Design, Synthesis and Biological Evaluation of Tasiamide Analogues as Tumor Inhibitors

Eighteen analogues of the marine cytotoxic linear peptide tasiamide were designed, synthesized and screened for their inhibitory activities against the growth of human nasopharyngeal carcinoma (KB) and human non-small cell lung tumor (A549) cell lines. The results indicated that minor modifications of the C-terminuswith aromatic groups were tolerated, with the IC50 values between 1.29 and 12.88 μM against these two cancer cell lines. Truncation, minor modifications at the N-terminus or elimination of the N-methyl groups in N-Me-d-Gln and/or N-Me-d-Phe residues resulted in inactive analogues.


Introduction
Marine cyanobacteria are a well-known prolific source of novel bioactive compounds [1][2][3], most of which are linear or cyclic (depsi)peptides with distinctive structures. Tasiamide ( Figure 1) is a linear peptide isolated from the marine cyanobacterium Symploca sp. in 2002, which showed moderate cytotoxicity against human nasopharyngeal carcinoma (KB) and human colon carcinoma (LoVo) cell lines with IC 50 values of 0.48 and 3.47 μg/mL, respectively [4]. The first total synthesis of tasiamide was reported by our group [5] in 2008. The result indicated that the previously assumed N-Me-L-glutamine residue should be N-Me-D-glutamine by comparing the physical data of the synthetic and natural products ( 1 H-NMR, 13 C-NMR and the optical rotation). As a part of our ongoing OPEN ACCESS efforts to find bioactive marine peptides [6][7][8], herein we report the design, synthesis and biological evaluation of some analogues of this natural product.

Chemistry
Tasiamide is an acyclic peptide composed of six amino acid residues and an α-hydroxy acid ). In order to determine the minimal active fragment of this natural product, we decided to design and prepare some truncated analogues ( Figure 2). Truncating the Hmp-Leu residues or the Hmp-Leu-N-Me-D-Gln residues from the N-terminus of tasiamide gave analogues S1 and S2, respectively. Truncating the N-Me-D-Phe-Pro residues or Gly-N-Me-D-Phe-Pro residues from the C-terminus resulted in analogues S3 and S4, respectively. The preparation of these four truncated analogues are shown in Scheme 1. Dipeptide 1 was prepared by coupling commercially available reagents H-Pro-OMe and Boc-N-Me-D-Phe-OH using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride/1-hydroxy-7-azabenzotriazole (EDC/HOAt) in dichloromethane (DCM). Removal of the Boc protecting group yielded 1a, which was used directly in the next cycle of coupling, giving the corresponding tripeptide 2 in good yield. Next, the N-Fmoc of 2 was removed under a mild condition by diethylamine (DEA), which was coupled with N,N-Me 2 -Ile-OH, leading to the desired analogue S2. On the other hand, the free amine 2a was coupled with Boc-Ile-OH, giving tetrapeptide 3 in a 73% yield. After revealing the amino group of compound 3, N,N-Me 2 -D-Gln(Trt)-OH was coupled with 3a to give the fully protected pentapeptide 4 in a yield of 95%. Removal of the Trt group under an acidic condition gave the desired truncated analogue S1 in a 97% yield. As shown in Scheme 2, dipeptides 5 and 6 were conveniently synthesized from commercially available amino acids. Deprotections of 5 with DEA and 6 through Pd-mediated hydrogenation liberated the amine and carboxylic acid, respectively, and was subsequently coupled to afford S3. Repeating similar procedures, compound S3a with carboxylic acid was coupled with H-Gly-OBn to yield target S4. Scheme 2. Reagents and conditions: (a) EDC/HOAt, N-methylmorpholine (NMM), DCM, 12 h, 92% for 5, 91% for 6, 85% for S3, 60% for S4; (b) DEA/CH 3 CN; (c) Pd-C, H 2 , EtOAc.
In order to determine the effect of C-terminus modifications, several full-length analogues were designed, with Pro residue being replaced by an amino group having different aromatic groups (C1-C9, Figure 3). Starting from Boc-N-Me-D-Phe-OH, the desired compounds were obtained by following the standard solution phase peptide synthesis procedure (Scheme 3). In order to investigate the role of the Hmp residue on the N-terminus, two analogues were designed. The α-hydroxyl or sec-butyl group was truncated to afford N1 or N2, respectively (Scheme 4). With the pentapeptide fragment 10d in hand, these two compounds were prepared easily in two steps. Structurally, there are two N-methyl amino acids in the parent compound. Usually, N-methyl amino acids are believed to play important roles in the conformational alteration and increasing stability over proteolytic enzymes [9]. In order to investigate the role of the N-methyl amino acids, three analogues were designed (M1, M2 and M3). Starting from easily obtained peptide fragments 11, 12 and some commercially available reagents, these three compounds were obtained smoothly, as shown in Scheme 5.

Biological Results and Discussion
All these eighteen tasiamide derivatives prepared above were evaluated for cytotoxicity against KB and A549 cancer cell lines using etoposide as the positive control.
As shown in Table 1, none of the truncated analogues (S1-S4) were effective against KB or A549 cell lines even at 50 μM. This result indicated that a certain length of the peptide might be necessary for cytotoxicity. Analogues with full length, but some minor modifications at the C-terminus and/or glutamine residue (C1-C8) showed moderate activities against cancer cell lines. According to the IC 50 values of eight compounds (C1-C8), L-Gln residue-containing analogues showed slightly better activity than their D-Gln counterparts (C1 vs. C2, C3 vs. C4, C5 vs. C6, C7 vs. C8). On the other hand, if Gln was replaced by Ala (C9), the cytotoxicity reduced dramatically. However, further structural optimizations of whether simplification on the Hmp residue (N1, N2) or modification on the N-methylated amino acids (M1-M3) led to inactive analogues.

Materials and Methods
Solvents were processed by conventional methods. Thin layer chromatography (TLC) was performed on pre-coated Merck silica gel 60 F 254 plates. Flash column chromatography was performed on silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd, Qingdao, China). Optical rotations were determined with a JASCO P-1020 polarimeter. NMR spectra were recorded on a Jeol JNM-ECP 600 MHz spectrometer (Jeol Ltd., Tokyo, Japan) with Me 4 Si as the internal standard, and chemical shifts were recorded in δ value. Mass spectra were obtained on a Q-TOF GIOBAL mass spectrometer (Waters, Wilford, MA, USA). (3) DEA (5 mL) was added to a solution of tripeptide 2 (428.4 mg, 0.75 mmol) in CH 3 CN (5 mL). The solution was concentrated in vacuo after stirring at room temperature (rt) for 2 h. Subsequently, the residue was then redissolved in CH 3 CN (5 mL), concentrated in vacuo again and dried under vacuum for 2 h. This free amine was dissolved in dry tetrahydrofuran (THF) (10 mL) and cooled with an ice-water bath for 10 min. Then, Boc-Ile-OH (208.0 mg, 0.9 mmol), EDC (173.0 mg, 0.9 mmol), HOAt (123.0 mg, 0.9 mmol) and NaHCO 3 (130.0 mg, 1.5 mmol) were added, respectively. The mixture was stirred at 0 °C for 2 h and then rt overnight. The solvent was removed, and the residue was diluted with EtOAc (200 mL), washed with 10% citric acid, 5% NaHCO 3 and brine. The organic layer was dried over Na 2 SO 4 , then concentrated in vacuo. The crude product was purified by flash chromatography providing Tetrapeptide (3)

Synthesis of Boc-Ile-Gly-N-Me-D-Phe-Pro-OMe
A solution of compound 3 (91 mg, 0.162 mmol) in HCl/EtOAc (4 mol/L, 2 mL) was concentrated in vacuo after stirring for 45 min at rt. The residue was redissolved in EtOAc (5 mL) and concentrated in vacuo again. The resulting yellow solid was dried under vacuum for 2 h and then dissolved in dry THF (10 mL). After being cooled with an ice-water bath for 10 min, N,N-Me 2 -D-Gln(Trt)-OH (81.1 mg, 0.195 mmol), EDC (46.6 mg, 0.243 mmol), HOAt (33.1 mg, 0.243 mmol) and NaHCO 3 (27.2 mg, 0.324 mmol) were added, respectively. The mixture was stirred at this temperature for 2 h and then at rt for another 12 h. The solvent was removed, and the residue was dissolved in EtOAc (100 mL) and washed with 10% citric acid, 5% NaHCO 3 and brine. The organic layer was dried over Na 2 SO 4 and then concentrated in vacuo. The residue was purified by flash column chromatography to give the desired pentapeptide 4 as a pale-yellow solid (133 mg, 95%).

Synthesis of HO-Hmp-Leu-N-Me-D-Gln-Ile-Gly-OBn (S4)
Hydrogenation of S3 (23.6 mg, 0.04 mmol) was carried out in EtOAc-EtOH (1:4, 10 mL) in the presence of a catalytic amount of Pd-C (10%) under hydrogen for 4 h. The Pd-C was removed by filtration, and the filtrate was concentrated in vacuo to yield the corresponding carboxylic acid S3a, which was used for next step without further purification.

General Procedure for the Preparation of Compounds 7a-d
To a solution of Boc-N-Me-D-Phe-OH (1.0 mmol) and amine (1.1 mmol of aniline, phenylmethanamine, tetrahydroisoquinoline or tetrahydroisoquinoline carboxylate) in DCM-DMF (1:1, 10 mL) at 0 °C were added EDC (1.2 mmol), HOAt (1.2 mmol) and NaHCO 3 (1.2 mmol), respectively. The mixture was stirred at this temperature for 2 h and then at rt overnight. The reaction mixture was diluted with 100 mL of EtOAc and washed with 10% citric acid, 5% NaHCO 3 and brine. The organic layer was dried over Na 2 SO 4 and then concentrated in vacuo. The residue was purified by flash column chromatography to give the desired compounds.

General Procedure for the Preparation of Compounds 8a-d
Compound 7a (or 7b-d) (0.13 mmol) was treated with HCl/EtOAc (4 mol/L, 2 mL) for 45 min then concentrated in vacuo. The residue was redissolved in EtOAc (5 mL) and concentrated in vacuo again. The resulting solid was dried under vacuum for 2 h and then dissolved in 4 mL of dry DCM-DMF (3:1). After being cooled with an ice-water bath for 10 min, Fmoc-Gly-OH (0.13 mmol), EDC (0.16 mmol), HOAt (0.16 mmol) and NaHCO 3 (0.16 mmol) were added, respectively. The mixture was stirred at this temperature for 2 h and then at rt for another 12 h. The mixture was diluted with 80 mL of EtOAc and washed with 10% citric acid, 5% NaHCO 3 , water and brine. The organic layer was dried over Na 2 SO 4 and then concentrated in vacuo. The residue was purified by flash column chromatography to give the desired compound 8a (or 8b-d).