In vivo evaluation of 44 on FR(+)-KB cells.

mL) and dried over anhydrous Na 2SO4, filtered, and concentrated. Product formation was confirmed by LC-MS. The aldehyde 7 was redissolved in CHCl3 (2 mL). To this solution was added ethanolamine (63.6 μL, 1.05 mmol) and anhydrous MgSO4 (600.00 mg, 4.98 mmol) at rt under argon atmosphere. The reaction was stirred for 1 h and filtered though a syringe filter. To the solution was added the mixed carbonate HCl salt 11 (607.10 mg, 1.58 mmol) in DCM (2 mL) and DIPEA (275.40 μL, 1.58 mmol). The reaction was kept at rt and stirred for additional 4 h. The product was isolated with prep-HPLC (10% MeCN to 100% MeCN with 50 mM NH4HCO3, pH = 7.4), to give 8 (97.2 mg, 40% yield for three steps). LC-MS: [M + H]+ m/z = 456. 1H NMR (CDCl3, 500 MHz): δ 8.45 (d, J = 3.3 Hz, 1H), 7.65 (m, 2H), 7.08 (t, J = 6.0 Hz, 1H), 5.20 (m, 1H), 4.32 (m, 3H), 4.08 (br, 1H), 3.88 (m, 2H), 3.40 (br, 1H), 3.31 (m, 2H), 3.05 (br, 2H), 1.80−2.00 (m, 5H), 1.40 (s, 9H). (R)-2-(Pyridin-2-yldisulfanyl)ethyl-2-((S)-pyrrolidin-2yl)oxazolidine-3-carboxylate (9). The Boc protecting group was removed from compound 8 (33.55 mg, 0.074 mmol) with TFA/DCM (1:1, 4 mL) at rt in 1 h. The solvent was then completely removed in vacuo, coevaporated with DCM (4 mL, 3×), and kept on the high vacuum for 1 h. LC-MS: [M + H]+ m/z = 356. 1H NMR (CDCl3, 500 MHz): δ 8.48 (s, 1H), 7.68 (s, 2H), 7.14 (d, J = 3.3 Hz, 1H), 5.20 (m, 1H), 4.42 (br, 2H), 4.15 (d, J = 4.5 Hz, 1H), 3.97 (d, J = 6.6 Hz, 1H), 3.77 (br, 1H), 3.49 (m, 2H), 3.09 (br, 2H), 2.87 (br, 2H), 2.21 (br, 1H), 2.06 (m, 3H). (R)-2-(Pyridin-2-yldisulfanyl)ethyl-2-((S)-1-(2-amino4,5-dimethoxybenzoyl)pyrrolidin-2-yl)oxazolidine-3carboxylate (10). 9 (0.074 mmol in DCM, 1 mL), and 2amino-4,5-dimethoxybenzoic acid (16.03 mg, 0.081 mmol, in DCM, 2 mL) were mixed and to this solution was added DIPEA (38.7 μL, 0.22 mmol) and PyBOP (115.5 mg, 0.22 mmol) sequentially at rt. The reaction was stirred under argon for 2 h. Then the solvent was removed in vacuo and the residue was redissolved in DMSO (4 mL) and purified with prepHPLC (10% MeCN to 100% MeCN with H2O) to provide the desired product 10 (10.0 mg, 25% yield). LC-MS: [M + H]+ m/z = 535. 1H NMR (CDCl3, 500 MHz): δ 8.46 (d, J = 3 Hz, 1H), 7.67 (m, 2H), 7.09 (t, J = 5.1 Hz, 1H), 6.75 (br, 1H), 6.20 (s, 1H), 5.32 (br, 1H), 4.83 (br, 1H), 4.35 (br, 5H), 3.83 (s, 3H), 3.78 (d, J = 4.0 Hz, 3H), 3.54 (br, 2H), 3.39 (m, 2H), 3.06 (br, 2H), 1.61−1.98 (m, 4H).

FA-Asp-Arg-Asp-Asp-Cys-Compound 10-Disulfide Exchange Product (13). Folate spacer 12 (24.0 mg, 0.023 mmol) was suspended in 2 mL water under Ar. To this suspension was added TEA (15.64 μL, 0.11 mmol) to make a clear solution. Compound 10 (10.0 mg, 0.019 mmol) was dissolved in DMSO (2 mL) and transferred into the solution of folate spacer 12. The reaction was stirred for 1 h and diluted with DMSO (2 mL), and loaded onto a prep-HPLC (5% MeCN to 50% MeCN in 50 mM NH4HCO3, pH = 7.4) and purified to give conjugate 13 (16.7 mg, 60% yield, 96% purity by UV (280 nm)). HRMS: [M+2H]2+ m/z = 735.244; calc. for C59H76N18O23S2 = 735.246. 1H NMR (DMSO-d6:D2O 9:1, 500 MHz): δ 8.61 (s, 1H), 7.56 (d, J = 9.0 Hz, 2H), 6.62 (d, J = 8.5 Hz, 2H), 6.56 (m, 1H), 6.31 (d, J = 7.5 Hz, 1H), 5.27 (br, 1H), 4.55−4.35 (m, 6H), 4.25−4.05 (m, 5H), 3.76 (br, 2H), 3.65 (s, 3H), 3.59 (s, 3H), 3.50−2.75 (m, 10H), 2.65−2.35 (m, 6H), 2.13 (t, J = 7.0 Hz, 2H), 2.05−1.70 (m, 6H), 1.65−1.30 (m, 4H). 13C NMR (DMSO-d6:D2O 9:1, 500 MHz): δ 175.46, 175.05, 174.08, 173.80, 173.52, 173.40, 173.30, 172.29, 172.03, 171.43, 171.09, 166.93, 162.51, 157.06 (2C), 155.56, 154.25 (2C), 150.98, 149.55, 149.14, 140.05, 129.17 (3C), 127.79, 121.74, 112.74, 111.98 (3C), 100.57 (2C), 63.46, 56.51, 56.50, 55.63 (3C), 53.84 (2C), 52.74, 50.71, 50.53, 50.28, 45.90, 42.03, 40.62, 37.53, 37.20 (3C), 32.14 (2C), 29.27, 28.23, 24.53, 21.68 (2C). 1,5-Di-(3-methoxy-4-oxy-methyl benzoate-4-yl)-pentane (21). Methyl vanillate 20 (6.84 g, 37.5 mmol) was dissolved in dry DMF (30 mL) and to this solution was added 1,5-dibromopentane (2.44 mL, 17.9 mmol) and K2CO3 (19.7 g, 142 mmol). The reaction was heated to 75 °C under Ar for 3 h. The reaction was cooled to rt and the solid was filtered out. The filtrate was diluted with ether (200 mL) and the resulting organic phase was washed with 10% aq. NH4Cl solution 3 times, with white ppt formation in the organic layer. Dichloromethane was added to the organic layer to dissolve white ppt, then washed again with 10% aq. NH4Cl solution to remove more DMF. Organic layer was dried over anhydrous sodium sulfate. After filtration the solution was evaporated under high vacuum. 21 (7.92g, 95% purity) was obtained in quantitative yield. HRMS: [M + H]+ m/z = 433.196; calc. for C23H28O8 = 433.186. 1H NMR (CDCl3, 500 MHz): δ 7.65 (dd, J = 2 Hz, 9 Hz, 2H)), 7.53 (d, J = 2 Hz, 2H), 6.86 (d, J = 9 Hz, 2H), 4.10 (d, J = 7 Hz, 4H), 3.90 (s, 6H), 3.89 (s, 6H), 1.95 (m, 4H), 1.69 (m, 2H). 13C NMR (CDCl3, 125 MHz): δ 166.9, G

DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

amine (81.4 μL, 1.35 mmol) dropwise, and stirred at rt under argon atmosphere. After 1 h, 11 (691.98 mg, 1.798 mmol) was added, followed by TEA (376.0 μL, 2.697 mmol) and stirred at rt for 2 h. The reaction was diluted with DCM and washed with water. Organic layer was dried over anhydrous Na2SO4 and concentrated to 0.5 mL, then purified by silica chromatography in 10−100% EtOAc/petroleum ether. 27 was obtained as a foamy white solid (193.4 mg, 46% yield over 3 steps). HRMS: [M + H]+ m/z = 468.177; calc. for C21H29N3O5S2 = 468.163. 1 H NMR (CDCl3, 500 MHz): δ 8.47 (d, J = 5 Hz, 1H), 7.66 (m, 2H), 7.09 (m, 1H), 5.16 (br, 1H), 4.97 (br, 2H), 4.38 (br, 3H), 4.05 (br, 2H), 3.85 (br, 3H), 3.20 (m, 1H), 3.06 (br, 2H), 2.85 (br, 1H), 2.52 (m, 1H), 1.55 (s, 3H), 1.43 (s, 9H). Bis(2-(pyridin-2-yldisulfanyl)ethyl)-2,2′-((2S,2′S)-1,1′(4,4′-(pentane-1,5-diylbis(oxy))-bis(2-amino-5-methoxybenzoyl))-bis(4-methylenepyrrolidine-2,1-diyl))-bis(oxazolidine-3-carboxylate) (29). 27 (44.6 mg, 0.095 mmol) was dissolved in a solution of TFA (1 mL) and DCM (1 mL), and stirred at rt under argon atmosphere for 1 h. The reaction was concentrated and coevaporated with DCM (1 mL) three times, affording crude 28 as a dark, reddish-brown oil. LC-MS: [M + H]+ m/z = 368. The solution of the mixture of 28 (20.0 mg, 0.0544 mmol) and intermediate 24 (10.7 mg, 0.0247 mmol) in DCM/DMF (0.25 mL/0.25 mL) was treated with DIPEA (17.2 μL, 0.0988 mmol). To the resulting clear solution was added PyBOP (28.3 mg, 0.0544 mmol) at rt under argon atmosphere and stirred overnight. The reaction was concentrated in vacuo and purified with prep-HPLC (10% MeCN to 100% MeCN in 50 mM NH4HCO3, pH = 7.4) to give the bis pro-PBD product 29 (24.5 mg, 88% yield). HRMS: [M + H]+ m/z = 1133.356; calc. for C53H64N8O12S4 = 1133.360. 1H NMR (CDCl3, 500 MHz, selected data): δ 8.41 (m, 2H), 7.61 (m, 4H), 7.07 (m, 2H), 6.82 (br, 1H), 6.71 (br, 1H), 6.20 (br s, 2H), 1.85 (m, 4H), 1.62 (m, 2H). 13C NMR (CDCl3, 125 MHz): δ 169.8, 159.7, 154.0, 151.3, 149.7, 144.6, 141.5, 137.2, 120.8, 119.8, 113.3, 109.7, 107.9, 106.8, 102.0, 88.3, 68.5, 66.0, 63.8, 57.2, 53.2, 49.0, 45.1, 37.6, 29.7, 28.8, 22.6. Sacchro-Peptidic Folate Spacer 30-Compound 29Monodisulfide Exchange Product (31). Folate spacer 30 (8.25 mg, 0.0049 mmol) was dissolved in DMSO/H2O (7:3, 0.4 mL) and added dropwise to a solution of compound 29 (6.13 mg, 0.0055 mmol) in DMSO (0.2 mL) at rt under argon. The reaction was treated with TEA (4.1 μL, 0.0294 mmol) and monitored with LC-MS. When the reaction was complete, it was purified with prep-HPLC (10% MeCN to 100% MeCN in 50 mM NH4HCO3, pH = 7.4) to provide the mono conjugate 31 (3.2 mg, 24% yield, 90% pure by UV (280 nm)) with recovery of 29. HRMS: [M+2H]2+ m/z = 1350.982; calc. for C113H157N23O46S4 = 1350.985. 1H NMR (DMSO-d6:D2O 9:1, 500 MHz, selected data): δ 8.59 (s, 1H), 8.28 (b, 1H), 7.69 (b, 1H), 7.62 (b, 1H), 7.54 (d, J = 8.3, 2H), 7.15 (b. 1H), 6.62 (d, J = 8.8, 2H), 6.56 (b, 2H), 6.32 (b, 2H). (S)-2-Amino-5-methoxy-4-((5-((7-methoxy-2-methylene-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)benzoic acid (34). Compound 25 was deprotected with TFA/DCM (1:4) as for compounds 8 and 27. The crude 32 (0.047 mmol) in DCM (1 mL) was added to the solution of 24 (36.5 mg, 0.084 mmol) in DMF (1 mL). The mixture was treated with DIPEA (32.8 μL, 0.188 mmol) and followed by PyBOP (25.7 mg, 0.049 mmol) at rt under argon. The coupling reaction was completed in 1.5 h, but the reaction mixture was kept stirring

152.5, 148.9, 132.1, 132.0, 131.9, 128.5, 128.4, 123.5, 122.6, 112.4, 111.5, 68.7, 56.0, 51.9, 28.7, 22.5, 21.9. 1,5-Di-(3-methoxy-4-oxy-2-nitro-methyl benzoate-4yl)-pentane (22). 21 (201.2 mg, 0.465 mmol) in Ac2O (1.2 mL) was cooled to 0 °C and then Cu(NO3)2·3H2O (280.3 mg, 1.16 mmol) was added slowly, and after 1 h, the ice-bath was removed. The reaction was stirred at rt for 4 h. The reaction was poured into ice water and stirred for 1 h until a yellow precipitate formed. The precipitate was collected by filtration. The solid was washed with more cold water (2 mL, 3×) and air-dried to give 22 (198.4 mg, 82% yield). HRMS: [M + H]+ m/z = 523.158; calc. for C23H26N2O12 = 523.156. 1H NMR (CDCl3, 500 MHz): δ 7.44 (s, 2H), 7.10 (s, 2H), 4.12 (t, J = 6.5 Hz, 4H), 3.95 (s, 6H), 3.90 (s, 6H), 1.97 (m, 4H), 1.71 (m, 2H). 13C NMR (CD2Cl2, 125 MHz): δ 166.1, 152.9, 150.0, 141.2, 121.3, 111.0, 108.0, 69.4, 56.6, 28.5, 22.5. Note: Carbon signal of methyl ester (prediction 51.6 ppm) likely obscured by solvent signal. 1,5-Di-(3-methoxy-4-oxy-2-nitro-benzoic acid-4-yl)pentane (23). 22 (198.4 mg) was dissolved in THF (2 mL) and treated with aq. NaOH (2 mL, 1 M) and heated to 40 °C for 3 h. The solvent was removed in vacuo. The aqueous phase was acidified to pH 1 with concentrated HCl to form a precipitate, which was collected by filtration and was washed with H2O (3 × 1 mL). The solid was air-dried to give the acid 23 (187.7 mg, quantitative yield). HRMS: [M + H]+ m/z = 495.122; calc. for C21H22N2O12 = 495.125. 1H NMR (DMSOd6, 500 MHz): δ 7.37 (s, 2H), 7.11 (s, 1H), 4.06 (t, J = 7 Hz, 4H), 1.80 (m, 4H), 1.56 (m, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 167.1, 151.8, 148.2, 141.5, 111.6, 107.8, 69.2, 56.5, 28.6, 22.5. 1,5-Di-(3-methoxy-4-oxy-2-amino-benzoic acid-4-yl)pentane (24). Acid 23 was dissolved in 0.5 M aq. NaOH (6 mL) and hydrogenation was carried out with Pd/C (10%, 4.82 mg) under H2 (45 psi) in the Parr hydrogenator. The reaction was shaken for 5 h and then filtered though a pad of Celite. The filtrate was adjusted to pH 2−3 with concentrated HCl with stirring and a precipitate formed. The precipitate was isolated by filtration and washed with H2O (3 × 1 mL). The solid was dried in a desiccator in the presence of P2O5 under high vacuum overnight. 24 was obtained as a brown solid (34.2 mg, 81% yield). HRMS: [M + H]+ m/z = 435.174; calc. for C21H26N2O8 = 435.177. 1H NMR (DMSO-d6, 500 MHz): δ 7.11 (s, 2H), 6.32 (s, 2H), 3.92 (t, J = 6.6 Hz, 4H), 3.61 (s, 6H), 1.78 (m, 4H), 1.57 (m, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 169.5, 154.5, 148.7, 139.7, 114.2, 101.0, 100.3, 68.2, 56.6, 28.7, 22.7. (R)-2-(Pyridin-2-yldisulfanyl)ethyl-2-((S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidin-2-yl)oxazolidine-3carboxylate (27). 25 (2.00 g, 8.3 mmol, GreenChemPharm) was dissolved in a solution of dichloromethane (10 mL) and toluene (30 mL), and then cooled to −78 °C with stirring. DIBAL in toluene (1.0 M, 16.6 mL, 16.6 mmol) was added slowly dropwise over 30 min and then the reaction was stirred for 4 h at −78 °C. Methanol (344 μL, 8.5 mmol) was added dropwise and the reaction mixture was stirred for 30 min at −78 °C. Hydrochloric acid solution (5%, 238 mL) was added to the reaction mixture and then extracted with ethyl acetate (106 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and evaporation to yield crude 26 as a clear oil. LC-MS: [M + H]+ m/z = 212. To a stirring solution of 26 (0.899 mmol, crude, from above) in DCM (2 mL) was added MgSO4 powder (300 mg, Mallinckrodt), then ethanolH

DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

acetone (dried though a pad of Na2SO4, 48.4 mL) and to this solution was added 1,5-dibromopentane (49.4 mL, 36.3 mmol) and K2CO3 (6.69 g, 48.4 mmol). The reaction was heated to reflux under Ar for 6 h. The reaction was cooled to rt and the solid was removed by filtration. The filtrate was concentrated and purified with silica chromatography in 0−30% EtOAc/ petroleum ether to obtain 37 (3.39 g, 84.5% yield) as a solid. HRMS: [M + H]+ m/z = 331.064; calc. for C14H19BrO4 = 331.055. 1H NMR (CDCl3, 500 MHz): δ 7.65 (dd, J = 8.5, 2.0 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.91 (s, 3H), 3.89 (s, 3H), 3.44 (t, J = 6.5 Hz, 2H), 2.00−1.90 (m, 4H), 1.65 (m, 2H). 13C NMR (CD3OD, 125 MHz): δ 167.1, 152.9, 149.0, 123.4, 122.2, 112.2, 111.6, 68.4, 55.1, 48.1, 32.8, 32.3, 27.9, 24.5. Methyl 4-((5-Bromopentyl)oxy)-5-methoxy-2-nitrobenzoate (38). 37 (3.39 g, 10.2 mmol) in Ac2O (52 mL) was cooled to 0 °C and treated with Cu(NO3)2·3H2O (2.97 g, 12.3 mmol) by slow addition. The reaction was stirred at 0 °C for 1 h then at rt for 2 h. After the reaction was completed, it was poured into ice water and stirred for 1 h. The resultant precipitate was collected by filtration. The product was washed with water (3×) and air-dried to give 38 (3.71 g, 96% yield). HRMS: [M + H]+ m/z = 376.040; calc. for C14H18BrNO6 = 376.040. 1H NMR (CDCl3, 500 MHz): δ 7.41 (s, 1H), 7.05 (s, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.94 (s, 3H), 3.89 (s, 3H), 3.42 (t, J = 7.0 Hz, 2H), 1.93 (m, 4H), 1.63 (m, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 165.8, 152.8, 150.1, 141.3, 120.7, 111.7, 108.6, 69.5, 57.0, 53.4, 35.5, 32.3, 27.9, 24.6. Methyl 5-Methoxy-4-((5-(4-(5-(4-methylpiperazin-1yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)pentyl)oxy)-2-nitrobenzoate (40). A solution of 38 (45 mg, 0.12 mmol) in DMF (1 mL) was added to a stirring solution of Hochest dye 39 (53 mg, 0.1 mmol) and K2CO3 (55 mg, 0.4 mmol) in DMF (1 mL) at 100 °C over the course of 3 h via syringe pump. The reaction was then stirred for an additional 2 h before allowing it to cool to rt. The reaction mixture was then concentrated, dissolved in DCM with a small amount of MeOH, and purified via silica chromatography in 0−20% MeOH/chloroform with 5% NH4OH to give 40 (33 mg, 46% yield). HRMS: [M + H]+ m/z = 720.321; calc. for C39H41N7O7 = 720.315. 1H NMR (DMSO-d6, 500 MHz): δ 8.35 (d, J = 10.4 Hz, 0.5H), 8.22 (d, J = 15.3 Hz, 0.5H), 8.14 (dd, J = 8.8, 2.9 Hz, 2H), 8.02 (d, J = 8.0 Hz, 0.5H), 7.96 (d, J = 8.5 Hz, 0.5H), 7.71 (d, J = 8.4 Hz, 0.5H), 7.65 (s, 1H), 7.60 (d, J = 8.4 Hz, 0.5H), 7.48 (d, J = 8.7 Hz, 0.5H), 7.36 (d, J = 8.3 Hz, 0.5H), 7.31 (s, 1H), 7.12 (d, J = 8.2 Hz, 2H), 6.92 (m, br, 2H), 4.16 (t, J = 6.5 Hz, 2H), 4.09 (t, J = 6.5 Hz, 2H), 3.92 (s, 3H), 3.82 (s, 3H), 3.13 (m, br, 4H), 2.50 (td, J = 4.0, 2.2 Hz, 5H), 2.24 (s, 3H), 1.83 (m, 4H), 1.64−1.55 (m, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 165.84, 160.73, 153.37, 153.13, 152.79, 150.17, 145.32, 144.61, 141.34, 138.60, 135.78, 128.71, 128.65, 122.72, 120.63, 119.07, 115.34, 113.76, 111.84, 111.72, 111.40, 109.13, 108.58, 97.64, 69.56, 68.08, 57.03, 55.33, 53.46, 50.71, 50.26, 46.24, 40.49, 40.32, 40.16, 39.99, 39.82, 39.65, 39.49, 28.72, 28.51, 22.55. 5-Methoxy-4-((5-(4-(5-(4-methylpiperazin-1-yl)1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)pentyl)oxy)-2-nitrobenzoic acid (41). Ester 40 (13.1 mg, 0.0182 mmol) was dissolved in THF/MeOH/H2O (3/1/1, 0.2 mL) and treated with aq. LiOH solution (1 M, 36 μL) for 4 h at rt under Ar. Most of the solvent was removed in vacuo and the aqueous phase was acidified with concentrated HCl to pH 2−3. The precipitate was collected by filtration. The filter cake was

for overnight to allow for lactam formation. The reaction was then concentrated in vacuo. The residue was purified with silica chromatography in 0−20% MeOH/DCM to afford 34 (21.2 mg, 86% yield for 3 steps). HRMS: [M + H]+ m/z = 526.232; calc. for C27H31N3O8 = 526.219. 1H NMR (CD3OD, 500 MHz): δ 7.22 (s, 1H), 7.12 (s, 1H), 6.69 (s, 1H), 6.32 (s, 1H), 5.07 (br, 2H), 4.24 (m, 1H), 4.21 (br, 1H), 4.02 (m, 1H), 4.00 (t, J = 7.0 Hz, 2H), 3.93 (t, J = 6.5 Hz, 2H), 3.76 (s, 3H), 3.62 (s, 3H), 3.18 (d, J = 15 Hz, 1H), 2.77 (m, 1H), 1.79 (m, 4H), 1.54 (m, 2H). 13C NMR (CD3OD, 125 MHz): δ 169.92, 169.07, 164.46, 154.04, 151.17, 148.28, 145.47, 142.77, 139.23, 130.82, 117.89, 113.72, 111.86, 107.90, 105.19, 100.60, 99.82, 68.26, 67.70, 56.35, 56.15, 55.68, 51.07, 31.41, 28.21, 28.14, 22.23. 2-(Pyridin-2-yldisulfanyl)ethyl-2-((S)-1-(2-amino-5methoxy-4-((5-(((S)-7-methoxy-2-methylene-5,11-dioxo2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)benzoyl)-4-methylenepyrrolidin-2-yl)oxazolidine-3-carboxylate (35). To the solution of compound 34 (25.3 mg, 0.048 mmol) and compound 28 (0.055 mol, crude from 27) in DCM (2 mL) was added DIPEA (33.5 μL, 0.192 mmol), followed by addition of PyBOP (49.95 mg, 0.096 mmol). The reaction was stirred for 3.5 h at rt. Then the reaction was concentrated in vacuo and the residue was purified with silica chromatography in 0−10% MeOH/DCM to provide 35 (24.3 mg, 58% yield). HRMS: [M + H]+ m/z = 875.323; calc. for C43H50N6O10S2 = 875.311. 1H NMR (CD3OD, 500 MHz): δ 8.37 (br, 1H), 7.78 (br, 2H), 7.33 (s, 1H), 7.19 (br, 1H), 6.73 (m, 1H), 6.64 (m, 1H), 6.35− 6.45 (m, 1H), 5.13 (d, J = 14 Hz, 2H), 5.05 (s, 1H), 5.00 (s, 1H), 4.95 (m, 1H), 4.22−4.38 (m, 4H), 4.18−3.96 (series of m, 6H), 3.85 (s, 3H), 3.72 (m, 7H), 3.35 (d, J = 16 Hz, 1H), 3.15 (m, 3H), 3.04 (br, 1H), 2.87 (m, 2H), 2.58 (m, 1H), 1.88 (m, 4H), 1.67 (m, 2H). FA-Asp-Arg-Asp-Asp-Cys-Compound 35-Disulfide Exchange Product (36). The solution of pure 35 (24.3 mg, 0.0278 mmol) in DMSO (2 mL) was added to a solution of 12 (36.3 mg, 0.0347 mmol) in DMSO (3 mL) at rt. Then to the reaction mixture was added TEA (23.3 μL, 0.167 mmol) and the reaction was stirred for 1 h. The product was isolated with prep-HPLC (10% MeCN to 100% MeCN in 50 mM NH4HCO3, pH = 7.4) to afford the conjugate 36 (28.8 mg, 57% yield, 96% pure by UV (280 nm)). HRMS: [M+2H]2+ m/ z = 905.331; calc. for C78H96N20O27S2 = 905.318. 1H NMR (DMSO-d6, 500 MHz, selected data): δ 8.60 (s, 1H), 8.35 (b, 1H), 8.25 (b, 1H), 8.08 (d, J = 7.5 Hz, 1H), 7.87 (b, 1H), 7.79 (b, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.46 (s, 1H), 7.34 (b, 2H), 7.20 (s, 1H), 7.05 (b, 1H), 6.93 (m, 1H), 6.71 (s, 1H), 6.59 (d, J = 6.0 Hz, 3H), 6.35 (s, 1H), 5.05 (b, 3H), 4.96 (d, J = 12.5 Hz, 2H), 4.48 (m, 3H), 4.36 (m, 1H), 4.23 (m, 4H), 4.20−3.85 (m, 10H), 3.77 (s, 3H), 3.63 (s, 4H). 13C NMR (DMSO-d6, 125 MHz): δ 176.18, 175.82, 175.18 (3C), 174.01, 173.88, 172.85, 172.57, 172.46, 172.16, 171.49, 170.88, 167.34, 166.00, 165.93, 162.76, 157.04, 154.30 (2C), 151.94, 151.11, 150.98, 149.89, 149.34, 146.11 (2C), 142.22 (2C), 130.97, 129.33 (2C), 127.81, 121.83, 118.09, 114.78, 112.22 (2C), 111.89, 109.27, 109.20, 105.56, 101.70, 101.56, 100.42, 69.04 (3C), 68.48, 68.44, 63.71, 57.18 (2C), 56.19 (3C), 54.30, 54.14, 51.59, 51.26, 50.83, 50.66, 46.58 (2C), 45.95, 40.70, 39.84 (2C), 36.92, 32.24, 31.70, 28.50, 28.41 (2C), 22.64, 22.07 (2C), 8.92. Methyl 4-((5-Bromopentyl)oxy)-3-methoxybenzoate (37). The phenol 20 (2.20 g, 12.1 mmol) was dissolved in I

DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

solution of 43 (5.0 mg, 0.0049 mmol) in DMSO (0.2 mL), followed by the addition of DIPEA (5.1 μL, 0.029 mmol). The reaction was stirred at rt under argon for 30 min. The reaction was purified with prep-HPLC (10 to 100% ACN in 50 mM NH4HCO3, pH = 7.4) to give the conjugate 44 (3.8 mg, 30% yield, 98% purity (mixture of diastereomers) at 280 nm). HRMS: [M+2H]2+ m/z = 1297.518; calc. for C114H153N25O41S2 = 1297.514. 1H NMR (DMSO-d6, D2O (9:1), 500 MHz, selected data): δ 8.62 (s, 1H), 8.27 (br s, 1H), 8.10 (d, J = 9 Hz, 2H), 7.95 (d, J = 8.5 Hz, 1H), 7.66 (br, 1H), 7.59 (d, J = 8 Hz, 2H), 7.45 (br d, J = 8.5 Hz, 1H), 7.10 (d, J = 9 Hz, 2H), 7.05 (br s, 1H), 6.95 (d, J = 9.5 Hz, 1H), 6.62 (d, J = 8 Hz, 2H), 6.36 (s, 1H), 5.0−4.9 (m, 3H), 4.46 (s, 2H). Cell Growth Inhibition Studies. KB cells were maintained in folate-free RPMI medium (FFRPMI) containing 10% heatinactivated fetal calf serum (HIFCS) at 37 °C in a 5% CO2/ 95% air-humidified atmosphere with no antibiotics. Exponentially growing cells were seeded in 24-well plates 24 h before treatment with drugs (n = 3). Cells receiving FR targeted drugs were pulsed for 2 h at 37 °C, rinsed 4 times with 0.5 mL of medium, and then chased in 1 mL of fresh medium for 72 h. Cells were treated with fresh medium containing 3H-thymidine for 2 h at 37 °C, washed with phosphate-buffered saline (PBS), and then treated with ice-cold 5% trichloroacetic acid (TCA). After 15 min, the TCA was aspirated and cells solubilized by the addition of 0.25 N sodium hydroxide for 15 min at room temperature. Each solubilized sample was transferred to scintillation vials containing EcoLume scintillation cocktail and counted in a liquid scintillation counter. In Vivo Antitumor Experiments. Four- to eight-week-old female nu/nu mice (Harlan Sprague−Dawley, Inc.) were maintained on a standard 12 h light−dark cycle and fed ad libitum with a low-folate chow (Harlan Teklad diet #TD.01013, Madison, WI) for the duration of dosing and 1 week post dosing schedule. Tumor cells (1 × 106 per nu/nu mouse) in 100 μL were injected in the subcutis of the dorsal medial area. Mice were divided into groups of 5, and freshly prepared test articles were injected through the lateral tail vein under sterile conditions in a volume of 200 μL of PBS. Intravenous treatments typically initiated on days 5−10 PTI. The mice in the control groups received no treatment. Growth of each subcutaneous tumor was followed by measuring the tumor 3 times per week during treatment and twice per week thereafter, until a volume of 1500 mm3 was reached. Tumors were measured in 2 perpendicular directions using Vernier calipers, and their volumes were calculated as V = 0.5 × L × W2, where L = measurement of longest axis in mm and W = measurement of axis perpendicular to L in mm. As a general measure of gross toxicity, changes in body weights were determined on the same schedule as tumor volume measurements.

washed with water (3×) and air-dried to give crude 41 (12.8 mg). A portion of this material was purified via silica chromatography (0 to 20% MeOH in CHCl3 with 0.5% ammonium hydroxide (concentrated)) to provide material for analysis. HRMS: [M + H]+ m/z = 706.301; calc. for C38H39N7O7 = 706.299. 1H NMR (DMSO-d6, 500 MHz) δ 8.32 (s, 1H), 8.11 (d, J = 8.8 Hz, 2H), 7.97 (dd, J = 8.4, 1.7 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.56 (d, 1H), 7.55 (s, 1H) 7.24 (s, 1H), 7.13 (m, 4H), 4.12 (t, J = 6.5 Hz, 2H), 4.09 (t, J = 6.3 Hz, 2H), 3.88 (m, 2H), 3.22 (m, 3H), 3.00 (s, 3H), 2.87 (s, 3H), 1.82 (p, J = 6.9 Hz, 4H), 1.58 (p, J = 7.9 Hz, 3H). 13C NMR (DMSO-d6, 125 MHz): δ 166.19, 161.57, 152.22, 149.86, 148.85, 148.59, 141.70, 129.74, 121.40, 115.52, 114.85, 111.59, 108.27, 99.47, 70.14, 69.54, 68.28, 57.02, 55.57, 52.50, 46.65, 42.47, 29.28, 24.84. (R)-2-(Pyridin-2-yldisulfanyl)ethyl-2-((S)-1-(2-amino-5methoxy-4-((5-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)pentyl)oxy)benzoyl)-4-methylenepyrrolidin-2-yl)oxazolidine-3-carboxylate (43). 41 (15.7 mg, 0.022 mmol) in MeOH (10 mL) was subjected to hydrogenation in a Parr shaker (10% wet Pd/ C, 5 wt %, 7.85 mg, H2 41 PSI) for 2 h. The catalyst was removed by filtration though a pad of Celite. The filtrate was concentrated in vacuo to give crude 42. A sample of this material was purified via silica chromatography (0 to 20% MeOH in CHCl3 with 0.5% ammonium hydroxide (concentrated)) to provide material for analysis. HRMS: [M + H]+ m/z = 676.331; calc. for C38H41N7O5 = 676.325. 1H NMR (DMSOd6, 500 MHz): δ 8.39 (s, 1H), 8.14 (d, J = 8.7 Hz, 2H), 7.99 (d, J = 8.4, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.30 (d, J = 9.1, 1H), 7.21 (d, J = 2.2 Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 7.13 (s, 1H), 6.34 (s, 1H), 4.11 (t, J = 6.3 Hz, 2H), 3.96 (t, J = 6.4 Hz, 2H), 3.63 (s, 3H), 3.23 (m, 2H), 3.05 (m, 2H), 2.88 (s, 2H), 2.51 (s, 3H), 1.81 (h, J = 7.1 Hz, 3H), 1.58 (m, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 168.92, 162.88, 153.99, 152.40, 149.11, 147.78, 141.48, 137.08, 133.58, 130.75, 126.61, 124.69, 119.61, 117.68, 116.88, 115.79, 115.30, 114.83, 114.36, 114.04, 104.26, 102.36, 99.26, 68.51, 68.40, 56.60, 52.45, 46.57, 42.31, 28.67, 22.59. The crude product in DMF (0.5 mL) was mixed with a solution of 28 (8.81 mg, 0.024 mmol) in DCM (2.0 mL). The reaction mixture was treated with PyBOP (20.8 mg, 0.04 mmol) and DIPEA (13.9 μL, 0.08 mmol) under Ar at rt. The reaction was stirred overnight and then purified with Prep-HPLC (Mobile phase A: 50 mM NH4HCO3 buffer, pH = 7.0; B = ACN. Method: 10−100 B% in 30 min) to afford 43 (17.4 mg, 85% yield over two steps). HRMS: [M + H]+ m/z = 1025.420; calc. for C54H60N10O7S2 = 1025.417. 1H NMR (CD3OD, 500 MHz, selected data): δ 8.36 (s, 1H), 8.23 (s, 1H), 8.03 (m, 2H), 7.96 (m, 1H), 7.77 (m, 3H), 7.69 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 9.0 Hz, 1H), 7.16 (m, 2H), 7.06 (m, 4H), 6.43 (m, 1H), 5.02 (m, 3H), 4.10 (m, 4H), 3.75 (m, 4H), 1.82 (m, 4H), 1.65 (m, 2H). 13C NMR (CD3OD, 125 MHz): δ 161.2, 159.7, 153.9, 152.4 (2C), 151.6 (2C), 149.0, 147.9 (2C), 144.6 (2C), 141.0, 137.7 (3C), 128.1 (3C), 124.2, 121.4, 121.0, 119.8, 115.0 (2C), 114.7 (3C), 114.5, 113.2 (4C), 101.6 (3C), 68.2 (2C), 65.5, 67.7, 63.2, 56.3 (3C), 54.6 (2C), 50.1 (2C), 44.3, 37.3, 28.5 (2C), 22.4. Notes: Carbonyl carbon expected at 168.9 not observed. N-methyl expected at 46.6 obscured by solvent signal. Sacchro Peptidic Spacer 30-Compound 43-Disulfide Exchange Product (44). 30 (10.24 mg, 0.006 mmol) was dissolved in DMSO (0.3 mL) and water (0.2 mL) and bubbled with Ar at rt in an amber vial. To this solution was added a



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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00476. General methods and instrumentation; 1H and 13C NMR spectra for selected compounds 13−44 (PDF)



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DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry ORCID

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Paul J. Kleindl: 0000-0003-2236-3999 Fei You: 0000-0001-9457-7916 Notes

The authors declare the following competing financial interest(s): All authors are/were employees and/or stockholders in Endocyte Inc.



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DOI: 10.1021/acs.bioconjchem.7b00476 Bioconjugate Chem. XXXX, XXX, XXX−XXX