Design, Synthesis, and Cytotoxic Activity of New Tubulysin Analogues

Hai Van Le; Loc Van Tran; Anh Tuan Tran; Thao Thi Phuong Tran; Sung Van Tran; Chien Van Tran

doi:10.1055/s-0041-1737139

Synlett, Table of Contents

Synlett 2022; 33(02): 187-195
DOI: 10.1055/s-0041-1737139

letter

Design, Synthesis, and Cytotoxic Activity of New Tubulysin Analogues

Hai Van Le

^aInstitute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

^cGraduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

,

Loc Van Tran

^aInstitute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

^cGraduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

,

Anh Tuan Tran∗

^bUniversity of Science and Technology of Hanoi, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

,

Thao Thi Phuong Tran

^aInstitute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

^cGraduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

,

Sung Van Tran

^aInstitute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

,

Chien Van Tran∗

^aInstitute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

^cGraduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

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Abstract

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Abstract

Synthesis of tubulysin analogues, containing an N-methyl substituent on tubuvaline-amide together with the replacement of either the hydrophobic N-terminal N-methyl pipecolic acid (Mep) or at both N- and C- terminal peptides with available heteroaromatic acids and an unsaturated tubuphenylalanine moiety, respectively, were described. The in vitro cytotoxic activity by SRB assay on five cancer cell lines for sixteen tubulysins was evaluated. Among them, five analogues exhibited strong cytotoxic activities against five human cancer cell lines, including human breast carcinoma (MCF7), human colorectal adenocarcinoma (HT-29), HL-60, SW-480, human lung adenocarcinoma (A459). Interestingly, one analogue showed the strongest cytotoxicity on all five tested cell lines even much higher toxicity than the reference compound ellipticine.

Key words

tubulysin analogues - tetrapeptide - cytotoxicity - myxobacterial

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References

References and Notes
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30 Synthesis of Tubulysin Analogue 31a–c Peptides 29b,c were synthesized in a similar procedure as described to the peptide 29a. These products were used for next steps after silica gel column chromatography, but they were not pure enough. A stirred solution of menthyl esters 29a–d (35 mg) in THF/H₂O (3 mL, 2:1) was treated with a solution of 10% KOH (10 equiv). The reaction mixture was warmed to 45 °C and stirred for 48 h. The reaction mixture was concentrated under reduced pressure and redissolved in H₂O, acidified with 5% HCl, and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with aqueous NaHCO₃, brine solution, and dried over Na₂SO₄. Removal of solvent and flash column chromatography on silica gel afforded acids 30a–c and the ester 30d which only showed the removal of acetyl group. For compounds 30b,c, we directly acetylated to obtain the corresponding final products 31b,c. Acetylation of Hydroxy Peptides Tetrapeptides 30a–c were treated with acetic anhydride (0.1 mL) in pyridine (1 mL) at room temperature overnight. Then, pyridine was evaporated under reduced pressure to dryness. The residue was dissolved in EtOAc and washed with 2% HCl, saturated aqueous solution of NaHCO₃, brine solution, and dried over Na₂SO₄. The crude material was subjected to column chromatography on silica gel eluted with DCM/MeOH (20:1) to obtain final products 31a–c. Compound 31a: yield 80% (12.5 mg); R_f = 0.22 (DCM/MeOH, 20:1); [α]_D ²⁵ –11.26 (c 0.12, MeOH). ¹H NMR (500 MHz, CD₃OD): δ = 8.11 (s, 1 H), 7.29–7.22 (m, 5 H), 5.70 (td, J = 10.5, 2.5 Hz, 1 H), 4.74 (overlap, 2 H), 4.58–4.53 (m, 1 H), 4.40 (br s, 1 H), 3.72–3.71 (m, 1 H), 3,.60–3.5 (m, 2 H), 3.23–3.18 (m, 2 H), 3.11 (s, 3 H), 2.94 (m, 1 H), 2.59–2.57 (m, 1 H), 2.35 (s, 3 H), 2.15 (s, 3 H, OAc), 2.30–2.25 (m, 2 H), 1.91–1.88 (m, 6 H), 1.69–1.58 (m, 4 H), 1.17–1.5 (d, 3 H), 1.07–1.04 (overlap, 3 H), 0.99–0.97 (overlap, 6 H), 0.85 (d, J = 6.5 Hz, 3 H), 0.80 (d, J = 7 Hz, 3 H). ¹³C NMR (125 MHz, CD₃OD): δ = 177.7, 174.9, 172.5, 171.7, 171.1, 162.6, 150.1, 139.4, 130.5, 130.5, 129.5, 127.4. 125.1, 75.6, 71.2, 69.6, 64.4, 56.4, 44.0, 40.6, 38.6, 35.7, 32.3, 31.4, 27.6, 25.3, 24.9, 23.2, 22.0, 20.0, 18.0, 16.7, 16.1, 13.13, 11.2, 9.2. HRMS-ESI: m/z calcd for C₃₈H₅₈N₅O₇S [M + H]⁺: 728.4057; found: 728.4052. Compound 31b: yield 45% (13.5 mg), over two steps; R_f = 0.30 (DCM/MeOH, 20:1); [α]_D ²⁵ .27 (c 0.15, MeOH). ¹H NMR (500 MHz, CD₃OD): δ = 8.57 (d, J = 9.5 Hz, 1 H), 8.40 (dd, J = 4.5, 1 Hz, 1 H), 7.88 (s, 1 H), 7.56–7.54 (m, 1 H), 7.31–7.20 (m, 5 H), 5.74 (td, J = 11.5, 2.5 Hz, 1 H), 4.97–4.95 (m, 1 H), 4.55–4.51 (m, 1 H), 3.47–3.43 (dd, J = 11.5, 3.5 Hz, 1 H), 3.22–3.19 (m, 2 H), 3.07 (s, 3 H), 2.98–2.94 (m, 1 H), 2.72–2.76 (m, 1 H), 2.70 (s, 3 H), 2.55 (m, 1 H), 2.41–2.39 (m, 1 H), 2.25–2.30 (m, 1 H), 2.18 (s, 3 H, OAc), 1.95–1.93 (m, 1 H), 1.78–1.73 (m, 2 H), 1.72–1.68 (m, 2 H), 1.55–1.49 (m, 1 H), 1.19 (d, J = 7 Hz, 3 H), 1.15 (d, J = 7 Hz, 2 H), 1.07 (d, J = 7 Hz, 3 H), 0.97 (d, J = 7 Hz, 3 H), 0.94 (overlap, 3 H), 0.76 (d, J = 7 Hz, 3 H). ¹³C NMR (125 MHz, CD₃OD): δ = 177.6, 173.4, 170.2, 169.2, 169.1, 165.6, 163.4, 149.4, 149.2, 147.2, 145.7, 140.5, 137.1, 136.6, 135.1, 129.7, 128.8, 127.3, 126.9, 125.5, 125.1, 69.7, 57.0, 56.3, 55.7, 53.7, 39.5, 39.0, 37.5, 36.6, 34.3, 31.9, 29.7, 24.1, 20.8, 16.2, 13.8, 11.2. HRMS-ESI: m/z calcd for C₃₈H₅₀N₅O₆S [M + H – H₂O]⁺: 704.3482; found: 704.3488. Compound 31c: yield 39% (11.5 mg), over two steps; R_f = 0.32 (DCM/MeOH, 20:1); [α]_D ²⁵ –26.75 (c 0.11, MeOH). ¹H NMR (500 MHz, CDCl₃): δ = 9.53 (d, J = 8.0 Hz, 1 H), 8.72 (d, J = 9.5 Hz, 1 H), 8.48 (d, J = 5.5 Hz, 1 H), 8.02 (s, 1 H), 7.83 (d, J = 8 Hz, 1 H), 7.78 (d, J = 8 Hz, 1 H), 7.71 (t, J = 7 Hz, 1 H), 7.66 (t, J = 7 Hz, 1 H), 7.29–7.27 (m, 3 H), 7.22–7.20 (m, 2 H), 5.73 (dd, J = 11.5, 2.5 Hz, 1 H), 5.10–5.05 (m, 1 H), 4.59–4.56 (m, 1 H), 4.39–4.38 (br s, 1 H), 3.15 (s, 3 H), 2.98–2.94 (m, 1 H), 2.92–2.83 (m, 1 H), 2.63–2.58 (m, 2 H), 2.35–2.34 (m, 1 H), 2.19 (s, 3 H, OAc), 2.05–1.97 (m, 2 H), 1.82–1.73 (m, 2 H), 1.68–1.62 (m, 2 H), 1.19–1.17 (d, J = 6.5 Hz, 3 H), 1.09 (d, J = 6.5 Hz, 3 H), 1.04 (d, J = 6.5 Hz, 3 H), 0.87 (d, J = 7 Hz, 3 H), 0.81 (d, J = 7 Hz, 3 H). ¹³C NMR (125 MHz, CDCl₃): δ = 175.7, 173.3, 170.1, 169.9, 165.7, 160.5, 150.2, 147.8, 140.5, 137.4, 131.3, 130.4, 129.7, 128.4, 127.1, 124.3, 122.3, 117.3, 74.2, 69.6, 53.9, 48.8, 47.0, 41.8, 334.7, 31.4, 30.1, 20.8, 20.1, 17.5, 16.1, 15.8. HRMS-ESI: m/z calcd for C₄₁H₅₂N₅O₇S [M + H]⁺: 758.3587; found: 758.3566.
31 Synthesis of Tubulysin Analogue 34a General Procedure To ethyl esters 33a,b (20 mg) in THF/H₂O (3 mL, 2:1) was added a solution of 5% LiOH (10 equiv) and stirred at room temperature for 24 h. The reaction mixture was concentrated under reduced pressure and redissolved in H₂O, acidified with 5% HCl, and extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with aqueous NaHCO₃, brine, and dried over Na₂SO₄. Removal of solvent and column chromatography on silica gel (DCM/MeOH, 25:1) afforded desired products 34a,b. Compound 34a: yield 76% (14 mg); R_f = 0.28 (DCM/MeOH, 25:1); [α]_D ²⁵ +1.91 (c 0.09, MeOH). ¹H NMR (500 MHz, CD₃OD): δ = 8.07 (d, J = 11 Hz, 1 H), 7.27–7.19 (m, 5 H, H-Ph), 6.61 (br s, 1 H), 5.13 (br s, 1 H), 4.70–4.68 (m, 3 H), 3.21–3.20 (m, 2 H), 3.18 (s, 3 H), 3.16–3.12 (m, 1 H), 2.96–2.94 (dd, J = 7.5, 3.5 Hz, 1 H), 2.37 (s, 3 H), 2.11–1.94 (br s, 2 H), 1.88 (m, 1 H), 1.76 (s, 3 H), 1.51–1.43 (m, 3 H), 1.31–1.16 (br s, 3 H), 1.14 (d, J = 7 Hz, 3 H), 1.09–0.99 (overlap, 6 H), 0.88 (d, J = 7 Hz, 3 H). HRMS-ESI: m/z calcd for C₃₆H₅₄N₅O₆S [M + H]⁺: 684.3795; found: 684.3773.
32 Tubulysin Analogue 34b Yield 82% (15 mg); R_f = 0.34 (DCM/MeOH, 20:1); [α]_D ²⁵ –19.13 (c 0.09, MeOH). ¹H NMR (500 MHz, CD₃OD): δ = 9.04 (d, J = 8.5 Hz, 1 H), 8.52 (d, J = 5.5 Hz, 1 H), 8.05 (s, 1 H), 7.99 (d, J = 8.5 Hz, 1 H), 7.95 (d, J = 8.5 Hz, 1 H), 7.80 (t, J = 7.5 Hz, 1 H), 7.66 (t, J = 7.5, 8 Hz, 1 H), 7.28 (m, 2 H), 7.17 (t, 1 H), 7.08–7.04 (m, 2 H), 6.53 (d, J = 9.5 Hz, 1 H), 5.08 (d, J = 8 Hz, 1 H), 4.71–4.66 (overlap, 1 H), 4.62–5.59 (d, J = 7.5 Hz, 1 H), 4.16 (t, J = 7.5 Hz, 1 H), 3.08–3.06 (m, 1 H), 2.95 (s, 3 H), 2.66–2.64 (m, 1 H), 2.52–2.49 (m, 1 H), 2.40 (m, 1 H), 2.31–2.25 (m, 2 H), 2.08–2.04 (m, 1 H), 1.73 (s, 3 H), 1.59–1.55 (m, 2 H), 1.12 (d, J = 7 Hz, 3 H), 1.04 (d, J = 6.5 Hz, 3 H), 0.94 (d, J = 7 Hz, 3 H), 0.88 (d, J = 7 Hz, 3 H). ¹³C NMR (125 MHz, CD₃OD): δ = 173.3, 170.1, 167.8, 165.4, 160.0, 149.7, 147.8, 140.2, 139.0, 137.5, 136.6, 130.5, 130.4, 129.6, 128.6, 128.5, 127.6, 127.0, 126.9, 126.8, 124.3, 123.7, 69.5, 60.3, 54.0, 48.7, 40.9, 37.4, 34.6, 30.0, 24.3, 20.8, 20.1, 19.6, 14.2, 12.7, 11.2. HRMS-ESI: m/z calcd for C₃₉H₄₇N₅O₆SNa [M + Na]⁺: 736.3145; found: 736.3125.
33 Synthesis of Tubulysin Analogue 37 N-Boc-protected tripeptide 35 or 36 (0.05 mmol) in DCM (2 mL) was treated with a solution of TFA/TIPS/H₂O (3 mL, 95/2.5/2.5) at 0 °C for 2 h, then concentrated under reduced pressure to dryness to yield an intermediate trifluoroacetate salt which was used for the next steps without further purification. To a stirred solution of the resulting trifluoroacetate salt (1 equiv), HATU (1.5 equiv), and 5-methyl-2-pyrazinecarboxylic acid (1.5 equiv) in DMF (2 mL), DIPEA (3.0 equiv) was added. After stirring at room temperature for 12 h, the reaction mixture was diluted with EtOAc (50 mL), washed with brine solution (3×), and dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography eluted with n-hexane/EtOAc (1:1) to afford peptides 37 and 38. Compound 37: yield 64% (21 mg); R_f = 0.4 (DCM/MeOH, 25:1); [α]_D ²⁵ –5.82 (c 0.10, MeOH). ¹H NMR (500 MHz, CDCl₃): δ = 9.21 (s, 1 H), 8.42 (s, 1 H), 8.06 (s, 1 H), 7.29–7.21 (m, 4 H, Ph), 7.18 (t, 1 H), 5.08 (d, J = 8 Hz, 1 H), 5.01–4.98 (m, 1 H), 4.59 (dd, J = 7.5, 2 Hz, 1 H), 3.73 (s, 3 H), 3.24 (t, J = 7.0 Hz, 3 H), 3.12 (s, 3 H), 2.66 (s, 3 H), 2.10–2.03 (m, 2 H), 1.87–1.85 (m, 2 H), 1.79–1.77 (m, 1 H), 1.67–1.62 (m, 2 H), 1.03 (t, J = 7.0 Hz, 3 H), 0.97 (d, J = 7 Hz, 3 H), 0.93 (d, J = 7 Hz, 3 H), 0.82 (d, J = 7 Hz, 3 H). HRMS-ESI: m/z calcd for C₃₃H₄₄N₆O₆SNa [M + Na]⁺: 675.2941; found: 675.2906.
34 Synthesis of Tubulysin Analogue 38 Yield 68% (24 mg); R_f = 0.56 (DCM/MeOH, 25:1); [α]_D ²⁵ –10.67 (c 0.10, MeOH). ¹H NMR (500 MHz, CDCl₃): δ = 9.21 (s, 1 H), 8.46 (s, 1 H), 8.21 (d, J = 9.5 Hz, 1 H), 8.06 (s, 1 H), 7.30–7.27 (m, 3 H, Ph), 7.18 (t, J = 7 Hz, 2 H, Ph), 5.66 (dd, J = 11.0, 2.5 Hz, 1 H), 5.06–4.99 (m, 2 H), 4.49 (br s, 1 H), 3.73 (s, 3 H), 3.24–3.22 (t, J = 4 Hz, 3 H), 3.05 (s, 3 H), 2.67 (s, 3 H), 2.35–2.29 (td, J = 9.5, 2.5 Hz, 1 H), 2.19 (s, 3 H, OAc), 2.09–2.06 (m, 2 H), 1.96–1.94 (m, 1 H), 1.79–1.77 (m, 1 H), 1.67–1.62 (m, 2 H), 1.03 (d, J = 7 Hz, 3 H), 0.97 (d, J = 7 Hz, 3 H), 0.91 (t, J = 7 Hz, 3 H), 0.76 (d, J = 7 Hz, 3 H). ¹³C NMR (125 MHz, CDCl₃): δ = 178.7, 173.2, 173.0, 171.7, 170.1, 160.3, 1586, 149.2, 142.9, 142.5, 135.9, 129.3, 128.5, 127.1, 124.1, 69.5, 53.9, 53.1, 52.3, 38.1, 37.4, 34.3, 29.9, 24.0, 21.4, 20.7, 20.1, 19.5, 16.1, 11.1. HRMS-ESI: m/z calcd for C₃₅H₄₇N₆O₇S [M + H]⁺: 695.3227; found: 695.3237.
35 Synthesis of Tubulysin Analogue 39 Ester 38 (25 mg, 0.035 mmol) in THF/H₂O (2:1, 2 mL) was treated with LiOH (17.5 mg, 0.73 mmol) for 12 h at room temperature and concentrated to dryness under reduced vacuum. The residue was acidified with HCl 5% to pH 4, extracted with EtOAc, and dried over Na₂SO₄. Pure product 39 (18 mg, 85%) was obtained by silica gel column chromatography (DCM/MeOH, 20:1) as a white foam. R_f = 0.36 (DCM/MeOH, 20/1); [α]_D ²⁵ –12.05 (c 0.12, MeOH). ¹H NMR (500 MHz, CD₃OD): δ = 9.08 (s, 1 H), 8.59 (s, 1 H), 8.07 (s, 1 H), 7.24–7.22 (m, 5 H, Ph), 5.03 (d, J = 7.5 Hz, 1 H), 4.66 (dd, J = 2, 9.5 Hz, 2 H), 3.30–3.29 (m, 1 H), 3.23–3.20 (m, 2 H), 3.15 (s, 3 H), 2.65 (s, 3 H), 2.24 (m, 1 H), 2.09–1.98 (m, 2 H), 1.96–1.94 (m, 1 H), 1.71–1.64 (m, 1 H), 1.31–1.37 (m, 2 H), 1.08 (d, J = 7 Hz, 3 H), 0.97 (d, J = 7 Hz, 3 H), 0.94 (t, J = 7 Hz, 3 H), 0.80 (d, J = 6.5 Hz, 3 H). ¹³C NMR (125 MHz, CDCl₃): δ = 176.5, 173.1, 171.2, 170.4, 160.6, 158.2, 149.2, 143.4, 142.7, 135.9, 129.6, 128.8, 127.1, 124.1, 69.5, 53.9, 53.1, 52.3, 38.1, 37.4, 34.3, 24.1, 21.4, 20.3, 20.1, 19.6, 15.7, 12.2. HRMS-ESI: m/z calcd for C₃₂H₄₂N₆O₆SNa [M + Na]⁺: 661.2784; found: 661.2770.
36 Tubulysin Analogue 42 To a solution of acid 41 (50 mg, 0.093 mmol), HATU (54 mg, 0.14 mmol), and DIPEA (0.1 mL, 0.48 mmol) in dry DMF (2 mL) was added 4-(2-aminoethyl)benzensulfonamide (30 mg, 0.14 mmol). The reaction mixture was stirred at room temperature for 18 h, then followed by removal of the solvent. The residue was subjected to silica gel column chromatography (DCM/MeOH, 15:1) to afford 42 (0.31 mg, 47%). R_f = 0.35 (DCM/MeOH, 15:1); [α]_D ²⁵ +3.65 (c 0.13, MeOH). ¹H NMR (500 MHz, CD₃OD): δ = 8.15 (s, 1 H), 7.85–7.83 (m, 2 H), 7.47 (dd, J = 3.5, 8.5 Hz, 2 H), 5.83 (dd, J = 3.5, 11 Hz, 1 H), 4.47–4.42 (m, 1 H), 3.69 (t, 3 H), 3.04 (t, 3 H), 2.91 (s, 3 H), 2.76–2.75 (d, 1 H), 2.39–2.34 (m, 1 H), 2.27–2.21 (m, 1 H), 2.13 (s, 3 H, OAc), 2.06 (s, 3 H), 1.85–1.80 (m, 1 H), 1.40 (t, 2 H), 1.34 (br s, 6 H), 1.08–1.02 (m, 3 H), 0.99 (d, J = 7 Hz, 3 H), 0.92 (t, 3 H), 0.87 (d, J = 6.5 Hz, 3 H). ¹³C NMR (125 MHz, CD₃OD): δ = 174.5, 171.9, 171.7 (OAc), 163.2, 161.5, 150.8, 145.2, 143.1, 130.5, 127.3, 125.2, 75.5, 70.6, 61.7, 57,4, 54,9, 41.4, 36.3, 35.6, 31.0, 30.7, 29.6, 21.9, 20.9, 20.3, 20.1, 18.7, 14.6, 11.5. HRMS-ESI: m/z calcd for C₃₄H₅₃N₆O₇S₂ [M + H]⁺: 721.3417; found: 721.3428.
37 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. J. Natl. Cancer Inst. 1990; 82: 1107
38 Biological Assay Human cell lines, HT29 (human colorectal adenocarcinoma), MCF7 (Human breast carcinoma), A549 (human lung adenocarcinoma), SW480 (human colon carcinoma), HL60 (human acute leukemia), were provided by Prof J. Maier, Milan University, Italia, and Prof J. M. Pezzuto, Rutgers University, USA. HL60 cells were maintained in RPMI medium with 10% fetal bovine serum (FBS) and gentamicin (50 μg/mL). The others were cultured in Eagle’s minimum essential medium (EMEM) containing 1% nonessential amino acids (NAA), gentamicin, and 10% FBS in the incubator at 37 °C and 5% CO₂. Cells were subcultured for 48 h. Cytotoxicity of tubulysin analogues against MFC7, A549, SW480, HL60, and HT29 human cancer cell lines was evaluated by sulforhodamine B (SRB) assay. Briefly, 3·10⁴ cells mL^–1 were seeded in 96-well microplates and treated with four different concentrations (100, 20, 4.0, 0.8 μg mL^–1) of each tested compound in DMSO. After incubation at 37 °C in 5% CO₂ for 48 h, cells were fixed with 20% trichloroacetic acid (TCA) for 30 min, washed three times with acetic acid, dried at room temperature, and then stained with SRB 0.4% for 30 min. The protein-binding SRB dye, which is related with cell viability, was dissolved in 10 mM unbuffered Tris base and shaken gently for 10 min. The optical density (OD) was recorded at 540 nm by ELISA plate reader (Biotek). Each concentration was tested in triplicate, and ellipticine was used as positive control. IC₅₀ values were determined based on using TableCurve 2Dv4 software.

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