Tanjungides A and B: New Antitumoral Bromoindole Derived Compounds from Diazona cf formosa. Isolation and Total Synthesis

Tanjungides A (1) (Z isomer) and B (2) (E isomer), two novel dibrominated indole enamides, have been isolated from the tunicate Diazona cf formosa. Their structures were determined by spectroscopic methods including HRMS, and extensive 1D and 2D NMR. The stereochemistry of the cyclised cystine present in both compounds was determined by Marfey’s analysis after chemical degradation and hydrolysis. We also report the first total synthesis of these compounds using methyl 1H-indole-3-carboxylate as starting material and a linear sequence of 11 chemical steps. Tanjungides A and B exhibit significant cytotoxicity against human tumor cell lines.


Introduction
Ascidians [1] are a rich source of bromoindole derived metabolites such as eudistomin [2], didemnimide [3], meridianin [4], coscinamide [5], rhopaladin [6], kottamide [7,8] and aplicyanin [9]. Most of these compounds exhibit antiviral, antibacterial and anti-inflammatory activity as well as cytotoxicity against tumor cell lines. The diazonamides isolated from Diazona angulata (originally misidentified as Diazona chinensis) [10] and Diazona sp. [11] provide a further example of secondary metabolites from ascidians. Strong cytotoxic activity has been reported for these compounds with IC 50 values in the nanomolar range. As part of work to study marine organisms from Indonesia, we have examined the constituents of the tunicate Diazona cf formosa collected off the coast of Tanjung Liarua and Toro Doro (Timor Island). In this paper we report the isolation, structure elucidation and synthesis of two new indole alkaloids Tanjungides A and B (1 and 2). Tanjungides are novel alkaloids containing a dibromoindole joined to a disulfide dipeptide by an enamide bond.

Isolation and Structure Elucidation
Cytotoxicity bioassay-guided fractionation of an organic extract of the organism, including VLC RP-18 chromatography followed by reverse-phase preparative HPLC of selected active fractions, led to the isolation of Tanjungides A and B.
Compound 1 was isolated as an optically active pale yellow amorphous solid with a pseudomolecular ion in the (+)-HRESIMS at m/z 518.9142 and an isotopic cluster consistent with the presence of two bromine atoms. The presence of 16 signals in the 13 C NMR spectrum (Table 1) was also in agreement with the molecular formula C 16  . The presence of a 3,5,6-trisubstituted indole in 1 ( Figure 1) was inferred by the existence of four characteristic signals in the low field region of the 1 H NMR spectrum in DMSO-d 6 , two doublets at δ H 7.78 (d, H-2, J = 2.4 Hz) and 11.78 (d, NH-1, J = 2.6 Hz) and two singlets at δ H 7.81 (s, H-7) and δ H 8.01 (s, H-4). In addition, the two bromine atoms contained in the molecular formula were located at C-5 and C-6 based on their 13 C chemical shifts. The intense 3-bond long range couplings between H-4 and C-6 at δ C 115.7 ppm and between H-7 and C-5 at δ C 113.5 ppm observed in the HMBC spectrum further confirmed the chemical shifts of these two quaternary carbons. The nature of the substituent at C-3 was deduced from analysis of additional signals in the low field region of the 1 H NMR spectrum and correlations observed in the COSY, HSQC and HMBC spectra. A spin system comprising two olefinic signals at δ H 6.06 ppm (H-8) and 6.68 ppm (H-9), and an interchangeable proton at δ H 9.60 ppm (NH-10) established the presence of an enamide. A coupling constant of 9.4 Hz between H-8 and H-9 confirmed a Z geometry for this double bond. Finally, HMBC correlations from H-9 to C-3 (δ C 109.2 ppm) and from H-8 to C-2 (δ C 126.8 ppm), and C-3a (δ C 127.6 ppm) indicated that the indole moiety was substituted at C-3 with a Z geometry enamide fragment. The remaining atoms, C 6 H 9 N 2 O 2 S 2 , comprised two carbonyl (δ C 169.9 and 167.1 ppm), two methine, (δ C 52.5/δ H 5.02 ppm and δ C 51.2/δ H 4.65 ppm) and two methylene groups (δ C 41.6/δ H 3.40 and 2.86 ppm and δ C 39.7/δ H 3.17 and 2.94 ppm) with three degrees of unsaturation being required for this molecular formula, including the two carbonyls mentioned previously. Analysis of the bidimensional spectra revealed the presence of a two spin system corresponding to two consecutive cysteine residues. Cross-peaks observed in the HMBC experiment between H-12 and H-16 and carbon C-14 at δ C 169.9 ppm ( Figure 2) confirmed this structural proposal. Furthermore, correlations observed in the HMBC experiment between H-9, NH-10, H-12 and H-17 to C-11, and a ROESY correlation between NH-10 and H-12, connected these cysteines residues to the enamide group through C-11. Finally, linkage of the two cysteine amino acids by a S-S bond to form a cyclic cystine explained the remaining unsaturation present and established the complete structure of Tanjungide A.   The absolute configuration of 1 was determined by converting the cyclized cystine into two alanines by Raney ® -Nickel desulfurization [12]. The absolute configuration of the resulting Ala amino acids was determined to be R by comparing the hydrolysis products of 1 with appropriate amino acid standards using HPLC-MS chromatography and after derivatization with Marfey's reagent L-FDAA (Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide) [13].
Compound 2 ( Figure 1) was isolated as an optically active pale yellow amorphous solid with the same molecular formula as 6 Hz corresponding to a E geometry for the double bond. The absolute configuration of the Cys residues was not determined due to the small amount of compound isolated and was assumed to be the same as in Tanjungide A (1). The validity of this assumption was later confirmed by total synthesis of the molecule.

Biological Activities of Tanjungides A and B
The cytotoxic activity of the new compounds ( Table 2) was tested against three human tumour cell lines, lung (A549), colon (HT29), and breast (MDA-MB-231), following a published procedure [14]. Tanjungide A (1) exhibited strong activity with GI 50 values in the range 0.19 to 0.33 μM, whereas Tanjungide B (2) displayed only mild cytotoxicity, with GI 50 values ranging from 1.00 to 2.50 μM.

Total Synthesis of Tanjungides A and B
In order to solve the supply problem for these two new marine chemical entities and progress pharmaceutical development and in vivo preclinical studies, we have completed the first total synthesis of Tanjungides A and B. This synthesis uses methyl 1H-indole-3-carboxylate as starting material and involves a linear sequence of 11 chemical steps. Key elements of our approach include selective dibromination of the indole, formylation by Vilsmeier reaction, Wittig olefination, stereoselective enamide formation and oxidation to create the disulfide bond ( Figure 3). The strategy uses vinyl iodide indole 8 as a common precursor to give both Tanjungides. The synthesis started, as outlined in Scheme 1, from the cheap commercially available methyl 1H-indole-3-carboxylate as this provided a high yielding route to a 5,6-dibrominated indole possessing an aldehyde moiety at C3, a highly versatile building block for the total synthesis of the two natural products. The slow addition of two equivalents of bromine to methyl 1H-indole-3-carboxylate in acetic acid at 23 °C yielded the corresponding 5,6-dibromo intermediate 3 as a single pure product in 66% yield [15]. Disappointingly, attempted methyl ester reduction of 3 to give aldehyde 6 directly was unsuccessful, and an alternative stepwise process to give aldehyde 6 was used involving hydrolysis and decarboxylation to give 5,6-dibromo-1H-indole 5 in good yield (90% over two steps) followed by Vilsmeier formylation using dimethylformamide and phosphorus oxychloride. After protection of the indole nitrogen as a tert-butyl carbamate, Wittig olefination with (iodomethyl)triphenylphosphonium iodide [16] gave the desired vinyl iodide indole 8 in 83% yield and as a 9:1 ratio of Z:E isomers [17].
With vinyl iodide 8 in hand, the next step involved coupling of the suitably-protected cysteine amino acids (Scheme 1). As described by Buchwald and co-workers [18], depending on the conditions used for the coupling reaction, vinyl iodide 8 provided access to both stereoisomers of enamide 10 and hence to both Tanjungide A and B. Specifically, copper-catalyzed reaction of 8 with N-allyloxycarbonyl-S-trityl-L-cysteine-amide 9, made in one step from commercially available S-trityl-L-cysteine-amide, gave enamide 10 in moderate yield (50%−60%) with use of Cs 2 CO 3 as base affording mainly enamide (Z)-10, which could be readily separated from the corresponding (E)-isomer by column chromatography, and K 2 CO 3 providing predominantly enamide (E)-10. Next, removal of the Alloc group of (Z)-10 or (E)-10 under neutral conditions using Pd(PPh 3 ) 4 and PhSiH 3 and coupling of the resulting primary amine with (N-(tert-butoxycarbonyl)-S-trityl-L-cysteine) by treatment with HATU and HOBt yielded the corresponding amide (Z)-12 or (E)-12. After substantial experimentation, the trityl group proved to be the best thiol protecting group for each of the cysteine amino acid building blocks. To complete the synthesis, the key cyclization of 12 to form the disulfide bond was accomplished using I 2 in CH 2 Cl 2 :CH 3 OH at high dilution to avoid undesired side-products [19,20] and subsequent simultaneously cleavage of both Boc protecting groups of 13 with TFA gave Tanjungides A (1) and B (2). All the spectroscopic data ( 1 H and 13 C NMR, optical rotation, IR, etc.), HPLC retention times and biological activities of the synthetic samples exactly matched those of the isolated natural products. The Supplementary Information provides more details.

General
Dry solvents were purchased and used without any extra processing. All reagents were used as purchased without further purification unless otherwise stated. All reactions were performed under an atmosphere of nitrogen in flame dried or oven dried glassware. Routine monitoring of reactions was performed using silica gel TLC plates (Merck 60 F254, Merck KGaA, Darmstadt, Germany). Spots were visualized by UV and/or dipping the TLC plate into an ethanolic phosphomolybdic acid solution and heating with a hot plate. Flash chromatography was carried out on silica gel 60 (200-400 mesh). 1 H and 13 C NMR were recorded on a Varian Unity 300 or 500 spectrometer at 300 or 500, and 75 or 125 MHz, respectively. Chemical Shifts (δ) are reported in parts per millions (ppm) referenced to CHCl 3 at 7.26 ppm for 1 H and CDCl 3 at 77.0 ppm for 13 C, to CH 3 OH at 3.30 ppm for 1 H and CD 3 OD at 49.0 ppm; and to (CH 3 ) 2 SO at 2.50 ppm for 1 H and (CD 3 ) 2 SO at 39.5 ppm for 13 C. Coupling constants are reported in Hertz (Hz), with the following abbreviations used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. When appropriate, the multiplicities are preceded with br, indicating that the signal was broad. Optical rotations were determined using a Jasco P-1020 polarimeter (Jasco Inc., Easton, MD, USA) with a sodium lamp and are reported as follows: [α] 25 D (c g/100 mL, solvent). (+)-HRESIMS was performed on an Applied Biosystems QStar pulsar Analyzer spectrometer (Applied Biosystems Inc., Foster City, CA, USA) employing 0.1% of formic acid in methanol as an ionic mobile phase. (+)-ESIMS were recorded using an Agilent 1100 Series LC/MSD spectrometer (Agilent Technologies, Santa Clara, CA, USA). UV spectra were performed using an Agilent 8453 UV-VIS spectrometer (Agilent Technologies). IR spectra were obtained with a Perkin Elmer Spectrum 100 FT-IR spectrometer (PerkinElmer Inc., Waltham, MA, USA) with ATR sampling.

Animal Material
The tunicate Diazona cf formosa (Order Phlebobranchia, Family Diazonidae, Genus Diazona) was collected by hand using a rebreather diving system in East Timor (08°25.637′S/126°22.849′E) at depths ranging between 6 and 80 m in June 2009. A sample of the specimen was deposited in the Center for the Advanced Studies of Blanes in Girona, Spain, with the reference code TISM-763.

Extraction and Isolation
A specimen of Diazona cf formosa (128 g) was triturated and exhaustively extracted with CH 3 OH:CH 2 Cl 2 (50:50, 3 × 200 mL). The combined extracts were concentrated to yield a crude mass of 5

Absolute Configuration of Cysteine Residues
Approximately 100 μL of Raney ® -Nickel (50% slurry in H 2 O, excess) was added to Tanjungide

Total Synthesis of Tanjungides A (1) and B (2)
3.5.1. 5,6-Dibromo-1H-indole-3-carboxylic Acid (4) To a stirred solution of methyl 5,6-dibromo-1H-indole-3-carboxylate (3) (12.5 g, 37.8 mmol) in CH 3 OH (124 mL) was added an aqueous solution of NaOH (188 mL, 2 M, 376 mmol). The suspension was refluxed for 2.5 h. After this time, the brown solution was cooled to 23 °C and the volatiles were evaporated. The aqueous phase was acidified with a 1 M solution of HCl until reached pH 2 and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to afford crude 4 (11.4 g, 95% yield) as a brown solid which was used in the next step without further purification. 1 (5) 5,6-Dibromo-1H-indole-3-carboxylic acid (4) (11.7 g, 36.7 mmol) was dissolved in pyridine (20.5 mL) and refluxed overnight. The solvent was concentrated in vacuo, the crude obtained was dissolved in CH 2 Cl 2 , precipitated with hexane and left at 5 °C overnight. The solid was filtered to yield crude 5 (9.6 g, 95% yield) which was used in the next step without further purification. 1

5,6-Dibromo-1H-indole-3-carbaldehyde (6)
To a stirred solution of DMF (46.8 mL) at 0 °C was dropwise added POCl 3 (12.0 mL, 131.3 mmol). The mixture was further stirred for 5 min at 0 °C and a solution of 5,6-dibromo-1H-indole (5) (7.22 g, 26.3 mmol) in DMF (70 mL) was slowly added. The reaction mixture was stirred 1 h at 35 °C, 1 h at 65 °C, and was left to reach 23 °C. An aqueous solution of NaOH (72.3 mL, 2 N) was added at 0 °C and the reaction mixture was stirred 5 min at 110 °C, left to reach 23 °C, and then added over an ice-water bath in order to precipitate 6. The reaction mixture was left overnight at 5 °C and filtered to obtain crude 6 (7.32 g, 92% yield) which was used in the next step without further purification. 1 (7) To a stirred solution of 5,6-dibromo-1H-indole-3-carbaldehyde (6) (9.2 g, 30.4 mmol) in 1,4-dioxane (152 mL) was added successively di-tert-butyldicarbonate (7.9 g, 36.4 mmol) and DMAP (370 mg, 3.0 mmol). After stirring for 2 h at 23 °C, the mixture was quenched with H 2 O and extracted with EtOAc. The combined organic phases were washed thoroughly with H 2 O, dried over Na 2 SO 4 , filtered and concentrated in vacuo to afford 7 (10.5 g, 86% yield) as a slightly brown solid that was used in the next steps without further purification. 1

Conclusions
In summary, we have isolated, determined the structure and completed the first total synthesis of Tanjungides A and B, two new bromoindole enamides with interesting cytotoxic properties from the tunicate Diazona cf formosa. The total synthesis confirmed the structural assignment and provides rapid access to these new natural products and related analogues for biological evaluation and drug development.