Antineoplastic Agents. 605. Isoquinstatins - Journal of Natural

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Antineoplastic Agents. 605. Isoquinstatins George R. Pettit,* Noeleen Melody, and Jean-Charles Chapuis Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871604, Tempe, Arizona 85287-1604, United States S Supporting Information *

ABSTRACT: In order to further explore quinoline-type structural modification of the powerful anticancer drug dolastatin 10, an Indian Ocean sea hare constituent and parent molecule of the very successful antibody drug conjugate (ADC) Adcetris, our recent quinstatin study has been extended by replacing the quinoline ring with an isoquinoline. The resulting isoquinstatins (4− 6) were modified to N-terminal desmethylisoquinstatins (7−9) and, in turn, bonded to appropriate linker units to give linkerdesmethylisoquinstatin conjugates 11−13 in preparation for eventual monoclonal antibody attachment. Comparison of the new isoquinstatins with their quinstatin counterparts against six human cancer cell lines indicated the isoquinstatins to have GI50 values that were comparable to or somewhat higher than those of the isomeric quinstatins. However, desmethylisoquinstatin 5 (7) was significantly more potent than its desmethylquinstatin 5 analogue. When evaluated against quinstatin 8, its isoquinstatin 8 (6) counterpart was somewhat less potent. In general, the isoquinstatins evaluated proved to be quite strong cancer cell growth inhibitors.

T

he long history of the quinoline ring system in contributing to useful bioactivity of certain natural products in essence began in the 17th century with the wider use of Cinchona bark, containing about 5% of the alkaloid quinine. This led much later to the well-known synthesis of the quinoline drugs such as chloroquine and mefloquine, with the former still in use for rheumatoid arthritis.2a−f The isoquinoline ring system is also well known in natural products such as the alkaloid papaverine. The quinoline ring is also the basis of some marine animal components such as an alkaloid from octopus ink.2e Very pertinent to the current research is the Streptoverticillium album microorganism constituent quinaldopeptin, an anticancer/ bisintercalating antibiotic drug incorporating two 3-hydroxyquinaldic acid units in a cyclic depsipeptide (1).2a−c The closely related luzopeptin A anticancer antibiotic is a cyclodepsipeptide with two 3-hydroxy-6-methylquinaldic acid units terminating both ends produced by Actinomadura luzonesis.2f Toward the objective of discovering an anticancer drug with the remarkable potency of dolastatin 10 (2),3 but possessing a wider therapeutic index, we have been pursuing a long-term structure−activity relationship endeavor. Some of these early results in 1992 led to our discovery of auristatin E, which was developed to Adcetris, a very successful antibody drug conjugate (ADC) now available in 65 countries.1a,b,c,5a−h A parallel long-term target has been an attempt to locate a dolastatin 10 derivative with such exceptional potency that it could be converted to an effective ADC. Our group has recently © 2017 American Chemical Society and American Society of Pharmacognosy

discovered the quinoline-based quinstatin series1c and converted them to their corresponding desmethylquinstatins with linkers suitable for later monoclonal bonding.1a Clearly, the Special Issue: Special Issue in Honor of Susan Horwitz Received: April 21, 2017 Published: September 19, 2017 451

DOI: 10.1021/acs.jnatprod.7b00352 J. Nat. Prod. 2018, 81, 451−457

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remote bonding of the quinstatin quinoline ring to the Nterminal segment of the dolastatin 10 peptide leads to exceptional cancer cell growth inhibition. Previously, when the dolastatin 10 unit was bonded directly, e.g., to the 2- and 6aminoquinoline positions (auristatins 2-AQ and 6-AQ, Figure 1), the anticancer activity was markedly reduced.4

Owing to the quinstatin structural success arising from bonding to (2-aminoethyl)quinolines, attention is directed now at exploring a representative of (2-aminoethyl)isoquinolines coupled to the same dolastatin 10 unit as for the quinstatins. For this purpose, isoquinolines with 2-aminoethyl groups at positions 5, 7, and 8 were selected for attachment to the truncated D-10 peptide unit. This allowed a comparison with three of the most powerful quinstatins obtained to date. Next, each of the isoquinoline 5, 7, and 8 analogues was modified by synthesizing an N-terminal desmethyl derivative and then onto a bonded linker suitable for eventual monoclonal antibody attachment. At this stage, another very positive result might be an increased hydrophilic property and thereby increased efficacy.5



RESULTS AND DISCUSSION The synthesis of the isoquinstatins 5, 7, and 8 and desmethylisoquinstatins 5, 7, and 8 (4−9) along with their respective linker conjugates (11−13) was accomplished using the methods described previously for the quinstatins1b and the desmethylquinstatins and their corresponding drug−linker conjugates.1b Briefly, coupling the 5, 7 and 8-(1′-ethyl-2′amino)-isoquinolines with N-Boc-dolaproine6a,b in the presence of diethyl cyanophosphonate (DEPC) and triethylamine in anhydrous CH2Cl2 gave the 5-, 7-, and 8-(1′-ethyl-2′-amidoBoc-Dap)-isoquinolines 3a−c. These three intermediates were then deprotected and similarly coupled with either Dov-ValDil-TFA6c or Fmoc-Meval-Val-Dil-tertiary butyl ester7a−c to give isoquinstatins 4−6 and in parallel the Fmoc desmethylisoquinolines, respectively. The latter were further treated with

Figure 1. Structures of auristatins 2-AQ and 6-AQ4 and quinstatins 2− 8.1c

Scheme 1. Synthesis of Isoquinstatins 5, 7, and 8 (4−6) and Desmethylisoquinstatins 5, 7, and 8 (7−9)

452

DOI: 10.1021/acs.jnatprod.7b00352 J. Nat. Prod. 2018, 81, 451−457

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Scheme 2. Synthesis of Mc-Val-Cit-PABC-desmethylisoquinstatins 5, 7, and 8 (11−13)

Table 1. Isoquinstatin 8 (6) Correlated 13C and 1H NMR Assignments in CDCl3 Solutiona

diethylamine to give, in turn, desmethylisoquinstatins 7−9 (Scheme 1). The linker Mc-Val-Cit-PABC-PNP (10) was next attached to 7, 8, and 9, respectively, using a previously described method.1b Purification was achieved by chromatography on Sephadex LH-20 eluting with methanol, which separated the drug−linker conjugates 11, 12, and 13 readily from the unreacted components (Scheme 2). Table 1 presents the 1H and 13 C NMR data for isoquinstatin 8 (6), which was assigned using 2D COSY, HSQC, and HMBC NMR data. Doubling of signals was observed in the NMR spectra due to conformational changes around the Dil−Dap bond.6c,d Electrospray ionization mass spectrometry (ESIMS) and 1H NMR spectroscopy were employed to confirm synthesis of the drug−linker conjugate. Clearly, the isoquinstatins illustrated by 4, 5, and 6 and the desmethylisoquinstatins (7, 8, and 9) represent a second very promising series of quinoline-type cytotoxic drugs (Table 2) now available for conversion to ADCs. As with the parent series of quinstatins,1b,c the isoquinstatins represent a group of remarkably potent cancer cell growth inhibitors. Their facile bonding to a classic ADC linker as shown by the ready conversion of desmethyl intermediates 7, 8, and 9 to the linker products 11, 12, and 13 indicates a smooth synthetic route.



position

EXPERIMENTAL SECTION

General Experimental Procedures. Both N-Boc-dolaproine, Dov-Val-Dil·TFA, and Fmoc-N-methylval-Val-Dil-O-tBu-ester were synthesized as described earlier.6a−c,7a−c The 5-, 7-, and 8-isoquinoline ethaneamines were purchased from WuXi Apptec (Wuhan) Co., Ltd. via LabNetwork (USA), and all were used as received. The linker MCVal-Cit-PABC-PNP (10) can now be obtained commercially such as from Fisher Healthcare, a Thermo Fisher Scientific brand. Other reagents including diethyl cyanophosphonate and anhydrous solvents were purchased from Acros Organics (Fisher Scientific) and Sigma− Aldrich Chemical Co. and were used as received. Melting points are uncorrected and were determined with a Fisher Scientific melting point apparatus. Optical rotations were measured by use of a Rudolph Research Autopol IV automatic polarimeter, and the [α]D values are given in 10−1 deg cm2 g−1. The 1H and 13C NMR spectra were recorded on Varian Unity INOVA 400 and 500 and Bruker 400 NMR instruments with deuterated solvents. High-resolution mass spectra were obtained using a JEOL LCMate instrument and a Bruker MicrOTOF-Q in the ESI positive mode (direct infusion with internal

δC (ppm)

δH [mult., J (Hz)]

1

149.2

9.58 (1H, s)

3 4 4a 5 6

142.9 121.1 136.5 127.7 130.2

8.53 (1H, d, 5.7) 7.64 (1H, m)b

7 8

128.2 136.9

7.47 (1H, d, 7)

8a 9 10

127.4 31.7 40.6

11 12

174.5

13 13a 14

44.7 15.1 82.0

14b

60.8

15 16

59.7 25.1

17

25.1

18

47.9

20

170.7

position

δC (ppm)

21

37.5

22 22b 23 23a 23b

78.5 58.1 61.4 33.3 25.9

23c 23d

10.9 19.7

24a 25 26

31.7 173.5 53.9

26a 26b

31.1 17.9

2.39 (1H, m)b 1.24 (3H, d, 7.1) 3.84 (1H, dd, 8.9, 1.6) 3.36 (3H, s)

26c 27 28

16.1 171.9

29

76.7

4.04 (1H, m)b 1.95 (1H, m)b, 1.75 (1H, m)b 1.95 (1H, m)b, 1.75 (1H, m)b 3.46 (1H, m)b, 3.35 (1H, m)b

29bc 30

7.71 (1H, m)b 7.62 (1H, m)b

3.45 (2H, m)b 3.72 (1H, m)b, 3.65 (1H, m)b 6.95 (1H, m)b

43.0 × 2 27.8

31

20.3

32

17.9

δH [mult., J (Hz)] 2.31 (1H, m)b, 2.39 (1H, m)b 4.08(1H, m)b 3.33 (3H, s) 3.38 (1H, m)b 1.68 (1H, m)b 1.37 (1H, m)b, 1.00 (1H, m)b 0.83 (3H, t, 7.1) 1.01 (3H, d, 6.7)b 3.00 (3H, s) 4.78 (1H, dd, J = 7.9, 6.7) 2.00 (1H, m)b 0.93 (3H, d, 6.8)b 0.97 (3H, d)b 6.93 (1H, m)b

2.45 (1H, d, 6.6 Hz) 2.25 (6H, s) 2.08 (1H, m) 1.01 (3H, d, 6.7)b 0.92 (3H, d)b

a

Signals assigned using 2D COSY, HSQC, and HMBC NMR data and ref 6e. bOverlapping signals.

calibration) in the Arizona State University CLAS High Resolution Mass Spectrometry Laboratory, and the data were provided by Dr. John C. Knight and Natalya Zolotova. For TLC, Analtech silica gel GHLF Uniplates were used and visualized with short-wave UV irradiation and an iodine chamber. For column chromatography, silica gel (230−400 mesh ASTM) from E. Merck (Darmstadt, Germany) 453

DOI: 10.1021/acs.jnatprod.7b00352 J. Nat. Prod. 2018, 81, 451−457

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Table 2. Human Cancer Cell Lines (GI50 μg/mL [nM]) Growth Inhibition of Isoquinstatins 5 (4), 7 (5), and 8 (6), Desmethylisoquinstatins 5 (7), 7 (8), and 8 (9), and Their Drug−Linker Conjugates 11, 12, and 13, with Comparsion Data for Quinstatins 5, 7, and 8, Dolastatin 10, Auristatin E, and Desmethylauristatin E1b,c cell linea compound dolastatin 103 (2) quinstatin 51c dmquinstatin 51b MC-Val-Cit-PABC- dmquinstatin 51b isoquinstatin 5 (4) dmisoquinstatin 5 (7) MC-Val-Cit-PABC- dmisoquinstatin 5 (11) quinstatin 7 isoquinstatin 7 (5) dmisoquinstatin 7 (8) MC-Val-Cit-PABC- dmisoquinstatin 7 (12) quinstatin 81c dmquinstatin 81b MC-Val-Cit-PABC-dmquinstatin 81b isoquinstatin 8 (6) dmisoquinstatin 8 (9) MC-Val-Cit-PABC-dmisoquinstatin 8 (13) auristatin E1c desmethylauristatin E1c a

BXPC-3

MCF-7

SF-268

NCI-H460

KM20L2

DU-145

0.000040 [0.051] 0.0021 [2.79] 0.02 [27.08] 0.40 [299.2] 0.0055 [7.31] 0.0081 [11.0] 0.41 [307] 0.0031 [4.12] 0.0033 [4.39] 0.033 [44.7] 0.20 [149] 0.00050 [0.425] 0.0085 [11.51] 0.60 [448.8] 0.0032 [4.25] 0.030 [40.6] 0.70 [524] >0.001 [>1.37] >0.001 [>1.39]

0.0000042 [0.005] 0.000073 [0.097] 0.00022 [0.298] 0.0070 [5.23] 0.00021 [0.279] 0.000070 [0.095] 0.029 [21.7] 0.000030 [0.039] 0.00014 [0.186] 0.00011 [0.149] 0.024 [18.0] 0.000040 [0.053] 0.000041 [0.06] 0.020 [14.96] 0.00010 [0.133] 0.00020 [0.271] 0.03 [22.4] 0.00017 [0.232] 0.00029 [0.404]

0.0000043 [0.006] 0.00011 [0.146] 0.00023 [0.311] 0.0040 [2.99] 0.00020 [0.266] 0.000098 [0.132] 0.013 [9.72] 0.000024 [0.030] 0.000051 [0.068] 0.00024 [0.325] 0.013 [9.72] 0.000023 [0.030] 0.000043 [0.06] 0.0047 [3.52] 0.000050 [0.07] 0.00010 [0.135] 0.022 [16.5] 0.00036 [0.492] 0.00031 [0.432]

0.00018 [0.229] 0.0048 [6.37] 0.028 [37.91] 0.33 [246.8] 0.013 [17.3] 0.031 [42.0] 0.53 [396] 0.011 [1.19] 0.0070 [9.30] 0.040 [54.2] 0.60 [448] 0.00090 [1.19] 0.011 [14.90] 0.60 [448.8] 0.0068 [9.03] 0.090 [0.122] 3.0 [2244] 0.00039 [0.533] 0.00049 [0.683]

0.0000080 [0.010] 0.00023 [0.305] 0.00043 [0.582] 0.015 [11.22] 0.00029 [0.385] 0.00020 [0.271] 0.041 [30.7] 0.000041 [0.054] 0.00030 [0.399] 0.00042 [0.569] 0.024 [32.2] 0.000040 [0.053] 0.00015 [0.203] 0.032 [23.94] 0.00011 [0.146] 0.00040 [0.542] 0.043 [32.2] 0.00036 [0.492] 0.00043 [0.599]

0.0000052 [0.007] 0.00013 [0.172] 0.00060 [0.812] 0.0060 [4.49] 0.0021 [2.79] 0.00050 [0.677] 0.036 [26.9] 0.000030 [0.040] 0.000025 [0.033] 0.000090 [0.122] 0.021 [15.7] 0.000040 [0.053] 0.000052 [0.070] 0.0080 [5.98] 0.000040 [0.053] 0.00031 [0.420] 0.031 [23.2] 0.00031 [0.424] 0.00030 [0.418]

Cancer cell lines in order: pancreas (BXPC-3); breast (MCF-7); CNS (SF-268); lung (NCI-H460); colon (KM20L2); prostate (DU-145). warming over time to room temperature (rt) for 18 h and then concentrated under reduced pressure to a light amber oil, which was separated by chromatography on a silica gel column. Elution with CH2Cl2−MeOH (97:3) gave the product as a colorless, frothy solid (0.24 g, 0.54 mmol, 78%): TLC Rf 0.22 (CH2Cl2−MeOH, 98:2); 1H NMR (CDCl3, 400 MHz) δ 9.55 (1H, s), 8.54 (1H, bs), 7.80−7.57 (3H, m), 7.46 (1H, m), 6.75, 6.17 (1H, bs, NH), 4.07−3.58 (4H, m), 3.48−3.30 (6H, m), 3.22 (1H, m), 2.39 (1H, m), 2.05−1.67 (4H, m), 1.61−1.37 (9H, m), 1.21 (3H, m); 13C NMR (CDCl3, 400 MHz) δ 174.6, 174.1, 155.1, 154.5, 148.8, 142.6, 136.9, 136.6, 130.4, 128.3, 125.7, 121.4, 84.1, 82.3, 79.9, 79.5, 77.4, 60.9, 59.4, 58.7, 47.0, 46.7, 44.4, 44.0, 40.5, 31.8, 28.7, 26.1, 25.4, 24.8, 24.3, 14.9, 14.4; (+)-HRAPCIMS m/z 442.2707 [M + H]+ (calcd for C25H36N3O4, 442.2706). 8-(1′-Ethyl-2′-amido-Dap)isoquinoline Trifluoroacetate Salt. A solution of the preceding amide 3c (0.075 g, 0.170 mmol) in CH2Cl2 (0.5 mL) was stirred at 0 °C under N2. Trifluoroacetic acid (TFA) (0.25 mL) was added, and the reaction mixture stirred at 0 °C for 2 h and monitored by TLC (CH2Cl2−MeOH, 96:4). The solvent was

Figure 2. Structure of isoquinstatin 8. and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) was employed. Isoquinstatin 8 (6). The isoquinstatins and desmethylisoquinstatins were synthesized as described herein for isoquinstatin 8 (6) and desmethylisoquinstatin 8 (9). 8-(1′-Ethyl-2′-amido-Boc-Dap)-isoquinoline (3c). To a stirred solution of Boc-Dap6a,b (0.2 g, 0.7 mmol) and 8-(1′-ethyl-2′amino)isoquinoline (0.14 g, 0.81 mmol, 1.16 equiv), in anhydrous CH2Cl2 (4 mL) at 0 °C under N2, was added triethylamine (TEA) (0.3 mL, 2.2 mmol, 2.7 equiv) and DEPC (0.15 mL, 0.99 mmol, l.41 equiv). The reaction mixture was stirred at 0 °C (ice bath) with 454

DOI: 10.1021/acs.jnatprod.7b00352 J. Nat. Prod. 2018, 81, 451−457

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153.3, 143.4, 134.9, 134.6, 130.9, 129.2, 127.0, 126.8, 117.0, 84.2, 82.3, 80.0, 79.6, 60.9, 59.4, 58.7, 47.1, 46.7, 44.6, 44.0, 40.4, 32.2, 28.7, 26.1, 25.3, 24.8, 24.3, 14.9, 14.4; (+)-HRAPCIMS m/z 442.2718 [M + H]+ (calcd for C25H36N3O4, 442.2706). 5-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)-isoquinoline (Isoquinstatin 5) (4). Straw-colored glass solid (31 mg, 24% from Dov-Val-DilTFA starting material): TLC Rf 0.4 (CH2Cl2−MeOH, 92:8); [α]24D −9.0 (c 0.30, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 9.24 (1H, s), 8.57 (1H, nm), 7.99 (1H, d, J = 5.9 Hz), 7.85 (1H, d, J = 8.0 Hz), 7.61−7.51 (2H, m), 6.94 (2H, m), 4.16−4.02 (2H, m), 3.84 (1H, dd, J = 8.9, 1.6 Hz), 3.72−3.43 (4H, m), 3.42−3.28 (11H, m), 3.37, 3.30 (s, OCH3), 3.03 (3H, s, OCH3), 2.54 (1H, m), 2.49−2.19 (9H, m), 2.14−1.90 (4H, m), 1.85−1.61 (3H, m), 1.33 (1H, m), 1.24 (3H, d, J = 7.1 Hz), 1.04−0.89 (16 H, m), 0.82 (3H, t, J = 7.3 Hz); 13C NMR (CDCl3, 400 MHz) δ 174.6, 170.7, 153.3, 143.5, 135.1, 130.9, 129.1, 127.0, 126.7, 117.2, 82.0, 78.7, 76.3, 61.7, 60.8, 59.8, 58.1, 54.0, 47.9, 45.8, 44.8, 42.9, 40.4, 37.6, 33.2, 32.1, 31.2, 27.9, 25.9, 25.1, 20.3, 19.7, 18.0, 16.0, 15.1, 10.9; (+)-HRAPCIMS m/z 753.5274 [M + H]+ (calcd for C42H69N6O6, 753.5279). Desmethylisoquinstatin 5 (7). 5-(1′-Ethyl-2′-amido-Dap-DilVal-N-Methylvaline)isoquinoline (Desmethylisoquinstatin 5) (7). Straw-colored, frothy solid [47 mg, 37% from 5-(1′-ethyl-2′-amidoBoc-Dap)-isoquinoline (3a)]: TLC Rf 0.27 (CH2Cl2−MeOH, 93:3); [α]25D −8.6 (c 0.28, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 9.22 (1H, s), 8.56 (1H, d, J = 5.7 Hz), 7.99 (1H, d, J = 5.8 Hz), 7.85 (1H, d, J = 8.3 Hz), 7.64−7.50 (3H, m), 6.98 (1H, m), 4.77 (1H, dd, J = 9.6, 7.0 Hz), 4.19−4.01 (2H, m), 3.85 (1H, dd, J = 8.9, 1.8 Hz), 3.70−3.43 (3H, m), 3.41−3.25 (10H, m), 3.38, 3.31 (3H, s, OCH3), 3.03 (3H, s), 2.75 (1H, d, J = 5 Hz), 2.47−2.22 (6H, m), 2.08−1.85 (5H, m), 1.83− 1.63 (3H, m), 1.37 (1H, m), 1.24 (3H, d, J = 7.1 Hz), 1.01−0.9 (16H, m), 0.83 (3H, t, J = 7.4 Hz); 13C NMR (CDCl3, 400 MHz) δ 174.6, 173.7, 170.7, 153.2, 143.4, 135.0, 130.8, 129.1, 127.0, 126.7, 117.1, 82.0, 78.6, 71.3, 61.6, 60.8, 59.7, 58.1, 53.9, 53.6, 47.8, 44.7, 40.4, 37.6, 36.0, 33.3, 32.1, 31.9, 31.6, 31.5, 31.2, 25.8, 25.0 23.7, 19.7, 19.6, 18.5, 18.1, 16.1, 15.1, 10.8; (+)-HRAPCIMS m/z 739.5117 [M + H]+ (calcd for C41H67N6O6, 739.5122). Isoquinstatin 7 (5). 7-(1′-Ethyl-2′-amido-Boc-Dap)-isoquinoline (3b). Straw-colored, frothy solid (0.22g, 70% yield from Boc-Dap): TLC Rf 0.19 (CH2Cl2−MeOH, 95:5); (+)-HRAPCIMS m/z 442.2701 [M + H]+ (calcd for C25H36N3O4, 442.2706); 1H NMR (CDCl3, 400 MHz) δ 9.16 (1H, s), 8.47 (1H, d, J = 5.7 Hz), 7.77 (2H, d, J = 8.2 Hz), 7.61−7.58 (2H, m), 6.59, 6.02 (1H, NH), 3.82 (1H, m), 3.76− 3.45 (4H, m), 3.36 (3H, s, OCH3), 3.17 (1H, m), 3.06 (2H, m), 2.33 (1H, m), 1.80 (2H, m), 1.68−1.53 (2H, m), 1.47 (9H, m), 1.20 (3H, m); 13C NMR (CDCl3, 400 MHz) δ 174.5, 174.1, 155.1, 152.2, 142.8, 138.7, 138.4, 134.8, 132.1, 128.9, 126.9, 120.4, 84.2, 82.1, 79.9, 79.6, 61.0, 59.3, 58.7, 47.0, 46.7, 44.6, 44.2, 40.4, 35.8, 28.8, 26.1, 25.3, 24.8, 24.3, 15.0, 14.4; (+)-HRAPCIMS m/z 442.2701 [M + H]+ (calcd for C25H36N3O4, 442.2706). 7-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)-isoquinoline (Isoquinstatin 7) (5). Straw-colored, glassy solid [42 mg, 21% from 7-(1′-ethyl-2′amido-boc-dap)-isoquinoline (3b)]: TLC Rf 0.32 (CH3COCH3); [α]24D −15.2 (c 0.24, CHCl3); 1H NMR (CDCl3, 400 MHz) peak splitting due to conformers observed δ 9.17 (1H, m), 8.45 (1H, m), 7.89−7.72 (2H, m), 7.67−7.47 (2H, m), 6.89−6.76 (2H, m), 4.78 (2H, m), 4.02−3.89 (1H, m), 3.88−3.77 (2H, m), 3.75−3.51 (2H, m), 3.43−3.23 (8H, m) 3.36, 3.32 (s, OCH3), 3.30, 3.27 (s, OCH3), 3.06 (2H, m), 3.02 (3H, NCH3), 2.49−2.30 (3H, m), 2.25 (6H, s), 2.19 (1H, m), 2.14−1.84 (5H, m), 1.67 (3H, m), 1.36 (1H, m), 1.21 (3H, m), 1.06−0.87 (16 H, m), 0.82 (3H, m); 13C NMR (CDCl3, 400 MHz) δ 174.6, 174.4, 173,6, 171,.8, 170.6, 170.5, 153.2, 152.1, 143.4, 142.7, 138.8, 134.9, 134.6, 132.1, 130.8, 126.8, 126.9, 126.7, 126.6, 120.4, 86.0, 82.0, 81.8, 78.6, 76.6, 61.7, 60.8, 60.77, 59.7, 59.6, 58.1, 58.0, 53.9, 47.8, 47.77, 44.8, 44.7, 43.0, 40.4, 40.3, 37.5, 37.4, 35.7, 33.2, 33.1, 32.1, 31.1, 27.8, 25.9, 25.0, 24.9, 20.3, 19.6, 18.3, 18.1, 17.9, 16.0, 15.2, 15.0, 10.8; (+)-HRAPCIMS m/z 753.5279 [M + H]+ (calcd for C42H69N6O6, 753.5279). Desmethylisoquinstatin 7 (8). 7-(1′-Ethyl-2′-amido-Dap-DilVal-N-Methyl-valine)isoquinoline, (Desmethylisoquinstatin 7) (8). Straw-colored glass [40 mg, 22% from from 7-(1′-ethyl-2′-amido-Boc-

removed under reduced pressure, and the residue dissolved in toluene and further concentrated (2×), then dried using a high vacuum for 16 h and used without further purification for the next step. 8-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)isoquinoline (Isoquinstatin 8) (6). The preceding TFA salt (0.17 mmol) was dissolved in CH2Cl2 (2 mL) and stirred at 0 °C. Then Dov-Val-Dil-TFA6c (0.092 g, 0.169 mmol, 1 equiv) was added followed by TEA (0.12 mL, 0.09 g, 0.86 mmol, 5 equiv) and DEPC (0.04 mL, 0.26 mmol, 1.5 equiv). The reaction mixture was stirred under N2 for 7 h at 0 °C and then concentrated under reduced pressure. The residue was dried under a high vacuum and separated on a silica gel column [gradient elution with CH2Cl2−MeOH, 97:3 → 95:5; column size (2 cm × 30 cm)]. This gave the product as a light yellow colored powder, 0.038 g, which was further purified on a pipet silica gel column (eluent: CH2Cl2− MeOH, 94:6) to give a colorless glass (0.024 g, 0.032 mmol, 19% yield): TLC Rf 0.15 (CH2Cl2−MeOH, 94:6); [α]23D −6 (c 0.2, CHCl3); 1H and 13 C NMR (CDCl3, 400 MHz) see Table 1; (+)-HRAPCIMS m/z 753.5278 (calcd for C42H69N6O6, 753.5279). Desmethylisoquinstatin 8 (9). Fmoc-tripeptide and 8-(1′-Ethyl2′-amido-Dap)isoquinoline Trifluoroacetate Mixture. To a stirred solution of Fmoc-tripeptide-OtBu ester7a−c (0.118 g, 0.170 mmol) and 8-(1′-ethyl-2′-amido-Boc-Dap)isoquinoline (3c) in dry CH2Cl2 (2.5 mL) at 0 °C under N2 was added TFA (0.75 mL), and stirring was continued for 18 h with warming to rt over time. The reaction mixture was concentrated under reduced pressure to remove CH2Cl2 and reconcentrated in the presence of toluene to remove excess TFA, then dried under a high vacuum for 6 h to yield an amber-colored residue. This material was used without further purification for the next reaction. 8-(1′-Ethyl-2′-amido-Dap-Dil-Val-N-Methylvaline)isoquinoline (Desmethylisoquinstatin 8) (9). The combined amide TFA salt residues obtained above were dissolved in dry CH2Cl2 (3 mL), and the solution was stirred under N2 and cooled to 0 °C (ice bath). TEA (0.125 mL, 5 equiv) and DECP (0.04 mL, 0.26 mmol, 1.1 equiv) were added. The solution was stirred for 18 h, then concentrated under a high vacuum to an amber oil. The oil was separated by flash silica gel chromatography using gradient elution with CH2Cl2−MeOH (97:3− 94:6) on a column (2 cm × 20 cm) to give the Fmoc-protected product as a colorless, foamy solid (0.12 g): TLC Rf 0.48 (CH2Cl2− MeOH, 94:6); (+)-HRAPCIMS m/z 961.5787 [M + H]+ (calcd for C56H77N6O8, 961.5803). The Fmoc-desmethylisoquinstatin 8 (0.12 g, 0.12 mmol) was dissolved in dry CH2Cl2 (1.5 mL) and deprotected in the presence of diethylamine (1.5 mL) for 18 h at rt under N2. The solvent was removed under reduced pressure, and the reaction products were separated using flash chromatography on silica gel, eluting with CH2Cl2−MeOH, 92:8 (column size: 1 cm × 20 cm). The product obtained was an off-white, frothy solid (36 mg, 41% from starting material): TLC Rf 0.23 (CH2Cl2−MeOH, 92:8); [α]23D −16.8 (c 0.28, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 9.56 (1H, bs), 8.52 (1H, bd, J = 4.3 Hz), 7.73−7.57 (4H, m), 7.47 (1H, d, J = 6.9 Hz), 6.94 (1H, m), 4.75 (1H, m), 4.16−3.99 (2H, m), 3.85 (1H, d, J = 8.6 Hz), 3.77− 3.68 (2H, m), 3.42 (3H, m), 3.35 (3H, s, OCH3), 3.38−3.34 (2H, m), 3.29 (3H, s, OCH3), 3.02 (3H, s, NCH3), 2.74 (1H, d, J = 4.9 Hz), 2.47−2.29 (5H, m), 2.08−1.87 (5H, m), 1.83−1.64 (3H, m), 1.37 (1H, m), 1.23 (3H, d, J = 7.1 Hz), 1.00−0.90 (17 H, m), 0.82 (3H, t, J = 7.45 Hz); 13C NMR (CDCl3, 100 MHz) δ 174.4, 173.7, 173.5, 170.7, 149.2, 143.0, 136.9, 136.5, 130.2, 128.2, 127.4, 125.7, 121.1, 82.0, 78.5, 71.4, 61.6, 60.8, 59.7, 58.4, 58.1, 53.9, 47.8, 46.7, 44.7, 40.6, 37.7, 36.0, 33.4, 31.7, 31.65, 31.2, 25.9, 25.1, 23.7, 19.8, 19.7, 18.5, 18.1, 16.1, 10.9; (+)-HRAPCIMS m/z 739.5129 [M + H]+ (calcd for C41H67N6O6, 739.5122). Isoquinstatin 5 (4). 5-(1′-Ethyl-2′-amido-Boc-Dap)-isoquinoline (3a). Straw-colored, frothy solid (0.26 g, 84% from Boc-Dap): TLC Rf 0.16 (CH2Cl2−MeOH, 95:5); 1H NMR (CDCl3, 400 MHz) δ 9.21 (1H, s), 8.54 (1H, d, J = 6.0 Hz), 7.95 (1H, d, J = 4.4 Hz), 7.85 (1H, d, J = 7.8 Hz), 7.60−7.49 (2H, m), 6.77, 6.13 (1H, NH), 3.88−3.72 (2H, m), 3.70−3.49 (2H, m), 3.39 (3H, s, OCH3), 3.39−3.15 (4H, m), 2.42−2.27 (1H, m), 1.87 (2H, m), 1.78−1.60 (2H, m), 1.48 (9H, m), 1.23 (3H, m); 13C NMR (CDCl3, 400 MHz) δ 174.7, 174.2, 155.2, 455

DOI: 10.1021/acs.jnatprod.7b00352 J. Nat. Prod. 2018, 81, 451−457

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Dap)-isoquinoline (3b)]: TLC Rf 0.14 (CH3COCH3); [α]24D −43.9 (c 0.23, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 9.17 (1H, bs), 8.49 (1H, bs), 7.84−7.71 (2H, m), 7.66−7.52 (3H, m), 6.80 (1H, m), 4.76 (1H, dd, J = 9.7, 7.0 Hz), 4.06 (1H, m), 3.94 (1H, m), 3.81 (1H, dd, J = 9.7, 1.7 Hz), 3.71 (1H, m), 3.58 (1H, m), 3.45−3.24 (8H, m), 3.07 (2H, t, J = 6.6 Hz), 3.03 (3H, s, NCH3), 2.75 (1H, d, J = 5.0 Hz), 2.41−2.28 (5H, m), 2.23 (1H, m), 2.13−1.81(6H, m), 1.73−1.52 (3H, m), 1.37 (1H, m), 1.21 (3H, d, J = 7.9 Hz), 1.02−0.90 (16 H, m), 0.84 (3H, t, J = 7.3 Hz); 13C NMR (CDCl3, 400 MHz), δ 174.5, 173.7, 173.5, 170.6, 152.2, 142.8, 138,9, 134.7, 132.2 × 2, 127.0, 126.8, 120.5, 86.0, 81.9, 78.6, 71.4, 61.7, 60.8, 59.7, 58.1, 54.0, 47.8, 44.9, 40.5, 37.6, 36.0, 35.8, 33.3, 31.7, 31.2, 25.9, 25.0, 19.8, 19.7, 18.5, 18.1, 16.1, 15.2, 10.7; (+)-HRAPCIMS m/z 739.5122 [M + H] + (calcd for C41H67N6O6, 739.5122). Mc-Val-Cit-PABC-desmethylisoquinstatin 8 (13). To a stirred solution of desmethylisoquinstatin 8 (9) (0.010 g, 0.014 mmol), McVal-Cit-PABC-PNP8 (10) (0.020 g, 0.027 mmol), and N-hydroxybenzytriazole (HOBt) (0.004 g, 0.030 mmol) in dimethylformamide (DMF) (0.20 mL) was added diisopropylethylamine (DIEA) (0.011 mL). After 16 h of stirring at rt, the DMF was removed under reduced pressure and the crude reaction product was separated by column chromatography using Sephadex LH-20 and by eluting with methanol to give the product as a colorless, amorphous solid [11 mg, 58% yield from desmethylquinstatin 8 (9)]: TLC Rf 0.10 (CH2Cl2−MeOH, 7%); 1 H NMR (CD3OD, 500 MHz) consistent with the proposed structure; (+)-HRESIMS m/z 1337.7825 [M + H]+ (calcd for C70H105N12O14, 1337.7868). Mc-Val-Cit-PABC-desmethylisoquinstatin 5 (11). 11 was synthesized as recorded for desmethylisoquinstatin 8 (9) above and separated by column chromatography using Sephadex LH-20, eluting with methanol, to give the product as a colorless, amorphous solid [9.2 mg, 49% yield from desmethylquinstatin 5 (7)]: TLC Rf 0.11 (CH2Cl2−MeOH, 7%); 1H NMR (CD3OD, 500 MHz) consistent with the proposed structure; (+)-HRESIMS m/z 1337.7818 [M + H]+ (calcd for C70H105N12O14, 1337.7868). Mc-Val-Cit-PABC-desmethylisoquinstatin 7 (12). 12 was synthesized as recorded for desmethylisoquinstatin 8 (9) above and separated by column chromatography using Sephadex LH-20 and eluting with methanol to give the product as a colorless, amorphous solid [12.5 mg, 67% yield from desmethylquinstatin 7 (8)]: TLC Rf 0.18 (CH2Cl2−MeOH, 7%); 1H NMR (CD3OD, 500 MHz) consistent with the proposed structure; (+)-HRESIMS m/z 1337.7825 [M + H]+ (calcd for C70H105N12O14, 1337.7868). Cancer Cell Line Procedures. Human cancer cell growth inhibition was measured using the standard sulforhodamine B assay of the U.S. National Cancer Institute, as previously described.8 Briefly, cells in a 5% fetal bovine serum/RPMI1640 medium were inoculated in 96-well plates and incubated for 24 h. That was followed by serial dilutions of the compounds added. Forty-eight hours later the plates were fixed with trichloroacetic acid, stained with sulforhodamine B, and read with an automated microplate reader. Next, growth inhibition of 50% (GI50, or the drug concentration causing a 50% reduction in the net protein increase) was calculated from optical density data with Immunosoft software.



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AUTHOR INFORMATION

Corresponding Author

*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@ asu.edu. ORCID

George R. Pettit: 0000-0001-8706-8929 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS With appreciation, we acknowledge and thank for financial support the Arizona Biomedical Research Commission, the J.W. Kiecknefer Foundation, and the Margaret T. Morris Foundation.



DEDICATION Dedicated to Dr. Susan Band Horwitz, of Albert Einstein College of Medicine, Bronx, NY, for her pioneering work on bioactive natural products.



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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.7b00352. 1 H and 13C NMR spectra for intermediates 3a−c and for isoquinstatin 5 (4) and 8 (6), desmethylisoquinstatin quinstatin 5, 7, and 8 (7−9), and Mc-Val-Cit-PABCdesmethylisoquinstatins 5, 7, and 8 (11−13); 1H and HSQC NMR spectra for isoquinstatin 7 (5) (PDF) 456

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