Synthesis of new hemiasterlin derivatives with α,β-Unsaturated carbonyl-thiophene groups in fragment A

Vietnam Journal of Chemistry, International Edition, 55(4): 484-488, 2017 DOI: 10.15625/2525-2321.2017-00495 484 Synthesis of new hemiasterlin derivatives with α,β-unsaturated carbonyl-thiophene groups in fragment A Pham The Chinh 1* , Pham Thi Tham 2 , Nguyen Van Tuyen 3 1 Thai Nguyen University of Science 2Hanoi University of Industry 3 Insitute of Chemistry, Vietnam Academy of Science and Technology Received 19 February 2017; Accepted for publication 28 August 2017 Abstract Hemiasterlin, an antimitotic tripeptide, exhibits cytotoxicity in the nanomolar range against a variety of cultured human and murine cell lines. For this reason, the synthesis of new hemiasterlin derivatives has attracted a lot of interest in the organic chemistry community recently. In this article, we synthesized new simplified derivatives of hemiasterlin in which the α,α-dimethylbenzylic group in fragment A is replaced by α,β-unsaturated carbonyl-thiophene group. These new compounds will be prepared by classical peptide coupling approach between the carboxylic acid fragments A 12 and dipeptide 13. We expect that this derivative will possess interesting biological activities due to the high reactivity of the α,β-unsaturated carbonyl group as Michael receptor with biological nucleophiles, such as DNA, RN5A, and enzymes. Keywords.Synthesis, hemiasterlin, α,β-unsaturated; tripeptides, nanomolar. 1. INTRODUCTION Hemiasterlins belong to a family of naturally occurring tripeptides from marine sponges [1]. The important derivatives of hemiasterlin are hemiasterlin A, hemiasterlin B, and hemiasterlin C, which were isolated from marine sponge Auletta and Cymbastella (figure 1) [2, 3]. These naturally occurring substances exhibited potent cytotoxicity in vitro against murine leukemia P388 and human breast, ovarian, colon, and lung cancer cell lines [2,3,5]. Hemiasterlins suppress microtubule depolymerization presumably by binding to the vinca alkaloid site of tubulin and causing mitotic arrest and cell death [4]. This active mechanism makes them very attractive molecules for new anticancer drugs. However, synthesis of the stereospecific amine group and especially the gem- dimethyl moiety in segment A was proved to be highly problematic. To overcome this difficulty, several studies explored the modifications of segment A that eliminated the gem-dimethyl moiety [6]. Some derivatives from this approach (7, 8) showed promising cytotoxic test results. Figure 1: Examples of hemiasterlin derivatives and modification of segment A of hemiasterli VJC, 55(4), 2017 Pham The Chinh et al. 485 Recently, Hayashi et al. and McMurray et al. prepared new synthetic peptides (7, 8) with α,β- unsaturated carbonyl systems which were thought to play a role in the remarkably strong cytotoxicity of these compounds [7, 8]. The α,β-unsaturated carbonyl moieties are well-known Michael type acceptors. They are particularly reactive and interact strongly with electron-rich biological macromolecules such as DNA, protein, and enzyme, resulting in a wide range of biological effects including general toxicity, allergenic reactions, mutagenicity, and carcinogenicity. In this article, we report the synthesis of new derivatives of hemiasterlin with a novel direction in the construction of fragment A that contained α,β- unsaturated-thiophene group. 2. EXPERIMENTAL 2.1. General information All reactions were performed in the appropriate oven-dried glass apparatus and under nitrogen atmosphere. Unless otherwise stated, solvents and chemicals were obtained from commercial sources and used without further purification. Column chromatography was performed using silica gel (60 Å, particle size 40-60 µm). NMR spectra were recorded on a Bruker Avance (500 MHz). Chemical shifts (δ) are given in parts per million (ppm) and coupling constants (J) in hertz (Hz). Mass spectrometry analysis (MS) were recorded on a Q- exactive or a Q-TOF2 instrument. IR analysis was recorded on Perkin Elmer Spectrum Two. 2.2. Synthesis of compound 11 A solution of N-acetylglycine (10) (500 mg, 4.27 mmol), thiophene-2-carbaldehyde (360 mg, 3.20 mmol), acetic anhydride (15 mL), and fused sodium acetate (350 mg, 4.27 mmol) was heated at 90 °C with stirring for 12 h. Afterwards, the acetic anhydride was evaporated in vacuum, the residue was extracted with CH2Cl2 and the combined CH2Cl2 extract was washed with brine, dried (MgSO4). The solvent was removed in vacuum to give crude azalactone 11 which was purified by column chromatography on silica gel (n- hexane/EtOAc 98/2) to afford pure azalactone 11 (328 mg, 53 %). M.p. 168-170 °C. IR (KBr): 3063; 1793; 1676; 1655; 1624; 1561; 1537; 1468; 1381; 1305; 1247; 1189; 1017; 1003; 941; 896; 873; 824; 766 cm -1 . 1 H-NMR (CDCl3, 500 MHz) δ ppm: 8.41 (1H, s, CH=C-); 7.66 (1H, brs, H-5’); 7.32 (1H, brs, H-3’); 6.95 (1H, brs, H-4’); 2.45 (3H, s, CH3). 2.3. Synthesis of compound 12 A solution of azalactone 11 (300 mg, 1.55 mmol) in NaOH 1 N (5 mL) was heated at 100 o C for 30 min, followed by addition of a solution of HCl 12 N (5 mL). After stirring for 4 h, the solvent was evaporated in vacuum to give a residue which was extracted with EtOAc and the combined EtOAc extract was washed with brine and dried (MgSO4). The solvent was removed in vacuum to give amide 12 which was purified by column chromatography on silica gel (n-hexane/EtOAc: 95/5) to afford the pure desired product 12 (200 mg, 61 %). M.p. 214- 215 °C. IR (KBr): 3422; 3288; 3121; 2915; 1687; 1640; 1624; 1558; 1527; 1471; 1519; 1302; 1244; 1216; 1190; 1024; 941; 886; 869; 767; 749; 695 cm -1 . 1 H-NMR (CDCl3, 500 MHz) δ ppm: 7.48 (1H, brs, H-4’); 7.15 (1H, brs, H-3’); 6.57 (1H, brs, H- 5’); 6.44 (1H, s, H-3); 2.11 (3H, s, CH3). 13 C-NMR (CDCl3, 125 MHz) δ ppm: 170.6 (CO, Ac), 166.4 (C=O), 149.1 (C-2’), 144.5 (C-5’), 121.7 (NH- C=CH); 120.6 (NH-C=CH), 115.5 (C-3’), 111.9 (C-4’), 22.0 (CH3). 2.4. Synthesis of compounds 14a,b To a solution of compound 13a (100 mg, 0.24 mmol), EDC (50 mg, 0.26 mmol), HOBt (35 mg, 0.26 mmol) and i-PrNHEt (124 mg, 0.48 mmol) in DMF (3 mL) was added compound 12 (55 mg, 0.26 mmol) and the solution was stirred at room temperature for 12 h. The reaction mixture was partitioned between water and EtOAc and extracted with EtOAc. The combined EtOAc extract was washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude product. This material was purified by column chromatography on silica gel (hexane/EtOAc: 80/20) to obtain pure compound 14a (59 mg, 50 %). The compound 14b (62 %) was prepared in the same way using acid 12 as starting material. Compound 14a. White solid in 50 % yield, m.p. 96-98 o C. IR (KBr): 3292; 2962; 2878; 1700; 1650; 1529; 1370; 1270; 1240; 1109; 752 cm -1 . 1 H-NMR (CDCl3, 500 MHz) δ ppm: 7.52 (1H, s, H-5’); 6.73 (1H, brs, H-3’); 6.62 (1H, brs, H-4’); 6.57 (1H, s, H- 15); 6.48 (1H, s, H-3); 4.54 (1H, s, H-10); 4.39 (1H, d, J = 9.0 Hz, H-4); 4.13-4.20 (2H, m, CH2 Et); 2.10 (3H, s, CH3 Ac); 1.92 (3H, s, CH3-C2); 1.77-1.82 (1H, m, H-5); 1.27 (3H, t, J = 7.0 Hz, CH3 Et); 0.97 (9H, s, H-12); 0.86 (6H, d, J = 7.5 Hz; H-6, H-7). 13 C-NMR (CDCl3, 125 MHz) δ ppm: 169.8 (C-9); 168.2 (C=O Ac); 164.6 (COOEt); 162.4 (C-13); 149.7 (C-2’); 143.9 (C-5’); 139.6 (C-14); 129.8 (C- 3); 127.1 (C-2); 114.3 (C-15); 113.7 (C-3’); 112.0 VJC, 55(4), 2017 Synthesis of new hemiasterlin derivatives with 486 (C-4’); 61.1 (C-10); 60.7 (C-4); 52.7 (CH2 Et); 36.4 (C-11); 34.9 (C-5); 32.7 (CH3 Ac); 26.6 (C-12); 18.6 (C-6, C-7); 14.1 (CH3 Et); 13.1 (CH3-C-2). HRMS (ESI) [M+Na] + : calc. for C25H37N3NaO5S: 514.2346; Found: 514.2349. Compound 14b. White solid in 62 % yield, m.p. 93-94 o C. IR (KBr): 3278; 2960; 2932; 2871; 1710; 1691; 1665; 1620; 1510; 1475; 1410; 1369; 1281; 1248; 1100; 1026; 981; 753 cm -1 . 1 H-NMR (CDCl3, 500 MHz) δ ppm: 7.51 (1H, brs, H-5’); 6.89 (1H, brs, H-3’); 6.78 (1H, brs, H-4’); 6.62 (1H, dd, J = 1.5, 9.0 Hz,H-3); 6.46 (1H, s, H-15); 5.01 (1H, s, H- 10); 4.92 (1H, d, J = 9.5 Hz, H-4); 4.16-4.22 (2H, m, CH2 Et); 3.0 (3H, s, H-8); 2.19 (3H, s, CH3 Ac); 1.91 (3H, s, CH3-C-2); 1.88-1.89 (1H, m, H-5); 1.27 (3H, t, J = 7.0 Hz, CH3 Et); 0.99 (9H, s, H-12); 0.84 (6H, d, J = 7.5; Hz, H-6, H-7). 13 C-NMR (CDCl3, 125 MHz) δ ppm: 170.2 (C-9); 167.7 (C=O Ac); 164.1 (COOEt); 162.0 (C-13); 149.8 (C-2’); 143.7 (C-5’); 138.2 (C-14); 132.6 (C-3); 127.0 (C-2); 121.2 (C- 15); 113.8 (C-3’); 112.4 (C-4’); 60.9 (C-10); 56.2 (C-4); 55.7 (CH2 Et); 35.2 (C-11); 34.5 (C-5); 31.1 (C-8); 29.9 (CH3 Ac); 26.4 (C-12); 18.7 (C-6, C-7); 14.3 (CH3 Et); 13.8 (CH3-C-2). HRMS (ESI) [M+Na] + : calc. for C26H39N3NaO5S: 528.2503; Found: 528.2511. 2.5. Synthesis of compound 15a,b To a cooled (0 o C) solution of 14a (50 mg, 0.102 mmol) in 2 mL MeOH/H2O (2:1) was added LiOH (25 mg, 1.02 mmol). The cooling bath was removed, and the resulting mixture was stirred at room temperature for 10 h. The solvent was removed in vacuum and the residue was dissolved in a small amount of H2O. The aqueous solution was cooled to 0 o C, and acidified to pH 5.5-6.0 with aqueous hydrochloride acid solution (pH = 2). Afterward, the reaction mixture was extracted with EtOAc. The combined EtOAc extract was washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give pure compound 15a (45 mg, 95 %). The compound 15b (97 %) was prepared in the same way from 14b. Compound 15a. White solid in 95 % yield, m.p. 120-122 o C. IR (KBr): 3410; 3279; 2963; 2874; 1649; 1519; 1385; 1370; 1268; 1242; 1150; 1021; 751 cm -1 . 1 H-NMR (CD3OD, 500 MHz) δ ppm: 7.69 (1H, d, J = 1.5 Hz, H-5’); 7.12 (1H, s, H-15); 6.73 (1H, d, J = 3.5 Hz, H-3’); 6.60 (1H, dd, J = 1.5, 10.0 Hz, H-3); 6.58 (1H, dd, J = 1.5, 3.5 Hz, H-4’); 4.50 (1H, s, H-10); 4.39 (1H, dd, J = 1.5, 10.0 Hz, H-4); 2.22 (3H, s, CH3 Ac); 1.92 (3H, s, CH3-C-2); 1.81- 1.84 (1H, m, H-5); 1.00 (9H, s, H-12); 0.96 (3H, d, J = 6.5 Hz, CH3 H-6); 0.91 (3H, d, J = 6.5 Hz, H-7). 13 C-NMR (CD3OD, 125 MHz) δ ppm: 173.4 (C-1); 171.8 (C-9); 166.5 (CO Ac); 149.1 (C-2’); 146.7 (C- 5’); 140.2 (C-3); 131.4 (C-14); 126.7 (C-3); 119.5 (C-15); 116.2 (C-4’); 113.2 (C-3’); 62.2 (C-10); 54.5 (C-4); 36.2 (C-11); 33.6 (C-5); 27.1 (C-12); 22.7 (CH3 Ac); 19.3 (C-6); 19.1 (C-7); 13.3 (CH3-C-2). HRMS (ESI) [M+Na] + : calc. for C23H33N3NaO5S: 486.2033; Found: 486.2042. Compound 15b. White solid in 97 % yield, m.p. 128-129 o C. IR (KBr): 3420; 3280; 2961; 2930; 2875; 1697; 1620; 1514; 1478; 1410; 1369; 1270; 1244; 1077; 1022; 980; 754 cm -1 . 1 H-NMR (CD3OD, 500 MHz) δ ppm: 7.60 (1H, d, J = 1.5 Hz, H-5’); 7.12 (1H, s, H-15); 6.76 (2H, dd, J = 1.0, 3.5 Hz, H- 3’, H-4’); 6.58 (1H, dd, J = 1.5, 9.0 Hz, H-3); 5.08 (1H, s, H-10); 4.97 (1H, d, J = 9.5 Hz, H-4); 3.06 (3H, s, H-8); 2.21 (3H, s, CH3 Ac); 1.99-2.03 (1H, m, H-5); 1.91 (3H, s, CH3-C-2); 1.02 (9H, s, 3CH3 H-12); 0.91 (3H, d, J = 6.5 Hz, H-6); 0.86 (3H, d, J = 6.5 Hz, H-7). 13 C-NMR (CDCl3, 125 MHz) δ ppm: 173.5 (C-1); 173.3 (C-9); 173.1 (C=O Ac); 166.7 (CO-NH); 151.0 (C-2’); 146.3 (C-5’); 139.5 (C-3); 133.8 (C-14); 126.6 (C-2); 119.6 (C-15); 116.2 (C- 4’); 113.1 (C-3’); 58.9 (C-10); 56.8 (C-4); 37.0 (C- 11); 31.7 (C-8); 30.9 (C-5); 26.8 (C-12); 22.7 (CH3 Ac); 19.7 (C-6); 19.2 (C-7); 14.0 (CH3-C-2). 3. RESULTS AND DISCUSSION Recently, we synthesized new hemiasterlin analogues in which the α,α-dimethylbenzylic group and amino NHMe moiety were replaced respectively by a α,β- unsaturated aryl and an amide NHAc group leading to the suppression of one chiral center [10]. However, derivatives of hemiasterlin containing 2-thiophenyl group moiety have not been investigated. As a part of our ongoing work, we continue to focus on the new hemiasterlin analogues. For this purpose, we investigated the synthesis of new hemiasterlin derivatives in which the α,α-dimethylbenzylic group in fragment A is replaced by α,β-unsaturated thiophen-2-yl group. These new compounds will be prepared by classical peptide coupling approach between the carboxylic acid fragments A 12 and dipeptide 13 [10]. A general procedure for the synthesis of compound 12 is outlined in Scheme 1 [10]. Compound 12 was synthesized from N-acetyl glycine (10) through two steps. The first step was condensation of N-acetyl glycine (10) with furan-2- carbaldehyde using sodium acetate in the presence of acetic anhydride at 90 °C for 12 h affording azalactone 11 in 80 % yield [9, 10]. Finally, azlactone 11 was hydrolyzed in aqueous sodium hydroxide, followed by treatment with hydrochloric acid (12 N) at 100 °C for 4 h to give compound 12 in 61 % yield. VJC, 55(4), 2017 Pham The Chinh et al. 487 Scheme 1 The hemiasterlins 15a,b were prepared in two steps after peptide coupling reactions of 13a,b with amides 12 followed by the saponification of ester using a 1N lithium hydroxide solution (scheme 2). The HRMS (ESI) of compound 14a showed a pseudo-molecular ion peak at m/z 514.2349, corresponding to the molecular formula of C25H37N3NaO5S. The 1 H-NMR showed signals of a thiophene ring at 7.51 (1H, brs, H-5’); 6.89 (1H, brs, H-3’) and 6.78 (1H, brs, H-4’). The 13C-NMR revealed its function groups, as follows: an thiophene heterocycles 149.7, 113.7 and 112.0; four carbonyls 169.8, 168.2, 164.6 and 162.4; two double bonds 139.6, 114.3, 129.8 and 126.6; seven metyls including one metyl of acetyl, one vinyl- metyl, a gem-dimethyl group, one isopropyl and one tert-butyl group. All spectral data thus confirmed the structure of hemiasterlin 14a. The structure of hemiasterlins 14b and 15a,b were elucidated the same way by IR, NMR and MS spectroscopic methods. Scheme 2 In conclusion, a successful synthesis of new modified hemiasterlin derivatives was achieved in which the α,α-dimethylbenzylic group and amino NHMe moiety were replaced by α,β-unsaturated carbonyl-thiophene and amide NH-Ac group, respectively. REFERENCES 1. Talpir R., Benayahu Y., Kashman Y., Pannell L., Schleyer M. Hemiasterlin and geodiamolide TA- two new cytotoxic peptides from the marine sponge Hemiasterella minor, Tetrahedron Lett., 35, 4453- 4456 (1994). 2. Gamble W. 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