Synthesis and Evaluation of Dolastatin 10 Analogues Containing

Sep 8, 2017 - Julien Dugal-Tessier, Stuart D. Barnscher†, Akira Kanai, and Brian A. Mendelsohn. Agensys Inc., an affiliate of Astellas Pharma Inc., ...
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Synthesis and Evaluation of Dolastatin 10 Analogues Containing Heteroatoms on the Amino Acid Side Chains Julien Dugal-Tessier, Stuart D. Barnscher,† Akira Kanai, and Brian A. Mendelsohn* Agensys Inc., an affiliate of Astellas Pharma Inc., 1800 Stewart Street, Santa Monica, California 90404, United States S Supporting Information *

ABSTRACT: Synthetic analogues of the natural occurring dolastatin 10 are of great interest in cancer due to their potent in vitro activity and their uses as payloads in antibody drug conjugates (ADCs). Modification of the dolastatin 10 core scaffold has mainly focused on modifications of the P1, N-terminus, and P5, C-terminus, with minimal attention to the P2 subunit. In this paper we discuss the introduction of heteroatoms to the P2 side chain, which results in potent activity in vitro. The most active compounds contained azides in the P2 unit and required a phenylalanine-derived P5 subunit.

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demonstrated sub-nanomolar activity in vitro toward a variety of cancer cell lines but were found to show no clinical benefit in a variety of indications.5−17 Potent natural products and their derivatives, despite their promise in vitro, have not been as successful in the clinic as their potency would suggest.3,18 As a consequence, dolastatin 10 has been modified to be used as a cytotoxic molecule in targeted therapies. One targeted therapy approach has used an auristatin E derivative, monomethyl auristatin E (MMAE), the active payload in Adcetris.19 Other synthetic dolastatin 10 derivatives, auristatins, are being investigated as the cytotoxic molecule in antibody drug conjugate (ADC) clinical trials.18 For this reason derivatives of dolastatin 10 are still of great interest. Dolastatin 10 is a pentapeptide; each amino acid subunit is sequentially named P1 to P5 starting from the N-terminus (Figure 1A). Figure 1B illustrates some of the modification of the dolastatin 10 around the P5 subunit.20,21 The most prevalent areas of modification have centered around the P1 and P5 subunits and the introduction of additional groups to extend the pentapeptide motif on either side of the linear peptide.20 Modifications of the P2−P4 subunits, the core structure, have been underrepresented, as changes to these subunits result in attenuated compound potency.21 In particular, modification of the P2 subunit has received little attention outside of substituting the natural valine residue with leucine or isoleucine.22 In this paper we discuss modification of the P2 side chain that allows the introduction of heteroatoms and other nonnatural amino acids while maintaining potent in vitro activity.

ancer is a leading cause of death worldwide, and as a growing burden on society it remains an active field of scientific research.1 One area that has been successful in the advancement of cancer treatment has been the search and isolation of natural products that have potent cell-based activity.2 Many of these natural products are highly cytotoxic against in vitro models, but when administered in vivo these natural products demonstrate systemic toxicity, which limits their use as single-agent therapeutics.3 One such natural product is dolastatin 10 (D10) isolated from the sea hare Dolabella auricularia from the Pettit lab (Figure 1).4 Dolastatin 10 and analogues (i.e., TZT-1027)

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RESULTS AND DISCUSSION In our recent report, we investigated the addition of heterocycles in the P5 subunit based on the reports from Pettit.23,24 These P5 Received: April 24, 2017 Published: September 8, 2017

Figure 1. Dolastatin derivatives. Dov, dolavaline; Dil, (3R,4S,5S)dolaisoleuine; Dap, (2R,3R,4S)-dolaproine; Doe, (S)-dolaphenine. © 2017 American Chemical Society and American Society of Pharmacognosy

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Scheme 1. General Synthesis of Auristatins

a

CMPI, iPrNEt2, EtOAc. bPiperidine, MeCN. cDolavaline, HATU, iPrNEt2, DMF. dTFA, CH2Cl2. eHATU, iPrNEt2, DMF. fTFA, CH2Cl2. gP4−P5, HATU, iPrNEt2, DMF.

P2 heteroatoms did not result in activity as potent as expected with the pyridine P5, we decided to explore different P5s with higher homology to the dolastatin 10 P5. We focused on L-phenylalanine (Phe) methyl ester based P5s that would allow the compounds to be more membrane permeable (4−6). These compounds were more potent than the corresponding pair (compounds 1−3) with a pyridine P5. Having obtained 1000 3.2 0.16 0.076 3.4 × 102 >1000 >1000 4.4 3.5 2.5 3.1 34 33 13 0.4 0.4 0.089 27 18

>1000 41.8 5.1 1.2 0.38 0.093 0.11 20 1.1 >1000 3.8 0.36 0.54 NC 6.1 × 102 >1000 19 5.0 11 4.7 50 40 34 1.7 1.2 0.045 41 33

Results are the average of two independent triplicate runs with compound purity above >90%.

Because the phenylalanine P5 was required for the P2 Abu(3N3) compounds to be active, we explored different phenylalanine derivatives including Phe-carboxamide (24), Phe-tert-butyl amide (25), Phe-tert-butyl ester (26), and two carboxylic acid mimics, a triazole (27) and a thiazole (28). All compounds showed potent activity in vitro except for the carboxylic acid mimics 27 and 28. This suggests that the presence of an ester or amide at P5 is important for the activity of these molecules in vitro. Compounds 27 and 28 do inhibit tubulin polymerization in a biochemical assay (Supporting Information), suggesting that other factors for in vitro potency are at play other than simply tubulin polymerization inhibition. The presence of bigger, bulkier P5 groups Phe-tert-butyl amide (25) and Phe-tert-butyl ester (26) gave similar results to the smaller amide and ester Phecarboxamide (24) and Phe-methyl ester (6). To summarize, compounds with a P2-modified side chain can be active in vitro; however, the modified P2 compounds follow a different P5 activity trend than valine-based P2 compounds (Figure 2). The replacement of the thiazole group with a proton resulted in loss of potency, in contrast with P2 valine auristatins, which are equipotent.21 The basis of this novel P2-modified auristatin SAR is currently under investigation but could be due to many factors such as binding to the tubulin dimer, conformation of the pentapeptide, or changes in active transport.25 Research conducted at Pfizer demonstrated that two

Figure 2. SAR conclusion with P2-modified auristatin.

conformations of PF-06380101 are found bound to tubulin, one productive and one not productive, and could be a cause of the differences observed.25 Tubulin polymerization assays have been performed with many of the compounds synthesized, but due to the high compound concentrations required, in our screening assay, no major differences were observed to 2486

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was dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. Fmoc-Ser(Bzl)-Dil-OtBu was isolated by flash chromatography on silica gel (40 μm, 60 Å, 3.0 × 17.0 cm) using 18% to 90% EtOAc in hexanes as the eluent. A total of 3.75 g of Fmoc-Ser(Bzl)-DilOtBu (5.69 mmol, 84%) was obtained. To a stirred rt solution of Fmoc-Ser(Bzl)-Dil-OtBu (1.79 g, 2.72 mmol) in MeCN (5 mL) was added piperidine (4 mL). After 5 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was extracted with hexanes, and the MeCN layer was concentrated in vacuo to yield crude H-Ser(Bzl)-Dil-OtBu, which was used without further purification. To a stirred rt suspension of crude H-Ser(Bzl)-Dil-OtBu and Dov (0.790 g, 5.44 mmol) in DMF (10 mL) was added DIEA (1.45 mL, 8.16 mmol), followed by HATU (2.07 g, 5.44 mmol). After 5 h, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The combined organic fractions were washed with brine, dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.882 g of Dov-Ser(Bzl)-Dil-OtBu (1.30 mmol, 48%) was obtained as the TFA salt. To a rt solution of Dov-Ser(Bzl)-Dil-OtBu (0.288 g, 0.425 mmol) in CH2Cl2 (5 mL) was added TFA (4 mL). After 10 h, analysis by LCMS showed the reaction was complete. Volatile organics were evaporated in vacuo to yield crude Dov-Ser(Bzl)-Dil-OH TFA salt, which was used without further purification. To a stirred rt suspension of crude Dov-Ser(Bzl)-Dil-OH TFA salt and H-Dap-Phe-OMe TFA salt (0.163 g, 0.467 mmol) in DMF (10 mL) was added DIEA (0.303 mL, 1.70 mmol), followed by HATU (0.323 g, 0.850 mmol). After 6 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The combined organic fractions were washed with brine, dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.217 g of Dov-Ser(Bzl)Dil-Dap-Phe-OMe (0.228 mmol, 54%) was obtained as the TFA salt. A stirred rt suspension of Dov-Ser(Bzl)-Dil-Dap-Phe-OMe TFA salt (0.217 g, 0.228 mmol) and palladium on activated charcoal (10% Pd basis, 0.174 g) in MeOH (5 mL) was hydrogenated under refluxing conditions. After 48 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was filtered over a pad of diatomaceous earth, and the filtrate was concentrated. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.104 g of the title compound was obtained as the TFA salt (0.121 mmol, 47%) as a white, amorphous solid: LCMS tR = 2.32 min (method A); ESIMS m/z 748.72 [M + H]+; HRESIMS m/z 748.4846 [M + H]+ (calcd for C39H66N5O9, 748.4855). Compound 5. To a stirred rt solution of Fmoc-Thr(Bzl)-OH (2.92 g, 6.76 mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) in EtOAc (15 mL) was added DIEA (2.17 mL, 12.2 mmol). The solution was cooled (0 °C) and stirred for 20 min. DIEA (2.17 mL, 12.2 mmol) was added to the reaction mixture. The solution was cooled (0 °C) and stirred for 20 min. CMPI (2.76 g, 10.8 mmol) was added to the reaction mixture, which was allowed to reach rt. After 12 h, analysis by LCMS showed the reaction was complete. The crude reaction was washed with 0.1 M HCl (150 mL × 2). The organic fraction was dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. Fmoc-Thr(Bzl)Dil-OtBu was isolated by flash chromatography on silica gel (40 μm, 60 Å, 3.0 × 17.0 cm) using 18% to 90% EtOAc in hexanes as the eluent. A total of 3.71 g of Fmoc-Thr(Bzl)-Dil-OtBu (5.51 mmol, 82%) was obtained. To a stirred rt solution of Fmoc-Thr(Bzl)-Dil-OtBu (3.71 g, 5.51 mmol) in MeCN (5 mL) was added piperidine (4 mL). After 5 h, analysis by LCMS showed the reaction was complete. The crude

explain the differences in activity seen in vitro (Supporting Information).

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CONCLUSION We have developed novel auristatins that have a P2 side chain modification. These auristatins demonstrate activity in vitro comparable to other compounds in this class, and they are efficacious as a drug in ADCs. We determined that modification of the P2 side chain required a specific aromatic P5 subunit (ester or amide), and these P2 modifications did not follow previously reported SAR trends for P2 valine-based auristatins.21 It is also important to note when modifying the auristatin core structure that different peripheral groups (i.e., P5) are necessary to determine if the changes were beneficial or not.

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EXPERIMENTAL SECTION

General Experimental Procedures. Reactions were carried out at ambient temperature with exposure to air, unless otherwise noted. All reagents and solvents were purchased from commercial sources and used as received. NMR spectra were obtained on a Bruker AV 500 MHz spectrometer at 25 °C. The NMR spectra were obtained from DMSO-d6 and were referenced to the residual solvent peak (1H: 2.50 ppm; 13C: 39.5 ppm). High-resolution mass spectrometry samples were injected without column onto a Dionex 3000-Orbitrap Velos LC-MS. Flash chromatography was carried out on a Yamazen purification system using prepacked Yamazen Universal columns. Preparative HPLC was carried out using a Phenomenex Gemini-NX 10 μm, C18 110 Å column (150 × 30 mm) using a 5% to 100% gradient of MeCN/0.05% aqueous TFA mixture over 13 min unless another column or solvent system is noted. Drug compounds purified by preparative HPLC were assumed to be salts containing one molecule of TFA or formic acid (FA). Liquid chromatography mass spectrometry (LCMS) retention times were acquired on an Acquity UPLC BEH C8 1.7 μm 2.1 × 50 mm column, 40 °C. Method A: 0−0.5 min: isocratic 80:10:10 H2O/ MeCN/1% FA in H2O; 0.5−3.5 min: 80:10:10 H2O/MeCN/1% FA in H2O to 90:10 MeCN/1% FA; 3.50−3.99 min: isocratic 90:10 MeCN/ 1% FA in H2O; 3.99−4.00 min: linear gradient 90:10 MeCN/1% FA in H2O to 80:10:10 H2O/MeCN/1% FA in H2O. Method B: 0−0.5 min: isocratic 85:5:10 H2O/MeCN/1% FA in H2O; 0.5−1.6 min: linear gradient 85:5:10 H2O/MeCN/1% FA in H2O to 98:2 MeCN/1% FA in H2O; 1.60−1.9 min: linear gradient 98:2 MeCN/1% FA in H2O to 85:5:10 H2O/MeCN/1% FA in H2O; 1.9−2.0 min: isocratic 85:5:10 H2O/MeCN/1% FA in H2O. Compound 4. To a stirred room-temperature (rt) suspension of Boc-Dap-OH dicyclohexylamine salt (8.00 g, 17.1 mmol) and H-PheOMe HCl salt (4.42 g, 20.5 mmol) in CH2Cl2 (20 mL) was added diisopropylethylamine (DIEA; 9.13 mL, 51.3 mmol), followed by diethylpyrocarbonate (DEPC; 5.15 mL, 34.2 mmol). After 10 h, analysis by LCMS showed the reaction was complete. Boc-Dap-Phe-OMe was isolated by flash chromatography on silica gel (40 μm, 60 Å, 3.0 × 17.0 cm) using 2% to 10% MeOH in CH2Cl2 as the eluent. A total of 7.45 g of Boc-Dap-Phe-OMe (16.61 mmol, 97%) was obtained. To a stirred rt solution of Boc-Dap-Phe-OMe (4.67 g, 10.4 mmol) in CH2Cl2 (10 mL) was added TFA (10 mL). After 10 h, analysis by LCMS showed the reaction was complete. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.52 g of H-Dap-Phe-OMe (6.23 mmol, 59%) was obtained as the TFA salt. To a stirred rt solution of Fmoc-Ser(Bzl)-OH (2.82 g, 6.76 mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) in EtOAc (15 mL) was added DIEA (2.17 mL, 12.2 mmol). The solution was cooled to 0 °C and stirred for 20 min. DIEA (2.17 mL, 12.2 mmol) was added to the reaction mixture. The solution was cooled to 0 °C and stirred for 20 min. 2-Chloro-1-methylpyridinium iodide (2.76 g, 10.8 mmol) was added to the reaction mixture, which was allowed to reach rt. After 12 h, analysis by LCMS showed the reaction was complete. The crude reaction was washed with 0.1 M HCl (150 mL × 2). The organic fraction 2487

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The organic fraction was dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo to yield crude Fmoc-Abu(3-N3)-Dil-OtBu (1.12 g, 1.84 mmol), which was used without further purification. To a stirred rt solution of Fmoc-Abu(3-N3)-Dil-OtBu (1.00 g, 1.65 mmol) in MeCN (10 mL) was added piperidine (2 mL). After 5 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was extracted with hexanes, and the MeCN layer was concentrated in vacuo to yield crude H-Abu(3-N3)-Dil-OtBu, which was used without further purification. To a stirred rt suspension of crude H-Abu(3-N3)-Dil-OtBu and Dov (0.478 g, 3.29 mmol) in DMF (10 mL) was added DIEA (0.880 mL, 4.94 mmol), followed by HATU (1.25 g, 3.29 mmol). After 6 h, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The combined organic fractions were washed with brine, dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.670 g of Dov-Abu(3-N3)-Dil-OtBu (1.07 mmol, 65%) was obtained as the TFA salt. To a rt solution of Dov-Abu(3-N3)-Dil-OtBu TFA salt (0.289 g, 0.425 mmol) in CH2Cl2 (5 mL) was added TFA (5 mL). After 12 h, analysis by LCMS showed the reaction was complete. Volatile organics were evaporated in vacuo to yield crude Dov-Abu(3-N3)-Dil-OH TFA salt, which was used without further purification. To a stirred rt suspension of crude Dov-Abu(3-N3)-Dil-OH TFA salt (1.04 g, 1.82 mmol) and H-Dap-Phe-OMe TFA salt (1.59 g, 3.44 mmol) in DMF (10 mL) was added DIEA (1.18 g, 1.60 mL, 9.11 mmol), followed by the addition of HATU (1.74 g, 4.56 mmol). After 10 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL × 3). The combined organic fractions were washed with brine, dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Gemini NX-C18 10 μ 110 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.1% aqueous formic acid. A total of 935 mg of the title compound was obtained as the formic acid salt (1.12 mmol, 49%) as a white, amorphous solid. 1H NMR (500 MHz, DMSO-d6, a complex spectrum was observed, presumably due to cis/trans conformational isomers) δ 9.18 (br s, 1H), [8.65−8.42 (m) 8.29 (dd, J = 8.1, 4.3 Hz) 1H], 7.20 (m, 5H), [5.05 (q, J = 7.0 Hz), 4.87 (dd, J = 9.4, 6.0 Hz) 1H], [4.71−4.63 (m), 4.56−4.43 (m), 2H], 4.10−3.93 (m, 2H), 3.76−3.70 (m, 2H), [3.66 (d, J = 4.0 Hz), 3.63 (d, J = 4.0 Hz), 3H], 3.59−3.24 (m, 3H), 3.24−3.12 (m, 6H), [3.07 (s), 2.98 (s) 3H], 2.93−2.83 (m, 2H), [2.80 (s), 2.78 (s), 6H], 2.46−2.18 (m, 3H), 1.87−1.32 (m, 7H), 1.27 (t, J = 7.2 Hz, 3H), 1.08−0.89 (m, 10H), 0.88−0.82 (m, 3H), 0.78 (q, J = 6.5, 5.9 Hz, 3H); 13C NMR (126 MHz, DMSO) δ 173.84, 173.53, 173.32, 173.11, 172.10, 171.96, 171.87, 170.57, 168.95, 168.56, 137.23, 137.04, 128.85, 128.82, 128.54, 128.02, 128.00, 126.30, 126.21, 85.23, 82.17, 81.28, 79.15, 76.93, 68.60, 61.02, 60.83, 60.31, 60.15, 58.37, 58.14, 57.82, 57.45, 57.27, 57.18, 53.32, 52.91, 52.00, 51.92, 51.81, 51.78, 46.92, 46.01, 43.00, 42.81, 42.74, 41.13, 37.18, 36.11, 34.09, 32.66, 30.61, 25.31, 25.25, 25.12, 24.20, 24.14, 23.05, 18.93, 18.60, 18.57, 17.23, 17.01, 15.71, 15.65, 15.32, 15.21, 15.12, 15.04, 14.72, 10.88, 10.65, 10.17; LCMS tR = 1.09 min (method B); ESIMS m/z 787.53 [M + H]+; HRESIMS m/z 787.5072 [M + H]+ (calcd for C40H67N8O8, 787.5076). Compound 7. To a stirred rt solution of Boc-β-azido-alanine dicyclohexylamine salt (1.02 g, 2.47 mmol) and H-Dil-OtBu hydrochloride (0.730 g, 2.74 mmol) in EtOAc (10 mL) was added DIEA (0.792 mL, 4.44 mmol). The solution was cooled (0 °C) and stirred for 20 min. Additional DIEA (0.792 mL, 4.44 mmol) was added to the reaction mixture and stirred for 20 min. CMPI (1.01 g, 3.95 mmol) was added to the reaction mixture, which was allowed to reach rt. After 12 h, analysis by LCMS showed the reaction was complete. The crude reaction was washed with 0.1 M HCl (100 mL × 2), followed by brine (20 mL × 2). The organic fraction was dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo to yield crude Boc-β-azido-Ala-Dil-OtBu (1.08 g, 2.29 mmol), which was used without further purification.

reaction mixture was extracted with hexanes, and the MeCN layer was concentrated in vacuo to yield crude H-Thr(Bzl)-Dil-OtBu, which was used without further purification. To a stirred rt suspension of crude H-Thr(Bzl)-Dil-OtBu and Dov (1.60 g, 11.0 mmol) in DMF (20 mL) was added DIEA (2.95 mL, 16.5 mmol), followed by HATU (4.19 g, 11.0 mmol). After 6 h, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The combined organic fractions were washed with brine, dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 1.09 g of Dov-Thr(Bzl)-Dil-OtBu (1.58 mmol, 29%) was obtained as the TFA salt. To a rt solution of Dov-Thr(Bzl)-Dil-OtBu TFA salt (0.331 g, 0.478 mmol) in CH2Cl2 (5 mL) was added TFA (4 mL). After 10 h, analysis by LCMS showed the reaction was complete. Volatile organics were evaporated in vacuo to yield crude Dov-Thr(Bzl)-Dil-OH TFA salt, which was used without further purification. To a stirred rt suspension of crude Dov-Thr(Bzl)-Dil-OH TFA salt and H-Dap-Phe-OMe TFA salt (0.183 g, 0.526 mmol) in DMF (10 mL) was added DIEA (0.341 mL, 1.92 mmol), followed by HATU (0.364 g, 0.957 mmol). After 24 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The combined organic fractions were washed with brine, dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.290 g of Dov-Thr(Bzl)Dil-Dap-Phe-OMe (0.300 mmol, 63%) was obtained as the TFA salt. A stirred rt suspension of Dov-Thr(Bzl)-Dil-Dap-Phe-OMe TFA salt (0.290 g, 0.300 mmol) and palladium on activated charcoal (10% Pd basis, 0.232 g) in MeOH (5 mL) was hydrogenated. After 24 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was filtered over a pad of diatomaceous earth, and the filtrate was concentrated. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.129 g of the title compound was obtained as the TFA salt (0.147 mmol, 43%) as a white, amorphous solid. 1H NMR (500 MHz, DMSO-d6, a complex spectrum was observed, presumably due to cis/trans conformational isomers) δ [9.04 (d, J = 7.8 Hz), 9.02 (d, J = 7.8 Hz) 1H], [8.50 (d, J = 8.4 Hz), 8.26 (d, J = 8.0 Hz) 1H], 7.27−7.11 (m, 5H), [4.74 (t, J = 8.2 Hz), 4.68 (t, J = 8.2 Hz) 1H], [4.66−4.57 (m), 4.48−4.43 (m) 2H], 3.98−3.92 (m, 1H), 3.87−3.78 (m, 1H), [3.76− 3.66 (m), 3.56−3.42 (m) 3H], [3.65 (s), 3.61 (s) 3H], 3.30−3.11 (m, 8H), 3.10−2.98 (m, 1H), [3.06 (s), 2.99 (s) 3H], 2.89−2.71 (m, 7H), 2.46−2.13 (m, 4H), 1.83−1.58 (m, 3H), 1.49−1.35 (m, 2H), 1.34−1.23 (m, 1H), [1.20 (d, J = 6.2 Hz), 1.11 (d, J = 6.2 Hz) 3H], [1.04 (d, J = 6.7 Hz), 1.01 (d, J = 6.7 Hz) 3H], 0.98−0.78 (m, 10H), 0.74 (td, J = 7.4, 2.5 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 174.06, 173.92, 172.65, 172.48, 171.52, 169.27, 169.13, 165.61, 165.59, 137.80, 137.61, 129.39, 129.17, 128.58, 126.88, 126.80, 85.67, 81.79, 78.07, 77.79, 71.77, 71.72, 67.53, 67.49, 61.39, 60.66, 58.96, 58.62, 57.58, 57.52, 56.11, 56.05, 53.52, 52.53, 52.33, 47.55, 46.62, 43.58, 43.35, 42.08, 42.04, 41.50, 37.48, 36.78, 36.68, 35.56, 32.38, 32.11, 31.83, 26.92, 25.78, 25.70, 24.75, 24.71, 23.56, 20.16, 19.93, 19.39, 16.88, 15.95, 15.70, 15.66, 15.28, 10.84, 10.74; LCMS tR = 2.40 min (method A); ESIMS m/z 762.75 [M + H]+; HRESIMS m/z 762.5009 [M + H]+ (calcd for C40H68N5O9, 762.5012). Compound 6. To a stirred rt solution of (2S,3S)-Fmoc-Abu(3-N3)OH (1.00 g, 2.74 mmol) and H-Dil-OtBu hydrochloride (0.810 g, 2.74 mmol) in EtOAc (10 mL) was added DIEA (0.880 mL, 4.93 mmol). The solution was cooled (0 °C) and stirred for 20 min. Additional DIEA (0.880 mL, 4.93 mmol) was added to the reaction mixture and stirred for 20 min. CMPI (1.12 g, 4.38 mmol) was added to the reaction mixture, which was allowed to reach rt. After 12 h, analysis by LCMS showed the reaction was complete. The crude reaction was washed with 0.1 M HCl (100 mL × 2), followed by brine (20 mL × 2). 2488

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(method A); ESIMS m/z 787.49 [M + H]+; HRESIMS m/z 787.5078 [M + H]+ (calcd for C40H67N8O8, 787.5076). Compound 12. To a stirred 25 °C solution of Boc-propargylGlyOH (1.00 g, 4.69 mmol) and H-Dil-OtBu HCl salt (1.15 g, 3.90 mmol) in EtOAc (10 mL) was added DIEA (1.49 mL, 9.38 mmol). The solution was cooled (0 °C) and stirred for 20 min. Additional DIEA (1.49 mL, 9.38 mmol) was added to the reaction mixture and stirred for 20 min. CMPI (1.80 g, 7.04 mmol) was added to the reaction mixture, which was allowed to reach rt. After 12 h, analysis by LCMS showed the reaction was complete. The crude reaction was washed with 0.1 M HCl (20 mL × 2), followed by brine (20 mL × 2). The organic fraction was dried using magnesium sulfate, filtered, and concentrated in vacuo to give crude product. A total of 2.05 g of Boc-propargylGly-Dil-OtBu was obtained (4.50 mmol, 96%): LCMS tR = 3.46 min (method A); ESIMS m/z 455.42 [M + H]+. To a stirred 25 °C solution of Boc-propargylGly-Dil-OtBu (2.05 g, 4.50 mmol) in CH2Cl2 (6 mL) was added TFA (6 mL). After 14 h, analysis by LCMS showed the reaction was complete. The volatile organics were concentrated in vacuo to give a crude product that was used without further purification. A total of 1.30 g of H-propargylGlyDil-OH was obtained as the TFA salt (3.16 mmol, 70%). To a stirred 25 °C solution of Dov (1.27 g, 8.71 mmol) in DMF (10 mL) was added DIEA (2.33 mL, 13.1 mmol), followed by HATU (3.32 g, 8.71 mmol). After 10 min, H-propargylGly-Dil-OH TFA salt (1.30 g, 3.16 mmol) in a DMF solution was added to the reaction. After 8 h, analysis by LCMS showed the reaction was complete. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 455 mg of DovpropargyGly-Dil-OH was obtained as the TFA salt (0.844 mmol, 27%) as a white, amorphous solid: LCMS tR = 1.65 min (method A); ESIMS m/z 425.95 [M + H]+. To a stirred rt solution of Dov-propargylGly-Dil-OH TFA salt (0.198 g, 0.367 mmol) and H-Dap-Phe-OMe TFA salt (0.156 g, 0.338 mmol) in DMF (10 mL) was added DIEA (0.222 mL, 1.40 mmol), followed by the addition of HATU (0.355 g, 0.931 mmol). After 10 h, analysis by LCMS showed the reaction was complete. The crude reaction was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL × 2). The combined organic fractions were washed with brine, dried using magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 10 mg of the title compound was obtained as the TFA salt (0.012 mmol, 3%) as a white, amorphous solid: LCMS tR = 2.50 min (method A); ESIMS m/z 756.44 [M + H]+; HRESIMS m/z 756.4902 [M + H]+ (calcd for C41H66N5O8, 756.4906). Compound 13. To a stirred rt solution of Dov-Abu(3-N3)-Dil-DapPhe-OMe TFA salt (10 mg, 0.011 mmol) in THF (0.10 mL) was added trimethyl phosphine in THF (1 M, 0.022 mL, 0.022 mmol). After 4 h, analysis by LCMS showed the reaction was complete and H2O (0.05 mL) was added to the reaction mixture. The crude reaction mixture was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 6.00 mg of the title compound was obtained as the TFA salt (0.007 mmol, 62%) as a white, amorphous solid: LCMS tR = 2.12 min (method A); ESIMS m/z 761.63 [M + H]+; HRESIMS m/z 761.5159 [M + H]+ (calcd for C40H69N6O8, 761.5171). Compound 24. To a stirred rt solution of Dov-Abu(3-N3)-Dil-DapPhe-OH formic acid salt (42.1 mg, 0.051 mmol), ammonium chloride (7.9 mg, 0.148 mmol), and TBTU (52.5 mg, 0.163 mmol) in DMF (0.2 mL) was added DIEA (0.045 mL, 0.258 mmol). After 16 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was purified by preparative RP-HPLC with a Phenomenex Gemini-NX 10 μ C-18 110 Å column (150 × 30 mm) using 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of 13.4 mg of the title compound was obtained (0.017 mmol, 33%) as a white, amorphous solid: 1H NMR (500 MHz, DMSO-d6) δ 9.17 (br s, 1H), [8.35−8.23 (m), 8.05−7.95 (m), 1H], [7.45−7.40 (m), 7.40−7.27

To a rt solution of Boc-β-azido-Ala-Dil-OtBu (1.08 g, 2.29 mmol) in CH2Cl2 (5 mL) was added TFA (3 mL). After 10 h, analysis by LCMS showed the reaction was complete. Volatile organics were evaporated in vacuo to yield crude H-β-azido-Ala-Dil-OH TFA salt that was used without further purification. To a stirred rt suspension of Dov (0.739 g, 5.09 mmol) in DMF (10 mL) was added DIEA (1.36 mL, 7.63 mmol), followed by HATU (1.95 g, 5.09 mmol). After 10 min crude H-β-azido-Ala-Dil-OH was added to the reaction mixture. After 6 h, analysis by LCMS showed that the reaction was complete. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.130 g of Dov-β-azido-Ala-Dil-OH (0.261 mmol, 10%) was obtained as the TFA salt. To a stirred rt suspension of Dov-β-azido-Ala-Dil-OH TFA salt (0.130 g, 0.261 mmol) and H-Dap-Phe-OMe TFA salt (0.205 g, 0.588 mmol) in DMF (10 mL) was added DIEA (0.140 mL, 0.783 mmol), followed by HATU (0.198 g, 0.522 mmol). After 6 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The combined organic fractions were washed with brine, dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.015 g of the title compound was obtained as the TFA salt (0.017 mmol, 7%) as a white, amorphous solid: LCMS tR = 2.36 min (method A); ESIMS m/z 773.48 [M + H]+; HRESIMS m/z 773.4916 (calcd for C39H65N8O8, 773.4920). Compound 9. To a stirred rt solution of Boc-4-azido-homoalanine dicyclohexylamine salt (1.01 g, 2.37 mmol) and H-Dil-OtBu hydrochloride (0.700 g, 2.37 mmol) in EtOAc (10 mL) was added DIEA (0.759 mL, 4.26 mmol). The solution was cooled (0 °C) and stirred for 20 min. Additional DIEA (0.759 mL, 4.26 mmol) was added to the reaction mixture and stirred for 20 min. CMPI (0.967 g, 3.79 mmol) was added to the reaction mixture, which was allowed to reach rt. After 12 h, analysis by LCMS showed the reaction was complete. The crude reaction was washed with 0.1 M HCl (100 mL × 2), followed by brine (20 mL × 2). The organic fraction was dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo to yield crude Boc-4-azidohomoAla-Dil-OtBu (1.09 g, 2.25 mmol), which was used without further purification. To a rt solution of Boc-4-azido-homoAla-Dil-OtBu (1.09 g, 2.25 mmol) in CH2Cl2 (5 mL) was added TFA (3 mL). After 10 h, analysis by LCMS showed the reaction was complete. Volatile organics were evaporated in vacuo to yield crude H-4-azido-homoAla-Dil-OH TFA salt, which was used without further purification. To a stirred rt suspension of Dov (0.652 g, 4.49 mmol) in DMF (10 mL) was added DIEA (1.20 mL, 6.73 mmol), followed by HATU (1.71 g, 4.49 mmol). After 10 min crude H-4-azido-homoAla-Dil-OH TFA salt was added to the reaction mixture. After 6 h, analysis by LCMS showed that the reaction was complete. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.127 g of Dov-4-azido-homoAla-Dil-OH (0.223 mmol, 10%) was obtained as the TFA salt. To a stirred rt suspension of Dov-4-azido-homoAla-Dil-OH TFA salt (0.127 g, 0.223 mmol) and H-Dap-Phe-OMe TFA salt (0.198 g, 0.568 mmol) in DMF (10 mL) was added DIEA (0.140 mL, 0.783 mmol), followed by HATU (0.198 g, 0.522 mmol). After 6 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The combined organic fractions were washed with brine, dried over a pad of magnesium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.015 g of the title compound was obtained as the TFA salt (0.017 mmol, 7%) as a white, amorphous solid: LCMS tR = 2.38 min 2489

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1.42 min (method B); ESIMS m/z 828.94 [M + H]+; HRESIMS m/z 829.5536 [M + H]+ (calcd for C43H73N8O8, 829.5546).

(m), 1H], 7.25−7.17 (m,, 4H), 7.13 (t, J = 6.3 Hz, 1H), 7.04 (br s, 1H), [5.05−4.97 (m), 4.87 (t, J = 8.4 Hz), 1H], [4.68−4.51 (m), 4.50−4.40 (m), 2H], 4.05−3.92 (m, 2H), 3.87−3.72 (m, 2H), 3.55−3.38 (m, 2H), 3.31−3.13 (m, 7H), 3.11−2.96 (m, 3H), 2.81−2.70 (m, 8H), 2.44 (d, J = 16.1 Hz, 1H), 2.35−2.17 (m, 2H), 1.88−1.60 (m, 1H), 1.47−1.40 (m, 1H), 1.37−1.29 (m, 3H), 1.28 (dd, J = 9.6, 6.5 Hz, 3H), 1.08−0.82 (m, 15H), 0.78 (t, J = 7.2 Hz, 3H); 13C NMR (126 MHz, DMSO) δ 173.35, 173.23, 173.05, 169.89, 169.50, 168.64, 168.58, 165.45, 138.05, 137.88, 128.88, 128.64, 127.91, 127.83, 127.77, 126.00, 125.87, 85.21, 81.31, 77.04, 71.11, 60.75, 60.11, 58.43, 58.02, 57.70, 57.48, 57.23, 57.16, 56.75, 53.46, 52.51, 52.14, 52.03, 46.93, 46.02, 43.44, 42.86, 41.09, 37.37, 37.24, 37.08, 35.25, 32.40, 31.49, 31.15, 26.48, 25.28, 25.13, 24.21, 24.05, 23.01, 18.98, 16.52, 15.64, 15.48, 15.29, 15.16, 14.60, 14.29, 10.54, 10.19; LCMS tR = 1.05 min (method B); ESIMS m/z 772.61 [M + H]+; HRESIMS m/z 772.5078 [M + H]+ (calcd for C39H66N9O7, 772.5080). Compound 25. To a stirred rt solution of Dov-Abu(3-N3)-Dil-DapPhe-OH formic acid salt (24.0 mg, 0.029 mmol), tert-butyl amine hydrochloride (7.9 mg, 0.072 mmol), and HATU (24.5 mg, 0.064 mmol) in DMF (0.2 mL) was added DIEA (0.022 mL, 0.124 mmol). After 18 h, analysis by LCMS showed the reaction was complete. The crude reaction mixture was purified by preparative RP-HPLC with a Phenomenex Gemini-NX 10 μ c-18 110 Å column (150 × 30 mm) using 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of 15.4 mg of the title compound was obtained (0.017 mmol, 59%) as a white, amorphous solid: 1H NMR (500 MHz, DMSO-d6, a complex spectrum was observed, presumably due to cis/trans conformational isomers) δ 8.50 (br s, 1H), [8.34 (d, J = 8.3 Hz), 8.13 (d, J = 8.3 Hz) 1H], 7.27−7.10 (m, 5H), [4.97−4.91 (m), 4.76 (t, J = 8.9 Hz) 1H], [4.67−4.48 (m), 4.33 (ddd, J = 10.2, 7.9, 5.2 Hz) 2H], 4.02−3.74 (m, 4H), 3.58−3.48 (m, 1H), 3.43−3.22 (m, 9H), 3.10−2.78 (m, 2H), [3.05 (s), 2.94 (s) 3H], 2.43−2.18 (m, 9H), 2.05−1.89 (m, 1H), 1.85−1.60 (m, 3H), 1.52−1.21 (m, 5H), [1.37 (s), 1.34 (s) 9H], 1.18−0.95 (m, 1H), [1.06 (d, J = 6.8 Hz), 1.02 (d, J = 6.8 Hz) 3H], 0.93−0.82 (m, 7H), 0.78−0.69 (m, 6H); 13C NMR (126 MHz, DMSO-d6) δ 173.89, 173.68, 171.23, 171.17, 169.53, 169.14, 137.95, 137.68, 129.42, 129.13, 128.53, 128.51, 126.77, 126.70, 85.77, 81.85, 81.20, 81.01, 78.15, 77.46, 72.97, 61.38, 60.74, 59.01, 58.80, 58.76, 58.49, 57.84, 57.79, 54.09, 52.78, 52.23, 52.17, 47.49, 46.58, 43.61, 43.34, 41.76, 37.84, 37.00, 36.85, 35.75, 33.34, 32.20, 31.77, 28.02, 27.18, 25.80, 25.70, 24.79, 24.72, 23.61, 19.95, 16.65, 16.24, 15.92, 15.41, 11.32, 10.79; LCMS tR = 1.27 min (method B); ESIMS m/z 828.8 [M + H]+; HRESIMS m/z 828.5671 [M + H]+ (calcd for C43H74N9O7, 828.5706). Compound 26. To a stirred 25 °C solution of Boc-Dap-OH dicyclohexylamine salt (6.47 g, 13.8 mmol) and H-Phe-OtBu (3.91 g, 15.2 mmol) in CH2Cl2 (20 mL) was added DIEA (8.76 mL, 55.2 mmol), followed by the addition of DEPC (3.12 mL, 20.7 mmol). After 8 h, analysis by LCMS showed the reaction was complete. The volatile organics were evaporated in vacuo to give a crude product that was used without further purification. A total of 5.35 g of Boc-Dap-Phe-OtBu was obtained (10.9 mmol, 79%): LCMS tR = 2.89 min (method A); ESIMS m/z 491.48 [M + H]+. To a stirred rt solution of Boc-Dap-Phe-OtBu (5.25 g, 10.7 mmol) in CH2Cl2 (10.0 mL) was added TFA (10.0 mL). After 12 h, analysis by LCMS showed the reaction was complete. The crude oil was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.85 g of H-Dap-Phe-OtBu was obtained as the TFA salt (5.65 mmol, 68%): LCMS tR = 1.82 min (method A); ESIMS m/z 391.02 [M + H]+. To a stirred 23 °C suspension of Dov-Abu(3-N3)-Dil-OH TFA salt (327 mg, 0.574 mmol) and H-Dap-Phe-OtBu TFA salt (0.340 g, 0.675 mmol) in DMF (10 mL) was added DIEA (0.370 g, 0.500 mL, 2.87 mmol) followed by the addition of HATU (0.550 g, 1.43 mmol). After 10 h, analysis by LCMS showed the reaction was complete. The crude reaction was purified by preparative RP-HPLC with a Phenomenex Gemini NX-C18 10 μ 110 Å column (150 × 30 mm) using 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 453 mg of the title compound was obtained as the formic acid salt (0.518 mmol, 72%) as a white, amorphous solid: LCMS tR =

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00359. Detailed experimental procedures, 1H and 13C NMR spectra for 3, 5, 6, 14, 16, 18, 21, 22, 24, 25, and tubulin assay results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 424-280-5607. ORCID

Brian A. Mendelsohn: 0000-0002-6339-2377 Present Address †

Zymeworks, 540-1385 West 8th Avenue, Vancouver, BC, Canada, V6H 3V9. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge the work of D. Jackson, M. Shiwalker, and J. Yin for in vitro work and T. Martin and A. Soohoo for technical assistance. The authors would like to acknowledge K. Morrison and D. R. Stover for manuscript review and helpful discussions.

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REFERENCES

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