2020 Volume 43 Issue 8 Pages 1154-1158
Pretubulysin is a bio-precursor of highly toxic tetrapeptide tubulysins. Although pretubulysin has a much simpler chemical structure, it has similar anti-mitotic potency. A series of 2-amino-thiazole-4-carboxamides were designed and synthesized based on the structure of cemadotin. These are all novel compounds and their structures are characterized by 1H-NMR, 13C-NMR, and high resolution (HR)MS. The antitumor activities of these compounds were screened using the methyl thiazolyl tetrazolium colorimetric (MTT) cell viability method in MCF7 (breast cancer) and NCI-H1650 (lung cancer) cells. All the synthesized compounds 6a–n showed moderate anti-proliferation activities. Compounds 6m exhibited antitumor activity with the IC50 value of 0.47 and 1.1 µM in MCF7 and NCI-H1650 cells, respectively.
The tubulysins are a family of anti-mitotic tetrapeptides isolated by Höfle and colleagues from Angiococcus disciformis in 2000.1) Tubulysin D, the most potent compound in the family, exhibits high cytotoxicity towards a wide range of tumor cells in the low nano- or even picomolar concentration range.2) However, synthesis and commercial supply of tubulysins present a challenge due to their complex structures.3,4) Pretubulysin (1, Fig. 1), a tubulysin precursor, shows equal anti-mitotic potency to tubulysins, but has a much simpler chemical structure.5,6) Therefore, there has been significant interest in modifying and simplifying the structure of pretubulysin, which has resulted in the synthesis of several promising analogues.7–10) Tubulysins and pretubulysin can bind to tubulin and subsequently inhibit its polymerization.2,11) These tubulin-binding agents (TBAs) were shown to be successful anti-cancer compounds.12)
Cemadotin, a synthetic analogue of dolastatin, is a linear penta-peptide that inhibits cell proliferation by suppressing microtubules.13,14) Pretubulysin and cemadotin share a structural similarity, as shown in Fig. 1. Previous studies on the structure–activity relationship (SAR) of tubulysin and pretubulysin suggested that the central N-methyl-L-Valyl was the key functional group for maintaining antitumor activity.15) The Mep and Tup structural sections in pretubulysin were found to tolerate significant modification.16) We designed and synthesized a series of 2-amino-thiazole-4-carboxamides based on our previous findings.17–19)
These compounds were hybrids of the key structure moieties of both pretubulysin and cemadotin, such as the N-methyl-L-Valyl group and the thiazole group. An amide group was used to connect the above two groups instead of the ethylene in pretubulysin.
Another notable feature of this series of compounds was that there were only two chiral centers, compared to five and six in cemadotin and pretubulysin, respectively.
The synthesis of these 2-amino-thiazole-4-carboxamides is shown in Chart 1. The commercially available compound ethyl 2-amino-4-carboxylate (3) was converted to intermediate amides 4a–c by a 3-step transformation. First, the amino of compound 3 was protected by di-tert-butyl dicarbonate. Second, the ester group of 3 was hydrolyzed to the corresponding acid. Third, coupling of the acid and a variety of amines, including methylamine, benzylamine, and phenylethylamine, yielded compounds 4a–c under classic peptide coupling conditions (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA)). Removal of the tert-butoxycarbonyl (Boc) group in compounds 4a–c, followed by coupling with (L)-9-fluorenylmethyloxycarbonyl (Fmoc)-N-Me-Val,20) gave compounds 5a–c. After removal of the Fmoc group of compounds 5a–c, the resulting amines were coupled with isocyanates to give 6a–i and with isonicotinic acid to give 6j–l.
Reagent and conditions; a) i. Boc2O, DCM, 92%; ii. LiOH, H2O/THF, 98%; iii. R1NH2, EDCI, HOBt, DIPEA, 93–97%; b) i.TFA, DCM; ii. EDCI, HOBt, DIPEA, Fmoc-N-Me-(L)-Val, 54–68%; c) i. Piperidine, MeOH, 85%; ii. R2-NCO, DCM, 80–93%, or isonicotinic acid, HOBt, EDCI, DMF, 71–92%.
The in vitro antitumor activities of compounds 6a–n were screened using the methyl thiazolyl tetrazolium colorimetric (MTT) cell viability method in MCF7 (breast cancer) and NCI-H1650 (lung cancer) cell lines. Taxol and pretubulysin were chosen as the control. The synthesized compounds 6a–n showed moderate anti-proliferation activities against MCF7 and NCI-H1650 tumor cells. Among these compounds, 6m showed the most significant inhibitory activity (IC50 = 0.47 and 1.1 µM, respectively) against MCF7 and NCI-H1650 cells. The anti-proliferation activities of the synthesized compounds are shown in Table 1.
| Compd. | IC50 (µM) | Compd. | IC50 (µM) | ||
|---|---|---|---|---|---|
| MCF7 | NCI-H1650 | MCF7 | NCI-H1650 | ||
| 6a | 3.2 ± 1.1 | 4.6 ± 2.5 | 6j | 23.1± 7.9 | 15.3 ± 7.6 |
| 6b | 13.5 ± 6.3 | 17.6 ± 8.2 | 6k | 33.2± 11.3 | 15.7 ± 6.9 |
| 6c | 42.5 ± 13.6 | 52.3 ± 18.7 | 6l | 48.2± 13.6 | 35.7± 14.7 |
| 6d | 7.6 ± 3.2 | 3.3 ± 0.9 | 6m | 0.47 ± 0.1 | 1.1 ± 0.46 |
| 6e | 12.3 ± 5.6 | 10.2 ± 4.5 | 6n | 5.3 ±2.5 | 9.6 ± 4.9 |
| 6f | 25.3± 9.6 | 18.3 ± 9.8 | Taxol | 0.03 ± 0.004 | 0.06 ± 0.01 |
| 6g | 1.1 ± 0.6 | 2.3 ± 0.9 | 1 | <0.001 | <0.001 |
| 6h | 3.6 ± 1.7 | 4.8± 2.2 | 2 | <0.001 | <0.001 |
| 6j | 18.6± 8.2 | 12.5 ± 5.8 | |||
The above biological screening results regarding the SAR of this series of compounds indicated the following: 1. Compounds with phenylethylamine (6a, d, g) in the Tup segment (R1 group) generally showed better antitumor activity than those with methylamine or benzylamine. Compounds with a phenylalanine (Phe)-OEt (an additional ester group attached to phenylethylamine) group (6m, n) also showed better antitumor activity. 2. Compounds with a urea group in the Mep segment (R2) generally showed better antitumor activity than those with an isonicotinic amide group (6j–l). Furthermore, compounds 6c and f, which contained benzamine groups, showed similar activities to compounds 6j–l.
Although most of the compounds in this series showed antitumor activity, they were less potent than taxol and pretubulysin. In order to investigate the mechanism of the observed activity, molecular docking was carried out using the co-crystal structure of microtubules and tubulysin M (PDB code: 4ZOL).21) The most and least potent compounds were selected; 6m and c, respectively, the docking results are shown in Fig. 2. Compound 6m had two hydrogen (H) bonds that interacted with the target microtubule, with a total score of 6.78, compared to tubulysin M, which had four hydrogen bonds and a score of 7.09. One H-bond was situated between the urea group and amino acid residue aspartic acid (Asp) 179, while another was between an amide group and residue tyrosine (Tyr) 224. Both residues were found to form H-bonds with tubulysin M. Compound 6c had two H-bonds that interacted with the target microtubule and a total score of 6.47. The lower docking score may explain why compounds 6m and c were less potent than tubulysins. The docking results also suggested that the urea group might play a key role in maintaining the antitumor activity
The urea group has an H-bond interaction with Asp 179; the amide group has an H-bond interaction with Tyr 224. The total score is 6.78, compared to 7.09 for Tubulysin M. b: The molecular docking results of compound 6c (light gray) The urea group has an H-bond interaction with Asn 329; the amide group has an H-bond interaction with Tyr 224. The total score is 6.47.
A series of 2-amino-thiazole-4-carboxamides were designed and synthesized. Their antitumor activities were screened using the MTT cell viability assay. All the synthesized compounds 6a–n showed anti-proliferative activities in MCF7 and NCI-H1650 tumor cells. Compounds 6m showed relatively better antitumor activity in MCF7 and NCI-H1650 tumor cells compared to the other compounds in the series, with IC50 values of 0.47 and 1.1 µM, respectively. Although this series of compounds were overall less potent than taxol, they may represent a promising lead for the further development of novel antitumor drugs.
All reactions were carried out under a nitrogen atmosphere with dry solvent under anhydrous conditions, unless otherwise noted. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise noted. The NMR spectra of the intermediates and final products in deuterated solvent were detected on a Bruker 400 or 600 MHz spectrometer. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, h = heptet, m = multiplet, b = broad. High-resolution (HR) MS were recorded on an Agilent 6210 ESI/TOF mass spectrometer. Melting points (MP) were recorded on a Büchi B-540 melting point apparatus and are uncorrected. Flash column chromatographic separation was achieved using a silica gel from Qingdao Ocean Chemical (200 to 300 mesh) with a particle size from 54 to 74 µm using ethyl acetate and hexane (or petroleum ether) as the eluent. Analytical TLC was carried out on Merck pre-coated silica gel 60 GF-254 using 0.25-mm-thick TLC plates.
General Procedure for Preparation of Compounds 5a–cTFA (10 mL) was added to a solution of compound 4b (3.30 g, 9.90 mmol) in 30 mL dichloromethane at room temperature (r.t.). The resulting reaction mixture was stirred at room temperature for 3h and then concentrated in vacuo. The residue was diluted with 15 mL DCM and 8 mL N,N-diisopropylethylamine to afford the amine solution. In another flask, a solution of Fmoc-N-Me-(L)-Val (3.5 g, 9.90 mmol), EDC (2.85 g, 14.86 mmol), and HOBt (1.34 g, 9.90 mmol) in 100 mL dichloromethane was stirred at r.t. for 30 min. The above amine solution was added to the aforementioned reaction mixture and stirred for a further 6h at r.t. Water was added to the reaction mixture and the aqueous solution was extracted with dichloromethane. The organic layers were combined, washed with water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified using column chromatography [V (PE) : V (EA) = 3 : 1] to extract compound 5b (3.13 g, 55.6%) as a colorless oil. 1H-NMR (400 MHz, CDCl3) δ: 10.23 (s, 1H), 7.74–7.18 (m, 14H), 4.55 (d, J = 8 Hz, 2H), 4.52 (d, J = 4 Hz, 1H), 4.46 (m, 2H), 4.16 (t, J = 8 Hz, 1H), 2.82 (s, 3H), 2.36 (m, 1H), 0.98 (d, J = 6.4 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H). 13C-NMR (100 MHz, CDCl3): 170.3, 162.7, 161.3, 154.0, 145.6, 143.9, 141.9, 136.6, 128.1, 127.3, 127.0, 126.5, 125.9, 120.0, 114.9, 79.2, 65.9, 47.4, 41.3, 28.3, 26.9, 19.0; HR MS Calcd for C32H33N4O4S [M + H]+ 569.2217, Found 569.2213.
General Procedure for Docking of Urea Group in Compounds 6a–i(S)-3-(4-Methoxybenzoyl)-1-methyl-1-(3-methyl-1-oxo-1-(4-(phenethylcarb Amoyl)thiazol-2-ylamino)butan-2-yl)urea (6a)Piperidine (1.5 mL) was added to a solution of compound 5 (2.6 g, 4.46 mmol) in 50 mL N,N-dimethylformamide and the reaction mixture was stirred at r.t. until TLC showed that the reaction was complete. The crude product was diluted with water (15 mL) and extracted with ethyl acetate (3 × 20 mL). The organic layers were collected, dried, filtered, and concentrated. The obtained residue was purified using flash column chromatography to provide pure free amine intermediate. This intermediate (100 mg, 0.28 mmol) was dissolved in DCM (20 mL), then 4-methoxyphenyl isocyanate (40 mg, 0.28 mmol) was added and the reaction mixture was stirred at r.t. for 3 h until TLC showed that the reaction was complete. The solid was filtered out, after which the filtrate was diluted with water and extracted with DCM (10 mL × 3). This was combined with the organic phase, dried over Na2SO4, filtered, and concentrated. The obtained residue was purified using flash column chromatography to yield product 6a (126.3 mg, yield: 89.3%) as a colorless oil. 1H-NMR (600 MHz, CDCl3) δ H 10.90 (br s, 1H), 7.58 (s, 1H), 7.15–7.26 (multiple, 8H), 6.70–6.78 (d, J = 12 Hz, 2H), 6.36 (s, 1H), 4.58–4.60 (m, 1H), 3.75 (s, 3H), 3.67–3.74 (m, 1H), 3.54–3.59 (m, 1H), 2.87–2.97 (multiple, 5H), 2.31–2.37 (m, 1H), 0.95–0.96 (d, J = 6.6 Hz, 3H), 0.88–0.90 (d, J = 6.6 Hz, 3H). 13C-NMR (150 MHz, CDCl3): 168.6, 160.1, 156.6, 155.3, 155.2, 143.1, 137.9, 129.2, 127.5, 127.3, 126.1, 122.9, 116.2, 112.6, 54.0, 39.5, 35.1, 34.3, 25.5, 17.8, 17.6; HR MS Calcd for C27H32N5O5S [M + H]+ 538.2119, Found 538.2117.
(S)-2-(2-(3-(4-Methoxyphenyl)-1-methylureido)-3-methylbutanamido)-N-methylthiazole-4-carboxamide (6b)White wax, 85.1%. 1H-NMR (600 MHz, CDCl3) δH 10.82 (br s, 1H), 7.60 (s, 1H), 7.20–7.26 (m, 2H), 7.13–7.14 (m, 1H), 6.78–6.79 (m, 2H), 6.51 (s, 1H), 4.59–4.61 (m, 1H), 3.75 (s, 3H), 3.09 (s, 3H), 2.95 (s, 3H), 2.41–2.43 (m, 1H), 0.90–0.99 (m, 6H). 13C-NMR (150 MHz, CDCl3):169.6, 162.0, 157.8, 156.7, 156.5, 144.6, 130.7, 127.6, 123.7, 117.4, 114.1, 55.4, 29.6, 26.7, 25.9, 19.5, 18.9; HR MS Calcd for C20H26N5O5S [M + H]+ 448.1649, Found 448.1644.
(S)-N-Benzyl-2-(2-(3-(4-methoxyphenyl)-1-methylureido)-3-methylbutanamido)thiazole-4-carboxamide (6c)White wax, 80%. 1H-NMR (400 MHz, CDCl3) δH 10.81 (br s, 1H), 7.63 (s, 1H), 7.43–7.46 (m, 1H), 7.27–7.34 (m, 5H),7.13–7.14 (m, 2H), 6.74–6.76 (m, 2H), 4.66–4.71 (m, 1H), 4.48–4.57 (m, 2H), 3.74 (s, 3H), 2.83 (s, 3H), 2.27–2.34 (m, 1H), 0.89–0.92 (m, 6H). 13C-NMR (100 MHz, CDCl3): 169.5, 162.7, 161.3, 157.4, 154.6, 144.7, 137.9, 131.5, 129.2, 128.7, 128.4, 120.8, 118.0, 113.8, 70.2, 60.6, 44.0, 29.9, 26.8, 19.3; HR MS Calcd for C25H30N5O4S [M + H]+ 469.2013, Found 469.2011.
(S)-3-(4-Chlorobenzoyl)-1-methyl-1-(3-methyl-1-oxo-1-(4-(phenethylcarbamoyl)thiazol-2-ylamino)butan-2-yl)urea (6d)White wax, 91.7%. 1H-NMR (600 MHz, CDCl3) δH 10.54 (br s, 1H), 7.62 (s, 1H), 7.21–7.29 (multiple, 10H), 6.56 (s, 1H), 4.55 (m, 1H), 3.60–3.71 (m, 2H), 2.98 (s, 3H), 2.90–2.92 (m, 2H), 2.38–2.40 (m, 1H), 0.95–0.98 (m, 6H). 13C-NMR (150 MHz, CDCl3): 169.8, 164.6, 162.8, 154.8, 153.4, 139.6, 138.6, 138.3, 133.4, 129.2, 128.7, 128.4 (2), 126.1, 113.0, 54.0, 41.5, 35.5, 34.1, 27.4, 19.3; HR MS Calcd for C26H29ClN5O4S [M + H]+ 542.1623, Found 542.1631.
(S)-2-(2-(3-(4-Chlorophenyl)-1-methylureido)-3-methylbutanamido)-N-meth Ylthiazole-4-carboxamide (6e)White wax, 86.7%. 1H-NMR (400 MHz, CDCl3) δH. 8.09 (s, 1H), 7.71 (s, 1H), 7.31–7.33 (m, 2H), 7.24–7.26 (m, 2H), 7.10 (br s, 1H), 4.47–4.50 (m, 1H), 3.04 (s, 3H), 2.97 (s, 3H), 2.45–2.49 (m, 1H), 0.96–1.02 (m, 6H). 13C-NMR (100 MHz, CDCl3):170.8, 169.1, 162.0, 144.7, 136.9, 133.8, 129.1, 127.3, 122.1, 113.6, 29.2, 28.7, 26.1, 17.3, 16.7; HR MS Calcd for C18H23ClN5O3S [M + H]+ 424.1205, Found 424.1201.
(S)-N-Benzyl-2-(2-(3-(4-chlorophenyl)-1-methylureido)-3-methylbutanamido)thiazole-4-carboxamide (6f)White wax, 85.8%. 1H-NMR (400 MHz, CDCl3) δH 10.73 (br s, 1H), 7.62 (s, 1H), 7.38–7.41 (m, 1H), 7.28–7.35 (m, 2H), 7.15–7.25 (m, 7H), 6.55 (s, 1H), 4.64–4.69 (m, 1H), 4.54–4.56 (m, 2H), 2.87 (s, 3H), 2.29–2.35 (m, 1H), 0.91–0.94 (m, 6H). 13C-NMR (100 MHz, CDCl3):172.6, 166.9, 162.3, 152.9, 142.9, 139.3, 138.6, 130.6, 129.5, 128.6, 127.9, 127.6, 121.5, 114.2, 62.1, 44.8, 36.1, 27.1, 19.4; HR MS Calcd for C24H27ClN5O3S [M + H]+ 500.1518, Found 500.1511.
(S)-2-(2-(3-(4-Chloro-3-(trifluoromethyl)phenyl)-1-methylureido)-3-methylbutanamido)-N-phenethylthiazole-4-carboxamide (6g)White wax, 92.5%. 1H-NMR (600 MHz, CDCl3) δH 10.30 (br s, 1H), 7.72 (s, 1H), 7.71 (s, 1H), 7.52–7.64 (m, 1H), 7.39–9.40 (m, 1H), 7.26–7.29 (m, 2H), 7.21–7.22 (m,2H), 7.15 (m, 1H), 6.74 (s, 1H), 4.52 (m, 1H), 3.61–3.71 (m, 2H), 3.02 (s, 3H), 2.38–2.40 (m, 1H), 0.96–0.98 (m, 6H). 13C-NMR (150 MHz, CDCl3):169.4, 161.8, 161.4, 157.3, 156.6, 144.7, 139.2, 136.7, 129.5 129.0, 128.9 (3C), 128.7, 126.5, 122.6 (2C), 117.9, 40.9, 35.9, 29.8, 19.6, 19.0; HR MS Calcd for C27H28ClF3N5O4S [M + H]+ 610.1497, Found 610.1491.
(S)-2-(2-(3-(4-Chloro-3-(trifluoromethyl)phenyl)-1-methylureido)-3-methylbutanamido)-N-methylthiazole-4-carboxamide (6h)White wax, 92.3%. 1H-NMR (400 MHz, CDCl3) δH 7.75 (s, 1H), 7.69 (s, 1H), 7.56–7.57 (m, 2H), 7.40–7.53 (m, 1H), 7.07 (br s, 1H), 6.85 (br s, 1H), 4.51–4.54 (m, 1H), 3.08 (s, 3H), 2.95 (s, 3H), 2.41–2.47 (m, 1H), 0.96–1.01 (m, 6H). 13C-NMR (100 MHz, CDCl3): 172.6, 163.0, 160.9, 155.6, 144.9, 133.9, 129.9, 129.2, 128.9, 128.3, 123.6, 118.2, 113.2, 69.1, 28.8, 26.7, 25.7, 18.4; HR MS Calcd for C19H22ClF3N5O3S [M + H]+ 492.1078, Found 492.1075.
(S)-N-Benzyl-2-(2-(3-(4-chloro-3-(trifluoromethyl)phenyl)-1-methylureido)-3-methylbutanamido)thiazole-4-carboxamide (6i)White wax, 75.8%. 1H-NMR (400 MHz, CDCl3) δH 10.51 (br s, 1H), 7.64 (s, 2H), 7.38–7.44 (m, 1H), 7.27–7.37 (multiple, 7H), 6.62 (br s, 1H), 4.67–4.71 (m, 1H), 4.48–4.56 (m, 2H), 2.92 (s, 3H), 2.32–2.36 (m, 1H), 0.93–0.95 (m, 6H). 13C-NMR (100 MHz, CDCl3): 171.9, 161.9, 161.2, 155.5, 144.5, 137.9, 133.8, 129.2, 128.1(2C), 127.8, 127.1, 126.2, 124.8, 122.8, 116.4, 112.5, 71.4, 39.4, 28.0, 25.7, 18.6; HR MS Calcd for C25H26ClF3N5O3S [M + H]+ 568.1391, Found 568.1388.
General Procedure for Synthesis of Compounds 6j–nPiperidine (1.5 mL) was added to a solution of compound 5 (2.6 g, 4.46 mmol) in 50 mL N,N-dimethylformamide (DMF) and the reaction mixture was stirred at r.t. until TLC showed that the reaction was complete. The reaction mixture was diluted with water (15 mL) and extracted with ethyl acetate (3 × 20 mL). The organic layers were collected, dried, filtered, and concentrated. The obtained residue was purified using flash column chromatography to provide pure free amine intermediate. This intermediate (10 mg, 0.14 mmol) was dissolved in DMF (10 mL). Meanwhile, isonicotinic acid (20 mg, 0.17 mmol) was added to a solution of 1-hydroxybenzotriazole (HOBt)(23 mg, 1.7 mmol) and EDCI (33 mg, 0.17 mmol) in DMF (10 mL) at r.t.. The reaction mixture was allowed to stir at r.t. for 0.5 h, then the above amine solution was added. The resulting reaction mixture was allowed to stir for 10 h till TLC showed that all starting material consumed. Water was added, and the aqueous solution was extracted with DCM (10 mL × 3). The combined organic layers were washed with water, brine, dried over Na2SO4, filtered, and concentrated. The obtained residue was purified using flash column chromatography to yield product 6j.
(S)-2-(3-Methyl-2-(N-methylisonicotinamido)butanamido)-N-phenethylthia Zole-4-carboxamide (6j)Pale yellow wax, 77.8%. 1H-NMR (400 MHz, CDCl3) δH 8.60 (br s, 1H), 7.79 (s, 1H), 7.46–7.47 (d, J = 4 Hz, 1H), 7.25–7.29 (multiple, 5H), 7.18–7.19 (m, 1H), 4.98–5.0 (m, 1H), 3.60–3.63 (m, 2H), 3.03 (s, 3H), 2.89–2.92 (m, 2H), 2.49–2.51 (m, 1H), 1.05–1.1 (m, 6H). 13C-NMR (100 MHz, CDCl3): 172.4, 165.7, 161.2, 153.8, 148.5, 146.2, 137.9, 137.2, 129.2, 127.8, 126.5, 116.6, 113.5, 62.7, 42.1, 35.0, 34.2, 27.9, 18.4; HR MS Calcd for C24H28N5O3S [M + H]+ 466.1907, Found 499.1903.
(S)-N-Methyl-2-(3-methyl-2-(N-methylisonicotinamido)butanamido)thiazole-4-carboxamide (6k)Pale yellow wax, 71.7%. 1H-NMR (400 MHz, CDCl3) δH 10.31 (br s, 1H), 7.72 (s, 2H), 7.28–7.31 (m, 2H), 7.11 (br s, 1H), 6.57–6.58 (m, 1H), 4.76–4.77(m, 1H), 3.60–3.63 (m, 2H), 3.04 (s, 3H), 2.97 (s, 3H), 2.43–2.49 (m, 1H), 0.93–1.02 (m, 6H). 13C-NMR (100 MHz, CDCl3):172.1, 167.9, 162.5, 161.2, 149.0, 144.8, 144.5, 121.9, 114.9, 60.2, 33.6, 27.1, 26.3, 18.7; HR MS Calcd for C17H22N5O3S [M + H]+ 376.1438, Found 376.1433.
(S)-N-Benzyl-2-(3-methyl-2-(N-methylisonicotinamido)butanamido)thiazole -4-carboxamide (6l)White wax, 76.9%. 1H-NMR (400 MHz, CDCl3) δH 9.9 (br s, 1H), 7.79 (s, 1H), 8.71–8.73 (m, 2H), 7.79 (s, 1H), 7.31–7.34 (multiple, 8H), 4.67–4.70 (m, 1H), 4.61–4.64 (m, 2H), 2.91 (s, 3H), 2.52–2.58 (m, 1H),1.06–1.08 (m, 6H). 13C-NMR (100 MHz, CDCl3): 171.9, 168.8, 162.8, 161.8, 149.0(C), 144.2, 137.9, 129.2, 127.1, 126.2, 122.8, 112.6, 62.5, 44.1, 35.6, 27.1, 18.7; HR MS Calcd for C23H26N5O3S [M + H]+ 452.1751, Found 452.1749.
(S)-Ethyl 2-(2-((S)-2-(3-(4-Methoxyphenyl)-1-methylureido)-3-methylbutan Amido) Thiazole-4-carboxamido)-3-phenylpropanoate (6m)White wax, 83.1%. 1H-NMR (400 MHz, CDCl3) δH 11.33 (s, 1H, NH), 7.65 (s, 1H), 7.54 (d, J = 8 Hz, 2H), 7.43 (s, 1H), 7.30–7.18 (m, 7H), 6.67 (d, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz, 1H), 5.04 (q, J = 4 Hz, 1H), 4.90 (d, J = 8 Hz, 1H), 4.13 (q, J = 4 Hz, 2H), 3.69 (s, 3H), 3.30 (q, J = 4 Hz, 1H), 3.23 (q, J = 4 Hz, 1H), 3.05 (s, 3H), 2.31–2.25 (m, 1H), 1.18 (t, J = 4 Hz, 3H), 1.01 (d, J = 4 Hz, 3H), 0.89 (d, J = 4 Hz, 3H); 13C-NMR (100 MHz, CDCl3): 171.9, 170.5, 163.7, 161.4, 158.3, 155.2, 145.1, 136.4, 132.1, 128.4, 127.5, 124.8, 119.7, 124.8, 119.7, 115.7, 113.2, 67.4, 62.5, 56.8, 53.1, 38.9, 29.1, 25.7, 18.1, 16.2; HRMS Calcd for C29H36N5O6S [M + H]+ 582.2389, Found 582.2391.
(S)-Ethyl 2-(2-((S)-3-Methyl-2-(1-methyl-3-(pyridin-4-yl)ureido)butanamido)thiazole-4-carboxamido)-3-phenylpropanoate (6n)Pale yellow wax, 76.3%. 1H-NMR (400 MHz, CDCl3) δH 9.9 (br s, 1H), 7.79 (s, 1H), 8.71–8.73 (m, 2H), 7.79 (s, 1H), 7.31–7.34 (multiple, 8H), 4.67–4.70 (m, 1H), 4.61–4.64 (m, 2H), 2.91 (s, 3H), 2.52–2.58 (m, 1H),1.06–1.08 (m, 6H). 13C-NMR (100 MHz, CDCl3): 171.9, 170.7, 162.0, 160.3, 155.5, 154.9, 150.2, 144.8, 136.6, 128.2, 122.8, 126.7, 125.0, 113.7, 110.6, 68.6, 61.6, 57.1, 36.9, 29.4, 25.7, 19.2, 18.1; HR MS Calcd for C27H33N6O5S [M + H]+ 553.2228, Found 553.2223.
The author thanks for the financial support by Doctor Foundation of Yantai University (No. YX13B04), and the Talent Development Project of Blue Economic Zone of Shandong Province (No. RS11YX).
The authors declare no conflict of interest.
Experimental procedures and spectral data (1H-NMR, 13C-NMR, HR MS), and copies of 1H-NMR and 13C-NMR for all compounds described in the paper. The online version of this article contains supplementary materials.
