Asymmetric Synthesis and Cytotoxicity Evaluation of Right-Half Models of Antitumor Renieramycin Marine Natural Products

A general protocol for the asymmetric synthesis of 3-N-arylmethylated right-half model compounds of renieramycins was developed, which enabled structure–activity relationship (SAR) study of several 3-N-arylmethyl derivatives. The most active compound (6a) showed significant cytotoxic activity against human prostate cancer DU145 and colorectal cancer HCT116 cell lines (IC50 = 11.9, and 12.5 nM, respectively).


Introduction
Natural products belonging to the bis-1,2,3,4-tetrahydroisoquinoline family, such as renieramycins, saframycins, and ecteinascidins, have attracted considerable attention due to their potent biological activities, structural diversity, and meager availability in nature ( Figure 1) [1]. We have discovered a number of renieramycin marine natural products having extraordinary structures from blue sponges collected in Thailand and the Philippines [2][3][4]. For example, renieramycin M (1m) isolated from the Thai blue sponge Xestospongia sp. has p-quinones in both terminal rings [2]. In contrast, renieramycins T (1t) and U (1u) share a common A-ring with ecteinascidin 743 (ET-743, 2), which has already been approved as an anticancer agent [3]. In addition, the A-ring of renieramycin Y (1y) has the same substituent pattern as the E-ring of 2 [4]. These renieramycins have similar structures to 2 and are expected to have similar potent antitumor activity. However, the amount obtainable from nature is scarce, and this has set back the implementation of detailed biological tests.

Introduction
Natural products belonging to the bis-1,2,3,4-tetrahydroisoquinoline family, such as renieramycins, saframycins, and ecteinascidins, have attracted considerable attention due to their potent biological activities, structural diversity, and meager availability in nature ( Figure 1) [1]. We have discovered a number of renieramycin marine natural products having extraordinary structures from blue sponges collected in Thailand and the Philippines [2][3][4]. For example, renieramycin M (1m) isolated from the Thai blue sponge Xestospongia sp. has p-quinones in both terminal rings [2]. In contrast, renieramycins T (1t) and U (1u) share a common A-ring with ecteinascidin 743 (ET-743, 2), which has already been approved as an anticancer agent [3]. In addition, the A-ring of renieramycin Y (1y) has the same substituent pattern as the E-ring of 2 [4]. These renieramycins have similar structures to 2 and are expected to have similar potent antitumor activity. However, the amount obtainable from nature is scarce, and this has set back the implementation of detailed biological tests. Under these circumstances, we have been developing a total synthesis of these fascinating marine natural products. We have succeeded in the total syntheses of renieramycins G-I, cribrostatin Under these circumstances, we have been developing a total synthesis of these fascinating marine natural products. We have succeeded in the total syntheses of renieramycins G-I, cribrostatin 4, and renieramycin T [5][6][7][8]. However, the long and tedious procedures for the total synthesis of these natural products have impeded detailed structure-activity relationship (SAR) studies.

Results
We have been trying to simplify the structures of renieramycins without impairing their biological activities. Several right-hand (CDE-ring system) and left-hand (ABC-ring system) model compounds were prepared, and their in vitro cytotoxic activities against human cancer cell lines were tested [9,10]. These efforts have yielded CDE-ring model compounds (±)-3 to (±)-6a (Figure 2), and the presence of amino nitriles was found to induce at nanomolar concentrations (Table 1) [9]. 3-N-benzyl derivative (±)-6a exhibited approximately five and nine times more potent cytotoxic activity against HCT116 and QG56, respectively, than 3-N-methylated derivative (±)-5, indicating the importance of the substituent at 3-nitrogen.

Results
We have been trying to simplify the structures of renieramycins without impairing their biological activities. Several right-hand (CDE-ring system) and left-hand (ABC-ring system) model compounds were prepared, and their in vitro cytotoxic activities against human cancer cell lines were tested [9,10]. These efforts have yielded CDE-ring model compounds (±)-3 to (±)-6a (Figure 2), and the presence of amino nitriles was found to induce at nanomolar concentrations (Table 1) [9]. 3-Nbenzyl derivative (±)-6a exhibited approximately five and nine times more potent cytotoxic activity against HCT116 and QG56, respectively, than 3-N-methylated derivative (±)-5, indicating the importance of the substituent at 3-nitrogen.  From the structure comparison of (±)-6a and 1m, we expected that 3-N-Bn would correspond to the A-ring of 1m. Thus, 6b having an arylmethyl group whose substituent pattern was similar to the A-ring of 1m, and 6c having the same A-ring as 1y were set as the new target molecules ( Figure 3). A summary of our previously reported synthesis of racemic 6a is shown in Scheme 1 [9,11]. Conversion of 1,2,4-Trimethoxybenzene (7) into piperadine-2-5-dione derivative 8 took seven steps, and treatment of 8 with NaH and BnBr gave N-benzyl compound 9. Racemic compound 6a was  From the structure comparison of (±)-6a and 1m, we expected that 3-N-Bn would correspond to the A-ring of 1m. Thus, 6b having an arylmethyl group whose substituent pattern was similar to the A-ring of 1m, and 6c having the same A-ring as 1y were set as the new target molecules ( Figure 3).  4, and renieramycin T [5][6][7][8]. However, the long and tedious procedures for the total synthesis of these natural products have impeded detailed structure-activity relationship (SAR) studies.

Results
We have been trying to simplify the structures of renieramycins without impairing their biological activities. Several right-hand (CDE-ring system) and left-hand (ABC-ring system) model compounds were prepared, and their in vitro cytotoxic activities against human cancer cell lines were tested [9,10]. These efforts have yielded CDE-ring model compounds (±)-3 to (±)-6a (Figure 2), and the presence of amino nitriles was found to induce at nanomolar concentrations (Table 1) [9]. 3-Nbenzyl derivative (±)-6a exhibited approximately five and nine times more potent cytotoxic activity against HCT116 and QG56, respectively, than 3-N-methylated derivative (±)-5, indicating the importance of the substituent at 3-nitrogen.  From the structure comparison of (±)-6a and 1m, we expected that 3-N-Bn would correspond to the A-ring of 1m. Thus, 6b having an arylmethyl group whose substituent pattern was similar to the A-ring of 1m, and 6c having the same A-ring as 1y were set as the new target molecules ( Figure 3). A summary of our previously reported synthesis of racemic 6a is shown in Scheme 1 [9,11]. Conversion of 1,2,4-Trimethoxybenzene (7) into piperadine-2-5-dione derivative 8 took seven steps, and treatment of 8 with NaH and BnBr gave N-benzyl compound 9. Racemic compound 6a was A summary of our previously reported synthesis of racemic 6a is shown in Scheme 1 [9,11]. Conversion of 1,2,4-Trimethoxybenzene (7) into piperadine-2-5-dione derivative 8 took seven steps, and treatment of 8 with NaH and BnBr gave N-benzyl compound 9. Racemic compound 6a was prepared from 9 in ten steps. As the 3-N-arylmethyl group was critical to generate strong antitumor activity, derivatives with different 3-N-arylmethyl groups were prepared in subsequent steps. In addition, it is very interesting to compare the biological activities of the racemic form and the optically active form [12]. Thus, in order to facilitate the synthesis of structural analogs, a new asymmetric synthetic route for preparing the 3-N-arylmethyl group in the later steps should be developed.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 17 prepared from 9 in ten steps. As the 3-N-arylmethyl group was critical to generate strong antitumor activity, derivatives with different 3-N-arylmethyl groups were prepared in subsequent steps. In addition, it is very interesting to compare the biological activities of the racemic form and the optically active form [12]. Thus, in order to facilitate the synthesis of structural analogs, a new asymmetric synthetic route for preparing the 3-N-arylmethyl group in the later steps should be developed.
An outline of an alternative synthetic strategy to facilitate the asymmetric synthesis of various 3-N-arylmethyl derivatives is shown in Figure 4. We envisioned that the final step in the asymmetric synthesis of 6 should involve a reductive cyanation of the lactam carbonyl followed by a two-step oxidation of the phenol into p-quinone from 10. An N-arylmethyl group, which would be important for the cytotoxic activity, should be installed on lactam 11. The C-ring formation proceeded automatically from the lactonization of the primary amine, which was generated by the deprotection of the N-Cbz protecting group of 12. The synthesis of 1,3-cis-1,2,3,4-tetrahydroisoquinoline 12 was accomplished via the regio-and diastereoselective Pictet-Spengler cyclization reaction of aminophenol (−)-13 with N-Cbz glyoxal 14 [13]. Starting material (−)-13 was easily prepared from L-tyrosine according to Liu's method [14].
Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 17 prepared from 9 in ten steps. As the 3-N-arylmethyl group was critical to generate strong antitumor activity, derivatives with different 3-N-arylmethyl groups were prepared in subsequent steps. In addition, it is very interesting to compare the biological activities of the racemic form and the optically active form [12]. Thus, in order to facilitate the synthesis of structural analogs, a new asymmetric synthetic route for preparing the 3-N-arylmethyl group in the later steps should be developed.
With tricyclic chiral model (−)-6a in hand, 6d having an arylmethyl group with more electron-rich trimethoxy substituents was prepared (Scheme 4). The phenol of (−)-17 was selectively protected with 1.2 equivalents each of NaH and BnBr in N,N-dimethylformamide (DMF) to give (−)-11, which could be used to prepare several kinds of 3-N-alkylated compounds. The reaction of (−)-11 with substituted benzyl bromide 22, which was obtained by a reported method [17], produced 3-N-arylmethylated (−)-23 in 55% yield. The conversion (−)-23 into (−)-25 was carried out using a similar three-step sequence to that shown above, and salcomine oxidation of (−)-25 gave p-quinone (+)-6d in 79% yield. treated with KCN and water to provide α-aminonitrile (−)-20 in 74% yield as a single diastereomer. Chemoselective O-debenzylation was achieved with BCl3 in the presence of pentamethylbenzene to give desired phenol (−)-21 in 89% yield [16]. Finally, oxidation of (−)-21 with O2 in the presence of salcomine afforded chiral 3-N-benzylated CDE-ring model compound (−)-6a. (−)-6a was confirmed to have 99%ee by high performance liquid chromatography (HPLC) analysis, proving that the chiral center in L-tyrosine did not cause any racemization in this synthetic route. With tricyclic chiral model (−)-6a in hand, 6d having an arylmethyl group with more electronrich trimethoxy substituents was prepared (Scheme 4). The phenol of (−)-17 was selectively protected  The preparation of right-half model compounds 6b and 6c whose A-ring substitution patterns correspond to those of 1m and 1y, respectively, was carried out as follows (Scheme 5). Benzyl bromide 27 was prepared by the Appel reaction of corresponding alcohol 26 [18] in 99% yield. Alkylation of the lactam nitrogen of (−)-11 with 27 gave (−)-28 in 72% yield. Reductive cyanation of (−)-28 generated aminonitrile (−)-29 in 71% yield. Debenzylation of (−)-29 by using BCl3 gave a crude product that was expected to contain iminium by-products produced by the cyano group elimination. Thus, the crude product without further purification was treated with KCN to furnish desired phenol (−)-30 in 85% yield. Finally, bisphenol (−)-30 was oxidized with two equivalents of salcomine in oxygen atmosphere to give (−)-6b and (+)-6c in 43% and 31% yields, respectively. This oxidation could be controlled by adjusting the proportion of salcomine, as shown in Table 2. The preparation of right-half model compounds 6b and 6c whose A-ring substitution patterns correspond to those of 1m and 1y, respectively, was carried out as follows (Scheme 5). Benzyl bromide 27 was prepared by the Appel reaction of corresponding alcohol 26 [18] in 99% yield. Alkylation of the lactam nitrogen of (−)-11 with 27 gave (−)-28 in 72% yield. Reductive cyanation of (−)-28 generated aminonitrile (−)-29 in 71% yield. Debenzylation of (−)-29 by using BCl 3 gave a crude product that was expected to contain iminium by-products produced by the cyano group elimination. Thus, the crude product without further purification was treated with KCN to furnish desired phenol (−)-30 in 85% yield. Finally, bisphenol (−)-30 was oxidized with two equivalents of salcomine in oxygen atmosphere to give (−)-6b and (+)-6c in 43% and 31% yields, respectively. This oxidation could be controlled by adjusting the proportion of salcomine, as shown in Table 2. Although the detailed molecular mechanism underlying the antitumor activities of renieramycin marine natural products were unclear, we had speculated that the cyano or hydroxyl substituent at C-21 position of renieramycin would be essential for the potent cytotoxic activity. Elimination of the functional group at C-21 produced an electrophilic iminium ion species that was implicated in the formation of covalent bonds with DNA [19]. In 2008, Avendaño and co-workers reported a series of 1,2,3,4-tetrahydroisoquinolines with antitumor activities that were attributed to both apoptosis in the G2/M checkpoint and cytostatic activity in the G1 phase [20]. In addition, we prepared a series of renieramycin left-half model compounds from phenylalanine derivatives, and re-confirmed the importance of the C-21 cyano group for favorable activity [10]. Four right-half chiral model compounds 6a-6d and racemic 6a and 6b, including natural renieramycin M (1m) as positive control, were tested in vitro for cytotoxicity toward two representative human cancer cell lines (prostate cancer DU145 and colorectal cancer HCT116) using the CCK-8 assay (Table 3). Interestingly, the structure of the E-ring was found to be important for the enhanced biological activity. In order to examine the influence of the E-ring on the bioactivity, the IC50 values of three compounds (6a, 20, 21), in which the 3-N-Bn substituent and the C-4 cyano group were fixed, were compared. p-Quinone 6a was the most active, phenol 21 had comparable activity to 6a, and benzyl ether 20 showed markedly Although the detailed molecular mechanism underlying the antitumor activities of renieramycin marine natural products were unclear, we had speculated that the cyano or hydroxyl substituent at C-21 position of renieramycin would be essential for the potent cytotoxic activity. Elimination of the functional group at C-21 produced an electrophilic iminium ion species that was implicated in the formation of covalent bonds with DNA [19]. In 2008, Avendaño and co-workers reported a series of 1,2,3,4-tetrahydroisoquinolines with antitumor activities that were attributed to both apoptosis in the G2/M checkpoint and cytostatic activity in the G1 phase [20]. In addition, we prepared a series of renieramycin left-half model compounds from phenylalanine derivatives, and re-confirmed the importance of the C-21 cyano group for favorable activity [10]. Four right-half chiral model compounds 6a-6d and racemic 6a and 6b, including natural renieramycin M (1m) as positive control, were tested in vitro for cytotoxicity toward two representative human cancer cell lines (prostate cancer DU145 and colorectal cancer HCT116) using the CCK-8 assay (Table 3). Interestingly, the structure of the E-ring was found to be important for the enhanced biological activity. In order to examine the influence of the E-ring on the bioactivity, the IC 50 values of three compounds (6a, 20, 21), in which the 3-N-Bn substituent and the C-4 cyano group were fixed, were compared. p-Quinone 6a was the most active, phenol 21 had comparable activity to 6a, and benzyl ether 20 showed markedly decreased activity. Then, the importance of the cyano group at C-4 position was also confirmed in the right-half models. A significant decrease in cytotoxic activity was observed when the lactam carbonyl at C-4 position was converted into an aminonitrile (i. e., conversion of 19 into 20, 23 into 24, and 28 into 29). However, it was interesting that 28 showed moderate activity even though C-4 had a lactam carbonyl. Next, on comparing the IC 50 values of 21, 25, and 30 having characteristic 3-N-arylmethyl groups, 30 was found to show the least potent activity, whereas 25 with a trimethoxy arylmethyl group exhibited more potent activity. In the case of 21, which has an unsubstituted arylmethyl group, very strong activity at nanomolar concentrations was observed. A similar tendency was also observed in the model compounds. Among compounds 6a, 6b, 6c, and 6d whose E-rings were a quinone, phenol 6c, which has a phenol in the A-ring exhibited the weakest activity, whereas 3-N-benzyl 6a showed the strongest activity.
Finally, the effect of optical activity on the biological activity was investigated. It was recently confirmed that optically active (−)-1m obtained from nature had approximately two to three times stronger activity than racemic (±)-1m [21]. Unlike 1m, the chirality of racemic 6a and 6b and their chiral counterparts had no effect on their cytotoxic activities. Worth noting was that 6a with p-quinone on the E-ring, a cyano group at C-4 position, and 3-N-benzyl was the most active compound against the two types of cancer cell lines, and had similar potency to natural 1m.

Chemistry
IR spectra were obtained with a Shimadzu IRAffinity-1 FT-IR spectrometer (Shimadzu Corporation, Kyoto, Japan). Optical rotations were measured with Horiba SEPA-500 polarimeters (Horiba Ltd., Kyoto, Japan). 1 H-and 13 C-NMR spectra were recorded on a JEOL JNM-AL 400 NMR spectrometer (JEOL Ltd., Tokyo, Japan) at 400 MHz for 1 H and 100 MHz for 13 C; and a JEOL JNM-AL 300 NMR spectrometer at 300 MHz for 1 H and 75 MHz for 13 C (ppm, J in Hz with tetramethylsilane (TMS) as internal standard). All proton and carbon signals were assigned by extensive NMR measurements using correlation spectroscopy (COSY), Heteronuclear Multiple-Bond Correlation (HMBC), and Heteronuclear Multiple Quantum Correlation (HMQC) techniques. Mass spectra were recorded on a JEOL JMS 700 instrument (JEOL Ltd., Tokyo, Japan) with a direct inlet system operating at 70 eV.

Biological Evaluation
A single-cell suspension of each cell line (2 × 10 3 cells/well) was added to the serially diluted test compounds in a 96-well microplate and cultured for 4 days. Cell viability was measured with Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). IC 50 was expressed as the concentration at which cell growth was inhibited by 50% compared with the untreated control.

Conclusions
We presented a short and efficient methodology for the preparation of the chiral right-half model compounds of renieramycins. The synthesized model compounds were screened for their cytotoxic activity against DU145 and HCT116. Compounds 6a and 21 bearing benzyl group at 3-nitrogen showed very strong activity with IC 50 at nanomolar concentrations. It was also found that chirality had no effect on the cytotoxic activities of the model compounds.