Unprecedented Biomimetic Homodimerization of Oroidin and

May 8, 2013 - Clarisse Lejeune, Hua Tian, Jérôme Appenzeller, Ludmila Ermolenko, Marie-Thérèse Martin, ... 91198 Gif-sur-Yvette, France. •S Supporting...
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Unprecedented Biomimetic Homodimerization of Oroidin and Clathrodin Marine Metabolites in the Presence of HMPA or Phosphonate Salt Tweezers Clarisse Lejeune, Hua Tian, Jérôme Appenzeller, Ludmila Ermolenko, Marie-Thérèse Martin, and Ali Al-Mourabit* Institut de Chimie des Substances Naturelles, UPR 2301, Centre de Recherches de Gif-sur-Yvette (CNRS), Avenue de la Terrasse, 91198 Gif-sur-Yvette, France S Supporting Information *

ABSTRACT: The first biomimetic homodimerization of oroidin and clathrodin was effected in the presence HMPA and diphosphonate salts, strong guanidinium and amide chelating agents. The intermolecular associations probably interfere with the entropically and kinetically favored intramolecular cyclizations. Use of oroidin·1/2 HCl salt or clathrodin·1/2HCl was indicative in the presence of the ambident nucleophilic and electrophilic tautomers of the 2aminoimidazolic oroidin and clathrodin precursors. Surprisingly, the homodimerization of oroidin led to the nagelamide D skeleton, while the homodimerization of clathrodin gave the benzene para-symmetrical structure 19. The common process was rationalized from tautomeric precursors I and III.

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sceptrin and its oxidized derivative benzosceptrin B. It should be noted that radical mechanisms were proposed by Lindel13 for the isomerization of (Z)-oroidin into (E)-oroidin and by Baran for the isomerization/expansion of the vinyl cyclobutane of the sceptrin dimeric member into ageliferin.14 Consequently, Baran proposed that sceptrin is the precursor of ageliferin and other congeners.15 How the dimerization involving the monomers 6−8 and the subtle chemoselectivity could occur in selected solvents represents an interesting subject of investigation in synthesis. So far, the dimerization of oroidin without enzymatic catalysis has not been achieved. It should be noted that a vinyl 2aminoimidazole model lacking the pyrrole carboxamide group was used by Horne in the total synthesis of mauritiamine through an oxidative heterodimerization.16 Here we disclose the first synthetic direct dimerization of oroidin and clathrodin to detectable dimeric P-2-AIs.

yrrole 2-aminoimidazole (P-2-AI) alkaloids isolated from marine sponges are excellent examples of the molecular diversity created in nature.1 Over 150 derivatives, including the dimeric members nagelamides (1) and D (2),2 mauritiamine (3),3 stylissazole A (4),4 and benzosceptrin (5)5 (Scheme 1), are believed to arise from monomeric precursors such as clathrodin (6),6 hymenidin (7),7 and oroidin (8),8 via various but controlled C−C and/or C−N dimerizations and further processes. The diversity of the known dimeric structures isolated so far suggests a key role for the modulation of the chemoselectivity during the dimerization process. The biosynthetic pathway leading to these compounds and the biomechanistic steps controlling the chemoselectivity are not known. Our earlier proposal9 was based on the ambident reactivity inherent to the tautomeric forms I−IV of the monomers 6-8 (Scheme 1). Subsequent investigations of the tautomeric equilibria by Zipse and Wei,10 including molecular mechanics calculations, concluded that the tautomeric interconversion of I−IV could only be explained in aqueous acidic conditions.11 Of particular interest is the process leading to the activation and the selection of the reacting tautomers in Nature. The dynamic hydrogenbonding interaction of the precursors 6−8 with the host enzymatic system is probably one of the most intriguing biochemical catalytic processes. Recently, Molinski and Romo succeeded in the first experimental evidence of bioconversion of 15N-labeled oroidin into P-2-AIs benzosceptrin C and nagelamide H.12 The authors used cell-free enzyme preparations from the marine sponges Agelas sceptrum and Stylissa caribica to achieve the homodimerization of oroidin. They proposed an enzymatic single-electron transfer (SET) transformation of 6−8 to the redox-neutral © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Assuming the challenge posed by the “one-pot” coexistence of nucleophilic and electrophilic oroidin tautomers, our objective was the homodimerization of oroidin itself into any dimer at detectable concentration without any oxidant. Keeping in mind that the influence on the tautomeric equilibrium in acidic conditions is probably crucial for the homodimerization, we started to study the solvation effects by varying the solvents and the reaction temperature. According to the preliminary results showing that oroidin decomposes under basic conditions to a Received: January 17, 2013 Published: May 8, 2013 903

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Scheme 1. Structures of Selected Dimeric P-2-AI Metabolites and the Tautomeric Forms of the Monomers 6−8 Depicted as Free Bases for Clarity

Scheme 2. Condition-Dependent Intramolecular Cyclizations/Decomposition of Oroidin (8)

We then directed our attention to the oroidin salt and the use of a chelating environment that would favor intermolecular reaction with kinetic preference for the dimerization process. After several unsuccessful trials using various oroidin salts and solvents,21 we focused on altering the intramolecular hydrogen bonds inherent to the coexistence of the guanidine and amide groups in oroidin. Inspired by the work of Schrader regarding the artificial receptors for guanidinium ions in arginine22 and oroidin,23 we explored the effect of the association of oroidin with HMPA and benzylic bisphosphonates. An anionic homodimerization of oroidin would presumably involve the ambident electrophilic/nucleophilic reactivity of the 2-aminoimidazole moiety. The latter reasoning suggested the heating of 1/2·salt oroidin hydrochloride (8·1/2HCl) in HMPA. Under these conditions, LC-MS analysis showed different reaction profiles indicating the presence of dimeric derivatives for the first time. From the complex reaction mixture, and after repetitive HPLC purifications, we could isolate the dimeric isomers 12a and 12b in 3% total yield along with the starting material oroidin (80%) (Scheme 3). When the dimerization was attempted in the presence of the molecular tweezer benzylic bisphosphonate tetrabutylammonium 1424 with oroidin (8·1/2HCl) in DMSO at 130 °C, dimers 16 and 17 (5%) along with 12a and 13b (4%) could be isolated. Monomeric oxazoline 9 and dihydrooroidin 15 were also obtained in 32% and 4% yields, respectively. The structure of 15 (isolated as a 30% mixture with oroidin) was determined by comparison of its spectroscopic data with the synthesized sample using our previous method.25 It is interesting to note that the 1H NMR spectrum of dihydrooroidin with 30% oroidin did not match with the reference sample even under the same conditions. The discrepancy in the imidazolic proton signal was solved by recording the 1H NMR spectrum (see Supporting Information) of a 1/1 mixture of the isolated dihydroroidin (15) with the reference sample. The mutual influence of the oroidin/dihydrooroidin mixture on the 1H NMR spectra confirms their high degree of interactivity. Gratifyingly, we found that compounds 12a, 12b, 16, and 17 all had the same

complex and intractable mixture, we started with acidic conditions. When oroidin17 was heated in methanesulfonic acid, LC-MS analysis of the reaction mixture showed only monomeric products. The major product corresponds to the oxazoline 9 (Scheme 2).18 The oxazoline unit has been reported as a natural structural element of the dimers nagelamide R19 and T.5 While exploring the homodimerization reaction, Lindel et al.20 obtained a different intramolecular cyclization of the formate salt of oroidin into cyclooroidin in ethanol in 93% yield. It seems that, even if the products of the later reactions are structurally different, the mode of cyclization is similar in the sense that the acidic conditions favor ionic cyclization. The contrast between the intermolecular entropically and kinetically favored cyclizations occurring through the amide oxygen into oxazoline 9 or through the pyrrolic nitrogen into cyclooroidin could be explained by the nature of the salt and the solvent influences. Interestingly, when the neutral oroidin was heated in HMPA in the presence of BF3·Et2O, the decomposition product dibromopyrrole carboxamide 11 was isolated. This observation is coherent if we consider the tautomer 8-II and the driving force induced by the high affinity of BF3·Et2O to the carbonyl of the carboxamide. It is important to note that more or less brominated carboxamides of type 11 were frequently isolated from marine sponges together with other members of P-2-AI metabolites. 904

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H-4′/H-8′ showed an E configuration for compound 12b. The hydrogenation of 12a in the presence of Pd(OH)2/C led to 13, which corresponds to the debrominated natural product nagelamide D (Scheme 3). The total synthesis of nagelamide D was achieved in 2009 by Lovely,26 and its NMR data did not match that of the natural nagelamide D isolated by Kobayashi.27 As far as we are concerned, the chemical shifts of our debrominated derivative 13 are closer to those of the synthetic compound described by Lovely. The planar structures of the diastereoisomers 16 and 17 were determined by 2D NMR spectroscopy, but their relative configurations remain unknown (Figure 1). The low yields due to the difficulty in purifying individual diastereoisomers by HPLC in sufficient quantities hampered further studies for the determination of the relative configurations. Finally, we decided to explore the influence of the bromines on the homodimerization by testing the debrominated derivative clathrodin under the same conditions. Clathrodin (6) was prepared25 and its hydrochloride 1/2·salt (6·1/2HCl) was heated in HMPA. Surprisingly, after 2 h at 120 °C, the bisbenzodiimidazole 19 was obtained as the major product in 10% yield. The symmetrical dimeric structure of 19 showed a mass of 461.2 and simple 1H and 13C NMR spectra. The important HMBC correlations of H-7 and C-6, H-7 and C-4′, and H-7 and C-5′ were observed as shown in Figure 2. At this stage, we

Scheme 3. Acid-Promoted Dimerization/Cyclization of Oroidin (8) into Nagelamide Derivatives in HMPA or in DMSO in the Presence of Complexing Tweezer 14

C-4−C-6′ connectivity as for nagelamides A, D, and R (Schemes 1 and 3). Despite the low yield of the dimerization, it suggests the important role played by the proposed tautomers I and III in this particular reaction. The structures of the separated isomers 12a and 12b on one hand, and 16 and 17 on the other hand were determined by their MS and NMR data, particularly COSY, HMBC, and ROESY spectra (see Supporting Information). For 12a and 12b, the C-4−C-6′ connection was confirmed in particular by the H-7′ and C-4 HMBC correlation (Figure 1). The ROESY correlations H-6/H-8′ and H-7/H-8′ indicated a Z configuration for compound 12a, and the correlations H6−H7′ and

Figure 2. Clathrodin (6·1/2HCl) dimerization in HMPA into the D2h symmetrical product 19 and its comparison with the alternative C2v symmetrical 20 and selected HMBC correlations for 19.

had initially considered the second possible structure 20, which is a close derivative of benzosceptrin A (Scheme 1), but the number of aromatic C-environments (two carbon signals δC) suggested D2h symmetry rather than C2v (three carbons). The influence of the bromines on reaction selectivity is an important observation. Beyond the steric hindrance that may orient the molecular association during the progress of the reaction, the modified electronic behavior of the carboxamide due to the presence/absence of the bromine atoms could also participate in the generation of the reactive tautomeric forms. The bromination degree of the pyrrole moiety seems important during the dynamic development of the dimeric forms (Scheme 4). The formation of dimers 12a, 12b, 16, 17, and 19 from clathrodin (6·1/2HCl) and oroidin (8·1/2HCl) through their homodimerization could be rationalized from tautomers I and

Figure 1. Selected COSY, ROESY, and HMBC correlations for 12a, 12b, 16, and 17. 905

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redox state seem to be important factors determining the impressive molecular diversity of this family of marine metabolites. Although our homodimerization still requires improvement in terms of yield and chemoselectivity, it could feature an exploratory way to a short biomimetic construction of the dimeric members directly from their corresponding monomers. The biomimetic strategy has the advantage of giving access to the similarly discovered P-2-AI metabolites and undiscovered skeletons. Synthetic studies of pyrrole-2-aminoimidazole marine alkaloids on the basis of this finding are in progress.

Scheme 4. Proposed Mechanism for the Homodimerization and Rationalization of the Dynamic Tautomeric Equilibria

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EXPERIMENTAL SECTION

General Experimental Procedures. Solvents and chemicals used for the reactions were purchased from commercial suppliers and were used without further purification. The products were identified by analysis of 1H NMR, 13C NMR, IR, and HRMS. The IR spectra were measured (neat) on a Perkin-Elmer BX-FT-IR spectrometer. NMR experiments were carried out on a Bruker Avance 600 MHz and DRX 500 MHz spectrometer. The chemical shifts were referenced to the residual solvent signal (DMF-d7, δH = 2.72, 2.93, and 8.04, δC = 29.8, 34.9, and 162.4, or methanol-d4, δH = 3.31 and δC = 49.1; TFA-d4, δH = 11.65 and 2.04 and δC = 179.0 and 20.0). HRMS data were obtained with a hybrid linear trap/Orbitrap mass spectrometer (LTQ-Orbitrap, Thermo Fisher) in electrospray ionization mode by direct infusion of the purified compounds. Preparative HPLC was performed on an Autoprep system (Waters 600 controller and Waters 600 pump with a Waters 996 photo diode array detector). Samples were injected by the Waters 2700 sample manager. A Waters Sunfire C18 5 μm 19 × 150 mm column was used for preparative HPLC purifications, and a Waters Sunfire C18 5 μm 4.6 × 150 mm column was used for analytical HPLC purifications. Oroidin Preparation. Oroidin was obtained as formic acid salt by extraction and purification of the marine sponge Agelas oroides. The freeze-dried sponge was extracted three times with DCM/MeOH (1/ 1) at room temperature. After solvent evaporation, the crude extract was partitioned between H2O and n-butanol. After filtration, the nBuOH-soluble materials were purified by a C18 HPLC (Waters Sunfire C18; eluent, H2O/MeOH/HCOOH, 85/15/0.1 to 00/100/0.1 over 65 min; flow rate, 17 mL/min; UV detection at 270 nm) to afford oroidin (10.5 min, 1.5%). Oroidin formic acid salt was filtered on a silica gel column using ammonia-saturated DCM/MeOH, 85/15, to give free oroidin base. The oroidin hydrochloride salt was prepared by adding a HCl dioxane solution to oroidin in MeOH. Solvent evaporation gave oroidin·HCl. Data for oroidin were previously published. Oroidin Dimerization into 12a and 12b in HMPA. Oroidin (8·HCl) (42 mg) and free oroidin base (38 mg) were dissolved in HMPA (150 μL) and heated at 120 °C for 5.5 h under an argon atmosphere. The reaction mixture was diluted with MeOH (3 mL) and purified by C18 preparative HPLC (Waters Sunfire C18; eluent, H2O/MeOH/HCOOH, 80/20/0.1 to 70/30/0.1 over 7 min and 70/ 30/0.1 to 40/60 over 50 min; flow rate, 17 mL/min; UV detection at 270 nm) to afford five fractions, A−E. Fractions B and C showed dimeric mass compounds. Fraction B was further purified on an analytical Waters Sunfire C18 column (5 μm, 4.6 × 150 mm column; eluent, H2O/MeOH/TFA, 63/37/0.1 1 min and then 63/37/0.1 to 58/42/0.1 over 40 min; flow rate, 1 mL/min; UV detection at 270 nm) to afford compound 12a as a TFA salt (tR = 13.6 min, 3.0 mg, 2.0%). Fraction C was further purified on an analytical Waters Sunfire C18 column (5 μm, 4.6 × 150 mm column; eluent, H2O/MeOH/TFA, 72/28/0.1 to 64/36/0.1 over 60 min; flow rate, 1 mL/min; UV detection at 270 nm) to afford compound 12b as TFA salt (tR =7.2 min, 1.5 mg, 1.0%) and oroidin. Compound 12a: yellow, amorphous solid; IR (neat) νmax 3360, 2935, 2654, 2281, 1687, 1641, 1440, 1419, 1335, 1203, 1139 cm−1; 1H NMR (methanol-d4, 500 MHz) δ 6.83 (1H, s), 6.77 (1H, s), 6.69 (1H, s), 6.20 (1H, t, J = 6.5 Hz), 4.01 (2H, d, J = 6.5 Hz), 3.30 (2H, m), 2.50 (2H, m), 1.85 (2H, m); 1H NMR (DMF-d7, 600 MHz, 273 K) δ

III (Scheme 4). The intermolecular reaction would be allowed by the geometrical forced alignment of the monomer due to the strong interaction with HMPA or the bisphosphonate tweezer 14 in DMSO. For both oroidin and clathrodin, the dimerization may begin with nucleophilic attack of the tautomer I on tautomer III of another molecule. The intermediate A can undergo either direct intramolecular cyclization (path a) into the oxazolines 16/17 or tautomeric changes into B (nagelamide A) and C, giving rise to compounds 19 (path c) and 12a/12b (path e), respectively. The isolation of dihydrooroidin from the reaction mixture indicates the facile redox changes within the P-2-AI compounds. Interestingly, the vinyl-2-aminoimidazole entity of oroidin derivatives could be a hydride acceptor (imine or azafulvene forms) or hydride source (enamine forms). The latter forms would allow changes in the oxidation levels as observed for the oroidin and dihydrooroidin redox systems. The vinyl-2-aminoimidazole of oroidin thus tends to disproportionate and acts as reducing agent, whereas the corresponding imine and azafulvene forms have few oxidizing properties. Presumably, the formation of the saturated compounds such as nagelamide D can be explained by this type of dismutation or intermolecular redox reactions within the family of 2-imidazolic metabolites. In conclusion, we have effected the homodimerization of oroidin and clathrodin. Strong guanidinium and amide chelating agents were used to interfere with the intramolecular preorganization that led to the entropically and kinetically favored intermolecular cyclization. The propensity of P-2-AI derivatives to form hydrogen bonds, their facile inversion of reactivity through the tautomeric equilibria, and their unstable 906

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identical to those from the literature, while dihydrooroidin 15 was compared to a synthesized sample of 15 (see Supporting Information). Compound 16: yellow, amorphous solid; IR (neat) νmax 3373, 2940, 2654, 2297, 2071, 1984, 1682, 1437, 1343, 1207, 1143 cm−1; 1H NMR (methanol-d4, 500 MHz) δ 6.89 (1H, s), 6.79 (1H, s), 6.42 (1H, s), 4.63 (1H, m), 3.88 (1H, dd, J = 14.0 Hz, 4.6), 3.61 (1H, t, J = 6.8 Hz), 3.53 (1H, d, J = 14.0 Hz), 3.29 (2H, m), 2.99 (1H, dd, J = 15.2 Hz, 14.2), 2.86 (1H, dd, J = 14.2 Hz, 4.0), 1.92 (1H, m), 1.78 (1H, m); 1 H NMR (DMF-d7, 500 MHz) δ 13.14 (1H, br s, H-1/H-3), 13.04 (1H, br s, H-1/H-3), 12.88 (1H, br s, H-3′), 12.71 (3H, br s, H-1′, H12, H-12′), 8.28 (1H, br s, H-9′), 7.86 (2H, br s, 2-NH2/2′-NH2), 7.68 (1H, br s, H-9), 6.92 (1H, s, H-15′), 6.79 (1H, s, H-15), 6.73 (1H, s, H-4′), 4.63 (1H, br s, H-7), 4.01 (1H, t, J = 7.4 Hz, H-6′), 3.78 (1H, d, J = 13.6 Hz, H-8a), 3.49 (1H, d, J = 13.6 Hz, H-8b), 3.26 (2H, m, H-8′a, H-8′b), 3.03 (1H, m, H-6a), 2.85 (1H, m, H-6b), 2.16 (1H, m, H-7′a), 2.03 (1H, m, H-7′b); 13C NMR (DMF-d7, 125 MHz) δ 159.7 (C-10′), 158.0 (C-10), 148.8 (C-2′), 148.6 (C-2), 128.7 (C11′), 126.9 (C-5′), 126.0 (C-11), 123.4 (C-5), 118.6 (C-4), 114.4 (C15′), 113.1 (C-15), 110.1 (C-4′), 106.1 (C-13), 104.6 (C-13′), 99.7 (C-14), 98.3 (C-14′), 53.4 (C-7), 42.6 (C-8), 37.3 (C-8′), 32.2 (C-7′), 29.9 (C-6′), 26.4 (C-6); ESIMS [(M + H)+] m/z 776.9, 778.9, 780.9; HRESIMS [(M + H)+] m/z 774.8765 (calcd for C22H23N10O279Br4, 774.8739). Compound 17: yellow, amorphous solid; IR (neat) νmax 3321, 2646, 2321, 2075, 1678, 1575, 1427, 1324, 1254, 1187, 1137 cm−1; 1H NMR (methanol-d4, 500 MHz) δ 6.70 (1H, s, H-15/H-15′), 6.69 (1H, s, H-15/H-15′), 6.41 (1H, s, H-4′), 4.55 (1H, m, H-7), 3.80 (1H, dd, J = 13.6 Hz, 4.2, H-8a), 3.49 (1H, d, J = 13.6 Hz, H-8b), 3.32 (1H, t, J = 7.9 Hz, H-6′), 3.10 (2H, t, J = 6.8 Hz, H-8′a, H-8′b), 2.93 (1H, m, H6a), 2.81 (1H, dd, J = 15.3, 4.9 Hz, H-6b), 2.01 (1H, m, H-7′a), 1.70 (1H, m, H-7′b); 13C NMR (methanol-d4, 125 MHz) δ 162.0 (C-10′), 161.8 (C-10), 149.1 (C-2′), 128.5 (C-11), 127.5 (C-5′), 126.1 (C11′), 124.0 (C-4), 119.8 (C-5), 116.9 (C-15′), 114.5 (C-15), 111.3 (C-4′), 106.5 (C-13′), 101.8 (C-13), 101.1 (C-14′), 100.1 (C-14), 54.1 (C-7), 44.8 (C-8), 38.2 (C-8′), 33.3 (C-7′), 30.7 (C-6′), 28.4 (C6); ESIMS [(M + H)+] m/z 776.9, 778.9, 780.9; HRESIMS [(M + H)+] m/z 774.8773 (calcd for C22H23N10O279Br4, 774.8739). Clathrodin 6 Dimerization into 19 in HMPA. Clathrodin 6 was synthesized following the protocol from dihydropyridine derivatives.25 The clathrodin (6·HCl) salt was prepared by adding HCl/dioxane solution to free clathrodin base in MeOH solution and drying under vacuum. Clathrodin salt (6·HCl, 20 mg) and free clathrodin base (20 mg) were dissolved into MeOH and concentrated under vacuum. HMPA (100 μL) was added to the oil residue and heated at 120 °C for 2 h under an argon atmosphere before cooling to room temperature. The mixture was purified by C18 preparative HPLC (Waters Sunfire C18; eluent, H2O/MeOH/HCOOH, 98/02/0.1 over 5 min, 98/02/0.1 to 95/05/0.1 over 10 min, 95/05/0.1 over 10 min, 95/05/0.1 to 50/ 50/0.1 over 15 min; flow rate, 17 mL/min; UV detection at 270 nm) to afford an impure fraction. Further purification by C18 preparative HPLC (Waters Sunfire C18; eluent, H2O/MeCN/HCOOH, 98/02/ 0.1 over 5 min, 98/02/0.1 to 85/15/0.1 over 10 min, 85/15/0.1 over 10 min; flow rate, 17 mL/min; UV detection at 270 nm) afforded compound 19 as a formic acid salt (4 mg, 10.2%). Compound 19: white, amorphous solid; IR (neat) νmax 3152, 1669, 1185, 1129 cm−1; 1H NMR (acetic acid-d4, 600 MHz) δ 6.96 (1H, br s, H-13), 6.78 (1H, d, J = 3.0 Hz, H-15), 6.19 (1H, t, J = 3.0 Hz, H14), 3.73 (1H, t, J = 6.4 Hz, H-8a, H-8b), 3.29 (1H, t, J = 6.4 Hz, H-7a, H-7b); 13C NMR (acetic acid-d4, 150.8 MHz) δ 164.2 (C-10), 152.6 (C-2), 126.8 (C-4, C-5), 125.5 (C-11), 124.0 (C-13), 112.9 (C-15), 110.9 (C-14), 106.8 (C-6), 39.6 (C-8), 27.5 (C-7); ESIMS [(M + H)+] m/z 461.2; HRESIMS [(M + H)+] m/z 461.2162 (calcd for C22H25N10O2, 461.2161).

13.74 (1H, br s, H-1′), 13.43 (1H, br s, H-1), 13.37 (1H, br s, H-3′), 13.06 (1H, s, H-12′), 13.02 (1H, br s, H-3), 12.97 (1H, s, H-12), 8.68 (1H, t, J = 5.4 Hz, H-9′), 8.44 (2H, br s, 2′-NH2), 8.39 (1H, t, J = 5.4 Hz, H-9), 8.31 (2H, br s, 2-NH2), 7.10 (1H, s, H-15′), 7.07 (1H, s, H4′), 7.05 (1H, s, H-15), 6.34 (1H, t, J = 7.3 Hz, H-7′), 4.04 (2H, m, H8′a, H-8′b), 3.31 (2H, m, H-8a, H-8b), 2.52 (2H, t, J = 7.2 Hz, H-6a, H-6b), 1.87 (2H, t, J = 7.2 Hz, H-7a, H-7b); 13C NMR (DMF-d7, 150.8 MHz, 273 K) δ 160.0 (C-10′), 159.3 (C-10), 149.6 (C-2′), 148.4 (C-2), 129.3 (C-7′), 128.8 (C-11′), 128.6 (C-11), 126.9 (C-5′), 126.6 (C-5), 118.2 (C-6′), 116.4 (C-4), 113.1 (C-15), 112.8 (C-15), 112.5 (C-4′), 104.8 (C-13′), 104.4 (C-13), 98.4 (C-14′), 98.3 (C-14), 38.4 (C-8′), 38.1 (C-8), 28.2 (C-7), 21.6 (C-6); 15N NMR (DMF-d7, 60.8 MHz, 273 K) δ 165.5 (N-12, N-12′), 139.9 (N-1), 137.1 (N-3), 136.0 (N-3′), 132.4 (N-1′), 107.3 (N-9), 105.8 (N-9′), 58.9 (2′-N), 56.9 (2-N); ESIMS [(M + H)+] m/z 776.9, 778.9, 780.9; HRESIMS [(M + H)+] m/z 774.8725 (calcd for C22H23N10O279Br4, 774.8739). Compound 12b: yellow, amorphous solid; IR (neat) νmax 3325, 3182, 2940, 2654, 2289, 1982, 1678, 1635, 1573, 1528, 1395, 13331, 1201, 1139 cm−1; 1H NMR (methanol-d4, 500 MHz) δ 6.94 (1H, s, H4′), 6.83 (1H, s, H-15′), 6.78 (1H, s, H-15), 5.92 (1H, t, J = 7.4 Hz, H7′), 4.12 (2H, d, J = 7.4 Hz, H-8′a, H-8′b), 3.25 (2H, m, H-8a, H-8b); 2.49 (2H, t, J = 6.9 Hz, H-6a, H-6b), 1.81 (2H, t, J = 6.9 Hz, H-7a, H7b); 13C NMR (methanol-d4, 125 MHz) δ 162.5 (C-10′), 161.4 (C10), 149.2 (C-2′), 132.4 (C-7′), 128.5 (C-11′), 127.5 (C-11), 126.5 (C-5), 123.4 (C-5′), 121.4 (C-4), 118.5 (C-6′), 114.7 (C-15′), 114.1 (C-15), 114.0 (C-4′), 106.8 (C-13′), 106.0 (C-13), 38.8 (C-8′), 38.7 (C-8), 29.4 (C-7), 21.5 (C-6); ESIMS [(M + H)+] m/z 776.9, 778.9, 780.9; HRESIMS [(M + H) + ] m/z 774.8770 (calcd for C22H23N10O279Br4, 774.8739). Reduction of 12b into Debromonagelamide 13. Compound 12a (2.5 mg, 0.0032 mmol, 1 equiv) was dissolved in 2 mL of MeOH, and Pd(OH)2/C (0.25 mg, 10%) was added. The reaction was allowed to proceed under a hydrogen atmosphere for 1.5 h. The catalyst was filtered on Celite, the cake was washed with MeOH, and the solvent was removed under reduced pressure. The reaction mixture, dissolved in MeOH, was purified by C18 analytical HPLC (Waters Sunfire C18; eluent, H2O/MeOH/HCOOH, 99/01/0.1 5 min and then 99/01/0.1 to 30/70/0.1 over 30 min; flow rate, 1 mL/min; UV detection at 270 nm) to afford debromonagelamide D (13) as a formic acid salt (tR = 13.3 min, 0.9 mg, 63%). Compound 13: yellow, amorphous solid; 1H NMR (methanol-d4, 500 MHz) δ 6.92 (2H, m, H-13, H-13), 6.75 (2H, m, H-15, H-15′), 6.60 (1H, s, H-4′), 6.17 (2H, m, H-14, H-14′), 3.99 (1H, m, H-6′), 3.42 (2H, m, H-8′a, H-8′b), 3.29 (2H, m, H-8a, H-8b), 2.53 (2H, t, J = 7.0 Hz, H-6a, H-6b), 2.27 (1H, m, H-7′a), 2.11 (1H, m, H-7′b), 1.81 (2H, t, J = 7.0 Hz, H-7a, H-7b); 13C NMR (methanol-d4, 125 MHz) δ 155.0 (C-10, C-10′), 148.5 (C-2′), 130.3 (C-5′), 125.2 (C-11, C-11′), 123.1 (C-5), 121.4 (C-13, C-13′), 110.3 (C-15, C-15′), 108.8 (C-14, C-14′, C-4′), 37.6 (C-8), 36.6 (C-8′), 32.1 (C-7′), 30.4 (C-6′), 28.9 (C-7), 19.8 (C-6); ESIMS [(M + H)+] m/z 465.0. Oroidin Dimerization into 12a, 12b, 16, and 17 in the Presence of Bisphosphonate 14. Bisphosphonate 14 (140 mg, 0.098 mmol, 1 equiv) was added to a solution of oroidin·1/2HCl (80 mg, 0.196 mmol) in DMSO (150 μL). The reaction mixture was stirred under an argon atmosphere at 120 °C for 6 h. The reaction was monitored by LC-MS (Waters Sunfire C18; eluent, H2O/MeOH/HCOOH, 85/15/ 0.1 to 50/50/0.1; flow rate, 1 mL/min; UV detection at 270 nm). After filtration and CH3OH addition for a 20 mg/mL concentration, the reaction mixture was purified by C18 preparative HPLC (Waters Sunfire C18; eluent, H2O/MeOH/TFA, 60/40/0.2 to 47/53/0.2 over 10 min and then 47/53/0.2 to 20/80/0.2 over 50 min; flow rate, 17 mL/min; UV detection at 270 nm) to afford compound 9 as a TFA salt (tR =7.1 min, 20 mg, 32%), compound 16 as a TFA salt (tR =19.0 min, 3 mg, 2%), compound 17 as a TFA salt (tR = 23.7 min, 3.6 mg, 3%), compound 12a as a TFA salt (tR = 28.6 min, 3.7 mg, 3%), compound 12b as a TFA salt (tR = 29.5 min, 1.1 mg, 1%), and compound 11 as a TFA salt (tR = 34.4 min, 4 mg, 7.5%). Having the same retention time (tR), dihydrooroidin was obtained with 25% of oroidin as TFA salts. Spectroscopic data of compounds 9 and 11 were

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ASSOCIATED CONTENT

S Supporting Information *

Experimental section, tables of NMR spectroscopic data, and NMR spectra (1H NMR, 1H−1H COSY, 13C NMR, 1H−13C HMQC, and HMBC) for compounds 12a, 12b, 13, 16, 17, and 907

dx.doi.org/10.1021/np400048r | J. Nat. Prod. 2013, 76, 903−908

Journal of Natural Products

Article

(19) Araki, A.; Kubota, T.; Aoyama, K.; Mikami, Y.; Fromont, J.; Kobayashi, J. Org. Lett. 2009, 11, 1785−1788. (20) Poeverlein, C.; Breckle, G.; Lindel, T. Org. Lett. 2006, 8, 819− 821. (21) Among the reaction conditions attempted with oroidin or clathrodin hydrochloride for which monomoric products were detected along with the starting material: toluene at 100 °C; DMSO at 120 °C, H2O at 90 °C; neutral oroidin in toluene and BF3·Et2O at 100 °C; neutral oroidin in HMPA and BF3·Et2O at 100 °C. In the latter conditions, dibromopyrrole carboxamide 11 was observed. As the reaction mixtures were complex, decomposition compounds were not systematically isolated. (22) Schrader, T. H. Chem.Eur. J. 1997, 3, 1537−1541. (23) Schrader, T. H. Tetrahedron Lett. 1998, 39, 517−520. (24) Schrader, T. H. J. Org. Chem. 1998, 63, 264−272. (25) (a) Schroif-Gregoire, C.; Travert, N.; Zaparucha, A.; AlMourabit, A. Org. Lett. 2006, 8, 2961−2964. (b) Sanchez-Salvatori, M. R.; Abou-Jneid, R.; Ghoulami, S.; Martin, M.-T.; Zaparucha, A.; AlMourabit, A. J. Org. Chem. 2005, 70, 8208−8211. (26) Bhandari, M. R.; Sivappa, R.; Lovely, C. J. Org. Lett. 2009, 11, 1535−1538. (27) Endo, T.; Tsuda, M.; Okada, T.; Mitsuhashi, S.; Shima, H.; Kikuchi, K.; Mikami, Y.; Fromont, J.; Kobayashi, J. J. Nat. Prod. 2004, 67, 1262−1267.

19. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (+33) 1-69824585. Fax: (+33) 1-6907-7247. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from ICSN-CNRS is gratefully acknowledged. We thank F. Pelissier and O. Thoison for HPLC assistance and J.-F. Gallard for NMR assistance. This work is supported by CNRS (France).

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REFERENCES

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dx.doi.org/10.1021/np400048r | J. Nat. Prod. 2013, 76, 903−908