Total Syntheses and Preliminary Biological Evaluation of Brominated Fascaplysin and Reticulatine Alkaloids and Their Analogues

A simple approach toward the synthesis of the marine sponge derived pigment fascaplysin was used to obtain the marine alkaloids 3-bromofascaplysin and 3,10-dibromofascaplysin. These compounds were used for first syntheses of the alkaloids 14-bromoreticulatate and 14-bromoreticulatine. Preliminary bioassays showed that 14-bromoreticulatine has a selective antibiotic (to Pseudomonas aeruginosa) activity and reveals cytotoxicity toward human melanoma, colon, and prostate cancer cells. 3,10-Dibromofascaplysin was able to target metabolic activity of the prostate cancer cells, without disrupting cell membrane’s integrity and had a wide therapeutic window amongst the fascaplysin alkaloids.

Remarkably, some derivatives of fascaplysin were found to have an increased therapeutic potential compared to the parental alkaloid. Thus, methylation of fascaplysin at C-9 results in the more potent Aβ aggregation inhibitor than alkaloid 1 [22]. The synthetic chloro derivative of fascaplysin (2) inhibited the VEGF-mediated microvessel sprouting with blood vessel formation in the matrigel plug of C57/BL6J mice and the tumor growth in ET (solid) mouse tumor model [23]. In addition, natural 3-and 10-bromofascaplysins (3,4) showed anti-cancer activity at submicromolar concentrations. This was, at least in part, mediated through the induction of caspase-8, -9, and -3dependent apoptosis [24]. Antitumor effects of 3-bromofascaplysin and 10-bromofascaplysin were comprehensively examined in an in vitro glioma C6 cell model. The cytotoxic efficiency of compounds 3 and 4 was higher than that of unsubstituted fascaplysin; 3-bromofascaplysin exhibited the best capacity to kill glioma C6 cells [25]. 3,10-Dibromofascaplysin (5)-the last representative of fascaplysin alkaloids was synthesized in eight steps from 6-bromoindole and 4-amino-2bromotoluene, but the therapeutic potential of that perspective compound has not been investigated yet [26].
Remarkably, some derivatives of fascaplysin were found to have an increased therapeutic potential compared to the parental alkaloid. Thus, methylation of fascaplysin at C-9 results in the more potent Aβ aggregation inhibitor than alkaloid 1 [22]. The synthetic chloro derivative of fascaplysin (2) inhibited the VEGF-mediated microvessel sprouting with blood vessel formation in the matrigel plug of C57/BL6J mice and the tumor growth in ET (solid) mouse tumor model [23]. In addition, natural 3and 10-bromofascaplysins (3,4) showed anti-cancer activity at submicromolar concentrations. This was, at least in part, mediated through the induction of caspase-8, -9, and -3-dependent apoptosis [24]. Antitumor effects of 3-bromofascaplysin and 10-bromofascaplysin were comprehensively examined in an in vitro glioma C6 cell model. The cytotoxic efficiency of compounds 3 and 4 was higher than that of unsubstituted fascaplysin; 3-bromofascaplysin exhibited the best capacity to kill glioma C6 cells [25]. 3,10-Dibromofascaplysin (5)-the last representative of fascaplysin alkaloids was synthesized in eight steps from 6-bromoindole and 4-amino-2-bromotoluene, but the therapeutic potential of that perspective compound has not been investigated yet [26].
Herein, we report the two-step method for the syntheses of 3-bromofascaplysin and 3,10-dibromofascaplysin, which has been previously used for the synthesis of fascaplysin. The similarity in structures lets us to use these compounds as starting materials for the first syntheses of several alkaloids of reticulatine group (compounds 6-12, Figure 1, [27]). Also, the bioactivities of the obtained compounds were investigated.

Chemistry
Several groups have synthesized fascaplysin and its naturally occurring analogs and more than 10 syntheses have been reported to date [28][29][30][31][32][33][34][35][36][37][38]. Among them the two-step scheme by Zhu et al. is the most suitable for the preparation of the target compounds [36]. To apply that synthetic scheme for the synthesis of 3,10-dibromofascaplysin, the reaction between 3-bromophenylhydrazine (13) and 4-bromobutanal (14) in an autoclave at 150 • C was used to prepare the mixture of 6-bromotryptamine (15) and 4-bromotryptamine (16) (Scheme 1). Thereafter, the obtained mixture and 2,4-dibromoacetophenone (17) were subjected to the cascade coupling protocol, previously developed by Zhu et al., which included the sequential iodination of the corresponding acetophenone, the Kornblum oxidation of the intermediate in the presence of DMSO to phenylglyoxal, and its Pictet-Spengler condensation with the derivative of tryptamine followed by the oxidation of the intermediate. After chromatography purification, two isomeric 1-benzoyl-β-carbolines (18,19) were obtained with the yields of 20% and 19%, respectively. These products were subsequently transformed to 3,10-dibromofascaplysin (5) and its isomer 20 according to the procedure reported by the group of Radchenko [31].

Chemistry
Several groups have synthesized fascaplysin and its naturally occurring analogs and more than 10 syntheses have been reported to date [28][29][30][31][32][33][34][35][36][37][38]. Among them the two-step scheme by Zhu et al. is the most suitable for the preparation of the target compounds [36]. To apply that synthetic scheme for the synthesis of 3,10-dibromofascaplysin, the reaction between 3-bromophenylhydrazine (13) and 4-bromobutanal (14) in an autoclave at 150 °C was used to prepare the mixture of 6-bromotryptamine (15) and 4-bromotryptamine (16) (Scheme 1). Thereafter, the obtained mixture and 2,4dibromoacetophenone (17) were subjected to the cascade coupling protocol, previously developed by Zhu et al., which included the sequential iodination of the corresponding acetophenone, the Kornblum oxidation of the intermediate in the presence of DMSO to phenylglyoxal, and its Pictet-Spengler condensation with the derivative of tryptamine followed by the oxidation of the intermediate. After chromatography purification, two isomeric 1-benzoyl-β-carbolines (18,19) were obtained with the yields of 20% and 19%, respectively. These products were subsequently transformed to 3,10-dibromofascaplysin (5) and its isomer 20 according to the procedure reported by the group of Radchenko [31]. 3-Bromofascaplysin was prepared in a similar manner from tryptamine (21) and 2,4dibromoacetophenone (17) with a total yield of 32%. Taking into account the high biological activity of synthetic chloro derivatives of fascaplysin, we obtained the corresponding derivative at C-2 (25) from tryptamine and 2,5-dichloroacetophenone (22) by a similar method (Scheme 2) [20]. 3-Bromofascaplysin was prepared in a similar manner from tryptamine (21) and 2,4-dibromoacetophenone (17) with a total yield of 32%. Taking into account the high biological activity of synthetic chloro derivatives of fascaplysin, we obtained the corresponding derivative at C-2 (25) from tryptamine and 2,5-dichloroacetophenone (22) by a similar method (Scheme 2) [20]. Previously zwitter-ionic β-carboline 26 was obtained from fascaplysin that was treated with aqueous solution of NaOH or 30% NH4OH [39]. After optimization of the reaction conditions 14bromoreticulatate (10) and its dibromo analog (27, not isolated from marine organisms) were obtained from compounds 3 and 5 in DMF at r.t. with 86% and 80% yields, respectively (Scheme 3). Different conditions for methylation of compounds 10 and 27 were investigated, including (i) the interaction with diazomethane; (ii) with POCl3 and following treatment with methanol; (iii) the reaction with dimethyl sulfate. In the latter case, best results were achieved. However, 7,14- Previously zwitter-ionic β-carboline 26 was obtained from fascaplysin that was treated with aqueous solution of NaOH or 30% NH 4 OH [39]. After optimization of the reaction conditions 14-bromoreticulatate (10) and its dibromo analog (27, not isolated from marine organisms) were obtained from compounds 3 and 5 in DMF at r.t. with 86% and 80% yields, respectively (Scheme 3). Different conditions for methylation of compounds 10 and 27 were investigated, including (i) the interaction with diazomethane; (ii) with POCl 3 and following treatment with methanol; (iii) the reaction with dimethyl sulfate. In the latter case, best results were achieved. However, 7,14-dibromoreticulatine (8) was not obtained after methylation of compound 27. Instead, the product of dimethylation (28) was obtained. Because of the insolubility of compound 28 in most solvents, only MS and 1 H NMR were used to identify its structure. The spectral characteristics of synthetic 3-bromofascaplysin, 3,10-dibromofascaplysin, 14-bromoreticulatate, and 14-bromoreticulatine were identical to those of the natural products. Previously zwitter-ionic β-carboline 26 was obtained from fascaplysin that was treated with aqueous solution of NaOH or 30% NH4OH [39]. After optimization of the reaction conditions 14bromoreticulatate (10) and its dibromo analog (27, not isolated from marine organisms) were obtained from compounds 3 and 5 in DMF at r.t. with 86% and 80% yields, respectively (Scheme 3). Different conditions for methylation of compounds 10 and 27 were investigated, including (i) the interaction with diazomethane; (ii) with POCl3 and following treatment with methanol; (iii) the reaction with dimethyl sulfate. In the latter case, best results were achieved. However, 7,14dibromoreticulatine (8) was not obtained after methylation of compound 27. Instead, the product of dimethylation (28) was obtained. Because of the insolubility of compound 28 in most solvents, only MS and 1 H NMR were used to identify its structure. The spectral characteristics of synthetic 3bromofascaplysin, 3,10-dibromofascaplysin, 14-bromoreticulatate, and 14-bromoreticulatine were identical to those of the natural products.

Biology
The bioactivities of obtained compounds were investigated using fascaplysin (1) as a standard. First, the cytotoxic effects of the compounds against human colorectal carcinoma (HT-29), human breast cancer (T-47D), and melanoma (SK-MEL-28) cell lines were evaluated by MTS assay ( Table 1). The cells were incubated with different concentrations of the respective compounds (0-5 µM) for 24 h. The concentration that caused inhibition of 50% of cell viability (IC50) was 5 µM for compound 1 against T-47D cells. Other investigated compounds were less cytotoxic against this type of cancer cells at concentrations up to 5 µM. However, the IC50 of 1, 3, and 7 were detected at concentrations ranging from 1.1 to 1.9 µM against SK-MEL-28 cells. Among the investigated cancer cells, the most

Biology
The bioactivities of obtained compounds were investigated using fascaplysin (1) as a standard. First, the cytotoxic effects of the compounds against human colorectal carcinoma (HT-29), human breast cancer (T-47D), and melanoma (SK-MEL-28) cell lines were evaluated by MTS assay ( Table 1). The cells were incubated with different concentrations of the respective compounds (0-5 µM) for 24 h. The concentration that caused inhibition of 50% of cell viability (IC 50 ) was 5 µM for compound 1 against T-47D cells. Other investigated compounds were less cytotoxic against this type of cancer cells at concentrations up to 5 µM. However, the IC 50 of 1, 3, and 7 were detected at concentrations ranging from 1.1 to 1.9 µM against SK-MEL-28 cells. Among the investigated cancer cells, the most resistant cell line to the cytotoxic effect of the compounds was found to be breast cancer cells T-47D, while the most sensitive were melanoma cells SK-MEL-28. It was shown that compounds 1 and 3 possessed comparable IC 50 against colorectal carcinoma cells HT-29. Our results indicated that the investigated compounds reveal selective cytotoxic effects to different cancer cell lines, with highest efficacy in melanoma cells SK-MEL-28. We have also investigated the effect of the synthesized compounds on the viability and the growth of human prostate cancer drug-resistant PC-3 and 22Rv1 cells. IC 50 s of the substances have been determined by both, MTT and trypan blue exclusion assay (ViCell assay) (Table 1, Figure 2). It is known that MTT assay accesses the metabolic activity of the cells, while the trypan blue exclusion assay shows the alive cells with either intact (non-stained) or disrupted (stained) membranes. Compound 20 was identified to be the most active among the tested fascaplysin derivatives. However, its cytotoxicity determined by MTT assay was within the range of compounds 3 and 25 and fascaplysin (1). Interestingly, compound 5, having a higher IC 50 of 0.69 ± 0.14 µM, had a very smooth cytotoxicity profile, suggesting a wide therapeutic window ( Figure 2). Moreover, for compound 5, the IC 50 determined by trypan blue exclusion assay was~8-fold higher than the IC 50 accessed using MTT test. In contrast, for the other compounds the difference of the IC 50 s generated by the two different methods was distinctly less pronounced. This may indicate an antimetabolic effect of compound 5 rather an effect on the cell membrane integrity (necrotic-like cell death). Compound 5 starts to suppress cancer cell viability/proliferation already at 0.1 µM, while the ranges of active concentrations for the other two tested compounds were rather narrow. Fascaplysin (1) started to suppress cancer cell viability/proliferation at 0.125 µM. Remarkably, for this compound no difference between IC 50 s generated with the two different methods was observed. The high potential of compound 5 for therapeutic assays was also confirmed by its low cytotoxity (IC 50 50 µM) against normal MRC-9 lung cells. IC50, the concentration of compounds that caused a 50% reduction in cell viability of tested normal and cancer cells; a MTS assay was used; b MTT assay was used; c ViCell assay (trypan blue exclusion) was used, n/d-not determined.
We have also investigated the effect of the synthesized compounds on the viability and the growth of human prostate cancer drug-resistant PC-3 and 22Rv1 cells. IC50s of the substances have been determined by both, MTT and trypan blue exclusion assay (ViCell assay) (Table 1, Figure 2). It is known that MTT assay accesses the metabolic activity of the cells, while the trypan blue exclusion assay shows the alive cells with either intact (non-stained) or disrupted (stained) membranes. Compound 20 was identified to be the most active among the tested fascaplysin derivatives. However, its cytotoxicity determined by MTT assay was within the range of compounds 3 and 25 and fascaplysin (1). Interestingly, compound 5, having a higher IC50 of 0.69 ± 0.14 µM, had a very smooth cytotoxicity profile, suggesting a wide therapeutic window ( Figure 2). Moreover, for compound 5, the IC50 determined by trypan blue exclusion assay was ~8-fold higher than the IC50 accessed using MTT test. In contrast, for the other compounds the difference of the IC50s generated by the two different methods was distinctly less pronounced. This may indicate an antimetabolic effect of compound 5 rather an effect on the cell membrane integrity (necrotic-like cell death). Compound 5 starts to suppress cancer cell viability/proliferation already at 0.1 µM, while the ranges of active concentrations for the other two tested compounds were rather narrow. Fascaplysin (1) started to suppress cancer cell viability/proliferation at 0.125 µM. Remarkably, for this compound no difference between IC50s generated with the two different methods was observed. The high potential of compound 5 for therapeutic assays was also confirmed by its low cytotoxity (IC50 50 µM) against normal MRC-9 lung cells. It is known that fascaplysins exhibit potent but nonselective antibiotic activities. To evaluate activity of reticulatines in comparison to known fascaplysin derivatives, compounds 1, 3, 7, 25 were studied in vitro for antibiotic activity against several microbes using the disk diffusion soft agar assay as shown in Table 2. 14-Bromoreticulatine (7) showed potent activity against Pseudomonas aeruginosa while it exhibited low activity or no activity at all against other tested microbes. As expected, high and non-selective antibiotic activities were demonstrated for the other tested compounds (1, 3, 25).

Chemistry
All starting materials are commercially available. Commercial reagents were used without any purification. The products were isolated by MPLC: Buchi B-688 pump, glass column C-690 (15 × 460 mm) with Silica gel (particle size 0.015-0.040 mm), UV-detector Knauer K-2001. The analytical examples were purified by Shimadzu HPLC system (model: LC-20AP) equipped with a RID detector (model: RID 10A) using Supelco C18 (5 µm, 4.6 × 250 mm) column using ACN:water (20:80, 50:50, 70:30) mobile phase by isocratic elution at flow rate of 1 mL/min. NMR spectra were recorded with a NMR instrument operating at 400 MHz ( 1 H) and 100 MHz ( 13 C). Proton spectra were referenced to TMS as internal standard, in some cases, to the residual signal of used solvents. Carbon chemical shifts were determined relative to the 13 C signal of TMS or used solvents. Chemical shifts are given on the δ scale (ppm). Coupling constants (J) are given in Hz. Multiplicities are indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br (broadened). The original spectra of the relative compounds could be found in Supplementary Materials. High-resolution mass spectra (HRMS) were obtained with a time-of-flight (TOF) mass spectrometer equipped with an electrospray source at atmospheric pressure ionization (ESI).

Preparation of Substituted 1-Benzoyl-β-Carbolines 18, 19, 23, 24
Corresponding acetophenone (0.458 mmol) and iodine (92 mg, 0.366 mmol) were added to 2 mL of DMSO, and the resulting solution was heated at 90 • C for 1 h. After that tryptamine, its derivative or their mixture (0.458 mmol) was added to the solution and this solution was stirred at the same temperature for 3-4 h till completion of reaction (monitored by TLC). Then the reaction mixture was cooled to room temperature followed by the addition of water (50 mL) and extraction with EtOAc (2 × 25 mL). The extract was washed with 10% Na 2 S 2 O 3 , dried over Na 2 SO 4 , filtered and evaporated under reduced pressure. The residue was purified by MPLC using benzene and benzene/hexanes as eluent to give the desired product.

Preparation of Fascaplysin Derivatives
Substituted 1-benzoyl-β-carboline (0.326 mmol) was heated in sealed tube at 220 • C for 15 min. After cooling, the reaction mixture was washed with EtOAc (3 × 3 mL) and H 2 O (3 × 10 mL). The combined aqueous layer was acidified with hydrochloric acid and evaporated under reduced pressure to give target product as a red powder.

Cytotoxicity Assays
MTS and MTT assays were used as an indicator of cell viability as determined by mitochondrial-dependent reduction of formazan or its salts. For MTS assay, the cells were seeded in density of 1.0 × 10 4 cells/200 µL of complete medium in 96-well plates. After incubation for 24 h attached cells were treated with various concentrations of the compounds (0.05; 0.1; 0.5; 1; 5 µM), while the control was treated with the complete McCoy, RPMI-1640, and DMEM medium only. Cells were cultured for additional 24 h at 37 • C in 5% CO 2 incubator. After incubation, MTS-reagent (20 µL) was added to each well, and then cells were incubated for 3 h at 37 • C in 5% CO 2 . Absorbance was measured at 490/630 nm by microplate reader (Power Wave XS, American). All tested samples were carried out in triplicates. MTT assay was performed as previously described with the 48 h drug treatment [42]. The trypan-blue-based viability assay (ViCell assay) was performed using Beckman Coulter Vi-CELL (Beckman Coulter, Krefeld, Germany) as has been described before [43].

Statistical Analysis
Statistical analyses were performed using GraphPad Prism software v. 5.01 (GraphPad Prism software Inc., La Jolla, CA, USA). Data are presented as mean ± SD. The unpaired Student's t-test was used for the comparison of two groups. Statistical significance was represented as * p < 0.05 and ** p < 0.01.

Conclusions
Thus, the two-step approach toward the synthesis of the marine sponge derived pigment fascaplysin was used to obtain the marine alkaloids 3-bromofascaplysin and 3,10-dibromofascaplysin. These compounds were used as the starting materials for first syntheses of the alkaloids 14-bromoreticulatine and 14-bromoreticulatate. Preliminary bioassays showed that 14-bromoreticulatine reveals selective antibiotic (to P. aeruginosa) and cytotoxic (to melanoma SK-MEL-28 cell line) activities. It was also demonstrated that 3,10-dibromofascaplysin was able to suppress the cell metabolism at concentrations at least 7 times lower than the cytotoxic concentrations, which induced cell membrane disruption. The examination of biological activity of the synthesized compounds showed that even minimal modification of fascaplysin structure has a significant effect on the bioactivity of this lead compound. At the present time, the biological activities of a large series of novel synthetic derivatives of fascaplysin are being investigated thoroughly. This should open new opportunities for the detailed studies of the structure-activity relationships among these potent and promising biologically active substances.